1. Department of Neurosciences, UC San Diego; Veterans Affairs Medical Center, San Diego, CA
Investigating the Functional Significance of Thalamocortical and Corticospinal Neurons in
Learning and Performance of Complex Behavioral Tasks in the
Adult Mammalian Motor System
C Hissom, JM Conner, Ph.D., MH Tuszynski, M.D./Ph.D.
Forelimb reach task exemplifies functional significance of TCT
and CST akin to motor performance
Figure 6: Eighteen rats were trained one week post injection in distal
forelimb reach task to obtain a sugar pellet by extending the forelimb
through panel opening. 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.
Results
Acknowledgments
Supported by the NIH in part by a MARC U-STAR Award (T34GM087193),
The Veterans Administration
LSAMP/CAMP
Academic Enrichment Programs.
References
Cruikshank SJ., Urabe H., Nurmikko Av., and Connors BW.(2009) Pathway-Specific Feedforwardcircuits between Thalamus
and Neocortex Revealed by Selective OpticalStimulation of Axons. Neuron 65.2: 230-45.
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.
Zhu, Hu, and Bryan L. Roth. "DREADD: a chemogenetic GPCR signaling platform." International Journal of
Neuropsychopharmacology 18.1 (2015): pyu007.
Zhan, Cheng, et al. "Acute and long-term suppression of feeding behavior by POMC neurons in the brainstem and
hypothalamus, respectively." The Journal of Neuroscience 33.8 (2013): 3624-3632.
In this study novel techniques for silencing and ablating specific brain circuitry in the
context were employed. Specifically, we examined how distinct populations of
neurons in the thalamocortical tract (TCT) and corticospinal tract (CST) contribute to
learning and performance of a skilled motor task in rodents. Prior studies attempting
to define the function of this specific system in skilled motor behavior have involved
lesion strategies that resulted in damage to non-target cell populations, thus
confounding the experimental findings. Using newly developed viral techniques,
thalamic projections specifically to primary (M1) and secondary (M2) motor cortex
and cortical projections specifically to cervical segment 4 (C4) and 8 (C8) were
selectively ablated (AC) or silenced (SC). Behavioral analysis revealed minimal
deficits in forelimb reach task learning and performance following either targeted AC
or SC of thalamocortical and corticospinal afferents. These results demonstrate that
the TCT and CST have minimal involvement in learning and performance of a well-
learned skilled motor task. In contrast, prior studies demonstrated that that non
selective damage to the motor thalamus produces drastic impairments in motor
function. We conclude that while this neuronal population may be required for
acquiring a new motor skill, it is not required for performing a previously learned
motor skill.
MethodsIntroduction
Figure 1: Primary motor cortex (M1) transmits several electrical impulses intracranially by way of the Basal ganglia
and Cerebellum that are pivotal for motor execution and sensory processing. These signals coalesce onto ventral
anterior/ ventral lateral (Va/Vl) thalamic nuclei. Basal ganglia facilitates initiation of motor commands by
disinhibition of thalamic neurons and cerebellum coordinates and governs appropriate motor performance. Motor
commands decends by way of the corticospinal tract and innervate cervical segments 4 & 8 for execution of distal
forelimb reach.
Striatum
GP
Thalamus
Va/Vl
CST to
C4/C8
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Rat 3
Rat 4
Rat 5
Rat 6
Rat 7
Rat 8
Rat 11
Rat 12
Rat 13
PercentHit
Day
Control: Rats 5-8
DT at day 19: Rats 3, 4, 11-13
Training
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Rat 1
Rat 2
Rat 9
Rat 10
Rat 5
Rat 6
Rat 7
Rat 8
PercentHit
Day
Control
DT prior to training
0%
20%
40%
60%
80%
100%
1 3 5 7 9 11 13 15 17 19 20 21 22 23
Rat 5
Rat 6
Rat 7
Rat 8
PercentHit
Day
Training
0%
20%
40%
60%
80%
100%
1 3 5 7 9 11 13 15 17 19 20 21 22 23
Rat 1
Rat 2
Rat 3
Rat 4
HitPercent
Day
Training
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 4 7 9 12 15 19 20 21 22 23
Rat 4
Rat 8
PercentHit
Day
CNO Rat 8 only
CNO Rat 4 Only
CNO Rat 4 & 8
Thalamocortical circuits and corticospinal tract impart and execute
motor comands information by way of the motor cortex
First Injection: Genetically engineered Adeno- associated virus (AAV) containing cre-recombinase transfer sequence
retrogradely infects neurons. Infected neuronal populations transcribe and express cre enzyme.
Second Injection: Cre-dependant viral vector coding for diphtheria toxin receptor (DTR) or designer receptors
exclusively activated by designer drug (DREADDs) were targeted specifically to motor cortex or thalamic nuclei.
Expression is controlled by double-floxed inverted orientation (DIO) sites loxP & lox2272.
The Adeno virus genome consists of envelope, transfer, and packaging plasmid.
Envelope plasmid is controlled by cytomeg-alovirus enhancer/ chicken β-actin promoter and encodes for FugG-C (Zhu
et al., 2015). Transfer plasmid encodes for DTR or DREADD as well as mCherry fluorescent protein, respectively. The
completed AAV- CAG- DIO+hDTR (or DREADD-Gq) vector is transferred into motor cortex or thalamic nuclei during
the same surgical session. Infected populations perform cre-recombinase and express DTR or DREADD.
Retrograde viral infection and FLEx system for selective ablation or
silencing of the thalamacortical or corticospinal tract in the rat
mammalian motor system.
TCT ablation or silencing specificity was achieved using a two part viral
injection
Figure 3&4: Fluorescent microscopy reveled
accuracy of targeted infection site. DREADD ( left-
top) . DTR (Left bottom) showed expression of
desired receptor system in Va/Vl of thalamus.
Figure 2: Stereotaxic intracranial injection of AAV-
cre and cre-dependant DREADD and DTR.
Anesthetized rats were placed in stereotaxic
apparatus for co-injections in M1/M2 and Va/Vl
thalamic nuclei.
CST ablation or silencing specificity was achieved using a two
part viral injection
Figure 5: Post histology
microscopy reveals infected
cells in motor cortex whose
descending projections to
c4/c8 were infected by AAV-
cre.
TCT silencing cohort forelimb performance reveals minimal deficits
in performance after intraperitoneal injection of
clozapine-N oxide
Rats were first co-infected with
Cre & DREADD (Gq). Post
operation training progressed
for 20 day at which point
performance accuracy was
constant among all 8 rats.
 Day 21: Rats 1-4 received IP
CNO while all other rats
received saline injections.
 Day 22: Rats 5-8 received IP
CNO while all other rats
received saline injections.
 Day 21,22 & 23: Rats 4 & 8
received offset injections.
Rats that received CNO
compared to saline (control)
show an overall insignificant loss
in performance (p=0.6).
TCT ablation cohort forelimb performance reveals minimal deficits
in performance after intraperitoneal injection of diptheria toxin
Rats were first co-infected with Cre & DTR.
Post operation training progressed for 21
day at which point performance accuracy
was constant among all 8 rats.
 Day 22: Rats 3,4,11-13 received IP
injection of diphtheria toxin (DT).
 Control rats 5-8 received saline
injections.
 Rats that received DTR compared to
saline (control) show an overall
insignificant loss in performance
(p=0.34).
Rats were first co-infected with Cre & DTR.
 Day 1: Rats 1-2, & 9-10 received DT prior to
training.
 Day 1: Rats 5-8 received saline injections.
 After 19 days performance for all rats plateaued.
 Variations in percent hit accuracy between DT and
saline groups were insignificant (p=0.5).
CST silencing cohort forelimb performance reveals minimal
deficits in performance after intraperitoneal injection of
clozapine-N oxide
Rats were first co-infected with Cre & DREADD (Gq). Post operation training progressed for 5 day
at which point performance accuracy was constant among all 5 rats.
 Day 6: Rats 1-3 received IP injection of CNO while all other rats received saline injections.
 Day 9: Rats 2 & 4 received IP injection of CNO while all other rats received saline injections.
 Day 10: Rats 1-2 & 5 received IP injection of CNO while all other tats received saline injections.
Rats that received CNO compared to saline (control) show significant loss in performance
(p=0.034).
Results, Cont.
CST ablation cohort forelimb performance reveals
minimal deficits in performance after intraperitoneal
injection of diptheria toxin
Rats were first co-infected with Cre & DTR. Post operation training
progressed for 17 day at which point performance accuracy was
constant among all 8 rats.
Cervical Segment 4 (C4)
 Day 17: Rats 3&6 received IP injection of diphtheria toxin (DT).
 Day 17: All other rat received IP injection of saline
 An insignificant variation in performance was observed (p=0.5).
Cervical Segment 8 (C8)
 Day 17: Rats 1-2,4-5,7-8 received IP injection of DT
 All other rats received IP injection of saline
 An insignificant variation in performance was observed
(p=0.3933).
Thalamocortical tract neurons dispersed within the Va/Vl nuclei recive input from a verity of brain
regions including basal ganglia and cerebellum. This tract is though to be a crucial relay station
required for learning and performance of complex motor tasks.
Results demonstrate that the TCT has minimal involvement in learning and performance of a
skilled motor task. In contrast, prior studies demonstrated that that non selective damage to the
motor thalamus produces drastic impairments in motor function.
We conclude that this neuronal population is not required for performing a previously learned
motor skill.
Corticospinal tract neurons residing in motor cortex are believed to play a crucial role in delivering
descending motor output for distal forelimb control. Ablation results suggest that this tract is not
required for performance of a learned motor task. However, silencing results demonstrate a
significant inability to preform a forelimb reach.
In an attempt to elucidate the discrepancy between all four cohorts succeeding histology will be
performed to verify viral infection location and strength.
In an attempt to back tract through the complex circuitry involved in distal forelimb reach,
succeeding basalthalamic and dentatethalimc ablations will be satisfied.
Conclusions and Future Work
0%
20%
40%
60%
80%
100%
Rat 1
Rat 2
Rat 3
Rat 4
Rat 5
Rat 6
Rat 7
Rat 8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
PercentHit
Day
Training
CNO Rats 1-4
CNO Rats 5-8
% Hit = Successful Reach
Total Reach
Intraperitoneal Injections
Of Clozapine N-Oxide (CNO)
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 10 11
Training
Rat #1
Rat #2
Rat #3
Rat #4
Rat #5
PercentHit
Day
Intraperitoneal Injections
Of Clozapine N-Oxide (CNO)
CNO Rats 1-3 CNO Rats 2 & 4 CNO Rats 1,3 & 5