project. thalamocortical

Christian Jaques HIssomSRP-MARC-Student Research

Investigating the functional significance of corticospinal and thalamocortical

neurons in learning and performance of complex behavioral tasks in the adult

mammalian motor system

C.Hissom, D.Pittman, J.Conner, M.Tuszynski. The Center for Neural Repair studies anatomical,

electrophysiological and functional plasticity

Abstract

Contrary to popular perception, our brains are not simply static systems that process and respond

to external stimuli, but are constantly adapting themselves in a dynamic fashion. One area of the

brain that has been an ideal model for studying learning and associated brain plasticity is the

primary motor cortex. In this study, our goal is to evaluate how distinct populations of neurons in

the thalamus and primary motor cortex contribute to learning and performance of skilled motor

tasks in rodents. Thalamocortical inputs to the motor cortex are postulated to relay information

from a variety of nuclei that mediate and coordinate motor performance, including the basal

ganglia and cerebellum. Corticospinal neurons are the sole source of neuronal output to the

spinal cord from the cortex and are postulated to play a direct role in skilled motor learning in

rats. Prior studies attempting to define the function of these neuronal systems in skilled motor

behavior have involved ablation strategies that resulted in damage to non-target cell populations,

thus confounding the interpretation of experimental findings. The goal of the present study is to

use a novel viral approach to selectively eliminate distinct populations of neurons implicated in

motor function and examine subsequent changes in motor performance. It is postulated that

behavioral changes will occur that restrict proper task performance and inhibit motor adaptation.

Introduction

Neuroplasticity is the antagonist of the once held dogma; that the brain, once fully developed,


remains stagnant in its ways and resilient to change. It is know now, however, that behavioral

changes and learning elicit new synaptic connections that rearrange neural pathways. This

phenomenon occurs throughout the entirety of the brain which makes it difficult to study.

However, we can isolate this phenomenon by observing changes that occur in the brain due to

motor learning.

The basal ganglia and cerebellum are two important brain regions necessary for learning and

motor performance. These brain regions do not communicate directly with primary motor cortex

but relay information to the primary motor cortex (M1) by way of the ventral lateral and ventral

anterior (Va/Vl)nuclei of thalamus (Cruikshank et al 2010). The basal ganglia has been

postulated to play a role in motivation, motor control, gain control, and motor learning. The

cerebellum corrects and plans motor commands and coordinates proper balance (Kaneko et al

2009). Output from these distinct brain centers converges onto Va/Vl and is then relayed to

primary motor cortex where thalamocortical inputs terminate monosynapticly onto corticospinal

motor neurons in Layer VB.

While prior studies ascertain the general function of these brain regions, the complexity and

significance of each individual connection that collaborates in the overall circuit of each brain

region, be it cerebellum or basal ganglia, remains incomplete.

Prior studies rely on electrolytic or cytotoxic lesions that not only damage the target but also

elicit collateral damage to surrounding areas Thus, a significant problem which stems from such

lesions—difficult as it may be to target a specific area in the brain, it is even more difficult to

target a specific connection and analyze a circuit in the brain.


The present experiment makes use of a novel method that allows for highly selective ablations of

a specific connection in the brain. A rabies pseudotyped lentivirus is used to retrogradely infect

cells providing afferent innervation of a specific brain region with a foreign receptor, the human

form of the IL2 receptor. Later, an immonotoxin selectively targeting the human IL2 receptor is

used to mediate selective ablation of one or more of the infected cell populations.

Using this technique we can hypothesize and observe the functional significance of the

thalamocortical tract akin to a complex motor behavior such as distal forelimb reach and

dexterity. In this manner, learning and performance related brain regions can be disassembled

and analyzed in order to ascertain the origins of motor learning and task performance.

Hypothesis: It is postulated that a thalamocortical ablation should result in behavioral

irregularities parallel to neurological disorders such as Parkinson’s and Huntington’s disease

ataxia, tremors, and dysdiadochokinesis, respectively. Therefore an overall decline in forelimb

reaching performance, inability to adapt to task altercations, and uncoordinated movements are

expected.

Experimental Procedures

Eighteen rats, weighing between 90-250g, were used in this experiment. Most rats received

collateral, retrograde virus (NeuRet), injections into primary motor cortex (M1) prior to training.

NeuRet infects neurons in the thalamocortical tract and transfers the gene for human IL-2α

receptor and GFP(Kato et al, 2011). Infected neurons now express a receptor whose presence is

negligible towards neuronal activity but critical for targeting specific cells for ablation. Later, the

receptor tagged cells will be the only population of cells afflicted by a secondary, immunotoxin

injection. Some rats receive only vehicle.


One week post injection, rats begin training in distal forelimb reaching task. In this task, rats are

enclosed in a plexi-glass cage and trained to obtain a sugar pellet by extending the forelimb out

of the cage through a slit in the wall (Wang and Conner, 2010). Performance is quantified by

recording successful attempts; extend forelimb and retract sugar pellet, and unsuccessful attempt;

reach but miss pellet or inability to retract pellet (Figure 1).

Training persists daily with one session per day and each individual rat has 5 minutes of training.

After ten to fourteens days of training their performance plateaus and all eighteen rats are able to

successfully retrieve the sugar pellet. It is important to note that rats with who received NeuRet

perform as well as rats that receive vehicle. This demonstrates that Neuret does not impair

neurological functions and its presence is negligible.

The trained rats are now ready to receive the neurotoxin, Saporin, which will be surgically

administered into the motor thalamic nuclei; nucleus ventralis lateralis and nucleus ventralis

anterior. The toxin will spread from the injection site and ablate only those neurons which have

been infected by the NeuRet virus and express the IL-2α receptor. Accordingly, only neurons in

the thalamocortical tract have been infected buy neuret and necrotized by saporin.

Post surgery the rats recover for up to a week prior to behavioral analysis. The rats are once

again assessed in the forelimb reaching task and their performance, pre and post surgery, is

compared and analyzed.

Methods

All procedures and animal care adhered to American Association for the Accreditation of

Laboratory Animal Care, Society for Neuroscience, and institutional guidelines for experimental

animal health, safety, and comfort.


Rabies Psuedotyped Lentivirus Production: Rabies psuedotyped lentivirus expressing the human

IL2-receptor alpha (IL2Ra) was generated according to published methods (Kobayashi et al.,

2011). NeuRet vector is a pseudotype HIV-1 Lentiviral vector with fused glycoprotein C type

(FuG-C). Envelope plasmid contains FuG-C cDNA which is controlled by cytomeg-alovirus

enhancer/ chicken β-actin promoter. Transfer plasmid contains cDNA that encodes for

Interleukin-2 receptor α-subunit (Il-2Rα) as well as green fluorescent protein (GFP). This forms

NeuRet-Il2R α -GFP Vector (Inoue et al., 2012). This vector will be transferred into M1 region

to C8 Corticospinal neurons for retrograde infection of thalamocortical channel.

Surgery: Male F344 rats were used for the study and were divided into 3 groups as follows:

Targeted CST ablation (n=6); targeted thalamocortical ablation (n=6) and controls (n=6; see

below for description). Rats weighing approximately 85 g (~ PD 35 (Harlan)), were anesthetized

with a cocktail (2 ml/kg) containing ketamine (25 mg/mL), xylazine (1.3 mg/mL), and

acepromazine (0.25 mg/mL). In rats, the C8 spinal cord segment contains lower motor neurons

that activate muscles controlling distal forelimb movements required for grasping. To

retrogradely infect corticospinal neurons projecting to the C8 cervical spinal cord, the overlying

dura between C7 and T1 was resected and a glass micropipette (tip < 40 µm) containing 1.5 µl

NeuRet virus was inserted into the dorsal horn of spinal cord (depth 0.75 mm, 0.55 mm lateral to

midline). A total volume of 1.5 µl virus was injected over 3 sites spaced ~0.3 mm apart. All rats

were injected bilaterally. To target thalamocortical cells providing afferent innervation of the

primary motor cortex, rats received a total volume of 1.0 µl NeuRet virus injected over 3 sites in

the primary motor cortex.

Immunotoxin Injections: Following acquisition of the skilled grasping task (see below) rats were

injected with an immounotoxin targeting the human form of the IL2 receptor (Advanced


Targeting Systems, San Diego, CA; IL2Ra-SAP). This toxin is comprised of an antibody

specific for the human form of the IL2Ra coupled to a ribosomal inactivating protein, saporin.

Pilot studies indicated that injection of the toxin kills >95% of IL2R expressing cells but does not

result in nonspecific cell loss around the injection site. The immunotoxin (total volume 0.7 µl)

was injected into the primary motor cortex for targeted ablation of CST neurons and into the

ventrolateral thalamus for targeted ablation of thalamocortical neurons providing afferent

innervation of motor cortex. Two experimental controls were included in the study: (i) to assess

possible nonselective effects of the NeuRet virus, some animals received injections of the

NeuRet virus but then received ACSF at the time the immunotoxin was delivered. (ii) to assess

possible nonspecific effects of the IL2Ra-SAP immunotoxin, additional animals received

injections of ACSF at the time of virus delivery and then received immunotoxin injections along

with experimental counterparts.

Skilled grasp training: Skilled forelimb reach training was carried out as previous described

(Conner et al 2003 Neuron). In brief, 5-7 days after virus injections, animals were acclimated to

the experimenter and testing chamber. The animals were handled for a total of 5 days before

initiating reaching. Animals were weighed and food restriction was initiated 2 days prior to

starting reaching. Animals were required to reach through a small opening to obtain a single

sucrose pellet located on an indented platform approximately 2mm beyond the reaching

chamber. Reach training was carried out across 10 continuous days and animals performed 40-

60 reaching trials per day. A successful trial was scored if animals successfully retrieved the

pellet and consumed it.


Assessment of Targeted Ablations: Two weeks following injection of the IL2Ra-SAP

immunotoxin animals were tested for 4 consecutive days in the skilled forelimb reaching task to

assess lesion-induced deficits.

Grip Strength: A mirror is placed behind the Grip Strength Meter (GSM) platform at an angle so

that the gripping bar is in full view. The animals are first habituated to handling, weighing and

the testing environment as follows: they are placed on the GSM platform and allow free

exploration for intervals of 2-3 minutes then gently grasped by the nape of the neck and

positioned so that the hind feet are standing on the GSM platform and the forepaws are held

outstretched facing the sensor bar. Then, slowly the whole body is moved forward allowing

either forepaw to grip the bar with all digits. Finally, while the animal is actively gripping the

sensor bar, in one fluid motion, the animal is pulled strait back away from the bar. The

movement is horizontal to the base-plate, and in line with the attachment axis of the bar. The

animal resists the pull by maintaining a grip on the bar. When the animal releases its grip, the

GSM will produce a reading of the maximum measured force of that grip. 4 trials/ forepaw/day,

1 day/ week

Montoya staircase retrieval task: Rats will be trained to reach with both front paws to retrieve

small food pellets resting on a series of platforms at increasing lower levels (more difficult to

retrieve). Motivation will be provided by prior food restriction. The number of pellets eaten,

lowest level of platform "stair" reached, and number of pellets displaced are recorded in 15

minute sessions, up to two sessions/day, up to 5 days/week.

Horizontal Ladder or Grid Walk: Rats will be trained to walk across the length of a 1 meter

horizontal ladder (wire rungs 1-3 cm spacing) or a 0.5 meter wire grid (2x2 cm squares) to reach


a food reward. Motivation may be provided by prior food restriction. The number of errors

(footfalls through rungs or grid) will be recorded. 4 trials/ day, 1 day/ week.

The CatWalk system: (Noldus, Noldus Information Technology, Inc., Leesburg, VA) is used to

record gait and motor co-ordination during continuous locomotion along a walkway. The

CatWalk apparatus consists of a plexiglass tunnel (4”x8”x36”) with a glass runway. The

CatWalk is set up in a darkened room so that internally reflected light illuminates the animal's

paws only at the points where they touch the glass floor, producing a bright paw print image. The

runway is filmed from below by a video camera equipped with a wide-angle objective while the

CatWalk software program records quantitative analysis of gait. (Adapted from Starkey) Before

any handling begins, animals are acclimated to small amounts of food rewards, such as peanuts,

sunflower seeds, and sweetened cereals, as approved by the VMO. Training begins 2-3 weeks

before the injury so the animals acclimate to the food rewards, testing environment and handlers.

During the first three days of training, the animals are taken to the testing room, and each animal

is allowed two fifteen-minute sessions alone to explore the CatWalk. The apparatus is “on” so

that the animals habituate to the internal illumination and other testing conditions. They are

encouraged to cross the entire length with food rewards near the tunnel exit. Each animal

receives training for a maximum of 30 minutes (two fifteen minute sessions) separated by fifteen

minutes of rest. The tunnel is cleaned with 70% Ethanol and rinsed with water between each

session. After animals are trained to run directly through the tunnel, baseline data is acquired.

Testing resumes two weeks following the first surgery

and continues until two weeks before sacrifice.

Results

Distal forelimb reach (Fig. 1): Rats were trained over a


10 day period and demonstrated a significant improvement in reaching success across time.

Group assignment was made based on the reaching performance averaged over the last 3 days of

acquisition testing. Two weeks after immunotoxin injection, rats were reassessed for skilled

reaching performance for 4 consecutive days (Fig. 2). Animals with targeted corticospinal

lesions or thalamocortical lesions

showed a significant decline in reaching performance in the post lesion period relative to their

pre lesion performance. In fact, thalamocortical lesioned animals were unable to execute a reach

at all over the 4 days pf post lesion testing. Corticospinal elsioned rats executed at least 40

reaches each day in the post lesion period but showed poor reaching accuracy. However,

corticospinal lesioned animals did show some improvement in reaching accuracy across the 4

days of postlesion testing. Importantly, control animals showed no decrement in reaching

accuracy from the prelesion to the postlesion periods.

Figure 2. On the left, the graph indicates a consecutive progression in forelimb reaching task performance over a 10 day training period. On the left, the graph indicates forelimb task performance post lesion over a 4 day period. Control (blue) is able to perform the task. CTS (green) is at first unable to perform the task but improves throughout the 4 day period. TC (red) shows an inability to perform and no improvement.


Grip strength: Post lesion rats were habituated to grip strength meter for 3 min prior to session.

Each session consisted of 4 trials to obtain max grip strength for each animal. Animals with both

corticospinal and thalamocortical ablations showed a decline in forelimb grip strength (Fig. 3).

Thalamocortical ablation animals had the lowest grip strength percent out of all three groups.

Grid walk: Post lesion rats were first trained on grid walk prior to sessions. A percent value is

obtained to demonstrate percent of successful steps out of total number of steps (Fig.4). Both

lesion groups exhibit a decline in their ability to successfully walk across the grid. Results

demonstrate that out of all three groups,

rats that received a thalamocortical

ablation have a greater difficulty walking

across the grid.

Figure 3. Grip strength comparison between all 3 groups demonstrates a decline in grip strength. While both lesion groups show a decline, TCT’s (green)show the most weak forelimb grip.

Figure 4. The percent of successful steps was calculated for each group post lesion. Corticospinal lesion rats (red) showed that 11% of their total steps resulted in a slip. Thalamocortical lesion rat (green) showed that 13% of their total steps resulted in a slip. 7% of all control rat steps resulted in a slip.


Cat walk: Post lesion rats were first acclimated than trained to walk across cat walk rig. A

camera records the rat’s stride and the software is able to produce a multitude of stride assays.

Animals that received thalamocortical ablations diverged from the rest of the group in both

forelimb stride contact and intensity. This group showed greater stride contact with diminished

stride intensity. Both control and corticospinal rats showed the opposite; greater stride intensity

than stride contact (Fig. 5, Fig 6).

Discussion

A novel method was used to selectively ablate neurons in the thalamocortical tract that allegedly

mediate and coordinate motor commands and thus influence the success and overall performance

akin to distal forelimb reaching task. Contrary to expected results, a thalamocortical ablation is

not a cause of decline in forelimb reaching performance but rather a complete inability to initiate

the learned task. Thus, the thalamocortical tract is empirically necessary for the commencement

of a forelimb reach.

Figure 5. Overall evaluation of both left and right forelimb stride contact. Both control (blue) and CST (red) show a contact of about 40. Max stride contact win TCT (green) is significantly higher (p=0.0109).

Figure 6. Overall evaluation of both left and right forelimb stride intensity. Both control (blue) and CST(red) show a contact intensity value of 200. TCT (green) animals diverge and demonstrate a significant decrease in overall stride intensity(p=.026).


Basal ganglia: Cortico-subcortical loop

The Cortico-subcortical loop, also known as the basal ganglia- thalamocortical circuit, terminates

on VA/VL motor thalamus and is completed by way of the thalamocortical tract. This circuit is

of considerable interest due to its inhibitory influence over thalamocortical input to primary

motor cortex (M1). In reverse, motor thalamus receives inhibitory signals from globus pallidus

internal, which is either excited or inhibited by the striatum. The striatum in turn receives input

from higher cortical areas as well as dopiminergic signals from the substantian nigra pars

compacta. These signals mediate two pathways of the striatum; those being the indirect and

direct pathway. Overall, the indirect pathway increases inhibition of motor commands and the

direct pathway initiates dis-inhibition of motor commands. Accordingly it is hypothesized that

proper motor control is orchestrated by inhibitory and dis-inhibitory sub-cortical commands. It

may be the case; however, that dis-inhibition of the thalamocortical tract is a prerequisite to

complex motor behavior. It would follow then that excitatory signals from motor thalamic

nuclei, which must be silent in the absence of the thalamocortical tract, either initiate or approves

of higher order motor command.

Cerebellum: Corticopontine Cerebelar loop

VA/VL nuclei of thalamus also receive afferents from deep cerebellar nuclei. The cerebellum

takes part in balance and orientation, online updating, comparing and correcting of ongoing

motor commands, and planning of impending motor commands. The performance of a complex

motor sequence consists of an orchestrated succession of motor commands.

The initial motor command of a motor sequence begins in higher brain areas and descends via

the Corticospinal tract to its destination. A simultaneous message is sent to the pontine grey

nucleus which is relayed to the intermediate lobe of cerebellum for corrections. Necessary


corrections are sent to the motor cortex by way of the thalamocortical tract in order to adjust

future motor command. The motor cortex implements these corrections by integrating them into

the planning of the subsequent motor commands that comprise the motor sequence.

The planning information of the second motor command is then sent from the motor cortex to the

lateral lobe of the cerebellum by way of the pontine grey nucleus. It is believed that the initial

learning of a motor command elicits synaptic rearrangement in the lateral lobes and thus aspect

of the memory trace reside in the lateral lobes. This region is the necessary for the retrieval and

storage of long term motor memories. The lateral lobe afferents to Va/Vl thalamus by way of the

dentate nucleus and thus takes part in the approval of the planned secondary motor command of

the motor sequence.

Overall, cerebellar input to M1 through Va/Vl helps correct and plan motor commands in order

to achieve a successful motor sequence. A thalamocortical ablation may result in an inability to

adapt to environmental perturbation by making necessary motor corrections, and it may also

result in an inability to complete a motor sequence. Presumably, the lack of input from the

cerebellum could be the cause of the observed behavioral irregularities which indicate an

inability to perform a forelimb reach.

Conclusion and prospective experiments

This experiment has produced valuable indications about the functional significance of the

thalamocortical tract. These indications will help shape future studies that can help explain the

vast array of behavioral irregularities. By using the viral vector ablation method, we can continue

to back track through these learning and performance circuits in order to isolate they key regions

of plasticity and preservation of motor memory.


Dentatothalamic tract ablation

As mentioned earlier, the lateral lobe assists in planning and sends this information through the

dentate nucleus to thalamus. The rat’s inability to perform a forelimb reaching task may be

further assessed by selectively targeting and ablating cells in this connection.

Corticostriatal ablation

The absence of this connection could also help explain the rat’s inability to perform the desired

task. Furthermore, the results of this ablation can be compared with the thalamocortical ablation

results in order to decipher the origin of the observed behavioral irregularities.

REFERENCES

Cruikshank SJ., Urabe H., Nurmikko Av., and Connors BW.(2009) Pathway-Specific

Feedforwardcircuits between Thalamus and Neocortex Revealed by Selective Optical

Stimulation of Axons. Neuron 65.2: 230-45.

Doyon J., Penhune V., Ungerleider LG. (2003) Distinct contribution of the cortico-striatal and

cortico cerebellar system to motor skilled learning. Neuropsychologia 41: 252-262.

Deniau JM., Kita H., Kitai ST. ( 1992) Patterns of termination of cerebellar and basal ganglia

efferents in the rat thalamus. Strictly segregated and partly overlapping projections.

Neuroscience Laboratoire 44: 202- 206.

Hoover JE., Strick PL. (1999) The organization of cerebellar and basal ganglia outputs to

primary motor cortext as revealed by retrograde transneuronal transport of herpes

simplex virus type 1. J Neuroscience 19.4: 1446-1463.

Inoue KI., Koketsu D., Kato S., Kobayashi K., Nambu A., Takada M. (2012) Immunotoxin


mediated tract targeting in the primate brain: selective elimination of the cortico

subthalamic ‘‘hyperdirect’’ pathway. PLoS ONE 7.6: e39149.

Kato S., Kuramochi M., Kobayashi K., Fukabori R., Okada K., Uchigashima M., Watanabe M.,

Tsutsui Y.,Kobayashi K. (2011) Selective neural pathway targeting reveals key roles of

thalamostriatal projection in the control of visual discrimination methods. J Neuroscience

31.47: 17169-17179.

Turner RS., Desmurget M. (2010) Basal ganglia contributions to motor control: a vigorous tutor.

Current opinion in Neurobiology 20:704-716.

Tlamsa, Aileen P., and Joshua C. Brumberg (2010) Organization and Morphology of

Thalamocortical Neurons of Mouse Ventral Lateral Thalamus. Somatosensory & Motor

Research 27.1: 34-43.

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