Targeting barrel field spiny stellate cells using a vesicular monoaminergic transporter 2-Cre mouse line

Abstract

Rodent primary somatosensory cortex (S1) is organized in defined layers, where layer IV serves as the main target for thalamocortical projections. Serotoninergic signaling is important for the organization of thalamocortical projections and consequently proper barrel field development in rodents, and the vesicular monoamine transporter 2 (VMAT2) can be detected locally in layer IV S1 cortical neurons in mice as old as P10, but the identity of the Vmat2-expressing neurons is unknown. We here show that Vmat2 mRNA and also Vmat2-Cre recombinase are still expressed in adult mice in a sub-population of the S1 cortical neurons in the barrel field. The Vmat2-Cre cells showed a homogenous intrinsically bursting firing pattern determined by whole-cell patch-clamp, localized radial densely spinous basal dendritic trees and almost exclusively lack of apical dendrite, indicative of layer IV spiny stellate cells. Single cell mRNA sequencing analysis showed that S1 cortical Vmat2-Cre;tdTomato cells express the layer IV marker Rorb and mainly cluster with layer IV neurons, and RNAscope analysis revealed that adult Vmat2-Cre neurons express Vmat2 and vesicular glutamate transporter 1 (Vglut1) and Vglut2 mRNA to a high extent. In conclusion, our analysis shows that cortical Vmat2 expression is mainly confined to layer IV neurons with morphological, electrophysiological and transcriptional characteristics indicative of spiny stellate cells.

The glycine receptor alpha 3 subunit mRNA expression shows sex-dependent differences in the adult mouse brain

“…The scRNA-seq data was obtained using the 10X Genomics method. The dataset was analyzed using SCANPY 1.9.1 [ 36 ] in Python 3.8.8 in similarity as described before [ 37 ] and the full code can be found at https://github.com/HannahMWeman/glra3-expression-analysis-in-the-nervous-system . Firstly, all annotated neurons were isolated from the dataset to be used for basic preprocessing, resulting in 74,539 neurons and 27,998 genes.…”

“…Fluorescent in situ hybridization was performed to target the expression of Glra3 in various tissues using the RNAscope Fluorescent Multiplex kit (cat#: 320850, ACD, USA) in accordance with ACD guidelines for fresh frozen tissues with minor modifications [ 24 ] and as described previously [ 37 ]. In brief: the slides to be used were taken from -80 °C and immediately fixated in room temperature 4% PFA in 1 × PBS (Histolab, Sweden) for 15 min before being washed in autoclaved 1 × PBS for 2 min.…”

“…The dataset ‘l5_all.loom’ was acquired from ( http://linnarssonlab.org/ ). The dataset was analyzed using SCANPY 1.9.1 [ 36 ] in Python 3.8.8 in similarity as described before [ 37 ] and the full code can be found at https://github.com/HannahMWeman/glra3-expression-analysis-in-the-nervous-system . All qRT-PCR data generated or analyzed during this study are included in this published article and its additional files.…”

Spinal Glycine Receptor Alpha 3 Cells Communicate Sensations of Chemical Itch in Hairy Skin

“…Adult Glra3 -Cre(+) mice microinjected with AAVDJ.EF1a-DIO-HTB (3 females, 2 males, 7–11 weeks) and mice included in the retrograde rabies tracing (1 female and 2 males, 14 weeks) in the L5/L6 spinal dorsal horn were subjected to similar procedures as described previously ( Freitag et al, 2021 ). The HTB protein is a histone-tagged GFP, and this virus was used since the fluorescence could be detected after RNAscope protocol.…”

“…The fluorescent in situ hybridization was performed using the RNAscope Fluorescent Multiplex kit (ACD, catalog #320850) in accordance with ACD guidelines for fresh frozen tissues, with minor modifications ( Wang et al, 2012 ) on sections from Glra3 -Cre.HTB and sensory stimulated C57BL/6J mice. In brief, as performed previously ( Freitag et al, 2021 ), the slides to be used were taken from −80°C and immediately fixated in 4% FA (Histolab) for 15 min at RT before being washed in autoclaved 1× PBS for 2 min. The tissues were thereafter dehydrated in a stepwise increase of EtOH concentration; 3 min in 50%, 3 min in 70%, and 2× 5 min in 100% (Merck KGaA).…”