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. Methods Introduction 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. M1/M2 Striatum GP SNr Dentate 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 R at3 R at4 R at5 R at6 R at7 R at8 R at11 R at12 R at13 P ercentH it D ay C ontrol:R ats 5-8 D T atday 19:R ats 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 R at1 R at2 R at9 R at10 R at5 R at6 R at7 R at8 PercentH it D ay C ontrol D T prior to training 0% 20% 40% 60% 80% 100% 1 3 5 7 9 11 13 15 17 19 20 21 22 23 R at5 R at6 R at7 R at8 PercentH it D ay Training 0% 20% 40% 60% 80% 100% 1 3 5 7 9 11 13 15 17 19 20 21 22 23 R at1 R at2 R at3 R at4 H itPercent D ay Training 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 4 7 9 12 15 19 20 21 22 23 R at4 R at8 PercentH it D ay CNO R at8 only C N O R at4 O nly CN O R at4 & 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% R at1 R at2 R at3 R at4 R at5 R at6 R at7 R at8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 PercentH it D ay 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 R at#1 R at#2 R at#3 R at#4 R at#5 PercentH it D ay Intraperitoneal Injections Of Clozapine N-Oxide (CNO) CNO R ats 1-3 CNO R ats 2 & 4 CNO R ats 1,3 & 5
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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 SystemC 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
AcknowledgmentsSupported by the NIH in part by a MARC U-STAR Award (T34GM087193),The Veterans AdministrationLSAMP/CAMPAcademic Enrichment Programs.
ReferencesCruikshank 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.
M1/M2
Striatum
GP
SNr
DentateThalamusVa/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 3Rat 4Rat 5Rat 6Rat 7Rat 8Rat 11Rat 12Rat 13
Per
cent
Hit
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 1Rat 2Rat 9Rat 10Rat 5Rat 6Rat 7Rat 8
Per
cent
Hit
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 5Rat 6Rat 7Rat 8P
erce
nt H
it
Day
Training
0%
20%
40%
60%
80%
100%
1 3 5 7 9 11 13 15 17 19 20 21 22 23
Rat 1Rat 2Rat 3Rat 4H
it P
erce
nt
Day
Training
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 4 7 9 12 15 19 20 21 22 23
Rat 4Rat 8
Per
cent
Hit
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 ofclozapine-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 1Rat 2Rat 3Rat 4Rat 5Rat 6Rat 7Rat 8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Per
cent
Hit
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
TrainingRat #1Rat #2Rat #3Rat #4Rat #5
Per
cent
Hit
Day
Intraperitoneal Injections Of Clozapine N-Oxide (CNO)
CNO Rats 1-3 CNO Rats 2 & 4 CNO Rats 1,3 & 5
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