Axo-axonic synaptic input drives homeostatic plasticity by tuning the axon initial segment structurally and functionally.

Homeostatic plasticity maintains the stability of functional brain networks. The axon initial segment (AIS), where action potentials start, undergoes dynamic adjustment to exert powerful control over neuronal firing properties in response to network activity changes. However, it is poorly understood whether this plasticity involves direct synaptic input to the AIS. Here we show that changes of GABAergic synaptic input from chandelier cells (ChCs) drive homeostatic tuning of the AIS of principal neurons (PNs) in the prelimbic (PL) region, while those from parvalbumin-positive basket cells do not. This tuning is evident in AIS morphology, voltage-gated sodium channel expression, and PN excitability. Moreover, the impact of this homeostatic plasticity can be reflected in animal behavior. Social behavior, inversely linked to PL PN activity, shows time-dependent alterations tightly coupled to changes in AIS plasticity and PN excitability. Thus, AIS-originated homeostatic plasticity in PNs may counteract deficits elicited by imbalanced ChC presynaptic input at cellular and behavioral levels. Teaser: Axon initial segment dynamically responds to changes in local input from chandelier cells to prevent abnormal neuronal functions.

Specific and comprehensive genetic targeting reveals brain-wide distribution and synaptic input patterns of GABAergic axo-axonic interneurons.

Axo-axonic cells (AACs), also called chandelier cells (ChCs) in the cerebral cortex, are the most distinctive type of GABAergic interneurons described in the neocortex, hippocampus, and basolateral amygdala (BLA). AACs selectively innervate glutamatergic projection neurons (PNs) at their axon initial segment (AIS), thus may exert decisive control over PN spiking and regulate PN functional ensembles. However, the brain-wide distribution, synaptic connectivity, and circuit function of AACs remains poorly understood, largely due to the lack of specific and reliable experimental tools. Here, we have established an intersectional genetic strategy that achieves specific and comprehensive targeting of AACs throughout the mouse brain based on their lineage (Nkx2.1) and molecular (Unc5b, Pthlh) markers. We discovered that AACs are deployed across essentially all the pallium-derived brain structures, including not only the dorsal pallium-derived neocortex and medial pallium-derived hippocampal formation, but also the lateral pallium-derived claustrum-insular complex, and the ventral pallium-derived extended amygdaloid complex and olfactory centers. AACs are also abundant in anterior olfactory nucleus, taenia tecta and lateral septum. AACs show characteristic variations in density across neocortical areas and layers and across subregions of the hippocampal formation. Neocortical AACs comprise multiple laminar subtypes with distinct dendritic and axonal arborization patterns. Retrograde monosynaptic tracing from AACs across neocortical, hippocampal and BLA regions reveal shared as well as distinct patterns of synaptic input. Specific and comprehensive targeting of AACs facilitates the study of their developmental genetic program and circuit function across brain structures, providing a ground truth platform for understanding the conservation and variation of a bona fide cell type across brain regions and species.

Cortical network and projection neuron types that articulate serial order in a skilled motor behavior.

Skilled motor behaviors require orderly coordination of multiple constituent movements with sensory cues towards achieving a goal, but the underlying brain circuit mechanisms remain unclear. Here we show that target-guided reach-grasp-to-drink (RGD) in mice involves the ordering and coordination of a set of forelimb and oral actions. Cortex-wide activity imaging of multiple glutamatergic projection neuron (PN) types uncovered a network, involving the secondary motor cortex (MOs), forelimb primary motor and somatosensory cortex, that tracked RGD movements. Photo-inhibition highlighted MOs in coordinating RGD movements. Within the MOs, population neural trajectories tracked RGD progression and single neuron activities integrated across constituent movements. Notably, MOs intratelencephalic, pyramidal tract, and corticothalamic PN activities correlated with action coordination, showed distinct neural dynamics trajectories, and differentially contributed to movement coordination. Our results delineate a cortical network and key areas, PN types, and neural dynamics therein that articulate the serial order and coordination of a skilled behavior.

Direct and indirect neurogenesis generate a mosaic of distinct glutamatergic projection neuron types in cerebral cortex.

Variations in size and complexity of the cerebral cortex result from differences in neuron number and composition, rooted in evolutionary changes in direct and indirect neurogenesis (dNG and iNG) that are mediated by radial glia and intermediate progenitors (IPs), respectively. How dNG and iNG differentially contribute to neuronal number, diversity, and connectivity are unknown. Establishing a genetic fate-mapping method to differentially visualize dNG and iNG in mice, we found that while both dNG and iNG contribute to all cortical structures, iNG contributes the largest relative proportions to the hippocampus and neocortex. Within the neocortex, whereas dNG generates all major glutamatergic projection neuron (PN) classes, iNG differentially amplifies and diversifies PNs within each class; the two pathways generate distinct PN types and assemble fine mosaics of lineage-based cortical subnetworks. Our results establish a ground-level lineage framework for understanding cortical development and evolution by linking foundational progenitor types and neurogenic pathways to PN types.

The progenitor basis of cortical projection neuron diversity.

Diverse glutamatergic projection neurons (PNs) mediate myriad processing streams and output channels of the cerebral cortex. Yet, how different types of neural progenitors, such as radial glia (RGs) and intermediate progenitors (IPs), produce PN diversity, and hierarchical organization remains unclear. A fundamental issue is whether RGs constitute a homogeneous, multipotent lineage capable of generating all major PN types through a temporally regulated developmental program, or whether RGs comprise multiple transcriptionally heterogenous pools, each fated to generate a subset of PNs. Beyond RGs, the role of IPs in PN diversification remains underexplored. Addressing these questions requires tracking PN developmental trajectories with cell-type resolution - from transcription factor-defined RGs and IPs to their PN progeny, which are defined not only by laminar location but also by projection patterns and gene expression. Advances in cell-type resolution genetic fate mapping, axon tracing, and spatial transcriptomics may provide the technical capability for answering these fundamental questions.

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models.

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

Long-range connectome of pyramidal neurons in the sensorimotor cortex.

The neocortex mediates information processing through highly organized circuitry that contains various neuron types. Distinct populations of projection neurons in different cortical regions and layers make specific connections and participate in distinct physiological functions. Herein, with the fluorescence micro-optical sectioning tomography (fMOST) and transgenetic mice that targeted intratelencephalic (IT) and pyramidal tract (PT) neurons at specific layers, we dissected the long-range connectome of pyramidal neurons in six subregions of the sensorimtor cortex. The distribution of the input neurons indicated that IT and PT neurons in the same region received information from similar regions, while the neurons in different subregions received from the preferred neuron populations. Both the input and projection areas of these six subregions showed the transverse and longitudinal correspondence in the cortico-cortical, cortico-thalamic, and cortico-striatal circuits, which indicated that the connections were topologically organized. This study provides a comprehensive resource on the anatomical connections of cortical circuits.

Cortical glutamatergic projection neuron types contribute to distinct functional subnetworks.

The cellular basis of cerebral cortex functional architecture remains not well understood. A major challenge is to monitor and decipher neural network dynamics across broad cortical areas yet with projection-neuron-type resolution in real time during behavior. Combining genetic targeting and wide-field imaging, we monitored activity dynamics of subcortical-projecting (PTFezf2) and intratelencephalic-projecting (ITPlxnD1) types across dorsal cortex of mice during different brain states and behaviors. ITPlxnD1 and PTFezf2 neurons showed distinct activation patterns during wakeful resting, during spontaneous movements and upon sensory stimulation. Distinct ITPlxnD1 and PTFezf2 subnetworks were dynamically tuned to different sensorimotor components of a naturalistic feeding behavior, and optogenetic inhibition of ITsPlxnD1 and PTsFezf2 in subnetwork nodes disrupted distinct components of this behavior. Lastly, ITPlxnD1 and PTFezf2 projection patterns are consistent with their subnetwork activation patterns. Our results show that, in addition to the concept of columnar organization, dynamic areal and projection-neuron-type specific subnetworks are a key feature of cortical functional architecture linking microcircuit components with global brain networks.

Programmable RNA sensing for cell monitoring and manipulation.

RNA is a central and universal mediator of genetic information underlying the diversity of cell types and cell states, which together shape tissue organization and organismal function across species and lifespans. Despite numerous advances in RNA sequencing technologies and the massive accumulation of transcriptome datasets across the life sciences1,2, the dearth of technologies that use RNAs to observe and manipulate cell types remains a bottleneck in biology and medicine. Here we describe CellREADR (Cell access through RNA sensing by Endogenous ADAR), a programmable RNA-sensing technology that leverages RNA editing mediated by ADAR to couple the detection of cell-defining RNAs with the translation of effector proteins. Viral delivery of CellREADR conferred specific cell-type access in mouse and rat brains and in ex vivo human brain tissues. Furthermore, CellREADR enabled the recording and control of specific types of neurons in behaving mice. CellREADR thus highlights the potential for RNA-based monitoring and editing of animal cells in ways that are specific, versatile, simple and generalizable across organ systems and species, with wide applications in biology, biotechnology and programmable RNA medicine.

A genetically defined insula-brainstem circuit selectively controls motivational vigor.

The anterior insular cortex (aIC) plays a critical role in cognitive and motivational control of behavior, but the underlying neural mechanism remains elusive. Here, we show that aIC neurons expressing Fezf2 (aICFezf2), which are the pyramidal tract neurons, signal motivational vigor and invigorate need-seeking behavior through projections to the brainstem nucleus tractus solitarii (NTS). aICFezf2 neurons and their postsynaptic NTS neurons acquire anticipatory activity through learning, which encodes the perceived value and the vigor of actions to pursue homeostatic needs. Correspondingly, aIC → NTS circuit activity controls vigor, effort, and striatal dopamine release but only if the action is learned and the outcome is needed. Notably, aICFezf2 neurons do not represent taste or valence. Moreover, aIC → NTS activity neither drives reinforcement nor influences total consumption. These results pinpoint specific functions of aIC → NTS circuit for selectively controlling motivational vigor and suggest that motivation is subserved, in part, by aIC's top-down regulation of dopamine signaling.

Recruitment and inhibitory action of hippocampal axo-axonic cells during behavior.

The axon initial segment of hippocampal pyramidal cells is a key subcellular compartment for action potential generation, under GABAergic control by the "chandelier" or axo-axonic cells (AACs). Although AACs are the only cellular source of GABA targeting the initial segment, their in vivo activity patterns and influence over pyramidal cell dynamics are not well understood. We achieved cell-type-specific genetic access to AACs in mice and show that AACs in the hippocampal area CA1 are synchronously activated by episodes of locomotion or whisking during rest. Bidirectional intervention experiments in head-restrained mice performing a random foraging task revealed that AACs inhibit CA1 pyramidal cells, indicating that the effect of GABA on the initial segments in the hippocampus is inhibitory in vivo. Finally, optogenetic inhibition of AACs at specific track locations induced remapping of pyramidal cell place fields. These results demonstrate brain-state-specific dynamics of a critical inhibitory controller of cortical circuits.

Genetic dissection of the glutamatergic neuron system in cerebral cortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels.

Single-cell alternative polyadenylation analysis delineates GABAergic neuron types.

BACKGROUND: Alternative polyadenylation (APA) is emerging as an important mechanism in the post-transcriptional regulation of gene expression across eukaryotic species. Recent studies have shown that APA plays key roles in biological processes, such as cell proliferation and differentiation. Single-cell RNA-seq technologies are widely used in gene expression heterogeneity studies; however, systematic studies of APA at the single-cell level are still lacking. RESULTS: Here, we described a novel computational framework, SAPAS, that utilizes 3'-tag-based scRNA-seq data to identify novel poly(A) sites and quantify APA at the single-cell level. Applying SAPAS to the scRNA-seq data of phenotype characterized GABAergic interneurons, we identified cell type-specific APA events for different GABAergic neuron types. Genes with cell type-specific APA events are enriched for synaptic architecture and communications. In further, we observed a strong enrichment of heritability for several psychiatric disorders and brain traits in altered 3' UTRs and coding sequences of cell type-specific APA events. Finally, by exploring the modalities of APA, we discovered that the bimodal APA pattern of Pak3 could classify chandelier cells into different subpopulations that are from different laminar positions. CONCLUSIONS: We established a method to characterize APA at the single-cell level. When applied to a scRNA-seq dataset of GABAergic interneurons, the single-cell APA analysis not only identified cell type-specific APA events but also revealed that the modality of APA could classify cell subpopulations. Thus, SAPAS will expand our understanding of cellular heterogeneity.

Retinal and Callosal Activity-Dependent Chandelier Cell Elimination Shapes Binocularity in Primary Visual Cortex.

In mammals with binocular vision, integration of the left and right visual scene relies on information in the center visual field, which are relayed from each retina in parallel and merge in the primary visual cortex (V1) through the convergence of ipsi- and contralateral geniculocortical inputs as well as transcallosal projections between two visual cortices. The developmental assembly of this binocular circuit, especially the transcallosal pathway, remains incompletely understood. Using genetic methods in mice, we found that several days before eye-opening, retinal and callosal activities drive massive apoptosis of GABAergic chandelier cells (ChCs) in the binocular region of V1. Blockade of ChC elimination resulted in a contralateral eye-dominated V1 and deficient binocular vision. As pre-vision retinal activities convey the left-right organization of the visual field, their regulation of ChC density through the transcallosal pathway may prime a nascent binocular territory for subsequent experience-driven tuning during the post-vision critical period.

A Genetically Defined Compartmentalized Striatal Direct Pathway for Negative Reinforcement.

The striosome compartment within the dorsal striatum has been implicated in reinforcement learning and regulation of motivation, but how striosomal neurons contribute to these functions remains elusive. Here, we show that a genetically identified striosomal population, which expresses the Teashirt family zinc finger 1 (Tshz1) and belongs to the direct pathway, drives negative reinforcement and is essential for aversive learning in mice. Contrasting a "conventional" striosomal direct pathway, the Tshz1 neurons cause aversion, movement suppression, and negative reinforcement once activated, and they receive a distinct set of synaptic inputs. These neurons are predominantly excited by punishment rather than reward and represent the anticipation of punishment or the motivation for avoidance. Furthermore, inhibiting these neurons impairs punishment-based learning without affecting reward learning or movement. These results establish a major role of striosomal neurons in behaviors reinforced by punishment and moreover uncover functions of the direct pathway unaccounted for in classic models.

Maternal Experience-Dependent Cortical Plasticity in Mice Is Circuit- and Stimulus-Specific and Requires MECP2.

The neurodevelopmental disorder Rett syndrome is caused by mutations in the gene Mecp2 Misexpression of the protein MECP2 is thought to contribute to neuropathology by causing dysregulation of plasticity. Female heterozygous Mecp2 mutants (Mecp2het ) failed to acquire a learned maternal retrieval behavior when exposed to pups, an effect linked to disruption of parvalbumin-expressing inhibitory interneurons (PV) in the auditory cortex. Nevertheless, how dysregulated PV networks affect the neural activity dynamics that underlie auditory cortical plasticity during early maternal experience is unknown. Here we show that maternal experience in WT adult female mice (WT) triggers suppression of PV auditory responses. We also observe concomitant disinhibition of auditory responses in deep-layer pyramidal neurons that is selective for behaviorally relevant pup vocalizations. These neurons further exhibit sharpened tuning for pup vocalizations following maternal experience. All of these neuronal changes are abolished in Mecp2het , suggesting that they are an essential component of maternal learning. This is further supported by our finding that genetic manipulation of GABAergic networks that restores accurate retrieval behavior in Mecp2het also restores maternal experience-dependent plasticity of PV. Our data are consistent with a growing body of evidence that cortical networks are particularly vulnerable to mutations of Mecp2 in PV neurons. Moreover, our work links, for the first time, impaired in vivo cortical plasticity in awake Mecp2 mutant animals to a natural, ethologically relevant behavior.SIGNIFICANCE STATEMENT Rett syndrome is a genetic disorder that includes language communication problems. Nearly all Rett syndrome is caused by mutations in the gene that produces the protein MECP2, which is important for changes in brain connectivity believed to underlie learning. We previously showed that female Mecp2 mutants fail to learn a simple maternal care behavior performed in response to their pups' distress cries. This impairment appeared to critically involve inhibitory neurons in the auditory cortex called parvalbumin neurons. Here we record from these neurons before and after maternal experience, and we show that they adapt their response to pup calls during maternal learning in nonmutants, but not in mutants. This adaptation is partially restored by a manipulation that improves learning.

High-Throughput Mapping of Long-Range Neuronal Projection Using In Situ Sequencing.

Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.

The diversity of GABAergic neurons and neural communication elements.

The phenotypic diversity of cortical GABAergic neurons is probably necessary for their functional versatility in shaping the spatiotemporal dynamics of neural circuit operations underlying cognition. Deciphering the logic of this diversity requires comprehensive analysis of multi-modal cell features and a framework of neuronal identity that reflects biological mechanisms and principles. Recent high-throughput single-cell analyses have generated unprecedented data sets characterizing the transcriptomes, morphology and electrophysiology of interneurons. We posit that cardinal interneuron types can be defined by their synaptic communication properties, which are encoded in key transcriptional signatures. This conceptual framework integrates multi-modal cell features, captures neuronal input-output properties fundamental to circuit operation and may advance understanding of the appropriate granularity of neuron types, towards a biologically grounded and operationally useful interneuron taxonomy.

Genetic Single Neuron Anatomy Reveals Fine Granularity of Cortical Axo-Axonic Cells.

Parsing diverse nerve cells into biological types is necessary for understanding neural circuit organization. Morphology is an intuitive criterion for neuronal classification and a proxy of connectivity, but morphological diversity and variability often preclude resolving the granularity of neuron types. Combining genetic labeling with high-resolution, large-volume light microscopy, we established a single neuron anatomy platform that resolves, registers, and quantifies complete neuron morphologies in the mouse brain. We discovered that cortical axo-axonic cells (AACs), a cardinal GABAergic interneuron type that controls pyramidal neuron (PyN) spiking at axon initial segments, consist of multiple subtypes distinguished by highly laminar-specific soma position and dendritic and axonal arborization patterns. Whereas the laminar arrangements of AAC dendrites reflect differential recruitment by input streams, the laminar distribution and local geometry of AAC axons enable differential innervation of PyN ensembles. This platform will facilitate genetically targeted, high-resolution, and scalable single neuron anatomy in the mouse brain.

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq).

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing platforms process all input cells indiscriminately. Sometimes, fluorescence-activated cell sorting (FACS) is used upstream to isolate a specifically labeled population of interest. A limitation of FACS is the need for high numbers of input cells with significantly labeled fractions, which is impractical for collecting and profiling rare or sparsely labeled neuron populations from the mouse brain. Here, we describe a method for manually collecting sparse fluorescently labeled single neurons from freshly dissociated mouse brain tissue. This process allows for capturing single-labeled neurons with high purity and subsequent integration with an in vitro transcription-based amplification protocol that preserves endogenous transcript ratios. We describe a double linear amplification method that uses unique molecule identifiers (UMIs) to generate individual mRNA counts. Two rounds of amplification results in a high degree of gene detection per single cell.