Neuron type-specific optogenetic stimulation for differential stroke recovery in chronic capsular infarct.

Cortical neuromodulation (CNM) is widely used to promote recovery after stroke. Despite the beneficial results of CNM, the roles played by different neuron types in the effects of current CNM techniques are unable to be differentiated. Our aim was to use selective optogenetic cortical stimulation to explore how different subpopulations of neuronal cells contribute to poststroke recovery. We transduced the sensory-parietal cortex (SPC) of rats with CamKII-ChR2 (pyramidal neurons), PV-ChR2 (parvalbumin-expressing inhibitory neurons), or hSyn-ChR2 (pan-neuronal population) before inducing photothrombotic capsular infarct lesions. We found that selective stimulation of inhibitory neurons resulted in significantly greater motor recovery than stimulation of excitatory neurons or the pan-neuronal population. Furthermore, 2-deoxy-2-[18F] fluoro-D-glucose microPET (FDG-microPET) imaging revealed a significant reduction in cortical diaschisis and activation of the corticostriatal neural circuit, which were correlated with behavioral recovery in the PV-ChR2 group. The spatial pattern of brain-derived neurotrophic factor (BDNF) expression was evident in the stimulated cortex and underlying cortico-subcortical circuit. Our results indicate that the plasticity of inhibitory neurons is crucial for functional recovery after capsular infarct. Modifying CNM parameters to potentiate the stimulation of inhibitory neurons could improve poststroke outcomes.

Tonic excitation by astrocytic GABA causes neuropathic pain by augmenting neuronal activity and glucose metabolism.

Neuropathic pain is a debilitating condition caused by the hyperexcitability of spinal dorsal horn neurons and is often characterized by allodynia. Although neuron-independent mechanisms of hyperexcitability have been investigated, the contribution of astrocyte-neuron interactions remains unclear. Here, we show evidence of reactive astrocytes and their excessive GABA release in the spinal dorsal horn, which paradoxically leads to the tonic excitation of neighboring neurons in a neuropathic pain model. Using multiple electrophysiological methods, we demonstrated that neuronal hyperexcitability is attributed to both increased astrocytic GABA synthesis via monoamine oxidase B (MAOB) and the depolarized reversal potential of GABA-mediated currents (EGABA) via the downregulation of the neuronal K+/Cl- cotransporter KCC2. Furthermore, longitudinal 2-deoxy-2-[18F]-fluoro-D-glucose microPET imaging demonstrated increased regional glucose metabolism in the ipsilateral dorsal horn, reflecting neuronal hyperexcitability. Importantly, inhibiting MAOB restored the entire astrocytic GABA-mediated cascade and abrogated the increased glucose metabolism and mechanical allodynia. Overall, astrocytic GABA-mediated tonic excitation is critical for neuronal hyperexcitability, leading to mechanical allodynia and neuropathic pain.

Clozapine-Induced Chemogenetic Neuromodulation Rescues Post-Stroke Deficits After Chronic Capsular Infarct.

Long-term disabilities induced by stroke impose a heavy burden on patients, families, caregivers, and public health systems. Extensive studies have demonstrated the therapeutic value of neuromodulation in enhancing post-stroke recovery. Among them, chemogenetic neuromodulation activated by clozapine-N-oxide (CNO) has been proposed as the potential tool of neuromodulation. However, recent evidence showed that CNO does not cross the blood - brain barrier and may in fact have low binding affinity for chemogenetic tool. Thus, clozapine (CLZ) has been suggested for use in chemogenetic neuromodulation, in place of CNO, because it readily crosses the blood-brain barrier. Previously we reported that low doses of CLZ (0.1 mg/kg) successfully induced neural responses without off-target effects. Here, we show that low-dose clozapine (0.1 mg/kg) can induce prolonged chemogenetic activation while avoiding permeability issues and minimizing off-target effects. In addition, clozapine-induced excitatory chemogenetic neuromodulation (CLZ-ChemoNM) of sensory-parietal cortex with hsyn-hM3Dq-YFP-enhanced motor recovery in a chronic capsular infarct model of stroke in rats, improving post-stroke behavioral scores to 56% of pre-infarct levels. Longitudinal 2-deoxy-2-[18F]-fluoro-D-glucose microPET (FDG-microPET) scans showed that a reduction in diaschisis volume and activation of corticostriatal circuits were both correlated with post-stroke recovery. We also found c-Fos increases in bilateral cortices and BDNF increases in the cortices and striatum after CLZ-ChemoNM, indicating an increase in neural plasticity. These findings suggest the translational feasibility of CLZ-ChemoNM for augmenting recovery in chronic stroke.

Excessive Astrocytic GABA Causes Cortical Hypometabolism and Impedes Functional Recovery after Subcortical Stroke.

Glucose hypometabolism in cortical structures after functional disconnection is frequently reported in patients with white matter diseases such as subcortical stroke. However, the molecular and cellular mechanisms have been poorly elucidated. Here we show, in an animal model of internal capsular infarct, that GABA-synthesizing reactive astrocytes in distant cortical areas cause glucose hypometabolism via tonic inhibition of neighboring neurons. We find that reversal of aberrant astrocytic GABA synthesis, by pharmacological inhibition and astrocyte-specific gene silencing of MAO-B, reverses the reduction in cortical glucose metabolism. Moreover, induction of aberrant astrocytic GABA synthesis by cortical injection of putrescine or adenovirus recapitulates cortical hypometabolism. Furthermore, MAO-B inhibition causes a remarkable recovery from post-stroke motor deficits when combined with a rehabilitation regimen. Collectively, our data indicate that cortical glucose hypometabolism in subcortical stroke is caused by aberrant astrocytic GABA and MAO-B inhibition and that attenuating cortical hypometabolism can be a therapeutic approach in subcortical stroke.

Optimizing clozapine for chemogenetic neuromodulation of somatosensory cortex.

Clozapine (CLZ) has been proposed as an agonist for Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to replace Clozapine-N-oxide (CNO); however, there are no reliable guidelines for the use of CLZ for chemogenetic neuromodulation. We titrated the optimal dose of CLZ required to evoke changes in neural activity whilst avoiding off-target effects. We also performed [18F]Fluoro-deoxy-glucose micro positron emission tomography (FDG-microPET) scans to determine the global effect of CLZ-induced hM3D(Gq) DREADD activation in the rat brain. Our results show that low doses of CLZ (0.1 and 0.01 mg/kg) successfully induced neural responses without off-target effects. CLZ at 1 mg/kg evoked a stronger and longer-lasting neural response but produced off-target effects, observed as changes in locomotor behavior and FDG-microPET imaging. Unexpectedly, FDG-microPET imaging failed to demonstrate an increase in regional glucose metabolism in the stimulated cortex during CLZ chemogenetic neuromodulation. Therefore, caution should be used when interpreting FDG-PET images in the context of cortical chemogenetic activation.

Ultrastructural Dendritic Changes Underlying Diaschisis After Capsular Infarct.

Diaschisis has been described as functional depression distant to the lesion. A variety of neuroscientific approaches have been used to investigate the mechanisms underlying diaschisis. However, few studies have examined the pathological changes in diaschisis at ultrastructural level. Here, we used a rat model of capsular infarct that consistently produces diaschisis in ipsilesional and contralesional motor and sensory cortices. To verify the occurrence of diaschisis and monitor time-dependent changes in diaschisis, we performed longitudinal 2-deoxy-2-[18F]-fluoro-d-glucose microPET (FDG-microPET) study. We also used light and electron microscopy to identify the microscopic and ultrastructural changes at the diaschisis site at 7, 14, and 21 days after capsular infarct modeling (CIM). FDG-microPET showed the occurrence of diaschisis after CIM. Light microscopic examinations revealed no significant histopathological changes at the diaschisis site except a mild degree of reactive astrogliosis. However, electron microscopy revealed swollen, hydropic degeneration of axial dendrites and axodendritic synapses, although the neuronal soma (including nuclear chromatin and cytoplasmic organelles) and myelinated axons were relatively well preserved up to 21 days after injury. Furthermore, number of axodendritic synapses was significantly decreased after CIM. These data indicate that a circumscribed subcortical white-matter lesion produces ultrastructural pathological changes related to the pathogenesis of diaschisis.

Transcortical photothrombotic pyramidotomy model with persistent motor deficits.

Traditional pyramidotomy models have a high mortality rate from breathing difficulties and show early recovery from the induced motor deficits. This study establishes a novel pyramidotomy technique in Sprague Dawley rats that generates persistent motor deficits and has a reduced mortality rate. We used viral neural tracing to identify the course and relative distribution of forelimb and hindlimb motor fibers (n = 9). On basis of the neural tracing data, the medullary pyramid was targeted dorsally from the cerebellar cortex for photothrombotic infarct lesioning (n = 18). The photothrombotic technique selectively destroyed the corticospinal fibers in the medullary pyramid with relative preservation of neighboring grey-matter tissue. MicroPET imaging using 2-deoxy-2-[18F]-fluoro-D-glucose (FDG-microPET) showed a decrease in regional cerebral glucose metabolism (rCGM) in the bilateral pyramid and ipsilateral sensory cortex (p < 0.001, FDR q < 0.05). In addition, the trapezoid bodies and superior olivary nuclei showed a decrease in rCGM, compatible with damage caused during the introduction of the optical fiber. Connected structures such as the inferior colliculi and auditory cortices also showed decreases in rCGM in both hemispheres (p < 0.001, FDR q < 0.05). There was a significant and persistent decrease in motor and sensory function in the contralateral limb following pyramidotomy, as demonstrated by performance in the single pellet reaching task and the foot-fault test. There was no operative mortality or loss of respiratory function in this study. These results indicate that photothrombotic pyramidotomy with a dorsal transcortical approach is a safe and reliable technique for generating a pyramidotomy model with persistent motor deficits.

An Experimental Infarct Targeting the Internal Capsule: Histopathological and Ultrastructural Changes.

BACKGROUND: Stroke involving the cerebral white matter (WM) has increased in prevalence, but most experimental studies have focused on ischemic injury of the gray matter. This study was performed to investigate the WM in a unique rat model of photothrombotic infarct targeting the posterior limb of internal capsule (PLIC), focusing on the identification of the most vulnerable structure in WM by ischemic injury, subsequent glial reaction to the injury, and the fundamental histopathologic feature causing different neurologic outcomes. METHODS: Light microscopy with immunohistochemical stains and electron microscopic examinations of the lesion were performed between 3 hours and 21 days post-ischemic injury. RESULTS: Initial pathological change develops in myelinated axon, concomitantly with reactive change of astrocytes. The first pathology to present is nodular loosening to separate the myelin sheath with axonal wrinkling. Subsequent pathologies include rupture of the myelin sheath with extrusion of axonal organelles, progressive necrosis, oligodendrocyte degeneration and death, and reactive gliosis. Increase of glial fibrillary acidic protein (GFAP) immunoreactivity is an early event in the ischemic lesion. WM pathologies result in motor dysfunction. Motor function recovery after the infarct was correlated to the extent of PLIC injury proper rather than the infarct volume. CONCLUSIONS: Pathologic changes indicate that the cerebral WM, independent of cortical neurons, is highly vulnerable to the effects of focal ischemia, among which myelin sheath is first damaged. Early increase of GFAP immunoreactivity indicates that astrocyte response initially begins with myelinated axonal injury, and supports the biologic role related to WM injury or plasticity. The reaction of astrocytes in the experimental model might be important for the study of pathogenesis and treatment of the WM stroke.

Remodeling of Neuronal Circuits After Reach Training in Chronic Capsular Stroke.

BACKGROUND: Subcortical capsular stroke has a poor prognosis, and it is not yet fully understood how and under what circumstances reach training contributes to motor recovery. Objective This study was performed to investigate changes in neuronal circuits and motor recovery in a chronic capsular stroke model in the presence or absence of reach training. METHOD: We generated photothrombotic capsular lesions in 42 Sprague-Dawley rats and evaluated motor recovery with or without daily training in a single-pellet reaching task (SPRT). We used 2-deoxy-2-[18F]-fluoro-D-glucose-microPET (positron emission tomography) to assess remodeling of neuronal circuits. RESULTS: SPRT training was selectively beneficial only for the group with incomplete capsular destruction (P < .05), suggesting the relevance of plasticity in the remaining capsular fibers for motor recovery. Groups that did not receive SPRT training showed no motor recovery at all. The microPET analysis demonstrated that motor recovery was correlated with a reduction in cortical diaschisis in ipsilesional motor and sensory cortices and in the contralesional sensory cortex (Pearson's correlation, P < .05). We also observed training-dependent subcortical activation in the contralesional red nucleus, the internal capsule, and the ventral hippocampus (P < .0025; false discovery rate q < 0.05). The groups without reach training did not show the same degree of reduction in diaschisis or activation of the red nucleus. CONCLUSIONS: Our results suggest that motor recovery and remodeling of neuronal circuits after capsular stroke depend on the magnitude of the capsular lesion and on the presence or absence of reach training. Task-specific training is strongly indicated only when there is incomplete destruction of the capsular fibers.

Sensory-parietal cortical stimulation improves motor recovery in severe capsular infarct.

The prevalence of subcortical white matter strokes in elderly patients is on the rise, but these patients show mixed responses to conventional rehabilitative interventions. To examine whether cortical electrical stimulation can promote motor recovery after white matter stroke, we delivered stimulation to a small or wide region of sensory-parietal cortex for two weeks in a rodent model of circumscribed subcortical capsular infarct. The sham-operated group (SOG) showed persistent and severe motor impairments together with decreased activation in bilateral sensorimotor cortices and striatum. In contrast, sensory-parietal cortex stimulation significantly improved motor recovery: final recovery levels were 72.9% of prelesion levels in the wide stimulation group (WSG) and 37% of prelesion levels in the small stimulation group (SSG). The microPET imaging showed reversal of cortical diaschisis in both groups: in both hemispheres for the WSG, and in the hemisphere ipsilateral to stimulation in the SSG. In addition, we observed activation of the corpus callosum and subcortical corticostriatal structures after stimulation. The results from the c-Fos mapping study were grossly consistent with the microPET imaging. Sensory-parietal cortex stimulation may therefore be a useful strategy for overcoming the limits of rehabilitative training in patients with severe forms of subcortical capsular infarct.