A Multifaceted Examination of the Central Processes

Underlying Vestibular Compensation

by

Raquel Sweezie

A thesis submitted in conformity with the requirements

for the degree of Philosophy Doctorate

Department of Physiology

University of Toronto

© Copyright by Raquel Sweezie 2011

ii

A Multifaceted Examination of the Central Processes

Underlying Vestibular Compensation

Raquel Sweezie

Philosophy Doctorate

Department of Physiology

University of Toronto

2011

Abstract

The vestibular system provides us with sensory information that is essential for

maintaining balance and stability. When sensory input is lost due to unilateral vestibular damage

(UVD), our ability to maintain stable gaze and posture becomes compromised. Over time,

vestibular function is partially restored through a process known as vestibular compensation,

which is associated with the rebalancing of activity in the vestibular nuclear complex (VNC) of

the brainstem. However, the physiological mechanisms associated with vestibular compensation

remain elusive. We addressed several different experimental objectives pertaining to plasticity

and sensory adaptation associated with vestibular compensation. First, we demonstrated that

systemic manipulation of γ-amino-butyric acid type B (GABAB) receptors altered the course of

vestibular behavioural recovery within the first several hours after UVD. Second, we showed

that immunohistochemical labeling of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid (AMPA) receptor subunit GluR4 was elevated in the VNC on the intact compared to

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lesioned side acutely following UVD. Third, we produced preliminary data suggesting that

excitatory responses to vestibular nerve stimulation may be acutely potentiated by UVD on the

intact side. Finally, we established that rapid sensory adaptation may increase the dynamic

ranges of vestibular neurons and perhaps improve limited vestibular reflex function in the long

term. Acutely following UVD, potentiation of vestibular nerve synapses appear to be associated

with an increase in GluR4 subunit expression in the contralesional VNC. Also, such potentiation

could be enhanced by acute modifications in pre-synaptic GABAB receptor activation. In the

long term, and independent of these plastic changes, sensory adaptation may enable the

vestibular system to overcome the persistent limitations imposed by UVD.

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Acknowledgments

This work was made possible with the support of many wonderful individuals. First of all, I am

especially grateful to my supervisor, Dr. Dianne Broussard, for her patience and for going that

extra mile to provide me with advice, guidance and constructive criticism. Next, I would like to

thank my advisory committee, Drs. Bob Harrison and Doug Tweed, for their invaluable

suggestions. Many thanks to my lab mate, Heather Titley, for her friendship, support, assistance

with my many experiments and for regularly waking up at 4 am to prepare my drugs for Chapter

2! I am also thankful to Yao-Fang Tan and Dr. Martin Wojtowicz for allowing me to use their

vibratome. The histology for Chapter 5 would not have turned out so well without it. Special

thanks to Chiping Wu, Dr. Joan S. Baizer and the Baizer lab in Buffalo for their extensive

contributions to Chapter 3. And, finally, I would like to express extreme gratitude toward those

who have worked in the Animal Resources Center at Toronto Western Hospital, especially

Donna Pires, Shawna Vandenburg, Tracey Robinson and Sarah Banks. This group of

exceptional individuals went above and beyond in making sure that my animals got the best

possible care. This work was financially supported by the Vision Science Research Program at

the Toronto Western Hospital and by the Ontario Graduate Scholarship Program.

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Table of Contents

Acknowledgments.......................................................................................................................... iv

Table of Contents .............................................................................................................................v

List of Tables ...................................................................................................................................x

List of Figures ................................................................................................................................ xi

List of Abbreviations ................................................................................................................... xiii

Chapter 1: General Introduction ................................................................................................1

1.1 The Vestibular Reflexes .......................................................................................................4

1.1.1 The Vestibular Labyrinth .........................................................................................4

1.1.2 The Horizontal Vestibulo-Ocular Reflex (VOR) .....................................................8

1.1.3 Neural Pathways for the VOR .................................................................................9

1.1.4 Vestibular Neurons Mediating the VOR in Vivo ..................................................14

1.1.5 Vestibular Neurons Mediating the VOR in Vitro ..................................................16

1.1.6 The Vestibulo-Spinal Reflexes (VSRs) .................................................................17

1.2 Compensation of the Vestibular Reflexes..........................................................................20

1.2.1 Static Compensation ..............................................................................................21

1.2.2 Dynamic Compensation of the VOR .....................................................................22

1.2.3 Dynamic Compensation of the VSR ......................................................................23

1.3 Neuronal Changes Associated with Vestibular Compensation .........................................24

1.3.1 Acute Neuronal Changes Associated with UVD ...................................................24

1.3.2 Overview of Neuronal Changes Associated with Static and Dynamic

Compensation ........................................................................................................25

1.3.3 Static and Dynamic Compensation Depend on Acute Changes in the Intrinsic

Excitability of VNC Neurons.................................................................................26

1.3.4 Static and Dynamic Compensation are Dependent on Cerebellar Inhibition ........27

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1.3.5 Dynamic but not Static Compensation Depends on Commissural Inputs .............28

1.4 Neurochemistry of Vestibular Compensation ....................................................................29

1.4.1 GABA and Normal Vestibular Function ...............................................................30

1.4.2 GABA in Static and Dynamic Compensation .......................................................31

1.4.3 GABA Receptors in Normal and Impaired Vestibular Function ...........................32

1.4.4 Glutamate in VNC During Normal and Impaired Vestibular Function .................36

1.4.5 Glutamate Receptors: Roles in Normal and Compromised Vestibular Function ..37

1.5 Plasticity in the VNC: The Foundation for Vestibular Compensation ..............................43

1.5.1 Synaptic Plasticity in the Normal VNC .................................................................43

1.5.2 Central Plasticity and Early-Stage Compensation .................................................45

1.5.3 Central Plasticity Associated with Late-Stage Compensation ...............................46

1.6 Sensory Adaptation in the VNC: Overcoming the Limits of Dynamic Compensation .....47

1.6.1 Sensory Adaptation in the VNC ............................................................................48

1.6.2 Adaptive Rescaling and its Implications for Dynamic Reflex Function after

UVD .......................................................................................................................49

1.6.3 Efferents, Afferents and Adaptive Rescaling.........................................................50

1.7 Final Remark ......................................................................................................................50

1.8 List of Hypotheses .............................................................................................................51

Chapter 2: GABAB Receptors Contribute to Early Recovery of Balance Following

Unilateral Vestibular Damage in Mice .....................................................................................52

2.1 Introduction ........................................................................................................................52

2.2 Methods..............................................................................................................................53

2.2.1 Animals ..................................................................................................................53

2.2.2 Determining Drug Dosages and Comparing Activity Levels ................................54

2.2.3 Experimental Protocol ...........................................................................................55

2.2.4 Surgical Procedure .................................................................................................55

2.2.5 Evaluating Static Vestibular Reflexes ...................................................................56

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2.2.6 Evaluating Dynamic Vestibular Reflexes ..............................................................56

2.2.7 Statistics .................................................................................................................59

2.2.8 Grip Test ................................................................................................................60

2.2.9 Termination ............................................................................................................60

2.3 Results ................................................................................................................................60

2.4 Discussion ..........................................................................................................................67

Chapter 3: GluR4-Containing AMPA Receptors are altered during the Acute Stage of

Vestibular Compensation in Mouse Vestibular Nuclei .............................................................72

3.1 Introduction ........................................................................................................................72

3.2 Methods..............................................................................................................................72

3.2.1 Animals ..................................................................................................................73

3.2.2 Collaboration..........................................................................................................73

3.2.3 Surgical Procedure .................................................................................................73

3.2.4 Evaluating Static Vestibular Reflexes ...................................................................73

3.2.5 Immunohistochemistry ..........................................................................................74

3.2.6 Data Analysis .........................................................................................................75

3.2.7 Statistics .................................................................................................................76

3.3 Results ................................................................................................................................76

3.4 Discussion ..........................................................................................................................79

Chapter 4: Excitatory and Inhibitory Synaptic Transmission during the Acute Stage of

Vestibular Compensation in the Mouse MVN ..........................................................................82

4.1 Introduction ........................................................................................................................82

4.2 Methods..............................................................................................................................82

4.2.1 Animals ..................................................................................................................83

4.2.2 Evaluating Static Vestibular Reflexes ...................................................................83

4.2.3 Survival Times .......................................................................................................83

4.2.4 Slice Preparation ....................................................................................................84

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4.2.5 Recording Method .................................................................................................85

4.2.6 Stimulation Method ...............................................................................................85

4.2.7 Stimulus Parameters...............................................................................................87

4.2.8 Data Analysis .........................................................................................................87

4.2.9 Statistics .................................................................................................................89

4.3 Results ................................................................................................................................89

4.3.1 Acute Effects of UVD at the NVIII Synapse in the Contralesional MVN ............90

4.3.2 Acute Effects of UVD at Commissural Synapses in the Ipsilesional MVN ..........93

4.4 Discussion ..........................................................................................................................96

Chapter 5: Adaptive Rescaling is Preserved in the Vestibular Nuclei and Extends the

Dynamic Ranges of Central Neurons after Unilateral Vestibular Damage ............................100

5.1 Introduction ......................................................................................................................100

5.2 Methods............................................................................................................................100

5.2.1 Animals ................................................................................................................100

5.2.2 Surgical Procedure ...............................................................................................101

5.2.3 Recording Eye Movements ..................................................................................102

5.2.4 Recording Single Unit Responses ........................................................................103

5.2.5 Data Analysis .......................................................................................................105

5.2.6 Statistics ...............................................................................................................108

5.3 Results ..............................................................................................................................109

5.4 Discussion ........................................................................................................................122

Chapter 6: Concluding Remarks ............................................................................................127

6.1 Acute Changes in GABAB Receptor Activation Following UVD ...................................127

6.2 Actions of GABAB Receptors and Acute Changes in Neurotransmission in the

Ipsilesional VNC ..............................................................................................................127

6.3 GABAB Receptors and Acute Changes in Excitatory Neurotransmission in the

Contralesional VNC Following UVD ..............................................................................130

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6.4 Sensitivity Rescaling in the VNC Following Compensation for UVD ...........................132

References ....................................................................................................................................133

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List of Tables

Table 2-1 Experimental Groups……………………………………………………………….53

Table 4-1 Effects of Cesium on Resting Membrane Properties……………………................92

Table 4-2 Summary of Input/Output Curves for each Response Type from Commissural

Stimulation Experiments……………………………………………………………94

Table 5-1 Summary of Response Types……………………………………………………..108

Table 5-2 Number of Cell Types Recorded for Each Lesion………………………..............110

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List of Figures

Figure 1-1 Projection of NVIII to the VNC…………………………………………................2

Figure 1-2 The Vestibular Labyrinth…………………………………………………………...5

Figure 1-3 The Semicircular Canal………………………………………………….................7

Figure 1-4 The VOR Network………………………………………………………...............10

Figure 1-5 Commissural Connections in the VOR Network………………………………….12

Figure 1-6 Cerebellar Connections in the VOR Network……………………………………..14

Figure 1-7 The VSR Network…………………………………………………………………18

Figure 2-1 The Beam Crossing Apparatus…………………………………………………….57

Figure 2-2 Methods for Analyzing Gait……………………………………………………….59

Figure 2-3 Timeline for Experimental Procedures Before Surgery…………………...............61

Figure 2-4 Effects of Saline-Based Test Substances on Static Signs…………………………62

Figure 2-5 Effects of Methylcellulose-Based Substances on Static Signs…………................64

Figure 2-6 Experimental Effects on Beam Crossing Ability………………………................65

Figure 2-7 Effects of UVD on Beam Crossing Ability……………………………………….66

Figure 2-8 Experimental Effects on Gait……………………………………………………..67

Figure 3-1 Examples of GluR4 Labeling……………………………………………………..76

Figure 3-2 Examples of GluR1 Labeling……………………………………………………...77

Figure 3-3 Effects of UVD on GluR4 Cell Densities…………………………………………78

Figure 3-4 Effects of UVD on GluR1 Cell Densities…………………………………………79

Figure 4-1 Time Course for Behavioural Recovery in Mouse Pups……………….................84

Figure 4-2 Placement of Electrodes in the Slice Preparation………………………………....86

Figure 4-3 Latency Distributions……………………………………………………………..88

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Figure 4-4 Input/Output Functions from Afferent Stimulation Experiments…………………90

Figure 4-5 EPSCs from Afferent Stimulation Experiments…………………………………..91

Figure 4-6 Effects of Cesium on the EPSC……………………………………………………93

Figure 4-7 Input/Output Functions from Commissural Stimulation Experiments……………94

Figure 4-8 Pure Excitatory and Inhibitory Post-Synaptic Currents……………………………95

Figure 4-9 Mixed Post-Synaptic Currents………………………………………….................96

Figure 5-1 Methods for Measuring Sensitivity………………………………………………107

Figure 5-2 Effect of Lesion Type on Rescaling……………………………………...............111

Figure 5-3 Summary of Sensitivity Measured Relative to Peak Head Velocity……………..113

Figure 5-4 Phase Relationship for Velocity and Acceleration……………………………….114

Figure 5-5 Summary of Phase Measured Relative to Peak Head Velocity………………….115

Figure 5-6 Summary of Sensitivity Measured Relative to Frequency……………………….116

Figure 5-7 Effect of Eye Movement Sensitivity on Rescaling………………………………117

Figure 5-8 Summary of Zero-Velocity Spike Density……………………………………….118

Figure 5-9 Summary of Response Linearity…………………………………………………119

Figure 5-10 Time Course for Sensitivity Changes…………………………………………….121

Figure 5-11 Summary of the VOR Before and After UVD…………………………...............122

Figure 6-1 Illustration of Acute Changes in Ipsilesional Commissural Synapses

Following UVD.....................................................................................................128

Figure 6-2 Illustration of Acute Changes in Contralesional NVIII Synapses

Following UVD.....................................................................................................131

xiii

List of Abbreviations

2-DG 2-deoxyglucose

AHP after-hyperpolarization

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AraC cytosine-β-D arabinofuranoside

BK big conductance potassium

CNS central nervous system

CsM cesium methanesulfonate

CP canal plug

DVN descending vestibular nucleus

EM eye movement

EMG electromyographic

EPSC excitatory post-synaptic current

EPSP excitatory post-synaptic potential

FTN flocculus target neuron

GABA γ-amino-butyric acid

GABAA γ-amino-butyric acid type A

GABAB γ-amino-butyric acid type B

HFS high-frequency stimuli

I/O input/output

IPSC inhibitory post-synaptic current

IPSP inhibitory post-synaptic potential

LFS low-frequency stimuli

LRN lateral reticular nucleus

LTD long-term depression

LTP long-term potentiation

LVN lateral vestibular nucleus

LVST lateral vestibule-spinal tract

mGluR metabotropic glutamate receptor

MVN medial vestibular nucleus

MVNmc magnocellular division of the medial vestibular nucleus

MVNpc parvocellular division of the medial vestibular nucleus

NMDA N-methyl-D-aspartate

NVIII eigth cranial nerve

SK small conductance potassium

SVN superior vestibular nucleus

THIP 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol

UL unilateral labyrinthectomy

UVD unilateral vestibular damage

UVN unilateral vestibular neurectomy

VNC vestibular nuclear complex

VO vestibular-only

VOR horizontal vestibulo-ocular reflex

VSR vestibulo-spinal reflex

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Chapter 1: General Introduction

The vestibular system operates to maintain balance and stabilize gaze whenever we move our

heads. Head motion is initially detected by a pair of vestibular labyrinths, one located in the

inner ear on each side of the head. Each labyrinth is composed of a group of sensory organs

which function collectively to detect head motion through three-dimensional space. Depending

on the type of motion we undertake, the sensory receptors in the vestibular labyrinth will cause

activation of the appropriate muscles in the eyes, neck and/or limbs in order to compensate for

the movement, keeping our eyes on target and our bodies upright and balanced. The appropriate

motor responses to motion are carried out through a network of neurons that link the inner ear

with motor neurons in the brainstem and spinal cord. The sensory receptors in the end organs are

linked to the CNS by way of the primary afferents. The axons of the primary afferents project

through the eighth cranial nerve (NVIII) and synapse onto neurons in the VNC in the brainstem

(Figure 1-1). All afferent axons enter the VNC on its most lateral aspect and branch throughout

(Lorente de No 1933). The VNC lies at the base of the fourth ventricle on either side of the

brainstem and contains four main divisions or nuclei – the medial vestibular nucleus (MVN), the

lateral vestibular nucleus (LVN), the descending vestibular nucleus (DVN) and the superior

vestibular nucleus (SVN). The neurons located in these nuclei transmit sensory information

received by the primary afferents to motor neurons or pre-motor neurons located upstream in the

oculomotor centers and/or downstream in the spinal cord. When the vestibular network is

disrupted by inflicting damage or disease upon the labyrinth unilaterally, gaze stabilization and

postural balance become heavily compromised. Such UVD can be neural or structural.

In cases where UVD manifests as neural damage, the hair cells or vestibular afferents are usually

affected (Brandt and Daroff 1980; Susilawati et al. 1997), resulting in a unilateral loss of neural

input to the VNC. Depending on the severity of the damage, VNC neurons on the damaged

(ipsilesional) side become less active or even completely silent (Shimazu and Precht 1966;

Markham et al. 1977; Smith and Curthoys 1988a; Smith and Curthoys 1988b; Newlands and

Perachio 1990a; Newlands and Perachio 1990b). This loss of input to the VNC therefore

disrupts the balance of neural activity between the ipsilesional VNC and the VNC on the intact

(contralesional) side and leads to an acute dysfunction of the vestibular reflexes. However,

2

depending on the species being affected, a balance in neural activity in the brainstem is re-

established within days to weeks and, by the end of this recovery period, vestibular function is

partially restored (Precht et al. 1966; Baarsma and Collewijn 1975; Lacour et al. 1976; Haddad et

al. 1977; Maioli et al. 1983; Paige 1983; Sirkin et al. 1984; Fetter and Zee 1988; Ott and Platt

1988; Black et al. 1989; de Waele et al. 1989; Halmagyi et al. 1990; Newlands and Perachio

1990a; Cass and Goshgarian 1991; Ris et al. 1997; Gilchrist et al. 1998; Hamann et al. 1998;

Broussard et al. 1999; Kaufman et al. 1999; Lasker et al. 1999; Lasker et al. 2000; Magnusson et

al. 2000; Galiana et al. 2001; Magnusson et al. 2002; Broussard and Hong 2003; Gliddon et al.

2004; Murai et al. 2004; Faulstich et al. 2006; Sadeghi et al. 2006; Beraneck et al. 2008;

Bergquist et al. 2008). This process of neural rebalancing between the bilateral VNC and

behavioural recovery is known as vestibular compensation. All animals and subjects that have

already undergone this process will therefore be referred to as ―compensated‖.

Figure 1–1: Projection of NVIII through each division of the VNC. The orange circles represent the cell bodies of

individual neurons. VNC = vestibular nuclear complex, SVN = superior vestibular nucleus, MVN = medial

vestibular nucleus, LVN = lateral vestibular nucleus, DVN = descending vestibular nucleus.

3

In cases where UVD manifests itself as structural damage, the labyrinthine structure is damaged

causing vertigo through inappropriate activation of the vestibular reflexes (Brandt and Daroff

1980; Furman and Cass 1999; Maroun and Megerian 2010). If conditions causing damage to the

labyrinthine structure are left untreated, episodes of vertigo would continue throughout the

patient’s life. When conservative treatment with medication or physical therapy fails in these

patients, surgical intervention to remove or block the affected labyrinth or end organ is indicated

(Sismanis 2010; Teufert and Doherty 2010). The complete unilateral loss of input through

surgery leads to the induction of compensation in these patients and relieves them of their

vertigo. Surgical treatments that are considered highly effective for treating vertigo include

unilateral vestibular neurectomy (UVN), which is a severing of NVIII (House 1961) or unilateral

labyrinthectomy (UL), destruction of the entire labyrinth (Pulec 1974). UL is usually the

preferred choice since it leads to a better recovery than UVN (Cass et al. 1991). The UL also

results in nearly complete silencing (Sirkin et al. 1984) and eventual degeneration of the primary

afferents (Schuknecht 1982). Another form of surgical UVD that can be quite effective for

treating certain conditions is canal plug (CP) surgery (Parnes and McClure 1990; Crane et al.

2008; Charpiot et al. 2010), which was originally developed by Ewald for experimental

investigation in 1892 (Ewald 1892). In this case, the lumen of one or more semicircular canals

(see Section 1-1-1 and Figure 1-3 for a description of the semicircular canal) is opened and

plugged with bone dust or bone wax. Unlike the UL or UVN, canal plugging has the benefit of

not destroying the hair cells or inactivating the primary afferents, rather it simply prevents

movement of the endolymph thereby blocking activation of the canal.

While all of the aforementioned surgical treatments have proven to be very successful at treating

vertigo, patients who undergo these procedures, as well as those who have suffered neural

damage, are left with limited vestibular function, even after compensation has taken place. This

is because, as mentioned earlier, the normal operation of the vestibular reflexes can only be

partially restored through compensaton (Curthoys and Halmagyi 1995). Symptoms associated

with a unilateral loss of neural input to the VNC can be observed while the patient is stationary

(static signs) or moving around (dynamic signs). Typically, compensation results in a

disappearance of static signs but only a partial resolution of dynamic signs (Smith and Curthoys

1989). Numerous studies have been conducted in humans and animals in attempt to understand

the mechanisms underlying static and dynamic compensation (Smith and Curthoys 1989;

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Curthoys and Halmagyi 1995; Darlington et al. 2002; Lacour and Tighilet 2010). The results

from such studies have greatly improved our knowledge of vestibular compensation, though

much about this topic still remains elusive. In this introductory chapter we will review the

literature and propose new hypotheses that could advance our understanding of the physiology of

vestibular compensation.

1.1 The Vestibular Reflexes

Before we begin our discussion of vestibular compensation, the vestibular reflexes will be

reviewed. In this section, we will provide background on the structure and function of the

vestibular labyrinth, in addition to relevant anatomy and physiology for neural pathways

mediating the VOR and VSRs.

1.1.1 The Vestibular Labyrinth

Activation of all vestibular reflexes begins in the vestibular labyrinth, where head motion is

detected. The vestibular labyrinth is comprised of five different end organs, three semicircular

canals and two otolith organs (Figure 1-2). The three canals are roughly orthogonal to one

another (Blanks et al. 1972). The horizontal canal responds best to rotation about an Earth-

vertical axis while the two vertical canals respond mostly to rotation about Earth-horizontal axes.

The two otolith organs are also approximately perpendicular to each other and respond to linear

motion generated by translation, which is movement from one point to another in a given plane

(Wilson and Jones 1979). The utricle and saccule respond, respectively, to horizontal and

vertical translational motion and both otolith organs respond to gravity.

5

Figure 1–2: Diagrammatic illustration of the vestibular labyrinth

The semicircular canals are each composed of a hollow ring that continues into a bulging region

known as the ampulla (Figure 1-3). The interior of the canal, or its lumen, is filled with a fluid

known as endolymph. Inside the ampulla a flexible mass known as the cupula spans its entire

cross-section from base to apex (Dohlman 1935). The cupula can be deflected back and forth

(Dohlman 1969). When the head moves, there is a change in the endolymphatic pressure

(Rabbitt et al. 1994), which forces the endolymph to move through the canal in the opposite

direction but the same rate as the head. The change in endolymphatic pressure and movement of

the endolymph then cause the cupula to deflect. Recordings from primary afferents in mammals

revealed that, during sinusoidal rotation, the signal being transmitted from the canal to the

primary afferents is head velocity, or the velocity at which the cupula is deflected (Fernandez

and Goldberg 1971; Keller 1976; Anderson et al. 1978). Much work has been done to determine

6

how cupular deflection leads to signal transmission in the primary afferent. Early work by

Loewenstein and Sand (Loewenstein and Sand 1936) demonstrated that the afferent axons

projecting from below the ampulla increased their firing rates when the cupula was deflected in

one direction and decreased their firing rates for cupular deflection in the opposite direction.

Recordings of the electrical potential in the ampulla of the horizontal canal (Trincker 1957) were

consistent with Loewenstein and Sand’s results. The electrical potential measured in the ampulla

became depolarized during cupular deflection toward the utricle and then hyperpolarized during

deflection in the opposite direction. How these changes in electric potential occur has become

more evident since early electron microscopy studies had revealed the structure of the sensory

receptors, or hair cells, located in the crista at the base of the ampulla (Wersaell 1956). Each hair

cell has a set of rod-like structures, the stereocilia and kinocilium, which protrude out into the

cupula (Figure 1-3). The stereocilia are arranged from shortest to tallest against the much thicker

and taller kinocilium. The position of the kinocilium determines the polarity of the hair cell.

When the stereocilia are deflected towards the kinocilium the hair cells themselves become

depolarized, but when the stereocilia are deflected in the opposite direction a hyperpolarization

results (Hudspeth and Corey 1977; Shotwell et al. 1981). In each canal the polarities of the hair

cells are all the same and a deflection of the cupula can either depolarize or hyperpolarize all hair

cells simultaneously. More specifically, the hair cells are depolarized during ipsilateral head

rotation and hyperpolarized during contralateral head rotation (ie. rotation toward and away from

the side of the canal, respectively).

7

Figure 1–3: Structural features of the semicircular canal. The double-headed arrow across the cupula indicates that

this flexible structure can be deflected in two directions.

Compared to the semicircular canals, the otolith organs are slightly more complex but operate

under similar principles. Each otolith organ forms a flat, plate-like structure that is made up of

two main layers. The bottom layer, the macula, is where all of the hair cells are located. Each

hair cell in the macula projects its stereocilia and kinocilium into the otolith membrane, the top

layer, which contains a high density of calcite (calcium carbonate) crystals (Carlstrom et al.

1953). When the head moves in a given direction, inertial forces cause the calcium carbonate

crystals, or otoconia, to shift, causing the otolith membrane to undergo a displacement. The

velocity of this displacement is then transmitted to the primary afferents (Fernández and

Goldberg 1976; Anderson et al. 1978). The otolith organs have hair cells that are arranged in

many different polarities (Flock 1964), which ensures a response to linear motion or gravity in

any direction within the plane of the macula. Gravitational forces, which are maximal in any

8

Earth-vertical plane, are placed on the otolith membrane whenever the macular plane is

positioned outside of the Earth-horizontal plane.

1.1.2 The Horizontal Vestibulo-Ocular Reflex (VOR)

The normal VOR activated by Earth-vertical axis rotation is well-documented and its operation

is well-understood. When the head rotates horizontally about an Earth-vertical axis, the eyes

respond reflexively by moving at a velocity (angular, in deg/s) that is approximately equal and

opposite to that of the head. During sinusoidal rotation, for example, the peak head velocity

would be exactly 180 degrees out of phase with the peak eye velocity. This reaction cancels the

effect of head velocity on the visual field. Such gaze stabilization has to occur over a wide range

of frequencies and velocities as the majority of humans can generate horizontal head movements

with velocities up to 170 deg/s and frequencies up to 3 Hz during locomotion (Grossman et al.

1988). The human VOR can actually produce compensatory eye movements during mid-

frequency (0.2-2 Hz) head rotation with velocities reaching as high as 350 deg/s (Pulaski et al.

1981). The response of the VOR in this range, as demonstrated by Paige (1983), is linear and its

performance, or gain (ratio of eye speed to head speed), is consistent over several different peak

velocities ranging from 40 to 360 deg/s. The VOR is also effective during more rapid head

movements with higher frequencies. During low-velocity rotation (10-20 deg/s) eye movements

were perfectly compensatory for frequencies up to 2 Hz in humans (Tabak and Collewijn 1994),

5 Hz in cats (Broussard et al. 1999), 6 Hz in Rhesus monkeys (Keller 1978) and 10 Hz in squirrel

monkeys (Minor et al. 1999). At higher rotational frequencies, the peak eye velocity starts to lag

behind the peak head velocity and gaze stabilization is no longer perfect. The timing of eye

velocity relative to head velocity is measured in terms of phase angle, in degrees, between the

peaks of the two sine functions.

In the range of frequencies and velocities in which compensatory eye movements are generated,

the gain of the VOR in the light is near unity. However, measurements of the VOR are usually

conducted in the dark since visual following mechanisms (ie. smooth pursuit and optokinetic

reflex) can contribute to gaze stabilization. The gain of the VOR in the dark can vary depending

on the frequency of rotation. Visual following mechanisms operate mainly at frequencies below

1 Hz (Martins et al. 1985), therfore the gain of the VOR measured in the dark is usually reduced

9

at low compared to high frequencies (Keller 1978; Minor et al. 1999). Overall, visual pathways

contribute only slightly to the overall gain value and the VOR still serves as the main channel for

gaze stabilization during head movements.

1.1.3 Neural Pathways for the VOR

The most basic reflex arc responsible for generating compensatory eye movements during head

rotation is composed of only three neurons (Szentagothai 1950). There are two pairs of muscles

responsible for carrying out horizontal eye movements, the medial rectus and lateral rectus. The

three-neuron arc describes an excitatory pathway from the horizontal canal to the contralateral

lateral rectus (Figure 1-4, black pathway) or an inhibitory pathway from the horizontal canal to

the ipsilateral lateral rectus (Figure 1-4, red pathway). The excitatory pathway consists of the

primary afferent projecting through NVIII from the horizontal canal to an excitatory second

order neuron in the ipsilateral MVN. This second order neuron then projects to the contralateral

abducens nucleus where it activates a lateral rectus motor neuron. The inhibitory pathway is

similar except the second order neuron in the ipsilateral MVN projects to the ipsilateral abducens

where it deactivates a lateral rectus motor neuron (Baker et al. 1969). Electrical stimulation of

the ipsilateral vestibular nerve also leads to the activation of the ipsilateral medial rectus (Ito et

al. 1976). Field potentials in the division of the ipsilateral oculomotor nucleus containing the

medial rectus motor neurons are generated by activation of the contralateral abducens nucleus

(Baker and Highstein 1975; Highstein and Baker 1978). Thus, activation of the ipsilateral

second order neurons also leads to activation of the ipsilateral medial rectus motor neurons

(Figure 1-3, pink pathway). As the agonist muscles contract, the antagonist muscles, the

ipsilateral lateral rectus and contralateral medial rectus, are inhibited and relaxed allowing the

eyes to be pulled away from the side of NVIII stimulation (Ito et al. 1976). The lateral and

medial rectus muscles are inhibited, respectively, by activation of the inhibitory neurons shown

in the red and blue pathways of Figure 1-4.

10

Figure 1–4: Illustration of some basic neural pathways involved in the control of the horizontal vestibulo-ocular

reflex. For rotation toward the black canal, the black pathway excites the LR and the blue pathway inhibits the MR.

For rotation in the opposite direction (ie. toward the pink canal), the pink pathway excites the MR while the red

pathway inhibits the LR. HSCC = horizontal semicircular canal; MVN = medial vestibular nucleus; Ab = abducens

nucleus; OMN = oculomotor nucleus; MR = medial rectus; LR = lateral rectus. Solid axons are excitatory and

dashed axons are inhibitory.

The VOR is modulated by excitatory and inhibitory input through crossed projections between

the vestibular nuclei on both sides of the brainstem (Figure 1-5, blue and purple pathways).

11

These crossed projections are collectively known as the vestibular commissure and they function

to adjust the depth at which responses of vestibular neurons are modulated during head

movement. In other words, they influence the sensitivities of vestibular neurons. Vestibular

fibers that cross the midline were first identified in cats through anatomical investigation of the

paths followed by degenerated neurons following a lesion to the vestibular nuclei on one side

(Gray 1926). Shimazu & Precht (Shimazu and Precht 1966) demonstrated that a subset of

neurons in the vestibular nucleus was excited by activation of the contralateral NVIII. Some of

these neurons were thought to be inhibitory interneurons that inhibit second order MVN neurons

during contralateral rotation (―i‖ in Figure 1-5). These results were confirmed through a later

investigation (Nakao et al. 1982). While it is well accepted that commissural inhibition of

contralateral second order neurons is carried out through contralateral inhibitory interneurons,

the second order neurons can also be directly inhibited as shown by the purple pathway in Figure

1-5 (Kasahara et al. 1968; Mano et al. 1968; Ito et al. 1970). Inhibitory projections connecting

the VNC on both sides were revealed by imaging sections obtained from mutant mice that

selectively express green fluorescent protein in their inhibitory neurons (Bagnall et al. 2007).

Even more recently it was shown in the frog that 70% of the inhibitory responses to contralateral

NVIII stimulation are in fact disynaptic (Malinvaud et al. 2010). This pathway is known as the

inhibitory vestibular commissure and it acts to increase the sensitivity of second order neurons to

head rotation (Kasahara and Uchino 1971).

12

Figure 1–5: Commissural connections in the horizontal vestibulo-ocular reflex network. MVN = medial vestibular

nucleus, i = inhibitory interneuron. Solid axons are excitatory and dashed axons are inhibitory.

A substantial amount of evidence also supports the existence of an excitatory vestibular

commissure. After Shimazu & Precht (1966) showed that contralateral NVIII stimulation can

induce excitatory responses in the MVN a number of other studies have confirmed their results

in a variety of different species [frog: (Ozawa et al. 1974); cat: (Farrow and Broussard 2003);

monkey: (Goldberg et al. 1987; Broussard and Lisberger 1992); guinea pig: (Babalian et al.

1997); turtle: (Ariel et al. 2004); mouse: (Broussard 2009)]. In some cases it was found that

13

second order vestibular neurons, not just interneurons, received direct excitatory inputs from the

contralateral canal (Broussard and Lisberger 1992; Farrow and Broussard 2003; Ariel et al.

2004). It has also been reported that some neurons in the MVN receive a mixture of inhibitory

and excitatory commissural inputs (Goldberg et al. 1987; Babalian et al. 1997).

The excitation and inhibition resulting from activation of the horizontal canals can be further

modulated at the level of the second order MVN neurons through a side loop that courses

through the cerebellum (Figure 1-6, red pathway). Stimulation of NVIII leads to activation of

Purkinje cells in the ipsilateral flocculus (Precht and Llinas 1969). Vestibular mossy fibers

project onto floccular granule cells which in turn synapse onto the Purkinje cells by way of

parallel fibers. Purkinje cells in the flocculus project directly into the MVN (Langer et al. 1985a;

Sekirnjak et al. 2003) where they inhibit second order vestibular neurons (Ito et al. 1970;

Shimazu and Smith 1971; Highstein 1973b; Lisberger and Pavelko 1988; Sato et al. 1988;

Sekirnjak et al. 2003). The MVN units that receive floccular inhibition are known as the

flocculus target neurons (FTNs) (Lisberger and Pavelko 1988). Unlike the majority of second

order neurons, the FTNs do not appear to receive input from the inhibitory commissure. Instead,

these neurons receive excitatory commissural inputs (Figure 1-6, blue pathway). This is

supported by a study in which stimulation of the contralateral NVIII produced either an

increase in the probability of firing or no response in the FTNs (Broussard and Lisberger 1992).

Also, FTNs are thought to belong to the group of second order vestibular neurons that send

inhibitory projections to the ipsilateral abducens nucleus (Baker et al. 1969; Scudder and Fuchs

1992; Lisberger et al. 1994). In fact, electrical stimulation of the flocculus leads to a reduction of

inhibitory field potentials in the ipsilateral abducens nucleus while the excitatory field potentials

in the contralateral abducens remain unaffected (Babalian and Vidal 2000). In general, the FTNs

form a unique class of second order vestibular neuron that functions in the adaptive modification

of VOR gain (Lisberger and Pavelko 1988).

14

Figure 1–6: Cerebellar connections in the horizontal vestibulo-ocular reflex network. MVN = medial vestibular

nucleus; FTN = flocculus target neuron; PC = Purkinje cell; GC = granule cell. Solid axons are excitatory and

dashed axons are inhibitory.

1.1.4 Vestibular Neurons Mediating the VOR in Vivo

It is evident, just from the description of the VOR pathways, that neurons in the MVN can serve

a variety of different functions and can receive inputs other than those provided by the horizontal

canal afferents. In fact, these neurons often send projections to other areas of the brain and

15

spinal cord, and they also receive and integrate information from more than one vestibular end

organ as well as from different regions in the central nervous system (Barmack 2003; Uchino et

al. 2005; Zwergal et al. 2009). Due to the large amount of signal processing that occurs in the

MVN, the timing of the peak responses of individual MVN neurons and primary afferents are not

usually perfectly matched. The peak responses of MVN neurons tend either to lag behind or to

lead ahead of the peak afferent responses and the degree to which the responses lead or lag

shows a large amount of variation between different MVN neurons (Dickman and Angelaki

2004). However, as a population, during mid-frequency rotation (0.2-2 Hz) where vestibular-

evoked eye movements are compensatory in most species, the MVN neurons usually respond

closely in phase with head velocity (Lisberger and Miles 1980; Newlands and Perachio 1990a;

Newlands and Perachio 1990b). That is, the maximum response for a population of vestibular

neurons will occur at almost the same time as peak head velocity during rotation.

Among the population of MVN neurons, differences also exist in response types, not just

dynamics. Four different groups of MVN neurons have been classified according to the

direction of head rotation to which they respond (Duensing and Schaeffer 1958). First, there are

Type I neurons for which the ―on‖ direction is rotation toward the ipsilateral canal. Thus, Type I

units increase their firing rate during ipsilateral rotation. Next, there are Type II neurons which

increase their firing during contralateral rotation. Finally, there are Type III and Type IV

neurons that, respectively, increase and decrease their discharge for both directions of rotation.

Type III and IV responses are not very common and are rarely observed at rotational frequencies

below 4 Hz (Broussard et al. 2004) however Type I and Type II responses to horizontal angular

rotation are frequently observed in the MVN of alert animals [monkeys: (Henn et al. 1974; Fuchs

and Kimm 1975; Keller and Daniels 1975; Lisberger and Miles 1980; Scudder and Fuchs 1992;

Lisberger et al. 1994; Dickman and Angelaki 2004) cats: (Cheron et al. 1996; Broussard et al.

2004) guinea pigs: (Smith and Curthoys 1988a; Smith and Curthoys 1988b) gerbils: (Newlands

and Perachio 1990a; Newlands and Perachio 1990b) mice: (Beraneck and Cullen 2007)]. Most

Type I units are second order neurons and they usually carry out the sensory-to-motor

transformation between the afferents and contralateral abducens motor neurons (Scudder and

Fuchs 1992). Type II units were originally thought to represent the inhibitory interneurons

responsible for commissural inhibition (Shimazu and Precht 1966), however it was later

16

discovered that some second order neurons, particularly FTNs, can also behave as Type II units

(Scudder and Fuchs 1992; Lisberger et al. 1994).

The responses of many MVN neurons are also shaped by eye movement inputs (Henn et al.

1974; Miles 1974; Fuchs and Kimm 1975; Keller and Daniels 1975; Waespe and Henn 1977;

Lisberger and Miles 1980). This is to be expected since, as we saw earlier, visual following

mechanisms contribute to the VOR in the light. Of the vestibular neurons that do respond to eye

movements, the majority of them are sensitive to the position of the eye in the orbit (Scudder and

Fuchs 1992). Collectively, these neurons are categorized as eye movement-sensitive (EM) cells

while those that respond only to head rotation are classified as vestibular-only (VO) cells

(Broussard et al. 2004; Dickman and Angelaki 2004; Beraneck and Cullen 2007).

1.1.5 Vestibular Neurons Mediating the VOR in Vitro

Much of what is currently known of MVN neuron physiology comes from in vitro studies of

MVN neurons in brain slice preparations. It will become apparent in later discussion that one of

the most important findings in vitro was the observation that MVN neurons are spontaneously

active when no longer under the influence of their synaptic inputs (Gallagher et al. 1985; Dutia et

al. 1992). Part of what enables the MVN neurons to spontaneously fire is their intrinsic ability

to generate action potentials in the absence of synaptic inputs. This intrinsic excitability of MVN

neurons is made possible by a variety of channels that control the flux of ions across their

membranes during action potential generation (Serafin et al. 1991a; Serafin et al. 1991b;

Johnston et al. 1994; Smith et al. 2002). Inward sodium currents passing through voltage-gated

channels are important for the generation and depolarization phase of the action potential spike,

while outward potassium currents passing through voltage-gated channels act to repolarize the

cell. The afterhyperpolarization (AHP) that follows the repolarization phase is dependent on a

combination of different outward potassium currents, some of which are dependent on calcium

for activation. The ability to generate action potentials in the absence of synaptic inputs may be

caused by persistent inward sodium currents that automatically bring a neuron’s membrane

above the action potential threshold after repolarization (Raman et al. 2000). Changes in the

level of intrinsic excitability of MVN neurons can be induced by changes in the activation of the

calcium-dependent small conductance (SK) and big conductance (BK) potassium channels when

17

the cell is under inhibitory influence. In MVN neurons, briefly hyperpolarizing the membrane

(1s current injection) during synaptic blockade is often followed by a short period (1 – 2 s) of

increased excitability modulated by SK channels (Sekirnjak and du Lac 2002). When a neurons

is hyperpolarized for a much longer period (5 minute current injection), a more persistent (30

minutes to 2.5 hours) BK channel-dependent increase in intrinsic excitability, known as firing

rate potentiation, ensues (Nelson et al. 2003).

1.1.6 The Vestibulo-Spinal Reflexes (VSRs)

The VSRs controlling the muscles involved in balance and limb motion during gait are

more complex than the simple horizontal VOR. Each VSR is activated through NVIII by input

originating from four out of the five vestibular end-organs [anterior canal: (Suzuki and Cohen

1964; Kitajima et al. 2006) posterior canal: (Suzuki and Cohen 1964; Kushiro et al. 2008)

utricle: (Sato et al. 1996) saccule: (Sato et al. 1997)]. A combination of behavioural and

neurophysiological evidence suggests that the horizontal canal does not provide any input to the

VSRs involved in limb control (Suzuki and Cohen 1964; Sugita et al. 2004). It is well-

established that the main pathway for the VSRs stems from neurons in the LVN, also known as

Deiters nucleus and passes through the lateral vestibulospinal tract (LVST) to all levels of the

spinal cord (Gray 1926; Pompeiano and Brodal 1957; Nyberg-Hansen and Mascitti 1964; Abzug

et al. 1973; Abzug et al. 1974; Shinoda et al. 1988). A similar pathway also originates in the

DVN (Sato et al. 1996), though most of what we know about the VSR pathway comes from the

study of Deiters nucleus. This pathway is illustrated in Figure 1-7. Often, axons from the largest

cells in the LVN, also known as Deiters neurons, will branch into multiple collaterals that can

terminate on several levels of the spinal cord (Shinoda et al. 1988). A large number of Deiters

neurons could be antidromically activated by stimulation of both the cervical and lumbar spinal

cord (Abzug et al. 1973; Abzug et al. 1974). A histological analysis showed that Deiters neurons

projecting as far as the lumbar region have only two axon collaterals, one terminating in the

cervical cord and another terminating in the lumbar cord (Shinoda et al. 1988). This termination

pattern suggests that some of the Deiters neurons projecting through the LVST to the lumbar

cord may be involved in complex limb coordination. In quadrupeds, most Deiters neurons

activate extensor motor neurons and inhibit flexor motor neurons of both the forelimbs and

hindlimbs (Wilson and Yoshida 1969; Grillner et al. 1970). Activation of the extensors by

18

Deiters neurons is an important part of maintaining both balance on an unstable surface (Schor

and Miller 1981) and a rhythmic step cycle during locomotion (Orlovsky 1972; Yu and

Eidelberg 1981).

Figure 1–7: The pathway from a Deiters neuron to the cervical and lumbar spinal cord. In this example, the axon

emanating from a Deiters neuron (red) in the LVN enters the dorsal horn at both levels where it synapses onto an

excitatory interneuron (yellow), which in turn synapses onto motor neurons (blue) in the ventral horn. LVN = lateral

vestibular nucleus, or Deiters nucleus; LVST = lateral vestibulospinal tract.

19

As with secondary neurons mediating the VOR, neurons in the VSR are under the influence of

the vestibular commissure and the cerebellum. Few studies have demonstrated the existence of

commissural input to vestibulospinal neurons. In the earliest of these investigations, the

responses of Deiters neurons were recorded during contralateral NVIII stimulation (Shimazu and

Smith 1971). In this study, an excitatory commissural pathway through the ventral brainstem,

possibly including the reticular formation, was revealed in cats that had midline incisions

through both the cerebellum and dorsal brainstem. The existence of excitatory inputs to

vestibulospinal neurons was later confirmed in another investigation in which vestibulospinal

neurons were activated by contralateral electrical NVIII stimulation in the cat (Uchino et al.

2001). More recently, an anatomical investigation using retrograde tracer methods in the frog

showed that neurons in the LVN receive inputs from the contralateral SVN, MVN and DVN but

not the contralateral LVN (Malinvaud et al. 2010). While evident in cats and frogs, a

commissural projection to the LVN in the rabbit was not found as revealed by retrograde tracer

injections into the contralateral VNC (Epema et al. 1988). Thus, existence and function of the

vestibular commissure in the VSR is not well understood.

Unlike for the commissure, the existence of cerebellar inputs to the VSR is well known. A large

body of evidence has shown a link between the large Deiters neurons and the cerebellar cortex.

Stimulation of Purkinje cells in lobules III, IV and V of the anterior lobe in the cerebellar vermis

leads to monosynaptic inhibition in Deiter’s neurons (Ito et al. 1966; Ito and Yoshida 1966; Ito et

al. 1968). Field potentials were evoked in the anterior lobe during electrical stimulation of both

the ipsilateral and contralateral vestibular nerves (Precht et al. 1977; Denoth et al. 1979). The

pathways between the vestibular labyrinth and the anterior lobe are not entirely clear. One

possible pathway exists between the ipsilateral lateral reticular nucleus (LRN) and the

contralateral anterior lobe (Wu et al. 1999). Lesion experiments have shown that the LRN,

which receives convergent input from the limb extensors and the vestibular labyrinth (Kubin et

al. 1980), is important for transmission of ipsilateral vestibular input to the contralateral vermis

(Precht et al. 1977). Other more direct pathways to the vermis could also exist. Some

anatomical evidence has demonstrated that many second order vestibular neurons (Precht et al.

1977) and even primary afferents (Kotchabhakdi and Walberg 1978) contact the vermis directly.

The cerebellar vermis also has a modulatory effect on the VSR (Orlovsky 1972). Recently it was

shown that adaptive changes in the VSR, induced by a combination of proprioceptive and

20

vestibular stimulation, were prevented by inactivation of Lobe V (Manzoni et al. 1994).

Inactivation of the anterior lobe also caused the reversal of previously induced adaptive changes

in the VSR (Andre et al. 2005).

1.2 Compensation of the Vestibular Reflexes

Having outlined the normal function of the vestibular reflexes and the neurons involved in their

control, we can now enter our discussion of vestibular compensation. However, before we can

discuss the physiology in more detail, we need to review the effects UVD has on the behavior of

the vestibular reflexes.

In subjects that have undergone surgery that causes UVD, vestibular reflex function at first

becomes disrupted but then gradually recovers through vestibular compensation. Since our focus

will be on compensation following surgical UVD, the term UVD will be used to reference

surgically-induced UVD through the remainder of this Introduction. Immediately following

UVD, disruption of the vestibular reflexes manifests itself as a combination of static and

dynamic signs. Static signs are behavioural characteristics that can be observed in the absence of

head movement, while dynamic signs are those observed only while moving around. As

described in some of the earliest studies (Bechterew 1880; Money and Scott 1962; Precht et al.

1966) on UVD in mammals, static signs typically include spontaneous nystagmus, which is a

persistent beating of the eyes with a quick phase toward the intact (contralesional) side and a

slow phase toward the lesioned (ipsilesional) side, and postural imbalances such as head and

body tilt, spontaneous circling, barrel rolling and falling, all directed toward the side of damage.

Static signs experienced in the absence of movement could reflect inappropriate activation of the

vestibular reflexes through neural imbalance in the brainstem. It is believed that a resting

imbalance in neural activity contributes to the development of static signs (Precht et al. 1966;

Luyten et al. 1986; Newlands and Perachio 1990a; Ris et al. 1997). It should be pointed out that

static signs may not appear after canal plug surgery (Money and Scott 1962; Broussard et al.

1999) and, when they do, they are much less severe than after UL or UVN. Unlike other forms

of UVD, the canal plug does not cause silencing of the afferents (Goldberg and Fernández 1975)

and therefore does not produce a neural imbalance in the VNC while the subject is stationary

(Abend 1978). Any static signs observed after a canal plug surgery are likely caused by transient

21

pressure changes in the canal resulting from insertion of the plug or loss of endolymph (Lasker et

al. 1999). Dynamic signs occur after all types of UVD and manifest as deficits in the vestibular

reflexes, which typically include asymmetries and/or reductions in the responses of the VOR and

VSR. Asymmetries are imbalances in the responses to ipsilesional and contralesional head

movement, with ipsilesional responses being comparatively smaller. The asymmetrical

responses are also thought to be the result of an imbalance in neural activity in the VNC (Smith

and Curthoys 1989). Immediately after UVD surgery, both static and dynamic signs are

observed and their recovery (referred to as static and dynamic compensation, respectively) varies

depending on species, extra-vestibular sensory input and age. In all species vestibular

compensation leads to almost complete abatement of static signs, however dynamic signs persist

even though they show some degree of recovery.

1.2.1 Static Compensation

In many species, static signs come close to being fully resolved through compensation. The rate

at which static signs subside is very much species-dependent. For instance, in gerbils (Newlands

and Perachio 1990a) and mice (Aleisa et al. 2007), the recovery of spontaneous nystagmus

measured in the light is very fast, with near complete disappearance within one day, while in

other species such as rats (Sirkin et al. 1984; Bergquist et al. 2008), guinea pigs (Gliddon et al.

2004), cats (Haddad et al. 1977) and monkeys (Lacour et al. 1976; Fetter and Zee 1988) it

persists for longer periods of time. It is important to note that the recovery from spontaneous

nystagmus can vary depending on whether it was measured in the light or in the dark. If

measured in the dark, spontaneous nystagmus is exaggerated and is detected over a longer period

of time (Fetter and Zee 1988). For example, in the rat, recovery from spontaneous nystagmus

measured in the light, along with recovery from most of the other static imbalances, takes about

2 days (Sirkin et al. 1984; Luyten et al. 1986; Bergquist et al. 2008), but when measured in the

dark recovery from spontaneous nystagmus can take between 4 and 5 days (Magnusson et al.

2002). This difference in recovery times occurs because visual fixation, carried out through

inputs from the flocculus, can act to suppress vestibular nystagmus (Takemori and Cohen 1974).

The recovery of postural imbalances also shows species-dependent variability. Postural

imbalances in goldfish (Ott and Platt 1988) and ipsilesional circling in gerbils (Kaufman et al.

22

1999) are minimized within one hour following UVD. Postural stability is also quickly resolved

in rhesus monkeys (Fetter and Zee 1988), within several hours after UVD, though in other

species such as cats (Precht et al. 1966; Maioli et al. 1983), rats (Sirkin et al. 1984; Luyten et al.

1986; Hamann et al. 1998; Magnusson et al. 2000; Bergquist et al. 2008), guinea pigs (de Waele

et al. 1989; Ris et al. 1997) and baboons (Lacour et al. 1976) it is regained only after a matter of

days. In humans postural recovery takes several weeks (Black et al. 1989; Cass et al. 1991). It

should be pointed out that in some instances head tilt can be slower to recover and more

persistent than other postural deficits (Money and Scott 1962; Precht et al. 1966; Lacour et al.

1976; Maioli et al. 1983; de Waele et al. 1989; Hamann et al. 1998).

1.2.2 Dynamic Compensation of the VOR

Dynamic recovery of the VOR has been of great interest to many vestibular physiologists and the

process is well-characterized (Curthoys and Halmagyi 1995). Dynamic compensation of the

VOR typically follows an exponential time course in which there is an early rapid recovery

followed by a more gradual and incomplete restoration of reflex function (Fetter and Zee 1988;

Murai et al. 2004; Faulstich et al. 2006; Beraneck et al. 2008). In some cases, the gain of the

VOR will also fluctuate over time, between high and low values, before reaching a stable,

recovered state (Broussard and Hong 2003). Acutely following UVD, the gain of the VOR

measured during ipsilesional rotation is often less than fifty percent of control values (Maioli et

al. 1983; Fetter and Zee 1988; Beraneck et al. 2008). Contralesional gains are also reduced

though they are typically higher than ipsilesional gains, creating an asymmetry in gain for the

two directions of rotation (Maioli et al. 1983; Fetter and Zee 1988; Lasker et al. 1999; Lasker et

al. 2000; Broussard and Hong 2003; Beraneck et al. 2008). Most of the recovery occurs during

the early, rapid stage of recovery (Fetter and Zee 1988; Lasker et al. 1999; Lasker et al. 2000;

Broussard and Hong 2003; Murai et al. 2004; Faulstich et al. 2006; Sadeghi et al. 2006;

Beraneck et al. 2008). Further restoration of gain and symmetry then proceeds slowly until it

reaches an asymptote where no further recovery occurs. At this point, the VOR has reached a

compensated state.

VOR recovery following UVD appears to be independent of lesion type since recovery was

found to be similar after CP and UL in the same species (Broussard et al. 1999; Lasker et al.

23

1999; Lasker et al. 2000). However, recovery does depend on species type. Across species,

there is a wide variation in the time course over which gain recovers. For low velocity

(<40deg/s) mid-frequency (0.2-2 Hz) rotations, maximum gain values were reached between two

weeks and one month following UVD in monkeys (Paige 1983; Fetter and Zee 1988; Lasker et

al. 1999; Lasker et al. 2000) and cats (Broussard and Hong 2003). However, by comparison,

mice (Murai et al. 2004; Faulstich et al. 2006; Beraneck et al. 2008) and rats (Magnusson et al.

2002) recover very rapidly. In fact, recovery is so rapid in rodents that maximum gain is

achieved within a just few days after surgery.

1.2.3 Dynamic Compensation of the VSR

Few studies have evaluated dynamic function of the VSR after UVD. Typically, dynamic signs

associated with a loss of input to the VSR include an abnormal gait and a reduced ability to

maintain balance or postural stability while in motion. These signs have been observed in

squirrel monkeys after both UL (Igarashi et al. 1970) and unilateral sectioning of the utricular

nerve (Igarashi et al. 1972). In both studies, the ability of the monkeys to cross a rotating rail

was evaluated. Immediately after UVD, balance was highly compromised and the monkeys

could not cross the rail without falling off. Within a few days after surgery, balance had

improved and the monkeys could successfully cross the rail but, in a given set of trials, they

experienced a greater frequency of falls than before surgery. Recovery continued for another 6

to 10 weeks after surgery, after which falling frequency returned to pre-operative levels. A

similar recovery pattern was shown on a much shorter time scale, within 1 day after surgery, in

rats (Hamann and Lannou 1987). More recently, an analysis of locomotion was performed on

cats while they crossed a cylindrical beam before and at several times after UVN (Lacour et al.

1997). Consistent with the findings of Igarashi et al. (1970, 1972), the cats were unable to cross

the beam in the first few days following surgery. Once the cats were able to cross the beam, they

walked across it in a crouched position with their centers of gravity shifted closer to the ground

than before surgery. The cats also had a tendency to walk more slowly and with shorter steps

than normal. While these locomotory patterns became less obvious over time, they persisted past

the end of the recovery period and normal locomotion was not completely restored.

24

The compensation of behavioural symptoms described above may result from the recovery of an

imbalance in the extensor muscles activated by the VSR. During unexpected falls, baboons

demonstrated asymmetric activation of the hindlimb extensors, as revealed through

electromyographic (EMG) recordings shortly after surgery for UVN (Lacour et al. 1979). On the

lesioned and intact sides, EMG activity was attenuated and elevated, respectively, compared to

normal. In the same group of animals, monosynaptic spinal reflexes activated by muscle spindle

stimulation were also tested during falls. Consistent with an imbalance in the activation of spinal

motor neurons, which exhibit reduced responsiveness on the lesioned side after UVN (Gernandt

and Thulin 1953), the activation of monosynaptic spinal reflexes was asymmetric with reduced

activation on the lesioned compared to intact side. By the end of a three-week period following

UVN, the asymmetries were reduced and extensor muscle function was partially restored.

1.3 Neuronal Changes Associated with Vestibular Compensation

Symptoms that manifest after UVD, while standing still (static) and while in motion (dynamic),

are associated with physiological changes that take place in the VNC. Most notably, UVD

induces changes in neural discharge when stationary (resting discharge) and in neural sensitivity

during head movement. Immediately after UVD, resting discharge and head movement

sensitivity are altered such that they are no longer the same bilaterally (Shimazu and Precht

1966; Hamann and Lannou 1987; Smith and Darlington 1988; Smith and Curthoys 1988a;

Newlands and Perachio 1990a; Newlands and Perachio 1990b). However, over time, recovery of

both resting discharge and sensitivity takes place and a balance in reflex control is partially

restored. It has been proposed that static compensation is largely associated with recovery of

resting rate while dynamic compensation is associated with restoration of both resting rate and

head movement sensitivity (Smith and Curthoys 1989).

1.3.1 Acute Neuronal Changes Associated with UVD

Acutely following UVD, the resting activities of vestibular neurons (see Section 1-1-4) are

dramatically altered. With the exception of canal plugging, which does not have an effect on

resting activity (Abend 1978), all forms of surgical UVD result in below-normal discharge rates

(Shimazu and Precht 1966; Xerri et al. 1983; Smith and Curthoys 1988a; Smith and Curthoys

25

1988b; Newlands and Perachio 1990a; Newlands and Perachio 1990b) and head movement

sensitivites (Shimazu and Precht 1966; Hamann and Lannou 1987; Smith and Curthoys 1988b;

Newlands and Perachio 1990a) in the ipsilesional VNC. This is due to silencing of primary

afferents in NVIII (Sirkin et al. 1984) and the subsequent elimination of excitatory afferent input

to the lesioned side. Many vestibular neurons are then left under the influence of inhibitory

inputs from the commissure (Shimazu and Precht 1966; Kasahara et al. 1968; Mano et al. 1968;

Ito et al. 1970) and cerebellum (Ito et al. 1968; Shimazu and Smith 1971; Highstein 1973b;

Lisberger and Pavelko 1988; Sato et al. 1988). Furthermore, the amount of tonic inhibition may

become increased on the lesioned side. A recent investigation reported elevated release of

inhibitory neurotransmitters into the ipsilesional MVN within one hour after UVD (Bergquist et

al. 2008).

1.3.2 Overview of Neuronal Changes Associated with Static and Dynamic Compensation

By the time static and dynamic compensation are complete, there is a moderate restoration of

resting activity (Precht et al. 1966; McCabe and Ryu 1969; Dieringer and Precht 1977; Smith

and Curthoys 1988a; Smith and Curthoys 1988b; Newlands and Perachio 1990a; Newlands and

Perachio 1990b) and head movement sensitivity (Hamann and Lannou 1987; Smith and Curthoys

1988b; Newlands and Perachio 1990a) in the ipsilesional VNC. Rebalancing of resting activity

appears to be at least partly associated with changes in static behaviour. Acute symptoms such

as spontaneous nystagmus and postural instability are usually detected in conjunction with a

bilateral imbalance in resting activity, though once neural activity is restored static signs are

rarely observed (Precht et al. 1966; Newlands and Perachio 1990a). In labyrinthectomized

guinea pigs a similar amount of time was required for recovery of both static signs and

ipsilesional resting activity (Ris et al. 1997). A similar result was produced in rats (Luyten et al.

1986). In both studies, more time was required for the restoration of neural activitiy in the

ipsilesional VNC than for the compensation of static signs , which suggests that rebalancing of

resting activity may also be associated with early dynamic recovery. An association between

these two processes is quite possible since the early stage of dynamic compensation overlaps

with static compensation (Hamann and Lannou 1987; Fetter and Zee 1988; Faulstich et al. 2006).

Also, the restoration of resting rate could permit an increase in head movement sensitivity. This

is because higher resting discharge rates would enable vestibular neurons to experience a greater

26

degree ofmodulation by the remaining inhibitory inputs. Once resting activity is restored, an

increase in the efficacies of inhibitory inputs to the ipsilesional VNC (Dieringer and Precht

1979b; Farrow and Broussard 2003) could act to further improve head movement sensitivity and,

ultimately, reflex function.

1.3.3 Static and Dynamic Compensation Depend on Acute Changes in the Intrinsic Excitability of

VNC Neurons

The early restoration of resting rate, which appears to be important for both static and dynamic

compensation, is associated with modifications in the intrinsic excitability of vestibular neurons.

Intrinsic excitability is the ability of a cell to fire spontaneously in the absence of synaptic input

(see section 1-1-5). The suggestion that intrinsic excitability might contribute to neural

rebalancing came from studies of VNC neurons in slice preparations where labyrinthine input

was absent. In the earliest of these studies (Darlington et al. 1989; Smith and Darlington 1992),

with the use of slices obtained from fully compensated guinea pigs, it was found that

spontaneous firing was elevated compared to normal in the ipsilesional MVN and unchanged

relative to normal on the contralesional MVN. In a later study (Cameron and Dutia 1997),

spontaneous firing of rat rostral MVN neurons was monitored bilaterally at 2, 4, 24 and 48 hours

after UL. In this series of experiments it was found that spontaneous firing of ipsilesional MVN

neurons was above normal in slices obtained between 4 and 48 hours after surgery, a time frame

in which static compensation would have been near completion (Sirkin et al. 1984; Bergquist et

al. 2008) and the early phase of dynamic compensation would have been well under way

(Hamann and Lannou 1987). Also, consistent with Darlington & Smith’s findings, there were no

observable increases in spontaneous firing on the contralesional side. In order to test whether

such increases in discharge rates were due to changes in spontaneous firing or to changes in

synaptic inputs, a similar study was conducted using guinea pig brainstem slices but with

synaptic transmission blocked (Ris et al. 2001). In this study, slices were obtained 48 hours and

1 week after UL, within the time frame for static compensation in the guinea pig (Ris et al.

1997). After synaptic blockade, the spontaneous firing rates of ipsilesional MVN neurons were

increased above control values at both times, indicating a greater degree of intrinsic excitability

after UVD. However one question remained: do changes in intrinsic excitability also participate

in the later stages of compensation, after static recovery is complete? This issue was addressed

27

in a recent study using rat brain slices obtained 4 hours, 48 hours and 1 week after UL (Guilding

and Dutia 2005). At each time, firing rates were measured in the rostral MVN before and after

synaptic transmission was blocked. The firing rates measured 4 hours after surgery were

significantly elevated compared to normal but were not altered when synaptic input was blocked

with a cocktail of receptor antagonists (synaptic blockade), indicating an increase in intrinsic

excitability at this time. At 48 hours and 1 week, firing rate in the presence of synaptic input

remained significantly elevated but intrinsic excitability, revealed through synaptic blockade,

was no longer different from control. This result suggests that, in the rat, a compensatory

increase in intrinsic excitability might play a role in both static compensation and the earliest

stage of dynamic compensation (ie. within the first 48 hours). A lesion-induced enhancement of

intrinsic excitability could be generated through increases in tonic inhibition. It has been shown

in normal slices that long periods of synaptic inhibition were capable of inducing long-lasting

increases in the intrinsic excitability of MVN neurons (Nelson et al. 2003). Also, in rats that had

bilateral removal of the flocculus prior to UL there were no compensatory increases in

spontaneous firing of neurons in the rostral MVN 4 hours after UL (Johnston et al. 2002). This

result suggests that inhibition from the flocculus could be important during the early stages of

compensation (see Section 1-3-3).

1.3.4 Static and Dynamic Compensation are Dependent on Cerebellar Inhibition

A role for the cerebellum in both static and dynamic compensation is well-supported. One of the

earliest reports suggesting that the cerebellum may participate in compensation was produced by

McCabe & Ryu in 1969. In this study, a complete cerebellectomy performed in cats within the

first week after UVD caused an elevation in the resting activity within the VNC on both sides.

However, one month after UL, removal of the cerebellum had no effect on resting activity. This

was the first indication that the cerebellum is important during the early stages of compensation.

Several studies have since confirmed the importance of the cerebellum in the recovery of static

behavior and early recovery of dynamic reflex function. In the first of these studies, spontaneous

nystagmus was monitored after unilateral destruction of NVIII in cats that had part of their

cerebellum, including the flocculus, removed bilaterally (Haddad et al. 1977). Partially

cerebellectomized cats exhibited a delayed recovery from spontaneous nystagmus compared to

cerebellum-intact cats. The importance of the cerebellum to static compensation was also

28

demonstrated by a study in the mouse (Beraneck et al. 2008). It was shown that

labyrinthectomized Lurcher mice, mutants which lack a functional cerebellum (Caddy and

Biscoe 1979), were compromised in their ability to recover from static signs compared to normal

wildtypes. While spontaneous nystagmus and head tilt had mostly subsided in wild types by the

tenth day after UL, these signs had resolved to a lesser degree in the Lurcher mutant. Even by

the third week after surgery, static signs in the Lurcher remained unresolved. A number of

results obtained from mutant mice also support a role for the cerebellum in dynamic

compensation after UVD. In the GluRδ2 knock-out mouse, a mutant which has a reduced

number of parallel fiber-Purkinje cell synapses (Kashiwabuchi et al. 1995), the ability to swim

showed far less recovery than in the wild-type after injection of sodium arsanilate, a toxin that

destroys hair cells, into the labyrinth on one side (Funabiki et al. 1995). Also, the restoration of

VOR gain was delayed compared to normal in the GluRδ2 mutant after UL (Murai et al. 2004).

No restoration of gain was observed in this mouse until about 15 days after surgery. Consistent

with this finding, there was also no recovery of VOR gain in the Lurcher mouse within the first

week after surgical UVD (Faulstich et al. 2006). Together, these results support a role for the

cerebellum in the early stages of recovery of the VOR. While a role for the flocculus has been

clearly demonstrated in compensation of vestibulo-ocular function, no studies to date have been

done to determine the role of the vermal anterior lobe in compensation of the VSR. However, it

has been proposed that a pathway through the contralesional LRN and ipsilesional anterior lobe

is involved at some point in the recovery of VSR function (Lacour et al. 1985).

1.3.5 Dynamic but not Static Compensation Depends on Commissural Inputs

Along with cerebellar inhibition, commissural inputs also play an important role in vestibular

compensation. The role of vestibular commissure, however, appears to be limited to dynamic

compensation since static compensation can proceed normally in the absence of commissural

inputs (Smith et al. 1986). Also, after UVD, many second order premotor neurons in the

ipsilesional VNC rely entirely on commissural inhibition for modulating their discharge rates

during head movement (Smith and Curthoys 1988b). The importance of the commissure to

dynamic compensation was exemplified by early experiments in frogs (Bienhold and Flohr

1978). Severing the commissure in compensated frogs produced an irreversible decompensation

of dynamic reflex function. Dynamic compensation is most likely associated with an increase in

29

the depth of modulation by the inhibitory commissure. Indeed, lesion-induced increases in the

efficacy of commissural inibition have been observed in the VNC. Experiments in

labyrinthectomized frogs showed that inhibitory commissural inputs to the lesioned side were

scarce in acute preparations but numerous in compensated animals (Dieringer and Precht 1979b).

Dynamic compensation is also associated with modifications to excitatory transmission through

the commissure. Many neurons excited by the commissure are inhibitory interneurons that

mediate commissural inhibition (Shimazu and Precht 1966), but some are also secondary VOR

interneurons, shown in red in Figure 1-6 (Baker et al. 1969; Scudder and Fuchs 1992; Lisberger

et al. 1994). Like commissural inhibition, commissural excitation is enhanced by compensation.

In a series of experiments on labyrinthectomized frogs, crossed excitatory transmission was

evaluated in the ipsilesional VNC during electrical stimulation of NVIII on the intact side

(Dieringer and Precht 1977; Dieringer and Precht 1979a). Compared to neurons from acutely

deafferented preparations, those from compensated frogs exhibited larger magnitude excitatory

responses during commissural activation. Increasing the efficacies of excitatory commissural

inputs could ultimately, by increasing the activity of inhibitory commissural interneurons (see

Figure 1-4), increase the inhibitory modulation of ipsilesional premotor neurons. It should be

pointed out that not all neurons which receive excitatory commissural inputs are inhibitory

interneurons. Some are actually inhibitory second order neurons that project ipsilaterally to the

abducens nucleus as illustrated by the FTN in Figure 1-4 (Broussard and Lisberger 1992). In this

type of neuron, compensation leads to a reduction in the efficacy of their excitatory commissural

inputs (Farrow and Broussard 2003). During contralesional rotation, excitation of these

inhibitory neurons would oppose the activation of abducens motor neurons and reduce

contralesional gain. Reducing the responsiveness of inhibitory second order neurons would

therefore permit contralesional gain to increase during dynamic compensation.

1.4 Neurochemistry of Vestibular Compensation

Clearly, vestibular compensation is associated with modifications to both inhibitory and

excitatory neurotransmission in the VNC. Normally, the responses of vestibular neurons

mediating the VOR and VSR in the brainstem are shaped by a combination of excitatory and

inhibitory inputs through multiple synaptic contacts on the cell bodies (somata) and dendrites.

30

As we have seen, these responses are highly plastic and can be modified when the balance

between excitatory and inhibitory inputs is altered after UVD. A plethora of neurotransmitters

and their many receptors participate in the generation, modulation and plasticity of responses in

the VNC. Two of the most common neurotransmitters in the VNC are γ amino butyric acid

(GABA) and glutamate. Both GABA and glutamate have attracted a lot of interest in the field

of vestibular physiology. These substances mediate neural transmission and plasticity through a

variety of transmembrane receptors and play an important role in static and dynamic

compensation.

1.4.1 GABA and Normal Vestibular Function

GABA is an inhibitory amino acid that is common in the CNS, including the areas of the

brainstem and cerebellum involved in vestibular function. Its presence in the VNC has long been

known. The first evidence that GABA was an inhibitory transmitter in the VNC came from

Obata et al. (1967) when they suppressed the responses of Deiters neurons to cerebellar

stimulation, both excitatory and inhibitory, with intravenous application of GABA. It is now

well established that GABA influences the responses of vestibular neurons to activation of the

cerebellum and is indeed the neurotransmitter released by Purkinje cells onto vestibular neurons

(Obata and Takeda 1969; ten Bruggencate and Engberg 1969; Curtis et al. 1970; Steiner and

Felix 1976; Houser et al. 1984; de Zeeuw and Berrebi 1995). Purkinje cells are the main source

of GABAergic inputs to Deiters neurons. The amount of punctate staining for glutamic acid

decarboxylase, an enzyme involved in the synthesis of GABA, was reduced by more than 70%

around Deiters neurons after ablation of the anterior lobe of the cerebellum (Houser et al. 1984).

This result indicated that the majority of presynaptic inputs to Deiters neurons originate from the

anterior cerebellar lobe. GABA has also been established as one of the transmitters mediating

inhibition through the commissure (Precht et al. 1973; Furuya et al. 1992; Holstein et al. 1999a;

Malinvaud et al. 2010) as well as through interneurons intrinsic to the VNC (Holstein et al.

1999b). Through these inhibitory influences, GABA release suppresses the firing rates of

vestibular neurons. This is reflected by the effect GABA has on the tonic activity of vestibular

neurons. In whole animals (Furuya et al. 1992) and in the slice (Smith et al. 1991; Dutia et al.

1992; Vibert et al. 1995), the resting discharge of vestibular neurons is reduced by the

application of GABA. GABAergic neurons are present in all divisions of the VNC (Nomura et

31

al. 1984; Kumoi et al. 1987). Among these are the neurons that project through the commissure

(Bagnall et al. 2007), the interneurons that project intrinsically within the VNC (Holstein et al.

1999b) and others that send axons to the spinal cord bilaterally (Blessing et al. 1987).

1.4.2 GABA in Static and Dynamic Compensation

GABA and its receptors are thought to be key players in both static and dynamic compensation.

A large amount of the compensation literature has been dedicated towards substantiating a role

for GABAergic neurotransmission in the recovery process. A very recent and elegant study has

shown that GABA release is altered in the MVN during static compensation (Bergquist et al.

2008). In this series of experiments, GABA levels were measured through continuous sampling

of the extracellular fluid in the MVN of rats. Following UL, it was found that ipsilesional

GABA began increasing after 3 hours, and peaked after 24 hours. After reaching its peak,

GABA release subsided after 2 days in conjunction with the abatement of static signs. Although

there was a dramatic reduction in GABA release over the second post-lesion day, it remained

elevated above normal, possibly in association with dynamic compensation. When the

extracellular fluid was perfused with a GABA re-uptake inhibitor, to prevent removal of GABA

from the extracellular space, it was found that ipsilesional GABA levels actually began rising as

early as 20 minutes after UL. In rats that had bilateral flocculectomy, the release of GABA was

suppressed during the first 1.5 hours but then gradually increased toward levels measured in rats

with intact flocculi. Static signs began to show decline 2 hours after UL, which suggests that the

initiation of static compensation is associated with GABA release from the flocculus (see Section

1-3-3), while further development of static recovery, and perhaps dynamic compensation as well,

is more closely associated with increased GABA release at commissural or interneuronal

synapses.

Evidence suggests that a lesion-induced increase in GABA release might result from a

multiplication in the number of GABAergic inputs to ipsilesional VNC neurons. This was first

shown in the frog preparation (Dieringer and Precht 1979b). Immediately after UL, crossed

inhibition to the ipsilesional VNC was nearly absent and was unchanged from what it typically is

in the normal frog (Ozawa et al. 1974). However, such inhibition, which was sensitive to the

GABA receptor antagonist picrotoxin, became more apparent 3 days later and had become quite

32

prominent after 2 months. Consistent with a gradual increase in numbers of crossed inhibitory

responses, a parallel increase in the numbers of GABAergic neurons and synaptic contacts was

found by immunolabeling in the VNC of neurectomized cats (Tighilet and Lacour 2001). The

numbers of neurons labeled for GABA were increased above normal within the first week afer

UN in the MVN and LVN. A year later, many more GABAergic neurons, but not synaptic

contacts, appeared in the MVN while the number of neurons had returned to control values in the

LVN. Double-labeling for glutamic acid decarboxylase (GAD), the enzyme that converts

glutamate into GABA, and 5-bromo-2-deoxyuridine, a marker for cell proliferation, revealed that

the lesion-induced generation of new GABAergic neurons begins 1 day after neurectomy, peaks

after 3 days and is complete within one week (Tighilet et al. 2007). It is possible that some of

these newly formed neurons may have formed synaptic contacts within the first week after

surgery since the number of punctate structures was also increased at this time in the MVN

(Tighilet and Lacour 2001). Due to the possibility that the newly formed cells could participate

in both static and dynamic compensation, behavioural recovery was evaluated in neurectomized

cats after an inhibitor to cell proliferation, cytosine-β-D arabinofuranoside (AraC), was

continuously infused into the extracellular fluids (Dutheil et al. 2009). It was found that AraC

cats and control cats did not exhibit a difference in the time over which spontaneous nystagmus

abated. However, more persistent postural imbalances, such as a shift in weight over the paws,

took much longer to recover than in controls. Also, recovery of dynamic VSR function, assessed

by the ability to cross a rotating beam, was completely suppressed until after AraC infusion was

stopped. These results suggest that newly formed GABAergic neurons may participate in both

static and dynamic compensation but that they might maintain a greater role in latter.

1.4.3 GABA Receptors in Normal and Impaired Vestibular Function

Along with changes in GABA release and the numbers of GABAergic inputs to VNC neurons,

compensation is also associated with changes in the tonic activation of GABA receptors. Two

types of receptors are activated by GABA: γ-amino-butyric acid type A (GABAA) and γ-amino-

butyric acid type B (GABAB). While GABAA receptors have been implicated in static

compensation (Gliddon 2005), we did not investigate their possible roles in this process.

Therefore, we will limit our discussion to the roles of GABAB receptors in vestibular function

before and after UVD.

33

1.4.3.1 GABAB Receptors and Their Roles in Normal Vestibular Function

GABAB receptors are found throughout the brain (Bowery et al. 1987) and may be located on

either the presynaptic axon terminal or the post-synaptic membrane. They are metabotropic G-

protein coupled receptors that function to modulate neurotransmission at both inhibitory (Davies

et al. 1991; Mouginot and Gahwiler 1996; Yamada et al. 1999) and excitatory synapses (Yamada

et al. 1999; Aroniadou-Anderjaska et al. 2000). The GABAB receptor is assembled through the

association of two subunits, GABAB1 and GABAB2 (Jones et al. 1998; Kaupmann et al. 1998; Ng

et al. 1999). The former binds GABA and has two known variants [GABAB1a and GABAB1b:

(Kaupmann et al. 1997)], while the latter couples with the G-protein (Robbins et al. 2001). No

other types of GABAB receptor subunits are known to exist. Both anatomical and physiological

evidence suggests that the GABAB1a subunits are localized mainly to the presynaptic terminal,

while GABAB1b subunits are mostly post-synaptic (Billinton et al. 1999; Perez-Garci et al. 2006;

Vigot et al. 2006). Through G-protein coupling, GABAB receptor activation can trigger a wide

range of intracellular events pre- and post-synaptically. Presynaptically, GABAB receptors

modulate the release of both GABA and glutamate (Ulrich and Bettler 2007). On the calyx of

Held, a large presynaptic terminal in the brainstem, GABAB receptors can suppress the release of

neurotransmitter through closure of calcium channels (Takahashi et al. 1998) or by activation of

second messenger pathways that slow down the release of neurotransmitter-containing synaptic

vesicles (Sakaba and Neher 2003). Post-synaptically, GABAB receptor activation can suppress

the activity of a neuron in several ways. In the supraoptic nucleus, the activation of GABAB

receptors can hyperpolarize the cell by reducing currents through select calcium channels

(Harayama et al. 1998). In other areas such as the cerebellum and hippocampus, GABAB

receptor activation leads hyperpolarization of the post-synaptic membrane through the opening

of G-protein-coupled potassium channels (Lüscher et al. 1997; Slesinger et al. 1997).

In the VNC, GABAB receptors are expressed throughout (Eleore et al. 2005). Visualization of

synaptic terminals with electron microscopy revealed both pre-and post-synaptic GABAB

receptors in the MVN (Holstein et al. 1992). No direct evidence exists for the specific location

of GABAB receptors at synapses formed by commissural or cerebellar projections in the VNC.

However, GABAB receptors influence the resting activity in the VNC. Application of baclofen

induced hyperpolarization (Vibert et al. 1995) and a reduction in spontaneous activity (Dutia et

34

al. 1992) of MVN neurons in the slice preparation. A balance in the tonic activity of GABAB

receptors is also important to the maintenance of the normal vestibular function. Unilateral

injections of baclofen, a GABAB receptor agonist, into the LVN of normal cats resulted in

postural imbalance (Luccarini et al. 1992).

1.4.3.2 GABAB Receptors and Their Roles in Static and Dynamic Compensation

Recordings from vestibular neurons in vitro and in vivo have indicated that GABAB receptor

function may be altered in both static and dynamic recovery. For instance, in rat brainstem

slices, the application of baclofen, a GABAB receptor agonist, was less effective than normal at

reducing spontaneous firing in ipsilesional MVN neurons 4 hours (Yamanaka et al. 2000) after

UL. At the same time, the effect of baclofen on spontaneous firing was enhanced compared to

normal on the contralesional side. The reduced efficacy of GABAB recptors in the ipsilesional

MVN was still observed in slices obtained 7 days (Johnston et al. 2001) after UL. Since static

compensation would have mostly taken place within the first couple of days after UL (Sirkin et

al. 1984; Luyten et al. 1986; Bergquist et al. 2008), this result suggests that GABAB receptors

may also be important for dynamic recovery, which would have still been under way at this time

(Magnusson et al. 2000). A role for GABAB receptors in dynamic compensation was further

supported by another study in which the effects of baclofen were tested 4 and 8 days after UL in

alert rats (Yu et al. 2009). In this study, the discharge rates of MVN neurons both before and

after systemic administration of baclofen were recorded. It was found that baclofen induced a

greater reduction in the resting activity of neurons in the contralesional compared to the

ipsilesional MVN. This result is consistent with the reduced efficacy of GABAB receptors

observed at 7 days in the slice (Johnston et al. 2001).

A possible role for GABAB receptors in both static and dynamic recovery was implicated by a

series of experiments done in labyrinthectomized rats (Magnusson et al. 1998; Magnusson et al.

2000; Magnusson et al. 2002). Systemic administration of the GABAB receptor antagonist

CGP56433A 30 minutes before each eye movement recording augmented spontaneous

nystagmus measured in the dark between 8 hours and 4 days after UL (Magnusson et al. 2002).

This drug also caused the exaggeration of postural imbalances. When the same experimental

protocol was used with baclofen the opposite effect, a decline in spontaneous nystagmus, was

35

observed. These results suggest that the activation of GABAB receptors may act to restore static

balance. Furthermore, systemic administration of baclofen 30 minutes before eye movement

recordings also resulted in the restoration of symmetry in VOR gain measured at several

different times between 3 days and 3 months after UL (Magnusson et al. 1998; Magnusson et al.

2000). At the same time, an antagonist to GABAB receptors, CGP36742, had the opposite effect

since its administration exacerbated the asymmetry in gain (Magnusson et al. 2000). These

results indicate the potential importance of GABAB receptor activation during the recovery of

dynamic reflex function. The idea that GABAB receptors are important to both static and

dynamic recovery has been contradicted by the results of a more recent study in humans (de

Valck et al. 2009). In this study, a battery of static and dynamic balance tests were performed on

two groups of patients that had undergone surgery for removal of a tumor in the vestibular nerve.

One group was treated with baclofen while the other served as a control. Baclofen was

administered orally and the treatment began after a minimum of one week following surgery.

The baclofen therapy, which lasted several months, never led to an improvement in postural

imbalances or dynamic reflex function, both of which were evaluated several times throughout

the year following surgery. In fact, during some of the balance tasks, baclofen reduced

performance. Baclofen is a known muscle relaxant often used to treat muscle spasticity (Kita

and Goodkin 2000) and may have interfered with the maintenance of balance in these subjects.

Within the first few hours or, depending on the species, days following UVD GABAB receptor

function may be crucial to behavioural recovery. It has been shown that the effectiveness of

GABAB receptor activation on behavioural recovery is reduced with time after UVD in the rat

(Magnusson et al. 2000), suggesting GABAB receptors are probably most important during the

earliest stages of compensation. As we have seen, the earliest time at which the effects of

GABAB receptors were tested on static recovery was 8 hours after UL (Magnusson et al. 2002).

However, it is likely that GABAB receptor function may be affected at earlier times since it was

shown in the rat brain slice that the effect of baclofen on resting firing rate is altered compared to

normal 4 hours after UL (Yamanaka et al. 2000). For dynamic compensation, the earliest time at

which the effects of GABAB receptor activation were evaluated was 3 days after the lesion

(Magnusson et al. 1998). However, it has been shown that the rat (Hamann and Lannou 1987)

and several other species (Fetter and Zee 1988; Lasker et al. 1999; Lasker et al. 2000; Broussard

and Hong 2003; Faulstich et al. 2006; Beraneck et al. 2008) start showing signs of dynamic

36

compensation at much earlier times, usually within less than two days, after surgery.

Considering that synaptic GABA release increases in the ipsilesional VNC within the first hour

after UVD (Bergquist et al. 2008), we hypothesized that during the most acute stage of

vestibular compensation, GABAB receptors play a role in the recovery of both static and

dynamic reflex function. We predicted that GABAB receptors would participate in both static

and dynamic recovery within the first several hours and then over the next couple of days

following UVD. Also, we have seen throughout this introduction that static and dynamic

recovery, while they overlap in the earliest stages (Fetter and Zee 1988; Faulstich et al. 2006),

may be associated with different physiological changes that take place after UVD. Thus, we

have also predicted that within the first several hours after UVD, GABAB receptor activation

may differentially affect static and dynamic recovery.

1.4.4 Glutamate in VNC During Normal and Impaired Vestibular Function

Glutamate is an excitatory amino acid and is one of the most common neurotransmitters in the

vestibular nuclei (de Waele et al. 1995; Smith 2000). The earliest evidence suggesting that

glutamate was a major excitatory neurotransmitter in the VNC came from a study by Raymond

& colleagues (Raymond et al. 1984) in which it was found that the uptake of glutamate was

reduced after sectioning the ipsilateral NVIII. Later, Cochran and colleagues (Cochran et al.

1987) revealed that excitation by the contralateral vestibular nerve was also presumably

mediated by glutamate. Many successful efforts have shown that glutamate is indeed the

neurotransmitter of the primary afferents (Lewis et al. 1989; Dememes et al. 1990; Carpenter and

Hori 1992; Reichenberger and Dieringer 1994; Yamanaka et al. 1997) as well as of many central

vestibular neurons (Walberg et al. 1990; Bagnall et al. 2007).

Glutamate release is altered for only a very brief period of time after UVD. In alert rats (Inoue et

al. 2003), periodic sampling of glutamate in the extracellular fluid of the MVN showed that

immediately after UVD glutamate levels are decreased and increased on the lesioned and intacts

sides, respectively. Within 4 hours, glutamate levels on the intact side had returned to normal

and after 12 hours there was no longer a difference between the two sides. This result suggests

that changes to glutamate release are associated with only the earliest stage of compensation.

37

1.4.5 Glutamate Receptors: Roles in Normal and Compromised Vestibular Function

Several different glutamate receptors have been discovered in the VNC. This group of receptors

consists of the ionotropic glutamate receptors, which include those that are selectively activated

by either α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) (Petralia and Wenthold

1992), N-methyl-D-aspartate (NMDA) (Monoghan and Cotman 1985) or kainate (Petralia et al.

1994), and a variety of metabotropic glutamate receptors (mGluRs), (Horii et al. 2001; Grassi et

al. 2002). Very little is known about the functional role of kainate receptors in the VNC. The

mGluRs can have a modulatory effect on the firing rates of vestibular neurons (Darlington and

Smith 1995) and a variety of different mGluRs participate in synaptic plasticity in the MVN

(Grassi et al. 1998; Grassi et al. 2002), however very little is known about their role in

compensation. The following discussion will therefore be limited to AMPA and NMDA

receptors and the evidence for their participation in the early stages of compensation.

1.4.5.1 AMPA Receptors: Basic Function and Roles in Normal Vestibular Function

In general, the ionotropic AMPA receptor participates in fast excitatory transmission. AMPA

receptors are tetrameric transmembrane proteins that, in the mature brain, are found almost

exclusively on the post-synaptic membrane (Martin et al. 1993). Each AMPA receptor is

structured from a combination of GluR subunits [GluR1, GluR2, GluR3 and GluR4: (Keinaenen

et al. 1990)], which varies throughout the brain (Martin et al. 1993). Together the GluR subunits

form a ligand-gated ion channel that is permeable to sodium, potassium and, in some cases,

calcium (Ozawa and Iino 1993). During excitatory synaptic activation, currents that pass

through AMPA channels can be readily observed during whole cell voltage clamp experiments

in which the cell’s membrane potential is kept constant. The excitatory post-synaptic current

(EPSC) observed during a voltage clamp experiment has a fast early phase (Nelson et al. 1986)

that is overlapped by a later slow phase (Forsythe and Westbrook 1988). AMPA channels

contribute to the early phase of the EPSC. Typically, the AMPA component of the EPSC is

inward when the cell’s membrane is held at -80 mV and outward when held at 40 mV. The

reversal potential for the AMPA current is just below 0 mV (Crunelli et al. 1984) and, usually,

the relationship between the EPSC amplitude and the clamped potential (current-voltage

relationship) is perfectly linear for AMPA channels (Nelson et al. 1986). The amplitude of the

38

AMPA current can be altered during synaptic plasticity. It has been shown in the hippocampus

that, under such circumstances, the number of AMPA receptors at the synaptic contacts on a

cell’s surface can change. When excitatory synapses in the hippocampus undergo long-term

potentiation (LTP), a condition where the efficacy of a synapse is increased, there is an increase

in the synthesis of AMPA receptor subunits (Nayak et al. 1998) and their insertion into the post-

synaptic membrane (Heynen et al. 2000; Moga et al. 2006; Williams et al. 2007). On the other

hand, when a hippoocampal excitatory synapse undergoes long-term depression (LTD) and its

efficacy is reduced, the number of AMPA receptor subunits found at the synaptic contact

decreases (Heynen et al. 2000; Moga et al. 2006).

In the VNC, the presence of all four AMPA channel subunits has been demonstrated through in

situ hybridization (Popper et al. 1997) and immunolabeling (Petralia and Wenthold 1992; Popper

et al. 1997; Chen et al. 2000) methods. Each subunit was found in all divisions of the VNC, with

GluR2 being dominant in each division. AMPA receptors participate in excitatory transmission

through primary afferent and commissural synapses in the VNC. Using DNQX, an antagonist to

AMPA channels, Kinney and colleagues (Kinney et al. 1994) were able to block the early phase

of the EPSC produced by ipsilateral vestibular nerve stimulation. In earlier studies (Doi et al.

1990; Carpenter and Hori 1992), the activation of MVN neurons by NVIII stimulation was

blocked by the application of CNQX, another AMPA channel antagonist. The application of

CNQX produced similar effects on direct excitatory transmission through the vestibular

commissure (Doi et al. 1990).

1.4.5.2 AMPA Receptors are Associated with Acute Behavioral Imbalances Following UVD

Very little work has been done to determine a role for AMPA receptors in vestibular

compensation. The few reports that have addressed this issue suggest that AMPA receptors are

associated only with the earliest stage of compensation. In the first of these studies (Hirate et al.

2000), AMPA receptors in the guinea pig were manipulated at several different times after UL

with systemic administration of kainate, a non-specific agonist to AMPA receptors. When

kainate was administered 3 hours after surgery, it significantly exaggerated spontaneous

nystagmus and postural imbalance. By the third day, kainate no longer produced any significant

effects. These results suggest that AMPA receptor function may be altered during static

39

compensation. There have been no other investigations into the effects of AMPA receptor

manipulation on behavioral recovery and it is not known how systemic administration of AMPA

receptor antagonists affects normal static balance.

Other evidence suggests that it is possible for lesion-induced static imbalances to be associated

with a disparity in AMPA receptor activity between the two sides of the brain stem. Compared

to the intact side, GluR2 gene transcription in the VNC was significantly downregulated on the

lesioned side 6 hours after UL in rats (Horii et al. 2001). A difference in gene expression was no

longer observed 2 days later. Consistent with these results, an acute asymmetry in GluR2 protein

expression was also observed in the rat VNC 10 hours after UL (King et al. 2002). Compared to

sham-operated animals, GluR2 protein content in labyrinthectomized rats, quantified through

Western blotting, was significantly increased bilaterally compared to sham operated animals.

GluR2 content was also elevated on the intact relative to the lesioned side. No differences were

observed when GluR2 content was evaluated a second time two weeks later. In the rat, lesion-

induced imbalances in AMPA receptor expression appear to be resolved within the first few days

after UVD. When the expression of GluR2, 3 and 4 subunit proteins was evaluated through

immunolabeling between 4 and 8 days after surgery, no lesion-induced effects were observed

(Rabbath et al. 2002).

The above results suggest that GluR2-containing AMPA receptors might be expressed

asymmetrically between the contralesional and ipsilesional VNC within the first day after UVD.

However, the earliest effects of UVD on the expression of GluR subunits, that is within the first

few hours after surgery, has not yet been investigated. In the ventral tegmental area of the

brainstem, GluR subunits can be synthesized and translocated to the post-synaptic membrane

within few hours after the induction of a plastic change is initialized (Mameli 2007; Argilli

2008). Therefore, we hypothesized that within the first few hours after UVD, the expression

of GluR subunits in the VNC becomes asymmetric.

1.4.5.3 General Function of NMDA Receptors and Their Roles in the Normal VNC

The NMDA receptor is an ionotropic tetrameric transmembrane protein that participates in fast

excitatory transmission. Some NMDA receptors are pre-synaptic but the majority of them are

located post-synaptically (Conti et al. 1997). All NMDA receptors are comprised of a

40

combination of several different subunits [NR1: (Moriyoshi et al. 1991); NR2A, NR2B, NR2C:

(Monyer et al. 1992); NR2D: (Ikeda et al. 1992)] that varies depending on their location in the

brain (Ishii et al. 1993; Monyer et al. 1994), though the NR1 subunit appears to be ubiquitous in

all brain areas (Moriyoshi et al. 1991). The NMDA channel has the same reversal potential as

the AMPA channel (Crunelli et al. 1984) but it differs from the AMPA channel in four ways.

First, it is highly permeable to calcium in addition to sodium (MacDermott et al. 1986). Second,

the NMDA receptor is co-activated and potentiated by glycine (Johnson and Ascher 1987;

Kleckner and Dingledine 1988). Third, the NMDA receptor channel is often dependent on

membrane potential due to the presence of a magnesium ion in the channel’s pore (Mayer et al.

1984; Nowak et al. 1984). The magnesium ion does not begin exiting the pore until membrane

potentials above -80 mV are reached and it does not completely leave the pore until the

membrane is depolarized above -40 mV. Thus the current-voltage relationship is nonlinear for

membrane potentials below -20 mV. Finally, because of this, currents through NMDA channels

have slow rise times and contribute to the slow phase of the EPSC (Forsythe and Westbrook

1988). The voltage dependence of NMDA receptors makes them highly susceptible to shunting

by inhibitory inputs (Collingridge et al. 1988). In this case, the cell membrane becomes

hyperpolarized to a level at which NMDA receptors remain blocked by the magnesium ion.

NMDA receptors are important for the induction (Citri and Malenka 2008) and possibly the

maintenance (Cavazzini et al. 2005) of long-term synaptic plasticity throughout the brain.

Which form of long-term plasticity, LTP or LTD, is induced depends on the amount of calcium

influx through NMDA receptors (Malenka and Nicoll 1993; Cummings et al. 1996). It also

depends on the stimulus activating the synapse. In hippocampal neurons, NMDA receptor-

dependent LTP can be induced by repetitive high- (Collingridge et al. 1988) or low- (Habib and

Dringenberg 2009) frequency stimulation of the presynaptic nerve terminal, while NMDA

receptor-dependent LTD is usually induced by repeated low-frequency stimulation (Dudek and

Bear 1992). In the CA1 region of the adult hippocampus, NMDA receptor-dependent LTP and

LTD are associated, respectively, with long-term increases (Heynen et al. 2000; Zhong et al.

2006) and decreases (Heynen et al. 2000) in the quantities of synaptic AMPA receptor subunits

GluR1 and GluR2. Similar observations have also been made for GluR2 in the adult dentate

gyrus (Williams et al. 2007) and, in abducens motor neurons, NMDA-dependent classical

conditioning induces a significant increase in synaptic GluR4 (Keifer 2001; Mokin and Keifer

41

2004) but it is unknown whether this change persists for more than 30 minutes. Some forms of

synaptic plasticity not only depend on NMDA receptor activation but also induce changes in the

surface expression of the NMDA receptors themselves. Long-term changes in the NR1 and

NR2B subunits have been observed, respectively, in the CA1 (Heynen et al. 2000; Zhong et al.

2006) and dentate gyrus (Williams et al. 2003; Williams et al. 2007) of the adult hippocampus.

Some plastic changes associated with NMDA receptor-dependent plasticity have also been

observed in the VNC.

The NMDA receptor has received much attention with regard to its expression and function in

the VNC. NMDA receptors colocalize with AMPA receptors on vestibular neurons (Chen et al.

2000) and, like the AMPA receptors, participate in excitatory transmission at NVIII (Doi et al.

1990; Kinney et al. 1994; Takahashi et al. 1994; Straka et al. 1996) and commissural (Knoepfl

1987; Doi et al. 1990) synapses. The NMDA receptors involved in excitatory transmission in the

VNC are built from different combinations of all five NR subunits (Sans et al. 1997). NR1 is the

most abundant subunit in the vestibular nuclei and, along with NR2A, it is ubiquitous in all

divisions. NR2B, NR2C and NR2D, on the other hand, are much less common and their

expression is limited to the MVN and LVN. Functionally, NMDA receptors appear to play

many roles in the VNC. Vestibular physiologists began to show interest in NMDA receptors

after the selective NMDA receptor antagonist APV was first tested in the frog VNC (Knoepfl

1987). APV significantly altered the shape of the excitatory post-synaptic potential (EPSP)

evoked by ipsilateral vestibular nerve stimulation but reduced the amplitude of the EPSP induced

by activating the commissure. A later study by Doi and colleagues (1990) further supported a

differential role for NMDA receptors at afferent and commissural synapses. In this study, APV

was far more effective at suppressing the responses to commissural stimulation than responses to

NVIII stimulation. In general, it would appear that excitatory transmission in the VNC requires

NMDA receptors, especially at commissural synapses. The participation of NMDA receptors in

excitatory transmission was further demonstrated by a study by Smith and colleagues (1990) in

which a large percentage of neurons in MVN slices reduced their firing rates after the application

of the NMDA receptor antagonists MK-801 or CPP (Smith et al. 1990).

In addition to their role in exctiatory transmission, NMDA receptors also appear to play a role in

the maintenance of excitability patterns in vestibular neurons (Serafin et al. 1992). In cells

recorded in brain slices, activating NMDA receptors while hyperpolarizing a cell’s membrane

42

induced an oscillatory behaviour in the cell’s spontaneous discharge. It was proposed by Serafin

and colleagues (Serafin et al. 1992) that NMDA receptors might contribute to the rhythmicity of

Deiter’s neurons during locomotion (Orlovsky 1972), which is influenced by inhibitory inputs

from the cerebellum (Orlovsky 1972). Finally, NMDA receptors are also involved in synaptic

plasticity in the VNC of normal and lesioned animals. The evidence implicating NMDA

receptors in vestibular plasticity is presented in Section 1-5.

1.4.5.4 NMDA Receptors in Vestibular Compensation

Evidence strongly supports a role for NMDA receptors in normal vestibular function. However,

it is unclear whether the NMDA receptor may be involved in either static or dynamic

compensation. The effects of NMDA receptor antagonists on static balance have been studied in

guinea pigs (Smith and Darlington 1988; Sansom et al. 1990; Sansom et al. 1992; Hirate et al.

2000) and rats (Kitahara et al. 1995) at different times after UVD. When the NMDA receptor

antagonist MK-801 was administered systemically within the first 12 hours after UVD, static

signs were alleviated (Sansom et al. 1992; Hirate et al. 2000). Unfortunately this result does not

necessarily indicate a change in NMDA receptor function. On the ipsilesional side,

glutamatergic afferent input is entirely removed and resting rates are acutely reduced (see

Section 1-3-1). Therefore, applying MK-801 shortly after UVD could partially block afferent

input and reduce resting activity on the intact side, which could improve balance in resting

activity between the two sides of the brain stem and reduce static signs (see Section 1-3-2).

When the same NMDA receptor antagonist was administered at later times, after the abatement

of static signs, either by systemic administration (Smith and Darlington 1988; Kitahara et al.

1995; Hirate et al. 2000) or by infusion into the VNC bilaterally (Sansom et al. 1990), static

signs reappeared. It is unknown how NMDA receptor antagonists affect normal static balance so

it is uncertain whether the effects of the antagonist on the maintenance of static balance after

UVD are actually different from the effects of MK-801 on normal static reflexes.

There has also been little insight into the physiology underlying the behavioural observations

described above. Only one study has so far examined the effects of NMDA receptor antagonists

on neurons in the VNC (Smith and Darlington 1992). This investigation was conducted on

brainstem slices obtained from normal and labyrinthectomized guinea pigs with the latter having

43

completed the course of static compensation. No difference in the effect of MK-801 on the

spontaneous firing of MVN neurons was observed between normal and ipsilesional brain stem

slices. However, there were three problems with this study. First, a temporal effect of MK-801

on behavior after static recovery (Kitahara et al. 1995) had not yet been reported and data from

labyrinthectomized slices obtained between 3 days and 2 months after surgery were pooled

together. Second, it was also not specified how many slices were obtained at which times.

Finally, the effects of UL on spontaneous firing within the first post-operative week (Ris et al.

2001) were not controlled for. Other studies have shown that gene expression for some NMDA

receptor subunits is transiently altered in the first couple of days after UVD (de Waele et al.

1994; Sans et al. 1997), but these studies could not provide any specific information about the

proteins being incorporated into functional NMDA receptors at the cell surface. The total

expression of two NMDA receptor proteins, NR1 and NR2A, in the VNC was subsequently

evaluated bilaterally in the rat (King et al. 2002). A comparison between labyrinthectomized and

sham-operated animals showed no difference in the quanitities of both subunits at either 10 hours

or 2 weeks after surgery. However, this investigation did not consider how the subunits may

have been redistributed between the intracellular space and cell surface. Thus, more work needs

to be done to determine how NMDA receptor function is altered after UVD.

1.5 Plasticity in the VNC: The Foundation for Vestibular Compensation

The neurochemical changes associated with both static and dynamic compensation could be

associated with one or more form(s) of plasticity. Several forms of synaptic plasticity have been

observed in the normal VNC and could serve as candidate mechanisms for the development of

static and dynamic balance after UVD. In addition, published evidence suggests that the

formation of new synapses onto VNC neurons could also contribute to both stages of recovery.

1.5.1 Synaptic Plasticity in the Normal VNC

Long-term synaptic plasticity can be expressed in many different ways in the VNC. For

instance, several forms of long-term plasticity have been documented at the NVIII synapse of

both VOR neurons and interneurons in the MVN. In an early report on this subject, it was shown

that high-frequency electrical stimuli (HFS) applied repeatedly to NVIII in rat brainstem slices

44

can induce potentiation of monosynaptic excitatory field potentials in the ventral portion of the

MVN (Capocchi et al. 1992; Caria et al. 2001), an area where many VOR neurons reside

(McCrea et al. 1987; Sekirnjak and du Lac 2006). In the same preparation, long-lasting

potentiation could also be triggered with repetitive low-frequency stimuli (LFS, <20 Hz) of

NVIII, but was usually less robust than when induced by HFS (Grassi et al. 1996). Both forms

of LTP were prevented after applying NMDA receptor antagonists to the slice and were therefore

shown to be NMDA-dependent (Capocchi et al. 1992; Grassi et al. 1996; Caria et al. 2001).

Very recently, an electrophysiological examination of MVN neurons projecting to other brain

regions, including the oculomotor centers, revealed two novel forms of long-term plasticity at the

NVIII synapse in the mouse (McElvain et al. 2010). First, a series of HFS applied to NVIII

produced an NMDA-dependent form of LTD in the projection neurons. In a second set of

experiments on the same cell-type, hyperpolarizing currents synchronized with HFS of NVIII,

were repeatedly applied to each cell. With this protocol, an NMDA-independent form of LTP

mediated by calcium-permeable AMPA receptors was produced. Interestingly, when the

calcium-permeable AMPA receptors were blocked during this experiment, NMDA-dependent

LTD was unmasked suggesting that long-term plasticity at the NVIII synapse is bidirectional.

A wide variety of long-term plasticity mechanisms have also been reported for the NVIII

synapse of MVN interneurons. In rat brainstem slices, HFS repeatedly applied to NVIII

occasionally led to a long-lasting potentiation of the monosynaptic field potentials in dorsal

portion of the MVN (Capocchi et al. 1992), a region containing many small interneurons (Epema

et al. 1988; Bagnall et al. 2007). However, in this region, a persistent depression of polysynaptic

field potentials was more frequently observed. This form of long-term depression was dependent

on NMDA receptors but was also found to depend on the activation of GABAB receptors (Grassi

et al. 1995). This implies that excitatory interneurons may develop depressed responses through

a suppression of glutamate release by presynaptic GABAB receptors at the NVIII synapse. The

former possibility may result from NMDA-dependent long-term potentiation of an upstream

inhibitory interneuron adjacent to the source of excitatory input (Grassi and Pettorossi 2001).

The latter assumption has been supported by slice experiments in which the application of the

GABAB receptor agonist baclofen produced a persistent pre-synaptic depression at the NVIII

synapse in rat MVN neurons (Peterson et al. 1996). Two other forms of long-term plasticity

have been observed at the NVIII synapse on MVN interneurons in the mouse (McElvain et al.

45

2010). The first was an NMDA-dependent LTD generated by HFS applied repetitively to NVIII,

either alone or synchronized with brief periods of hyperpolarization applied to the interneuron by

current injection. The second was an NMDA-independent form of LTP induced only by

combining HFS with hyperpolarization when the NMDA-dependent LTD was prevented with an

NMDA receptor antagonist. These long-term plasticity mechanisms were similar to those

observed at the NVIII synapse on projection neurons. The only difference was that, in the

interneurons, NMDA-dependent LTD was stronger than NMDA-independent LTP.

1.5.2 Central Plasticity and Early-Stage Compensation

Long-term synaptic plasticity is a candidate mechanism for both static and dynamic

compensation. One study investigated how the induction of long-term plasticity at the NVIII

synapse in the contralesional MVN is altered within the first couple of hours after UL (Pettorossi

et al. 2003). In both the ventral and dorsal portions of the MVN, compared to sham-operated

animals, there was a reduced probability of inducing long-term potentiation of monosynaptic

field potentials with repeated HFS applied to NVIII. In the dorsal portion, the ability to induce

polysynaptic depression was unchanged after UL. It is possible that most of the NVIII synapses

in the contralesional MVN may have already been potentiated at the time this experiment was

conducted. After inducing LTP at excitatory synapses in the hippocampus, it has been shown

that there are several hours during which no further potentiation can take place (Frey et al. 1995).

Assuming that UVD induces a similar early phase of LTP, which could have lowered the

probability of eliciting HFS-induced potentiation at the NVIII synapse acutely after UL

(Pettorossi et al. 2003), we hypothesized that within the first few hours after UVD, excitatory

transmission at the NVIII synapse is altered from normal in the contralesional MVN. We

predicted that the NVIII synapse becomes potentiated in the contralesional MVN within the first

several hours following UVD. Also, since it is well-established that long-term plasticity at the

NVIII synapse can be mediated by NMDA receptors in labyrinthine-intact animals (Capocchi et

al. 1992; Grassi et al. 1996; Caria et al. 2001; Grassi et al. 2001; McElvain et al. 2010), we

sought to determine the relative contribution of NMDA receptors to EPSCs induced by NVIII

activation in both normal and acutely lesioned mice.

46

While plasticity at the NVIII synapse has been well-documented, not much is known about the

specific mechanisms of long-term plasticity that take place at commissural synapses. Some

evidence from the frog suggests that UVD induces plasticity at both excitatory and inhibitory

commissural synapses in the ipsilesional VNC. Frogs that had been labyrinthectomized two

months earlier exhibited a significant potentiation in excitatory transmission in the ipsilesional

VNC compared to normal frogs and those which were lesioned no more than 12 hours earlier

(Dieringer and Precht 1977; Dieringer and Precht 1979a). Also in this preparation, which

normally lacks commissural inhibition (Ozawa et al. 1974), inhibition through the commissure

developed two months after UL (Dieringer and Precht 1979b). Responses to commissural

stimulation have never been thoroughly evaluated within the first hour or two following UVD, so

the most immediate effects of UVD on commissural synaptic transmission are unknown. Since

plastic changes may have already taken place prior to Dieringer & Precht’s (1977, 1979a, 1979b)

earliest post-lesion recordings, we were interested in whether UVD might have an immediate,

short-lasting effect on commissural synaptic transmission. Therefore, we hypothesized that

acutely following UVD, responses to commissural inputs are altered from normal in the

ipsilesional MVN.

1.5.3 Central Plasticity Associated with Late-Stage Compensation

During the later stages of compensation, synaptic transmission could be altered in combination

with the formation of new synapses and neurons (see Section 1-4-2). The first indication for

post-lesion synaptogenesis came from a study comparing commissural EPSPs recorded in

normal, acutely labyrinthectomized and compensated frogs (Dieringer and Precht 1977;

Dieringer and Precht 1979a). It was found that these EPSPs were of significantly shorter

duration and reached their peak faster in compensated frogs than in either the acute or intact

preparation. These data were compared to theoretical predictions made by Rall and colleagues

(Rall et al. 1967), which state that EPSPs generated by synapses on the cell soma and proximal

dendrites exhibit faster rise times than those generated at synapses on the distal dendrites.

Basing their results on this theory, Dieringer and Precht (1979a) proposed that compensation

leads to the formation of additional commissural synapses on the cell bodies of ipsilesional VNC

neurons.

47

Direct evidence for synaptogenesis in the post-lesion VNC has been subsequently produced

through a series of histological and biochemical investigations. The first anatomical evidence for

post-lesion synaptogenesis came from an examination of the cat SVN five days after UVD

(Korte and Friedrich 1979). Through electron microscopy, new synapses, which were

structurally unique compared to those found in the SVN of intact cats, were found on the side of

the lesion. The idea that compensation leads to the formation of new synapses was further

supported by the results of a recent gel electrophoresis study (Paterson et al. 2006). The effects

of UVD and sham surgery on hundreds of different possible MVN proteins were studied

bilaterally. Only a few of these proteins were reported altered by the lesion without also having

been affected by sham sugery. Interestingly, each of these proteins had been previously

implicated in neuronal growth and metabolism, plus they were affected bilaterally, which

suggested that the formation of new synapses was likely taking place on both sides of the

brainstem. While it is clear that synaptogenesis takes place during compensation, the origin of

these synapses remains uncertain.

1.6 Sensory Adaptation in the VNC: Overcoming the Limits of Dynamic Compensation

So far, we have focused on the restoration of vestibular function during acute compensation. We

will now switch our attention to a limition in dynamic reflex function that persists once the

recovery period is over. Normally, the VOR responds linearly (Paige 1983) and can stabilize

gaze over a very wide range of head velocities, up to 350 deg/s (Pulaski et al. 1981), during mid-

frequency (0.2-2 Hz) rotation. However, after UVD, the linear range is reduced and gaze

becomes unstable (Paige 1983; Fetter and Zee 1988; Halmagyi et al. 1990; Gilchrist et al. 1998;

Lasker et al. 1999; Lasker et al. 2000; Sadeghi et al. 2006). At high rotational velocities

(>40deg/s) the VOR’s response function (eye velocity as a function of head velocity) becomes

nonlinear and typically saturates in the direction of ipsilesional rotation (Maioli et al. 1983; Paige

1983; Fetter and Zee 1988; Halmagyi et al. 1990; Gilchrist et al. 1998; Lasker et al. 1999; Lasker

et al. 2000; Galiana et al. 2001). Over the course of recovery, the saturating nonlinearity does

not completely resolve (Paige 1983; Fetter and Zee 1988).

It is possible that persistence of nonlinear dynamic reflex function after UVD reflects a limitation

in the dynamic ranges of central neurons mediating the VOR. Many vestibular neurons exhibit

48

nonlinear responses and are often subject to inhibitory cutoff, which is the cessation of firing

over part of the cycle of rotation in their ―off‖ directions (Melvill Jones and Milsum 1970; Fuchs

and Kimm 1975; Newlands and Perachio 1990a; Newlands and Perachio 1990b; Escudero et al.

1992; Chen-Huang and McCrea 1999; Broussard et al. 2004; Newlands et al. 2009). Cutoff

responses have also been observed in some primary afferents (Dickman and Correia 1989; Hullar

et al. 2005), which could induce nonlinear responses in their target neurons in the VNC. In

normal animals the VOR is linear despite the nonlinear responses of many vestibular neurons,

probably because there is an equal balance of input orignating from two complementary

labyrinths. However, when this balance is disrupted by UVD, nonlinearities in the responses of

vestibular neurons may become unmasked and expressed in the behaviour of the VOR. Thus, a

means for extending the dynamic ranges of vestibular neurons could help to maximize the linear

range of the VOR after UVD. In this section, we will introduce a rapid form of sensory

adaptation that could be implicated in optimizing dynamic reflex function after UVD.

1.6.1 Sensory Adaptation in the VNC

In general, sensory adaptation is the ability of a neuron to adjust its response to the state of a

given stimulus (ie. constant or changing). It is different from sensorimotor adaptation, in which

a neuron's response is shaped by errors in stimulus detection. Sensory adaptation was first

described in a study of the responses of stretch receptors while applying a weight stimulus to a

muscle (Adrian 1926). After weight application, a stretch receptor would at first increase its

discharge rate and then gradually reduce its firing until the weight was either removed or

changed. Such adaptation has also been observed in the VNC. When a monkey was rotated at a

constant velocity in one direction, there was a dramatic increase in the firing rate of central

vestibular neurons at the start of rotation but, after the stimulus had been maintained after about

1 minute, the firing rate had reduced to pre-rotation levels (Waespe and Henn 1977). This type

of adaptation, which we will refer to as temporal adaptation, depends on the length of time over

which a rotational stimulus is presented and is typically not observed during sinusoidal rotation

at frequencies above 0.2 Hz. Some evidence suggests that a different form of sensory adaptation,

one that depends on the intensity rather than duration of the stimulus, may take place in the VNC

at higher rotational frequencies. For instance, during rotation at a fixed frequency between 0.25

and 1 Hz, Melvill Jones and Milsum (1970) reported a decrease in the senstivity of VNC neurons

49

each time peak head velocity was increased. The velocity-dependent sensitivity changes

observed by Melvill Jones & Milsum may be analogous to a form of rapid sensory adaptation

known as adaptive rescaling.

1.6.2 Adaptive Rescaling and its Implications for Dynamic Reflex Function after UVD

Adaptive rescaling has been characterized in several different sensory systems (Brenner et al.

2000; Fairhall et al. 2001; Kim and Rieke 2001; Dean et al. 2005; Nagel and Doupe 2006). It

matches the dynamic range of a neuron to the dynamic range of its stimulus (Brenner et al.

2000). This can be explained with an example from the fly’s visual system (Brenner et al. 2000;

Fairhall et al. 2001). While in flight, the velocity of movement in a fly’s visual field is

constantly fluctuating about a zero mean and the variance of these fluctuations changes from

moment to moment. The ever-changing movement in the visual field is detected by the H1

motion-sensitive neuron. When a fly suddenly changes its in-flight behaviour the variance

detected by H1 can go from large to small or vice versa. Typically, larger variances are

associated with a reduction in sensitivity and an extension of the dynamic range of the H1

receptor (ie. the range of velocities to which the H1 detector can respond). In addition,

sensitivity is usually adjusted in such a way that the amount of information transmitted is

maximized. The time course of adaptive rescaling is very rapid, usually less than 1 second

(Fairhall et al. 2001), and can be completed in as soon as 100 ms after a change in stimulus

variance (Nagel and Doupe 2006). With its rapid time course, adaptive rescaling could be an

efficient and effective means through which the dynamic ranges of vestibular neurons may be

extended after UVD. In our laboratory, a former student gathered preliminary evidence

suggesting that adaptive rescaling may occur after UVD. During this pilot study, responses of

vestibular neurons were recorded in alert cats that had compensated from canal plugging. It was

found that the response sensitivities of vestibular neurons decreased as the peak head velocity

was increased during sinusoidal rotation at 1 Hz. Interestingly, there is published evidence

sugggesting that adaptive rescaling might also occur in the normal VNC (Melvill Jones and

Milsum 1970). Therefore we predicted that adaptive rescaling is a property of central vestibular

responses that is preserved after UVD and hypothesized that in compensated subjects, adaptive

rescaling extends the dynamic ranges of vestibular neurons during high-velocity rotation.

50

1.6.3 Efferents, Afferents and Adaptive Rescaling

It is worth mentioning that rescaling of neural sensitivity has also been reported in primary

vestibular afferents. In particular, the sensitivities of primary afferents can be rescaled through

activation of the vestibular efferents. The efferents are a group of neurons that are typically

located between the abducens nucleus and the VNC in the brainstem (Warr 1974; Goldberg and

Fernandez 1980; Carpenter et al. 1987; Perachio and Kevetter 1989). Efferent neurons project

either to the ipsilateral or contralateral labyrinth (Perachio and Kevetter 1989) where they form

synaptic contacts with both hair cells and primary afferents (Nakajima and Wang 1974; Sans and

Highstein 1984). In both toadfish and monkeys, efferent stimulation alters the responses of

primary afferents. More specifically, electrical stimulation of the efferents elicits both an

increase in afferent discharge rate (Highstein and Baker 1985; Boyle and Highstein 1990; Boyle

et al. 2009) and a reduction in afferent sensitivity (Goldberg and Fernandez 1980; Boyle and

Highstein 1990; Boyle et al. 2009).

1.7 Final Remark

Vestibular compensation induced by surgical UVD can be studied on a variety of levels from

behavioural to cellular to molecular. Throughout this Introduction, the literature for surgically-

induced compensation was reviewed on all levels, with specific focus on lesion –induced

changes in the CNS. In addition, new hypotheses about the central mechansims underlying static

and dynamic compensation were proposed. In the following chapters, four experimental

objectives will be addressed specifically: 1) The role of GABAB receptors in static and dynamic

behavioural compensation, 2) The short-term effects of surgical UVD on the bilateral surface

expression of AMPA receptors in the VNC, 3) Short-term, lesion-induced plasticity at excitatory

and inhibitory synapses onto single VNC neurons, 4) Rapid adaptation of VNC neurons in the

compensated animal and how it could be of benefit to dynamic compensation. All of these

topics are of importance to understanding the central mechanisms and limitations of vestibular

compensation. These issues were addressed by studying compensation in two different types of

vestibular reflex systems: the vestibulo-ocular reflex (VOR) activated during rotation about an

Earth-vertical axis and the vestibulo-spinal reflexes (VSRs) activated during gait and balance.

51

1.8 List of Hypotheses

During the most acute stage of vestibular compensation, GABAB receptors play a role in the

recovery of both static and dynamic reflexes.

Within the first few hours after UVD, the expression of GluR subunits in the VNC becomes

asymmetric.

Within the first few hours after UVD, excitatory transmission at the NVIII synapse is altered

from normal in the contralesional MVN.

Acutely following UVD, responses to commissural inputs are altered from normal in the

ipsilesional MVN.

In compensated subjects, adaptive rescaling extends the dynamic ranges of vestibular neurons

during high-velocity rotation.

52

Chapter 2: GABAB Receptors Contribute to Early

Recovery of Balance Following Unilateral Vestibular

Damage in Mice

2.1 Introduction

Several studies have demonstrated the involvement of GABAB receptors in both static and

dynamic compensation (Magnusson et al. 1998; Magnusson et al. 2000; Magnusson et al. 2002).

However, whether GABAB receptors participate in compensation within the first few hours after

UVD remains unknown. A recent investigation has revealed that there is an increase in synaptic

GABA release in the ipsilesional VNC within the first hour after UVD (Bergquist et al. 2008),

therefore we hypothesized that GABAB receptors play a role in behavioural recovery during

the earliest stage of vestibular compensation. Since the time courses of static and dynamic

compensation are quite different (Fetter and Zee 1988; Lasker et al. 1999; Lasker et al. 2000;

Magnusson et al. 2000; Magnusson et al. 2002; Faulstich et al. 2006; Beraneck et al. 2008), with

dynamic compensation occurring at a much slower rate, it is possible that these two processes are

being driven by different neural mechanisms. Considering this possibility, we also predicted that

within the first several hours after UVD, the effects of GABAB receptor activation may

differentially affect static and dynamic recovery. We tested our hypotheses in mice by

evaluating static and dynamic compensation while pharmacologically manipulating GABAB

receptor activation between 1 and 48 hours after UVD. The number of active GABAB receptors

was either reduced with a GABAB receptor antagonist, CGP56433A, or increased with the

GABAB receptor agonist baclofen. Since baclofen is a known muscle relaxant (Kita and

Goodkin 2000) and may interfere with the muscle control during static and/or dynamic balance,

we also tested the effects of a positive allosteric modulator to GABAB receptors, CGP7930

(Urwyler et al. 2001). This compound, which has no known side effects, binds to a site on the

GABAB receptor that is different from the GABA binding site. The simultaneous binding of

CGP7930 and GABA causes a change in the structural conformation of the GABAB receptor and

amplifies its effect on intracellular second messenger pathways. The results of our experiments

implicate a role for GABAB receptors in the early compensation for static balance after UVD.

53

2.2 Methods

2.2.1 Animals

A total of 68 young male C57Bl/6 mice (1 to 3 months old) were used in this study. Mice were

bred in our facility or supplied to us by Taconic Farms (New York). All animals were kept on a

normal light cycle and always had unlimited access to food and water. Experiment start times

were always between 7:30 AM and 10:30 AM. Guidelines set by the Canadian Council for

Animal Care were strictly followed and all procedures were approved by the Animal Care

Committee at the University Health Network.

A weight restriction was placed on the experimental groups and only mice that weighed between

15 and 23 grams were selected for study. This size range was chosen because smaller

individuals recovered more quickly and more consistently from the anesthetic. Altogether, thirty-

six mice were included in the experimental groups. Another 14 mice either died during surgery

or were excluded after testing (see Table 1 for details). Additional mice were used for

determining dosages, monitoring dose-related activity levels after anesthesia, and/or testing for

training effects, as described below. A breakdown of how all mice were used is shown in Table

2-1.

Sample

Size

Treatment or condition Dosage Injection

volume (ml)

Injection vehicle Data shown in

figures

4 immunohistochemistry -- -- -- 1

6 CGP56433A 5 mg/kg 0.34 + 0.05 saline 2, 4, 5, 6

6 R-baclofen 1 mg/kg 0.24 + 0.03 saline 2, 4, 5, 6

6 CGP7930 25 mg/kg 0.27 + 0.02 methylcellulose 3, 4, 5, 7

6 saline -- 0.28 + 0.04 -- 2, 4, 5, 6

12* methylcellulose -- 0.29 + 0.01 -- 3, 4. 5, 7

1 died during surgery –

CGP56433A

5 mg/kg 0.37 saline --

3 died during surgery – R-

baclofen

1 mg/kg 0.27 + 0.01 salne --

1 died during surgery –

CGP7930

25 mg/kg 0.29 methylcellulose --

2 died during surgery - saline -- 0.28 + 0.00 -- --

4 Died during surgery -

methylcellulose

-- 0.26 + 0.03 -- --

1 air injection ineffective – R-

baclofen

1 mg/kg 0.23 saline --

1 air injection ineffective –

CGP7930

25 mg/kg 0.26 methylcellulose --

1 activity measurements

following isoflurane – R-

1 mg/kg 0.26 saline --

54

baclofen

2 activity measurements

following isoflurane –

CGP56433A

5 mg/kg 0.34 + 0.01 saline --

1 activity measurements

following isoflurane – saline

-- 0.23 --

1 activity measurements

following isoflurane –

methylcellulose

-- 0.29 -- --

1 training effects -- -- -- --

1** training effects, determining

CGP56433A drug dosing

5 mg/kg 0.35 saline --

2** training effects, determining

CGP7930 drug dosing

25-30

mg/kg

0.25-0.3 methylcellulose --

4† determining R-baclofen drug

doses

0.5–1.5

mg/kg

0.08- 0.2 saline --

1 Control (no drug) for

determining drug dosing

-- -- -- --

Table 2-1: Experimental groups. All mice used in this study are included in the table, including the five drug-

treatment and vehicle groups, failures, and additional controls. * This group originally consisted of two experimental

groups that were later combined. ** This group was used for training effects and then subsequently used for

determining drug doses. † The concentration of drug used in this group was lower than for the test groups.

2.2.2 Determining Drug Dosages and Comparing Activity Levels

Recovery from UVD depends on how physically active the animal is after the lesion (Mathog

and Peppard 1982). We considered the possible effects of an interaction between our drugs and

the isoflurane anesthetic could have on the activity levels of our mice. Previously published

experiments in mice showed that baclofen can enhance the effects of isoflurane (Sugimura et al.

2002) while a study in rats demonstrated that CGP56433A can increase activity under certain

conditions (Slattery et al. 2005). For each test substance, the maximum dose that did not

significantly alter activity levels following isoflurane anesthesia was selected for experimental

trials. In preliminary experiments, experimental doses were determined in a separate group of 5

mice (Table 2-1). Each mouse in this group was anesthetized for 1.75 hours followed

immediately by drug administration. The mice were then observed for another 3.5 hours and any

obvious differences in activity were noted. The range of doses tested is shown for each

substance in Table 2-1. For CGP56433A, only one dose (5 mg/kg) was tested since it both met

our requirements and exceeded the dose known to exacerbate the symptoms of UVD

(Magnusson et al. 2002).

55

Once doses were selected, we confirmed that the drugs had no effect on normal activity in

another group of 5 mice (Table 2-1). In this group, the doses were administered as planned for

the experimental groups. Therefore, mice were given repeated doses 2 hrs before and 2.25 and

4.25 hrs following the start of a 1.75-hour period of isoflurane anesthesia at surgical levels (2%).

For each mouse, the total time spent walking during each 5-minute interval was recorded and

then averaged across all intervals over a 3.5-hour post-anesthetic observation period. No

differences in activity levels were observed in these mice.

2.2.3 Experimental Protocol

To investigate the effects of our substances on static and dynamic recovery, mice were divided

into four groups of 6 and one group of 12 (Table 2-1). All 5 groups were tested randomly and

the experimenter (R.H.-S.) was blind to all drug administration. Another student prepared all

drug solutions and administered all injections. Each group of mice received one of 5 test

substances in repeated doses. The test substances were a GABAB receptor antagonist,

CGP56433A (5 mg/kg, dissolved in saline); a GABAB receptor agonist, R-baclofen (1 mg/kg,

dissolved in saline), a GABAB receptor positive allosteric modulator CGP7930 (25 mg/kg,

suspended in 0.5% methylcellulose); methylcellulose alone; or saline alone. The route of

administration for all test substances was by subcutaneous injection. Altogether, each mouse

received six doses given at the following times: 3.25 hrs pre-UVD and 1, 3, 7, 19, and 31 hrs

post-UVD. The frequency of dose administration was reduced so that we could evaluate the

possibility of a lasting effect for each test substance. Damage to the labyrinth on one side was

always completed 1.25 hrs after the start of a 1.75 hr anesthetic period and the first three

injection times were well-matched with those set during experiments where activity levels were

measured.

2.2.4 Surgical Procedure

Vestibular damage was generated using the air injection method (Faulstich et al. 2006). Prior to

surgery, mice were anesthetized with isoflurane and then given an analgesic, meloxicam (2

mg/kg, sc), and sterile lactated Ringer’s solution (1 ml, sc). The head was stabilized by attaching

a nail to the skull with cyanoacrylate and then securing the tip of the nail to a magnetic stand.

56

The stand was adjusted so as to position the right side of head under a surgical microscope

(Zeiss, OPMI 1 FC) and the right ear was pulled forward so that a second incision could be made

behind the pinna. The right horizontal semicircular canal was exposed by blunt dissection and

then opened using a power drill (Foredom TXH Flex Shaft Kit, Gesswein) fitted with a 0.3-mm

carbide bur. After wicking away the excess endolymph, a blunt 30 ga. needle was inserted into

the opening and three ml of air were injected repeatedly until no more endolymph was emitted.

A #15 dental paper point (Patterson Dental) was then inserted into the opening, trimmed and

sealed in with bone wax. The time of paper point insertion was recorded as the time of UVD for

each mouse. Lactated Ringer’s was administered again at the end of the surgery and meloxicam

(1 mg/kg, sc) was administered again 24 hours later. In all except 3 mice (Table 1; ―air injection

ineffective‖), clear signs of vestibular deficit were evident in the post-operative behaviour.

2.2.5 Evaluating Static Vestibular Reflexes

Behaviour was monitored continuously from the end of the anesthetic period up until 4 hours

after air injection. Throughout this observation period, the mouse remained in its cage. From

the moment each mouse recovered from the anesthetic, it was gently pulled by its tail across the

diagonal of its cage once every 5 minutes to keep arousal levels consistent. In the 5-minute

intervals between tail-pullings, static signs were scored as follows: 1 point for each barrel roll,

fall, or circle toward the side of the lesion; 3 points for 5 minutes of continuous head tilt; 6 points

for 5 minutes of body tilt (leaning). Head and body tilts were always toward the lesioned side.

We were unable to successfully monitor spontaneous nystagmus in these mice without an eye

tracker or field coil, therefore only balance and gait were evaluated. The lesion was considered

effective only if a mouse achieved a minimum score of 6 during at least one of the 5-minute

intervals.

2.2.6 Evaluating Dynamic Vestibular Reflexes

Dynamic vestibular function was evaluated using both a beam-crossing test and a footprint-based

gait analysis. For each mouse, these tests were conducted at the following times: 3.5 and 1.5 hrs

before UVD, and at 4 hrs, 19 hrs and 44 hrs after UVD. The 19-hour measurement was made

immediately before the 19-hour dose as shown in Figure 2.6. The beam apparatus consisted of a

57

30 cm x 2 cm wooden dowel raised 15 cm above the surface (Figure 2-1). The beam abruptly

dropped off at one end and led to the opening (3cm x 3cm) of an enclosed box (8.5 x 11 x 8 cm)

containing a sample of peanut butter at the other end. Before each trial, the mouse was placed at

the start of the beam as shown in Figure 1 and allowed to explore the apparatus. After 1 minute

of exploration, the mouse was returned to the starting point and the time required either to reach

the opening or fall off the beam was recorded. Crossing times were highly variable, especially

after the lesion, therefore we evaluated performance on each trial by assigning a score as follows:

0-5 s, 1 point; 6-10 s, 2 points; 11-15 s, 3 points; 16-20 s, 4 points; 21-25 s, 5 points; >25 s, 6

points; fall, 10 points. Crossing times greater than 25 s were not commonly observed prior to the

lesion. We considered the possibility that repeated trials could introduce a learning effect on

beam crossing ability so we tested this in a separate group of normal mice (n=4; see Table 2-1).

Each of these mice crossed the beam once a day for 5 consecutive days and no differences were

observed between the scores obtained in any of the trials (p>0.05, paired Student’s t-test).

Figure 2-1: Beam crossing apparatus.

Gait was evaluated with a footprint analysis method modified from Klapdor et al. 1997 (Klapdor

et al. 1997). In each trial, mice had their front and hind paws painted with different colours of

India ink. They were then placed on a flat sheet of white paper (11‖x17‖) and allowed to roam

freely. From each set of footprints generated, we measured right and left front-paw stride length,

right and left hind-paw stride length, and front- and hind-paw stride width according to published

58

methods (Zhao et al. 2008). The methods are illustrated in Figure 2-2. Stride length (1) for each

of the four paws was measured along a line connecting two successive prints from a given paw.

The figure shows the stride length being measured for the right rear paw. Front (2) and hind

stride widths were measured by a line drawn between the left paw print and the point where the

line formed a perpendicular intersection with the adjacent line connecting two right paws. If the

angle (3) between the lines connecting two successive pairs of paw prints exceeded 45 degrees,

the stride width at that turn was not included in the analysis since sharp changes in direction

would often produce a very wide or narrow stride-width. In addition, to eliminate artifacts

caused by sudden stops, all stride-lengths below 50% of the maximum within-trial value were

excluded from further analysis. Measurements for each parameter within a single trial (n = 3.97

± 2.38 per trial) were averaged, and the mean for each trial was used in the data analysis. There

were no significant differences between right and left stride lengths, front and hind stride lengths,

or front and hind stride widths at any time point (p>0.05, 1-way ANOVA). Thus, all stride

lengths were pooled together, and stride widths were also pooled. Lengths and widths were

normalized as a percentage of the mean value measured at the start of the experiment.

59

Figure 2-2: Methods for measuring stride length and stride width. Orientation of paw prints are as follows: left

hind = solid black, left front = solid gray, right front = checkered gray, right hind = checkered black.

2.2.7 Statistics

All scored data (static recovery and beam crossing) was evaluated using non-parametric

statistics. For the three saline-based test groups, within-group comparisons (effect of UVD)

were performed using the Friedman test for multiple comparisons and between-group

comparisons (effect of test substance) were performed using the Kruskal-Wallis test for multiple

comparisons. Both types of multiple comparisons tests were followed up with a Schaich-

Hamerle post-hoc (Schaich 1984). For the two methylcellulose-based test groups, within-group

comparisons were performed using the Friedman test for multiple comparisons and between-

group comparisons were performed with the Mann-Whitney U test (p<0.05).

60

For gait data obtained from the saline-based test groups, both within-group and between-group

comparisons were perfomed using a 1-way ANOVA followed by the Tukey-Kramer post-hoc.

For gait data acquired from the methylcellulose-based test groups, within-group comparisons

were performed with a 1-way ANOVA followed by the Tukey-Kramer post-hoc and between-

group comparisons were performed with unpaired t-tests. The numbers of samples obtained for

each repeated measure were not equal since some of the mice did not produce any gait data

during some of the post-UVD evaluations. Therefore, a multi-factor ANOVA was not possible

in this case.

2.2.8 Grip Test

It was possible that damage could have been done to the muscles controlling the right front limb

during surgery. To ensure that none of the body tilts I observed resulted from muscle damage on

the lesioned side, the ability to grip a wire mesh and hang upside down was measured. For each

mouse, this was the amount of time it could hold on, up to a maximum of 1 minute. No failures

on the grip test were observed at any time in the experiment.

2.2.9 Termination

All mice that had successfully completed the experiment were euthanized 48 hours following

surgery. This was done by placing the mouse in a chamber and slowly filling it with CO2. The

temporal bone was inspected postmortem to ensure that the bone-wax seal remained intact,

which it did for all of the mice tested.

2.3 Results

The effects of our different treatments on normal dynamic balance and gait were tested according

to the schedule outlined in Figure 2-3. None of the test substances produced any balance deficits

in our mice before surgery. Static signs were absent and each mouse could successfully cross the

balance beam. No significant deficits in beam crossing times were observed in any of the groups

prior to surgery (Figure 2-6; p>0.05, Kruskal-Wallis test for multiple comparisons). Prelesion

61

stride parameters were not affected by any of the drug treatments (Figure 2-8; p>0.05, 1-way

ANOVA).

Figure 2-3: Timeline for experimental procedures prior to UVD.

The recovery of static balance following UVD was significantly altered through the manipulation

of GABAB receptors. Figure 2-4 illustrates the time course of compensation under the influence

of a GABAB receptor antagonist (CGP56433A, 5mg/kg), an agonist (baclofen, 1 mg/kg) and an

equal volume of physiological saline. Treatment times are indicated by the dotted vertical lines

in the left-hand panels of Figure 2-4. All mice were able to stand on all fours and walk around

their cages within 30 minutes after stopping the anesthetic. At this point, all mice exhibited

severe static balance deficits directed toward the lesioned side, including circular walking, falling

over, barrel rolling, and head and body tilts. The left-hand panels of Figure 2-4 show binned

balance scores, expressed as means and standard deviations, obtained in the first 4 postoperative

hours. A score of zero was assigned when the mice could not yet stand at the earliest

postoperative times.

CGP56433A impaired the compensation of static signs. Left-Hand Panels: The recovery of

static balance over the first 4 hours after UVD. The times at which test substances were

administered are indicated by the dotted vertical lines. Scores reflect the amount of impairment

observed. Scores were binned into 20 minute intervals for each mouse and the binned values

were averaged across mice in each group. Values of zero indicate times at which none of the

mice had yet recovered from the anesthetic. Data are presented as means and the errors as

62

standard deviations. Significant differences (p<0.05; Kruskal-Wallis test for multiple

comparisons) for the CGP56433A group compared to the saline and baclofen groups are

indicated by * and X, respectively. Right-Hand Panels: Baclofen increases the rate of recovery

as shown by single exponential fits to the raw data. Raw scores are shown across the first 80

minutes after the start of recovery, the time at which the highest score for each mouse was

obtained. Individual mice are represented by different symbols. Inset: Recovery rates obtained

from exponential fits (S = saline, C = CGP56433A, B = baclofen). Error bars represent the 95%

confidence intervals. Significance is indicated by * for non-overlapping 95% confidence

intervals.

Figure 2-4: CGP56433A impaired the compensation of static signs. Left-Hand Panels: The recovery of static

balance over the first 4 hours after UVD. The times at which test substances were administered are indicated by the

dotted vertical lines. Scores reflect the amount of impairment observed. Scores were binned into 20 minute

intervals for each mouse and the binned values were averaged across mice in each group. Values of zero indicate

times at which none of the mice had yet recovered from the anesthetic. Data are presented as means and the errors

as standard deviations. Significant differences (p<0.05; Kruskal-Wallis test for multiple comparisons) for the

63

CGP56433A group compared to the saline and baclofen groups are indicated by * and X, respectively. Right-Hand

Panels: Baclofen increases the rate of recovery as shown by single exponential fits to the raw data. Raw scores are

shown across the first 80 minutes after the start of recovery, the time at which the highest score for each mouse was

obtained. Individual mice are represented by different symbols. Inset: Recovery rates obtained from exponential

fits (S = saline, C = CGP56433A, B = baclofen). Error bars represent the 95% confidence intervals. Significance is

indicated by * for non-overlapping 95% confidence intervals.

Beginning 90 minutes after UVD, mice receiving CGP56433A (n=6) achieved significantly

higher scores than mice being treated with either saline (p<0.05, Kruskal-Wallis test with

multiple comparisons) or baclofen (p<0.05; Kruskal-Wallis test for multiple comparisons).

Scores for the CGP56433A mice remained significantly higher than for the other two groups

over the entire observation period. Although significant differences could not be detected

between saline and baclofen scores, the baclofen mice exhibited the fastest rate of recovery. For

each group the rate of recovery was determined by fitting the raw static scores obtained over the

first 90 post-operative minutes (right-hand panels of Figure 2-4) with the exponential decay

function

f(t) = a*e-b*t

where t is time, a is the initial value at t=0 and b is the rate of decay, which we refer to as the rate

of recovery. The baclofen group had a recovery rate that was (bbac = 0.27 mins-1

) more than

double of that measured for either the CGP56433A (bCGP56433A = 0.13 mins-1

) and saline (bsaline =

0.13 mins-1

) groups. This difference in recovery rate was significant as the 95% confidence

intervals surrounding baclofen’s recovery rate did not overlap with those surrounding the

recovery rates measured for saline or CGP56433A mice (Figure 2-4 inset). These results imply

that the earliest stage of static compensation is hampered by blocking GABAB receptors and

slightly accelerated when the number of active GABAB receptors is increased.

The positive allosteric modulator (CGP7930, 25 mg/kg) was also tested since it has the potential

to improve compensation without the side-effects of baclofen. Mice receiving the modulator

were compared to another group that received an equal volume of methylcellulose. Figure 2-5

exemplifies the lack of any difference in score between the two groups at any of the post-

operative times (p>0.05; Mann-Whitney U Test). The recovery rates for the methylcellulose

group (bmethylcellulose = 0.2 mins-1

; inset of Figure 2-5) and the CGP7930 group (bCGP7930 = 0.15

64

mins-1

) were not significantly different as their surrounding 95% confidence intervals

overlapped.

Figure 2-5: The compensation of static signs was not altered by CGP7930. Left-Hand Panels: The recovery of

static balance over the first 4 hours after UVD. No significant differences were observed between the two test

groups at any time after UVD (p>0.05 Kruskal-Wallis test for multiple comparisons). The times at which test

substances were administered are indicated by the dotted vertical lines. Scores are binned into 20 minute intervals

(the sum of 4 raw scores per mouse in each bin). Values of zero indicate times at which none of the mice had yet

recovered from the anesthetic. Data are presented as means and standard deviations. Right-Hand Panels: Single

exponentials are fit to the raw scores across the first 80 minutes after the start of recovery. Individual mice are

represented by different symbols. Inset: Recovery rates obtained from exponential fits (M = methylcellulose, C =

CGP7930). Error bars represent the 95% confidence intervals.

Unlike the recovery of static balance, compensation for dynamic balance was not significantly

affected by GABAB receptor manipulation. A full assessment of dynamic balance is shown for

each experimental group in Figure 2-6. Scores were assigned based on whether the mouse

stayed on the beam and, if it did, how quickly it crossed. The right-hand panels of Figure 2-6

illustrate times at which manipulations were made between measurements. A total of 6 doses,

each indicated by the dotted vertical lines, were administered before and after UVD (solid

vertical line at t=0). At 4 hours post-op, beam crossing scores were elevated in the baclofen and

CGP56433A groups compared to controls, as all the mice in these two groups fell off the beam

65

and achieved a score of 10 at this time. However, these drug effects were not significant

(Figures 2-6, top left; p>0.05, Kruskal-Wallis test for multiple comparisons). By 19 hours,

differences in score were no longer apparent between groups. CGP7930 did not produce any

effect compared to the control at any time after UVD (Figure 2-6, bottom left; p>0.05, Mann-

Whitney U Test).

Figure 2-6: Beam crossing ability was not significantly affected by GABAB receptor manipulation. Top Left:

CGP56433A and baclofen did not significantly affect beam crossing scores compared to the saline vehicle at 4 hours

after UVD (p>0.05; Krusal-Wallis test for multiple comparisons). Bottom Left: CGP7930 has no impact on beam

crossing ability compared to methylcellulose at any time after UVD. Right-Hand Panels: The data from A & B

are presented on a linear time scale. The solid vertical line indicates the time of UVD and the dashed vertical lines

indicate times at which test substances were administered. None of the drugs had any effect on the ability to cross

the beam before UVD. All data are presented as means and the errors as standard deviations.

While beam crossing scores were not affected by our test substances at any time during the

experiment, they were influenced significantly by UVD. Since scores were not significantly

different between groups at any time after UVD (p>0.05, Kruskal-Wallis test for multiple

comparisons), the data were pooled to evaluate the effect of UVD on beam crossing ability

(Figure 2-7). The effect of the UVD was significant at 4 and 19 hours (p<0.01, Friedman test

66

with multiple comparisons) indicating an impairment in dynamic balance at these times. By the

end of the experiment, scores were no longer significantly different from baseline values

(p>0.05, Friedman test for multiple comparisons) suggesting that all mice compensated for

dynamic balance by this time.

Figure 2-7: Beam crossing deficits underwent compensation within 44 hours after UVD. Compensation was

clearly demonstrated when beam crossing deficit scores were pooled across all mice at each time after UVD. The

data are presented as means and the errors as standard deviations. † indicates significant differences (p<0.01;

Friedman test for multiple comparisons) for the scores tallied at 4 and 19 hours after UVD compared to the scores

tallied at -1.5 hours.

We also observed an effect of UVD in our gait parameters. In all five groups, stride length

measured at 4 and 19 hours was significantly shorter compared to pre-lesion values (Figure 2-8,

top panels; p<0.01; 1-way ANOVA). By 44 hours, stride length was no longer significantly

different from normal in three of the five test groups (baclofen, CGP65433A, CGP7930; p<0.01;

1-way ANOVA). Baclofen was the only substance to produce an effect on stride length and it

exaggerated the effect of the lesion by significantly reducing stride length (Figure 2-8, top left;

p<0.01; 1-way ANOVA). No effect of baclofen was observed at 19 and 44 hours post-UVD.

Beyond 4 hours post-op, the dose rate was reduced from 4 hours between doses to 12 hours

between doses. This change in dose administration might have reduced systemic baclofen to

levels insufficient for producing effects on dynamic compensation at 19 and 44 hours post-UVD,

which may be why we observed no drug effects at these times. However, it appeared that

GABAB receptor activation does not participate in dynamic compensation within the first 4 hours

67

after surgery. Also, our evidence is consistent with static and dynamic compensation being

carried out by independent mechanisms since the course of dynamic compensation in the

CGP56433A group, as evaluated by the balance beam test and gait analysis, was never impeded

by interference with static compensation.

Figure 2-8: Drug effects on stride length (Top Panels) but not stride width (Bottom Panels). Baclofen exacerbated

the effect of UVD on stride length (Top Left) and CGP7930 induced a post-lesion effect on stride width (Bottom

Right). All data were normalized as a percentage of the baseline value obtained at -3.5 hours. The data are

presented as means and the errors as standard deviations. Significance differences between test substances at each

time point are indicated by * for p<0.05 (Kruskal-Wallis test for multiple comparisons).

Stride width was not significantly affected by UVD (Figure 2-8, bottom panels; p>0.05; 1-way

ANOVA). Baclofen did not produce any effects on stride width but, surprisingly, CGP7930

caused a significant increase in this parameter at 4 and 19 hours post-op (Figure 2-8, bottom

right; p<0.05, unpaired t-test).

2.4 Discussion

68

The manipulation of GABAB receptors following UVD produced a significant effect on the

recovery of static postural balance. We found that antagonizing GABAB receptors with

CGP56433A produced a significant deficit on the compensation of static signs. In the baclofen

group, enhancement of GABAB receptor activation produced a more rapid abatement of static

signs than in the saline controls. Baclofen produced no effect after the scores had reached

asymptote, probably because scores in the control group were already close to zero by this time

and could not be improved any further. These results suggest that an increase in GABAB

receptor activation may be important for static compensation.

Baclofen is known to produce an antinociceptive effect on post-operative pain (Goudet et al.

2009). A reduction in pain in our baclofen mice may have influenced their rate of recovery by

increasing their willingness to be active. However, published evidence suggests that

antinociceptive effects were unlikely in our experiments. Analgesic effects in rodents were

reported with subcutaneous and intraperitoneal doses of baclofen greater than the 1 mg/kg dose

we administered (Cutting and Jordan 1975; Franek et al. 2004). Also, in humans, pain is

typically treated by injecting baclofen directly into the spinal cord (Sanders et al. 2009).

Our results for static recovery suggest that there may be an increase in the tonic activation of

GABAB receptors in the VNC, which could result from a rise in endogenous GABA on the

lesioned side (Bergquist et al. 2008). The maintenance of balance requires input from Deiters’

neurons in the LVN (Schor and Miller 1981) therefore the drugs we administered may have

affected GABAB receptor activation in these cells. Deiters’ neurons in the LVN express an

abundance of GABAB receptors (Eleore et al. 2005) and may have been a major target for

pharmacological manipulation in our experiments. The LVN receives input from the vermis of

the anterior lobe (Ito et al. 1966; Ito and Yoshida 1966; Ito et al. 1968), which provides the main

source of GABA (Houser et al. 1984) to Deiters’ neurons. In addition, the anterior vermis is

known to participate in the plasticity of the vestibulo-spinal reflexes that control balance and

posture (Manzoni et al. 1994; Andre et al. 2005). While cerebellar influences on the LVN have

not yet been evaluated after UVD, studies in the MVN demonstrated that the cerebellum

participates in the restoration of neural balance within the first few hours after UVD (Johnston et

al. 2002). Also in the MVN, the rise in GABA release originates almost exclusively from the

cerebellum in the first few hours after UVD (Bergquist et al. 2008).

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Unlike CGP56433A or baclofen, the modulator CGP7930 did not have any effect on static

balance. We were forced to administer CGP7930 as a suspension rather than a solution since it

cannot easily be dissolved in aqueous solutions (Urwyler et al. 2001). CGP7930 can be

dissolved in DMSO or ethanol but both substances could potentially have toxic effects on

vestibular sensory function (Brandt 1991; Authier et al. 2002). In experiments with CGP7930,

nicotine addiction was suppressed at a minimum dose of 30 mg/kg administered subcutaneously

(Paterson et al. 2008) and anxiolytic effects were seen at 100 mg/kg through oral administration

(Jacobson and Cryan 2008). Thus it is possible that the drug dose of 25 mg/kg administered in

our study was insufficient to produce an effect on vestibular compensation.

Static balance recovered very quickly in our mice. Circling, falling, and barrel rolling were

almost completely abolished within 90 minutes post-op. A very fast rate of recovery has also

been reported in gerbils. It was reported that circling behaviour in gerbils typically abates within

1 hour post-UVD (Kaufman et al. 1999). However, a comparably fast rate of recovery was not

previously reported in C57Bl/6 mice. In these mice, circling and barrel rolling were found to

take between three and five days to disappear after UL (Gacek and Khetarpal 1998). It is

possible that our mice recovered faster because the air injection method was less damaging to the

labyrinth than UL. The air injection is designed to damage the sensory epithelia but, because the

labyrinth is not being physically removed as in UL, the air injection may destroy fewer hair cells

and silence fewer afferents in NVIII. In this case, the resting activity of the VNC on the lesioned

side would be greater after air injection than after UL and the neural imbalance between the two

sides of the brainstem would be less.

In addition to the recovery of static balance, we also evaluated the effects of UVD on dynamic

balance and gait, which require activation of the dynamic vestibulo-spinal reflexes (VSRs).

While the effects of GABAB receptor manipluation on VOR compensation have been

investigated, no study has yet looked into the role of GABAB receptors in the recovery of the

VSRs after UVD. The VSRs are typically activated while standing on an unstable surface and

during gait. Under these conditions stimulation of proprioceptors in the hindlimbs and forelimbs

activates the Deiters’ neurons and causes reflexive activation of the hindlimb and forelimb

extensors to maintain balance (Schor and Miller 1981) and a rhythmic step cycle (Orlovsky

1972; Yu and Eidelberg 1981). The vestibulo-spinal reflexes were first assessed with a beam

crossing task. We showed that beam crossing and stride length, but not stride width, were

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significantly altered by UVD at 4 hrs after surgery but not significantly different from normal 2

days later, indicating compensation of balance and gait by the fourth post-operative hour. This

result is consistent with a dependency of both balance and gait on dynamic vestibular function

previously reported in monkeys (Igarashi et al. 1970) and cats (Lacour et al. 1997). Immediately

after surgery for UVD both species became highly ataxic and could not successfully cross a

rotating beam, though balance and gait were eventually restored after several weeks. In addition,

cats demonstrated a shorter stride length after UVD (Lacour et al. 1997), consistent with our

finding of a lesion-induced reduction in stride length in mice.

In our study, the ability to cross a beam was not significantly affected by any of the test

substances at any post-operative time but, with the exception of CGP56433A, the drugs did

produce significant effects on gait. Baclofen exaggerated the decrease in stride length 4 hours

after UVD but it did not have any effect on subsequent measurements. It is possible that

baclofen compromised gait since this drug is a known muscle relaxant (Kita and Goodkin 2000)

and may have interfered with muscle control during locomotion in our experiments. A similar,

unfavourable effect was also observed after baclofen administration in human UVD patients (de

Valck et al. 2009). Subjects administered a course of baclofen as part of their treatment did not

perform as well as controls in dynamic gait and balance tests. However, all mice in our

experiments demonstrated successful performance in the grip test (see Methods), suggesting that

muscle tone was probably not affected by any of our test substances.

The effect of the GABAB receptor positive allosteric modulator, CGP7930, on stride width was

quite surprising considering it had no effect on any other parameters we measured. CGP7930

can enhance GABAB receptor activation by GABA by binding to a different site on the receptor

and changing its conformation (Urwyler et al. 2001). It is possible that extra-vestibular pathways

involved in gait control were disrupted by the effects of this substance. For instance, the control

of paw placement during gait is co-ordinated by a pathway coursing through the lateral

hemispheral cortex and dentate nucleus of the cerebellum (Marple-Horvat et al. 1998). This

pathway through the lateral hemisphere of the cerebellum is not known to be involved in

vestibular function but it might have been influenced by CGP7930 in our experiments. The

molecular layer of the cerebellar cortex has one of the highest densities of GABAB receptors in

the brain (Bowery et al. 1987), which would make it an ideal target for the actions of CGP7930.

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Static and dynamic vestibular compensation were differentially affected by our test substances.

While the GABAB receptor antagonist CGP56433A produced a marked deficit in static

compensation, it had no effect on dynamic function at any time after the lesion. While these

results suggest that short-term static and long-term dynamic compensation may be carried out

through separate, independent mechanisms, it is possible that our measures of dynamic reflex

function were simply not sensitive enough.

GABAB receptors have been implicated in the rebalancing of neural activity in the VNC

(Yamanaka et al. 2000; Johnston et al. 2001; Johnston et al. 2002; Yu et al. 2009). While there is

no direct evidence for the participation of GABAB receptors in lesion-induced plasticity, there is

preliminary evidence suggesting that a role for GABAB receptors in synaptic plasticity of the

intact vestibular system may exist (Peterson et al. 1996). Therefore, the specific mechanisms

through which GABAB receptors participate in compensation remains open to future study.

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Chapter 3: GluR4-Containing AMPA Receptors are

altered during the Acute Stage of Vestibular

Compensation in Mouse Vestibular Nuclei

3.1 Introduction

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function is altered

within the first few hours after UVD, as shown by interference with static compensation when

AMPA receptor antagonists are administered systemically within the first several hours after

UVD (Hirate et al. 2000). By 10 hours after UVD, the total number of GluR2 subunits in the

VNC change such that they are bilaterally increased, but with more GluR2 subunits on the intact

compared to lesioned side (King et al. 2002). In other regions of the brainstem, synthesis of

GluR subunits can take place very rapidly, within just a few hours, following the induction of a

plastic change (Mameli 2007; Argilli 2008). It is unknown how UVD specifically affects GluR

subunits expression within the first few hours after UVD. Therefore, we hypothesized that UVD

induces an acute asymmetry in the quantity AMPA receptor subunits in the VNC. Based

on the results of King et al. (2002), we predicted that, within the first few hours after UVD, GluR

subunits become elevated on the contralesional relative to ipsilesional side. We were also

interested in whether the newly expressed GluR subunits had been inserted into the post-synaptic

membrane at this time. Therefore, we tested our hypothesis by immunolabelling non-

permeabilized mouse brainstem sections with antibodies to the extracellular domains of a

selection of GluR subunits. The permeability of the cells in our sections was reduced by

eliminating treatment with Triton X-100, a detergent used in most immunolabeling procedures.

This compound partially breaks down the cell membrane (Helenius and Simons 1975) and

permits large, labeled antibodies to enter the intracellular space (de Graaf et al. 1991; Humbel

1998). We also selected antibodies to target the extracellular domains of GluR1 and GluR4.

Antibodies to the extracellular domains of GluR2 and GluR3 are not currently available so these

subunits were not evaluated in our study. The results of our experiments demonstrate that cell

surface GluR4 but not GluR1 is altered in the VNC during the first two hours after UVD.

3.2 Methods

73

3.2.1 Animals

A total of 4 female C57Bl/6 mice were used in this study. The sources of these mice,

experimental times, ages and weight restrictions were all identical to those described in the

Methods section of Chapter 2. Mice underwent surgery for UVD (performed by R.H.-S.),

followed by behavioural monitoring (performed by R.H.-S.) and then transcardial perfusion with

fixative (performed by Chiping Wu, a research associate at the University of Toronto). All

procedures followed guidelines set by the Canadian Council for Animal Care and were approved

by the Animal Care Committee at the University Health Network.

3.2.2 Collaboration

This study was a combined effort with Dr. Joan S. Baizer’s laboratory at the University at

Buffalo. All tissue samples were shipped to Buffalo by R.H.-S. Subsequent tissue preparation,

sectioning and immunolabeling were performed by J.S.B. and data were analyzed by her students

(Jishun Zhao, Marni Bleichfeld, and Nicholas Paolone) in collaboration with R.H.-S. Data

analysis was performed by J.Z., M.B., and N.P. on 3 out of the 4 mice. Data from the fourth

mouse was analyzed by R.H.-S.

3.2.3 Surgical Procedure

Each mouse underwent surgery for right UVD (n=4). Surgery followed the same procedure

descbribed in the Methods section of Chapter 2.

3.2.4 Evaluating Static Vestibular Reflexes

The degree of recovery from UVD was assessed through evaluation of static vestibular reflexes.

Static signs were continuously monitored and scored (see Chapter 2 Methods) during the first

five minutes after each mouse had fully recovered from anesthesia and then again over the last 5

minutes of the survival period. Throughout the survival period, arousal levels were maintained

as described in the Methods section of Chapter 2. We assumed a lack of recovery for UVD mice

74

if the score at the end was more than half of the score assigned at the beginning. UVD mice

showing poor recovery were excluded from the study since compensation may have been flawed

in these animals. Mice were also eliminated if they did not exhibit a minimum score of 6 after

UVD.

3.2.5 Immunohistochemistry

The extracellular domains of the AMPA receptor subunits GluR1 and GluR4 were labeled in all

4 mice using immunohistochemistry. Each mouse, regardless of its weight, was anesthetized

with 0.1 ml of sodium pentobarbital (10 mg/ml) and then perfused transcardially, at a flow rate

of 6 ml/min, with 30 ml of 0.1 M phosphate-buffered saline (PBS) followed by 30 ml of 4%

paraformaldehyde in PBS. For the perfusions, solutions were kept at room temperature. After

the brain was removed, it was stored at 4ºC in 4% paraformaldehyde in PBS for 24 hours. After

post-fixing the brain, a shallow cut was first made across the ventral aspect of the right brainstem

to mark the lesioned side. Next, it was cryoprotected in 15% sucrose/PBS and then shipped

overnight to Buffalo. During shipping, the brain was kept chilled in a vial of 15% sucrose/PBS

contained in a well-insulated ice-filled box. When it reached Buffalo, the brain was further

cryoprotected in 30% sucrose/PBS at 4ºC for an additional 24 hours.

After cryoprotection, the brains were prepared for immunolabeling. Each brain was frozen at -

70ºC and coronal sections through the brainstem, 40 µm thick, were cut on a sliding freezing

microtome (American Optical). The sections were then rinsed in PBS (all rinses were 3 x 10 min

at room temperature with gentle agitation) and incubated for 30 minutes in a diluted serum

solution consisting of 0.1M PBS with 1% bovine serum albumin, 1.5% normal goat serum

(Vectastain Elite ABC Kit, Vector Laboratories, Burlingame CA). Rabbit antiserum to either the

GluR1 subunit (Santa Cruz Biotechnology, GluR1 sc-7608, 1:500) or GluR4 subunit (Santa Cruz

Biotechnology, GluR4 sc-31394, 1:400) was subsequently added to the diluted serum solution

and incubated overnight at 4°C. Incubation in the diluted serum solution was done to prevent

binding of the primary antibody to non-specific tissue elements. Next, sections were rinsed and

incubated with the Vector anti-rabbit IgG secondary antibody (0.5%, following manufacturer’s

instructions) in PBS and normal goat serum (same concentrations as above), and then in the

Vector ABC reagent, according to manufacturer’s instructions, to enhance visualization of the

75

antibody complex. Immunoreactivity was visualized with the glucose oxidase-DAB-nickel

method (Shu et al. 1988). Each brain was labeled with both primary antibodies on alternating

sections. Control sections in which the primary antibody was omitted from the first incubation

solution were otherwise processed identically. The control sections were used to ensure that

non-specific labeling with the secondary antibody did not take place. When the labeling was

complete, sections were mounted on slides, air-dried, dehydrated in graded ethanol solutions,

cleared with Histosol (National Diagnostics) and coverslipped. Digital images of the sections

were captured at 2x and 10x magnification with a SPOT camera mounted on a Leitz Dialux 20

microscope. No immunostaining was seen on the control sections.

3.2.6 Data Analysis

Sections located between 5.5 and 7.1 mm caudal to Bregma were selected for analysis. The

distance from Bregma was approximated by using a mouse brain atlas (Paxinos and Franklin

2001). If at 2x magnification labeling appeared blotchy, cells could not be discerned, or there

were tears within the vicinity of the VNC, then the section was excluded from our analysis.

Altogether, 6 divisions of the VNC were analyzed: the parvocellular division of the MVN

(MVNpc), the magnocellular division of the MVN (MVNmc), the caudal MVN, the descending

vestibular nucleus (DVN), the lateral vestibular nucleus (LVN), and the superior vestibular

nucleus (SVN). According to Paxinos & Franklin (2001), the MVN has magno-and

parvocellular divisions at levels less than 6.64 mm caudal to Bregma. As described by Epema et

al. (1988), the MVNmc contains many large neurons while the MVNpc consists mostly of very

small neurons. Further caudal, the large neurons disappear and there is no longer a clear

distinction between MVNpc and MVNmc. A comparison of rostral and caudal sections could

not be made for all VN divisions. Therefore, for each divison of the VNC, data acquired across

the entire rostro-caudal axis were pooled.

For each section analyzed, label intensity was first measured for all divisions within the VN on

both sides of the brainstem at 10x magnification. All measurements were made using ImageJ

software (National Institute of Health). For right and left sides, all divisions were first selected

as regions of interest (ROIs). Next, the area and mean gray value were automatically measured

for each ROI. In ImageJ, lighter stains had larger mean gray values. The mean label intensity

76

was therefore estimated as 255 – mean gray value (255 was the maximum gray value or lightest

stain that could be assigned in ImageJ).

Next, the number of labeled cells in each region of interest was evaluated. For counting cells, a

threshold label intensity was first selected and only those cells above threshold were counted. A

cell was also counted if its approximate diameter fell within the range of diameters found in the

vestibular nucleus of the rat (Suárez et al. 1993). Only for the medial vestibular nucleus (MVN)

have these parameters been evaluated in mice (Bagnall et al. 2007) and they were very similar to

those of the rat. All cells between 10 and 30 µm were counted. In our sections, most structures

smaller than 10 µm in diameter were not neurons. All cells above 30 µm in diameter were

excluded to reduce the probability of cross-sectional staining. Each cell count was converted

into a cell density measurement (number of cells/ROI area) to compensate for any variability in

ROI size.

3.2.7 Statistics

For each antibody, contralesional-to-ipsilesional comparisons of the mean labeled cell densities

and mean label intensities within each ROI were performed with 1-tailed paired t-tests (p<0.05).

3.3 Results

Typical examples of labeled sections are shown in Figures 3-1 (GluR4) and 3-2 (GluR1). In

each figure, all divisions of the VNC, except the caudal MVN, are shown at 10x magnification.

Labeling in the caudal MVN (not shown) was similar in appearance to MVNpc.

Figure 3-1: Examples of 40 µm sections labeled for GluR4 subunits as they appear at 10x magnification. All

sections were taken from the contralesional VNC of Mouse 60. The distance from Bregma in mm is indicated in the

77

bottom right corner of each image. Negative numbers indicate a posterior location. (A) The parvocellular (pc) and

magnocellular (mc) divisions of the medial vestibular nucleus (MVN), (B) the lateral vestibular nucleus (LVN) and

the descending vestibular nucleus (DVN) and (C) the superior vestibular nucleus (SVN). The scale bar is indicated

in the bottom left corner of C. Scale bar = 250 µm.

Figure 3-2: Examples of 40 µm sections labeled for GluR1 subunits as they appear at 10x magnification. All

sections were taken from the contralesional VNC of Mouse 60. The distance from Bregma in mm is indicated in the

bottom right corner of each image. Negative numbers indicate a posterior location. (A) The parvocellular (pc) and

magnocellular (mc) divisions of the medial vestibular nucleus (MVN), (B) the lateral vestibular nucleus (LVN), (C)

the descending vestibular nucleus (DVN) and (D) the superior vestibular nucleus (SVN). The scale bar is indicated

in the bottom left corner of D. Scale bar = 250 µm.

Two hours after UVD, asymmetric labeling for GluR4 subunits was observed in the MVNmc

and DVN. In both divisions, labeling was significantly greater in the contralesional compared to

ipsilesional side (Figure 3-3, middle and top right; p<0.01, 1-tailed paired t-test). A similar

difference was also noted in the SVN and caudal MVN, though it was not significant in either of

these divisions. GluR4 label intensity was not different between the intact and lesioned sides

(p>0.05, 1-tailed paired t-test). We conclude that UVD causes an acute asymmetry in GluR4-

containing AMPA receptors in the MVNmc and DVN.

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Figure 3-3: An asymmetry in GluR4 cell densities was found between the intact and lesioned sides two hours after

UVD. GluR4 cell densities were greater on the contralesional compared to ipsilesional side in both the DVN and

MVNmc. The numbers of mice from which the data were acquired are indicated in the top left corner of each panel.

For one of the mice, no intact sections containing the SVN were obtained. Data are presented as means and standard

deviations. * indicates significance of p<0.05 (1-tailed paired t-test).

Acutely following UVD, the numbers of GluR1-labeled cells on the lesioned and intact sides

remained symmetric (Figures 3-4; p>0.05, 1-tailed paired t-test). Also, UVD induced no

difference in GluR1 label intensity between the lesioned and intact sides (data not shown, p>0.05

Mann-Whitney U test). Thus, UVD caused no asymmetries in the labeling of GluR1-containing

AMPA receptors in the VNC.

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Figure 3-4: Cell densities for GluR1 were bilaterally similar after UVD. Box plots are shown for contralesional and

ipsilesional label intensities (A) and cell densities (B) for the different subdivisions of the VNC. No significant

differences were detected between ipsilesional and contralesional sides (p>0.05, 1-tailed paired t-test). The numbers

of mice from which the data were acquired are indicated in the top left corner of each panel. Some of the mice did

not produce intact sections containing the caudal MVN (n=1) or the SVN (n=2).

3.4 Discussion

This is the first time GluR subunit expression in the VNC has been evaluated just two hours

following UVD. We found that the density of cells labeled for GluR4 was significantly greater

on the intact compared to lesioned side two hours after UVD in the MVNmc and DVN. While

we did not observe an asymmetry in the expression of GluR1, we cannot rule out the possibility

that UVD had an effect on the bilateral expression of this subunit since we did not compare it

with labeling in the VNC of sham-operated mice.

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Our results suggest that rapid synthesis of GluR4 subunits may take place in the contralesional

VNC during the most acute phase of vestibular compensation. The rapid synthesis of GluR

subunits associated with LTP was recently reported in the ventral tegmental area (VTA), a region

in the brainstem involved in reward response (Mameli 2007; Argilli 2008). These investigations

demonstrated that GluR subunits are synthesized and inserted into the post-synaptic membrane

within less than three hours following a potentiating stimulus. Evidence obtained from acutely

labyrinthectomized rats has suggested that UVD may induce LTP at the NVIII synapse in the

contralesional MVN (Pettorossi et al. 2003). In addition, King et al (2002) found that the

quantity of GluR2 subunits measured from Western blots was elevated on the contralesional

compared to ipsilesional side in the VNC of rats that had been labyrinthectomized 10 hours

earlier. Together, our results and the published evidence suggest that the induction of LTP

associated with the rapid syntheisis of GluR subunits may take place during the acute stage of

vestibular compensation.

To our knowledge, this was also the first attempt to evaluate labeling for AMPA receptor

subunits at the cell surface using immunolabeling in the VNC. We assumed that most labeling

was at the cell surface because Triton X-100 was omitted from our protocol. Triton X-100

partially breaks down lipid membranes (Helenius and Simons 1975), which would make the cells

more permeable to the labeled antibodies (de Graaf et al. 1991). However, it is quite likely that

some of the labeled antibodies gained access to the intracellular space through cross-sectioned

cells. Such contamination was unavoidable and therefore confounds the conclusion that our

results were due to changes in GluR subunits belonging to functional cell-surface receptors.

The effects of UVD on GluR4 were found only in our cell density measurements. We did not

find any significant differences in overall label intensity. Therefore, compared to the

surrounding neuropil, the contribution of labeled cell bodies to the overall label intensity was

probably too small to produce any significant differences. Axon terminals, glial cells and

dendritic processes are the main constituents of the neuropil. AMPA receptors are typically not

found pre-synaptically (Martin et al. 1993) so it is unlikely that GluR4 at axon terminals made

any contribution to label intensity. Astrocytes and microglia, which express GluR4 (Condorelli

et al. 1993; Noda 2000), appeared in the cat VNC after UVN (de Waele et al. 1996; Tighilet et

al. 2007). While a few astrocytes and microglia were visible one day after surgery, most of them

were not seen until at least 2 post-operative days in those studies. A similar glial cell response

81

probably takes place in the mouse VNC as well. In rodents, brainstem astrocytes (Hydman

2005) and microglia (Rappert 2004; Hydman 2005) remain sparse in the neuropil up until several

days after peripheral nerve injury. Our mice were perfused only two hours after UVD so it is

unlikely that astrocytic or microglial GluR4 made a significant contribution to label intensity.

Throughout the CNS, the majority of AMPA receptor subunits, including GluR4, are found at

synapses located on the post-synaptic membranes of distal dendrites (Tachibana et al. 1994; Farb

et al. 1995; Vissavajjhala et al. 1996; Ye and Westlund 1996; Petralia et al. 1997; He et al. 1998;

Kessler and Baude 1999; Montague and Greer 1999; Beckerman and Glass 2011). Therefore,

distal dendritic synapses were probably the main source of GluR4 labeling in our study. Since

label intensity was not significantly altered within 2 hours after UVD, it is unlikely that dendritic

GluR4 was acutely affected by the lesion. It is possible that UVD may have acutely altered

GluR4 at dendritic shafts or at the cell body, however the exact site for lesion-induced changes in

GluR4 could not be determined in our study.

In summary, our evidence suggests that UVD may acutely alter the function of GluR4-containing

AMPA receptors in the VNC. Our results are consistent with the effects of AMPA receptor

manipulation on reflex recovery in rats (Hirate et al. 2000). In the rat study, the AMPA receptor

antagonist, kainate, exaggerated lesion-induced deficits in static balance when administered

systemically three hours after UL. It is possible that at least some of the AMPA receptors

blocked in the rat contained GluR4 subunits since GluR4 is very common in all regions of the

normal VNC (Petralia and Wenthold 1992; Chen et al. 2000). While it is not known whether

GluR4 is normally incorporated into functional AMPA receptors in the VNC, GluR4-containing

AMPA receptors are normally found in other areas of the brainstem (Rubio and Wenthold 1997;

Wang et al. 1998).

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Chapter 4: Excitatory and Inhibitory Synaptic

Transmission during the Acute Stage of Vestibular

Compensation in the Mouse MVN

4.1 Introduction

Immediately after UVD, deficits in static and dynamic vestibular reflexes are associated with a

bilateral imbalance in the resting activities and head movement sensitivities of VNC neurons

(Shimazu and Precht 1966; Hamann and Lannou 1987; Smith and Curthoys 1988a; Smith and

Curthoys 1988b; Newlands and Perachio 1990a; Newlands and Perachio 1990b). A few authors

have proposed that synaptic plasticity may be involved in the restoration of neural activity and

the recovery of vestibular reflexes after UVD. Their evidence suggests that plastic changes may

take place at the NVIII synapse in the MVN on the intact side (Pettorossi et al. 2003) and the

commissural synapses on the lesioned side (Dieringer and Precht 1977; Dieringer and Precht

1979a). These studies also indicated that plastic changes could take place acutely following

UVD. However, it is unknown how soon after UVD these plastic changes take place. Long-

lasting plastic changes can take place within a matter of minutes following a persistent change in

pre-synaptic input in the normal VNC (Capocchi et al. 1992; Peterson et al. 1996; Grassi et al.

2001; Nelson et al. 2003; McElvain et al. 2010). UVD causes an immediate loss of excitatory

input to neurons on the ipsilesional side (see Section 1-3-1) and, consequently, loss of

commissural inputs to neurons on the contralesional side (Smith and Curthoys 1988a; Newlands

and Perachio 1990a; Newlands and Perachio 1990b). Therefore, we hypothesized that within

the first few hours after UVD synaptic transmission in the VNC is altered from normal at

the contralesional NVIII synapse and the ipsilesional commissural synapses. To test our

hypothesis, we used mouse brainstem slices obtained at 2 and 4 hours after UVD. We then

recorded responses of MVN neurons to NVIII and commissural stimulation on the intact and

lesioned sides, respectively. Through our experiments, we produced preliminary evidence

suggesting that both NVIII and commissural synapses are altered acutely following UVD.

4.2 Methods

83

4.2.1 Animals

C57/Bl6 mouse pups (post-natal days 14-19) of both genders were used in this study. All pups

were bred in our animal facility and kept on a normal light cycle. Two sets of experiments were

performed in which brainstem slices were prepared from each pup used. In the first series of

experiments, responses of MVN neurons to NVIII stimulation were studied and in the second we

investigated the responses to midline (commissural) stimulation. In each set of experiments, a

subset of pups underwent surgery for UVD by the air injection method (see "Surgical Procedure"

in Methods section of Chapter 2). All surgeries were performed by R.H.-S. Procedures followed

guidelines set by the Canadian Council for Animal Care and were approved by the Animal Care

Committee at the University Health Network.

4.2.2 Evaluating Static Vestibular Reflexes

The amount of recovery from UVD was assessed through evaluation of static vestibular reflexes.

For every pup, static signs were monitored as described earlier (performed by R.H.-S., see

―Evaluating Static Vestibular Reflexes‖ in Methods of Chapter 2) over the first five minutes after

it had fully recovered from anesthesia and then again over the last 5 minutes of the survival

period. A subset of pups used in each type of experiment was monitored throughout the entire

survival period. A lack of recovery was assumed if the score at the end was more than half of

the score assigned at the beginning. Pups that met this criterion were excluded from the study.

Pups showing poor recovery were excluded from the study since compensation may have been

flawed in these animals. Pups were also eliminated if they did not exhibit a minimum score of 6

in the first 5 minutes of recovery.

4.2.3 Survival Times

For air-injected pups, different survival times, which always began immediately after the air

injection, were used for each set of experiments. In the first set of experiments, NVIII

stimulation, a survival time of 4 hours was selected. The pups used for these experiments were

also subjected to a 2.5 hour period of anesthesia during surgery and usually took at least an hour

84

to recover from the anesthetic. Once pups recovered from the anesthetic, static signs reached

their peak and behavioural recovery (ie. the abatement of static signs) began (see Figure 4-1,

left). The abatement of static signs followed an exponential time course and reached asymptote

by 2.5 hours after UVD. We assumed that physiological changes associated with the abatement

of static signs could be detected as soon as behavioural recovery reached asymptote. For

commissural stimulation experiments (Figure 4-1, right), we shortened the survival time from 4

hours to 2 hours (vertical dashed line) based on the rapid behavioral recovery that was observed.

Figure 4-1: Time course for behavioural recovery (ie. the abatement of static signs) after UVD. Behavioural scores

recorded every 5 minutes were summed into 20 minute bins. Binned scores are shown for pups that survived 4 hrs

(left) and 2 hrs (right) after surgery for UVD. The vertical dotted lines indicate when the anesthetic was stopped.

The vertical dashed line in B indicates when 2 hours has past from the time of the lesion.

4.2.4 Slice Preparation

All slice preparation and subsequent recording was carried out by my supervisor, Dr. Broussard.

Pups were deeply anesthetized with isoflurane and decapitated. The brainstem and cerebellum

were removed from the skull by dissection and placed in a bath of ice cold artificial

cerebrospinal fluid (ACSF). The bath had an osmolarity of 330mOsm/L and it contained (in

mM) 124 NaCl, 5 KCl, 26 NaHCO3, 1.3 MgSO4, 2.5 CaCl2, 1 NaH2PO4, and 11 dextrose.

During all procedures the ACSF was maintained at pH 7.4 and was continuously infused with

95% O2 and 5% CO2. Coronal slices, 400 μm thick, were made between 5.9 and 6.8 mm caudal

to Bregma. The slices were cut in ice cold ACSF using breakable razor blades (Fine Science

Tools, Vancouver BC) on a Vibratome 1000 (Vibratome, Bannockburn, IL). Immediately after

being cut, slices were incubated at room temperature for a minimum of 2 hours. After they were

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transferred to the slice chamber for recording, a thermistor was used to keep the slices at 30 ºC.

In the slice chamber, strychnine (1 µm) and picrotoxin (10 µm) were added to the bath during

NVIII stimulation so as to block all synaptic inhibition.

4.2.5 Recording Method

The whole-cell recording method was used. Glass pipettes, each with a resistance of 5-6MΩ,

were filled with an intracellular solution containing (in mM) 8 NaCl, 10 HEPES, 2 ATP, 0.3

GTP, 0.8 QX314. QX314 was used to block sodium channels and prevent action potentials

during recording, and cesium methanesulfonate (CH3O3SCs), which we will abbriviate as CsM,

was used to block potassium channels in most cases. During NVIII stimulation, CsM was not

included in the recording protocol for the first set of cells (pre-lesion n=22, post-lesion n=6). It

was later added to the bath (pre-lesion n=9, post-lesion n=4) to improve the voltage clamp and

was used during all commissural stimulation experiments. With the aid of a dissecting

microscope (Control Optics), each pipette was positioned in the dorsal or parvocellular division

of the medial vestibular nucleus (MVN), just below the floor of the 4th

ventricle (Figure 4-2).

Cell recordings were made between 0 and 300 μm from the slice surface.

All recordings were performed with an Axoclamp 2B amplifier and carried out both in current

clamp and voltage clamp modes. Measured Vm and the electric potential measured with a

reference electrode outside the cell (Ve) were used to calculate Vm (Vi - Ve). In the voltage clamp

experiments, measured Vm was held constant by injecting current into the cell. Junction

potentials were zeroed in the bath before each recording and checked again after removing the

electrode from the cell. All measurements of membrane potential were also adjusted for a liquid

junction potential of 14 mV (Sekirnjak and du Lac 2002). Resting membrane potential and input

resistance were checked periodically to ensure that the cell was still healthy and the seal was

intact. Input resistance was measured using 500ms current pulses ranging from -200 to +50 pA.

If there was a dramatic fall in input resistance and/or if the membrane potential became

depolarized above -30mV, the recording was excluded from the analysis.

4.2.6 Stimulation Method

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For all experiments, stimulation was conducted using a pair of platinum-iridium electrodes

(0.002-inch diameter) one on the top surface of the slice and the other on the bottom surface

(Figure 4-2). Where the electrodes made contact with the slice, the insulation was stripped so

that a constant voltage could be applied between the two electrodes across the width of the slice

(Broussard 2009). This method was chosen since it was the most effective for generating

responses in the MVN. For activating the primary afferents, the stimulating electrodes were

positioned at the lateral aspect of the slice (Figure 4-2 left). The electrodes were centered on the

lateral vestibular nucleus (LVN) since most afferent axons projecting to the MVN traverse it

(Lorente de No 1933). For midline stimulation, the electrode pair was placed just contralateral to

the midline (Figure 4-2 right). This position was selected to avoid stimulation of the medial

longitudinal fasciculus (MLF). In all cases, direct stimulation of the vestibular nerve root was

not chosen since passage of the afferent axons from the root of NVIII into the VNC does not

follow a path within the plane of the slice (Lorente de No 1933). NVIII enters the VNC at a

more rostral location that was not included in the slice preparation.

Figure 4-2: Bipolar electrode placements for stimulating the primary afferents of the intact side (left) and the

vestibular commissure (right). The solid and dashed vertical lines represent the electrode poles on the top and

bottom surfaces of the slice, respectively. Biphasic pulses of constant voltage (0.1 ms duration) of various

amplitudes were applied. The recording electrode was always positioned in the contralesional medial vestibular

nucleus (MVN) for recording responses to afferent stimulation and the ipsilesional MVN for recording responses to

commissural stimulation. For the afferent stimulation protocol, the electrode was positioned over the lateral

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vestibular nucleus (LVN) since afferent projections to the MVN cross through this nucleus (Lorente de No, 1933).

The commissure was activated by placing the electrode slightly off the midline on the contralesional side.

4.2.7 Stimulus Parameters

Bipolar pulses at constant voltage (0.05 to 10 V, 0.1 ms duration) were applied. We chose

constant voltage because the electrodes had been stripped of their insulation and constant current

was not possible due to the low impedance of the electrodes. All constant voltage pulses were

generated with a stimulus isolation unit driven by a computer-controlled custom-built pulse

generator. In each experiment, a series of pulses was first applied in order of increasing intensity

during recording in current clamp mode. For each cell, the stimulus voltage at threshold was

determined and then increased as a multiple of 2n, where n is a whole number, up to a maximum

of 10V. At each stimulus voltage tested, a set of 10 biphasic pulses was applied to NVIII or the

midline at 5s intervals. During recordings in voltage clamp mode, the holding potential was set

to different values between -94 and +26 mV. At each holding potential, always beginning with -

94mV, biphasic pulses at twice threshold were applied to NVIII or the midline at 5 s intervals.

4.2.8 Data Analysis

Voltage traces, proportional to membrane potential (mV) or current (pA), were generated

through the Axoclamp 2B amplifier and acquired using Labview (National Instruments, Austin

TX) on a Pentium II computer. Input resistance was measured from linear fits of steady state

voltage against current within the linear range, and repeated measurements were averaged. From

recordings of membrane potential and current at least four good voltage traces were averaged.

Examples of averaged voltage traces for membrane potential and current can be found in Figure

4-4A and 4-5A. From the averaged traces, post-synaptic potential and post-synaptic current

amplitudes were calculated as the difference between the peak and baseline measurements. The

latencies for post-synaptic potentials were also measured as the time between the end of the

stimulus artifact (not shown in Figure 4-4A) and the start of the deflection from baseline. For

NVIII stimulation experiments, only those cells that responded with monosynaptic latencies were

included in the analysis. The range of monosynaptic latencies was determined from the latency

distribution for normal cells (Figure 4-3, left). All latencies to the left of the first gap in this

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distribution were considered monosynaptic. The average monosynaptic latency was 1.77 ± 0.48

ms, which is consistent with published values (Cochran et al. 1987; Doi et al. 1990; Kinney et al.

1994; Broussard 2009).

Figure 4-3: Latency distributions for excitatory post-synaptic potentials (EPSPs) recorded from cells in normal

(left) and lesioned (right) brainstem slices. Solid gray bars = monosynaptic latencies, hatched gray bars =

polysynaptic latencies.

For each cell, an input-output (I/O) curve was generated by plotting EPSP or IPSP amplitude

against stimulus voltage. If a cell’s I/O curve contained no EPSP or IPSP amplitudes greater

than 1 mV, the cell was said to be unresponsive and was excluded from the analysis of current

clamp data. For responsive cells, the slope of the I/O curve was measured by linear regression.

Current-voltage relationships were constructed by plotting EPSC or IPSC amplitude against

holding potential for each cell. Post-synaptic current was measured at holding potentials of -94,

-74, -54, -34, -14, +6, and +26 mV. If post-synaptic current did not exceed 1 pA throughout the

entire recording prototol, then the cell was considered unresponsive and its current-voltage

relationship was excluded from the analysis. The type of response (inhibitory, excitatory or

mixed) for each cell was determined by the reversal potentials measured from the current-voltage

relationship. The holding potential at which the smallest EPSC or IPSC amplitudes were

generated was taken as the reversal potential for the cell. If the reversal potential was

approximately 0 mV (EGlutamate Receptor Channel), the response was considered excitatory. For

excitatory responses, the contributions of AMPA and NMDA receptors were measured. The

current passed through AMPA receptors was taken as the peak of the EPSC. The NMDA

current, on the other hand, was measured 25 ms after the stimulus artifact (see Figure 4-5A), a

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point at which current through AMPA receptors is nearly zero (Kinney et al. 1994; Grassi et al.

2009; McElvain et al. 2010). The relative contribution of each receptor was determined by

calculating the NMDA to AMPA ratio. This ratio was determined by dividing the NMDA

current measured at a holding potential of +26 mV by the AMPA current measured at a holding

potential of -74mV. We chose -74 mV as the holding potential for the AMPA current since

NMDA currents are close to zero at membrane potentials below -40 mV (Kinney et al. 1994;

Grassi et al. 2009; McElvain et al. 2010).

Besides excitatory responses, inhibitory and mixed responses were also observed. An inhibitory

response was identified if the reversal potential was -74 mV (ECl). Mixed responses were

confirmed if the reversal potential was between -74 and +6 mV. For mixed responses, the

weights of excitatory and inhibitory inputs were assessed. The excitatory component was taken

as the peak of the post-synaptic current measured at a holding potential of -74 mV, where the

chloride current through glycine and GABA receptors is near reversal and therefore near zero.

The inhibitory component, on the other hand, was taken as the post-synaptic current measured at

a holding potential of +6 mV (see Figure 4-9A), which is approximately the reversal potential for

ionic currents through glutamatergic receptor channels. The relative contributions of each were

then evaluated by taking the ratio of the excitatory current measured at a -74 mV holding

potential to the inhibitory current measured at a +6 mV holding potential.

4.2.9 Statistics

For all data sets in which both the normal and post-UVD groups had n ≥ 4, comparisons between

the two groups were performed with the Mann-Whitney U test (p<0.05) since the distributions

for these data were non-normal. For all data sets in which either one or both of the groups being

compared had n < 4, a power analysis for comparisons of the means was performed. For these

data, the minimum sample size to fall within each distribution being compared and produce a

statistical power of 0.8 was reported. A power of 0.8 indicates that there is a 4 in 5 chance that

the means will be significantly different (p<0.05).

4.3 Results

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4.3.1 Acute Effects of UVD at the NVIII Synapse in the Contralesional MVN

Our first aim was to determine whether UVD induces acute potentiation at the NVIII synapse in

the contralesional MVN. We recorded responses to NVIII stimulation from a total of 41

neurons, 31 from normal slices and another 10 from slices obtained 4 hours after UVD. For each

of the neurons we included in our analysis (nNormal = 28, nUVD = 8), an I/O function was

constructed from the responses to increasing stimulus voltage. We predicted that an increase in

synaptic efficacy would produce an increase in the slope of the I/O function but UVD had no

significant effect on slope (Figure 4-4B, p>0.05, Mann-Whitney U test).

Figure 4-4: Responses to afferent stimulation in normal and UVD pups. The slopes of the input/output functions

generated by contralesional afferent stimulation are not significantly altered by UVD. (A) EPSP traces obtained

from a single normal MVN neuron in response to a series of voltage pulses (max. amplitude = 10.0V). (B) Slopes of

the I/O curves for MVN neurons recorded in normal slices and in slices obtained from mice that had undergone

surgery for UVD 4 hours earlier. Filled circles represent the outliers in each sample. The mean and median for each

sample are indicated by the dashed and solid lines. The bottom and top of each box mark the 25th and 75th

percentiles, while the bottom and top whiskers mark the 5th and 95th percentiles. Pre-lesion and post-lesion slope

distributions were not significantly different (p>0.05, Mann-Whitney U test). (C) Input/output (I/O) curves for all

MVN neurons included in B.

To evaluate the effects of UVD on glutamate receptor function at the NVIII synapse, we

evaluated post-synaptic current through AMPA and NMDA receptor channels in a subset of our

cells (nNormal = 6, nUVD = 3). For each cell, two current-voltage relationships were constructed to

depict AMPA and NMDA currents separately. Individual normalized current-voltage

relationships are shown in Figure 4-5C (AMPA) and Figure 4-5D (NMDA). Within both sets of

curves, an overlap between the normal and UVD groups was apparent. However, despite the

similarity in the current-voltage relationships for normal and UVD neurons, a difference in the

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NMDA to AMPA ratio was evident between the two groups (Figure 4-5B). Four hours after

UVD, this ratio was reduced compared to normal, which might indicate an increase in the

number of AMPA receptors recruited to the post-synaptic membrane at this time. As determined

by a power analysis (see Section 4-2-9) this difference would be significantly different (p<0.05)

if each distribution contained a minimum of just 10 cells. Therefore, our preliminary results

suggest that UVD may induce acute potentiation at the NVIII synapse on the intact side.

Figure 4-5: Excitatory post-synaptic currents (EPSCs) in response to afferent stimulation before and after UVD. A,

series of excitatory post-synaptic currents (EPSCs) obtained from a single normal MVN neuron at different holding

potentials. The current through AMPA receptors was measured from the peak of each EPSC. 25 ms indicates the

point on each EPSC at which NMDA currents were measured. B, NMDA to AMPA ratios for normal and post-UVD

neurons. This data set would require a minimum of only 10 samples in each distribution for the means to be

significantly different (p<0.05). Values were determined from the ratio of the NMDA current measured at a holding

potential of 26 mV and the AMPA current measured at a holding potential of -74mV. Box plots are set up as

described for Figure 4-4B. However, for samples sizes of n < 6, neither the 5th and 95th percentiles or the outliers

could be shown. The normalized AMPA (C) and NMDA (D) currents are shown for each neuron as a function of

holding potential. AMPA currents were normalized against the average value measured at a holding potential of -

34mV for normal and post-UVD neurons. NMDA currents were normalized against the average values measured at

a holding potential of -74mV. The vertical dashed lines indicate the measurements on each curve used for

determining the NMDA to AMPA ratio.

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Of the cells that responded with monosynaptic latencies to NVIII stimulation, 12 were studied in

the presence of CsM (nNormal = 9, nUVD = 3), a potassium channel blocker. The effects of CsM

were evaluated by comparing the responses of two separate groups of normal cells. In the first

group (n=9), responses were recorded only in the presence of CsM and the other responses were

recorded in the absence (n=19) of CsM. Input resistance was significantly increased (Table 4-1,

p<0.05, Mann-Whitney U test) and membrane potential was significantly depolarized (Table 4-1,

p<0.05, Mann-Whitney U test) in cells recorded in CsM. To ensure that responses to NVIII

stimulation were not also influenced by CsM, its effects on I/O curve slope, EPSP amplitude and

post-synaptic current were evaluated. Neither I/O curve slope nor EPSP amplitude were

significantly affected by CsM (Table 4-1, p>0.05, Mann-Whitney U test).

No CsM CsM

Mean S.D. n Mean S.D n Significance

Rin 214.02 106.03 18 307.87 132.00 9 <0.05

Vm -65.51 20.30 19 -46.65 7.50 9 <0.05

I/O slope 2.56 4.36 17 4.33 4.64 8 >0.05

Table 4-1: The effects of potassium channel block on normal resting membrane properties and responses to NVIII

stimulation recorded in current clamp mode. CsM = cesium methanesulfonate.

CsM also appeared to have very little effect on post-synaptic current (Figure 4-6). The

NMDA:AMPA ratio for cell recorded in CsM did not appear very different and a power analysis

revealed that each distribution would require a minimum of 120 samples in order for there to be

a significant difference. Therefore, it is unlikely that CsM had any influence on our results.

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Figure 4-6: Potassium channel block had very little effect on the NMDA to AMPA ratio in normal cells. For the

mean ratios to be considered significantly different (ie. power ≥0.8), a minimum of 120 cells would be required

within each distribution. CsM = cesium methanesulfonate.

4.3.2 Acute Effects of UVD at Commissural Synapses in the Ipsilesional MVN

The second aim of our study was to determine whether UVD acutely alters the responses of

ipsilesional MVN neurons to commissural inputs. We recorded responses to midline sitmulation

in 28 healthy neurons, 17 from normal slices and 11 from slices obtained 2 hours after UVD.

Responses to midline stimulation were classified as pure inhibitory, pure excitatory or mixed

(exhibiting both inhibitory and excitatory components). A response was considered mixed if it

exhibited excitatory and inhibitory responses that could be clearly distinguished in current clamp

and/or voltage clamp experiments.

To evaluate whether commissural inputs were altered after UVD, we constructed I/O curves for

the inhibitory and excitatory commissural responses (Figure 4-7A & C). The numbers of cells

for which I/O curves were evaluated is shown in Table 4-2 for each response type. Due to the

small numbers of cells for which I/O curves were generated, mixed and pure responses were

pooled together in Figure 4-7. The slopes of the input-output curves generated after UVD were

not significantly different from normal for either response type (Figure 4-7B & D, p>0.05,

Mann-Whitney U test).

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Response Type Number of Cells - Normal Number of Cells – Post-UVD

Pure Excitatory 3 4

Pure Inhibitory 9 3

Mixed Excitatory 2 2

Mixed Inhibitory 1 3

Table 4-2: The number of cells of each response type for which I/O curves were generated.

Figure 4-7: Responses to commissural inputs in the ipsilesional MVN. A & C: Input/output functions generated by

midline stimulation are not significantly altered by UVD on the ipsilesional side. B & D: Slopes corresponding to

the different I/O curves in A. No significant differences were detected between slopes from normal and post-UVD

I/O curves (p>0.05, Mann Whintey U test). The sample sizes for each group are listed above and below the top and

bottom panels, respectively. In the normal group, mixed responses contributed n=1 each to the excitatory and

inhibitory populations. In the post-UVD groups, mixed responses contributed n=2 to the excitatory population and

n=3 to the inhibitory population. Box plots are set up as described for Figure 4-4B. However, for samples sizes of n

< 6, the outliers could be shown.

Further examination of the responses in voltage clamp revealed that UVD may have an acute

effect on commissural synaptic transmission. For cells receiving pure excitatory commissural

input, the NMDA:AMPA ratio was reduced after UVD compared to normal (Figure 4-8 left).

The sample size was very small though a power analysis revealed that a minimum of 6 samples

would be required in each distribution for the means to be significantly different (p<0.05).

However, for cells receiving pure inhibitory inputs, there were no apparent differences between

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those from normal and post-UVD mice at any holding potential tested (Figure 4-8 right). Also,

very large sample sizes would be required for the means at any of the holding potentials to be

signficantly different. For cells with mixed inputs, the post-synaptic current measured in post-

UVD neurons appeared to be increased compared to normal at a holding potential of +6 mV

(Figure 4-9 left). Since excitatory currents are at a minimum at a holding potential of +6 mV

(see Section 4-2-8), our data suggest that inhibitory currents are potentiated in cells receiving

mixed inputs. Also, post-UVD mixed neurons exhibited a reduced excitatory-to-inhibitory ratio

compared to normal (Figure 4-9 right) within 2 hours after UVD. Together, our results suggest

that UVD may induce acute potentiation at excitatory and some inhibitory commissural synapses

in the ipsilesional MVN.

Figure 4-8: The effect of UVD on post-synaptic current for responses to pure excitatory and pure inhibitory

commissural inputs. Left: NMDA:AMPA ratio for normal and post-UVD neurons receiving excitatory commissural

inputs. For this data set, a minimum sample size of 6 would be required for the means to be significantly different.

Right: Average currents measured in cells receiving inhibitory inputs (nNormal = 7, nUVD = 2). Data are not shown for

more positive holding potentials since IPSCs from only one of the post-UVD neurons were recorded at holding

potentials above -14mV. No apparent differences are observed and a power analysis revealed that each distribution

would require very large sample sizes for the means at each holding potential to be significantly different (n>80).

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Figure 4-9: The relative contribution of inhibitory commissural inputs to mixed responses before and after UVD.

Left: Illustration of how the excitatory:inhibitory ratio was calculated. The ratio was determined by dividing the

current measured at a holding potential of -74mV by the current measured at a holding potential of +6 mV for each

cell (see methods for details). Right: The calculated ratios measured after UVD are reduced compared to normal.

The distributions for each group would require only 9 samples each for the means to be significantly different

(p<0.05).

4.4 Discussion

Our data suggest that synaptic plasticity takes place in the parvocellular division of the MVN

within the first few hours after UVD. Our preliminary data suggest that contralesional excitatory

NVIII synapses, ipsilesional excitatory commissural synapses, and some ipsilesional inhibitory

commissural synapses may become potentiated shortly after surgery. Only a few published

studies have provided evidence for synaptic plasticity in the VNC after UVD. Evidence for post-

UVD potentiation of the contralesional NVIII synapse was provided by a recent study in rats

(Pettorossi et al. 2003). In that study, high frequency stimulation, a procedure that can induce

long-term potentiation of NVIII field potentials in the MVN under normal conditions (Capocchi

et al. 1992), was ineffective at potentiating NVIII synapses in the MVN of acutely

labyrinthectomized rats, suggesting that the NVIII synapse was already potentiated by the time

high frequency stimulation was applied. Although this result is consistent with our findings, the

exact time at which the slices were obtained in the rat study was not specified. A few studies

have shown that commissural synapses could be altered acutely following UVD. First, a series

of investigations in the frog reported single-cell responses to commissural stimulation 12 hours

after UL (Dieringer and Precht 1977; Dieringer and Precht 1979a; Dieringer and Precht 1979b).

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In this study, excitatory responses were evaluated. Compared to normal (Ozawa et al. 1974),

excitatory responses were slightly potentiated when measured 12 hours after UVD (Dieringer

and Precht 1979a). A recent study in rats suggested that the release of the inhibitory

neurotransmitter GABA, possibly from ipsilesional commissural synapses, is increased between

2 and 3 hours after UL (Bergquist et al. 2008). The findings in the frog and mouse are consistent

with our results.

On the ipsilesional side, potentiation of excitatory and some inhibitory commissural synapses

could only be detected in voltage clamp experiments. No potentiation of EPSPs or IPSPs was

observed. This could have occurred for two reasons. First, we pooled I/O curve data for mixed

and pure responses in an attempt to increase our sample size, especially in the post-UVD group.

In the case of mixed responses, some of the IPSPs may have been partly cancelled by

overlapping EPSPs, which could mask potentiation of the IPSP. Second, we did not use any

blockers to excitatory or inhibitory receptors in the commissural stimulation experiments.

Therefore it is possible that any cells which were categorized as having pure inhibitory responses

but were not evaluated in voltage clamp mode may have actually been mixed responses. We

chose not to use receptor antagonists since we were interested in whether mixed responses to

commissural stimulation, which have been previously reported in the mouse MVN (Broussard

2009), were altered after UVD. Using the ratio of excitatory to inhibitory current (di Marco et al.

2009), our preliminary analysis suggested that commissural inhibition was increased relative to

commissural excitation in cells with mixed responses. These preliminary measurements are the

first indication that the relative amounts of commissural inhibition and excitation to a single cell

may be altered acutely following UVD. One possible confound on our results was that we did

not measure the exact reversal potential of either inhibitory or excitatory inputs for each cell.

We always assumed that the reversal potentials for inhibitory and excitatory currents were those

of ideal chloride (-70 mV) and glutamate receptor (0 mV) channels.

Another possible confound in our midline stimulation experiments was the placement of our

stimulating electrodes, whose contact with the slice extended beyond the vestibular commissural

fibers. Such positioning may have caused some neurons in our preparation to be activated by

axons arising from non-vestibular regions within the plane of the slice. Care was taken not to

position the electrodes either between the prepositus nuclei or over top the medial longitudinal

fasciculus (MLF), both of which could send projections to the ipsilesional MVN within the plane

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of the slice (Carleton and Carpenter 1983; McCrea and Baker 1985). However, we could not

control for current spread to these regions. The only other structure in the slice that could have

contributed to the responses we recorded is the reticular formation. Our stimulating electrode

was positioned over the gigantocellular portion of this structure, which does project to the MVN

bilaterally (Hoddevik et al. 1975; Carleton and Carpenter 1983).

On the contralesional side, potentiation of the NVIII synapse was associated with a decrease in

the NMDA:AMPA ratio. There are two possible explanations for this result. First, it could

indicate a decrease in post-synaptic NMDA receptor function. It is well established that

potentiation of the NVIII synapse can depend on NMDA receptors (Capocchi et al. 1992; Grassi

et al. 1996; Caria et al. 2001; Grassi et al. 2001; McElvain et al. 2010). However, this possibility

is unlikely since the induction of NMDA receptor-dependent potentiation usually requires an

increase in current through post-synaptic NMDA receptors (Malenka and Nicoll 1993). Second,

and more likely, a lesion-induced reduction in the NMDA:AMPA ratio could reflect an increase

in current though post-synaptic AMPA receptors. Recent evidence obtained in the mouse MVN

suggests that a form of potentiation associated with increased currents through calcium-

permeable AMPA receptors can also take place at the NVIII synapse independent of NMDA

receptors (McElvain et al. 2010). It is possible that an increase in current through AMPA

receptors might reflect an increase in the quantity of AMPA receptors on the post-synaptic

membrane during the acute stage of compensation. On the post-synaptic cell, the number of

synaptic AMPA receptors might be increased either by their translocation from the intracellular

space to the cell surface (Gerges et al. 2006) or by lateral diffusion from the extrasynaptic to

synaptic membrane (Tardin et al. 2003). AMPA receptors translocated to the post-synaptic

membrane after UVD could either be obtained from pools of receptors formed prior to the lesion

or be newly assembled after the lesion. In the normal VNC, there are large numbers of AMPA

receptor subunits, particularly of GluR2, 3 and 4, in all divisions, including the MVN (Petralia

and Wenthold 1992; Chen et al. 2000). Therefore, it is likely that there is an abundance of

AMPA receptors available to contribute to lesion-induced plasticity. Some evidence also

suggests that new subunits may be formed acutely following UVD. Within the first 10 hours, the

total quantity of GluR2 subunits is increased bilaterally following UL in rats (King et al. 2002).

In summary, our data suggest that plastic changes take place along the disynaptic pathway

between the intact labyrinth and the parvocellular MVN on the lesioned side. This pathway

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contains excitatory NVIII synapses on the contralesional side and inhibitory commissural

synapses on the ipsilesional side. In mammals, most commissural projection neurons are located

in the parvocellular division of the MVN (Epema et al. 1988; Newlands et al. 1989) and many of

these cells project directly onto neurons in the parvocellular MVN on the opposite side (Epema

et al. 1988). Potentiation of the inhibitory commissural pathway to the parvocellular MVN

could have functional implications for vestibular compensation. It was shown that long periods

of inhibition can cause a cell to undergo firing rate potentiation, which is its ability to increase its

resting rate without changing its synaptic input (Nelson et al. 2003). More recently, it was

proposed that firing rate potentiation at the most acute stage of vestibular compensation could be

a mechanism through which neural activity can be rebalanced in the VNC (Gittis and du Lac

2006). It is possible that commissural inhibition could contribute to firing rate potentiation and

enable the rebalancing of neural activity. It is currently unknown whether firing rate potentiation

does in fact take place in the VNC after UVD though some evidence (see Section 1-3-3) supports

the possibility that it does (Guilding and Dutia 2005).

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Chapter 5: Adaptive Rescaling is Preserved in the

Vestibular Nuclei and Extends the Dynamic Ranges of

Central Neurons after Unilateral Vestibular Damage

5.1 Introduction

After unilateral vestibular damage (UVD), gaze stability is reduced as the VOR becomes

nonlinear and saturates during rotation toward the lesioned side (Maioli et al. 1983; Paige 1983;

Fetter and Zee 1988; Halmagyi et al. 1990; Gilchrist et al. 1998; Lasker et al. 1999; Lasker et al.

2000; Galiana et al. 2001). The behaviour of the VOR after UVD may reflect a limited dynamic

range exhibited by many neurons in the VNC (Melvill Jones and Milsum 1970; Fuchs and Kimm

1975; Newlands and Perachio 1990a; Newlands and Perachio 1990b; Escudero et al. 1992;

Chen-Huang and McCrea 1999; Broussard and Kassardjian 2004; Newlands et al. 2009).

Adaptive rescaling can adjust the dynamic range over which a neuron responds (Brenner et al.

2000) and could function as a means for optimizing dynamic vestibular function after UVD.

Adaptive rescaling might be a property of central vestibular responses (Melvill Jones and

Milsum 1970) and we predicted that this property is preserved after UVD. We hypothesized

that, after recovery from UVD, adaptive rescaling extends the dynamic ranges of vestibular

neurons during high-velocity rotation. We tested our hypothesis by recording the responses of

central vestibular neurons in the alert cat and provided the first evidence that adaptive rescaling

takes place in the VNC after UVD.

5.2 Methods

5.2.1 Animals

In this study, three young (1-2 yr-old) neutered male cats were used. My supervisor, Dr. Dianne

Broussard, collected all data from one animal (C) and another student, Karl Farrow, contributed

a very small amount of data from another cat (J). The data from cat O were gathered by R. H. S.,

who also analyzed all of the data presented here.

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Before starting any data collection, all three cats were trained to sit with their heads fixed in the

experimental apparatus. The UVD in cats C and J was caused by plugging the horizontal

semicircular canal (CP) whereas in cat O UVD was generated by unilateral labyrinthectomy

(UL). We had originally planned for cat O to be a canal-plugged animal, but one of the vertical

canals was damaged during the procedure and we decided to remove the labyrinth. All surgical

procedures were carried out by my supervisor, Dr. Dianne Broussard. Eye movements were

recorded repeatedly in each cat before, and again more than 30 days after UVD. Single unit

recordings were started at least 60 days after UVD. All surgical and experimental methods

complied with guidelines set by the Canadian Council for Animal Care and were approved by

the Animal Care Committee at the University Health Network.

5.2.2 Surgical Procedure

Each cat was fitted with several implants including a head holder (for attaching the cat's head to

the experimental apparatus), a scleral search coil (for recording eye movements) and a recording

cylinder (for electrode placement). Details of the implantation methods have been described

elsewhere (Broussard et al. 1999; Broussard et al. 2004). Briefly, cats were pre-medicated,

through subcutaneous injection (s.c), with a mixture of Demerol, atropine, and acepromazine.

Next, they were anesthetized with isoflurane and maintained at intra-operative levels between 1.5

and 2%. Before surgery, buprenorphine (0.01 mg/kg, s.c.) was administered as an analgesic.

During the first surgery, each cat was positioned in a stereotaxic frame and the head holder was

implanted first. Each head holder, a small steel cylinder, was secured to the head using dental

acrylic on top of a base built from fixation plates and cortical screws. Afterwards, a metal search

coil was sutured on to the sclera of one eye and linked to a small connector secured at the base of

the head holder. The recording cylinder (20-mm diameter, Crist Instruments) was implanted in a

second surgery. The cylinder was centered, pitched 20 degrees back from the stereotaxic vertical,

and then attached to the base of the head holder with dental acrylic. Buprenorphine (0.005 mg

/kg, s.c.) was administered by subcutaneous injection for postoperative analgesia. On the

following three days, ketoprofen was administered for analgesia once a day, first by

subcutaneous injection (1 mg/kg) and then by oral tablets (1 mg tablet).

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The procedures for CP have also been previously described (Broussard et al. 1999).

Buprenorphine was not administered before surgery in this case. While there is no published

evidence to support this claim, we have observed a lack of compensation following CP after

administering buprenorphine in our laboratory. Ketoprofen (2 mg/kg, s.c.) and a local injection

of lidocaine were instead administered while under isoflurane anesthesia. The horizontal

semicircular canal was exposed by carefully drilling down the temporal bone and opening the

middle ear. The canal was opened by sanding down the bone at its estimated position relative to

the incus, and tightly plugged with a piece of periosteum. The procedure for UL was similar

except, instead of simply plugging the horizontal canal, the sensory epithelia of the utricle,

saccule and ampullae of all three canals were removed. The muscle and skin were then sutured

closed and ketoprofen (1 mg/kg, s.c.) was once again administered for analgesia. Ketoprofen

administration was continued over the next three days as described above.

The UL is a more severe lesion than the CP since irreversible damage to the labyrinth silences

most of the primary afferents. The sensory epithelia are relatively intact after CP so the afferents

remain spontaneously active (Goldberg and Fernández 1975) and the tonic excitatory input to

secondary neurons is greater in plugged cats. However, despite this difference in tonic inputs,

the neurons we recorded from showed no differences in adaptive rescaling after CP versus after

UL. Therefore data for UL and CP were described and discussed together.

5.2.3 Recording Eye Movements

The VOR was activated by sinusoidal rotations about an earth-vertical axis. This was carried out

in complete darkness using a rate table (Neurokinetics). A velocity feedback signal, measured

from a tachometer built into the table, was digitized and recorded as head velocity. The

experimental apparatus, or cat chair, was positioned on top of the rate table. The cat was secured

inside by placing it in a close-fitting box. Its head was then fixed in space by inserting a steel

post into the head holder and then bolting it to an arch above the box. The cat’s interaural line

was always centered on the axis of rotation and the pitch angle of the head was adjusted. For

most recordings, we positioned the head 22o nose-down from the horizontal stereotaxic plane so

that the horizontal canals were roughly in the plane of rotation. In each experiment, the animal

was rotated at 1 Hz and a peak velocity of 10, 20, 40, 80 and 120 deg/s.

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Eye movements were recorded using a search coil apparatus, consisting of a phase detector

(CNC Engineering) with vertical and horizontal 17‖ field coils (see Figure 1). The midpoint

between the cat’s eyes was centered in both magnetic fields. The vertical and horizontal eye

position signals were digitized at either 1 KHz (cats C and J) or 4 KHz (cat O) using Labview

software (National Instruments). Signals were digitally low-pass filtered with a cutoff of 55 Hz

and eye velocity was calculated using a 5-point differentiation algorithm. Before recording eye

movements, the eye coil implanted in each cat was calibrated by rotating the cat at a constant

velocity for 400 ms in the light. Under these conditions, visual following mechanisms were

active so we could assume that fixation was optimal and eye velocity was equal in magnitude to

head velocity. We therefore assigned each cat a calibration factor independent of the magnetic

search coil system. This meant that even if the search coil system changed slightly we could

make adjustments to it without having to recalculate the calibration factor of the cats’ eye coils.

Eye movements were then recorded across at least 30 (cats C and J) or 60 cycles (cat O) of

rotation at each test stimulus. To prevent drowsiness during VOR recordings, cats were kept alert

by sound and touch.

5.2.4 Recording Single Unit Responses

Isolated single unit activity was recorded using glass-insulated platinum-iridium microelectrodes

with impedances of 2-8 M. Electrodes were positioned at different Cartesian coordinates using

a head-mounted micromanipulator (Narishige) that fit over top the recording cylinder. A

microdrive (Fred Haer Corp.) was used to push the electrode through the dura and into the

brainstem. Periodically, the cat had to be sedated with an intramuscular injection of ketamine

(20 mg/kg) so that scar tissue could be gently peeled away from the dura. This procedure

improved the chances that microelectrodes would pass through the dura without being damaged.

No recordings were made until at least 1 day after the dura was peeled. The target recording

sites were the medial and ventrolateral vestibular nuclei and their locations were determined with

reference to the coordinates at which the abducens nucleus was found. Most recordings were

made while the horizontal canal was in the plane of rotation (22 degrees nose-down). To ensure

that the cells were not encoding any vertical canal signals, an additional set of recordings were

made after the head was repositioned 5 degrees nose-up. If a cell’s sensitivity to rotation

increased during nose-up rotation, it was considered a vertical canal neuron and was excluded

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from the analysis. Cells that changed their response polarity during nose-up rotation were also

eliminated.

Cells were located with a search stimulus of 1 Hz rotation at a peak velocity of either 10 or 20

deg/s. All neurons had a nonzero resting discharge rate. Once a unit was isolated, trigger pulses

were generated by a window discriminator (Bak Electronics) each time the unit fired an action

potential. In cats J and C the times of the trigger pulses were recorded to the nearest 10 s using

a counter; in Cat O the trigger pulses were digitized at 10 kHz. In both cases, the trigger pulses

were used to calculate spike density (see below). In cat O, the extracellular voltage was also

digitized at a sample rate of 60 KHz. This was done to ensure that no spikes from the recorded

unit had gone undetected. Any spikes that were missed by the window discriminator were

assigned a trigger pulse offline.

Neuronal responses were first recorded in complete darkness during a frequency series (1, 2, 4

and 8 Hz) at a peak velocity of 10 deg/s. This was followed by a velocity series during which

recordings were made in total darkness at 1 Hz and peak velocities of 10, 20, 40, 80 and 120

deg/s. Isolation was frequently lost at 120 deg/s so a number of cells were missing data for this

stimulus. At each peak velocity, data was collected across 30 cycles of continuous rotation. The

peak velocities were presented in increasing order for Cats J and O but for Cat C, the 20 deg/s

file was acquired first (see Figure 10). The table was stopped only momentarily for 5-10

seconds and the light turned on in between velocities to maintain alertness. In cells where

isolation was maintained, rotation at 10 deg/s was repeated in the nose-up position.

For all cats, there was a period during which they remained stationary with the lights on. During

this period, discharge rates were recorded as the cat shifted its gaze to various targets around the

recording room. These recordings were used to determine eye position sensitivity. In Cat O, but

not in Cats C or J, there was a period between the frequency and velocity series during which the

animal was not rotated and remained stationary. During this time, cell discharges were recorded

in complete darkness. Stationary recordings in the dark were used to determine the resting

discharge of each cell. If a cell remained isolated after a complete velocity series (ie. including

120 deg/s) the resting discharge was recorded for a second time while animal remained

stationary in the dark.

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When the experiments were over, electrolytic marking lesions were made at one recording site

on each side of the brainstem by applying 5 A of DC current for 10 min. At least 4 days later,

the cat was sedated with 20 mg/kg ketamine i.m., deeply anesthetized with intravenous sodium

pentobarbital (100 mg/kg) and perfused transcardially with physiological saline followed by 10%

buffered formalin. Brains were processed for histology using a freezing microtome or vibratome

and stained with cresyl violet. Based on where the marking lesions were found, it was

determined that the recording sites of all neurons were in the medial or the ventral lateral

vestibular nucleus.

5.2.5 Data Analysis

VOR data were prepared for analysis by averaging at least 320 cycles of head and eye velocity

from Cat O and Cat C. These data were collected over the course of 8 separate recording

sessions with consistent eye movement responses. A large number of cycles were used so that

noise in the recordings could be removed by averaging. Prior to averaging, all quick phases and

saccades were manually removed from the eye velocity traces. After averaging, eye velocity was

plotted against head velocity and the cycle was divided at a zero breakpoint so that ipsi-and

contralesional half-cycles could be analyzed separately. For each half-cycle, the mean squared

error of each fit was minimized to correct for any phase differences, which were used as a

measure of phase lag or lead.

For recordings of single unit activity, spike densities were calculated by convolving pulse trains

with a Gaussian probability density function whose standard deviation was set at 15% of each

cycle (Broussard et al. 2004). In the recordings made during sinusoidal rotation, mechanical

artifacts in head velocity, caused by the initial acceleration of the table, were always found in the

first cycle. Therefore, the first cycle was eliminated and the next 10 to 30 cycles were averaged.

If triggering was lost during a cycle, that cycle was also discarded. Cells that provided fewer than

10 good cycles of data at any single velocity were entirely excluded from further analysis.

All cells were classified based on the type of response pattern they exhibited. Altogether, two

types of response patterns were observed - Type I or Type II. Type I neurons increased their

firing rate for ipsilateral rotation and type II neurons increased their firing rate for contralateral

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rotation. Next, based on how sensitive they were to eye position (Broussard et al. 2004), cells

were classified as having either a combination of eye movement and vestibular sensitivity (EM)

or vestibular sensitivity only (V-Only). Eye position sensitivity was determined by linear

regression of discharge rate against eye position. Among those analyzed, neurons with

sensitivities to horizontal eye position ≥ 0.5 sp/s per degree were classified as EM cells while the

rest were categorized as V-Only.

Sensitivity and linearity. To measure the sensitivities of neuronal responses, spike density was

plotted against head velocity as shown in Figure 5-1. First, the spike density trace was

temporally shifted to remove any phase lead or lag, optimizing the fit and reducing the combined

MSE of the fit. Next, two lines were fit to the data and their point of intersection, or breakpoint,

was moved along the x-axis until the combined MSE was minimized. If the slopes of the two

lines had non-overlapping 95% confidence intervals, the response was considered nonlinear

(Figure 5-1, top & middle). The breakpoint was defined as the head velocity measured at the

intersection of the fitted lines. If the slopes of the two lines were not significantly different, then

the response was said to be linear (Figure 5-1, bottom).

We defined three different response types (cutoff, saturating and linear) among the recorded

cells, and the methods for measuring sensitivity were specific for each type. For cutoff

responses, sensitivity was always the slope of the line fitted along the cell’s on-direction, i.e.,

above the breakpoint (arrow in Figure 5-1, top). For saturation-type responses, the sensitivity

was the slope of the line below the breakpoint (arrow in Figure 5-1, middle). After calculating

sensitivity, we set a post-hoc criterion for response sensitivity to ensure that the measurements

were reliable. Cells were included in this report only if the ratio of the mean standard error of

the linear fit to the slope of the linear fit (in spikes/s per deg/s) was less than 5 for at least one of

the peak rotational velocities tested. The number of recorded cells exhibiting each type of

response is summarized in Table 5-1.

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Figure 5-1: Methods for determining sensitivity of cells with different response types. All sensitivities were

measured from two-line fits to the average spike density traces recorded at each peak velocity. Top: sensitivity for

cells with cutoff-type responses was measured from the slope of the line (arrow) above the breakpoint (vertical

dashed line). Middle: the sensitivity for cells with saturating responses was measured from the line below the

breakpoint. Bottom: Linear response sensitivity was measured as the average slope for both lines.

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Side Lesion Cat(s) Cell Type Response type Total (n)

ipsi either O,J I Cutoff 1

ipsi either O,J I Sat. 3

ipsi either O,J I Linear 1

ipsi either O,J II Cutoff 10

ipsi either O,J II Sat. 1

ipsi either O,J II Linear 2

contra either O,C I Cutoff 14

contra either O,C I Sat. 3

contra either O,C I Linear 4

contra either O,C II Cutoff 2

contra either O,C II Sat. 1

contra either O,C II Linear 2

Table 5-1: Summary of response types. Shown here is the number of cells of each type that exhibited cutoff,

saturating, or linear responses, for both the ipsilesional and contralesional sides.

Sensitivity measurements were also used to calculate a rescaling index (RI), which was used as a

relative measure of how a cell’s sensitivity changed with peak head velocity:

RI = S10/S80

S10 and S80 are the sensitivities in spikes/s per deg/s to rotational velocity during 1 Hz rotation

with peak velocities of 10 deg/s and 80 deg/s, respectively. In general, the RI indicated whether

or not the cell rescaled its sensitivity with peak velocity. An RI of one would indicate that

rescaling was not a property of the cell's response.

Zero-velocity spike density (SDH=0) was the mean spike density measured from the phase-

adjusted response function at zero head velocity. This measurement was used as an estimate of

the resting discharge rate during rotation. The use of this parameter was justified by comparing

the resting discharge rates measured from Cat O before the velocity series and during rotation at

10 deg/s. There was no difference between these measurements obtained from either Type I or

Type II cells (P>0.5, paired Student’s t-test). This is illustrated in the top two panels of Figure 5-

8.

5.2.6 Statistics

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The parameters measured from responses to rotation at the lowest (10 deg/s) and highest (80

deg/s or 120 deg/s) peak velocities were evaluated for significant differences. These

comparisons, which were made within each group of cells, were performed with paired t-tests

(p<0.05 or p<0.01).

5.3 Results

The responses of 44 neurons isolated in the medial and ventral lateral vestibular nuclei were

recorded from 3 cats. All cells were tested for responses to rotation with peak velocities of 10,

20, 40 and 80 deg/s. A subset of neurons was also tested at 120 deg/s. Neural responses were

recorded on both sides of the brainstem a minimum of one month after surgery. Neurons

rescaled their sensitivities irrespective of the type of lesion or the side of the brainstem on which

they were located.

A summary of cell types and their numbers is shown in Table 5-2. All cells were spontaneously

active and two different response types were observed. Responses were classified as Type I or

Type II based on their polarities with respect to head rotation (see Methods for details). On the

ipsilesional side, there was a prevalence of Type II (56%) compared to Type I (17%) responses.

The remaining 27% of cells recorded on the ipsilesional side exhibited Type III and Type IV

responses (see Chapter 1-1-4), which were not included in our analysis. The opposite was found

on the contralesional side where Type I responses (75%) were predominant. The distributions

of Type I and Type II responses we observed on each side of the brainstem are consistent with

published findings (Shimazu and Precht 1966; Smith and Curthoys 1988a; Smith and Curthoys

1988b; Newlands and Perachio 1990a; Newlands and Perachio 1990b).

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Side Lesion Cat(s) Cell Type Sample (n) Total (n) Total for side % of type

ipsi UL O I 4 -- -- --

ipsi UL O II 12 -- -- --

ipsi CP J I 0 -- -- --

ipsi CP J II 1 -- -- --

ipsi either O,J I -- 4 18 22

ipsi either O,J II -- 13 18 78

contra UL O I 1 -- -- --

contra UL O II 1 -- -- --

contra CP C I 20 -- -- --

contra CP C II 4 -- -- --

contra either O,C I -- 21 26 81

contra either O,C II -- 5 26 19

Table 5-2: Cell types and lesion conditions. Shown are the samples subdivided according to neuronal type, that

were obtained from each side of the brain in each cat. The rows in bold type summarize all the data for each side of

the brain. Ipsi = ispilesional, contra = contralesional, UL = unilateral labyrinthectomy, CP = canal plug.

In most of the cells, we measured lower sensitivities with increasing peak head velocity.

Examples are shown for two cutoff-type cells in Figures 5-2a and b. In both cells, sensitivity

was reduced at higher peak velocities and, consequently, the dynamic range, or range of

velocities over which the cells respond in a linear fashion, increased. The reduced sensitivity

with increasing head velocity will be referred to as ―rescaling‖.

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Figure 5-2: Adaptive rescaling in vestibular neurons does not depend on the type of lesion or on which side of the

brainstem the neuron is located. A & B: Examples of rescaling observed during 1 Hz sinusoidal stimuli under

different conditions – ipsilesional to UL (A) and contralesional to CP (B). Spike density is plotted against head

velocity and the different colours represent different peak velocities. C & D: Rescaling index (RI) is plotted as a

function of sensitivity for all Type I (C) and all Type II (D) neurons. Filled circles = ipsilesional UL, open circles =

ipsilesional CP, filled triangles = contralesional CP, open triangles = contralesional UL.

The amount of rescaling was measured using a rescaling index (RI). Rescaling indices are

shown for Type I and Type II neurons in Figures 5-2c and d, respectively. There was little

correlation between sensitivity and RI in either group. A significant difference in RI could not

be detected between ipsilesional and contralesional neurons (P>0.5, unpaired t-test). There were

also no significant differences between Type I and Type II neurons (P>0.5, unpaired t-test). The

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mean RI for all cells was 1.86 . In other words, increasing the stimulus velocity by a factor of 8

resulted in a 2-fold reduction in sensitivity on average. The majority of the cells had RIs that

ranged from 1.0 (no rescaling, dotted line) to 4.0. A small group of cells had RIs less than 1.0.

Overall, rescaling was similar across all groups of cells.

Figure 5-3 illustrates how sensitivity rescaled with peak rotation velocity in our data. All

sensitivity measurements were acquired from the linear portion of the input-output functions for

each cell. The results from four different groups of cells are shown: Type I and Type II from the

ipsilesional and contralesional sides. Data obtained from the UL and the canal plug cats were

combined since we found that the type of lesion had no effect on the amount of rescaling (Figure

5-2C & D). For each group, mean sensitivities across cells tested in the 10-80 deg/s range of

peak velocities (heavy lines) as well as a subset of cells that were also tested at 120 deg/s

(symbols and lines) are shown. In all four groups, sensitivity was reduced with increasing peak

velocity and those with the highest sensitivities demonstrated the largest amount of rescaling.

The most sensitive neurons, ipsilesional Type II and contralesional Type I, were also the most

abundant and represented the largest samples. Significant decreases were detected in the

sensitivities of ipsilesional Type II (paired t-test, p<0.01, n=13) and contralesional Type I (paired

t-test, p<0.01, n=21) neurons when peak velocity was increased from 10 to 80 deg/s. A subset

of contralesional Type I neurons also exhibited significant sensitivity decreases at 120 deg/s

(p<0.01, paired t-test). The less sensitive and less abundant cells, ipsilesional Type I and

contralesional Type II, did not demonstrate any significant rescaling (paired t-test, p>0.05) in

either of these groups.

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Figure 5-3: Sensitivity changes with peak stimulus velocity. Sensitivity (gain) as a function of peak velocity is

displayed for four different groups of cells. In each panel, the black lines and symbols specify cells tested at

velocities up to 80 deg/s and the gray lines and symbols specify a subset of cells that were also tested at 120 deg/s.

Top left, ipsilesional (Ipsi) Type I neurons (n10 to 80=5 and n10 to 120=3); Top right, ipsilesional Type II neurons

(n10 to 80=13 and n10 to 120=7); Bottom left, contralesional Type I neurons at 80 (n10 to 80=2 and n10 to 120=19)

deg/s; Bottom right, contralesional (Contra) Type II neurons (n10 to 80=5 and n10 to 120=3) deg/s. Error bars

represent standard deviation (s.d.). Pairs of data that were significantly different (p<0.01, paired t-test), as measured

with a paired student’s t-test, are indicated by the solid arrows (cells tested up to 80 deg/s) and the dotted arrows

(cells tested up to 120 deg/s).

In order to confirm that we were measuring changes in sensitivity to head velocity and not

acceleration, phase angles were measured between spike density and head velocity. Since

acceleration is the derivative of velocity, a pure acceleration function starts from zero a quarter

cycle (90 degrees) earlier than a pure velocity function (Figure 5-4). Therefore, if our cells led

head velocity by 90 degrees, then we would assume that they were encoding acceleration rather

than velocity. This would apply for both Type I and Type II neurons. Phase differences for all

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four groups of cells are shown in Figure 5-5. Positive values indicate where spike density led

head velocity. Phase differences for most of the cells were relatively small (< 45 degrees),

suggesting that these neurons carried predominantly head velocity signals.

Figure 5-4: The phase relationship between velocity and acceleration. Acceleration (red) is the derivative of

velocity (black) as illustrated by the sine and cosine curves. The cosine function is the derivative of the sine

function. Temporally, acceleration (cosine) is advanced by one quarter cycle (90 degrees) with respect to velocity

(sine), as shown by the dashed portion of the acceleration curve. This equates to a 90 degree phase difference,

indicated by the double-headed arrow, in which acceleration leads velocity.

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Figure 5-5: Phase lead (mean ± s.d.) as a function of peak head velocity. Phase lead has a slight dependence on

peak velocity but changes in phase lead were significant (p<0.01, paired t-test) in only the Contra Type I cells.

Positive values indicate where discharge rate led head velocity. Phase data are from the same cells shown in Figure

5-3. Symbols, lines, arrows and sample sizes are also as outlined in Figure 5-3.

Larger phase leads were associated with higher peak velocities in all four groups but this change

was not significant in most cases. A significant increase in phase lead from 10 to 80 deg/s was

only observed in contralesional Type I neurons (p<0.01, paired t-test). The phase differences

shown in Figure 5-5, while small, were nonzero so there still remains the possibility that the

phase leads were caused by acceleration signals. To ensure that neural responses did not rescale

with acceleration, two subsets of cells from the ipsilesional Type II and contralesional Type I

groups were tested over a range of frequencies (1-8 Hz) while keeping the peak head velocity

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constant at 10 deg/s. The results are shown in Figure 5-6. When peak head velocity is constant,

an increase in frequency would produce an increase in peak acceleration. Sensitivity did not

rescale with frequency therefore it is unlikely that our sensitivity rescaled with acceleration.

Figure 5-6: Sensitivity is minimally affected by frequency in most vestibular neurons. Bode plots are shown for

the sensitivities of ipsilesional Type II (n=13, top) and contralesional Type I (n=10, bottom) neurons. The gray

lines represent individual neurons while the solid black lines and symbols are the means and standard deviations for

each sample.

Up until now, we have discussed only the responses to head movement, which is not the only

type of signal impinging on vestibular neurons. Many cells in the vestibular nuclei respond to

eye movements as well. In addition to having a sensitivity to head velocity, these cells could

also have sensitivity to eye velocity and/or eye position (Scudder and Fuchs 1992). For cells that

respond to eye movements, their sensitivities measured during head movement are the sum of

eye position, eye velocity and head velocity sensitivities (Keller and Daniels 1975). Therefore,

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we considered the possibility that eye movement signals could contribute to rescaling by

comparing the amount of rescaling in eye movement (EM) and vestibular only (V-Only) cells.

We were unable to measure eye velocity sensitivity (see Discussion) so neurons were

categorized as EM or VO cells based only on their eye position sensitivity. Neurons were placed

in the EM category (n=17) if their sensitivity to eye position was greater than 0.5 sp/s/degree.

Cells with lower eye position sensitivities (n=19) were classified as vestibular only (V-Only).

Rescaling was apparent in both groups. The mean RI for the entire population of EM cells was

not significantly different from that of V-Only cells (p>0.05, unpaired t-test). In Figure 5-7, the

RI of each cell is shown against sensitivity for the EM (filled symbols) and V-Only (open

symbols) classes.

Figure 5-7: Eye position sensitivity does not affect the amount of rescaling in vestibular neurons. Rescaling index

(RI) is plotted as a function of the sensitivity for all of the EM (filled symbols) and V- Only (open symbols) cells

shown in Figure 5. The sensitivity was measured at 10 deg/s peak velocity.

Besides rescaling their sensitivities in a velocity-dependent manner, vestibular neurons also

changed their resting discharge rate with peak rotational velocity. An increase in the discharge

rate at zero velocity could act to bring the cell further away from cutoff and increase its dynamic

range. In Figure 5-2b, an increase in the spike density at 0 deg/s can be seen at high peak

velocities. The effect of peak velocity on resting discharge rate, estimated by SDH=0, is outlined

in Figure 5-8 for the same cells that were shown in Figures 5-3 and 5-5. Significant changes in

SDH=0 were observed in Type I neurons, both ipsilesional and contralesional (p<0.01, paired t-

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test). Type II neurons did not change their zero-velocity discharge rates at velocities below 80

deg/s. SDH=0 did increase for Type II neurons between 80 and 120 deg/s though not

significantly (p>0.05, paired t-test).

Figure 5-8: Zero velocity spike density (SDH=0) is elevated by peak head velocity. SDH=0 is an estimate of the

resting discharge rate during rotation. Data were obtained from the same cells shown in Figures 5-3 and 5-5. The

square symbols represent the resting discharge before (open) and after (filled) the rotation series. All other symbols,

error bars, lines, arrows and sample sizes are as outlined in Figures 5-3. Significance is for p<0.01, paired t-test.

To be certain that changes in SDH=0 were not due to recording artifacts, such as cell injury or

sudden changes in the electrical properties of our electrodes, the resting discharge rate was

measured immediately after the rotation series. Post-rotation resting rates (open symbols in

Figure 5-8) were recorded from a subset of ipsilesional cells (nTypeI = 3, nTypeII = 7) tested at 120

deg/s in Cat O. A comparison of the resting rates measured before (filled symbols) and after

rotation revealed no significant differences (p>0.05, paired t-test) for either Type I or Type II

neurons.

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Velocity-dependent changes in sensitivity and spontaneous rate caused changes in the dynamic

ranges of our cells. Vestibular neurons can exhibit cutoff-type responses beyond a given head

velocity in the off direction. The examples in Figures 5-2a and b suggest that cutoff-type cells

can partially overcome their limitations at high peak velocities by increasing their dynamic

ranges. To quantify changes in dynamic range, cutoff-type cells with clear breakpoints were

selected (n=10), all of which were either ipsilesional Type II (n=6) or contralesional Type I

(n=4). The estimated velocity at the breakpoint was used as an estimate of the velocity at cutoff

and is plotted against peak velocity in Figures 5-9a and b. Breakpoint velocities climbed

upwards as peak velocity increased in both groups of cells, reflecting an increase in the dynamic

range. A significant change in the breakpoint was observed in ipsilesional Type II cells when

peak velocity reached 80 deg/s (p<0.01, paired t-test).

Figure 5-9: The linear range, as measured by the breakpoint (mean ± s.d.), is increased at high peak velocities.

Breakpoints were measured only from cells that had pronounced cutoff responses. A: Breakpoint measurements for

contralesional Type I neurons tested at velocities up to 80 deg/s (n=4) and 120 deg/s (n=3). Symbols, lines and

arrows are as shown in Figures 2 and 3. B: Breakpoints for ipsilesional Type II neurons at 80 deg/s (n=5) and 120

120

deg/s (n=3). C and D: the percentage of neurons that exhibited linear responses in each sample. Significant

differences are indicated by the double-headed errows (p<0.01, paired t-test).

If the dynamic range over which vestibular neurons can respond increases with peak velocity,

then then we would expect consistency in the number of linear responses recorded across all

peak velocities. To evaluate this, the percentage of linear responses observed across all cells is

shown against head velocity in Figures 5-9c and d. On both sides of the brainstem, nonlinearities

were observed even at the lowest peak velocities as less than half of the responses were linear at

10 deg/s. Among the contralesional cells, there was no change in the percentage of linear

responses as peak velocity was increased from 10 to 80 deg/s and the number of linear responses

decreased only slightly at 120 deg/s. The situation was quite different on the ipsilesional side

where the percentage of linear responses dropped from 40 % at 10 deg/s to only 10% at the

highest peak velocity tested. Taken together, these observations suggest that more neurons

extended their dynamic ranges on the contralesional compared to ipsilesional side.

Adaptive rescaling is a relatively rapid process (see Discussion). To ensure that our results were

not due to some other form of adaptation that takes place on a longer time scale, the time course

for sensitivity changes was roughly estimated in a subset of neurons (n=9). The spike density

records for these cells were divided into 5-cycle segments, and the cycles in each segment were

averaged. The first cycle of rotation at each peak velocity was not included in the analysis (see

Methods for details) and if a segment was lost due to poor isolation or problems with data

acquisition, the cell was excluded from the analysis. Figure 5-10 illustrates the time course for

sensitivity changes over the entire experimental protocol, including all peak velocities, for

ipsilesional Type II (n=5) and contralesional Type I (n=4) cells. The order in which the

velocities were tested was different in the two groups but revealed that rescaling is actually

dependent on peak velocity rather than time. The sensitivity to rotation at 20 deg/s was always

lower than at 10 deg/s, even though it was the first velocity tested in contralesional Type I cells.

During each period of continuous rotation sensitivity remained relatively constant and

differences in sensitivity only appeared between peak velocities. Our results suggest that

rescaling in vestibular neurons likely requires less than 5 seconds to complete since, at each peak

velocity, sensitivity measured across the first and second segments was rarely different.

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Figure 5-10: Time course of sensitivity changes. Each symbol (mean ± s.d.) represents the sensitivity measured

from consecutive 5-cycle averages of spike density for a subset of ipsilesional Type II (n=5, top) and contralesional

Type I (n=4, bottom) neurons. Different symbols indicate 1 Hz stimuli with different peak velocities. Regardless of

which order the stimuli were presented in, the sensitivity measured at 10 deg/s peak velocity was always the highest.

The possible functional significance of adaptive rescaling is suggested by the performance of the

VOR during high velocity rotation after UVD. Figure 5-11 shows response functions for the

VOR measured during 1-Hz horizontal rotation before UVD and toward the damaged side after

UVD, at peak velocities between 10 and 120 deg/s in the cat from which the ipsilesional cells

were recorded. Prior to UVD, the VOR response functions appeared linear at all peak velocities

(Figure 5-11a). In contrast, when the VOR was recorded after UVD (Figure 5-11b), the

responses appeared to be linear between 0 and 20 deg/s but then showed saturation at the higher

velocities. The velocities at which saturating responses were observed in the ipsilesional VOR

coincided with the range of velocities over which nonlinear responses were found in vestibular

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neurons. There was no rescaling in the slopes of the response functions. However, although

there was no change in slope, the range over which the VOR appeared linear increased (compare

curves obtained at 80 deg/s and 120 deg/s in Figure 5-11).

Figure 5-11: The horizontal VOR before and after a vestibular lesion. Average slow phase eye velocity for Cat O

during 1 Hz rotation is plotted against head velocity. A: Traces obtained before UL. Only rightward half-cycles are

shown and different colours indicate different peak head velocities. Each average contains at least 169 cycles

collected over 4 days. B: Traces obtained two months after UL. Each average contains at least 488 cycles collected

over 8 days. Inset: A whole cycle of 1 Hz rotation with a peak velocity of 120 deg/s is shown with the average

slow phase eye velocity recorded before and after UL. Gaps in the eye velocity traces indicate where saccades and

quick phases had been removed.

5.4 Discussion

Our results suggest that adaptive rescaling may occur in the central vestibular neurons of alert

cats. We produced the first evidence for adaptive rescaling in the VNC after compensation for

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UVD (Heskin-Sweezie et al. 2007). Sensitivity rescaling has also been described in the VNC of

labyrinthine-intact animals, including decerebrate cats (Melvill Jones and Milsum 1970) and

alert rhesus monkeys (Newlands et al. 2009). In normal cats, an 8-fold increase in peak head

velocity caused neural sensitivity to decrease by a factor of 2 (Melvill Jones and Milsum 1970).

This result was identical to what we have reported in labyrinthine-deficient cats, suggesting that

adaptive rescaling remains preserved after UVD. In association with sensitivity rescaling, we

also found a significant velocity-dependent increase in the dynamic range of the neural response,

although the dynamic range of the neural output did not perfectly match the dynamic range of the

stimulus. Ideally, adaptive rescaling will match the dynamic range of a cell’s output to the

dynamic range of its input (Brenner et al. 2000). However, even though the dynamic ranges of

vestibular neurons were only partially extended, we still found that the VOR response functions

became more linear with increasing peak velocity after UVD.

Adaptive rescaling is a very rapid form of adaptation. Studies in other sensory systems have

shown that adaptive rescaling is complete within less than one second of initiating a change in

stimulus variance (Fairhall et al. 2001; Nagel and Doupe 2006). While we did not measure the

exact time course of sensitivity rescaling, we were able to show that it is complete within one

second after a change in peak head velocity. The rapidity of sensitivity rescaling indicates that

this form of adaptation is unique compared to other forms previously observed in the vestibular

system, including motor learning (Broussard and Kassardjian 2004), habituation (Jager and Henn

1981) and peripheral adaptation (Fernandez and Goldberg 1971; Blanks et al. 1975; Rabbitt et al.

2004). It is unlikely that either motor learning or habituation contributed to our results.

Peripheral adaptation has a time course that is comparable to that of adaptive rescaling.

Peripheral adaptation is the attenuation of hair cell and primary afferent responses that occurs

during prolonged periods of constant velocity or acceleration (Fernandez and Goldberg 1971;

Blanks et al. 1975; Rabbitt et al. 2004). In some species, primary afferents can fully adapt (ie.

return their discharge rates to pre-rotational values) in less than two seconds (Rabbitt et al.

2004). However, in the cat, the shortest time constant for primary afferent adaptation is 3.4s so

peripheral adaptation would take much longer (at least 11s) in this species (Blanks et al. 1975).

In addition, we used a sinusoidal stimulus with a frequency of 1 Hz in our experiments, which

means that neither velocity nor acceleration could have remained constant for more than a

fraction of a second at any given time. Therefore, our stimulus could not permit enough time for

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peripheral adaptation to take place. Also, in cats, peripheral adaptation causes very little

reduction in the sensitivities of central vestibular neurons at frequencies above 0.5 Hz (Melvill

Jones and Milsum 1971).

We considered Type I and Type II neurons separately since these two groups consist of different

classes of vestibular neurons that perform different functions in the vestibular network. For

example, a large majority of Type I neurons are secondary premotor neurons (Scudder and Fuchs

1992) while it is believed that many Type II neurons are small inhibitory interneurons that

receive polysynaptic inputs from the contralateral labyrinth (Shimazu and Precht 1966). We had

to consider the possibility that these different types of neurons might also differ in their ability to

rescale. However, we found that adaptive rescaling was not influenced by response polarity

(Type I vs Type II).

We also took into account the effect eye movement sensitivity could have on rescaling. We

found no difference between eye movement (EM) and vestibular-only (VO) neurons, suggesting

that rescaling was not affected by eye movement sensitivity. However, these neurons were

classified based only on measurements of eye position sensitivity. It should be pointed out that

there is a sub-class of EM neurons that respond to eye velocity in addition to eye position

(Scudder and Fuchs 1992). These neurons were not considered in our study since we were

unable to measure eye velocity sensitivity. Eye velocity sensitivity can simply be measured

while the subject tracks a moving target (Scudder and Fuchs 1992) but our cats were not trained

to do such a task. The alternative means for measuring eye velocity sensitivity requires the

measurement of head movement sensitivity during a VOR cancellation protocol (Scudder and

Fuchs 1992). The first step of the VOR cancellation procedure is to rotate the cat in the light at

a low frequency (0.2 Hz) with a visual target centered in front of the animal and moving exactly

in phase with the rate table. Reflexive eye movements are inhibited as the cat fixates on the

target, preventing any measurement of eye velocity sensitivity. Next, recordings of head

movement sensitivity would then be made while rotating the cat at 0.2 Hz in the dark. Finally, to

obtain eye velocity sensitivity, the sensitivity measured during cancellation would be subtracted

from the sensitivity measured during cancellation. We intended to include this procedure at the

end of our experimental protocol but none of our cells remained isolated long enough.

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Another factor to consider is whether rescaling of the responses of vestibular neurons originates

in the vestibular nuclei or is acquired from the primary afferents, which also exhibit sensitivity

rescaling in response to efferent stimulation. After UVD, primary afferents on the lesioned side

are silenced, though afferent responses on the intact side could potentially produce a downstream

effect on central neurons bilaterally. Primary afferents rescale their responses during activation

of the efferent vestibular system (Highstein and Baker 1985; Boyle and Highstein 1990).

Therefore, an influence from the efferent vestibular system may be responsible for our

observations. The efferents are a group of neurons located outside the VNC (Warr 1974;

Goldberg and Fernandez 1980; Carpenter et al. 1987; Perachio and Kevetter 1989) that form

synaptic contacts with both hair cells and primary afferents (Nakajima and Wang 1974; Sans and

Highstein 1984). While efferent control of the primary afferents may seem like a candidate

mechanism for adaptive rescaling in the VNC, it should be treated with caution since a thorough

examination of afferent response dynamics has shown that there is generally no dependency of

afferent sensitivity on peak rotational velocity (Goldberg et al. 1982; Hullar and Minor 1999;

Hullar et al. 2005).

In general, adaptive rescaling might enable the VOR to overcome performance limitations

brought on by UVD. When both labyrinths are intact, the response of the VOR to mid-

frequency rotation (0.2-2 Hz) is linear for head velocities up to 360 deg/s (Pulaski et al. 1981;

Paige 1983). This implies that adaptive rescaling may not be important for normal VOR

function. Nonlinear responses are common in the normal VNC, even at low rotational velocities

(Newlands et al. 2009), but they do not affect the normal operation of the VOR. However,

adaptive rescaling may become beneficial to VOR function when labyrinthine input is lost from

one side. After UL (Fetter and Zee 1988; Gilchrist et al. 1998; Lasker et al. 2000; Galiana et al.

2001), UVN (Maioli et al. 1983) or unilateral canal plugging (Paige 1983; Lasker et al. 1999),

the VOR develops a saturating nonlinearity that manifests in humans, monkeys, and cats during

ipsilesional rotation and at head velocities above 40 deg/s. We confirmed that the saturating

nonlinearity is evident in cats after both UL and canal plug surgery and it could reflect the

limited dynamic range of vestibular neurons exhibiting cutoff or saturating responses.

Interestingly, we found that the number of nonlinear responses was kept constant across all

stimuli on the contralesional side. This was in constrast to the velocity-dependent decrease in

linear responses we observed on the lesioned side, which is consistent with what has been found

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in the VNC of animals with intact labyrinths as well (Newlands et al. 2009). It is possible that,

at least on the contralesional side, adaptive rescaling might maintain response linearity in an

effort to maximize signal transmission in the VNC and improve VOR performance.

In addition to extending the dynamic range of a neuron, adaptive rescaling also functions to

maximize the amount of information that a neuron transmits about any given stimulus. This is a

distinguishing feature of adaptive rescaling. Several reports on adaptive rescaling in other

sensory systems show that a neuron will consistently rescale its response to maximize the

amount of information it transmits about a stimulus (Brenner et al. 2000; Fairhall et al. 2001;

Dean et al. 2005). Whether rescaling in vestibular neurons is associated with optimized

information transmission remains to be determined.

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Chapter 6: Concluding Remarks

Vestibular compensation is a complex process associated with neurophysiological changes

within the vestibular nuclei. Our multi-faceted examination of vestibular compensation has shed

new light on the plasticity mechanisms that may take place acutely following UVD, both in the

ipsilesional and contralesional VNC. We also demonstrated that, following vestibular

compensation, vestibular neurons on both sides of the brainstem exhibited rapid sensitivity

rescaling in association with increases in their dynamic ranges. Compared to normal, such

rescaling does not appear to have been affected by plastic changes that take place acutely

following UVD and, in the long term, it could benefit dynamic reflex function, which remains

compromised during high velocity head movements.

6.1 Acute Changes in GABAB Receptor Activation Following UVD

We first showed that GABAB receptors participate in the acute stage of vestibular compensation.

We found that systemic administration of a GABAB receptor antagonist compromised static

compensation in mice over the first few hours following UVD. This result led us to believe that

the activation of GABAB receptors immediately following UVD is necessary for static

compensation.

A recent investigation demonstrated that there is an acute change in GABA release in the

VNC following UVD (Bergquist et al. 2008). Compared to normal, GABA release was

increased in the ipsilesional VNC and decreased in the contralesional VNC. The changes in

GABA release could affect the activation of GABAB receptors, possibly causing increased and

decreased GABAB receptor activation on the lesioned and intact sides, respectively.

6.2 Actions of GABAB Receptors and Acute Changes in Neurotransmission in the Ipsilesional

VNC

128

On the lesioned side, increased GABAB receptor activation could play a role in cell

hyperpolarization, which may contribute to an increase in the resting rates of vestibular neurons

after UVD (see Section 1-3-3). The combined activation of post-synaptic and presynaptic

GABAB receptors located on the membrane just outside (Kulik 2002; Kulik 2003) inhibitory and

excitatory synapses could hyperpolarize a cell through the pathways shown in Figure 6-1. On

the post-synaptic membrane at both inhibitory (Figure 6-1A) and excitatory synapses (Figure 6-

1B), G-proteins activated by GABAB receptors can lead to the opening of potassium channels

(Lüscher et al. 1997; Slesinger et al. 1997) or the closure of calcium channels (Harayama et al.

1998), both of which could contribute to the hyperpolarization of a cell. Also at excitatory

synapses (Figure 6-1B), pre-synaptic GABAB receptor activation could reduce the amount of

excitatory neurotransmitter being released (Takahashi et al. 1998; Sakaba and Neher 2003).

Such an action could also promote hyperpolarization of the post-synaptic neuron by reducing the

amount of depolarization through glutamate receptors.

Figure 6-1: Diagramatic illustration of potential physiological changes that may take place at ipsilesional

commissural synapses acutely following UVD. A: Hypothetical effects of post-synaptic GABAB receptor

(GABABR) activation at a GABAergic commissural synapse. Post-synaptic GABAB receptors may cause the

closure of calcium (Ca2+

) channels and/or the opening of potassium (K+) channels. Both actions could theoretically

lead to hyperpolarization of the cell, in addition to chloride (Cl-) currents through activated GABAA receptors

129

(GABAAR) (Barker and Harrison 1988). B: Hypothetical effects of pre- and post-synaptic GABAB receptor

activation at an excitatory commissural synapse. An increase in endogenous GABA (pink) in the ipsilesional VNC

(Bergquist et al. 2008) could lead to an increase in GABAB receptor activation. This could reduce the amount of

glutamate (orange) released into the synapse and the amount of post-synaptic depolarization through glutamate

receptors (AMPAR & NMDAR).

By recording responses to commissural stimulation in the ipsilesional MVN of the mouse in

vitro, we produced preliminary data suggesting that there was an increase in inhibitory

transmission at some commissural synapses 2 hours after UVD. We were unable to conclude

whether this change was due to GABAB receptors. In fact this result was more likely caused by

the activation of ionotropic GABAA or glycine receptors (Lim et al. 2010). Since the data we

obtained from our in vitro experiments are preliminary, additional experiments would need to be

performed in order to confirm the hypothesis that post-synaptic GABAB receptor activation takes

place at inhibitory commissural synapses in the VNC acutely following UVD. First,

confirmation of this hypothesis would require testing the effects of GABAB receptor

manipulation on the responses to commissural stimulation in order to determine whether the

activation of GABAB receptors at these synapses is altered compared to normal. Second, other

inhibitory synapses besides those from the commissure should be considered. During our

behavioural experiments, test substances were administered systemically so it is very likely that

they acted on GABAB receptors located at other synapses either in the VNC or in other brain

areas. Inhibitory synapses formed between vestibular neurons and neurons located in the

cerebellum, thalamus or cerebral cortex are all candidate sites for GABAB receptor activation in

the VNC since all contain high quantities of the GABAB receptor (Bowery et al. 1987).

In our in vitro experiments, we did not observe any reduction in the excitatory responses to

commissural stimulation in the ipsilesional VNC. Rather, we found that some ipsilesional cells

exhibited potentiated responses to commissural stimulation compared to normal. It is possible

that the sensitivities of pre-synaptic GABAB receptors may have been reduced at the synapses

producing these excitatory responses. Some published evidence suggests that this may be the

case in the ipsilesional VNC. It was found that the ability of the GABAB receptor agonist

baclofen to reduce the firing rates of ipsilesional vestibular neurons was decreased from normal

within the first several hours following UVD (Yamanaka et al. 2000). A reduction in the

sensitivity of presynaptic GABAB receptors could lead to an increase in glutamate release and, in

130

turn, an increase in the endogenous glutamate levels. Increasing endogenous glutamate could

therefore lead to potentiation of the post-synaptic neuron (Grassi et al. 2001).

6.3 GABAB Receptors and Acute Changes in Excitatory Neurotransmission in the

Contralesional VNC Following UVD

On the contralesional side, a reduction in pre-synaptic GABAB receptor activation could promote

potentiation at the NVIII synapse in the VNC. The activation of GABAB receptors can depress

the responses of vestibular neurons to NVIII stimulation (Peterson et al. 1996), most likely

through pre-synaptic inhibition of glutamate release (Figure 6-2).

In the contralesional VNC, our recordings of responses to NVIII stimulation in mouse brain stem

in vitro suggested that synaptic transmission was increased at the NVIII synapse acutely

following UVD. Our recordings also suggested that the increase in synaptic transmission may

have been associated with an increase in synaptic current through AMPA receptor channels. Our

immunohistochemical analysis of the mouse VNC revealed an increase in expression of the

AMPA receptor protein, GluR4, in the contralesional relative to ipsilesional VNC 2 hours after

UVD. Together, our preliminary data suggest that acute potentiation at the contralesional NVIII

synapse may be associated with an increase in the synthesis of GluR4 proteins and their

subsequent insertion into the post-synaptic membrane (Figure 6-2).

131

Figure 6-2: Diagramatic illustration of potential physiological changes that may take place at NVIII synapse in the

contralesional VNC acutely following UVD. Reduced endogenous GABA (pink) in the contralesional VNC could

reduce the activation of pre-synaptic GABAB receptors (GABABR). This could lead to an an increase in glutamate

(orange) release, followed by the activation of NMDA receptors (NMDAR) and subsequent LTP at the synapse

(Grassi et al. 2001). LTP at this synapse may be associated with an increase in the insertion of GluR4-contianing

AMPA receptors (AMPAR) into the post-synaptic membrane.

In many areas of the brain, long-term potentiation (LTP) is associated with an increase in the

insertion of AMPA receptors into the post-synaptic membrane (Heynen et al. 2000; Moga et al.

2006; Williams et al. 2007; Kessels 2009). It is also well-accepted that long-term potentiation is

also associated with the expression of specific types of GluR subunits. For example, in the

hippocampus, some forms of LTP lead to an increase in the insertion of GluR1-and GluR2-

containing AMPA receptors (Heynen et al. 2000), while in the abducens nucleus LTP leads to an

increase in post-synaptic GluR4 (Mokin and Keifer 2004). In addition, several recent

investigations have shown that NMDA receptor-dependent LTP is associated with the rapid

synthesis of GluR proteins, and their insertion into the post-synaptic membrane, within just a few

hours after a potentiating stimulus is applied (Mameli 2007; Argilli 2008). More experiments

would need to be done to confirm our preliminary results and additional experiments would be

132

required to determine whether potentiation at the contralesional NVIII synapse is in fact

associated with rapid protein synthesis.

6.4 Sensitivity Rescaling in the VNC Following Compensation for UVD

Following vestibular compensation, we found that the sensitivity of vestibular neurons rescaled

with the peak velocity of rotation in the alert cat. We also found that sensitivity rescaling was

associated with increases in the neurons’ dynamic ranges. Rescaling was similar to that which

was previously reported in normal cats (Melvill Jones and Milsum 1970). Therefore, it is

possible that the plastic changes that take place during the acute stage of vestibular compensation

likely had no lasting effect, if any, on sensitivity rescaling. Whether rescaling of neuronal

sensitivity is altered acutely following UVD would require further investigation.

It is plausible that the sensitivity rescaling we observed is a form of rapid sensory adaptation,

also known as adaptive rescaling, that operates to extend a neuron’s dynamic range (Brenner et

al. 2000). However, it remains open to question whether rescaling of sensitivity might simply be

the result of non-linear behavior that may be inherent to vestibular neurons. In either case,

because the vestibular reflexes remain compromised at high velocities in the long term, the effect

of rescaling on the dynamic range of vestibular neurons could be of benefit to the vestibular

reflexes during high velocity rotation.

Our results should lead to new directions in vestibular research focusing on the mechanisms for

neural plasticity and sensory adaptation associated with vestibular compensation. Such research

could ultimately lead to improvements in therapy for vestibular patients. Currently, there are

several different exercise-based, non-pharmacological approaches for improving balance and

stability (Horak 2010). However, any improvement of sensory perception through exercise must

rely on neural plasticity. Therefore, a better understanding of the specific cellular mechanisms

associated with plasticity, as well as adaptation, could improve therapy and the quality of life for

those inflicted by vestibular pathology resulting in UVD.

133

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