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The hippocampus contains many unique cell types, which serve the structure's specialized functions, including learning, memory and cognition. These cells have distinct spatial organization, morphology, physiology, and connectivity, highlighting the importance of transcriptome-wide profiling strategies that retain cytoarchitectural organization. Here, we generated spatially-resolved transcriptomics (SRT) and single-nucleus RNA-sequencing (snRNA-seq) data from adjacent tissue sections of the anterior human hippocampus in ten adult neurotypical donors to define molecular profiles for hippocampal cell types and spatial domains. Using non-negative matrix factorization (NMF) and label transfer, we integrated these data by defining gene expression patterns within the snRNA-seq data and inferring their expression in the SRT data. We identified NMF patterns that captured transcriptional variation across neuronal cell types and indicated that the response of excitatory and inhibitory postsynaptic specializations were prioritized in different SRT spatial domains. We used the NMF and label transfer approach to leverage existing rodent datasets, identifying patterns of activity-dependent transcription and subpopulations of dentate gyrus granule cells in our SRT dataset that may be predisposed to participate in learning and memory ensembles. Finally, we characterized the spatial organization of NMF patterns corresponding to non- cornu ammonis pyramidal neurons and identified snRNA-seq clusters mapping to distinct regions of the retrohippocampus, to three subiculum layers, and to a population of presubiculum neurons. To make this comprehensive molecular atlas accessible to the scientific community, both raw and processed data are freely available, including through interactive web applications., Competing Interests: Conflict of Interest: Co-Author Erik Nelson is now a full-time employee at GSK, which is unrelated to the contents of this manuscript. His contributions to this manuscript were made while previously enrolled as a student at Johns Hopkins University and performing research at Lieber Institute for Brain Development (LIBD). Co-Author Joel E. Kleinman is a consultant on a Data Monitoring Committee for an antipsychotic drug trial for Merck & Co., Inc. All other authors have no declared conflicts of interests.

Over the past decade, single-cell genomics technologies have allowed scalable profiling of cell-type-specific features, which has substantially increased our ability to study cellular diversity and transcriptional programs in heterogeneous tissues. Yet our understanding of mechanisms of gene regulation or the rules that govern interactions between cell types is still limited. The advent of new computational pipelines and technologies, such as single-cell epigenomics and spatially resolved transcriptomics, has created opportunities to explore two new axes of biological variation: cell-intrinsic regulation of cell states and expression programs and interactions between cells. Here, we summarize the most promising and robust technologies in these areas, discuss their strengths and limitations and discuss key computational approaches for analysis of these complex datasets. We highlight how data sharing and integration, documentation, visualization and benchmarking of results contribute to transparency, reproducibility, collaboration and democratization in neuroscience, and discuss needs and opportunities for future technology development and analysis., Competing Interests: Competing interests: S.A.L. declares a financial interest in AstronauTx and Synapticure. All other authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Maternal smoking during pregnancy (MSDP) is associated with significant cognitive and behavioral effects on offspring. While neurodevelopmental outcomes have been studied for prenatal exposure to nicotine, the main psychoactive component of cigarette smoke, its contribution to MSDP effects has never been explored. Comparing the effects of these substances on molecular signaling in the prenatal and adult brain may provide insights into nicotinic and broader tobacco consequences that are developmental-stage specific or age-independent. Pregnant mice were administered nicotine or exposed to chronic cigarette smoke, and RNA-sequencing was performed on frontal cortices of postnatal day 0 pups born to these mice, as well as on frontal cortices and blood of the adult dams. We identified 1,010 and 4,165 differentially expressed genes (DEGs) in nicotine and smoking-exposed pup brains, respectively (FDR<0.05, Ns = 19 nicotine-exposed vs 23 vehicle-exposed; 46 smoking-exposed vs 49 controls). Prenatal nicotine exposure (PNE) alone was related to dopaminergic synapses and long-term synaptic depression, whereas MSDP was associated with the SNARE complex and vesicle transport. Both substances affected SMN-Sm protein complexes and postsynaptic endosomes. Analyses at the transcript, exon, and exon-exon junction levels supported gene level results and revealed additional smoking-affected processes. No DEGs at FDR<0.05 were found in adult mouse brain for any substance (12 nicotine-administered vs 11 vehicle-administered; 12 smoking-exposed vs 12 controls), nor in adult blood (12 smoking-exposed vs 12 controls), and only 3% and 6.41% of the DEGs in smoking-exposed pup brain replicated in smoking-exposed blood and human prenatal brain, respectively. Together, these results demonstrate variable but overlapping molecular effects of PNE and MSDP on the developing brain, and attenuated effects of both smoking and nicotine on adult versus fetal brain., Competing Interests: CONFLICT OF INTEREST Andrew E. Jaffe (AEJ) is currently a full-time employee at Neumora Therapeutics. AEJ’s current work is unrelated to the contents of this manuscript, and his contributions to this manuscript were made while previously employed at LIBD. No other authors have financial relationships with commercial interests, and the authors declare no competing interests.

Pathophysiology of many neuropsychiatric disorders, including schizophrenia (SCZD), is linked to habenula (Hb) function. While pharmacotherapies and deep brain stimulation targeting the Hb are emerging as promising therapeutic treatments, little is known about the cell type-specific transcriptomic organization of the human Hb or how it is altered in SCZD. Here we define the molecular neuroanatomy of the human Hb and identify transcriptomic changes in individuals with SCZD compared to neurotypical controls. Utilizing Hb-enriched postmortem human brain tissue, we performed single nucleus RNA-sequencing (snRNA-seq; n=7 neurotypical donors) and identified 17 molecularly defined Hb cell types across 16,437 nuclei, including 3 medial and 7 lateral Hb populations, several of which were conserved between rodents and humans. Single molecule fluorescent in situ hybridization (smFISH; n=3 neurotypical donors) validated snRNA-seq Hb cell types and mapped their spatial locations. Bulk RNA-sequencing and cell type deconvolution in Hb-enriched tissue from 35 individuals with SCZD and 33 neurotypical controls yielded 45 SCZD-associated differentially expressed genes (DEGs, FDR < 0.05), with 32 (71%) unique to Hb-enriched tissue. eQTL analysis identified 717 independent SNP-gene pairs (FDR < 0.05), where either the SNP is a SCZD risk variant (16 pairs) or the gene is a SCZD DEG (7 pairs). eQTL and SCZD risk colocalization analysis identified 16 colocalized genes. These results identify topographically organized cell types with distinct molecular signatures in the human Hb and demonstrate unique genetic changes associated with SCZD, thereby providing novel molecular insights into the role of Hb in neuropsychiatric disorders., One Sentence Summary: Transcriptomic analysis of the human habenula and identification of molecular changes associated with schizophrenia risk and illness state.

Drugs of abuse activate defined neuronal ensembles in brain reward structures such as the nucleus accumbens (NAc), which are thought to promote the enduring synaptic, circuit, and behavioral consequences of drug exposure. While the molecular and cellular effects arising from experience with drugs like cocaine are increasingly well understood, the mechanisms that sculpt NAc ensemble participation are largely unknown. Here, we leveraged unbiased single-nucleus transcriptional profiling to identify expression of the secreted glycoprotein Reelin (encoded by the Reln gene) as a marker of cocaine-activated neuronal ensembles within the rat NAc. Multiplexed in situ detection confirmed selective expression of the immediate early gene Fos in Reln+ neurons after cocaine experience, and also revealed enrichment of Reln mRNA in Drd1 + medium spiny neurons (MSNs) in both the rat and human brain. Using a novel CRISPR interference strategy enabling selective Reln knockdown in the adult NAc, we observed altered expression of genes linked to calcium signaling, emergence of a transcriptional trajectory consistent with loss of cocaine sensitivity, and a striking decrease in MSN intrinsic excitability. At the behavioral level, loss of Reln prevented cocaine locomotor sensitization, abolished cocaine place preference memory, and decreased cocaine self-administration behavior. Together, these results identify Reelin as a critical mechanistic link between ensemble participation and cocaine-induced behavioral adaptations.

The molecular organization of the human neocortex historically has been studied in the context of its histological layers. However, emerging spatial transcriptomic technologies have enabled unbiased identification of transcriptionally defined spatial domains that move beyond classic cytoarchitecture. We used the Visium spatial gene expression platform to generate a data-driven molecular neuroanatomical atlas across the anterior-posterior axis of the human dorsolateral prefrontal cortex. Integration with paired single-nucleus RNA-sequencing data revealed distinct cell type compositions and cell-cell interactions across spatial domains. Using PsychENCODE and publicly available data, we mapped the enrichment of cell types and genes associated with neuropsychiatric disorders to discrete spatial domains.

The dentate gyrus of the hippocampus is important for many cognitive functions, including learning, memory, and mood. Here, we investigated age-associated changes in transcriptome-wide spatial gene expression in the human dentate gyrus across the lifespan. Genes associated with neurogenesis and the extracellular matrix were enriched in infants, while gene markers of inhibitory neurons and cell proliferation showed increases and decreases in post-infancy, respectively. While we did not find evidence for neural proliferation post-infancy, we did identify molecular signatures supporting protracted maturation of granule cells. We also identified a wide-spread hippocampal aging signature and an age-associated increase in genes related to neuroinflammation. Our findings suggest major changes to the putative neurogenic niche after infancy and identify molecular foci of brain aging in glial and neuropil enriched tissue.

Background: Cellular deconvolution of bulk RNA-sequencing (RNA-seq) data using single cell or nuclei RNA-seq (sc/snRNA-seq) reference data is an important strategy for estimating cell type composition in heterogeneous tissues, such as human brain. Computational methods for deconvolution have been developed and benchmarked against simulated data, pseudobulked sc/snRNA-seq data, or immunohistochemistry reference data. A major limitation in developing improved deconvolution algorithms has been the lack of integrated datasets with orthogonal measurements of gene expression and estimates of cell type proportions on the same tissue sample. Deconvolution algorithm performance has not yet been evaluated across different RNA extraction methods (cytosolic, nuclear, or whole cell RNA), different library preparation types (mRNA enrichment vs. ribosomal RNA depletion), or with matched single cell reference datasets., Results: A rich multi-assay dataset was generated in postmortem human dorsolateral prefrontal cortex (DLPFC) from 22 tissue blocks. Assays included spatially-resolved transcriptomics, snRNA-seq, bulk RNA-seq (across six library/extraction RNA-seq combinations), and RNAScope/Immunofluorescence (RNAScope/IF) for six broad cell types. The Mean Ratio method, implemented in the DeconvoBuddies R package, was developed for selecting cell type marker genes. Six computational deconvolution algorithms were evaluated in DLPFC and predicted cell type proportions were compared to orthogonal RNAScope/IF measurements., Conclusions: Bisque and hspe were the most accurate methods, were robust to differences in RNA library types and extractions. This multi-assay dataset showed that cell size differences, marker genes differentially quantified across RNA libraries, and cell composition variability in reference snRNA-seq impact the accuracy of current deconvolution methods., Competing Interests: Competing interests The authors declare that they have no competing interests.

Relative cell type fraction estimates in bulk RNA-sequencing data are important to control for cell composition differences across heterogenous tissue samples. Current computational tools estimate relative RNA abundances rather than cell type proportions in tissues with varying cell sizes, leading to biased estimates. We present lute , a computational tool to accurately deconvolute cell types with varying sizes. Our software wraps existing deconvolution algorithms in a standardized framework. Using simulated and real datasets, we demonstrate how lute adjusts for differences in cell sizes to improve the accuracy of cell composition. Software is available from https://bioconductor.org/packages/lute., Competing Interests: Competing interests The authors declare that they have no competing interests.

Norepinephrine (NE) neurons in the locus coeruleus (LC) make long-range projections throughout the central nervous system, playing critical roles in arousal and mood, as well as various components of cognition including attention, learning, and memory. The LC-NE system is also implicated in multiple neurological and neuropsychiatric disorders. Importantly, LC-NE neurons are highly sensitive to degeneration in both Alzheimer's and Parkinson's disease. Despite the clinical importance of the brain region and the prominent role of LC-NE neurons in a variety of brain and behavioral functions, a detailed molecular characterization of the LC is lacking. Here, we used a combination of spatially-resolved transcriptomics and single-nucleus RNA-sequencing to characterize the molecular landscape of the LC region and the transcriptomic profile of LC-NE neurons in the human brain. We provide a freely accessible resource of these data in web-accessible and downloadable formats., Competing Interests: LW, HD, SK, AS, KM, MT, RB, JK, SP, TH, LC, KM, KM, SH No competing interests declared, MT The authors declare that they have no competing interests. Matthew N. Tran (MNT) is now a full-time employee at 23andMe and whose current work is unrelated to the contents of this manuscript. His contributions to this manuscript were made while previously employed at the Lieber Institute for Brain Development (LIBD), (© 2023, Weber, Divecha et al.)

Deconvolution of cell mixtures in "bulk" transcriptomic samples from homogenate human tissue is important for understanding disease pathologies. However, several experimental and computational challenges impede transcriptomics-based deconvolution approaches using single-cell/nucleus RNA-seq reference atlases. Cells from the brain and blood have substantially different sizes, total mRNA, and transcriptional activities, and existing approaches may quantify total mRNA instead of cell type proportions. Further, standards are lacking for the use of cell reference atlases and integrative analyses of single-cell and spatial transcriptomics data. We discuss how to approach these key challenges with orthogonal "gold standard" datasets for evaluating deconvolution methods., (© 2023. The Author(s).)

Spatially resolved transcriptomics (SRT) is a growing field that links gene expression to anatomical context. SRT approaches that use next-generation sequencing (NGS) combine RNA sequencing with histological or fluorescent imaging to generate spatial maps of gene expression in intact tissue sections. These technologies directly couple gene expression measurements with high-resolution histological or immunofluorescent images that contain rich morphological information about the tissue under study. While broad access to NGS-based spatial transcriptomic technology is now commercially available through the Visium platform from the vendor 10× Genomics, computational tools for extracting image-derived metrics for integration with gene expression data remain limited. We developed VistoSeg as a MATLAB pipeline to process, analyze and interactively visualize the high-resolution images generated in the Visium platform. VistoSeg outputs can be easily integrated with accompanying transcriptomic data to facilitate downstream analyses in common programing languages including R and Python. VistoSeg provides user-friendly tools for integrating image-derived metrics from histological and immunofluorescent images with spatially resolved gene expression data. Integration of this data enhances the ability to understand the transcriptional landscape within tissue architecture. VistoSeg is freely available at http://research.libd.org/VistoSeg/., Competing Interests: A.E.J. is now a full-time employee at Neumora Therapeutics, a for-profit biotechnology company, which is unrelated to the contents of this manuscript. J.L.C. is now a full-time employee at Delfi Diagnostics, a for-profit biotechnology company, which is unrelated to the contents of this manuscript. Their contributions to the manuscript were made while previously employed by the Lieber Institute for Brain Development. All other authors declare no competing interests., (© The Author(s) 2023.)

We define and identify a new class of control genes for next-generation sequencing called total RNA expression genes (TREGs), which correlate with total RNA abundance in cell types of different sizes and transcriptional activity. We provide a data-driven method to identify TREGs from single-cell RNA sequencing data, allowing the estimation of total amount of RNA when restricted to quantifying a limited number of genes. We demonstrate our method in postmortem human brain using multiplex single-molecule fluorescent in situ hybridization and compare candidate TREGs against classic housekeeping genes. We identify AKT3 as a top TREG across five brain regions., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Neuropathological lesions in the brains of individuals affected with neurodegenerative disorders are hypothesized to trigger molecular and cellular processes that disturb homeostasis of local microenvironments. Here, we applied the 10x Genomics Visium Spatial Proteogenomics (Visium-SPG) platform, which couples spatial gene expression with immunofluorescence protein co-detection, to evaluate its ability to quantify changes in spatial gene expression with respect to amyloid-β (Aβ) and hyperphosphorylated tau (pTau) pathology in post-mortem human brain tissue from individuals with Alzheimer's disease (AD). We identified transcriptomic signatures associated with proximity to Aβ in the human inferior temporal cortex (ITC) during late-stage AD, which we further investigated at cellular resolution with combined immunofluorescence and single molecule fluorescent in situ hybridization (smFISH). The study provides a data analysis workflow for Visium-SPG, and the data represent a proof-of-principal for the power of multi-omic profiling in identifying changes in molecular dynamics that are spatially-associated with pathology in the human brain. We provide the scientific community with web-based, interactive resources to access the datasets of the spatially resolved AD-related transcriptomes at https://research.libd.org/Visium&#95;SPG&#95;AD/., Competing Interests: Author disclosure statement SRW and MM are employees of 10x Genomics. All other authors declare no conflicts of interest.

Activity-regulated gene (ARG) expression patterns in the hippocampus (HPC) regulate synaptic plasticity, learning, and memory, and are linked to both risk and treatment responses for many neuropsychiatric disorders. The HPC contains discrete classes of neurons with specialized functions, but cell type-specific activity-regulated transcriptional programs are not well characterized. Here, we used single-nucleus RNA-sequencing (snRNA-seq) in a mouse model of acute electroconvulsive seizures (ECS) to identify cell type-specific molecular signatures associated with induced activity in HPC neurons. We used unsupervised clustering and a priori marker genes to computationally annotate 15,990 high-quality HPC neuronal nuclei from N = 4 mice across all major HPC subregions and neuron types. Activity-induced transcriptomic responses were divergent across neuron populations, with dentate granule cells being particularly responsive to activity. Differential expression analysis identified both upregulated and downregulated cell type-specific gene sets in neurons following ECS. Within these gene sets, we identified enrichment of pathways associated with varying biological processes such as synapse organization, cellular signaling, and transcriptional regulation. Finally, we used matrix factorization to reveal continuous gene expression patterns differentially associated with cell type, ECS, and biological processes. This work provides a rich resource for interrogating activity-regulated transcriptional responses in HPC neurons at single-nuclei resolution in the context of ECS, which can provide biological insight into the roles of defined neuronal subtypes in HPC function., (© 2023 The Authors. Hippocampus published by Wiley Periodicals LLC.)

High-resolution and multiplexed imaging techniques are giving us an increasingly detailed observation of a biological system. However, sharing, exploring, and customizing the visualization of large multidimensional images can be a challenge. Here, we introduce Samui, a performant and interactive image visualization tool that runs completely in the web browser. Samui is specifically designed for fast image visualization and annotation and enables users to browse through large images and their selected features within seconds of receiving a link. We demonstrate the broad utility of Samui with images generated with two platforms: Vizgen MERFISH and 10x Genomics Visium Spatial Gene Expression. Samui along with example datasets is available at https://samuibrowser.com., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2023.)

Deconvolution of cell mixtures in "bulk" transcriptomic samples from homogenate human tissue is important for understanding the pathologies of diseases. However, several experimental and computational challenges remain in developing and implementing transcriptomics-based deconvolution approaches, especially those using a single cell/nuclei RNA-seq reference atlas, which are becoming rapidly available across many tissues. Notably, deconvolution algorithms are frequently developed using samples from tissues with similar cell sizes. However, brain tissue or immune cell populations have cell types with substantially different cell sizes, total mRNA expression, and transcriptional activity. When existing deconvolution approaches are applied to these tissues, these systematic differences in cell sizes and transcriptomic activity confound accurate cell proportion estimates and instead may quantify total mRNA content. Furthermore, there is a lack of standard reference atlases and computational approaches to facilitate integrative analyses, including not only bulk and single cell/nuclei RNA-seq data, but also new data modalities from spatial -omic or imaging approaches. New multi-assay datasets need to be collected with orthogonal data types generated from the same tissue block and the same individual, to serve as a "gold standard" for evaluating new and existing deconvolution methods. Below, we discuss these key challenges and how they can be addressed with the acquisition of new datasets and approaches to analysis., Competing Interests: Competing interests The authors declare that they have no competing interests.

High-resolution and multiplexed imaging techniques are giving us an increasingly detailed observation of a biological system. However, sharing, exploring, and customizing the visualization of large multidimensional images can be a challenge. Here, we introduce Samui, a performant and interactive image visualization tool that runs completely in the web browser. Samui is specifically designed for fast image visualization and annotation and enables users to browse through large images and their selected features within seconds of receiving a link. We demonstrate the broad utility of Samui with images generated with two platforms: Vizgen MERFISH and 10x Genomics Visium Spatial Gene Expression. Samui along with example datasets is available at https://samuibrowser.com., Competing Interests: Competing interests The authors declare that they have no competing interests.

Generation of a molecular neuroanatomical map of the human prefrontal cortex reveals novel spatial domains and cell-cell interactions relevant for psychiatric disease. The molecular organization of the human neocortex has been historically studied in the context of its histological layers. However, emerging spatial transcriptomic technologies have enabled unbiased identification of transcriptionally-defined spatial domains that move beyond classic cytoarchitecture. Here we used the Visium spatial gene expression platform to generate a data-driven molecular neuroanatomical atlas across the anterior-posterior axis of the human dorsolateral prefrontal cortex (DLPFC). Integration with paired single nucleus RNA-sequencing data revealed distinct cell type compositions and cell-cell interactions across spatial domains. Using PsychENCODE and publicly available data, we map the enrichment of cell types and genes associated with neuropsychiatric disorders to discrete spatial domains. Finally, we provide resources for the scientific community to explore these integrated spatial and single cell datasets at research.libd.org/spatialDLPFC/., Competing Interests: Competing Interests AB is a consultant for Third Rock Ventures, LLC and a shareholder in Alphabet, Inc.

The lateral septum (LS) is a basal forebrain GABAergic region that is implicated in social novelty. However, the neural circuits and cell signaling pathways that converge on the LS to mediate social behaviors aren't well understood. Multiple lines of evidence suggest that signaling of brain-derived neurotrophic factor (BDNF) through its receptor TrkB plays important roles in social behavior. BDNF is not locally produced in LS, but we demonstrate that nearly all LS GABAergic neurons express TrkB. Local TrkB knock-down in LS neurons decreased social novelty recognition and reduced recruitment of neural activity in LS neurons in response to social novelty. Since BDNF is not synthesized in LS, we investigated which inputs to LS could serve as potential BDNF sources for controlling social novelty recognition. We demonstrate that selectively ablating inputs to LS from the basolateral amygdala (BLA), but not from ventral CA1 (vCA1), impairs social novelty recognition. Moreover, depleting BDNF selectively in BLA-LS projection neurons phenocopied the decrease in social novelty recognition caused by either local LS TrkB knockdown or ablation of BLA-LS inputs. These data support the hypothesis that BLA-LS projection neurons serve as a critical source of BDNF for activating TrkB signaling in LS neurons to control social novelty recognition., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Background: Multispectral fluorescence imaging coupled with linear unmixing is a form of image data collection and analysis that allows for measuring multiple molecular signals in a single biological sample. Multiple fluorescent dyes, each measuring a unique molecule, are simultaneously measured and subsequently "unmixed" to provide a read-out for each molecular signal. This strategy allows for measuring highly multiplexed signals in a single data capture session, such as multiple proteins or RNAs in tissue slices or cultured cells, but can often result in mixed signals and bleed-through problems across dyes. Existing spectral unmixing algorithms are not optimized for challenging biological specimens such as post-mortem human brain tissue, and often require manual intervention to extract spectral signatures. We therefore developed an intuitive, automated, and flexible package called SUFI: spectral unmixing of fluorescent images., Results: This package unmixes multispectral fluorescence images by automating the extraction of spectral signatures using vertex component analysis, and then performs one of three unmixing algorithms derived from remote sensing. We evaluate these remote sensing algorithms' performances on four unique biological datasets and compare the results to unmixing results obtained using ZEN Black software (Zeiss). We lastly integrate our unmixing pipeline into the computational tool dotdotdot, which is used to quantify individual RNA transcripts at single cell resolution in intact tissues and perform differential expression analysis, and thereby provide an end-to-end solution for multispectral fluorescence image analysis and quantification., Conclusions: In summary, we provide a robust, automated pipeline to assist biologists with improved spectral unmixing of multispectral fluorescence images., (© 2023. The Author(s).)

Objective: Posttraumatic stress disorder (PTSD) is a debilitating neuropsychiatric disease that is highly comorbid with major depressive disorder (MDD) and bipolar disorder. The overlap in symptoms is hypothesized to stem from partially shared genetics and underlying neurobiological mechanisms. To delineate conservation between transcriptional patterns across PTSD and MDD, the authors examined gene expression in the human cortex and amygdala in these disorders., Methods: RNA sequencing was performed in the postmortem brain of two prefrontal cortex regions and two amygdala regions from donors diagnosed with PTSD (N=107) or MDD (N=109) as well as from neurotypical donors (N=109)., Results: The authors identified a limited number of differentially expressed genes (DEGs) specific to PTSD, with nearly all mapping to cortical versus amygdala regions. PTSD-specific DEGs were enriched in gene sets associated with downregulated immune-related pathways and microglia as well as with subpopulations of GABAergic inhibitory neurons. While a greater number of DEGs associated with MDD were identified, most overlapped with PTSD, and only a few were MDD specific. The authors used weighted gene coexpression network analysis as an orthogonal approach to confirm the observed cellular and molecular associations., Conclusions: These findings provide supporting evidence for involvement of decreased immune signaling and neuroinflammation in MDD and PTSD pathophysiology, and extend evidence that GABAergic neurons have functional significance in PTSD.

Background: Spatially-resolved transcriptomics has now enabled the quantification of high-throughput and transcriptome-wide gene expression in intact tissue while also retaining the spatial coordinates. Incorporating the precise spatial mapping of gene activity advances our understanding of intact tissue-specific biological processes. In order to interpret these novel spatial data types, interactive visualization tools are necessary., Results: We describe spatialLIBD, an R/Bioconductor package to interactively explore spatially-resolved transcriptomics data generated with the 10x Genomics Visium platform. The package contains functions to interactively access, visualize, and inspect the observed spatial gene expression data and data-driven clusters identified with supervised or unsupervised analyses, either on the user's computer or through a web application., Conclusions: spatialLIBD is available at https://bioconductor.org/packages/spatialLIBD . It is fully compatible with SpatialExperiment and the Bioconductor ecosystem. Its functionality facilitates analyzing and interactively exploring spatially-resolved data from the Visium platform., (© 2022. The Author(s).)

Background: Electroconvulsive therapy (ECT) efficacy is hypothesized to depend on induction of molecular and cellular events that trigger neuronal plasticity. Investigating how electroconvulsive seizures (ECS) impact plasticity in animal models can help inform our understanding of basic mechanisms by which ECT relieves symptoms of depression. ECS-induced plasticity is associated with differential expression of unique isoforms encoding the neurotrophin, brain-derived neurotrophic factor (BDNF)., Hypothesis: We hypothesized that cells expressing the Bdnf exon 1-containing isoform are important for ECS-induced structural plasticity in the piriform cortex, a highly epileptogenic region that is responsive to ECS., Methods: We selectively labeled Bdnf exon 1-expressing neurons in mouse piriform cortex using Cre recombinase dependent on GFP technology (CRE-DOG). We then quantified changes in dendrite morphology and density of Bdnf exon 1-expressing neurons., Results: Loss of promoter I-derived BDNF caused changes in spine density and morphology in Bdnf exon 1-expressing neurons following ECS., Conclusions: Promoter I-derived Bdnf is required for ECS-induced dendritic structural plasticity in Bdnf exon 1-expressing neurons., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Single-cell gene expression technologies are powerful tools to study cell types in the human brain, but efforts have largely focused on cortical brain regions. We therefore created a single-nucleus RNA-sequencing resource of 70,615 high-quality nuclei to generate a molecular taxonomy of cell types across five human brain regions that serve as key nodes of the human brain reward circuitry: nucleus accumbens, amygdala, subgenual anterior cingulate cortex, hippocampus, and dorsolateral prefrontal cortex. We first identified novel subpopulations of interneurons and medium spiny neurons (MSNs) in the nucleus accumbens and further characterized robust GABAergic inhibitory cell populations in the amygdala. Joint analyses across the 107 reported cell classes revealed cell-type substructure and unique patterns of transcriptomic dynamics. We identified discrete subpopulations of D1- and D2-expressing MSNs in the nucleus accumbens to which we mapped cell-type-specific enrichment for genetic risk associated with both psychiatric disease and addiction., Competing Interests: Declaration of interests A.E.J. is employed by a for-profit biotechnology startup company (company name pending), which is unrelated to the content of this manuscript. The remaining authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with schizophrenia risk. Integration of RNA-sequencing data from postmortem human brains with these risk SNPs identified transcripts associated with increased schizophrenia susceptibility, including a class of exon 9-spliced isoforms of Sorting nexin-19 (SNX19 d9 ) and an isoform of Arsenic methyltransferase (AS3MT) splicing out exons 2 and 3 (AS3MT d2d3 ). However, the biological function of these transcript variants is unclear. Defining the cell types where these risk transcripts are dominantly expressed is an important step to understand function, in prioritizing specific cell types and/or neural pathways in subsequent studies. To identify the cell type-specific localization of SNX19 and AS3MT in the human dorsolateral prefrontal cortex (DLPFC), we used single-molecule in situ hybridization techniques combined with automated quantification and machine learning approaches to analyze 10 postmortem brains of neurotypical individuals. These analyses revealed that both pan-SNX19 and pan-AS3MT were more highly expressed in neurons than non-neurons in layers II/III and VI of DLPFC. Furthermore, pan-SNX19 was preferentially expressed in glutamatergic neurons, while pan-AS3MT was preferentially expressed in GABAergic neurons. Finally, we utilized duplex BaseScope technology, to delineate the localization of SNX19 d9 and AS3MT d2d3 splice variants, revealing consistent trends in spatial gene expression among pan-transcripts and schizophrenia risk-related transcript variants. These findings demonstrate that schizophrenia risk transcripts have distinct localization patterns in the healthy human brains, and suggest that SNX19 transcripts might disrupt the normal function of glutamatergic neurons, while AS3MT may lead to disturbances in the GABAergic system in the pathophysiology of schizophrenia., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited part of Springer Nature.)

We used the 10x Genomics Visium platform to define the spatial topography of gene expression in the six-layered human dorsolateral prefrontal cortex. We identified extensive layer-enriched expression signatures and refined associations to previous laminar markers. We overlaid our laminar expression signatures on large-scale single nucleus RNA-sequencing data, enhancing spatial annotation of expression-driven clusters. By integrating neuropsychiatric disorder gene sets, we showed differential layer-enriched expression of genes associated with schizophrenia and autism spectrum disorder, highlighting the clinical relevance of spatially defined expression. We then developed a data-driven framework to define unsupervised clusters in spatial transcriptomics data, which can be applied to other tissues or brain regions in which morphological architecture is not as well defined as cortical laminae. Last, we created a web application for the scientific community to explore these raw and summarized data to augment ongoing neuroscience and spatial transcriptomics research ( http://research.libd.org/spatialLIBD ).

Background: Context fear memory dysregulation is a hallmark symptom of several neuropsychiatric disorders, including generalized anxiety disorder and posttraumatic stress disorder. The hippocampus (HC) and prelimbic (PrL) subregion of the medial prefrontal cortex have been linked with context fear memory retrieval in rodents, but the mechanisms by which HC-PrL circuitry regulates this process remain poorly understood., Methods: Spatial and genetic targeting of HC-PrL circuitry was used for RNA sequencing (n = 31), chemogenetic stimulation (n = 44), in vivo calcium imaging (n = 20), ex vivo electrophysiology (n = 8), and molecular regulation of plasticity cascades during fear behavior (context fear retrieval) (n = 16)., Results: We showed that ventral HC (vHC) neurons with projections to the PrL cortex (vHC-PrL projectors) are a transcriptomically distinct subpopulation compared with adjacent nonprojecting neurons, and we showed complementary enrichment for diverse neuronal processes and central nervous system-related clinical gene sets. We further showed that stimulation of this population of vHC-PrL projectors suppresses context fear memory retrieval and impairs the ability of PrL neurons to dynamically distinguish between distinct phases of fear learning. Using transgenic and circuit-specific molecular targeting approaches, we demonstrated that unique patterns of activity-dependent gene transcription associated with brain-derived neurotrophic factor signaling within vHC-PrL projectors causally regulated activity in excitatory and inhibitory PrL neurons during context fear memory retrieval., Conclusions: Together, our data show that activity-dependent brain-derived neurotrophic factor release from molecularly distinct vHC-PrL projection neurons modulates postsynaptic signaling in both inhibitory and excitatory PrL neurons, modifying activity in discrete populations of PrL neurons to suppress freezing during context fear memory retrieval., (Copyright © 2020 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Multiplex single-molecule fluorescent in situ hybridization (smFISH) is a powerful method for validating RNA sequencing and emerging spatial transcriptomic data, but quantification remains a computational challenge. We present a framework for generating and analyzing smFISH data in complex tissues while overcoming autofluorescence and increasing multiplexing capacity. We developed dotdotdot (https://github.com/LieberInstitute/dotdotdot) as a corresponding software package to quantify RNA transcripts in single nuclei and perform differential expression analysis. We first demonstrate robustness of our platform in single mouse neurons by quantifying differential expression of activity-regulated genes. We then quantify spatial gene expression in human dorsolateral prefrontal cortex (DLPFC) using spectral imaging and dotdotdot to mask lipofuscin autofluorescence. We lastly apply machine learning to predict cell types and perform downstream cell type-specific expression analysis. In summary, we provide experimental workflows, imaging acquisition and analytic strategies for quantification and biological interpretation of smFISH data in complex tissues., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro, they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Specific cell populations may have unique contributions to schizophrenia but may be missed in studies of homogenate tissue. Here laser capture microdissection followed by RNA sequencing (LCM-seq) was used to transcriptomically profile the granule cell layer of the dentate gyrus (DG-GCL) in human hippocampus and contrast these data to those obtained from bulk hippocampal homogenate. We identified widespread cell-type-enriched aging and genetic effects in the DG-GCL that were either absent or directionally discordant in bulk hippocampus data. Of the ~9 million expression quantitative trait loci identified in the DG-GCL, 15% were not detected in bulk hippocampus, including 15 schizophrenia risk variants. We created transcriptome-wide association study genetic weights from the DG-GCL, which identified many schizophrenia-associated genetic signals not found in transcriptome-wide association studies from bulk hippocampus, including GRM3 and CACNA1C. These results highlight the improved biological resolution provided by targeted sampling strategies like LCM and complement homogenate and single-nucleus approaches in human brain.

Brain-derived neurotrophic factor (BDNF) signals through its cognate receptor tropomyosin receptor kinase B (TrkB) to promote the function of several classes of inhibitory interneurons. We previously reported that loss of BDNF-TrkB signaling in cortistatin (Cort)-expressing interneurons leads to behavioral hyperactivity and spontaneous seizures in mice. We performed bulk RNA sequencing (RNA-seq) from the cortex of mice with disruption of BDNF-TrkB signaling in cortistatin interneurons, and identified differential expression of genes important for excitatory neuron function. Using translating ribosome affinity purification and RNA-seq, we define a molecular profile for Cort-expressing inhibitory neurons and subsequently compare the translatome of normal and TrkB-depleted Cort neurons, revealing alterations in calcium signaling and axon development. Several of the genes enriched in Cort neurons and differentially expressed in TrkB-depleted neurons are also implicated in autism and epilepsy. Our findings highlight TrkB-dependent molecular pathways as critical for the maturation of inhibitory interneurons and support the hypothesis that loss of BDNF signaling in Cort interneurons leads to altered excitatory/inhibitory balance., (Copyright © 2020 Maynard et al.)

Brain-derived neurotrophic factor (BDNF) signaling regulates synaptic plasticity in the hippocampus (HC) and prefrontal cortex (PFC), and has been extensively linked with fear memory expression in rodents. Notably, disrupting BDNF production from promoter IV-derived transcripts enhances fear expression in mice, and decreases fear-associated HC-PFC synchrony, suggesting that Bdnf transcription from promoter IV plays a key role in HC-PFC function during fear memory retrieval. To better understand how promoter IV-derived BDNF controls HC-PFC connectivity and fear expression, we generated a viral construct that selectively targets cells expressing promoter IV-derived Bdnf transcripts ("p4-cells") for tamoxifen-inducible Cre-mediated recombination (AAV8-p4Bdnf-ER T2 CreER T2 -PEST). Using this construct, we found that ventral hippocampal (vHC) p4-cells are recruited during fear expression, and that activation of these cells causes exaggerated fear expression that co-occurs with disrupted vHC-PFC synchrony in mice. Our data highlight how this novel construct can be used to interrogate genetically defined cell types that selectively contribute to BDNF-dependent behaviors.

Signaling of brain-derived neurotrophic factor (BDNF) via tropomyosin receptor kinase B (TrkB) plays a critical role in the maturation of cortical inhibition and controls expression of inhibitory interneuron markers, including the neuropeptide cortistatin (CST). CST is expressed exclusively in a subset of cortical and hippocampal GABAergic interneurons, where it has anticonvulsant effects and controls sleep slow-wave activity (SWA). We hypothesized that CST-expressing interneurons play a critical role in regulating excitatory/inhibitory balance, and that BDNF, signaling through TrkB receptors on CST-expressing interneurons, is required for this function. Ablation of CST-expressing cells caused generalized seizures and premature death during early postnatal development, demonstrating a critical role for these cells in providing inhibition. Mice in which TrkB was selectively deleted from CST-expressing interneurons were hyperactive, slept less and developed spontaneous seizures. Frequencies of spontaneous excitatory post-synaptic currents (sEPSCs) on CST-expressing interneurons were attenuated in these mice. These data suggest that BDNF, signaling through TrkB receptors on CST-expressing cells, promotes excitatory drive onto these cells. Loss of excitatory drive onto CST-expressing cells that lack TrkB receptors may contribute to observed hyperexcitability and epileptogenesis.

Brain-derived neurotrophic factor (BDNF) is a key neuropeptide in the central regulation of energy balance. The Bdnf gene contains nine promoters, each producing specific mRNA transcripts that encode a common protein. We sought to assess the phenotypic outcomes of disrupting BDNF production from individual Bdnf promoters. Mice with an intact coding region but selective disruption of BDNF production from Bdnf promoters I, II, IV, or VI (Bdnf-e1 -/- , -e2 -/- , -e4 -/- , and -e6 -/- ) were created by inserting an enhanced green fluorescent protein-STOP cassette upstream of the targeted promoter splice donor site. Body composition was measured by MRI weekly from age 4 to 22 wk. Energy expenditure was measured by indirect calorimetry at 18 wk. Food intake was measured in Bdnf-e1 -/- and Bdnf-e2 -/- mice, and pair feeding was conducted. Weight gain, lean mass, fat mass, and percent fat of Bdnf-e1 -/- and Bdnf-e2 -/- mice (both sexes) were significantly increased compared with wild-type littermates. For Bdnf-e4 -/- and Bdnf-e6 -/- mice, obesity was not observed with either chow or high-fat diet. Food intake was increased in Bdnf-e1 -/- and Bdnf-e2 -/- mice, and pair feeding prevented obesity. Mutant and wild-type littermates for each strain (both sexes) had similar total energy expenditure after adjustment for body composition. These findings suggest that the obesity phenotype observed in Bdnf-e1 -/- and Bdnf-e2 -/- mice is attributable to hyperphagia and not altered energy expenditure. Our findings show that disruption of BDNF from specific promoters leads to distinct body composition effects, with disruption from promoters I or II, but not IV or VI, inducing obesity.

Brain-derived neurotrophic factor ( Bdnf ) transcription is controlled by several promoters, which drive expression of multiple transcripts encoding an identical protein. We previously reported that BDNF derived from promoters I and II is highly expressed in hypothalamus and is critical for regulating aggression in male mice. Here we report that BDNF loss from these promoters causes reduced sexual receptivity and impaired maternal care in female mice, which is concomitant with decreased oxytocin ( Oxt) expression during development. We identify a novel link between BDNF signaling, oxytocin, and maternal behavior by demonstrating that ablation of TrkB selectively in OXT neurons partially recapitulates maternal care impairments observed in BDNF-deficient females. Using translating ribosome affinity purification and RNA-sequencing we define a molecular profile for OXT neurons and delineate how BDNF signaling impacts gene pathways critical for structural and functional plasticity. Our findings highlight BDNF as a modulator of sexually-dimorphic hypothalamic circuits that govern female-typical behaviors., Competing Interests: KM, JH, BP, AG, SR, CW, AR, JS, AJ, KM No competing interests declared, (© 2018, Maynard et al.)

Background: Electroconvulsive therapy (ECT) is a rapid and effective treatment for major depressive disorder. Chronic stress-induced depression causes dendrite atrophy and deficiencies in brain-derived neurotrophic factor (BDNF), which are reversed by anti-depressant drugs. Electroconvulsive seizures (ECS), an animal model of ECT, robustly increase BDNF expression and stimulate dendritic outgrowth., Objective: The present study aims to understand cellular and molecular plasticity mechanisms contributing to the efficacy of ECS following chronic stress-induced depression., Methods: We quantify Bdnf transcript levels and dendritic spine density and morphology on cortical pyramidal neurons in mice exposed to vehicle or corticosterone and receiving either Sham or ECS treatment., Results: ECS rescues corticosterone-induced defects in spine morphology and elevates Bdnf exon 1 and exon 4-containing transcripts in cortex., Conclusions: Dendritic spine remodeling and induction of activity-induced BDNF in the cortex represent important cellular and molecular plasticity mechanisms underlying the efficacy of ECS for treatment of chronic stress-induced depression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) is an activity-dependent neurotrophin critical for neuronal plasticity in the hippocampus. BDNF is encoded by multiple transcripts with alternative 5' untranslated regions (5'UTRS) that display activity-induced targeting to distinct subcellular compartments. While individual Bdnf 5'UTR transcripts influence dendrite morphology in cultured hippocampal neurons, it is unknown whether Bdnf splice variants impact dendrite arborization in functional classes of neurons in the intact hippocampus. Moreover, the contribution of Bdnf 5'UTR splice variants to dendritic spine density and shape has not been explored. We analyzed the structure of CA1 and CA3 dendrite arbors in transgenic mice lacking BDNF production from exon (Ex) 1, 2, 4, or 6 splice variants (Bdnf-e1, -e2, -e4, and -e6 -/- mice) and found that loss of BDNF from individual Bdnf mRNA variants differentially impacts the complexity of apical and basal arbors in vivo. Consistent with the subcellular localization studies, Bdnf Ex2 and Ex6 transcripts significantly contributed to dendrite morphology in both CA1 and CA3 neurons. While Bdnf-e2 -/- mice showed increased branching proximal to the soma in CA1 and CA3 apical arbors, Bdnf-e6 -/- mice showed decreased apical and basal dendrite complexity. Analysis of spine morphology on Bdnf-e6 -/- CA1 dendrites revealed changes in the percentage of differently sized spines on apical, but not basal, branches. These results provide further evidence that Bdnf splice variants generate a spatial code that mediates the local actions of BDNF in distinct dendritic compartments on structural and functional plasticity.

Posttraumatic stress disorder is characterized by hyperarousal, sensory processing impairments, sleep disturbances and altered fear regulation; phenotypes associated with changes in brain oscillatory activity. Molecules associated with activity-dependent plasticity, including brain-derived neurotrophic factor (BDNF), may regulate neural oscillations by controlling synaptic activity. BDNF synthesis includes production of multiple Bdnf transcripts, which contain distinct 5' noncoding exons. We assessed arousal, sensory processing, fear regulation and sleep in animals where BDNF expression from activity-dependent promoter IV is disrupted (Bdnf-e4 mice). Bdnf-e4 mice display sensory hyper-reactivity and impaired electrophysiological correlates of sensory information processing as measured by event-related potentials (ERP). Utilizing electroencephalogram, we identified a decrease in slow-wave activity during non-rapid eye movement sleep, suggesting impaired sleep homeostasis. Fear extinction is controlled by hippocampal-prefrontal cortical BDNF signaling, and neurophysiological communication patterns between the hippocampus (HPC) and medial prefrontal cortex (mPFC) correlate with behavioral performance during extinction. Impaired fear extinction in Bdnf-e4 mice is accompanied by increased HPC activation and decreased HPC-mPFC theta phase synchrony during early extinction, as well as increased mPFC activation during extinction recall. These results suggest that activity-dependent BDNF signaling is critical for regulating oscillatory activity, which may contribute to altered behavior.

Brain-derived neurotrophic factor (BDNF) regulates diverse biological functions ranging from neuronal survival and differentiation during development to synaptic plasticity and cognitive behavior in the adult. BDNF disruption in both rodents and humans is associated with neurobehavioral alterations and psychiatric disorders. A unique feature of Bdnf transcription is regulation by nine individual promoters, which drive expression of variants that encode an identical protein. It is hypothesized that this unique genomic structure may provide flexibility that allows different factors to regulate BDNF signaling in distinct cell types and circuits. This has led to the suggestion that isoforms may regulate specific BDNF-dependent functions; however, little scientific support for this idea exists. We generated four novel mutant mouse lines in which BDNF production from one of the four major promoters (I, II, IV, or VI) is selectively disrupted (Bdnf-e1, -e2, -e4, and -e6 mice) and used a comprehensive comparator approach to determine whether different Bdnf transcripts are associated with specific BDNF-dependent molecular, cellular, and behavioral phenotypes. Bdnf-e1 and -e2 mutant males displayed heightened aggression accompanied by convergent expression changes in specific genes associated with serotonin signaling. In contrast, BDNF-e4 and -e6 mutants were not aggressive but displayed impairments associated with GABAergic gene expression. Moreover, quantifications of BDNF protein in the hypothalamus, prefrontal cortex, and hippocampus revealed that individual Bdnf transcripts make differential, region-specific contributions to total BDNF levels. The results highlight the biological significance of alternative Bdnf transcripts and provide evidence that individual isoforms serve distinct molecular and behavioral functions.

Background: Neurogenesis continues throughout life in the hippocampal dentate gyrus. Chronic treatment with monoaminergic antidepressant drugs stimulates hippocampal neurogenesis, and new neurons are required for some antidepressant-like behaviors. Electroconvulsive seizures (ECS), a laboratory model of electroconvulsive therapy (ECT), robustly stimulate hippocampal neurogenesis., Hypothesis: ECS requires newborn neurons to improve behavioral deficits in a mouse neuroendocrine model of depression., Methods: We utilized immunohistochemistry for doublecortin (DCX), a marker of migrating neuroblasts, to assess the impact of Sham or ECS treatments (1 treatment per day, 7 treatments over 15 days) on hippocampal neurogenesis in animals receiving 6 weeks of either vehicle or chronic corticosterone (CORT) treatment in the drinking water. We conducted tests of anxiety- and depressive-like behavior to investigate the ability of ECS to reverse CORT-induced behavioral deficits. We also determined whether adult neurons are required for the effects of ECS. For these studies we utilized a pharmacogenetic model (hGFAPtk) to conditionally ablate adult born neurons. We then evaluated behavioral indices of depression after Sham or ECS treatments in CORT-treated wild-type animals and CORT-treated animals lacking neurogenesis., Results: ECS is able to rescue CORT-induced behavioral deficits in indices of anxiety- and depressive-like behavior. ECS increases both the number and dendritic complexity of adult-born migrating neuroblasts. The ability of ECS to promote antidepressant-like behavior is blocked in mice lacking adult neurogenesis., Conclusion: ECS ameliorates a number of anxiety- and depressive-like behaviors caused by chronic exposure to CORT. ECS requires intact hippocampal neurogenesis for its efficacy in these behavioral indices., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Globally, the largest contributors to neonatal mortality are preterm birth, intrapartum complications and infection. Many of these deaths could be prevented by providing temperature stability, respiratory support, hydration and nutrition; preventing and treating infections; and diagnosing and treating neonatal jaundice and hypoglycaemia. Most neonatal health-care technologies which help to accomplish these tasks are designed for high-income countries and are either unavailable or unsuitable in low-resource settings, preventing many neonates from receiving the gold standard of care. There is an urgent need for neonatal health-care technologies which are low-cost, robust, simple to use and maintain, affordable and able to operate from various power supplies. Several technologies have been designed to meet these requirements or are currently under development; however, unmet technology needs remain. The distribution of an integrated set of technologies, rather than separate components, is essential for effective implementation and a substantial impact on neonatal health. Close collaboration between stakeholders at all stages of the development process and an increased focus on implementation research are necessary for effective and sustainable implementation.

The developmental increase in the strength of inhibitory synaptic circuits defines the time window of the critical period for plasticity in sensory cortices. Conceptually, plasticity of inhibitory synapses is an attractive mechanism to allow for homeostatic adaptation to the sensory environment. However, a brief duration of visual deprivation that causes maximal change in excitatory synapses produces minimal change in inhibitory synaptic transmission. Here we examined developmental and experience-dependent changes in inhibition by measuring miniature IPSCs (mIPSCs) in layer 2/3 pyramidal neurons of mouse visual cortex. During development from postnatal day 21 (P21) to P35, GABAA receptor function changed from fewer higher-conductance channels to more numerous lower-conductance channels without altering the average mIPSC amplitude. Although a week of visual deprivation did not alter the average mIPSC amplitude, a subsequent 2 h exposure to light produced a rapid rebound potentiation. This form of plasticity is restricted to a critical period before the developmental change in GABAergic synaptic properties is completed, and hence is absent by P35. Visual experience-dependent rebound potentiation of mIPSCs is accompanied by an increase in the open channel number and requires activity-dependent transcription of brain-derived neurotrophic factor (BDNF). Mice lacking BDNF transcription through promoter IV did not show developmental changes in inhibition and lacked rebound potentiation. Our results suggest that sensory experience may have distinct functional consequences in normal versus deprived sensory cortices, and that experience-dependent BDNF expression controls the plasticity of inhibitory synaptic transmission particularly when recovering vision during the critical period., (Copyright © 2014 the authors 0270-6474/14/3410770-10$15.00/0.)

Down syndrome cell adhesion molecule, or DSCAM, has been implicated in many neurodevelopmental processes including axon guidance, dendrite arborization, and synapse formation. Here we show that DSCAM plays an important role in regulating the morphogenesis of cortical pyramidal neurons in the mouse. We report that DSCAM expression is developmentally regulated and localizes to synaptic plasma membranes during a time of robust cortical dendrite arborization and spine formation. Analysis of mice that carry a spontaneous mutation in DSCAM (DSCAM(del17)) revealed gross morphological changes in brain size and shape in addition to subtle changes in cortical organization, volume, and lamination. Early postnatal mutant mice displayed a transient decrease in cortical thickness, but these reductions could not be attributed to changes in neuron production or cell death. DSCAM(del17) mutants showed temporary impairments in the branching of layer V pyramidal neuron dendrites at P10 and P17 that recovered to normal by adulthood. Defects in DSCAM(del17) dendrite branching correlated with a temporal increase in apical branch spine density and lasting changes in spine morphology. At P15 and P42, mutant mice displayed a decrease in the percentage of large, stable spines and an increase in the percentage of small, immature spines. Together, our findings suggest that DSCAM contributes to pyramidal neuron morphogenesis by regulating dendrite arborization and spine formation during cortical circuit development.

Developmental guidance cues act to direct growth cones to their correct targets in the nervous system. Recent experiments also demonstrate that developmental cues are expressed in the adult mammalian nervous system, although their function in the brain is not yet clear. The semaphorin gene family has been implicated in the growth of dendrites and axons in a number of different species. While the expression of semaphorin and its influence on tibial pioneer neurons in the developing limb bud have been well characterized in the grasshopper, the expression of semaphorin 2a (sema2a) has not been explored in the adult insect. In this study we used polymerase chain reaction (PCR) with degenerate and gene-specific primers to clone part of the secreted form of sema2a from Gryllus bimaculatus. Using in situ hybridization and immunohistochemistry, we confirmed that sema2a mRNA and protein expression patterns in the embryonic cricket were similar to that seen in the grasshopper. We also showed that tibial neuron development in crickets was comparable to that described in grasshopper. An examination of both developing and adult cricket brains showed that sema2a mRNA and protein were expressed in the Kenyon cells in mushroom bodies, an area involved in learning and memory. Sema2a expression was most obvious near the apex of the mushroom body in a region surrounding the neurogenic tip, which produces neurons throughout the life of the cricket. We discuss the role of neurogenesis in learning and memory and the potential involvement of semaphorin in this process.

This study demonstrates that whilst some DNA-repair deficiencies can be detected using host cell reactivation of cisplatin (CDDP)-treated adenovirus (Ad5), not all repair deficiencies affected replication of CDDP-treated Ad5 in human cells. A line of fibroblasts (XP25), derived from a patient with a UV-hypersensitive syndrome xeroderma pigmentosum (XP), was found, as previously reported [1], to be deficient in reactivating the treated virus when compared to the apparently repair-proficient human tumor cell lines established from bladder and ovarian carcinomas. However, a testicular teratoma cell line (SuSa), shown previously to be deficient in the repair of guanine-guanine (G-G) intrastrand crosslinks, adenine-guanine (A-G) intrastrand crosslinks and interstrand crosslinks [2], was found to reactivate the treated virus to a similar extent as the repair-proficient ovarian tumor cell line and the similarly repair-proficient RT112 cell line derived from a bladder carcinoma. Therefore, not all repair-deficient cell lines were deficient at CDDP-treated Ad5 reactivation. However, the HCR technique may still prove to be useful as a rapid screen for DNA-repair deficiencies in CDDP-sensitive cells of unknown repair capacity. A CDDP-sensitive ovarian tumor cell line (TR175) was deficient in reactivating CDDP-treated Ad5, whilst another ovarian cell line (TR170) of intermediate CDDP sensitivity reactivated the virus to a marginally higher extent than the other more CDDP-resistant repair proficient ovarian cell line (SKOV3). In addition, sublines of either the SuSa cells or the RT112 cells expressing approximately two-fold levels of resistance or increased sensitivity to CDDP, showed no change in their abilities to reactivate this CDDP-treated virus, compared to their parental lines. CDDP-treated Ad5 was also used as a lethal probe to obtain cell lines specifically deficient in DNA repair. One such deficient line (SKOV3-C3A), derived from the SKOV3 ovarian carcinoma cell line, displayed an unusual biphasic curve for reactivation of the CDDP-treated virus. Further cell lines derived in this novel manner may prove useful in analysing the genetics of CDDP-repair.

The feasibility of using adenovirus 5 as an in vitro probe for chemosensitivity in short-term cultures of human tumors was evaluated using human melanoma cell lines and primary cultures of melanoma biopsies. A convenient immunoperoxidase method was developed for quantitating viral replication 2 days after infection. Two different approaches were explored: the host cell reactivation assay (HCR) using drug-treated virus; and the viral capacity assay using drug-treated cells. The HCR assay detected sensitivity to 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC) in Mer- (methyl excision repair deficient) cell lines as decreased ability of the cells to replicate MTIC-treated virus. This test should be applicable to DNA-damaging agents and repair-deficient tumors. Adenovirus replicated readily in nonproliferating primary cultures of melanoma biopsies; application of the HCR assays to this material identified one Mer- sample of 11 tested. Herpes viruses were not suitable for use in HCR because herpes simplex virus type 1 failed to distinguish Mer- from Mer+ melanoma cells; and nonproductive infection of MTIC-sensitive lymphoid cells with Epstein-Barr virus yielded an MTIC-resistant cell line. The second assay (viral capacity) involved determination of the inhibition of replication of untreated virus in treated cells. This approach correctly predicted sensitivity to hydroxyurea and deoxyadenosine in melanoma cell lines when compared with clonogenic survival assay. Viral capacity was also inhibited by cytosine arabinoside, fluorouracil, vincristine, adriamycin, 6-mercaptopurine and ionising radiation, and may therefore be useful for detecting sensitivity to a wide range of antitumor agents.