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In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

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The brainstem is a hub for sensorimotor integration, which mediates crucial innate behaviors. This brain region is characterized by a rich population of GABAergic inhibitory neurons, required for the proper expression of these innate behaviors. However, what roles these inhibitory neurons play in innate behaviors and how they function are still not fully understood. Here, we show that inhibitory neurons in the nucleus of the optic tract and dorsal-terminal nuclei (NOT-DTN) of the mouse can modulate the innate eye movement optokinetic reflex (OKR) by shaping the tuning properties of excitatory NOT-DTN neurons. Specifically, we demonstrate that although these inhibitory neurons do not directly induce OKR, they enhance the visual feature selectivity of OKR behavior, which is mediated by the activity of excitatory NOT-DTN neurons. Moreover, consistent with the sharpening role of inhibitory neurons in OKR behavior, they have broader tuning relative to excitatory neurons. Last, we demonstrate that inhibitory NOT-DTN neurons directly provide synaptic inhibition to nearby excitatory neurons and sharpen their tuning in a sustained manner, accounting for the enhanced feature selectivity of OKR behavior. In summary, our findings uncover a fundamental principle underlying the computational role of inhibitory neurons in brainstem sensorimotor circuits.

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Vasoactive intestinal polypeptide (VIP) is known to be present in a subclass of cortical interneurons. Here, using three different antibodies, we demonstrate that VIP is also present in the giant layer 5 pyramidal (Betz) neurons which are characteristic of the limb and axial representations of the marmoset primary motor cortex (cytoarchitectural area 4ab). No VIP staining was observed in smaller layer 5 pyramidal cells present in the primary motor facial representation (cytoarchitectural area 4c), or in the premotor cortex (e.g. the caudal subdivision of the dorsal premotor cortex, A6DC), indicating the selective expression of VIP in Betz cells. VIP in Betz cells was colocalized with neuronal specific marker (NeuN) and a calcium-binding protein parvalbumin (PV). PV also intensely labelled axon terminals surrounding Betz cell somata. VIP-positive interneurons were more abundant in the superficial cortical layers and constituted about 5-7% of total cortical neurons, with the highest density observed in area 4c. Our results demonstrate the expression of VIP in the largest excitatory neurons of the primate cortex, which may offer new functional insights into the role of VIP in the brain, and provide opportunities for genetic manipulation of Betz cells.

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Anxiety after surgery can be a major factor leading to postoperative cognitive dysfunction, particularly in elderly patients. The role of inhibitory neurons in the basolateral amygdala (BLA) in anxiety-like behaviors in aged mice following isoflurane anesthesia remains unclear. Therefore, the present study aimed to investigate the role of inhibitory neurons in isoflurane-treated mice. A total of 30 C57BL/6 mice (age, 13 months) were allocated into the control and isoflurane anesthesia groups (15 mice/group) and were then subjected to several neurological assessments. Behavioral testing using an elevated plus maze test showed that aged mice in the isoflurane anesthesia group displayed significant anxiety-like behavior, since they spent more time in the closed arm, exhibited more wall climbing behavior and covered more distance. In addition, whole-cell patch-clamp recording revealed that the excitability of the BLA excitatory neurons was notably increased following mice anesthesia with isoflurane, while that of inhibitory neurons was markedly reduced. Following mice treatment with diazepam, the excitability of the BLA inhibitory neurons was notably increased compared with that of the excitatory neurons, which was significantly attenuated. Overall, the results of the current study indicated that anxiety-like behavior could occur in aged mice after isoflurane anesthesia, which could be caused by a reduced excitability of the inhibitory neurons in the BLA area. This process could enhance excitatory neuronal activity in aged mice, thus ultimately promoting the onset of anxiety-like behaviors.

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The orbitofrontal cortex (ORB), a region crucial for stimulus-reward association, decision-making, and flexible behaviors, extensively connects with other brain areas. However, brain-wide inputs to projection-defined ORB neurons and the distribution of inhibitory neurons postsynaptic to neurons in specific ORB subregions remain poorly characterized. Here we mapped the inputs of five types of projection-specific ORB neurons and ORB outputs to two types of inhibitory neurons. We found that different projection-defined ORB neurons received inputs from similar cortical and thalamic regions, albeit with quantitative variations, particularly in somatomotor areas and medial groups of the dorsal thalamus. By counting parvalbumin (PV) or somatostatin (SST) interneurons innervated by neurons in specific ORB subregions, we found a higher fraction of PV neurons in sensory cortices and a higher fraction of SST neurons in subcortical regions targeted by medial ORB neurons. These results provide insights into understanding and investigating the function of specific ORB neurons.

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Detecting novelty is ethologically useful for an organism's survival. Recent experiments characterize how different types of novelty over timescales from seconds to weeks are reflected in the activity of excitatory and inhibitory neuron types. Here, we introduce a learning mechanism, familiarity-modulated synapses (FMSs), consisting of multiplicative modulations dependent on presynaptic or pre/postsynaptic neuron activity. With FMSs, network responses that encode novelty emerge under unsupervised continual learning and minimal connectivity constraints. Implementing FMSs within an experimentally constrained model of a visual cortical circuit, we demonstrate the generalizability of FMSs by simultaneously fitting absolute, contextual, and omission novelty effects. Our model also reproduces functional diversity within cell subpopulations, leading to experimentally testable predictions about connectivity and synaptic dynamics that can produce both population-level novelty responses and heterogeneous individual neuron signals. Altogether, our findings demonstrate how simple plasticity mechanisms within a cortical circuit structure can produce qualitatively distinct and complex novelty responses.

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Post-traumatic stress disorder (PTSD) is the most common psychiatric disorder after a catastrophic event; however, the efficacious treatment options remain insufficient. Increasing evidence suggests that cannabidiol (CBD) exhibits optimal therapeutic effects for treating PTSD. To elucidate the cell-type-specific transcriptomic pathology of PTSD and the mechanisms of CBD against this disease, we conducted single-nucleus RNA sequencing (snRNA-seq) in the hippocampus of PTSD-modeled mice and CBD-treated cohorts. We constructed a mouse model by adding electric foot shocks following exposure to single prolonged stress (SPS+S) and tested the freezing time, anxiety-like behavior, and cognitive behavior. CBD was administrated before every behavioral test. The PTSD-modeled mice displayed behaviors resembling those of PTSD in all behavioral tests, and CBD treatment alleviated all of these PTSD-like behaviors (n = 8/group). Three mice with representative behavioral phenotypes were selected from each group for snRNA-seq 15 days after the SPS+S. We primarily focused on the excitatory neurons (ExNs) and inhibitory neurons (InNs), which accounted for 68.4% of the total cell annotations. A total of 88 differentially upregulated genes and 305 differentially downregulated genes were found in the PTSD mice, which were found to exhibit significant alterations in pathways and biological processes associated with fear response, synaptic communication, protein synthesis, oxidative phosphorylation, and oxidative stress response. A total of 63 overlapping genes in InNs were identified as key genes for CBD in the treatment of PTSD. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the anti-PTSD effect of CBD was related to the regulation of protein synthesis, oxidative phosphorylation, oxidative stress response, and fear response. Furthermore, gene set enrichment analysis (GSEA) revealed that CBD also enhanced retrograde endocannabinoid signaling in ExNs, which was found to be suppressed in the PTSD group. Our research may provide a potential explanation for the pathogenesis of PTSD and facilitate the discovery of novel therapeutic targets for drug development. Moreover, it may shed light on the therapeutic mechanisms of CBD.

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Low-voltage fast (LVF) seizure-onset is one of the two frequently observed temporal lobe seizure-onset patterns. Depth electroencephalogram profile analysis illustrated that the peak amplitude of LVF onset was deep temporal areas, e.g., hippocampus. However, the specific dynamic transition mechanisms between normal hippocampal rhythmic activity and LVF seizure-onset remain unclear. Recently, the optogenetic approach to gain control over epileptic hyper-excitability both in vitro and in vivo has become a novel noninvasive modulation strategy. Here, we combined biophysical modeling to study LVF dynamics following changes in crucial physiological parameters, and investigated the potential optogenetic intervention mechanism for both excitatory and inhibitory control. In an Ammon's horn 3 (CA3) biophysical model with light-sensitive protein channelrhodopsin 2 (ChR2), we found that the cooperative effects of excessive extracellular potassium concentration of parvalbumin-positive (PV+) inhibitory interneurons and synaptic links could induce abundant types of discharges of the hippocampus, and lead to transitions from gamma oscillations to LVF seizure-onset. Simulations of optogenetic stimulation revealed that the LVF seizure-onset and morbid fast spiking could not be eliminated by targeting PV+ neurons, whereas the epileptic network was more sensitive to the excitatory control of principal neurons with strong optogenetic currents. We illustrate that in the epileptic hippocampal network, the trajectories of the normal and the seizure state are in close vicinity and optogenetic perturbations therefore may result in transitions. The network model system developed in this study represents a scientific instrument to disclose the underlying principles of LVF, to characterize the effects of optogenetic neuromodulation, and to guide future treatment for specific types of seizures.

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Epilepsy is a brain disorder affecting up to 1 in 26 individuals. Despite its clinical importance, the molecular mechanisms of epileptogenesis are still far from clarified. Our previous study showed that disruption of Clock in excitatory neurons alters cortical circuits and leads to generation of focal epilepsy. In this study, a GAD-Cre;Clockflox/flox mouse line with conditional Clock gene knockout in inhibitory neurons was established. We observed that seizure latency was prolonged, the severity and mortality of pilocarpine-induced seizure were significantly reduced, and memory was improved in GAD-Cre;Clockflox/flox mice. We hypothesize that mice with CLOCK knockout in inhibitory neurons have increased threshold for seizure, opposite from mice with CLOCK knockout in excitatory neurons. Further investigation showed Clock knockout in inhibitory neurons upregulated the basal protein level of ARC, a synaptic plasticity-associated immediate-early gene product, likely through the BDNF-ERK pathway. Altered basal levels of ARC may play an important role in epileptogenesis after Clock deletion in inhibitory neurons. Although sEPSCs and intrinsic properties of layer 5 pyramidal neurons in the somatosensory cortex exhibit no changes, the spine density increased in apical dendrite of pyramidal neurons in CLOCK knockout group. Our results suggest an underlying mechanism by which the circadian protein CLOCK in inhibitory neurons participates in neuronal activity and regulates the predisposition to epilepsy.

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Since more than a century, neuroscientists have distinguished excitatory (glutamatergic) neurons with long-distance projections from inhibitory (GABAergic) neurons with local projections and established layer-dependent schemes for the ~ 80% excitatory (principal) cells as well as the ~ 20% inhibitory neurons. Whereas, in the early days, mainly morphological criteria were used to define cell types, later supplemented by electrophysiological and neurochemical properties, nowadays. single-cell transcriptomics is the method of choice for cell type classification. Bringing recent insight together, we conclude that despite all established layer- and area-dependent differences, there is a set of reliably identifiable cortical cell types that were named (among others) intratelencephalic (IT), extratelencephalic (ET), and corticothalamic (CT) for the excitatory cells, which altogether comprise ~ 56 transcriptomic cell types (t-types). By the same means, inhibitory neurons were subdivided into parvalbumin (PV), somatostatin (SST), vasoactive intestinal polypeptide (VIP), and "other (i.e. Lamp5/Sncg)" subpopulations, which altogether comprise ~ 60 t-types. The coming years will show which t-types actually translate into "real" cell types that show a common set of multimodal features, including not only transcriptome but also physiology and morphology as well as connectivity and ultimately function. Only with the better knowledge of clear-cut cell types and experimental access to them, we will be able to reveal their specific functions, a task which turned out to be difficult in a part of the brain being so much specialized for cognition as the cerebral cortex.

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Cortical inhibitory interneurons (cINs) are born in the ventral forebrain and migrate into the cortex where they make connections with locally produced excitatory glutamatergic neurons. Cortical function critically depends on the number of cINs, which is also key to establishing the appropriate inhibitory/excitatory balance. The final number of cINs is determined during a postnatal period of programmed cell death (PCD) when ~40% of the young cINs are eliminated. Previous work shows that the loss of clustered gamma protocadherins (Pcdhgs), but not of genes in the Pcdha or Pcdhb clusters, dramatically increased BAX-dependent cIN PCD. Here, we show that PcdhγC4 is highly expressed in cINs of the mouse cortex and that this expression increases during PCD. The sole deletion of the PcdhγC4 isoform, but not of the other 21 isoforms in the Pcdhg gene cluster, increased cIN PCD. Viral expression of the PcdhγC4, in cIN lacking the function of the entire Pcdhg cluster, rescued most of these cells from cell death. We conclude that PcdhγC4 plays a critical role in regulating the survival of cINs during their normal period of PCD. This highlights how a single isoform of the Pcdhg cluster, which has been linked to human neurodevelopmental disorders, is essential to adjust cIN cell numbers during cortical development.

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In the neocortex, neural activity is shaped by the interaction of excitatory and inhibitory neurons, defined by the organization of their synaptic connections. Although connections among excitatory pyramidal neurons are sparse and functionally tuned, inhibitory connectivity is thought to be dense and largely unstructured. By measuring in vivo visual responses and synaptic connectivity of parvalbumin-expressing (PV+) inhibitory cells in mouse primary visual cortex, we show that the synaptic weights of their connections to nearby pyramidal neurons are specifically tuned according to the similarity of the cells' responses. Individual PV+ cells strongly inhibit those pyramidal cells that provide them with strong excitation and share their visual selectivity. This structured organization of inhibitory synaptic weights provides a circuit mechanism for tuned inhibition onto pyramidal cells despite dense connectivity, stabilizing activity within feature-specific excitatory ensembles while supporting competition between them.

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Breathing requires distinct patterns of neuronal activity in the brainstem. The most critical part of the neuronal network responsible for respiratory rhythm generation is the preBötzinger Complex (preBötC), located in the ventrolateral medulla. This area contains both rhythmogenic glutamatergic neurons and also a high number of inhibitory neurons. Here, we aimed to analyze the activity of glycinergic neurons in the preBötC in anesthetized mice. To identify inhibitory neurons, we used a transgenic mouse line that allows expression of Channelrhodopsin 2 in glycinergic neurons. Using juxtacellular recordings and optogenetic activation via a single recording electrode, we were able to identify neurons as inhibitory and define their activity pattern in relation to the breathing rhythm. We could show that the activity pattern of glycinergic respiratory neurons in the preBötC was heterogeneous. Interestingly, only a minority of the identified glycinergic neurons showed a clear phase-locked activity pattern in every respiratory cycle. Taken together, we could show that neuron identification is possible by a combination of juxtacellular recordings and optogenetic activation via a single recording electrode.

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The present review aims to provide a short update of our understanding of the inhibitory interneurons of the cerebellum. While these cells constitute but a minority of all cerebellar neurons, their functional significance is increasingly being recognized. For one, inhibitory interneurons of the cerebellar cortex are now known to constitute a clearly more diverse group than their traditional grouping as stellate, basket, and Golgi cells suggests, and this diversity is now substantiated by single-cell genetic data. The past decade or so has also provided important information about interneurons in cerebellar nuclei. Significantly, developmental studies have revealed that the specification and formation of cerebellar inhibitory interneurons fundamentally differ from, say, the cortical interneurons, and define a mode of diversification critically dependent on spatiotemporally patterned external signals. Last, but not least, in the past years, dysfunction of cerebellar inhibitory interneurons could also be linked with clinically defined deficits. I hope that this review, however fragmentary, may stimulate interest and help focus research towards understanding the cerebellum.

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Cortical interneurons shape network activity in cell type-specific ways, and are also influenced by interactions with other cell types. These specific cell-type interactions are understudied, as transgenic labeling methods typically restrict labeling to one neuron type at a time. Although recent methods have enabled post-hoc identification of cell types, these are not available to many labs. Here, we present a method to distinguish between two red fluorophores in vivo, which allowed imaging of activity in somatostatin (SOM), parvalbumin (PV), and putative pyramidal neurons (PYR) in mouse association cortex. We compared population events of elevated activity and observed that the PYR network state corresponded to the ratio between mean SOM and PV neuron activity, demonstrating the importance of simultaneous labeling to explain dynamics. These results extend previous findings in sensory cortex, as activity became sparser and less correlated when the ratio between SOM and PV activity was high.

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Electrical stimulation is an effective tool for mapping and altering brain connectivity, with applications ranging from treating pharmacology-resistant neurological disorders to providing sensory feedback for neural prostheses. Paramount to the success of these applications is the ability to manipulate electrical currents to precisely control evoked neural activity patterns. However, little is known about stimulation-evoked responses in inhibitory neurons nor how stimulation-evoked activity patterns depend on ongoing neural activity. In this study, we used 2-photon imaging and cell-type specific labeling to measure single-cell responses of excitatory and inhibitory neurons to electrical stimuli in the visual cortex of awake mice. Our data revealed strong interactions between electrical stimulation and pre-stimulus activity of single neurons in awake animals and distinct recruitment and response patterns for excitatory and inhibitory neurons. This work demonstrates the importance of cell-type-specific labeling of neurons in future studies.

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The entorhinal cortex (EC, A28) is linked through reciprocal pathways with nearby perirhinal and visual, auditory, and multimodal association cortices in the temporal lobe, in pathways associated with the flow of information for memory processing. The density and laminar organization of these pathways is not well understood in primates. We studied interconnections within the ventral temporal lobe in young adult rhesus monkeys of both sexes with the aid of neural tracers injected in temporal areas (Ts1, Ts2, TE1, area 36, temporal polar area TPro, and area 28) to determine the density and laminar distribution of projection neurons within the temporal lobe. These temporal areas can be categorized into three different cortical types based on their laminar architecture: the sensory association areas Ts1, Ts2, and TE1 have six layers (eulaminate); the perirhinal limbic areas TPro and area 36 have an incipient layer IV (dysgranular); and area 28 lacks layer IV (agranular). We found that (1) temporal areas that are similar in laminar architecture by cortical type are strongly interconnected, and (2) the laminar pattern of connections is dependent on the difference in cortical laminar structure between linked areas. Thus, agranular A28 is more strongly connected with other agranular/dysgranular areas than with eulaminate cortices. Further, A28 predominantly projected via feedback-like pathways that originated in the deep layers, and received feedforward-like projections from areas of greater laminar differentiation, which emanated from the upper layers. Our results are consistent with the Structural Model, which relates the density and laminar distribution of connections to the relationship of the laminar structure between the linked areas. These connections were viewed in the context of the inhibitory microenvironment of A28, which is the key recipient of pathways from the cortex and of the output of hippocampus. Our findings revealed a higher population of calretinin (CR)-expressing neurons in EC, with a significantly higher density in its lateral division. Medial EC had a higher density of CR neurons in the deep layers, particularly in layer Va. In contrast, parvalbumin (PV) neurons were more densely distributed in the deep layers of the lateral subdivisions of rostral EC, especially in layer Va, whereas the densities of calbindin (CB) neurons in the medial and lateral EC were comparable in all layers, except for layer IIIa, in which medial EC had a higher CB population than the lateral. The pattern of connections in the inhibitory microenvironment of EC, which sends and receives input from the hippocampus, may shed light on signal propagation in this network associated with diverse aspects of memory, and disruptions in neurologic and psychiatric diseases that affect this region.

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Debate remains around anatomic origins of specific brain cell subtypes and lineage relationships within the human forebrain. Thus, direct observation in the mature human brain is critical for a complete understanding of the structural organization and cellular origins. Here, we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific Mosaic Variant Barcode Analysis. From four hemispheres from two different human neurotypical donors, we identified 287 and 780 mosaic variants (MVs), respectively that were used to deconvolve clonal dynamics. Clonal spread and allelic fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted compared with resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome-transcriptome analysis at both a cell-type-specific and single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of MVs across 17 locations within one parietal lobe reveals restrictions of clonal spread in the anterior-posterior axis precedes that of the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus cell-type resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

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The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

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The superior colliculus (SC) is a midbrain structure that receives inputs from retinal ganglion cells (RGCs). The SC contains one of the highest densities of inhibitory neurons in the brain but whether excitatory and inhibitory SC neurons differentially integrate retinal activity in vivo is still largely unknown. We recently established a recording approach to measure the activity of RGCs simultaneously with their postsynaptic SC targets in vivo, to study how SC neurons integrate RGC activity. Here, we employ this method to investigate the functional properties that govern retinocollicular signaling in a cell type-specific manner by identifying GABAergic SC neurons using optotagging in VGAT-ChR2 mice. Our results demonstrate that both excitatory and inhibitory SC neurons receive comparably strong RGC inputs and similar wiring rules apply for RGCs innervation of both SC cell types, unlike the cell type-specific connectivity in the thalamocortical system. Moreover, retinal activity contributed more to the spiking activity of postsynaptic excitatory compared to inhibitory SC neurons. This study deepens our understanding of cell type-specific retinocollicular functional connectivity and emphasizes that the two major brain areas for visual processing, the visual cortex and the SC, differently integrate sensory afferent inputs.

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