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Post-stroke optogenetic stimulation has been shown to enhance neurovascular coupling and functional recovery. Neuronal nitric oxide synthase (nNOS) has been implicated as a key regulator of the neurovascular response in acute stroke; however, its role in subacute recovery remains unclear. We investigated the expression of nNOS in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examined the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. Optogenetically stimulated stroke mice demonstrated significant improvement on the horizontal rotating beam task at post-stroke days 10 and 14. nNOS mRNA and protein expression was significantly and selectively decreased in the contralesional primary motor cortex (cM1) of cLCN-stimulated mice. The nNOS expression in cM1 was negatively correlated with improved recovery. nNOS inhibitor (ARL 17477)-treated stroke mice exhibited a significant functional improvement in speed at post-stroke day 10, when compared to stroke mice receiving vehicle (saline) only. Our results show that optogenetic stimulation of cLCN and systemic nNOS inhibition both produce functional benefits after stroke, and suggest that nNOS may play a maladaptive role in post-stroke recovery.

HYPOTHESIS: A terra cotta plaque [LMU 2551] from the Neo-Babylonian period (c.629-539 BCE), housed in the museum of the Archaeology Center at Loyola Marymount University, Los Angeles, is a representation of right peripheral facial paralysis. BACKGROUND: Ancient representations of pathology are rare and often difficult to identify. This is particularly true of Assyrian-Babylonian cultures where, despite numerous surviving medical texts, artistic examples of disease are almost non-existent. METHODS: Precise caliper measurements and archaeological analysis of LMU 2551 were used to confirm the authors' hypothesis. RESULTS: The facial distortions portrayed in LMU 2551 are not accidental. Measurements show a pronounced asymmetry of the lower face where the length from the mid-philtrum to the oral commissure and from the lateral edge of the ala nasi to the mid-ipsilateral nasolabial fold are twice as long in the left than in the right side. The left eye is closed, whereas the right is widely open. CONCLUSION: The described plaque is among the oldest representations of facial paralysis on record. It correlates with contemporary Babylonian texts describing neurological disorders but its function is unknown.

Background and Purpose— Many restorative therapies have been used to study brain repair after stroke. These therapeutic-induced changes have revealed important insights on brain repair and recovery mechanisms; however, the intrinsic changes that occur in spontaneously recovery after stroke is less clear. The goal of this study is to elucidate the intrinsic changes in spontaneous recovery after stroke, by directly investigating the transcriptome of primary motor cortex in mice that naturally recovered after stroke. Methods— Male C57BL/6J mice were subjected to transient middle cerebral artery occlusion. Functional recovery was evaluated using the horizontal rotating beam test. A novel in-depth lesion mapping analysis was used to evaluate infarct size and locations. Ipsilesional and contralesional primary motor cortices (iM1 and cM1) were processed for RNA-sequencing transcriptome analysis. Results— Cluster analysis of the stroke mice behavior performance revealed 2 distinct recovery groups: a spontaneously recovered and a nonrecovered group. Both groups showed similar lesion profile, despite their differential recovery outcome. RNA-sequencing transcriptome analysis revealed distinct biological pathways in the spontaneously recovered stroke mice, in both iM1 and cM1. Correlation analysis revealed that 38 genes in the iM1 were significantly correlated with improved recovery, whereas 74 genes were correlated in the cM1. In particular, ingenuity pathway analysis highlighted the involvement of cAMP signaling in the cM1, with selective reduction of Adora2a (adenosine receptor A2A), Drd2 (dopamine receptor D2), and Pde10a (phosphodiesterase 10A) expression in recovered mice. Interestingly, the expressions of these genes in cM1 were negatively correlated with behavioral recovery. Conclusions— Our RNA-sequencing data revealed a panel of recovery-related genes in the motor cortex of spontaneously recovered stroke mice and highlighted the involvement of contralesional cortex in spontaneous recovery, particularly Adora2a, Drd2, and Pde10a-mediated cAMP signaling pathway. Developing drugs targeting these candidates after stroke may provide beneficial recovery outcome.

Background: Functional neuroimaging studies have reported increased activation in the contralesional primary motor cortex (cM1) in both experimental and clinical settings of stroke. However, the cellular resolution of cM1 activity and the role of cM1 activation in post-stroke recovery is unclear. In this study we characterized the time course of neuronal activation in cM1 during post-stroke recovery using activity-dependent markers. Furthermore, we investigated the role of cM1 activation in post-stroke recovery using the chemogenetic approach to silence the neuronal activity in cM1. Methods: Adult male C57Bl6 mice (6-7 weeks) were subjected to stereotaxic injection of AAV-CamKIIa-hM4D-mCherry into the cM1. Ischemic stroke was induced by transient MCAO. For time-course study, brains collected at post-stroke days (PD) 3, 5, 14 and 28 were processed for immunostaining using activity-dependent marker pCREB. For cM1 neuronal silencing, clozapine-N-oxide (CNO) was used to induce inhibitory effects via hM4D. Stroke mice received either CNO or saline intraperitoneally during early phase (PD5-14) or late phase (PD15-28). Functional recovery was evaluated using the rotating beam test at pre-stroke baseline and PD 4, 7, 10 and 14, 21 and 28. Results: Timecourse analysis showed that pCREB expression was predominantly observed in cM1 when compared to iM1, as early as PD5 and gradually increased at PD14. While neuronal silencing of cM1 during early phase (PD5-14) did not have significant effect on recovery, neuronal silencing of cM1 during late phase (PD 15-28) showed a trend of beneficial effect on recovery (CNO group n=5, SAL group n=4), suggesting a time-dependent role of cM1 activation during post-stroke recovery. Conclusions: Our results showed that basal cM1 neuronal activity increased after stroke and interhemispheric imbalance persisted until at least PD14. Chemogenetic neuronal silencing studies suggest that inhibiting cM1 activation during late phase of recovery may be beneficial. Ongoing studies are addressing the time dependent role of cM1 activation, and elucidating the neuronal subtypes (excitatory vs inhibitory) that are activated in cM1 after stroke.

Stroke induces network-wide changes in the brain, affecting the excitability in both nearby and remotely connected regions. Brain stimulation is a promising neurorestorative technique that has been shown to improve stroke recovery by altering neuronal activity of the target area. However, it is unclear whether the beneficial effect of stimulation is a result of neuronal or non-neuronal activation, as existing stimulation techniques nonspecifically activate/inhibit all cell types (neurons, glia, endothelial cells, oligodendrocytes) in the stimulated area. Furthermore, which brain circuit is efficacious for brain stimulation is unknown. Here we use the optogenetics approach to selectively stimulate neurons in the lateral cerebellar nucleus (LCN), a deep cerebellar nucleus that sends major excitatory output to multiple motor and sensory areas in the forebrain. Repeated LCN stimulations resulted in a robust and persistent recovery on the rotating beam test, even after cessation of stimulations for 2 weeks. Furthermore, western blot analysis demonstrated that LCN stimulations significantly increased the axonal growth protein GAP43 in the ipsilesional somatosensory cortex. Our results demonstrate that pan-neuronal stimulations of the LCN is sufficient to promote robust and persistent recovery after stroke, and thus is a promising target for brain stimulation.

Summary Synaptic connections undergo activity-dependent plasticity during development and learning, as well as homeostatic re-adjustment to ensure stability. Little is known about the relationship between these processes, particularly in vivo. We addressed this with novel quantal resolution imaging of transmission during locomotive behavior at glutamatergic synapses of the Drosophila larval neuromuscular junction. We find that two motor input types, Ib and Is, provide distinct forms of excitatory drive during crawling and differ in key transmission properties. Although both inputs vary in transmission probability, active Is synapses are more reliable. High-frequency firing "wakes up" silent Ib synapses and depresses Is synapses. Strikingly, homeostatic compensation in presynaptic strength only occurs at Ib synapses. This specialization is associated with distinct regulation of postsynaptic CaMKII. Thus, basal synaptic strength, short-term plasticity, and homeostasis are determined input-specifically, generating a functional diversity that sculpts excitatory transmission and behavioral function.

Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity.

Introduction: Spontaneous recovery after stroke occurs in both human and animals. However, the underlying molecular mechanisms driving spontaneous recovery are still unclear. This study investigates the molecular mechanisms driving spontaneous recovery after unilateral experimental stroke using RNA sequencing (RNAseq). Methods: Ischemic stroke was induced in C57BL/6J adult male mice by transient MCAO. Neurological score, vertical pole, and rotating horizontal beam test were performed at baseline and post stroke days (PD) 4, 8, and 14. All mice were sacrificed at PD15 and processed for immunohistochemistry or Hi-seq. Infarcts were visualized by T2WI at PD2 using 7T MR scanner or histology at PD15. All stroke mice included in the study have comparable cortico-striatal infarcts and exhibit similar prestroke baseline performance and PD4 deficits. These mice were further categorized into spontaneously recovered and non-recovered groups based on their rotating beam performance during recovery. Results: Out of 35 stroke mice with abovementioned similar conditions, 9 (26%) were categorized into spontaneously recovered group and 26 (74%) were categorized into non-recovered group. Hierarchical clustering analysis supported this categorization. At PD14, the spontaneously recovered group exhibited significant improvement in beam performance in distance traveled and speed (p Conclusions: Our study demonstrates that stroke mice with similar cortico-striatal infarct size can exhibit significant differences in their behavioral recovery outcome. The transcriptome profile in iM1-cM1 network at 2 weeks post-stroke highlighted several molecular pathways that were significantly different between spontaneously recovered and non-recovered mice. Validation of key molecular candidates from the RNAseq data would provide insights into the molecular mechanisms mediating spontaneous recovery after stroke.

Stroke is one of the leading contributors to morbidity, mortality, and health care costs in the United States. Although several preclinical strategies have shown promise in the laboratory, few have succeeded in the clinical setting. Optogenetics represents a promising molecular tool, which enables highly specific circuit-level neuromodulation. Here, the conceptual background and preclinical body of evidence for optogenetics are reviewed, and translational considerations in stroke recovery are discussed.