Project. Thalamocortical

ChristianJaquesHIssom
SRP-MARC-StudentResearch
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 1. Rats were trained in forlimb reaching
exercise. Trained rat performed successfulforelimb
reach through plexi-glass opening by obtaining and
retrieving sugarpellet.
Figure 2. On the left, the graph
indicates a consecutive progression in
forelimb reaching taskperformance
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 taskbut
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
showa 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)
showa 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
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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
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