Gata2, Nkx2-2 and Skor2 form a transcription factor network regulating development of a midbrain GABAergic neuron subtype with characteristics of REM sleep regulatory neurons

The midbrain reticular formation is a mosaic of diverse GABAergic and glutamatergic neurons that have been associated with a variety of functions, including the regulation of sleep. However the molecular characteristics and development of the midbrain reticular formation neurons are poorly understood. As the transcription factor Gata2 is required for the development of all GABAergic neurons derived from the embryonic mouse midbrain, we hypothesized that the genes expressed downstream of Gata2 could contribute to the diversification of GABAergic neuron subtypes in this brain region. Here, we show that Gata2 is indeed required for the expression of several lineage-specific transcription factors in post-mitotic midbrain GABAergic neuron precursors. These include a homeodomain transcription factor Nkx2-2 and a SKI family transcriptional repressor Skor2, which are co-expressed in a restricted group of GABAergic precursors in the midbrain reticular formation. Both Gata2, and Nkx2-2 function is required for the expression of Skor2 in GABAergic precursors. In the adult mouse as well as rat midbrain, the Nkx2-2 and Skor2 expressing GABAergic neurons locate at the boundary of the ventrolateral periaqueductal gray and the midbrain reticular formation, an area shown to contain REM-off neurons regulating REM sleep. In addition to the characteristic localization, the Skor2 positive cells increase their activity upon REM sleep inhibition, send projections to a pontine region associated with sleep control and are responsive to orexins, consistent with the known properties of the midbrain REM-off neurons.

Single-cell alternative polyadenylation analysis delineates GABAergic neuron types

Background Alternative polyadenylation (APA) is emerging as an important mechanism in the post-transcriptional regulation of gene expression across eukaryotic species. Recent studies have shown that APA plays key roles in biological processes, such as cell proliferation and differentiation. Single-cell RNA-seq technologies are widely used in gene expression heterogeneity studies; however, systematic studies of APA at the single-cell level are still lacking. Results Here, we described a novel computational framework, SAPAS, that utilizes 3′-tag-based scRNA-seq data to identify novel poly(A) sites and quantify APA at the single-cell level. Applying SAPAS to the scRNA-seq data of phenotype characterized GABAergic interneurons, we identified cell type-specific APA events for different GABAergic neuron types. Genes with cell type-specific APA events are enriched for synaptic architecture and communications. In further, we observed a strong enrichment of heritability for several psychiatric disorders and brain traits in altered 3′ UTRs and coding sequences of cell type-specific APA events. Finally, by exploring the modalities of APA, we discovered that the bimodal APA pattern of Pak3 could classify chandelier cells into different subpopulations that are from different laminar positions. Conclusions We established a method to characterize APA at the single-cell level. When applied to a scRNA-seq dataset of GABAergic interneurons, the single-cell APA analysis not only identified cell type-specific APA events but also revealed that the modality of APA could classify cell subpopulations. Thus, SAPAS will expand our understanding of cellular heterogeneity.

HGG-04. TARGETING GABAERGIC NEURON-GLIOMA SYNAPSES IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) THROUGH ANTI-EPILEPTIC DRUG REPURPOSING

Pediatric high-grade gliomas, including diffuse intrinsic pontine glioma (DIPG), are the leading cause of brain cancer-related death in children. While enormous progress has been made in recent years for many forms of cancer, high-grade gliomas remain seemingly intractable, indicating that fundamental aspects of glioma growth are not yet sufficiently understood. Neuronal activity drives glioma growth both through paracrine signaling and through direct neuron-to-glioma synapses. Recently glutamatergic, AMPA receptor-dependent synapses were discovered between microenvironmental neurons and malignant glioma cells. The depolarizing current that results from synaptic and other forms of electrical neuron-glioma signaling promotes pediatric high-grade glioma proliferation and regulates growth. Neuron-glioma cell synapses mediated by other neurotransmitters remain largely unexplored, though glioma cells express genes encoding neurotransmitter receptors such as GABAA receptor subunits. Using whole-cell patch clamp electrophysiology in patient-derived DIPG xenografts, we have identified functional GABAergic neuron-to-glioma synapses mediated by GABAA receptors. GABAergic input has a depolarizing effect on glioma cells, but the magnitude of depolarization is heterogeneous between high-grade glioma subtypes and between patient-derived DIPG xenograft models. As membrane depolarization increases glioma proliferation, depolarizing GABAergic inputs to glioma cells could promote DIPG progression. Drugs that stimulate GABA signaling, such as benzodiazepines, are often given to pediatric glioma patients to treat nausea, seizures or anxiety. In patient-derived DIPG xenograftn models, lorazepam, a benzodiazepine that increases GABAA receptor conductance, increases glioma growth. Conversely, levetiracetam, an anti-epileptic drug that reduces synaptic transmission including at GABAergic neuron-glioma synapses, reduces glioma proliferation in patient-derived DIPG xenografts. This emerging understanding of brain cancer neurophysiology reveals new therapeutic targets and highlights commonly used drugs about which more study is required in this disease context.

Total number and ratio of GABAergic neuron types in the mouse lateral and basal amygdala

GABAergic neurons are key circuit elements in cortical networks. In spite of growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types have been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22 %) is significantly higher than in the LA (16 %) in both male and female mice. No difference was observed between the right and left hemispheres in either sexes. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5-6 %), basket cells expressing parvalbumin (17-20 %) or cholecystokinin (7-9 %), dendrite-targeting inhibitory cells expressing somatostatin (10-16 %), NPY-containing neurogliaform cells (14-15 %), VIP and/or calretinin-expressing interneuron-selective interneurons (29-38 %) and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (nNOS, 5.5-8 %). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathological conditions, and to model functioning amygdalar networks in health and disease.