Department of Neurosciences, UC San Diego; Veterans Affairs Medical Center, San Diego, CA

An Anatomical and Functional Analysis of Corticostriatal Projections and Corticospinal Collaterals

C Hissom, JM Conner, Ph.D., MH Tuszynski, M.D./Ph.D.

Forelimb reach task was used to evaluates motor control.

Figure 6: Rats are trained to reach through Plexiglas opening andobtain sugar pellet.

Acquisition group: Rats 1-4 received DTR prior to training.

Performance group: Rats 5-7 received intraperitoneal CNO injections after d17 of forelimb training.

%𝐻𝑖𝑡 =𝑆𝑢𝑐𝑐𝑒𝑠𝑓𝑢𝑙𝑙 𝑅𝑒𝑎𝑐ℎ𝑒𝑠

𝑇𝑜𝑡𝑎𝑙 𝑅𝑒𝑎𝑐ℎ𝑒𝑠

Results, Anatomical

Acknowledgments

Dr. James Conner and Dr. Tuszynski Lab This research was funded by Stars program MARC program The Veterans Administration

LSAMP/CAMP

Academic Enrichment Programs (AEP)

ReferencesAlstermark, Bror, and Tadashi Isa. "Circuits for skilled reaching and grasping."Annual review of neuroscience 35 (2012): 559-578

Bury SD, Jones TA (2002) Unilateral sensorimotor cortex lesions in adult rats facilitate motor skill learning with the “unaffected” forelimb and training-induced

dendritic structural plasticity in the motor cortex. J Neurosci 22:8597–8606.

Kress, Geraldine J., et al. "Convergent cortical innervation of striatal projection neurons." Nature neuroscience 16.6 (2013): 665-667.

Kiritani, Taro, et al. "Hierarchical connectivity and connection-specific dynamics in the corticospinal–corticostriatal microcircuit in mouse motor cortex." The Journal

of Neuroscience 32.14 (2012): 4992-5001.

Karpova, Alla Y., et al. "Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons." Neuron 48.5 (2005): 727-735

Shepherd, Gordon MG. "Corticostriatal connectivity and its role in disease." Nature Reviews Neuroscience 14.4 (2013): 278-291.

Wall, Nicholas R., et al. "Differential innervation of direct-and indirect-pathway striatal projection neurons." Neuron 79.2 (2013): 347-360.

Zhu, Hu, and Bryan L. Roth. "DREADD: a chemogenetic GPCR signaling platform." International Journal of Neuropsychopharmacology 18.1 (2015): pyu007.

Wang, Ling, et al. "Structural plasticity within highly specific neuronal populations identifies a unique parcellation of motor learning in the adult brain. "Proceedings of

the National Academy of Sciences 108.6 (2011): 2545-2550.

Methods, Anatomical Introduction

Anatomical and Behavioral studies were achieved by first

injecting an Adeno-Assosiated Virus (AAV) expressing Cre

recombinase

Figure 2b: Cre recombinase activates CREdependent genes

B: Gene expression is controlled bydouble-floxed inverted orientation (DIO)sites loxP & lox2272.

C: In the presence of Cre recombinase, thedesired inverted gene is excised andinserted back into the host genome in thecorrect orientation.

Viral intersect strategy was used to selectively map axonal

projections and specify the location of postsynaptic cells

Behavior Cont.

Conclusions and Future Work

Methods, Behavior Results, Behavior

Corticostriatal projections (CStr) and corticospinal collaterals (CStc) are integral

to motor control. It’s widely-projected that a greater understanding of these

elusive neural formations may shed light on key components of developing

treatments for neuropsychiatric and movement disorders. Its been postulated that

hyperactivity of corticospinal neurons seen in amyotrophic lateral sclerosis

(ALS) might arise from or cause damage to striatal neurons (Shepherd, 2013).

Viral tracing strategies were used to elucidate the interaction between CStr

and CStc at the anatomical level and behavioral assays were used to analyze

the functional significance of CStr.

We hypothesized that, within the striatum, CStr and CStc innervate distinct

postsynaptic targets, wherein one’s removal will induce deficits in acquisition

and performance.

Figure 1: Viral tracing strategy was used to target corticostriatal projections in the rat brain.Immunohistochemistry was used to amplify native florescence of tracer protein which

revealed bilateral corticostriatal projections from unilateral injection sites.

A

B C

Firgure 2a: Genetically engineered Adeno-associated virus (AAV) containing Cre-recombinase transfer sequence retrogradelyinfects neurons (A).Infected neuronal populations transcribe andexpress cre enzyme.

Figure 4a: Viral intersect strategy was used to first infect C4/C8 projecting neurons with Cre-recombinase and later express Cre –Dependent tracer or HSV.

Tracer (red): Cre-dependent tdtomato stains axonal fibers

HSV (blue): Cre-dependent Herpes Simplex Virus/ Tdtomato stains post-synaptic cells

Figure 3a: Viral intersect strategy was used to first infect striatal projecting neurons with Cre-recombinaseand later express Cre dependent tracer or HSV in CStr projecting neurons.

Viral intersect strategy was used to specifically ablate or silence

striatal connections

Figure 5: Viral intersect strategy was used to first infect CStr projecting neurons with Cre-recombinase and laterspecifically express Cre-dependent DTR or DREADD.

Diphtheria Toxin Receptor (DTR): Diphtheria toxin was administered prior to behavioral training to ablate CStr neurons and evaluate the functional significance of this neuronal tract for learning.

Designer Receptor Exclusively Activated by Designer Drug (DREADD): temporarily silenced CStrconnections in the precedence of CNO (Clozapine N-Oxide).

HSV damage

Anatomical analysis of corticospinal tracts reviled striatal collateral

Corticostriatal axonal projections synapse onto multiple locations

Tracer (red): Cre-dependent tdtomato stains axonal fibersHSV (blue): Cre-dependent Herpes Simplex Virus/ Tdtomato

stains post-synaptic cells Corticostriatal projection tracing

Corticospinal

projection tracing

Figure 3b: Post perfusion tissue was processed using immunohistochemistry to amplify native florescence for both CStr fiber traced and HSV experimental groups. (A)Fiber trace: Results demonstrate CStr fibers traverse motor related nuclei indicating potential synaptic

connections. (B)HSV: Results expose CStr postsynaptic partners and revealed an extensive circuit.

Figure 4b: Tissue processing forboth CStc fiber traced and HSVexperimental groups.

(A & B) Fiber trace: Descending CStc fibers pass through striatal nuclei

(C) HSV: CStc fibers communicate

with striatal nuclei

Results showed no decline no significant decline in acquisition or

performance of forelimb reach task

Histology revealed significant DREADD expression in

corticostriatal tract neurons

A

B C

D

E

A: Overall progression of forelimb reach success rate (%Hit) for rats 1-7 from day 1-20

B: Close up of Acquisition group (rats 1-4) shows no significant change in learning capacity when

compared to control (rat #3- control).

C: Close up of Performance group (rats 5-7) shows on-off delivery (red) of DREADD from d17-20

D: Shows performance group %Hit average prior to CNO(dark blue), with CNO (blue), and with vehicle

(light blue)

E: Wilcoxon matched pairs test revealed significant decline in performance when rats received vehicle

(saline).

Figure:7: Behavioral results for forelimb reach task for both Acquisition and Performance groups

Figure 8: Rats 5-7 were perfused and the tissue was processed using

immunohistochemistry to amplify DREADD native florescence. Confocal imaging shows

strong viral construct expression in targeted CStr nuclei.

Conclusion: CSts: Axonal fiber and HSV trace shows that axons branch to and synapse onto the striatum CStr: Axonal fiber and HSV trace shows that axons branch to and synapse onto the striatum

and multiple other locations throughout the brainFuture anatomical work: Identify if both tracts synapse onto to the same cells within the striatum by staining sections

for dopamine D1 and D2 cells Generate 3d model

Conclusion: Acquisition group: CStr tract ablation shows that this tract does not play a critical part in

motor learning. Performance group: CStr tract silencing shows that this tract does not play a critical part in

motor performance.Future work: Test performance using DT instead of DREADD Use different behavioral assays Quantify CStr cell population

Behavior analysis

Anatomical analysis

Cre-dependent viral intersect strategy was used to

selectively express desired receptor