Single-nucleus RNA sequencing of mouse auditory cortex reveals critical period triggers and brakes

Abstract

<jats:p>Auditory experience drives neural circuit refinement during windows of heightened brain plasticity, but little is known about the genetic regulation of this developmental process. The primary auditory cortex (A1) of mice exhibits a critical period for thalamocortical connectivity between postnatal days P12 and P15, during which tone exposure alters the tonotopic topography of A1. We hypothesized that a coordinated, multicellular transcriptional program governs this window for patterning of the auditory cortex. To generate a robust multicellular map of gene expression, we performed droplet-based, single-nucleus RNA sequencing (snRNA-seq) of A1 across three developmental time points (P10, P15, and P20) spanning the tonotopic critical period. We also tone-reared mice (7 kHz pips) during the 3-d critical period and collected A1 at P15 and P20. We identified and profiled both neuronal (glutamatergic and GABAergic) and nonneuronal (oligodendrocytes, microglia, astrocytes, and endothelial) cell types. By comparing normal- and tone-reared mice, we found hundreds of genes across cell types showing altered expression as a result of sensory manipulation during the critical period. Functional voltage-sensitive dye imaging confirmed GABA circuit function determines critical period onset, while Nogo receptor signaling is required for its closure. We further uncovered previously unknown effects of developmental tone exposure on trajectories of gene expression in interneurons, as well as candidate genes that might execute tonotopic plasticity. Our single-nucleus transcriptomic resource of developing auditory cortex is thus a powerful discovery platform with which to identify mediators of tonotopic plasticity.</jats:p>

Experience-independent transformation of single-cell 3D genome structure and transcriptome during postnatal development of the mammalian brain

“…We detected an average of 12.8 k mRNA molecules (also known as unique molecule identifiers (UMIs); SD = 8.4 k, minimum = 1.7 k, maximum = 49.8 k) and 3.4 k genes (SD = 1.3 k, minimum = 1.0 k, maximum = 7.8 k) per cell ( Figure S1). This was an order of magnitude higher than any previous studies that probed part of postnatal cortical development in mice: 1.0 k UMIs and 0.7 k genes with SPLiT-seq (Rosenberg et al, 2018), and 1.9 k UMIs and 1.2 k genes with inDrops (Kalish et al, 2020). MALBAC-DT was also more accurate, because it avoided a UMI-overcounting issue that plagued most other methods (Chapman et al, 2020).…”

“…Second, the postnatal dynamics of single-cell transcriptome has not been comprehensively studied in the mammalian brain. Although transcriptional diversity has been extensively characterized in the adult brain (Tasic et al, 2016;Tasic et al, 2018;Zeisel et al, 2018) and more recently in the embryonic brain (Di Bella et al, 2020;La Manno et al, 2020), only a few studies have probed its postnatal dynamics, each with a limited set of time points (in mice): postnatal days (P) 2 and 11 for the whole brain (Rosenberg et al, 2018), P0, 4, 7, and 10 for the cerebellum (Carter et al, 2018), P1, 7, 90 for dopamine neurons (Tiklova et al, 2019), P10, 15, and 20 for the auditory cortex (Kalish et al, 2020), and P4, 8, 14, and 45 for the hypothalamus (Kim et al, 2020). Furthermore, most of these studies used low-sensitivity (split-pool or dropletbased) methods, which can only detect a small number of transcripts per cell and thus compromise data quality.…”

Shifting Developmental Trajectories During Critical Periods of Brain Formation

“…The role of non-neuronal brain cells in critical period plasticity has been less addressed. Oligodendrocyte-neurons interaction through Nogo-66 receptor drive the maturation of intracortical myelination necessary for the closure of OD and auditory plasticity (McGee et al, 2005 ; Kalish et al, 2020 ) and might be also a hallmark of critical period conclusion outside sensory modalities. Regions such as the PFC undergo a change in oligodendrocyte maturation and myelination following deprivation of social behavior in mice after weaning (Makinodan et al, 2012 , 2016 ).…”

“…Of special relevance are single cell RNA sequencing approaches that are accelerating our understanding of the molecular programs implemented by all brain cell types during critical periods. A pioneer effort employed single-cell RNAseq analysis during critical period for tonotopic topography in the primary auditory cortex and provides with a detailed transcriptomic profile of each neuronal and non-neuronal cell type during this critical period for auditory plasticity (Kalish et al, 2020 ). Following on previous studies describing the role of astrocyte maturation and microglia function in OD plasticity (Müller, 1990 ; Singh et al, 2016 ; Sipe et al, 2016 ), the study of Kalish et al ( 2020 ) substantiates evidences that activation of astrocytes and microglia contribute to critical period plasticity in the neocortex.…”

“Single-nucleus RNA-seq2 reveals a functional crosstalk between liver zonation and ploidy”

“…Single-nucleus RNA-seq (snRNA-seq) has emerged as a complementary approach to study complex tissues at a single-cell level [20,21], including brain [21][22][23][24][25][26][27], lung [28], kidney [29][30][31][32] and heart [33,34] in mouse and human frozen samples [35,36]. However, there are no snRNA-seq methods tailored for frozen liver tissues.…”