The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Cortical astrocyte N-methyl-D-aspartate receptors influence whisker barrel activity and sensory discrimination in mice.

Astrocytes express ionotropic receptors, including N-methyl-D-aspartate receptors (NMDARs). However, the contribution of NMDARs to astrocyte-neuron interactions, particularly in vivo, has not been elucidated. Here we show that a knockdown approach to selectively reduce NMDARs in mouse cortical astrocytes decreases astrocyte Ca2+ transients evoked by sensory stimulation. Astrocyte NMDAR knockdown also impairs nearby neuronal circuits by elevating spontaneous neuron activity and limiting neuronal recruitment, synchronization, and adaptation during sensory stimulation. Furthermore, this compromises the optimal processing of sensory information since the sensory acuity of the mice is reduced during a whisker-dependent tactile discrimination task. Lastly, we rescue the effects of astrocyte NMDAR knockdown on neurons and improve the tactile acuity of the animal by supplying exogenous ATP. Overall, our findings show that astrocytes can respond to nearby neuronal activity via their NMDAR, and that these receptors are an important component for purinergic signaling that regulate astrocyte-neuron interactions and cortical sensory discrimination in vivo.

Sensitive Quantitative In Vivo Assay for Evaluating the Effects of Biomolecules on Hair Growth and Coloring Using Direct Microinjections into Mouse Whisker Follicles.

Many people suffer from hair loss and abnormal skin pigmentation, highlighting the need for simple assays to support drug discovery research. Current assays have various limitations, such as being in vitro only, not sensitive enough, or unquantifiable. We took advantage of the bilateral symmetry and large size of mouse whisker follicles to develop a novel in vivo assay called "whisker follicle microinjection assay". In this assay, we plucked mouse whiskers and then injected molecules directly into one side of the whisker follicles using microneedles that were a similar size to the whiskers, and we injected solvent on the other side as a control. Once the whiskers grew out again, we quantitatively measured their length and color intensity to evaluate the effects of the molecules on hair growth and coloring. Several chemicals and proteins were used to test this assay. The chemicals minoxidil and ruxolitinib, as well as the protein RSPO1, promoted hair growth. The effect of the clinical drug minoxidil could be detected at a concentration as low as 0.001%. The chemical deoxyarbutin inhibited melanin production. The protein Nbl1 was identified as a novel hair-growth inhibitor. In conclusion, we successfully established a sensitive and quantitative in vivo assay to evaluate the effects of chemicals and proteins on hair growth and coloring and identified a novel regulator by using this assay. This whisker follicle microinjection assay will be useful when investigating protein functions and when developing drugs to treat hair loss and abnormal skin pigmentation.

VIP interneurons in sensory cortex encode sensory and action signals but not direct reward signals.

Vasoactive intestinal peptide (VIP) interneurons in sensory cortex modulate sensory responses based on global exploratory behavior and arousal state, but their function during non-exploratory, goal-directed behavior is not well understood. In particular, whether VIP cells are activated by sensory cues, reward-seeking actions, or directly by reinforcement is unclear. We trained mice on a Go/NoGo whisker touch detection task that included a delay period and other features designed to separate sensory-evoked, action-related, and reward-related neural activity. Mice had to lick in response to a whisker stimulus to receive a variable-sized reward. Using two-photon calcium imaging, we measured ΔF/F responses of L2/3 VIP neurons in whisker somatosensory cortex (S1) during behavior. In both expert and novice mice, VIP cells were strongly activated by whisker stimuli and goal-directed actions (licking), but not by reinforcement. VIP cells showed somatotopic whisker tuning that was spatially organized relative to anatomical columns in S1, unlike lick-related signals which were spatially widespread. In expert mice, lick-related VIP responses were suppressed, not enhanced, when a reward was delivered, and the amount of suppression increased with reward size. This reward-related suppression was not seen in novice mice, where reward delivery was not yoked to licking. These results indicate that besides arousal and global state variables, VIP cells are activated by local sensory features and goal-directed actions, but not directly by reinforcement. Instead, our results are consistent with a role for VIP cells in encoding the expectation of reward associated with motor actions.

The analysis of Period1 gene expression in vivo and in vitro using a micro PMT system.

To detect a small amount of Period1 (Per1) expression, we developed a micro-photomultiplier tube (µPMT) system which can be used both in vivo and in vitro. Using this system, we succeeded in detecting Per1 gene expression in the skin of freely moving mice over 240 times higher compared with that of the tissue contact optical sensor (TCS) as previously reported. For in vitro studies, we succeeded in detecting elevated Per1 expression by streptozotocin (STZ) treatment in the scalp hairs at an early stage of diabetes, when glucose content in the blood was still normal. In addition, we could detect elevated Per1 expression in a single whisker hair at the time of diabetes onset. These results show that our µPMT system responds to minute changes in gene expression in freely moving mice in vivo and in mice hair follicles in vitro. Furthermore, Per1 in the hair can be used for a marker of diabetic aggravation.

Electrophysiological and anatomical characterization of synaptic remodeling in the mouse whisker thalamus.

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).

Non-canonical role for Lpar1-EGFP subplate neurons in early postnatal mouse somatosensory cortex.

Subplate neurons (SPNs) are thought to play a role in nascent sensory processing in neocortex. To better understand how heterogeneity within this population relates to emergent function, we investigated the synaptic connectivity of Lpar1-EGFP SPNs through the first postnatal week in whisker somatosensory cortex (S1BF). These SPNs comprise of two morphological subtypes: fusiform SPNs with local axons and pyramidal SPNs with axons that extend through the marginal zone. The former receive translaminar synaptic input up until the emergence of the whisker barrels, a timepoint coincident with significant cell death. In contrast, pyramidal SPNs receive local input from the subplate at early ages but then - during the later time window - acquire input from overlying cortex. Combined electrical and optogenetic activation of thalamic afferents identified that Lpar1-EGFP SPNs receive sparse thalamic innervation. These data reveal components of the postnatal network that interpret sparse thalamic input to direct the emergent columnar structure of S1BF.

Chromatin remodeler Arid1a regulates subplate neuron identity and wiring of cortical connectivity.

Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.

Transcriptional Profiling of Whisker Follicles and of the Striatum in Methamphetamine Self-Administered Rats.

Methamphetamine (MA) use disorder is a chronic neuropsychiatric disease characterized by recurrent binge episodes, intervals of abstinence, and relapses to MA use. Therefore, identification of the key genes and pathways involved is important for improving the diagnosis and treatment of this disorder. In this study, high-throughput RNA sequencing was performed to find the key genes and examine the comparability of gene expression between whisker follicles and the striatum of rats following MA self-administration. A total of 253 and 87 differentially expressed genes (DEGs) were identified in whisker follicles and the striatum, respectively. Multivariate and network analyses were performed on these DEGs to find hub genes and key pathways within the constructed network. A total of 129 and 49 genes were finally selected from the DEG sets of whisker follicles and of the striatum. Statistically significant DEGs were found to belong to the classes of genes involved in nicotine addiction, cocaine addiction, and amphetamine addiction in the striatum as well as in Parkinson's, Huntington's, and Alzheimer's diseases in whisker follicles. Of note, several genes and pathways including retrograde endocannabinoid signaling and the synaptic vesicle cycle pathway were common between the two tissues. Therefore, this study provides the first data on gene expression levels in whisker follicles and in the striatum in relation to MA reward and thereby may accelerate the research on the whisker follicle as an alternative source of biomarkers for the diagnosis of MA use disorder.

Developmental divergence of sensory stimulus representation in cortical interneurons.

Vasocative-intestinal-peptide (VIP+) and somatostatin (SST+) interneurons are involved in modulating barrel cortex activity and perception during active whisking. Here we identify a developmental transition point of structural and functional rearrangements onto these interneurons around the start of active sensation at P14. Using in vivo two-photon Ca2+ imaging, we find that before P14, both interneuron types respond stronger to a multi-whisker stimulus, whereas after P14 their responses diverge, with VIP+ cells losing their multi-whisker preference and SST+ neurons enhancing theirs. Additionally, we find that Ca2+ signaling dynamics increase in precision as the cells and network mature. Rabies virus tracings followed by tissue clearing, as well as photostimulation-coupled electrophysiology reveal that SST+ cells receive higher cross-barrel inputs compared to VIP+ neurons at both time points. In addition, whereas prior to P14 both cell types receive direct input from the sensory thalamus, after P14 VIP+ cells show reduced inputs and SST+ cells largely shift to motor-related thalamic nuclei.

In vivo imaging of neural circuit formation in the neonatal mouse barrel cortex.

Higher brain function in mammals primarily relies on complex yet sophisticated neuronal circuits in the neocortex. In early developmental stages, neocortical circuits are coarse. Mostly postnatally, the circuits are reorganized to establish mature precise connectivity, in an activity-dependent manner. These connections underlie adult brain function. The rodent somatosensory cortex (barrel cortex) contains a barrel map in layer 4 (L4) and has been considered an ideal model for the study of postnatal neuronal circuit formation since the first report of barrels in 1970. Recently, two-photon microscopy has been used for analyses of neuronal circuit formation in the mammalian brain during early postnatal development. These studies have further highlighted the mouse barrel cortex as an ideal model. In particular, the unique dendritic projection pattern of barrel cortex L4 spiny stellate neurons (barrel neurons) is key for the precise one-to-one functional relationship between whiskers and barrels and thus an important target of studies. In this article, I will review the morphological aspects of postnatal development of neocortical circuits revealed by recent two-photon in vivo imaging studies of the mouse barrel cortex and other related works. The focus of this review will be on barrel neuron dendritic refinement during neonatal development.

Increase in IGF-1 Expression in the Injured Infraorbital Nerve and Possible Implications for Orofacial Neuropathic Pain.

Insulin-like growth factor-1 (IGF-1) is upregulated in the injured peripheral nerve bundle and controls nociceptive neuronal excitability associated with peripheral nerve injury. Here, we examined the involvement of IGF-1 signaling in orofacial neuropathic pain following infraorbital nerve injury (IONI) in rats. IONI promoted macrophage accumulation in the injured ION, as well as in the ipsilateral trigeminal ganglion (TG), and induced mechanical allodynia of the whisker pad skin together with the enhancement of neuronal activities in the subnucleus caudalis of the spinal trigeminal nucleus and in the upper cervical spinal cord. The levels of IGF-1 released by infiltrating macrophages into the injured ION and the TG were significantly increased. The IONI-induced the number of transient receptor potential vanilloid (TRPV) subfamily type 4 (TRPV4) upregulation in TRPV subfamily type 2 (TRPV2)-positive small-sized, and medium-sized TG neurons were inhibited by peripheral TRPV2 antagonism. Furthermore, the IONI-induced mechanical allodynia was suppressed by TRPV4 antagonism in the whisker pad skin. These results suggest that IGF-1 released by macrophages accumulating in the injured ION binds to TRPV2, which increases TRPV4 expression in TG neurons innervating the whisker pad skin, ultimately resulting in mechanical allodynia of the whisker pad skin.

Nuclear localization of Meis1 in dermal papilla promotes hair matrix cell proliferation in the anagen phase of hair cycle.

The dermal papilla (DP) is a key mesenchymal compartment of hair follicles that orchestrates mesenchymal-epithelial interaction regulating hair growth cycles. In the present study, we demonstrate that a TALE-family transcription factor, Meis1, is selectively localized in the nucleus of the DP in the anagen phase of the hair cycle. By using an ex vivo organ culture of vibrissae follicles, conditional Meis1 loss causes retardation in hair growth, accompanied by defects in cell proliferation of hair matrix cells. This cell proliferation defect is partly rescued by the addition of culture supernatants derived from Meis1-sufficient but not -deficient DP cells. These findings indicate that nuclear Meis1 in DP activate genes involved in secretion of some unknown factors, which promote proliferation of hair matrix cells in the anagen phase of the hair cycle.

The Sensorimotor Basis of Whisker-Guided Anteroposterior Object Localization in Head-Fixed Mice.

Active tactile perception combines directed motion with sensory signals to generate mental representations of objects in space. Competing models exist for how mice use these signals to determine the precise location of objects along their face. We tested six of these models using behavioral manipulations and statistical learning in head-fixed mice. Trained mice used a whisker to locate a pole in a continuous range of locations along the anteroposterior axis. Mice discriminated locations to ≤0.5 mm (<2°) resolution. Their motor program was noisy, adaptive to touch, and directed to the rewarded range. This exploration produced several sets of sensorimotor features that could discriminate location. Integration of two features, touch count and whisking midpoint at touch, was the simplest model that explained behavior best. These results show how mice locate objects at hyperacute resolution using a learned motor strategy and minimal set of mentally accessible sensorimotor features.

Developmental milestones and behavior of infant rats: The role of sensory input from whiskers.

Developmental milestones are behavioral and physical skills which are considered as markers of neurodevelopment. In rodents, sensory input from whiskers plays a crucial role in development of brain functions. Development of whisker system in rats includes the early period of passive whisker touch (PN1-8) before the onset of coordinated whisker movements which underlie active sensing. Inasmuch as transitioning from passive to active sensing requires a strong sensorimotor integration, we assume that the effect of whisker deprivation during the period of passive touch is unfavorable for neurodevelopment, but deprivation after the onset of active sensing might elicit less harmful effect due to compensatory neuroplalstic changes. Here we examined the effect of complete whisker trimming (WT) in WAG/Rij rats during PN1-8 and PN9-16 (active sensing) on achieving developmental milestones (e.g., eyelid opening, walking, self-grooming, rearing activity, physical maturation of forelimbs), locomotor activity and body weight. Control groups underwent sham trimming during the same periods. WT during PN1-8 caused a delay in achieving all investigated milestones, but WT during PN9-16 delayed only self-grooming. Both WT/sham trimming during PN9-16 caused a delay in explorative behavior, but accelerated self-grooming. These changes are likely to link with the effect of manipulations during PN9-16 in previously unhandled pups, but not specifically with WT. In general, developmental milestones appeared to be an informative tool to access neurodevelopment in rat pups and might have a translational value for studying developmental disorders during early life.

Functional properties of mechanoreceptors in mouse whisker hair follicles determined by the pressure-clamped single-fiber recording technique.

Several types of mechanoreceptors have been identified anatomically in rodent whisker hair follicles, but their functional properties have not been fully studied. Here we developed a pressure-clamped single-fiber recording technique to record impulses on mouse whisker hair follicle afferent nerves following displacements of whisker hair follicles. On the basis of the patterns of impulses evoked by the mechanical stimulation, three functional types of mechanoreceptors were identified, including rapidly adapting (RA), slowly adapting type 1 (SA1), and slowly adapting type 2 (SA2) mechanoreceptors. Impulses of all these mechanoreceptors were almost completely abolished by 30 nM TTX, and were largely suppressed by cooling temperatures at 15°C. Tested at different displacement distances as different stimulation intensity, RA mechanoreceptors showed a limited capacity for stimulation intensity encoding, but both SA1 and SA2 mechanoreceptors displayed linear increases of impulse numbers with increased stimulation intensity. Tested with different ramp speed of displacements, RA impulses were only evoked by rapid ramp stimulation but SA1 and SA2 impulses could be evoked by both rapid and slow ramp stimulation. Tested with different stimulation frequency, all three types of mechanoreceptors well followed the stimulation at 10-100 Hz. Taken together, this study revealed some important functional properties of RA, SA1 and SA2 mechanoreceptors, which helps better understand the encoding of tactile information by different types of low-threshold mechanoreceptors.

The effect of orofacial complete Freund's adjuvant treatment on the expression of migraine-related molecules.

BACKGROUND: Migraine is a neurovascular primary headache disorder, which causes significant socioeconomic problems worldwide. The pathomechanism of disease is enigmatic, but activation of the trigeminovascular system (TS) appears to be essential during the attack. Migraine research of recent years has focused on neuropeptides, such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP1-38) as potential pathogenic factors and possible therapeutic offensives. The goal of present study was to investigate the simultaneous expression of CGRP and precursor of PACAP1-38 (preproPACAP) in the central region of the TS in a time-dependent manner following TS activation in rats. METHODS: The right whisker pad of rats was injected with 50 µl Complete Freund's Adjuvant (CFA) or saline. A mechanical allodynia test was performed with von Frey filaments before and after treatment. Transcardial perfusion of the animals was initiated 24, 48, 72 and 120 h after injection, followed by the dissection of the nucleus trigeminus caudalis (TNC). After preparation, the samples were stored at - 80 °C until further use. The relative optical density of CGRP and preproPACAP was analyzed by Western blot. One-way ANOVA and Kruskal-Wallis followed by Tukey post hoc test were used to evaluate the data. Regression analysis was applied to explore the correlation between neuropeptides expression and hyperalgesia. RESULTS: Orofacial CFA injection resulted in significant CGRP and preproPACAP release in the TNC 24, 48, 72 and 120 h after the treatment. The level of neuropeptides reached its maximum at 72 h after CFA injection, corresponding to the peak of facial allodynia. Negative, linear correlation was detected between the expression level of neuropeptides and value of mechanonociceptive threshold. CONCLUSION: This is the first study which suggests that the expression of CGRP and preproPACAP simultaneously increases in the central region of activated TS and it influences the formation of mechanical hyperalgesia. Our results contribute to a better understanding of migraine pathogenesis and thereby to the development of more effective therapeutic approaches.

Optogenetic and transcriptomic interrogation of enhanced muscle function in the paralyzed mouse whisker pad.

The functional state of denervated muscle is a critical factor in the ability to restore movement after injury- or disease-related paralysis. Here we used peripheral optogenetic stimulation and transcriptome profiling in the mouse whisker system to investigate the time course of changes in neuromuscular function following complete unilateral facial nerve transection. While most skeletal muscles rapidly lose functionality after lower motor neuron denervation, optogenetic muscle stimulation of the paralyzed whisker pad revealed sustained increases in the sensitivity, velocity, and amplitude of whisker movements, and reduced fatigability, starting 48 h after denervation. RNA-seq analysis showed distinct regulation of multiple gene families in denervated whisker pad muscles compared with the atrophy-prone soleus, including prominent changes in ion channels and contractile fibers. Together, our results define the unique functional and transcriptomic landscape of denervated facial muscles and have general implications for restoring movement after neuromuscular injury or disease. NEW & NOTEWORTHY Optogenetic activation of muscle can be used to noninvasively induce movements and probe muscle function. We used this technique in mice to investigate changes in whisker movements following facial nerve transection. We found unexpectedly enhanced functional properties of whisker pad muscle following denervation, accompanied by unique transcriptomic changes. Our findings highlight the utility of the mouse whisker pad for investigating the restoration of movement after paralysis.

Transcriptome profiling of whisker follicles in methamphetamine self-administered rats.

Methamphetamine (MA) is a highly addictive psychostimulant that disturbs the central nervous system; therefore, diagnosis of MA addiction is important in clinical and forensic toxicology. In this study, a MA self-administration rat model was used to illustrate the gene expression profiling of the rewarding effect caused by MA. RNA-sequencing was performed to examine changes in gene expression in rat whisker follicles collected before self-administration, after MA self-administration, and after withdrawal sessions. We identified six distinct groups of genes, with statistically significant expression patterns. By constructing the functional association network of these genes and performing the subsequent topological analysis, we identified 43 genes, which have the potential to regulate MA reward and addiction. The gene pathways were then analysed using the Reactome and Knowledgebase for Addiction-Related Gene database, and it was found that genes and pathways associated with Alzheimer's disease and the heparan sulfate biosynthesis were enriched in MA self-administration rats. The findings suggest that changes of the genes identified in rat whisker follicles may be useful indicators of the rewarding effect of MA. Further studies are needed to provide a comprehensive understanding of MA addiction.

Stimulation of mouse vibrissal follicle growth by recombinant human fibroblast growth factor 20.

OBJECTIVES: To explore potential effects of recombinant human fibroblast growth factor 20 (rhFGF20) in the growth of cultured mouse vibrissal follicles. RESULTS: The growth of cultured mouse vibrissal follicles was significantly induced by rhFGF20 in a dose dependent pattern in the in vitro vibrissal follicle organ culture model. However, too high concentration of rhFGF20 could inhibit the growth of vibrissal follicles. We further demonstrated that rhFGF20 stimulated the proliferation of hair matrix cells and activated Wnt/ß-catenin signaling pathway. CONCLUSIONS: The rhFGF20 might be a potential therapeutic agent to treat hair loss disorders.