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Maps of the nervous system inspire experiments and theories in neuroscience. Advances in molecular biology over the past decades have revolutionized the definition of cell and tissue identity. Spatial transcriptomics has opened up a new era in neuroanatomy, where the unsupervised and unbiased exploration of the molecular signatures of tissue organization will give rise to a new generation of brain maps. We propose that the molecular classification of brain regions on the basis of their gene expression profile can circumvent subjective neuroanatomical definitions and produce common reference frameworks that can incorporate cell types, connectivity, activity, and other modalities. Here we review the technological and conceptual advances made possible by spatial transcriptomics in the context of advancing neuroanatomy and discuss how molecular neuroanatomy can redefine mapping of the nervous system.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice., Molecular diversity of proprioceptive neuron types (Ia, Ib and II PNs) is unclear. Here, the authors characterized the functional organization and development of eight subtypes of PNs in mice. Importantly, Ia subtypes are plastic, suggesting a role in adaptive proprioception during motor behavior.

High-throughput, spatially resolved gene expression techniques are poised to be transformative across biology by overcoming a central limitation in single-cell biology: the lack of information on relationships that organize the cells into the functional groupings characteristic of tissues in complex multicellular organisms. Spatial expression is particularly interesting in the mammalian brain, which has a highly defined structure, strong spatial constraint in its organization, and detailed multimodal phenotypes for cells and ensembles of cells that can be linked to mesoscale properties such as projection patterns, and from there, to circuits generating behavior. However, as with any type of expression data, cross-dataset benchmarking of spatial data is a crucial first step. Here, we assess the replicability, with reference to canonical brain subdivisions, between the Allen Institute’s in situ hybridization data from the adult mouse brain (Allen Brain Atlas (ABA)) and a similar dataset collected using spatial transcriptomics (ST). With the advent of tractable spatial techniques, for the first time, we are able to benchmark the Allen Institute’s whole-brain, whole-transcriptome spatial expression dataset with a second independent dataset that similarly spans the whole brain and transcriptome. We use regularized linear regression (LASSO), linear regression, and correlation-based feature selection in a supervised learning framework to classify expression samples relative to their assayed location. We show that Allen Reference Atlas labels are classifiable using transcription in both data sets, but that performance is higher in the ABA than in ST. Furthermore, models trained in one dataset and tested in the opposite dataset do not reproduce classification performance bidirectionally. While an identifying expression profile can be found for a given brain area, it does not generalize to the opposite dataset. In general, we found that canonical brain area labels are classifiable in gene expression space within dataset and that our observed performance is not merely reflecting physical distance in the brain. However, we also show that cross-platform classification is not robust. Emerging spatial datasets from the mouse brain will allow further characterization of cross-dataset replicability ultimately providing a valuable reference set for understanding the cell biology of the brain., Spatial expression is particularly interesting in the mammalian brain, but benchmarking of spatial data is a crucial first step in its use. This study uses cross-dataset modeling to determine the degree to which spatially-resolved transcriptomics data from the mouse brain capture canonical brain sub-divisions, with a focus on the replication of reference datasets.

Whole-brain spatial transcriptomics redefines the neuroanatomical classification of the adult mouse brain., Brain maps are essential for integrating information and interpreting the structure-function relationship of circuits and behavior. We aimed to generate a systematic classification of the adult mouse brain based purely on the unbiased identification of spatially defining features by employing whole-brain spatial transcriptomics. We found that the molecular information was sufficient to deduce the complex and detailed neuroanatomical organization of the brain. The unsupervised (non-expert, data-driven) classification revealed new area- and layer-specific subregions, for example in isocortex and hippocampus, and new subdivisions of striatum. The molecular atlas further supports the characterization of the spatial identity of neurons from their single-cell RNA profile, and provides a resource for annotating the brain using a minimal gene set—a brain palette. In summary, we have established a molecular atlas to formally define the spatial organization of brain regions, including the molecular code for mapping and targeting of discrete neuroanatomical domains.

GPR151 is a G‐protein coupled receptor for which the endogenous ligand remains unknown. In the nervous system of vertebrates, its expression is enriched in specific diencephalic structures, where the highest levels are observed in the habenular area. The habenula has been implicated in a range of different functions including behavioral flexibility, decision making, inhibitory control, and pain processing, which makes it a promising target for treating psychiatric and neurological disease. This study aimed to further characterize neurons expressing the Gpr151 gene, by tracing the afferent connectivity of this diencephalic cell population. Using pseudotyped rabies virus in a transgenic Gpr151‐Cre mouse line, monosynaptic afferents of habenular and thalamic Gpr151‐expressing neuronal populations could be visualized. The habenular and thalamic Gpr151 systems displayed both shared and distinct connectivity patterns. The habenular neurons primarily received input from basal forebrain structures, the bed nucleus of stria terminalis, the lateral preoptic area, the entopeduncular nucleus, and the lateral hypothalamic area. The Gpr151‐expressing neurons in the paraventricular nucleus of the thalamus was primarily contacted by medial hypothalamic areas as well as the zona incerta and projected to specific forebrain areas such as the prelimbic cortex and the accumbens nucleus. Gpr151 mRNA was also detected at low levels in the lateral posterior thalamic nucleus which received input from areas associated with visual processing, including the superior colliculus, zona incerta, and the visual and retrosplenial cortices. Knowledge about the connectivity of Gpr151‐expressing neurons will facilitate the interpretation of future functional studies of this receptor.

Once generated, neurons are thought to permanently exit the cell cycle and become irreversibly differentiated. However, neither the precise point at which this post-mitotic state is attained nor the extent of its irreversibility is clearly defined. Here we report that newly born neurons from the upper layers of the mouse cortex, despite initiating axon and dendrite elongation, continue to drive gene expression from the neural progenitor tubulin α1 promoter (Tα1p). These observations suggest an ambiguous post-mitotic neuronal state. Whole transcriptome analysis of sorted upper cortical neurons further revealed that neurons continue to express genes related to cell cycle progression long after mitotic exit until at least post-natal day 3 (P3). These genes are however down-regulated thereafter, associated with a concomitant up-regulation of tumor suppressors at P5. Interestingly, newly born neurons located in the cortical plate (CP) at embryonic day 18-19 (E18-E19) and P3 challenged with calcium influx are found in S/G2/M phases of the cell cycle, and still able to undergo division at E18-E19 but not at P3. At P5 however, calcium influx becomes neurotoxic and leads instead to neuronal loss. Our data delineate an unexpected flexibility of cell cycle control in early born neurons, and describe how neurons transit to a post-mitotic state.Cell Research advance online publication 21 June 2016; doi:10.1038/cr.2016.76.

A major long-term goal of systems neuroscience is to identify the different roles of neural subtypes in brain circuit function. The ability to causally manipulate selective cell types is critical to meeting this goal. This protocol describes techniques for optically stimulating specific populations of excitatory neurons and inhibitory interneurons in vivo in combination with electrophysiology. Cell type selectivity is obtained using Cre-dependent expression of the light-activated channel Channelrhodopsin-2. We also describe approaches for minimizing optical interference with simultaneous extracellular and intracellular recording. These optogenetic techniques provide a spatially and temporally precise means of studying neural activity in the intact brain and allow a detailed examination of the effect of evoked activity on the surrounding local neural network. Injection of viral vectors requires 30–45 min, and in vivo electrophysiology with optogenetic stimulation requires 1–4 h., National Institutes of Health (U.S.), National Science Foundation (U.S.), Simons Foundation, National Institutes of Health (U.S.). Pioneer Award, National Eye Institue (K99 Award), Knut and Alice Wallenberg Foundation (Postdoctoral Fellowship), Brain & Behavior Research Foundation. Young Investigator Award, Thomas F. Petersen

Cortical gamma oscillations (20–80 Hz) predict increases in focused attention, and failure in gamma regulation is a hallmark of neurological and psychiatric disease. Current theory predicts that gamma oscillations are generated by synchronous activity of fast-spiking inhibitory interneurons, with the resulting rhythmic inhibition producing neural ensemble synchrony by generating a narrow window for effective excitation. We causally tested these hypotheses in barrel cortex in vivo by targeting optogenetic manipulation selectively to fast-spiking interneurons. Here we show that light-driven activation of fast-spiking interneurons at varied frequencies (8–200 Hz) selectively amplifies gamma oscillations. In contrast, pyramidal neuron activation amplifies only lower frequency oscillations, a cell-type-specific double dissociation. We found that the timing of a sensory input relative to a gamma cycle determined the amplitude and precision of evoked responses. Our data directly support the fast-spiking-gamma hypothesis and provide the first causal evidence that distinct network activity states can be induced in vivo by cell-type-specific activation.

DNA methylation is an epigenetic mechanism involved in gene regulation and implicated in the functioning of the nervous system. The de novo DNA methyltransferase Dnmt3a is expressed in neurons, but its specific role has not been clarified. Dnmt3a is activated around embryonic day 10.5 in mouse neuronal precursor cells and remains active in postmitotic neurons in the adult. We assessed the role of neuronal Dnmt3a by conditional gene targeting. Mice lacking functional Dnmt3a in the nervous system were born healthy, but degenerated in adulthood and died prematurely. Mutant mice were hypoactive, walked abnormally, and underperformed on tests of neuromuscular function and motor coordination. Loss of Dnmt3a also led to fewer motor neurons in the hypoglossal nucleus and more fragmented endplates in neuromuscular junctions of the diaphragm muscle. Our results implicate a role for Dnmt3a in the neuromuscular control of motor movement.

Recent studies have shown that cells expressing neuronal antigens can be derived from a bone marrow transplant. A new report lends support to and extends these previous results by presenting compelling morphological evidence for the generation and integration of highly differentiated bone marrow–derived neurons.

The striatal microcircuitry consists of a vast majority of projection neurons, the medium spiny neurons (MSNs), and a small yet diverse population of interneurons. To understand how activity is orchestrated within the striatum, it is essential to unravel the functional connectivity between the different neuronal types. Fast-spiking (FS) interneurons provide feedforward inhibition to both direct and indirect pathway MSNs and are important in sculpting their output to downstream basal ganglia nuclei. FS interneurons are also interconnected with each other via electrical and chemical synapses; however, whether and how they inhibit other striatal interneuron types remains unknown. In this study we combined multineuron whole-cell recordings with optogenetics to determine the target selectivity of feedforward inhibition by striatal FS interneurons. Using transgenic and viral approaches we directed expression of channelrhodopsin 2 (ChR2) to FS interneurons to study their connectivity within the mouse striatal microcircuit. Optogenetic stimulation of ChR2-expressing FS interneurons generated strong and reliable GABAA-dependent synaptic inputs in MSNs. In sharp contrast, simultaneously recorded neighboring cholinergic interneurons did not receive any synaptic inputs from photostimulated FS cells, and a minority of low-threshold spiking (LTS) interneurons responded weakly. We further tested the synaptic connectivity between FS and LTS interneurons using paired recordings, which showed only sparse connectivity. Our results show that striatal FS interneurons form a feedforward inhibitory circuit that is target selective, inhibiting projection neurons while avoiding cholinergic interneurons and sparsely contacting LTS interneurons, thus supporting independent modulation of MSN activity by the different types of striatal interneurons.

How neurons develop their morphology is an important question in neurobiology. Here we describe a new pathway that specifically affects the formation of basal dendrites and axonal projections in cortical pyramidal neurons. We report that thousand-and-one-amino acid 2 kinase (TAOK2), also known as TAO2, is essential for dendrite morphogenesis. TAOK2 downregulation impairs basal dendrite formation in vivo without affecting apical dendrites. Moreover, TAOK2 interacts with Neuropilin 1 (Nrp1), a receptor protein that binds the secreted guidance cue Semaphorin 3A (Sema3A). TAOK2 overexpression restores dendrite formation in cultured cortical neurons from Nrp1(Sema-) mice, which express Nrp1 receptors incapable of binding Sema3A. TAOK2 overexpression also ameliorates the basal dendrite impairment resulting from Nrp1 downregulation in vivo. Finally, Sema3A and TAOK2 modulate the formation of basal dendrites through the activation of the c-Jun N-terminal kinase (JNK). These results delineate a pathway whereby Sema3A and Nrp1 transduce signals through TAOK2 and JNK to regulate basal dendrite development in cortical neurons.

Adult neurogenesis arises from neural stem cells within specialized niches. Neuronal activity and experience, presumably acting on this local niche, regulate multiple stages of adult neurogenesis, from neural progenitor proliferation to new neuron maturation, synaptic integration and survival. It is unknown whether local neuronal circuitry has a direct impact on adult neural stem cells. Here we show that, in the adult mouse hippocampus, nestin-expressing radial glia-like quiescent neural stem cells (RGLs) respond tonically to the neurotransmitter γ-aminobutyric acid (GABA) by means of γ2-subunit-containing GABAA receptors. Clonal analysis of individual RGLs revealed a rapid exit from quiescence and enhanced symmetrical self-renewal after conditional deletion of γ2. RGLs are in close proximity to terminals expressing 67-kDa glutamic acid decarboxylase (GAD67) of parvalbumin-expressing (PV+) interneurons and respond tonically to GABA released from these neurons. Functionally, optogenetic control of the activity of dentate PV+ interneurons, but not that of somatostatin-expressing or vasoactive intestinal polypeptide (VIP)-expressing interneurons, can dictate the RGL choice between quiescence and activation. Furthermore, PV+ interneuron activation restores RGL quiescence after social isolation, an experience that induces RGL activation and symmetrical division. Our study identifies a niche cell–signal–receptor trio and a local circuitry mechanism that control the activation and self-renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience.

SummarySeveral distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.

The mechanisms underlying the normal development of neuronal morphology remain a fundamental question in neurobiology. Studies in cultured neurons have suggested that the position of the centrosome and the Golgi may predict the site of axon outgrowth. During neuronal migration in the developing cortex, however, the centrosome and Golgi are oriented toward the cortical plate at a time when axons grow toward the ventricular zone. In the current work, we usein situlive imaging to demonstrate that the centrosome and the accompanying polarized cytoplasm exhibit apical translocation in newborn cortical neurons preceding initial axon outgrowth. Disruption of centrosomal activity or downregulation of the centriolar satellite protein PCM-1 affects axon formation. We further show that downregulation of the centrosomal protein Cep120 impairs microtubule organization, resulting in increased centrosome motility. Decreased centrosome motility resulting from microtubule stabilization causes an aberrant centrosomal localization, leading to misplaced axonal outgrowth. Our results reveal the dynamic nature of the centrosome in developing cortical neurons, and implicate centrosome translocation and microtubule organization during the multipolar stage as important determinants of axon formation.

Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury.

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury., Author Summary Spinal cord injuries occur in more than 30.000 individuals each year worldwide and result in significant morbidity, with patients requiring long physical and medical care. The recent identification of resident stem cells in the adult spinal cord has opened up for the possibility of pharmacological manipulation of these cells to produce cell types promoting recovery after injury. We have employed genetic tools to specifically address the identity and reaction to injury of a spinal cord subpopulation of cells known as ependymal cell. Genetic labeling of this putative stem cell population allows for the evaluation of stem cell activity in vitro and in vivo. We found that ependymal cells lining the central canal act as neural stem cells in vitro and contribute extensively to the glial scar in vivo. Interestingly, injury induces proliferation of ependymal cells and migration of ependyma-derived progeny towards the site of injury. Moreover, ependymal cell progeny differentiate and give rise to astrocytes as well as myelinating oligodendrocytes. In summary, our results point to ependymal cells as an attractive candidate population for non-invasive manipulation after injury., Spinal cord injury induces the proliferation of putative stem cells derived from the ependyma that migrate towards the site of injury. Ependymal cell progeny differentiate and give rise to astrocytes as well as myelinating oligodendrocytes.

Recent studies suggest that non-neural cells can generate neurons in mice and humans. There is no lack of excitement about these results: they challenge our view of cellular differentiation and the prospect of being able to generate neurons from, for example, blood is very attractive. But is it too good to be true?

Corrigendum: Autism spectrum disorder susceptibility gene TAOK2 affects basal dendrite formation in the neocortex