Neuronal Identity Research Articles

A comprehensive spatio-cellular map of the human hypothalamus.

The hypothalamus is a brain region that plays a key role in coordinating fundamental biological functions1. However, our understanding of the underlying cellular components and neurocircuitries have, until recently, emerged primarily from rodent studies2,3. Here we combine single-nucleus sequencing of 433,369 human hypothalamic cells with spatial transcriptomics, generating a comprehensive spatio-cellular transcriptional map of the hypothalamus, the 'HYPOMAP'. Although conservation of neuronal cell types between humans and mice, asbased on transcriptomic identity, is generally high, there are notable exceptions. Specifically, there are significant disparities in the identity of pro-opiomelanocortin neurons and in the expression levels of G-protein-coupled receptors between the two species that carry direct implications for currently approved obesity treatments. Out of the 452 hypothalamic cell types, we find that291 neuronal clusters are significantly enriched for expression of body mass index (BMI) genome-wide association study genes. This enrichment is driven by 426 'effector' genes. Rare deleterious variants in six of these (MC4R, PCSK1, POMC, CALCR, BSN and CORO1A) associate with BMI at population level, and CORO1A has not been linked previously to BMI. Thus, HYPOMAP provides a detailed atlas of the human hypothalamus in a spatial context and serves as an important resource to identify new druggable targets for treating a wide range of conditions, including reproductive, circadian and metabolic disorders.

Assessment of PRMT6-dependent alternative splicing in pluripotent and differentiating NT2/D1 cells.

Protein arginine methyltransferase 6 (PRMT6) is a well-characterized epigenetic regulator that methylates histone H3 at arginine 2 (H3R2me2a) in both promoter and enhancer regions, thereby modulating transcriptional initiation. We report here that PRMT6 also regulates gene expression at the post-transcriptional level in the neural pluripotent state and during neuronal differentiation of NT2/D1 cells. PRMT6 knockout causes widespread alternative splicing changes in NT2/D1 cells, most frequently cassette exon alterations. Most of the PRMT6-dependent splicing targets are not transcriptionally affected by the enzyme and regulated in an H3R2me2a-independent manner. However, for a small subset of splicing events, the PRMT6-mediated deposition of H3R2me2a overlaps with the splice site, suggesting a potential dual function in both transcriptional and co-/post-transcriptional regulation. The splicing targets of PRMT6 include ribosomal proteins, splicing factors, and chromatin-modifying enzymes such as PRMT4, DNMT3B, and ASH2L, some of which are associated with differentiation decisions. Taken together, our results in NT2/D1 cells show that PRMT6 exerts predominantly H3R2me2a-independent functions in RNA splicing, which may contribute to pluripotency and neuronal identity.

Trithorax regulates long-term memory in Drosophila through epigenetic maintenance of mushroom body metabolic state and translation capacity.

The role of epigenetics and chromatin in the maintenance of postmitotic neuronal cell identities is not well understood. Here, we show that the histone methyltransferase Trithorax (Trx) is required in postmitotic memory neurons of the Drosophila mushroom body (MB) to enable their capacity for long-term memory (LTM), but not short-term memory (STM). Using MB-specific RNA-, ChIP-, and ATAC-sequencing, we find that Trx maintains homeostatic expression of several non-canonical MB-enriched transcripts, including the orphan nuclear receptor Hr51, and the metabolic enzyme lactate dehydrogenase (Ldh). Through these key targets, Trx facilitates a metabolic state characterized by high lactate levels in MBγ neurons. This metabolic state supports a high capacity for protein translation, a process that is essential for LTM, but not STM. These data suggest that Trx, a classic regulator of cell type specification during development, has additional functions in maintaining underappreciated aspects of postmitotic neuron identity, such as metabolic state. Our work supports a body of evidence suggesting that a high capacity for energy metabolism is an essential cell identity characteristic for neurons that mediate LTM.

P0159 Effects of Transcranial Direct Current Stimulation (tDCS) on enteric nervous system (ENS) gene expression and intestinal barrier in chronic DSS colitis

Abstract Background Chronic visceral pain is a clinical challenge in >50% of Inflammatory Bowel Disease (IBD) cases. A randomized controlled trial (RCT) revealed that transcranial direct current stimulation (tDCS) can modulate brain activity and effectively alleviate chronic abdominal pain associated with IBD. To elucidate the underlying mechanism of tDCS, we implemented this technique in a mouse model of inflammatory, chronic DSS colitis. Methods The tDCS protocol (10 stimulations) was deduced from human tDCS studies. We administered an anodal direct current (0.5 mA, 20 min) to both primary motor cortices over two separate periods (5 consecutive days of stimulation per period separated by a two-day break). The study was controlled by a sham-tDCS group. For determining mucosal barrier integrity murine intestinal mucosa was mounted to Ussing chambers. Histology was done (H&E staining of FFPE colon sections). Murine activity after tDCS was assessed by mouse tracking in home cages (RFID-chipped mice). Myenteric plexus (MP) cells from the murine terminal ileum, were isolated and cultivated for 10 days. Immunostaining and bulk-RNA sequencing (NovaSeq Xplus system, read length 150 bp, read depth 30 M copies/run) were performed. Results DSS caused an inflammation-driven thickening of the colon wall, resulting in an increased transmural resistance. This effect was ameliorated by tDCS (mean±SEM DSS+sham-tDCS: 72.2±8.2 Ω·cm²; DSS+tDCS: 48.1±2.1 Ω·cm²; MWU: p=0.007). Induction of a DSS colitis was associated with a reduced murine activity, which was partially recovered by tDCS (mean±SEM DSS+sham-tDCS: 70.7±15.2 rel.dist.U/day; DSS+tDCS: 205.9 ± 40.4 rel.dist.U/day; MWU: p=0.029). Neuronal cell identity was confirmed in isolated murine MP cells by immunostaining (b3-tubulin, S100B). Bulk-RNAseq significantly separated tDCS- from sham-tDCS treated DSS colitis mice as revealed by PCA. Consecutively, 739 differentially expressed genes were identified in tDCS-treated colitis mice compared to sham-tDCS-treated DSS colitic mice (622 upregulated, 117 downregulated genes; log2FC≥1.5, p-adj<0.05). KEGG and GO pathway analysis included neuroactive ligand-receptor interaction (p=7.94·10-3), gated channel activity (p=2.4·10-7), substrate-specific channel activity (p=1.99·10-7), leukocyte migration (p=3.99·10-7). Conclusion In colitis, tDCS has the capacity to modulate activity of DSS-treated mice and alters gene expression in MP cells. This reveals another layer of evidence for the impact of central pathways on the brain-gut-axis. References Neeb et al., Brain Stimulation, 2019

Specification of claustro-amygdalar and palaeocortical neurons and circuits.

The ventrolateral pallial (VLp) excitatory neurons in the claustro-amygdalar complex and piriform cortex (PIR; which forms part of the palaeocortex) form reciprocal connections with the prefrontal cortex (PFC), integrating cognitive and sensory information that results in adaptive behaviours1-5. Early-life disruptions in these circuits are linked to neuropsychiatric disorders4-8, highlighting the importance of understanding their development. Here we reveal that the transcription factors SOX4, SOX11 and TFAP2D have a pivotal role in the development, identity and PFC connectivity of these excitatory neurons. The absence of SOX4 and SOX11 in post-mitotic excitatory neurons results in a marked reduction in the size of the basolateral amygdala complex (BLC), claustrum (CLA) and PIR. These transcription factors control BLC formation through direct regulation of Tfap2d expression. Cross-species analyses, including in humans, identified conserved Tfap2d expression in developing excitatory neurons of BLC, CLA, PIR and the associated transitional areas of the frontal, insular and temporal cortex. Although the loss and haploinsufficiency of Tfap2d yield similar alterations in learned threat-response behaviours, differences emerge in the phenotypes at different Tfap2d dosages, particularly in terms of changes observed in BLC size and BLC-PFC connectivity. This underscores the importance of Tfap2d dosage in orchestrating developmental shifts in BLC-PFC connectivity and behavioural modifications that resemble symptoms of neuropsychiatric disorders. Together, these findings reveal key elements of a conserved gene regulatory network that shapes the development and function of crucial VLp excitatory neurons and their PFC connectivity and offer insights into their evolution and alterations in neuropsychiatric disorders.

Data-driven synapse classification reveals a logic of glutamate receptor diversity.

The rich diversity of synapses facilitates the capacity of neural circuits to transmit, process and store information. We used multiplex super-resolution proteometric imaging through array tomography to define features of single synapses in mouse neocortex. We find that glutamatergic synapses cluster into subclasses that parallel the distinct biochemical and functional categories of receptor subunits: GluA1/4, GluA2/3 and GluN1/GluN2B. Two of these subclasses align with physiological expectations based on synaptic plasticity: large AMPAR-rich synapses may represent potentiated synapses, whereas small NMDAR-rich synapses suggest "silent" synapses. The NMDA receptor content of large synapses correlates with spine neck diameter, and thus the potential for coupling to the parent dendrite. Overall, ultrastructural features predict receptor content of synapses better than parent neuron identity does, suggesting synapse subclasses act as fundamental elements of neuronal circuits. No barriers prevent future generalization of this approach to other species, or to study of human disorders and therapeutics.

Long somatic DNA-repeat expansion drives neurodegeneration in Huntington's disease.

In Huntington's disease (HD), striatal projection neurons (SPNs) degenerate during midlife; the core biological question involves how the disease-causing DNA repeat (CAG)n in the huntingtin (HTT) gene leads to neurodegeneration after decades of biological latency. We developed a single-cell method for measuring this repeat's length alongside genome-wide RNA expression. We found that the HTT CAG repeat expands somatically from 40-45 to 100-500+ CAGs in SPNs. Somatic expansion from 40 to 150 CAGs had no apparent cell-autonomous effect, but SPNs with 150-500+ CAGs lost positive and then negative features of neuronal identity, de-repressed senescence/apoptosis genes, and were lost. Our results suggest that somatic repeat expansion beyond 150 CAGs causes SPNs to degenerate quickly and asynchronously. We conclude that in HD, at any one time, most neurons have an innocuous but unstable HTT gene and that HD pathogenesis is a DNA process for almost all of a neuron's life.

Temporal and Notch identity determine layer targeting and synapse location of medulla neurons.

How specification mechanisms that generate neural diversity translate into specific neuronal targeting, connectivity, and function in the adult brain is not understood. In the medulla region of the Drosophila optic lobe, neural progenitors generate different neurons in a fixed order by sequentially expressing a series of temporal transcription factors as they age. Then, Notch signaling in intermediate progenitors further diversifies neuronal progeny. By establishing the birth order of medulla neurons, we found that their temporal identity correlates with the depth of neuropil targeting in the adult brain, for both local interneurons and projection neurons. We show that this temporal identity-dependent targeting of projection neurons unfolds early in development and is genetically determined. By leveraging the Electron Microscopy reconstruction of the adult fly brain, we determined the synapse location of medulla neurons in the different optic lobe neuropils and find that it is significantly associated with both their temporal identity and Notch status. Moreover, we show that all the putative medulla neurons with the same predicted function share similar neuropil synapse location, indicating that ensembles of neuropil layers encode specific visual functions. In conclusion, we show that temporal identity and Notch status of medulla neurons can predict their neuropil synapse location and visual function, linking their developmental patterning with their specific connectivity and functional features in the adult brain.

Electrochemical imaging of neurotransmitter release with fast-scan voltammetric ion conductance microscopy.

Understanding the dynamic spatial and temporal release of neurotransmitters can help resolve long-standing questions related to chemical modulation of neurological circuits. Dopamine modulates function in a range of physiological processes and is key to transmission in addiction and neurological disorders. Studies at subcellular scales promise to help develop a broader understanding of dopamine release, diffusion, and receptor activation and how these processes lead to functional outcomes. Electrochemical measurements of dopamine release at individual cells have proven especially informative. We describe incorporation of fast-scan cyclic voltammetry for detection of dopamine release with subcellular spatial resolution and millisecond time resolution. The platform is benchmarked with standard redox probes and then applied to imaging stimulated release from subcellular locations of a coculture of dopaminergic neurons and astrocytes. Voltammetry reveals heterogeneity in release based on time, location, and neuron identity. We believe that this platform ultimately offers a window to understanding neurotransmission in pathophysiological models of disease where cell-cell communication is key.

Molecular patterns of evolutionary changes throughout the whole nervous system of multiple nematode species.

One avenue to better understand brain evolution is to map molecular patterns of evolutionary changes in neuronal cell types across entire nervous systems of distantly related species. Generating whole-animal single-cell transcriptomes of three nematode species from the Caenorhabditis genus, we observed a remarkable stability of neuronal cell type identities over more than 45 million years of evolution. Conserved patterns of combinatorial expression of homeodomain transcription factors are among the best classifiers of homologous neuron classes. Unexpectedly, we discover an extensive divergence in neuronal signaling pathways. While identities of neurotransmitter-producing neurons (glutamate, acetylcholine, GABA and several monoamines) remain stable, ionotropic and metabotropic receptors for all these neurotransmitter systems show substantial divergence, resulting in more than half of all neuron classes changing their capacity to be receptive to specific neurotransmitters. Neuropeptidergic signaling is also remarkably divergent, both at the level of neuropeptide expression and receptor expression, yet the overall dense network topology of the wireless neuropeptidergic connectome remains stable. Novel neuronal signaling pathways are suggested by our discovery of small secreted proteins that show no obvious hallmarks of conventional neuropeptides, but show similar patterns of highly neuron-type-specific and highly evolvable expression profiles. In conclusion, by investigating the evolution of entire nervous systems at the resolution of single neuron classes, we uncover patterns that may reflect basic principles governing evolutionary novelty in neuronal circuits.

Analysis of a shark reveals ancient, Wnt-dependent, habenular asymmetries in vertebrates

The mode of evolution of left-right asymmetries in the vertebrate habenulae remains largely unknown. Using a transcriptomic approach, we show that in a cartilaginous fish, the catshark Scyliorhinus canicula, habenulae exhibit marked asymmetries, in both their medial and lateral components. Comparisons across vertebrates suggest that those identified in lateral habenulae reflect an ancestral gnathostome trait, partially conserved in lampreys, and independently lost in tetrapods and neopterygians. Asymmetry formation involves distinct mechanisms in the catshark lateral and medial habenulae. Medial habenulae are submitted to a marked, asymmetric temporal regulation of neurogenesis, undetectable in their lateral counterparts. Conversely, asymmetry formation in lateral habenulae results from asymmetric choices of neuronal identity in post-mitotic progenitors, a regulation dependent on the repression of Wnt signaling by Nodal on the left. Based on comparisons with the mouse and the zebrafish, we propose that habenular asymmetry formation involves a recurrent developmental logic across vertebrates, which relies on conserved, temporally regulated genetic programs sequentially shaping choices of neuronal identity on both sides and asymmetrically modified by Wnt activity.

Notch Inhibition Enhances Morphological Reprogramming of microRNA-Induced Human Neurons.

The role of Notch signaling in direct neuronal reprogramming remains unknown despite its importance to brain development in vivo. Here, we use microRNA-induced neurons that are directly reprogrammed from human fibroblasts to determine how Notch signaling contributes to neuronal identity. We found that Notch inhibition during the first week of reprogramming was both necessary and sufficient to enhance neurite outgrowth at a later timepoint, indicating an important role in erasure of the original cell identity. Accordingly, transcriptomic analysis showed that the effect of Notch inhibition was likely due to improvements in fibroblast fate erasure and silencing of non-neuronal genes. To this effect, we identify MYLIP, whose downregulation in response to Notch inhibition significantly promoted neurite outgrowth. Moreover, Notch inhibition resulted in cells with neuronal transcriptome signature defined by expressing long genes at a faster rate than the control, demonstrating the effect of accelerated fate erasure on neuronal fate acquisition. Our results demonstrate the antagonistic role of Notch signaling to the pro-neuronal microRNAs 9 and 124 and the benefits of its inhibition to the acquisition of neuronal morphology.

TMIC-33. MICROENVIRONMENT-DRIVEN CHANGES IN GLIOBLASTOMA CELL STATE SIGNATURES EXAMINED USING REGION-SPECIFIC HUMAN BRAIN ORGANOIDS

Abstract Cell state plasticity is retained by all stem-like and differentiated cells within glioblastoma (GBM) tumors. GBM cells can exploit these state-shifting capacities to evade and resist therapeutics. Thus, there is a need to identify and target the regulators of GBM cell state transitions. To address this question, we use a fully human platform where hiSPC-derived organoids serve as a recipient environment to host cells engrafted directly from surgically resected GBMs. This allows us to precisely examine how microenvironmental variables impact the molecular signatures of GBM cells. We acutely isolate GBM cells from surgical resections, label cells with a GFP-lentivirus for 4 hours, and then engraft these tumor cells into parallelly patterned region-specific organoids that mimic dorsal forebrain, ventral forebrain, midbrain, and hindbrain identities. These regions were chosen as they capture derivatives of the three primary brain vesicles and because these regions contain unique cell types and signaling molecules. We performed paired single-cell RNA sequencing on tumor cells pre-engraftment and 14 days post-engraftment. Universal to all regional organoids, we find that GBM cells shift towards a neural progenitor-like and neuron-like signature within organoids, regardless of the cell state of the parent tumor prior to engraftment. To support our findings, we also observed that patient-derived glioma cell lines converge on a neural progenitor-like state within organoids. Thus, the dominance of this shared GBM cell state within organoids implies the existence of shared extrinsic factors in these immature (neurogenic) organoids that favor a progenitor-like or neuron-like identity across all four regions. We are now asking whether this bias towards neuronal and/or neural progenitor identity is a result of engrafting into young neurogenic organoids, or if the same phenomenon could be true in late-stage (post-gliogenic) mature organoids. We are also using this valuable platform and data to identify the extrinsic signals that GBM cells rely on to fulfill their plastic potential, adapt to the microenvironment, and resist therapeutics.

CCRG-02. MICRORNA TARGETING LOGIC IN DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMA

Abstract microRNA (miR)-124 is one of the main drivers of mitotic exit during neurogenesis, which is recapitulated in miR-9/9* and -124 (miR-9/9*-124)-mediated direct neuronal reprogramming. In the reprogramming of various cell types, miR-9/9*-124 induce non-neuronal cell fate erasure before activating the neuronal program. Regardless of the different starting transcriptome landscapes, a core gene regulatory network (GRN) is always targeted by miR-9/9*-124, with cell cycle progression as the most significant pathway. We reason that miR-9/9*-124 can induce cell fate erasure in cancerous cells by repressing similar GRNs, using glioblastoma multiforme (GBM) as a paradigm. Mechanistic insights into GBM fate erasure by miR-9/9*-124 while inducing the postmitotic state would facilitate the understanding of GRNs that govern the tumorigenic identity of GBMs and potentially offer novel therapeutic implications. Indeed, miR-9/9*-124 readily induces mitotic exit in patient-derived GBM cell lines, represses GBM cell fates, and promotes neuronal identity over time as assessed by RNA-seq. Mice receiving GBM intracerebral xenografts with miR-9/9*-124 survived significantly longer than those receiving non-specific control miRNA. Global profiling of miRNA targeting activity by AGO2-eCLIP reveals that miR-124 binds to transcripts enriched in G1/S transition and mitogen response. Conversely, miR-506—a miR-124 homolog—despite of sharing identical seed sequences, has significantly less targeting activity and fails to stop cell cycle progression. We are currently dissecting how the difference in the miRNA 3’ sequence renders contrasting biological outcomes. Preliminary data suggests that the length of miRNA alone alters targeting specificity to cell cycle transcripts. However, different permutations of miRNA 3’ sequence, though sufficient to induce mitotic exit, are not all competent to achieve neuronal reprogramming, indicating that mitotic exit and neurogenic activities of miR-124 could be decoupled. We hope that understanding the grammar logic of miRNA targeting would provide deeper insights into how miRNAs can be leveraged to manipulate GBM identity.

A neurotransmitter atlas of C. elegans males and hermaphrodites

Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the Caenorhabditis elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells, most notably in gonadal cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel sites of monoaminergic neurotransmitter uptake. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.

A neurotransmitter atlas of C. elegans males and hermaphrodites.

Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the Caenorhabditis elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells, most notably in gonadal cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel sites of monoaminergic neurotransmitter uptake. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.

Expansion of the neocortex and protection from neurodegeneration by in vivo transient reprogramming

Yamanaka factors (YFs) can reverse some aging features in mammalian tissues, but their effects on the brain remain largely unexplored. Here, we induced YFs in the mouse brain in a controlled spatiotemporal manner in two different scenarios: brain development and adult stages in the context of neurodegeneration. Embryonic induction of YFs perturbed cell identity of both progenitors and neurons, but transient and low-level expression is tolerated by these cells. Under these conditions, YF induction led to progenitor expansion, an increased number of upper cortical neurons and glia, and enhanced motor and social behavior in adult mice. Additionally, controlled YF induction is tolerated by principal neurons in the adult dorsal hippocampus and prevented the development of several hallmarks of Alzheimer's disease, including cognitive decline and altered molecular signatures, in the 5xFAD mouse model. These results highlight the powerful impact of YFs on neural proliferation and their potential use in brain disorders.

Claustrum and dorsal endopiriform cortex complex cell-identity is determined by Nurr1 and regulates hallucinogenic-like states in mice

The Claustrum/dorsal endopiriform cortex complex (CLA) is an enigmatic brain region with extensive glutamatergic projections to multiple cortical areas. The transcription factor Nurr1 is highly expressed in the CLA, but its role in this region is not understood. By using conditional gene-targeted mice, we show that Nurr1 is a crucial regulator of CLA neuron identity. Although CLA neurons remain intact in the absence of Nurr1, the distinctive gene expression pattern in the CLA is abolished. CLA has been hypothesized to control hallucinations, but little is known of how the CLA responds to hallucinogens. After the deletion of Nurr1 in the CLA, both hallucinogen receptor expression and signaling are lost. Furthermore, functional ultrasound and Neuropixel electrophysiological recordings revealed that the hallucinogenic-receptor agonists’ effects on functional connectivity between prefrontal and sensorimotor cortices are altered in Nurr1-ablated mice. Our findings suggest that Nurr1-targeted strategies provide additional avenues for functional studies of the CLA.

Distinct brain-wide presynaptic networks underlie the functional identity of individual cortical neurons.

Neuronal connections provide the scaffolding for neuronal function. Revealing the connectivity of functionally identified individual neurons is necessary to understand how activity patterns emerge and support behavior. Yet, the brain-wide presynaptic wiring rules that lay the foundation for the functional selectivity of individual neurons remain largely unexplored. Cortical neurons, even in primary sensory cortex, are heterogeneous in their selectivity, not only to sensory stimuli but also to multiple aspects of behavior. Here, to investigate presynaptic connectivity rules underlying the selectivity of pyramidal neurons to behavioral state 1-12 in primary somatosensory cortex (S1), we used two-photon calcium imaging, neuropharmacology, single-cell based monosynaptic input tracing, and optogenetics. We show that behavioral state-dependent neuronal activity patterns are stable over time. These are minimally affected by neuromodulatory inputs and are instead driven by glutamatergic inputs. Analysis of brain-wide presynaptic networks of individual neurons with distinct behavioral state-dependent activity profiles revealed characteristic patterns of anatomical input. While both behavioral state-related and unrelated neurons had a similar pattern of local inputs within S1, their long-range glutamatergic inputs differed. Individual cortical neurons, irrespective of their functional properties, received converging inputs from the main S1-projecting areas. Yet, neurons that tracked behavioral state received a smaller proportion of motor cortical inputs and a larger proportion of thalamic inputs. Optogenetic suppression of thalamic inputs reduced behavioral state-dependent activity in S1, but this activity was not externally driven. Our results revealed distinct long-range glutamatergic inputs as a substrate for preconfigured network dynamics associated with behavioral state.

Epigenetic Dynamics in Reprogramming to Dopaminergic Neurons for Parkinson's Disease.

Direct lineage reprogramming into dopaminergic (DA) neurons holds great promise for the more effective production of DA neurons, offering potential therapeutic benefits for conditions such as Parkinson's disease. However, the reprogramming pathway for fully reprogrammed DA neurons remains largely unclear, resulting in immature and dead-end states with low efficiency. In this study, using single-cell RNA sequencing, the trajectory of reprogramming DA neurons at multiple time points, identifying a continuous pathway for their reprogramming is analyzed. It is identified that intermediate cell populations are crucial for resetting host cell fate during early DA neuronal reprogramming. Further, longitudinal dissection uncovered two distinct trajectories: one leading to successful reprogramming and the other to a dead end. Notably, Arid4b, a histone modifier, as a crucial regulator at this branch point, essential for the successful trajectory and acquisition of mature dopaminergic neuronal identity is identified. Consistently, overexpressing Arid4b in the DA neuronal reprogramming process increases the yield of iDA neurons and effectively reverses the disease phenotypes observed in the PD mouse brain. Thus, gaining insights into the cellular trajectory holds significant importance for devising regenerative medicine strategies, particularly in the context of addressing neurodegenerative disorders like Parkinson's disease.