Dataset Information

Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons.

ABSTRACT: Subcellular RNAseq promises to dissect transcriptional dynamics but is not well characterized. Furthermore, FACS may introduce bias but has not been benchmarked genome-wide. Finally, D1 and D2 dopamine receptor-expressing medium spiny neurons (MSNs) of the nucleus accumbens (NAc) are fundamental to neuropsychiatric traits but have only a short list of canonical surface markers. We address these gaps by systematically comparing nuclear-FACS, whole cell-FACS, and RiboTag affinity purification from D1- and D2-MSNs. Using differential expression, variance partitioning, and co-expression, we identify the following trade-offs for each method. RiboTag-seq best distinguishes D1- and D2-MSNs but has the lowest transcriptome coverage. Nuclear-FACS-seq generates the most differentially expressed genes and overlaps significantly with neuropsychiatric genetic risk loci, but un-annotated genes hamper interpretation. Whole cell-FACS is more similar to nuclear-FACS than RiboTag, but captures aspects of both. Using pan-method approaches, we discover that transcriptional regulation is predominant in D1-MSNs, while D2-MSNs tend towards cytosolic regulation. We are also the first to find evidence for moderate sexual dimorphism in these cell types at baseline. As these results are from 49 mice (nmale?=?39, nfemale?=?10), they represent generalizable ground-truths. Together, these results guide RNAseq methods selection, define MSN transcriptomes, highlight neuronal sex differences, and provide a baseline for D1- and D2-MSNs.

SUBMITTER: Kronman H

PROVIDER: S-EPMC6554355 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons.

Kronman Hope H Richter Felix F Labonté Benoit B Chandra Ramesh R Zhao Shan S Hoffman Gabriel G Lobo Mary Kay MK Schadt Eric E EE Nestler Eric J EJ

Scientific reports 20190606 1

Subcellular RNAseq promises to dissect transcriptional dynamics but is not well characterized. Furthermore, FACS may introduce bias but has not been benchmarked genome-wide. Finally, D1 and D2 dopamine receptor-expressing medium spiny neurons (MSNs) of the nucleus accumbens (NAc) are fundamental to neuropsychiatric traits but have only a short list of canonical surface markers. We address these gaps by systematically comparing nuclear-FACS, whole cell-FACS, and RiboTag affinity purification from ...[more]

PMID: 31171808

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Project description:Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, from where they project to many brain areas including the nucleus accumbens (NAc), a brain area that integrates diverse monoaminergic inputs to coordinate motivated behaviours. While the NAc expresses various histamine receptor subtypes, the mechanisms by which histamine modulates NAc activity are still poorly understood. Using whole-cell patch-clamp recordings, we found that pharmacological activation of histamine 2 (H2) receptors elevates the excitability of NAc medium spiny neurons (MSNs), while activation of H1 receptors failed to significantly affect MSN excitability. The evoked firing of MSNs increased after seconds of local H2 agonist administration and remained elevated for minutes. H2 receptor (H2R) activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential afterhyperpolarization and increased the action potential half-width. The increased excitability was protein kinase A-dependent and associated with decreased A-type K+ currents. In addition, selective pharmacological inhibition of the Kv4.2 channel, the main molecular determinant of A-type K+ currents in MSNs, mimicked and occluded the increased excitability induced by H2R activation. Our results indicate that histaminergic transmission in the NAc increases MSN intrinsic excitability through H2R-dependent modulation of Kv4.2 channels. Activation of H2R will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of goal-induced behaviours. KEY POINTS: Histamine is synthesized and released by hypothalamic neurons of the tuberomammillary nucleus and serves as a general modulator for whole-brain activity including the nucleus accumbens. Histamine receptors type 2 (HR2), which are expressed in the nucleus accumbens, couple to Gαs/off proteins which elevate cyclic adenosine monophosphate levels and activate protein kinase A. Whole-cell patch-clamp recordings revealed that H2R activation increased the evoked firing in medium spiny neurons of the nucleus accumbens via protein kinase A-dependent mechanisms. HR2 activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential medium after-hyperpolarization and increased the action potential half-width. HR2 activation also reduced A-type potassium current. Selective pharmacological inhibition of the Kv4.2 channel mimicked and occluded the increased excitability induced by H2R activation.

Dataset Information

Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons.

Publications

Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons.

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