In the central nervous system, developmental and pathophysiologic conditions cause a large-scale reorganization of functional connectivity of neural circuits. Here, by using a mouse model for peripheral sensory nerve injury, we present a protocol for combined electrophysiological and anatomical techniques to identify neural basis of synaptic remodeling in the mouse whisker thalamus. Our protocol provides comprehensive approaches to analyze both structural and functional components of synaptic remodeling. For complete details on the use and execution of this protocol, please refer to Ueta and Miyata, (2021).

Nociception, a somatic discriminative aspect of pain, is, like touch, represented in the primary somatosensory cortex (S1), but the separation and interaction of the two modalities within S1 remain unclear. Here, we show spatially distinct tactile and nociceptive processing in the granular barrel field (BF) and adjacent dysgranular region (Dys) in mouse S1. Simultaneous recordings of the multiunit activity across subregions revealed that Dys neurons are more responsive to noxious input, whereas BF neurons prefer tactile input. At the single neuron level, nociceptive information is represented separately from the tactile information in Dys layer 2/3. In contrast, both modalities seem to converge on individual layer 5 neurons of each region, but to a different extent. Overall, these findings show layer-specific processing of nociceptive and tactile information between Dys and BF. We further demonstrated that Dys activity, but not BF activity, is critically involved in pain-like behavior. These findings provide new insights into the role of pain processing in S1.

Whisker deafferentation in mice disrupts topographic connectivity from the brainstem to the thalamic ventral posteromedial nucleus (VPM), which represents whisker map, by recruiting "ectopic" axons carrying non-whisker information in VPM. However, mechanisms inducing this plasticity remain largely unknown. Here, we show the role of region-specific microglia in the brainstem principal trigeminal nucleus (Pr5), a whisker sensory-recipient region, in VPM whisker map plasticity. Systemic or local manipulation of microglial activity reveals that microglia in Pr5, but not in VPM, are necessary and sufficient for recruiting ectopic axons in VPM. Deafferentation causes membrane hyperexcitability of Pr5 neurons dependent on microglia. Inactivation of Pr5 neurons abolishes this somatotopic reorganization in VPM. Additionally, microglial depletion prevents deafferentation-induced ectopic mechanical hypersensitivity. Our results indicate that local microglia in the brainstem induce peripheral nerve injury-induced plasticity of map organization in the thalamus and suggest that microglia are potential therapeutic targets for peripheral nerve injury-induced mechanical hypersensitivity.

Peripheral nerve injury induces functional and structural remodeling of neural circuits along the somatosensory pathways, forming the basis for somatotopic reorganization and ectopic sensations, such as referred phantom pain. However, the mechanisms underlying that remodeling remain largely unknown. Whisker sensory nerve injury drives functional remodeling in the somatosensory thalamus: the number of afferent inputs to each thalamic neuron increases from one to many. Here, we report that extrasynaptic γ-aminobutyric acid-type A receptor (GABAAR)-mediated tonic inhibition is necessary for that remodeling. Extrasynaptic GABAAR currents were potentiated rapidly after nerve injury in advance of remodeling. Pharmacological activation of the thalamic extrasynaptic GABAARs in intact mice induced similar remodeling. Notably, conditional deletion of extrasynaptic GABAARs in the thalamus rescued both the injury-induced remodeling and the ectopic mechanical hypersensitivity. Together, our results reveal a molecular basis for injury-induced remodeling of neural circuits and may provide a new pharmacological target for referred phantom sensations after peripheral nerve injury.

Dysregulation of cortical excitability crucially involves in behavioral and cognitive deficits of neurodegenerative and neuropsychiatric diseases. Electroconvulsive shock (ECS) changes neuronal excitability and has been used in the therapy of major depressive disorder and mood disorders. However, the action and the targets of the ECS in the cortical circuits are still poorly understood. Here we show that the ECS differently changes intrinsic properties of pyramidal cells (PCs) among superficial and deep layers. In layer 2/3 PCs, the ECS induced membrane hyperpolarization and the reduction of input resistances. In layer 5 PCs, the ECS also induced membrane hyperpolarization but had little effects on input resistances. In layer 6 PCs, the ECS had no effects on both of resting membrane potentials and input resistances. In addition, the ECS reduced the firing frequency of PCs in layer 2/3 but not in both layers 5 and 6. We further examined the ECS-induced changes in the influx of Ca2+ currents, and observed an enhanced Ca2+ currents in PCs of both layers 2/3 and 5 but not of layer 6. Thus, this study suggests the layer-specific suppression of PC excitability which will underlie the mechanism of the ECS action on the cortical activity.

Primary motor cortex (M1) infarctions sometimes cause sensory impairment. Because sensory signals play a vital role in motor control, sensory impairment compromises the recovery and rehabilitation of motor disability. However, the neural mechanism of the sensory impairment is poorly understood. We show that sensory processing in mouse primary somatosensory cortex (S1) was impaired in the acute phase of M1 infarctions and recovered in a layer-specific manner in the subacute phase. This layer-dependent recovery process and the anatomical connection pattern from M1 to S1 suggested that functional connectivity from M1 to S1 plays a key role in the sensory processing impairment. A simulation study demonstrated that the loss of inhibition from M1 to S1 in the acute phase of M1 infarctions could impair sensory processing in S1, and compensation for the inhibition could recover the temporal coding. Consistently, the optogenetic activation of M1 suppressed the sustained response in S1. Taken together, we revealed how focal stroke in M1 alters the cortical network activity of sensory processing, in which inhibitory input from M1 to S1 may be involved.

The frontal cortex plays an important role in the initiation and execution of movements via widespread projections to various cortical and subcortical areas. Layer 2/3 (L2/3) pyramidal cells in the frontal cortex send axons mainly to other ipsilateral/contralateral cortical areas. Subpopulations of layer 5 (L5) pyramidal cells that selectively project to the pontine nuclei or to the contralateral cortex [commissural (COM) cells] also target diverse and sometimes overlapping ipsilateral cortical areas. However, little is known about target area-dependent participation in ipsilateral corticocortical (iCC) connections by subclasses of L2/3 and L5 projection neurons. To better understand the functional hierarchy between cortical areas, we compared iCC connectivity between the secondary motor cortex (M2) and adjacent areas, such as the orbitofrontal and primary motor cortices, and distant non-frontal areas, such as the perirhinal and posterior parietal cortices. We particularly assessed the laminar distribution of iCC cells and fibers, and identified the subtypes of pyramidal cells participating in those projections. For connections between M2 and frontal areas, L2/3 and L5 cells in both areas contributed to reciprocal projections, which can be viewed as "bottom-up" or "top-down" on the basis of their differential targeting of cortical lamina. In connections between M2 and non-frontal areas, neurons participating in bottom-up and top-down projections were segregated into the different layers: bottom-up projections arose primarily from L2/3 cells, while top-down projections were dominated by L5 COM cells. These findings suggest that selective participation in iCC connections by pyramidal cell subtypes lead to directional connectivity between M2 and other cortical areas. Based on these findings, we propose a provisional unified framework of interareal hierarchy within the frontal cortex, and discuss the interaction of local circuits with long-range interareal connections.

Polyglycolic acid (PGA) nerve conduits, an artificial biodegradable nerve regeneration-inducing tube currently used in clinical practice, are effective in regenerating peripheral nerves. Dedifferentiated fat (DFAT) cells differentiate into various cells including adipocytes, osteoblasts, chondrocytes, skeletal muscle cells, and myofibroblasts, when cultured in appropriate differentiation-inducing conditioned culture medium. This study made a hybrid artificial nerve conduit by filling a PGA conduit with DFAT cells, applied the conduit to a rat facial nerve defect model, and investigated the facial nerve regenerative ability of the conduit.

Feedback projections from the secondary motor cortex (M2) to the primary motor and sensory cortices are essential for behavior selection and sensory perception. Intratelencephalic (IT) cells in layer 5 (L5) contribute feedback projections to diverse cortical areas. Here we show that L5 IT cells participating in feedback connections to layer 1 (L1) exhibit distinct projection patterns, genetic profiles, and electrophysiological properties relative to other L5 IT cells. An analysis of the MouseLight database found that L5 IT cells preferentially targeting L1 project broadly to more cortical regions, including the perirhinal and auditory cortices, and innervate a larger volume of striatum than the other L5 IT cells. We found experimentally that in upper L5 (L5a), ER81 (ETV1) was found more often in L1-preferring IT cells, and in IT cells projecting to perirhinal/auditory regions than those projecting to primary motor or somatosensory regions. The perirhinal region-projecting L5a IT cells were synaptically connected to each other and displayed lower input resistance than contra-M2 projecting IT cells including L1-preferring and nonpreferring cells. Our findings suggest that M2-L5a IT L1-preferring cells exhibit stronger ER81 expression and broader cortical/striatal projection fields than do cells that do not preferentially target L1.

Abnormalities in GABAergic inhibitory circuits have been implicated in the aetiology of autism spectrum disorder (ASD). ASD is caused by genetic and environmental factors. Several genes have been associated with syndromic forms of ASD, including FOXG1. However, when and how dysregulation of FOXG1 can result in defects in inhibitory circuit development and ASD-like social impairments is unclear. Here, we show that increased or decreased FoxG1 expression in both excitatory and inhibitory neurons results in ASD-related circuit and social behavior deficits in our mouse models. We observe that the second postnatal week is the critical period when regulation of FoxG1 expression is required to prevent subsequent ASD-like social impairments. Transplantation of GABAergic precursor cells prior to this critical period and reduction in GABAergic tone via Gad2 mutation ameliorates and exacerbates circuit functionality and social behavioral defects, respectively. Our results provide mechanistic insight into the developmental timing of inhibitory circuit formation underlying ASD-like phenotypes in mouse models.