Benjamin W. Okaty

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 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.

Cell-type-specific transcriptomics in the brain

Sequencing of multiple mammalian genomes, together with the development of whole-transcriptome profiling technologies, have opened the door to an unprecedented ability to study gene expression in the brain. Transcriptomics refers to a class of high-throughput methods, such as microarray (gene chip

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.

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.

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.

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

Significance Neuroanatomy is experiencing a renaissance due to the study of gene-expression data covering the entire mouse brain and the entire genome that have been recently released (the Allen Atlas). On the other hand, some cell types extracted from the mouse brain have been characterized by their genetic activity. However, given a cell type, it is not known in which brain regions it can be found. We propose a computational model using the Allen Atlas to solve this problem, thus estimating previously unidentified cell-type–specific maps of the mouse brain. The model can be used to define brain regions through genetic data. 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.

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.

The Maturation of Firing Properties of Forebrain GABAergic Interneurons

GABAergic interneurons display diverse cellular morphology, connectivity, and intrinsic membrane properties, allowing different interneuron subtypes to exert unique effects on neural activity. Although cell fates may be specified much earlier, distinctive firing types emerge after migration from the ganglionic eminences as interneurons integrate into forebrain circuits. We review evidence suggesting that maturation of interneuron cell-type-specific firing properties and ion channel expression arise from the interplay between cell-autonomous gene regulatory networks as well as cell-extrinsic factors, such as brain-derived neurotrophic factor (BDNF) signaling and neural activity.