Embracing diversity in the 5-HT neuronal system.

Neurons that synthesize and release 5-hydroxytryptamine (5-HT; serotonin) express a core set of genes that establish and maintain this neurotransmitter phenotype and distinguish these neurons from other brain cells. Beyond a shared 5-HTergic phenotype, these neurons display divergent cellular properties in relation to anatomy, morphology, hodology, electrophysiology and gene expression, including differential expression of molecules supporting co-transmission of additional neurotransmitters. This diversity suggests that functionally heterogeneous subtypes of 5-HT neurons exist, but linking subsets of these neurons to particular functions has been technically challenging. We discuss recent data from molecular genetic, genomic and functional methods that, when coupled with classical findings, yield a reframing of the 5-HT neuronal system as a conglomeration of diverse subsystems with potential to inspire novel, more targeted therapies for clinically distinct 5-HT-related disorders.

Altered oligodendroglia and astroglia in chronic traumatic encephalopathy.

Chronic traumatic encephalopathy (CTE) is a progressive tauopathy found in contact sport athletes, military veterans, and others exposed to repetitive head impacts. White matter rarefaction and axonal loss have been reported in CTE but have not been characterized on a molecular or cellular level. Here, we present RNA sequencing profiles of cell nuclei from postmortem dorsolateral frontal white matter from eight individuals with neuropathologically confirmed CTE and eight age- and sex-matched controls. Analyzing these profiles using unbiased clustering approaches, we identified eighteen transcriptomically distinct cell groups (clusters), reflecting cell types and/or cell states, of which a subset showed differences between CTE and control tissue. Independent in situ methods applied on tissue sections adjacent to that used in the single-nucleus RNA-seq work yielded similar findings. Oligodendrocytes were found to be most severely affected in the CTE white matter samples; they were diminished in number and altered in relative proportions across subtype clusters. Further, the CTE-enriched oligodendrocyte population showed greater abundance of transcripts relevant to iron metabolism and cellular stress response. CTE tissue also demonstrated excessive iron accumulation histologically. In astrocytes, total cell numbers were indistinguishable between CTE and control samples, but transcripts associated with neuroinflammation were elevated in the CTE astrocyte groups compared to controls. These results demonstrate specific molecular and cellular differences in CTE oligodendrocytes and astrocytes and suggest that white matter alterations are a critical aspect of CTE neurodegeneration.

Cell-type-based model explaining coexpression patterns of genes in the brain.

Spatial patterns of gene expression in the vertebrate brain are not independent, as pairs of genes can exhibit complex patterns of coexpression. Two genes may be similarly expressed in one region, but differentially expressed in other regions. These correlations have been studied quantitatively, particularly for the Allen Atlas of the adult mouse brain, but their biological meaning remains obscure. We propose a simple model of the coexpression patterns in terms of spatial distributions of underlying cell types and establish its plausibility using independently measured cell-type-specific transcriptomes. The model allows us to predict the spatial distribution of cell types in the mouse brain.

Cell-type-specific repression by methyl-CpG-binding protein 2 is biased toward long genes.

Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome and related autism spectrum disorders (Amir et al., 1999). MeCP2 is believed to be required for proper regulation of brain gene expression, but prior microarray studies in Mecp2 knock-out mice using brain tissue homogenates have revealed only subtle changes in gene expression (Tudor et al., 2002; Nuber et al., 2005; Jordan et al., 2007; Chahrour et al., 2008). Here, by profiling discrete subtypes of neurons we uncovered more dramatic effects of MeCP2 on gene expression, overcoming the "dilution problem" associated with assaying homogenates of complex tissues. The results reveal misregulation of genes involved in neuronal connectivity and communication. Importantly, genes upregulated following loss of MeCP2 are biased toward longer genes but this is not true for downregulated genes, suggesting MeCP2 may selectively repress long genes. Because genes involved in neuronal connectivity and communication, such as cell adhesion and cell-cell signaling genes, are enriched among longer genes, their misregulation following loss of MeCP2 suggests a possible etiology for altered circuit function in Rett syndrome.

Altered 5-HT2A/C receptor binding in the medulla oblongata in the sudden infant death syndrome (SIDS): Part II. Age-associated alterations in serotonin receptor binding profiles within medullary nuclei supporting cardiorespiratory homeostasis.

The failure of chemoreflexes, arousal, and/or autoresuscitation to asphyxia may underlie some sudden infant death syndrome (SIDS) cases. In Part I, we showed that some SIDS infants had altered 5-hydroxytryptamine (5-HT)2A/C receptor binding in medullary nuclei supporting chemoreflexes, arousal, and autoresuscitation. Here, using the same dataset, we tested the hypotheses that the prevalence of low 5-HT1A and/or 5-HT2A/C receptor binding (defined as levels below the 95% confidence interval of controls-a new approach), and the percentages of nuclei affected are greater in SIDS versus controls, and that the distribution of low binding varied with age of death. The prevalence and percentage of nuclei with low 5-HT1A and 5-HT2A/C binding in SIDS were twice that of controls. The percentage of nuclei with low 5-HT2A/C binding was greater in older SIDS infants. In >80% of older SIDS infants, low 5-HT2A/C binding characterized the hypoglossal nucleus, vagal dorsal nucleus, nucleus of solitary tract, and nuclei of the olivocerebellar subnetwork (important for blood pressure regulation). Together, our findings from SIDS infants and from animal models of serotonergic dysfunction suggest that some SIDS cases represent a serotonopathy. We present new hypotheses, yet to be tested, about how defects within serotonergic subnetworks may lead to SIDS.

Multiomic Analysis of Neuroinflammation and Occult Infection in Sudden Infant Death Syndrome.

Importance: Antemortem infection is a risk factor for sudden infant death syndrome (SIDS)-the leading postneonatal cause of infant mortality in the developed world. Manifestations of infection and inflammation are not always apparent in clinical settings or by standard autopsy; thus, enhanced resolution approaches are needed. Objective: To ascertain whether a subset of SIDS cases is associated with neuroinflammation and occult infection. Design, Setting, and Participants: In this case-control study, postmortem fluids from SIDS cases and controls collected between July 2011 and November 2018 were screened for elevated inflammatory markers, specifically cerebrospinal fluid (CSF) neopterin and CSF and serum cytokines. CSF, liver, and brain tissue from SIDS cases with elevated CSF neopterin were subjected to metagenomic next-generation sequencing (mNGS) to probe for infectious pathogens. Brainstem tissue from a subset of these cases was analyzed by single-nucleus RNA sequencing (snRNAseq) to measure cell type-specific gene expression associated with neuroinflammation and infection. All tissue and fluid analyses were performed from April 2019 to January 2023 in a pathology research laboratory. Included was autopsy material from infants dying of SIDS and age-matched controls dying of known causes. Exposures: There were no interventions or exposures. Main Outcomes and Measures: CSF neopterin levels were measured by high-performance liquid chromatography. Cytokines were measured by multiplex fluorometric assay. mNGS was performed on liver, CSF, brain, and brainstem tissue. snRNAseq was performed on brainstem tissue. Results: A cohort of 71 SIDS cases (mean [SD] age, 55.2 [11.4] postconceptional weeks; 42 male [59.2%]) and 20 controls (mean [SD] age, 63.2 [16.9] postconceptional weeks; 11 male [55.0%]) had CSF and/or serum available. CSF neopterin was screened in 64 SIDS cases and 15 controls, with no exclusions. Tissues from 6 SIDS cases were further analyzed. For CSF neopterin measures, SIDS samples were from infants with mean (SD) age of 54.5 (11.3) postconceptional weeks (38 male [59.4%]) and control samples were from infants with mean (SD) age of 61.5 (17.4) postconceptional weeks (7 male [46.7%]). A total of 6 SIDS cases (9.3%) with high CSF neopterin were identified, suggestive of neuroinflammation. mNGS detected human parechovirus 3 (HPeV3) in tissue and CSF from 1 of these 6 cases. snRNAseq of HPeV3-positive brainstem tissue (medulla) revealed dramatic enrichment of transcripts for genes with predominately inflammatory functions compared with 3 age-matched SIDS cases with normal CSF neopterin levels. Conclusions and Relevance: Next-generation molecular tools in autopsy tissue provide novel insight into pathogens that go unrecognized by normal autopsy methodology, including in infants dying suddenly and unexpectedly.

Transcriptional and electrophysiological maturation of neocortical fast-spiking GABAergic interneurons.

Fast-spiking (FS) interneurons are important elements of neocortical circuitry that constitute the primary source of synaptic inhibition in adult cortex and impart temporal organization on ongoing cortical activity. The highly specialized intrinsic membrane and firing properties that allow cortical FS interneurons to perform these functions are attributable to equally specialized gene expression, which is ultimately coordinated by cell-type-specific transcriptional regulation. Although embryonic transcriptional events govern the initial steps of cell-type specification in most cortical interneurons, including FS cells, the electrophysiological properties that distinguish adult cortical cell types emerge relatively late in postnatal development, and the transcriptional events that drive this maturational process are not known. To address this, we used mouse whole-genome microarrays and whole-cell patch clamp to characterize the transcriptional and electrophysiological maturation of cortical FS interneurons between postnatal day 7 (P7) and P40. We found that the intrinsic and synaptic physiology of FS cells undergoes profound regulation over the first 4 postnatal weeks and that these changes are correlated with primarily monotonic but bidirectional transcriptional regulation of thousands of genes belonging to multiple functional classes. Using our microarray screen as a guide, we discovered that upregulation of two-pore K(+) leak channels between P10 and P25 contributes to one of the major differences between the intrinsic membrane properties of immature and adult FS cells and found a number of other candidate genes that likely confer cell-type specificity on mature FS cells.

A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

Region-specific spike-frequency acceleration in layer 5 pyramidal neurons mediated by Kv1 subunits.

Separation of the cortical sheet into functionally distinct regions is a hallmark of neocortical organization. Cortical circuit function emerges from afferent and efferent connectivity, local connectivity within the cortical microcircuit, and the intrinsic membrane properties of neurons that comprise the circuit. While localization of functions to particular cortical areas can be partially accounted for by regional differences in both long range and local connectivity, it is unknown whether the intrinsic membrane properties of cortical cell types differ between cortical regions. Here we report the first example of a region-specific firing type in layer 5 pyramidal neurons, and show that the intrinsic membrane and integrative properties of a discrete subtype of layer 5 pyramidal neurons differ between primary motor and somatosensory cortices due to region- and cell-type-specific Kv1 subunit expression.

Molecular Profiling Defines Evolutionarily Conserved Transcription Factor Signatures of Major Vestibulospinal Neuron Groups.

Vestibulospinal neurons are organized into discrete groups projecting from brainstem to spinal cord, enabling vertebrates to maintain proper balance and posture. The two largest groups are the lateral vestibulospinal tract (LVST) group and the contralateral medial vestibulospinal tract (cMVST) group, with different projection lateralities and functional roles. In search of a molecular basis for these differences, we performed RNA sequencing on LVST and cMVST neurons from mouse and chicken embryos followed by immunohistofluorescence validation. Focusing on transcription factor (TF)-encoding genes, we identified TF signatures that uniquely distinguish the LVST from the cMVST group and further parse different rhombomere-derived portions comprising the cMVST group. Immunohistofluorescence assessment of the CNS from spinal cord to cortex demonstrated that these TF signatures are restricted to the respective vestibulospinal groups and some neurons in their immediate vicinity. Collectively, these results link the combinatorial expression of TFs to developmental and functional subdivisions within the vestibulospinal system.

Serotonergic Modulation of Aggression in Drosophila Involves GABAergic and Cholinergic Opposing Pathways.

Pathological aggression is commonly associated with psychiatric and neurological disorders and can impose a substantial burden and cost on human society. Serotonin (5HT) has long been implicated in the regulation of aggression in a wide variety of animal species. In Drosophila, a small group of serotonergic neurons selectively modulates the escalation of aggression. Here, we identified downstream targets of serotonergic input-two types of neurons with opposing roles in aggression control. The dendritic fields of both neurons converge on a single optic glomerulus LC12, suggesting a key pathway linking visual input to the aggression circuitry. The first type is an inhibitory GABAergic neuron: its activation leads to a decrease in aggression. The second neuron type is excitatory: its silencing reduces and its activation increases aggression. RNA sequencing (RNA-seq) profiling of this neuron type identified that it uses acetylcholine as a neurotransmitter and likely expresses 5HT1A, short neuropeptide F receptor (sNPFR), and the resistant to dieldrin (RDL) category of GABA receptors. Knockdown of RDL receptors in these neurons increases aggression, suggesting the possibility of a direct crosstalk between the inhibitory GABAergic and the excitatory cholinergic neurons. Our data show further that neurons utilizing serotonin, GABA, ACh, and short neuropeptide F interact in the LC12 optic glomerulus. Parallel cholinergic and GABAergic pathways descending from this sensory integration area may be key elements in fine-tuning the regulation of aggression.

Identification of Serotonergic Neuronal Modules that Affect Aggressive Behavior.

Escalated aggression can have devastating societal consequences, yet underlying neurobiological mechanisms are poorly understood. Here, we show significantly increased inter-male mouse aggression when neurotransmission is constitutively blocked from either of two subsets of serotonergic, Pet1+ neurons: one identified by dopamine receptor D1(Drd1a)::cre-driven activity perinatally, and the other by Drd2::cre from pre-adolescence onward. Blocking neurotransmission from other Pet1+ neuron subsets of similar size and/or overlapping anatomical domains had no effect on aggression compared with controls, suggesting subtype-specific serotonergic neuron influences on aggression. Using established and novel intersectional genetic tools, we further characterized these subtypes across multiple parameters, showing both overlapping and distinct features in axonal projection targets, gene expression, electrophysiological properties, and effects on non-aggressive behaviors. Notably, Drd2::cre marked 5-HT neurons exhibited D2-dependent inhibitory responses to dopamine in slices, suggesting direct and specific interplay between inhibitory dopaminergic signaling and a serotonergic subpopulation. Thus, we identify specific serotonergic modules that shape aggression.

Upregulation of µ3A Drives Homeostatic Plasticity by Rerouting AMPAR into the Recycling Endosomal Pathway.

Synaptic scaling is a form of homeostatic plasticity driven by transcription-dependent changes in AMPA-type glutamate receptor (AMPAR) trafficking. To uncover the pathways involved, we performed a cell-type-specific screen for transcripts persistently altered during scaling, which identified the µ subunit (µ3A) of the adaptor protein complex AP-3A. Synaptic scaling increased µ3A (but not other AP-3 subunits) in pyramidal neurons and redistributed dendritic µ3A and AMPAR to recycling endosomes (REs). Knockdown of µ3A prevented synaptic scaling and this redistribution, while overexpression (OE) of full-length µ3A or a truncated µ3A that cannot interact with the AP-3A complex was sufficient to drive AMPAR to REs. Finally, OE of µ3A acted synergistically with GRIP1 to recruit AMPAR to the dendritic membrane. These data suggest that excess µ3A acts independently of the AP-3A complex to reroute AMPAR to RE, generating a reservoir of receptors essential for the regulated recruitment to the synaptic membrane during scaling up.

Multi-Scale Molecular Deconstruction of the Serotonin Neuron System.

Serotonergic (5HT) neurons modulate diverse behaviors and physiology and are implicated in distinct clinical disorders. Corresponding diversity in 5HT neuronal phenotypes is becoming apparent and is likely rooted in molecular differences, yet a comprehensive approach characterizing molecular variation across the 5HT system is lacking, as is concomitant linkage to cellular phenotypes. Here we combine intersectional fate mapping, neuron sorting, and genome-wide RNA-seq to deconstruct the mouse 5HT system at multiple levels of granularity-from anatomy, to genetic sublineages, to single neurons. Our unbiased analyses reveal principles underlying system organization, 5HT neuron subtypes, constellations of differentially expressed genes distinguishing subtypes, and predictions of subtype-specific functions. Using electrophysiology, subtype-specific neuron silencing, and conditional gene knockout, we show that these molecularly defined 5HT neuron subtypes are functionally distinct. Collectively, this resource classifies molecular diversity across the 5HT system and discovers sertonergic subtypes, markers, organizing principles, and subtype-specific functions with potential disease relevance.

A quantitative comparison of cell-type-specific microarray gene expression profiling methods in the mouse brain.

Expression profiling of restricted neural populations using microarrays can facilitate neuronal classification and provide insight into the molecular bases of cellular phenotypes. Due to the formidable heterogeneity of intermixed cell types that make up the brain, isolating cell types prior to microarray processing poses steep technical challenges that have been met in various ways. These methodological differences have the potential to distort cell-type-specific gene expression profiles insofar as they may insufficiently filter out contaminating mRNAs or induce aberrant cellular responses not normally present in vivo. Thus we have compared the repeatability, susceptibility to contamination from off-target cell-types, and evidence for stress-responsive gene expression of five different purification methods--Laser Capture Microdissection (LCM), Translating Ribosome Affinity Purification (TRAP), Immunopanning (PAN), Fluorescence Activated Cell Sorting (FACS), and manual sorting of fluorescently labeled cells (Manual). We found that all methods obtained comparably high levels of repeatability, however, data from LCM and TRAP showed significantly higher levels of contamination than the other methods. While PAN samples showed higher activation of apoptosis-related, stress-related and immediate early genes, samples from FACS and Manual studies, which also require dissociated cells, did not. Given that TRAP targets actively translated mRNAs, whereas other methods target all transcribed mRNAs, observed differences may also reflect translational regulation.

Activity-dependent changes in the firing properties of neocortical fast-spiking interneurons in the absence of large changes in gene expression.

The diverse cell types that comprise neocortical circuits each have characteristic integrative and firing properties that are specialized to perform specific functions within the network. Parvalbumin-positive fast-spiking (FS) interneurons are a particularly specialized cortical cell-type that controls the dynamics of ongoing activity and prevents runaway excitation by virtue of remarkably high firing rates, a feature that is permitted by narrow action potentials and the absence of spike-frequency adaptation. Although several neuronal intrinsic membrane properties undergo activity-dependent plasticity, the role of network activity in shaping and maintaining specialized, cell-type-specific firing properties is unknown. We tested whether the specialized firing properties of mature FS interneurons are sensitive to activity perturbations by inactivating a portion of motor cortex in vivo for 48 h and measuring resulting plasticity of FS intrinsic and firing properties with whole-cell recording in acute slices. Many of the characteristic properties of FS interneurons, including nonadapting high-frequency spiking and narrow action potentials, were profoundly affected by activity deprivation both at an age just after maturation of FS firing properties and also a week after their maturation. Using microarray screening, we determined that although normal maturation of FS electrophysiological specializations is accompanied by large-scale transcriptional changes, the effects of deprivation on the same specializations involve more modest transcriptional changes, and may instead be primarily mediated by post-transcriptional mechanisms.