manipulations of spinal cord excitability evoke developmentally-dependent compensatory changes in...

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Journal of Comparative Physiology ANeuroethology, Sensory, Neural, andBehavioral Physiology ISSN 0340-7594Volume 198Number 1 J Comp Physiol A (2012) 198:25-41DOI 10.1007/s00359-011-0683-0

Manipulations of spinal cord excitabilityevoke developmentally-dependentcompensatory changes in the lampreyspinal cord

Ria Mishaal Cooke, Sophie Luco & DavidParker

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ORIGINAL PAPER

Manipulations of spinal cord excitability evoke developmentally-dependent compensatory changes in the lamprey spinal cord

Ria Mishaal Cooke • Sophie Luco • David Parker

Received: 26 May 2011 / Revised: 9 September 2011 / Accepted: 14 September 2011 / Published online: 29 October 2011

� Springer-Verlag 2011

Abstract We have examined homeostatic or compensa-

tory plasticity evoked by tonic changes in spinal cord

excitability in the lamprey, a model system for investi-

gating spinal cord function. In larval animals, reducing

excitability by incubating in tetrodotoxin or the glutamate

receptor antagonists CNQX or CNQX/AP5 for 20–48 h

resulted in a diverse set of cellular and synaptic changes

that together were consistent with an increase in spinal cord

excitability. Similar changes occurred to a tonic increase in

excitation evoked by incubating in high potassium physi-

ological solution (i.e. responses were unidirectional). We

also examined developmental influences on these effects.

In animals developing from the larval to adult form effects

were reduced or absent, suggesting that at this stage the

spinal cord was more tolerant of changes in activity levels.

Responses had returned in adult animals, but they were

now bi-directional (i.e. opposite effects were evoked by an

increase or decrease in excitability). The spinal cord can

thus monitor and adapt cellular and synaptic properties to

tonic changes in excitability levels. This should be con-

sidered in analyses of spinal cord plasticity and injury.

Keywords Spinal cord � Compensatory plasticity �Lamprey � Excitability

Abbreviations

AP5 (2R)-Amino-5-phosphonovaleric acid

CNQX 6-Cyano-7-nitroquinoxaline-2,3-dione

EPSP Excitatory postsynaptic potential

IPSP Inhibitory postsynaptic potential

NMDA N-methyl-D-aspartate

PP Paired pulse

PSD Postsynaptic density

RMP Resting membrane potential

TTX Tetrodotoxin

Introduction

Nervous systems must be plastic and able to adapt their

outputs to different requirements. However, plasticity must

be regulated to ensure that it does not disrupt ongoing

function (‘‘the stability-plasticity dilemma’’; Abraham and

Robins 2005; Davis 2006). This regulation is suggested to

involve homeostatic or compensatory plasticity that

maintains activity levels within set limits. Homeostatic

plasticity is a conserved feature of vertebrate and invertebrate

nervous systems and may be triggered during development,

as a result of learning, or in response to injury (Marder

et al. 1996; Turrigiano et al. 1998; Turrigiano 1999;

Golowasch et al. 1999; Davis and Bezprozvanny 2001;

Mizrahi et al. 2001; Desai et al. 2002; Thoby-Brisson and

Simmers 2000, 2002; Davis 2006; Marder and Goaillard

2006; Echegoyen et al. 2007; Wilhelm and Wenner 2008;

Zhang et al. 2009; Feldman 2009; Knogler et al. 2010;

Turrigiano 2011). While it has become a major focus of

R. M. Cooke � S. Luco � D. Parker

Department of Zoology, University of Cambridge,

Cambridge, UK

R. M. Cooke � S. Luco � D. Parker

Department of Physiology, Development and Neuroscience,

University of Cambridge, Cambridge, UK

Present Address:R. M. Cooke (&)

Department of Cell Physiology and Pharmacology,

University of Leicester, Leicester, UK

e-mail: [email protected]

123

J Comp Physiol A (2012) 198:25–41

DOI 10.1007/s00359-011-0683-0

Author's personal copy

research, as with other forms of plasticity (Lisman et al.

2003), there is marked variability in homeostatic effects

and uncertainty over their underlying mechanisms (see

Turrigiano 2007). This may reflect differences in the type of

manipulation performed; the system, developmental state, or

components examined; or changes introduced by tissue

preparation (McMahon and Nicholls 1990; Gundersen et al.

1995; Kuenzi et al. 2000; Fiala et al. 2003).

The lamprey spinal cord is a useful model system for

investigating nervous system function (Rovainen 1979;

Cohen et al. 1992; Buchanan 2001; Grillner 2003). Various

aspects of the spinal cord locomotor network and its

descending and sensory inputs have been investigated in

different developmental stages (Rovainen 1979; Cohen

et al. 1992; Hagevik and McClellan 1999; Buchanan 2001;

Grillner 2003; Brodin and Shupliakov 2006). Neuromod-

ulator-evoked plasticity of the locomotor network has been

examined extensively, predominantly in mature adults (see

Grillner 2003), while functional recovery after spinal injury

(typically the regeneration of axons across lesion sites) has

been studied extensively in larval or juvenile adults

(Rovainen 1979; Cohen et al. 1988; McClellan 1994).

Despite being recognized and examined across a range of

invertebrate and vertebrate nervous systems, compensatory

or homeostatic plasticity has not been studied in the lam-

prey. This could present a significant gap in our under-

standing of this system, as homeostatic effects could be an

important influence on normal function, as well as influ-

encing the changes evoked by neuromodulation or spinal

cord injury. Evidence for potential compensatory responses

is provided by the changes in functional properties after

acute or chronic spinal cord lesions in larval and adult

animals (Cooke and Parker 2009; Hoffman and Parker

2010). However, because lesions can cause several

uncontrolled effects it is not clear if these changes reflect

the homeostatic-like plasticity seen in other systems.

We sought evidence for potential compensatory effects

here by examining cellular, synaptic, network, and ultra-

structural changes evoked by tonically manipulating spinal

cord excitability. The results show significant changes in

cellular and synaptic properties that depended on the type

of manipulation performed and the developmental stage

examined.

Methods

Larval and juvenile sea lampreys (Petromyzon marinus)

were obtained from the United States, and adult river

lampreys (Lampetra fluviatilis) from the UK. The species

used reflected availability. While there are developmental

differences in these animals, there is no evidence for spe-

cies differences (Parker and Grillner 1998; Parker et al.

1998; Parker and Gilbey 2007). Animals were kept at 6�C.

Maintenance and experimental procedures conformed to

UK Home Office regulations.

Animals were anaesthetised in MS222 and three pieces

of spinal cord were dissected from the trunk region

(between the last gill and the dorsal fin) in each develop-

mental stage (each piece was *10–15 segments). While

there is no evidence for regional differences, we varied the

regions of the spinal cord used in control and for incubation

to avoid potential regional effects. The spinal cord was

kept at 4�C in oxygenated physiological solution contain-

ing (in mM) 138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4

glucose, 2 HEPES, 0.5 L-glutamine, and pH 7.4 (adjusted

with 1 M NaOH). Incubated pieces of spinal cord were

kept for 20–48 h at 4�C in physiological solution con-

taining either TTX (1.5 lM) to block all spike-evoked

activity; CNQX (10 lM) or CNQX (10 lM) and AP5

(50 lM) to reduce non-NMDA and NMDA-evoked gluta-

matergic inputs; or high potassium physiological solution

(standard lamprey physiological solution but with

10.5 mM potassium) to increase excitability. These

manipulations are routinely used to study homeostatic

plasticity (e.g. see Turrigiano et al. 1998; Knogler et al.

2010). After incubation, the spinal cord was isolated from

the notochord and superfused with physiological solution at

10–12�C. Cellular and synaptic properties were examined

after washing in normal physiological solution for 30 min

and thus reflected incubation-evoked changes, not direct

drug effects. Washing for 30 min resulted in a plateau

recovery of spinal cord stimulation-evoked synaptic

responses that did not change after washing for up to 2.5 h

(data not shown). Longer wash times were avoided as they

could have led to reversal of any incubation-evoked effects.

Changes in spinal cord excitability were examined by

electrically stimulating the cell body region of the spinal

cord with a glass suction electrode placed on the surface of

the cord (30 Hz stimulation for 500 ms every 30 s). The

stimulation intensity was set at 1.5 times the threshold for a

single spike to control for variability in electrode location

relative to the cord surface in different experiments.

Activity recorded from ventral roots three segments below

the stimulation site was quantified by rectifying, integrat-

ing, and then averaging the traces (n = 5).

Intracellular recordings were made using thin-walled

glass micropipettes filled with 3 M potassium acetate and

0.1 M potassium chloride (resistances of 40–60 MX). As

the aim was to assess the potential for compensatory

effects, in common with most studies in lamprey we have

only examined motor neurons and unidentified cells. The

unidentified cells were unlikely to be smaller interneurons

(Buchanan 2001; Parker 2003) as recordings were sta-

ble over long times, and they were not the large contra-

lateral caudal interneurons or lateral interneurons as no

26 J Comp Physiol A (2012) 198:25–41

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extracellular spikes were recorded on the ipsilateral or

contralateral region of the cord 5–10 segments caudal to

the recorded cell (Parker 2003): they were thus probably

motor neurons, the only other large cells in this region of

the cord. There were no differences in effects in identified

motor neurons or unidentified cells and so cells were

grouped. An Axoclamp 2B amplifier (Axon Instruments

Inc., Foster City, CA, USA) was used for amplification and

in discontinuous current clamp mode for current injection

(the sampling rate was 2–3 KHz: this was monitored to

ensure compete voltage settling before voltage measure-

ments). The input resistance and excitability were exam-

ined from a set potential of -70 mV to ensure that any

changes were not simply due to membrane potential dif-

ferences between cells (note that the actual membrane

potential is reported). Input resistance was measured by

injecting a 100-ms -1nA current pulse, and excitability

assessed from the proportion of cells that spiked, and the

number of spikes evoked to a 100 ms ?1nA current pulse.

Data were acquired, stored, and analysed on computer

using an analogue-to-digital interface (Digidata 1200,

Axon Instruments, California) and Axon Instruments soft-

ware (pClamp 9).

Monosynaptic glutamatergic EPSPs were examined

using paired recordings from reticulospinal axons and

spinal cord neurons. These EPSPs can have electrical and

chemical components, which can be clearly separated (see

Control trace on inset in Fig. 4a). However, in other cases

the EPSP is smooth with a single peak. In this, either the

electrical EPSP is absent or it is obscured by the chemical

EPSP. Electrical EPSPs were only measured when they

were clearly visible. The axons were stimulated with a train

of pulses every 20 s (20 Hz for 1 s followed by four

recovery test pulses at 0.5 Hz). The trains were averaged

(n = 10), the baseline preceding each EPSP adjusted to

zero, and the electrical and/or chemical peak of the EPSP

measured relative to baseline. The initial EPSP, paired-

pulse plasticity (EPSP2/EPSP1), and responses over the 2nd

to 5th EPSPs (EPSP2-5/EPSP1), 6th to 10th EPSPs (EPSP6-

10/EPSP1), and 11th to 20th EPSPs (EPSP11-20/EPSP1)

were examined. Synaptic properties were also examined

using spontaneous tetrodotoxin (TTX)-resistant miniature

EPSPs (mEPSPs; miniature IPSPs were not examined as

they are rarely seen in control or after any incubation even

when the membrane potential is depolarised to move fur-

ther away from the chloride reversal potential). Baseline

activity was recorded in each cell for 70 s at -70 mV after

action potential-evoked release was blocked by TTX

(1.5 lM). mEPSPs were identified using a template search

in Clampfit. Traces were checked to ensure that no events

were missed and that detected events met mEPSP identi-

fication criteria (decay time at least 1.5 times the rise time).

There were two types of events: single mEPSPs with a

smooth rising and decay phase; and summed mEPSPs with

two peaks on their rising or decay phases (to be summed,

the second event had to fall before the 50% decay point of

the first event). Single and summed events were analysed

and cross-checked to ensure that no events were counted

twice.

Because we examined several treatments in three

developmental stages, we devised a manipulation index

(MI) to compare the effects evoked by different treatments

across developmental stages. This was derived as follows:

MI ¼ RMPC � RMPInc

RMPC

� �x10þ IRInc � IRC

IRC

� �

þ ExciteInc � ExciteC

ExciteC

� �þ SynInc � SynC

SynC

� �

RMP is resting membrane potential, IR is input

resistance (see above), and excite is the excitability

(measured from the number of spikes evoked; see above).

The synaptic input was measured from the summation of

the first five EPSPs during the spike train in control and

incubation to take into account the amplitude, duration, and

activity-dependent plasticity of the EPSPs:

SynInc � SynC

SynC

� �¼P5

i¼1 EPSPIncubation �P5

i¼1 EPSPControlP5i¼1 EPSPControl

C represents control, Inc effects after incubation. The

normalised individual effects were added. This ignores

potential threshold effects or interactions between the

different factors. While this is simplistic, as the aim was

only to compare changes based on treatment or

developmental stage and because of the general lack of

insight into these interactions and the many assumptions

that would have to be made, we felt that the simplest

approach was a better first approximation. The RMP

change was multiplied by 10 to give it the same weight as

the other effects (without this even large RMP changes

(*10 mV) give values of (*0.1), while input resistance

and excitability effects are an order of magnitude greater).

The manipulation index was calculated using mean values

of all treatments across all cells. It was examined using

absolute values of effects and also when non-significant

trends were removed: in this case control values were used

for incubation measures to give a zero value for effects.

Electron microscopy

Synaptic ultrastructure was investigated in control and

TTX incubated spinal cords from larval animals. The spinal

cord was isolated from the muscle and notochord and fixed

overnight in 3% glutaraldehyde (Agar Scientific) in 0.1 M

phosphate buffer (40.5 ml 0.2 M Na2HPO4; 9.5 ml 0.2 M

NaH2PO4 at 25�C, pH 7.4), pH 7.4 at 4�C. The fixed tissue

J Comp Physiol A (2012) 198:25–41 27

123


was then dissected transversely into 1-mm3 sections and

post fixed in buffered 1% OsO4 (Taab Ltd./Agar Scientifc)

with subsequent dehydration in a series of solutions of

35–100% acetone in water. The sections were treated in

saturated acetone and then embedded in Araldite CY212

(Agar Scientific). Ultrathin sections (80 nm) were cut on an

Ultracut E ultramicrotome (Reichert-Jung) and mounted on

uncoated 3.05 mm diameter copper mesh grids (Type

G200HH, Gilder). The sections were mounted from below

to reduce folds or distortions. Mounted sections were

counterstained with uranyl acetate (2% uranyl acetate in

50% methanol) and lead citrate (1.33 g lead nitrate; 1.76 g

trisodium citrate in 30 ml deionised water, and cleared

with NaOH), and viewed and photographed using a Philips

CM100 transmission electron microscope.

Transverse single sections of the whole spinal cord were

used. Single sections have been used for comparative

studies of synapse morphology (Buchs and Muller 1996;

Tyler and Pozzo-Miller 2001; Bevan and Parker 2004), but

they can introduce errors in measurement, for example in

vesicle numbers if sections are thicker or thinner than the

vesicle diameter. However, if there is less than 10% vari-

ability in the thicknesses of control and test sections and the

mean vesicle profile is smaller than the average section

thickness (so that single vesicles are not present in more

than one section), then single sections provide a rapid

method of comparative synaptic analyses. Two quantitative

TEM techniques were used to measure section thickness,

the small-fold technique, and the electron scattering method

(De Groot 1988), using an Iso-tech IDM63 digital mul-

timeter. The mean section thicknesses varied from 104.5 to

114 nm: the smallest was thus 91.67% of the largest, and

thus section thickness varied less than 10%. The smallest

mean vesicle profile was 48.75 ± 1.5 nm and the largest

56.6 ± 1.74 nm. These are smaller than the estimated

section thickness, and so it can be confidently assumed that

counting vesicle profiles in single sections will not result in

an overestimation of the number of vesicles.

The cell body region of the spinal cord of each section was

searched for synapses (20 per animal).In this region synapses

are made onto cell bodies and dendrites. The incidence of

synapses per section was low, so the whole cell body region

was covered. Although there is no regionalisation of neurons

or synapses in the cell body region, this ensured against any

bias to a particular region. Specific neurons were not labelled

as this can prevent analysis of presynaptic structures

(Buchanan et al. 1989). The TEM negatives were scanned

using an Epson 2450 Photo Scanner at a resolution of 1,200

dots per inch and imported into Adobe Photoshop 7 (Adobe

Systems Inc.) for the analysis of synaptic structure. The

following aspects were examined: the number of vesicles per

synaptic profile (the number of vesicles adjacent to the pre-

synaptic membrane); the number of docked vesicles (vesicles

apposed to the presynaptic membrane); the mean vesicle

diameter (the average of the cross section diameter of five

randomly chosen vesicles); the ratio of the number of docked

vesicles to the total number of vesicles; the width of the

synaptic gap (measured as the mean average of three mea-

surements made perpendicular to the membrane at each

synapse); the length of the postsynaptic density (PSD; mea-

sured as the length of the darkened region of the postsynaptic

membrane); the number of docked vesicle profiles per nm of

postsynaptic density; the vesicle type (round, flattened or

pleomorphic); the synapse shape (unwrapped or wrapped,

where the presynaptic terminal encloses the postsynaptic

terminal); synapse symmetry (symmetric or asymmetric);

and the synapse curvature (whether the postsynaptic density

is straight, concave, or convex).

Statistical analyses

N numbers in the text reflect the numbers of cells or syn-

apses examined. In physiological analyses at most three

cells or synapses were examined in each piece of cord.

Incubation values were compared with control values (i.e.

non-incubated cords taken within 30 min to 4 h after dis-

section). Statistical significance was calculated using

parametric t tests or Analysis of Variance (ANOVA, with

Dunnett’s post hoc test), or non-parametric tests Mann-

Whitney U or Kruskal-Wallis. A repeated measures

ANOVA was used to compare the EPSP amplitudes across

the 20-Hz train. Frequency data were analysed using v2

tests. Cumulative frequency curves were plotted for the

amplitude and time between consecutive of mEPSPs. All

events from all cells were pooled for each condition. When

calculating the time between events over 70 s the starting

and final time points were included, so the time between

0 s and the first event and the last event and 70 s were

represented in the data sets. Data were grouped into

0.05 mV (mEPSPs) or categories above for amplitude and

200 ms categories for time between events. Kolmogorov–

Smirnov tests were performed on the grouped data. Values

quoted in the text represent the mean ± SEM. Ultrastruc-

tural data were examined for differences between individual

animals in each condition, as well as differences between

conditions. Parametric two-way ANOVAs (general linear

models) or non-parametric Scheirer-Ray-Hare tests were

used. The statistical results for interaction or animal are only

stated if they were significantly different.

Results

We examined homeostatic plasticity by manipulating

spinal cord excitability levels. Excitability was reduced by

blocking all spike-evoked activity with TTX (1.5 lM) or

28 J Comp Physiol A (2012) 198:25–41

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by reducing glutamatergic inputs with the NMDA and non-

NMDA receptor antagonists AP5 (50 lM) and CNQX

(10 lM; together these reduced spinal cord-evoked EPSPs

by 82.2 ± 6.5%, n = 4), and increased using high potas-

sium physiological solution. The effects seen were specific

to the type of incubation performed and thus did not simply

reflect time-dependent changes. This was confirmed in

spinal cords that were left for 20–24 h in normal physio-

logical solution: in this case there were no significant

changes in the resting membrane potential (Fig. 1a), input

resistance (Fig. 1b), cellular excitability (Fig. 1c), or spinal

cord excitability (see Fig. 6c) compared with cords taken

30 min–4 h after dissection. The data reported here are

predominantly from incubations for 20–24 h: where effects

were examined after longer incubation periods (48 h) they

did not differ significantly from the effects seen at this time

(see Fig. 2a, b). We examined effects after washing for

30 min and for up to 2.5 h after the start of wash-off. While

we have not systematically examined the reversal of any

incubation-evoked changes, there was no indication of

reversal of effects over this time.

Cellular and synaptic changes in larval spinal cords

Blocking all spinal cord activity by incubating in TTX

resulted in a significant depolarisation of the resting

membrane potential of larval spinal cord neurons (Fig. 2a;

Table 1). However, the input resistance was not signifi-

cantly affected (Fig. 2b; Table 2). Despite the failure to

change the input resistance, TTX significantly increased

the excitability of larval neurons as shown by the increased

fraction of cells that generated one or more spikes in

response to a 1nA depolarizing current pulse (Fig. 2c) and

the increase in the number of spikes evoked (Fig. 2d).

Incubation in the glutamate receptor antagonists CNQX

and AP5 significantly depolarized the resting membrane

potential of larval neurons (Fig. 2a; Table 1) and increased

the input resistance (Fig. 2b; Table 2). However, neither

effect was significant after incubation in CNQX (10 lM)

alone (Fig. 2a, b; Tables 1, 2). This may reflect a greater

reduction of glutamatergic-evoked excitation after incubation

in both CNQX and AP5 than in only CNQX (82.2 ± 6.5%

compared with 54 ± 1.9 mV, respectively; n = 4). How-

ever, CNQX incubation did significantly increase the pro-

portion of cells that spiked and the number of spikes evoked by

a 1nA depolarizing current pulse. Both effects were absent

following incubation in both CNQX and AP5 (Fig. 2c, d),

suggesting a potential NMDA-dependent effect.

High potassium physiological solution was used to

increase the excitability of the spinal cord. This depolarised

cells by 15 ± 4 mV (n = 4) at the start of incubation, and

by 11.5 ± 4 mV (n = 3) after incubation for 24 h. It also

increased excitability shown by the increased spiking in

response to depolarizing current pulses (data not shown),

suggesting against a reduction of excitability due to

depolarisation-induced sodium channel block. Incubating

in high potassium physiological solution for 20–24 h did

not significantly affect the resting membrane potential of

larval neurons (Fig. 2a; Table 1). However, it significantly

increased the input resistance (Fig. 2b; Table 2) and

excitability (both the proportion of cells that spiked and the

number of spikes evoked; Fig. 2c, d). An increase in

excitability in response to a manipulation that increased

excitability was not consistent with negative feedback-

driven bi-directional plasticity.

0

1

2

3

4

Control 20-24h

20-24h

20-24h

Control

Control

-70

-68

-66

-64

-62

-60

0

10

20

a

b

c

RM

P(m

V)

Inpu

t res

ista

nce

(MΩ

)N

umbe

r of

spi

kes

Fig. 1 Cellular properties in larval spinal cords left for 20–24 h

without any incubation to examine if there were significant time-

dependent changes. The resting membrane potential (a n = 24 in

control and n = 23 after 20–24 h), input resistance (b n = 19

in control and n = 21 after 20–24 h), and excitability (c; n = 13 in

control and n = 8 after 20–24 h) were not significantly altered

(p [ 0.05) over this time

J Comp Physiol A (2012) 198:25–41 29

123


Incubation-induced changes in synaptic effects were

examined using spontaneous TTX-resistant miniature

EPSPs (mEPSP). While incubation in TTX tended to increase

the mEPSP frequency, there was no significant change in

either the mEPSP frequency or amplitude (p [ 0.05,

n = 8, Fig. 3a, b). There was, however, a significant

)V

m(P

MR

*

*

*

-75

-70

-65

-60

-55

*

*

0

10

20

30

40

50

**

*

sek ips fo rebm

uN

0

1

2

3

4

5* * *

0

20

40

60

80

100

)%( sllec gnikips fo noit ropor

P

)M( ecnatsiser t upnI

20-2

4hT

TX

XT

Th

84

20-2

4h5

PA/

XQ

NC

20-2

4h XQ

NC

K h

giH

+

lort

no

C 20-2

4hT

TX

XT

Th

84

20-2

4h5

PA/

XQ

NC

20-2

4h XQ

NC

K h

giH

+

lort

no

C

h4

2-0

2 TT

X

20-2

4h5

PA /

XQ

NC

h4

2-0

2X

QN

C

K h

giH

+

lort

no

C 20-2

4hT

TX

20-2

4h5

PA/

XQ

NC

20-2

4h XQ

NC

K h

giH

+

Con

trol

a b

c d

Fig. 2 Changes in cellular

properties in larval neurons after

incubation in TTX (1.5 lM),

CNQX (10 lM), CNQX

(10 lM) and AP5 (50 lM), or

high potassium physiological

solution for 20–24 or 48 h.

Effects on a resting membrane

potential, b input resistance,

c the proportion of spiking cells,

and d the number of action

potentials evoked by a 1nA

depolarising current pulse. On

all graphs the symbols above the

bars indicate significant

changes (p \ 0.05)

Table 1 Values for resting potential measurements in control and in different incubations for 20–24 h in adult, transformer, and larval animals

Larval (mV) Transformer (mV) Adult (mV)

Control -69.7 ± 0.98 (n = 24) -67.6 ± 0.9 (n = 38) -66.9 ± 0.9 (n = 52)

TTX -64.7 ± 1.18 (n = 20)* -65 ± 1.2 (n = 25) -63.6 ± 0.9 (n = 37)*

CNQX -66.2 ± 2.4 (n = 14) -58 ± 1.2 (n = 15)* -58.6 ± 0.7 (n = 37)*

CNQX/AP5 -63.6 ± 1.5 (n = 16)* -61.3 ± 1 (n = 31)* -63.9 ± 1.4 (n = 26)

High potassium -71 ± 1.1 (n = 12) -67.6 ± 1.1 (n = 20) -70.5 ± 1.5 (n = 51)*

Numbers in the brackets indicate the sample sizes. The symbols indicate significant differences to control (p \ 0.05)

Table 2 Input resistance values in control and in different incubations for 20–24 h in adult, transformer, and larval animals

Larval (MX) Transformer (MX) Adult (MX)

Control 9.9 ± 1.1 (n = 19) 17.7 ± 1.9 (n = 38) 10.30 ± 1 (n = 34)

TTX 8.6 ± 1.5 (n = 10) 14 ± 1.9 (n = 17) 12.8 ± 2.6 (n = 11)

CNQX 15 ± 4.3 (n = 9) 18 ± 0.9 (n = 8) 27.8 ± 3.4 (n = 28)*

CNQX/AP5 36.8 ± 6.2 (n = 12)* 22 ± 2.5 (n = 15) 9.5 ± 1.4 (n = 15)

High potassium 23.5 ± 3.2 (n = 12)* 16.8 ± 3 (n = 21) 15.8 ± 2.7 (n = 42)*

Numbers in the brackets indicate the sample sizes. The symbols indicate significant differences to control (p \ 0.05)

30 J Comp Physiol A (2012) 198:25–41

123


increase in the mEPSP half-width (Fig. 3c) without a

change in the mEPSP rise time (Fig. 3d). Incubation in

CNQX or in CNQX/AP5 also did not significantly affect

the frequency or amplitude of mEPSPs (p [ 0.05, n = 7;

Fig. 3a, b), but in CNQX/AP5 the mEPSP half-width was

again significantly increased (p \ 0.05, n = 7) without any

change in the mEPSP rise time (Fig. 3c, d).

Incubation in high potassium physiological solution did

not affect the mEPSP frequency (Fig. 3a). However, in this

case there was a significant increase in the mEPSP

amplitude (n = 7; p \ 0.05; Fig. 3b). This increase in

glutamatergic inputs again argued against bi-directional

plasticity. High potassium physiological solutionalso sig-

nificantly reduced the mEPSP half-width and rise time

(Fig. 3c, d). These effects could in principle help to reduce

EPSP summation and consequently reduce overall network

excitability levels. However, while the frequency of single

mEPSPs was unaffected, high potassium physiological

solution significantly increased the frequency, but not

amplitude, of summed mEPSPs (Fig. 3e, f). This was sur-

prising as mEPSPs are assumed to reflect the spontaneous

release of single vesicles. Summed events would occur if

two or more spontaneous events occurred together, but for

this to happen significantly more often after incubation it

would be assumed that there would be an increase in the

frequency or duration of single mEPSPs, neither of which

occurred here.

To examine if the number of summed events was related

to the number of summed events we assumed that every

summed event resulted from the summation of two random

single events (summed events usually had two peaks; see

inset Fig. 3f), the fraction of the total number of events that

were summed was calculated as

Number of summed events

Number of single events þ 2 number of summed eventsð Þ

There was a significant increase in the fraction of the

total number of summed events after incubation in high

potassium physiological solution (control 0.0210 ± 0.014

(n = 9); high potassium physiological solution

0.22 ± 0.046 (n = 7); Mann-Whitney U p \ 0.05). The

percentage of neurons that exhibited summed events

significantly increased from 22.22% in control to 100%

after incubation in high potassium physiological solution

(v2 = 10.578, p = 0.001; control n = 9, high potassium

physiological solution n = 7).

We also examined synaptic effects by making paired

recordings from presynaptic reticulospinal neurons and

* *

*

Control TTX CNQXCNQX/AP5

CNQX High K +

High K +

High K +

High K +

High K +High K +

0.0

0.2

0.4

0.6

0.8

1.0

Control TTX CNQX/AP5

)zH(

ycneuqerfP

SP

Em

*


0.000.050.100.150.200.250.300.35

)V

m(edutilp

maP

SP

Em

0

1

2

3

4

5

6)s

m(htdi

w-f laH

*


0.0

0.5

1.0

1.5

2.0

2.5

)sm(

emit

esiR

0.4mV40ms

*


0.0

0.5

1.0

1.5

2.0

)zH(

ycneuqerF


0.3

0.4

0.5

0.6

0.7

0.8

0.9

)V

m(edutilp

mA

a b

c d

e f

Fig. 3 Changes in TTX

resistant mEPSPs after

incubation in larval spinal cords

for 20–24 h. Bar graphs show

changes in the a frequency,

b amplitude, c half-width, and

d rise-time of single mEPSPs. In

addition to single mEPSPs, we

also saw changes in summed

mEPSPs. There was no

significant change in the

amplitude in any incubation (e),

but there was a significant

increase in the frequency of

summed events after incubation

in high potassium physiological

solution (f). The traces on the

graph show examples of

mEPSPs in control and in high

potassium physiological

solution (right). The dashed boxhighlights a summed mEPSP

J Comp Physiol A (2012) 198:25–41 31

123


postsynaptic spinal cord neurons. Because this is more

difficult than examining spontaneous inputs we only

examined the effects of TTX and high potassium physio-

logical solution, treatments that reduce and increase

excitability, respectively. Reticulospinal EPSPs can consist

of an electrical and a chemical component (see control

trace on the inset in Fig. 4a). In TTX, despite the lack of a

significant effect on the mEPSP amplitude or frequency,

there was a significant increase in the chemical component

of the initial EPSP amplitude in the spike train (Fig. 4a)

and a significant increase in the EPSP half-width (p \ 0.05,

n = 4, data not shown). An electrical component was only

present in one connection after TTX incubation, and we

could thus not compare changes in this component. In

control connections inputs over the spike train depressed

(Fig. 4b, c). The depression was increased after incubation

in TTX, although only significantly over Train2–5 (Fig. 4b).

In high potassium physiological solution there was also an

increase in the amplitude of the chemical (p \ 0.05, n = 4;

Fig. 4a, c), but not the electrical component of the EPSP

(p [ 0.05, n = 4; Fig. 4a, c), and a reduction of the half-

width of the chemical component (data not shown; see

Fig. 4c). However, after incubation in high potassium

physiological solution the input facilitated during spike

trains (Fig. 4b, c). Note that this effect was associated with

a relative depolarization of the baseline (Fig. 4c). This will

amplify the relative EPSP amplitude over the spike train,

but as the EPSP amplitude was measured from the baseline

preceding each EPSP (see ‘‘Methods’’), there was also a

direct increase in the amplitude of each EPSP.

Analyses of synaptic ultrastructure

Electron microscopy was used to determine if the physio-

logical effects of incubation were associated with changes

in synaptic ultrastructure. Because of the large number of

synapses that have to be examined we have only examined

the effects of TTX incubation. This was chosen as it

allowed us to examine an incubation that has commonly

been used to examine homeostatic effects both physiolog-

ically and ultrastructurally in other systems (see Burrone

and Murthy 2003), and one that had a significant synaptic

effect here (see Fig. 4).

There were few ultrastructural effects of TTX incuba-

tion (n = 60; 20 synapses from each of three animals for

control and TTX-incubated; Fig. 5a). There were no sig-

nificant differences in the number of vesicles per synaptic

profile (Fig. 5b), the number of docked vesicles (Fig. 5c),

the mean vesicle diameter (data not shown), the ratio of the

number of docked vesicles to the total number of vesicles

(data not shown), the width of the synaptic gap (Fig. 5d),

the length of the postsynaptic density (PSD; Fig. 5e), or the

number of docked vesicle profiles per nm of postsynaptic

density (Scheirer-Ray-Hare test, p [ 0.05; Fig. 5f). The

distribution of vesicle types also did not differ significantly

after TTX incubation (v2 test: v2 = 2.43, p [ 0.05; data

not shown), and the synapse shape, symmetry, and position

all did not differ significantly when all synapses were

grouped or split into categories based on symmetry or

vesicle type (Scheirer-Ray-Hare test, p [ 0.05; data not

0.0

0.5

1.0

1.5

2.0

2.5

)V

m( e

dutil

pm

aP

SP

E

lortn oC

tcelE lort no

Cmeh

C

TT

Xmeh

C

K hgiH

+tcel

E

Control

TTX

High K+

PP Train2-5 Train 6-10 Train 11-20Rec

laitinI/nia rT

PS

PE

PS

PE

K hg iH

+meh

C

Highpotassium

Control

0.0

0.5

1.0

1.5

TTXControl

**

200ms

20ms

1mV

1mVa

b

c

Fig. 4 Effects of incubation on reticulospinal-evoked EPSPs in

larvae after incubation for 20–24 h. a Bar graph showing the

amplitude of the electrical and chemical component of reticulospinal-

evoked EPSPs after incubation in TTX or high potassium physiolog-

ical solution (the number of electrical connections in TTX was low

(n = 1) and this information is not included on the graph). The inset

shows averaged (n = 10) EPSPs in control and TTX. b Graph

showing changes in the paired pulse plasticity (PP) and the plasticity

over different regions of the spike train after incubation. c A train of

EPSPs evoked at 20 Hz in control and after incubation in high

potassium physiological solution

32 J Comp Physiol A (2012) 198:25–41

123


shown). Only the curvature of synaptic profiles differed:

after incubation in TTX they were predominantly straight

as opposed to predominantly convex in control (v2,

p [ 0.0001; Fig. 5g).

Functional effects of incubation-evoked changes

The significant changes in cellular and synaptic properties

after the various incubations that increased or decreased

spinal cord excitability were compared (see ‘‘Methods’’;

Fig. 6a, b). In each case the simple summed values were

positive for all treatments and thus the effects were uni-

directional. To confirm this prediction, we examined how

spinal cord responses to electrical stimulation were altered

after incubation (see ‘‘Methods’’). This allowed us to

determine the global effects of the cellular and synaptic

changes and also whether the manipulation index could

predict effects at the network level. We compared

responses to incubation to control cords that were left for

20–24 h but not incubated in any drugs (n = 14). These

control cords did not differ significantly to control cords

examined soon after dissection (n = 5; Fig. 6c). While

spiking and synaptic responses to spinal cord stimulation

occurred in the cells we recorded from after wash-out of

TTX for 30 min–2 h (see above), no ventral root spiking

could be evoked (n = 6 of 6; data not shown). We cannot

explain this, except to say that while the cells that we had

recorded from (identified and putative motor neurons) had

shown some recovery from the effects of TTX, longer

wash-off times may be needed to ensure complete removal

Fig. 5 EM analysis of synaptic

ultrastructure in larval spinal

cords. a Electron micrographs

of a synapse from a control (left)and a TTX incubated spinal

cord (right). The solid blackarrow indicates the postsynaptic

density. Both images were taken

at the same magnification. Scalebar 1 lm. Graphs showing b the

number of vesicles per synaptic

profile, c the number of docked

vesicles, d the width of the

synaptic gap, e the length of the

postsynaptic density (PSD), the

number of docked vesicle

profiles per nm of postsynaptic

density (f). Only the curvature

of synaptic profiles (g) differed

significantly

J Comp Physiol A (2012) 198:25–41 33

123


of TTX from other cells needed to generate the stimulation-

evoked responses (e.g. excitatory network interneurons).

For each of the other treatments [CNQX (n = 11), CNQX

and AP5 (n = 13), and high potassium physiological

solution (n = 13)] there was a significant increase in the

cord stimulation-evoked response compared with control

(Fig. 6c, d). Increased activity in response to treatments

that reduced or increased excitability mirrored the effects

shown by the manipulation index in suggesting against

bi-directional homeostatic responses.

Developmental differences in incubation effects

While we have focused on changes in larval animals, we

also investigated developmental influences by examining

incubation effects in animals undergoing the transforma-

tion from the larval to adult stage (‘‘transformers’’) and

mature adults, as these developmental stages have been

used to examine specific aspects of spinal cord function

and plasticity (see ‘‘Introduction’’).

In transformers, incubation in TTX or high potassium

physiological solution did not significantly affect the rest-

ing membrane potential (Fig. 7a; Table 1), input resistance

(Fig. 7b; Table 2), or excitability (Fig. 7c, d). CNQX or

CNQX/AP5 incubation significantly depolarized the rest-

ing membrane potential (Fig. 7a; Table 1), but did not

significantly affect the input resistance (Fig. 7b; Table 2).

Incubation in CNQX or CNQX/AP5 did not affect the

proportion of cells that spiked (Fig. 7c), but CNQX/AP5

incubation did increase the number of spikes evoked

(Fig. 7d). There were no significant differences in the

mEPSP amplitude, frequency, rise time, or half-width after

any incubation in transformers (p [ 0.05; control, n = 10;

TTX, n = 17; CNQX, n = 8; CNQX/AP5, n = 8; high

potassium physiological solution, n = 10; data not shown).

We again examined effects on evoked EPSPs after

incubation in TTX and high potassium physiological

solution by making paired recordings from presynaptic

reticulospinal neurons and postsynaptic spinal cord neu-

rons. Neither incubation significantly altered the half-width

Larval Transformer Adult

TTXCNQXCNQX+AP5High K+

No incubation

xedni noitalupinaM

-5

0

5

10

xedni noitalupinaM

Larval Transformer Adult-5

0

5

10

esnopser toor lartne v deta rgetnI lortn oC

lortnoC

24h X

QN

C

5P

A/X

QN

C

K hg iH

+

0

50

100

150 *

*

*

20s

a b

c d

Fig. 6 Functional effects of the incubations. a Graph showing the

manipulation index when the mean values of all the different

parameters were included irrespective of whether the changes were

significant or not, and b when only significant effects were included.

Where effects were insignificant they were given the same value as

control. Larval animals showed the largest responses, but note that the

effects were unidirectional (i.e. increasing or decreasing network

excitability resulted in an increase in excitation). In mature adults

bidirectional effects were seen (an increase or decrease in excitability

occurred when network activity was reduced or increased, respec-

tively). In mature adults there was also evidence for NMDA-

dependent plasticity, as the changes were absent in AP5. c Graph

showing the effects of incubation on network responses evoked by

cord stimulation compared with control responses. d Traces showing

raw and rectified and integrated ventral root responses in control and

after incubation in CNQX

34 J Comp Physiol A (2012) 198:25–41

123


(data not shown) or amplitude of the electrical or chemical

component of low frequency-evoked reticulospinal-evoked

EPSPs (n = 19 control, n = 6 TTX, n = 10 high potas-

sium; Fig. 8a). However, both altered the activity-depen-

dent plasticity of the chemical EPSP during spike trains

(Fig. 8b; the electrical EPSP was unchanged, data not

shown): connections usually depressed in control, but

facilitated after both incubations. While this again argued

against bidirectional plasticity, a bidirectional effect was

revealed when the relationship between the initial EPSP

amplitude and plasticity over different regions of the spike

train was examined. In control there was no significant

correlation between the initial EPSP amplitude and the

plasticity over any part of the spike train (r2 = 0.15–0.3,

p [ 0.05; data not shown). However, there was a signifi-

cant correlation between the initial EPSP amplitude and

plasticity over Train2-5 after TTX incubation (r2 = 0.72

(n = 6), p \ 0.05; Fig. 8c), but a significant negative

correlation after incubation in high potassium physiologi-

cal solution (r2 = 0.40 (n = 10) p \ 0.05; Fig. 8d). TTX

incubation would thus amplify and high potassium physi-

ological solution would reduce the effects of larger EPSPs.

In mature adults, TTX incubation significantly depo-

larised the membrane potential (Fig. 9a; Table 1), but did

not significantly affect the input resistance (Fig. 9b;

Table 2) or excitability of spinal cord neurons (Fig. 9c).

CNQX incubation also significantly depolarized the

membrane potential (Fig. 9a; Table 1), and significantly

increased both the input resistance and excitability

(Fig. 9b, c; Table 2). However, all effects of CNQX were

absent following incubation in both CNQX and AP5

(Fig. 9a–d; Tables 1, 2), suggesting a potential NMDA-

dependence. High potassium physiological solution had the

opposite effects: it significantly hyperpolarized the resting

membrane potential (Fig. 9a; Table 1), increased the input

resistance (Fig. 9b; Table 2), but reduced the excitability

(Fig. 9c). Thus, in contrast to larvae, effects were bidi-

rectional in adults.

TTX incubation did not significantly affect the ampli-

tude of the electrical or chemical component of low-fre-

quency reticulospinal-evoked EPSPs (1.16 ± 0.1 mV

(n = 48) in control compared with 0.94 ± 0.1 mV

(n = 19) after incubation in TTX, p [ 0.05; Fig. 10a).

However, it significantly reduced the EPSP half-width

(from 10.26 ± 0.6 ms (n = 46) in control to

6.78 ± 0.71 ms (n = 18) in TTX, p \ 0.05; Fig. 10b), and

significantly increased in the plasticity ratio over Train2–5,

Train6–10, and Train11–20 (i.e. increased facilitation;

Fig. 10c, d). In contrast to transformers, there was no

significant relationship between the initial EPSP amplitude

and the train plasticity in control or after incubation in

TTX, which suggested a general facilitation, irrespective of

the initial EPSP amplitude (Fig. 10e). High potassium

physiological solution did not affect the amplitude of

evoked EPSPs (1.16 ± 0.1 mV (n = 48) in control com-

pared with 0.94 ± 0.2 (n = 14) after incubation, p [ 0.05;

lortnoC

/X

QN

C5

PA

TT

X XQ

NC

+K hgi

H

lortnoC

/X

QN

C5

PA

TT

X XQ

NC

+K h gi

H

MΩ

)( ecnatsis er tupnI

*

*

0

10

20

30

-70

-65

-60

-55

lortnoC

/X

QN

C5

PA

TT

X XQ

NC

+K hgi

H

*

0

1

2

3

4

sekips fo rebm u

N

l ortnoC

/X

QN

C5

PA

TT

X XQ

NC

+K hgi

H

0

20

40

60

80

100)%( sllec g nikips fo noitropor

P)

Vm(

PM

R

a b

c d

Fig. 7 Changes in cellular

properties in transformers after

incubation for 20–24 h. Graphs

show changes in a resting

membrane potential (RMP),

b input resistance, c proportion

of spiking cells, and d the

number of spikes evoked by a

1nA depolarising current pulse

J Comp Physiol A (2012) 198:25–41 35

123


Fig. 10a), or the PP or plasticity over the spike train

(Fig. 10c, e). However, it significantly reduced the EPSP

half-width (from 10.26 ± 0.6 (n = 46) in control com-

pared with 6.0 ± 0.5 (n = 14) in high potassium physio-

logical solution, p \ 0.05; Fig. 10b).

The relative reduction in effects after incubation in

transformers was supported by the reduced manipulation

index values compared with larvae (Fig. 6a, b). This sug-

gested either a spinal cord more tolerant of tonic changes in

the level of excitability, or one in which homeostatic-like

mechanisms is absent. In adults the responses to tonic

manipulations of excitability had returned (Fig. 6a, b), but

unlike larvae the effects were bi-directional.

Discussion

While various aspects of spinal cord function have been

examined in lamprey (e.g. Grillner 2003), compensatory or

homeostatic plasticity, a widely recognized feature of

nervous systems (see Introduction for references), had not

previously been addressed. We have shown significant

compensatory-like changes in cellular and synaptic prop-

erties that depended on the type of manipulation performed

and on the developmental stage examined. This suggests

that the spinal cord can monitor and respond to tonic

changes in excitability levels.

While homeostatic changes have been reported in many

systems, in most cases the underlying mechanisms remain

unclear (see Turrigiano 2007; Rich and Wenner 2007).

Currently, we have only addressed the question of whether

these effects occur and have little insight into their under-

lying mechanisms. Various cellular (resting potential, input

resistance, excitability) and synaptic changes (spontaneous

TTX-resistant mEPSPs and evoked responses) occurred in

response to incubation-imposed changes in excitability.

While these effects should be linked functionally, the

changes did not necessarily follow simple associations (e.g.

resting potential changes occurred independently of changes

in input resistance). Given the range of mechanisms that

could underlie these effects the absence of simple assumed

linkages is not surprising (e.g. see Jiang et al. 1994).

Mandatory links are also probably not desirable, as they

would reduce the potential flexibility of the responses.

0.0

0.5

1.0

1.5

2.0

2.5

)V

m( edutilpma

PS

PE

lortnoC

t celE

TT

Xtcel

E lortnoC

m ehC

TT

Xmeh

C

K hgiH

tcelE

+

Control

TTX

High K+

PP Train2-5 Train6-10 Train11-20 Rec

laitinI/niarT

PS

PE

PS

PE

K hgiH

+meh

C

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7110

120

130

140

150

160

Initial EPSP amplitude (mV)

yticitsalpP

P

TTX r =0.722

0.00 0.25 0.50 0.75 1.000

100

200

Initial EPSP amplitude (mV)

yticitsalpP

P

High K+ r =0.402

0

0.5

1.0

1.5a b

c d

Fig. 8 Effects of incubation on reticulospinal-evoked EPSPs in

transformers after incubation for 20–24 h. a Bar graph showing the

amplitude of the electrical and chemical component of reticulospinal-

evoked EPSPs after incubation in TTX or high potassium physiolog-

ical solution. b Graph showing changes in the paired pulse (PP)

plasticity and the plasticity over different regions of the spike train

after incubation. The inset shows part of a train of EPSPs evoked at

20 Hz in control (black line) and after incubation in TTX (grey line):

scale bar 50 ms, 1 mV. c Graph showing the relationship of the initial

EPSP amplitude to the change in the PP ratio after incubation in TTX.

The positive correlation between these effects shows that larger initial

EPSPs exhibited larger increases in the PP ratio. d Graph showing the

relationship of the initial EPSP amplitude to the change in the PP ratio

after incubation in high potassium physiological solution. In this case

there was a significant negative correlation, which suggests that larger

initial EPSPs will exhibit a reduced change in the PP plasticity

36 J Comp Physiol A (2012) 198:25–41

123


Molecular and genetic analyses of the drosophila neu-

romuscular junction have provided detailed insight into

presynaptic homeostatic regulation of synaptic efficacy

(Davis and Goodman 1998; Paradis et al. 2001; Frank et al.

2006). Ultrastructural evidence for presynaptic changes has

also been reported in some mammalian studies (e.g.

Burrone and Murthy 2003). Changes in the amplitude and

frequency of TTX-resistant mEPSCs have typically been

used to support presynaptic or postsynaptic effects (change

in frequency or amplitude of MEPSCs, respectively; Kirov

et al. 1999; Murthy et al. 2001; Burrone and Murthy 2003),

but these traditional criteria no longer provide unequivocal

support for assumed presynaptic or postsynaptic sites (e.g.

see Karunanithi et al. 2002; Franks et al. 2003; Rich and

Wenner 2007). While we have found evidence for func-

tional changes in synaptic properties, both spontaneous

TTX-resistant mEPSPs and evoked inputs using paired

recordings, we found little evidence for changes in pre-

synaptic ultrastructure (see also Cooke and Parker 2005).

Note that significant ultrastructural changes after recovery

from spinal lesions were detected using the methods fol-

lowed here (Cooke and Parker 2009).

The increase in the frequency of summed mEPSPs fol-

lowing incubation in high potassium physiological solution

was surprising, as in the classical quantal model TTX-

resistant mEPSPs are assumed to reflect the random

spontaneous presynaptic release of individual vesicles (Fatt

and Katz 1952; Gage and Hubbard 1965). Summed events

could occur as a result of the summation of single spon-

taneous events, but this should require an increase in the

frequency or duration of single mEPSPs, which did not

occur. The analysis instead suggested that summed events

reflected the correlated spontaneous release of vesicles

rather than an increased probability of two independent

events summating, which would require a significant

change in synaptic release properties after incubation.

While we do not know the basis for this effect at present, it

could reflect synaptic vesicle ‘‘drag’’ (Martin and Pilar

1964), where release of one vesicle increases the likelihood

of another vesicle being released. After high potassium

physiological solution incubation evoked monosynaptic

EPSPs sat on a relatively depolarized baseline, an effect

not seen in control or TTX-incubated cords. This is con-

sistent with a synaptic drag mechanism that increases

asynchronous release to contribute to slow synaptic depo-

larisations (Iremonger and Bains 2007).

Potential functional role of the compensatory changes

The functional effects of homeostatic plasticity are often

unclear. This arises at least in part because these effects

cannot be examined in the tissue slices or cultured cells

often used experimentally (see Wilhelm and Wenner

2008). However, even where functional effects are exam-

ined the relevance of homeostatic effects can remain

uncertain. For example, in the developing chick spinal cord

glutamate receptor blockade abolishes spontaneous activ-

ity, which then recovers over several hours. Recovery is

associated with synaptic scaling, but this effect only

occurred after spontaneous activity had recovered

(Wilhelm and Wenner 2008; Wilhelm et al. 2009). Syn-

aptic scaling of mEPSPs after TTX or CNQX injection also

occurred in zebrafish embryos (there were no changes in

glycinergic inputs, and no changes in cellular properties).

)V

m(P

MR

lortnoC

/X

QN

C

5P

A

TT

X XQ

NC

+K hgi

H

-75

-70

-65

-60

-55

*

*

lortnoC

/X

QN

C

5P

A

TT

X XQ

NC

+K h gi

H

0

10

20

30

40

*

*

*

lortnoC

/X

QN

C

5P

A

TT

X XQ

NC

+K hgi

H

0

1

2

3

4

sekips fo rebmu

N

*

*

a

b

c

Inpu

t res

ista

nce

(MΩ

)

Fig. 9 Effects of incubation for 20–24 h on adult cellular properties.

Changes in a resting membrane potential, b input resistance, c the

number of spikes evoked by a 1nA depolarising current pulse

J Comp Physiol A (2012) 198:25–41 37

123


However, this synaptic scaling had no effect on swimming

behaviour (Knogler et al. 2010). We examined functional

effects by recording ventral root activity evoked by elec-

trical stimulation of the spinal cord. Locomotor network

activity in lamprey can be evoked by glutamate receptor

agonists, but this activity varies markedly even in pieces of

spinal cord from the same animal (Parker et al. 1998;

Zhang and Grillner 2000). As this would complicate

analyses of compensatory effects, cord stimulation-evoked

responses were used as they provided a stable control

baseline from which to compare the effects of incubation.

There were significant increases in ventral root activity

after incubation in CNQX, CNQX/AP5, and high potas-

sium physiological solution, which matched the predictions

of the summed cellular and synaptic changes in supporting

unidirectional response to excitability changes.

Developmental influences

While the analysis was focused on larval animals, we also

compared effects across developmental stages. Larval

neurons exhibited the largest changes and effects were

unidirectional: manipulations that increased or decreased

excitability evoked changes consistent with an increase in

excitability, suggesting both negative and positive feed-

back responses. Negative feedback control of fixed set-

points is often considered synonymous with homeostasis

(Burrone and Murthy 2003; Rich and Wenner 2007;

Turrigiano 2007), failure to see these effects being con-

sidered ‘‘anti-homeostatic’’ (Carrasco et al. 2007). How-

ever, this offers a very limited view of homeostasis, which

in its original formulation could include positive feedback

effects and altered set points at certain levels that act to

0.0

0.5

1.0

1.5

Control TTX High K+

)V

m( edutilpma

PS

PE laitinI

High K+

Control

TTX

laitinI/ni arT

PS

PE

PS

PE

PP Train2-5 Train6-10 Train11-20

0.0

0.5

1.0

1.5

Rec 0 5 10 15 20 250.0

0.5

1.0

1.5edutilpma

PS

PE desila

m roN

Stimulus number

Control

TTX

0 1 2 3 40

1

2 Control

TTX

High K+

yticitsalpP

P

Initial EPSPamplitude (mV)

0

5

10

15

)sm( htdi

w-f laH

**

lortnoC

t celE lortno

Cmeh

C

TT

Xtcel

E

TT

Xm eh

C

K hgiH

tcelE

+

K hg iH

+meh

C

a b

c

e

d

Fig. 10 Changes in the

properties of adult

reticulospinal-evoked EPSPs

after incubation in TTX or high

potassium physiological

solution for 20–24 h. a Bargraph showing the amplitude of

low frequency-evoked EPSPs in

control and after incubation.

b Bar graph showing the half-

width of EPSPs in control and

after incubation. c Changes in

the paired pulse (PP) plasticity

and plasticity over the spike

train. d Graph showing the

activity-dependent plasticity of

a single reticulospinal

connection in control and after

incubation in TTX. e Graph

showing the lack of a

relationship between the initial

EPSP amplitude and the change

in PP plasticity in control or

after incubation

38 J Comp Physiol A (2012) 198:25–41

123


maintain overall system function (see Carpenter 2004).

Effects considered anti-homeostatic at the cellular level

(e.g. the increased excitability after incubation in high

potassium physiological solution) could thus still be

homeostatic when considered at other levels. While we do

not know why the larval animal should exhibit the positive

feedback effect in high potassium physiological solution,

larvae do have markedly different behaviours to the free-

swimming adults, as they are largely sessile and filter feed

while burrowed in the substrate (Hardisty and Potter 1971).

Larvae only swim in daylight when disturbed, and then for

relatively short periods unless there is constant disturbance

or changes in environmental conditions (e.g. low oxygen

tensions; see Hardisty and Potter 1971). While speculative,

the positive feedback in high potassium physiological

solution could reflect an adaptation to an imposed increase

in activity in a normally sessile animal that helps to

maintain activity levels and promote swimming and sur-

vival in the face of constant stimulation caused by envi-

ronmental conditions (Hardisty and Potter 1971). In

transformers changes were small or absent. This may

reflect a more malleable spinal cord that is tolerant of

change, which may be a necessary adaptation in animals

that are undergoing the marked functional changes that

allow them to develop from the sessile larval to active adult

form. In adults effects had returned, but they were now bi-

directional (i.e. positive and negative manipulation index

values when excitability was reduced or increased,

respectively). The bi-directional effect in adults is that

typically associated with homoeostatic plasticity and may

suggest a window of excitability for optimal spinal cord

function. The diverse effects of glutamate receptor block in

CNQX were also all abolished in the presence of AP5,

which suggests that in adults these changes were either

monitored by or mediated through NMDA-dependent

processes.

Different developmental stages of the lamprey have

been used as model systems for investigating various

aspects of spinal cord function and plasticity. Neuromod-

ulation of the locomotor network has typically been

examined in adult animals, while analyses of functional

recovery after spinal injury (focusing on axonal regenera-

tion) have typically been examined in larvae or trans-

formers (Rovainen 1979; Cohen et al. 1988; McClellan

1994). The developmentally dependent compensatory

effects shown here could also be triggered by neuromod-

ulation or lesions and may in turn influence the ultimate

functional changes. The effects shown here are generally in

line with the changes we have seen following spinal cord

lesions: 8–10 weeks after lesioning, when most animals

exhibit functional recovery, there are changes in cellular

and synaptic properties below the lesion site that together

suggest an increase in excitability (Cooke and Parker

2009). The relevance of these changes to recovery are

unknown, but as the spinal cord can monitor and adjust to

tonic changes in activity levels they could be a compen-

satory response to the reduction of descending excitation to

the network. Insight into the mechanisms underlying these

changes and their relevance to network activity could thus

provide insight into the ways in which the spinal cord

responds to changes in intrinsic activity levels, either as a

result of injury or as a result of neuromodulator-evoked

plasticity.

Acknowledgments This work was supported by a Royal Society

University Research fellowship awarded to DP. RMC was supported

by a UK MRC studentship.

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