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Transcript of SACNAS poster
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