Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.

A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.

SUMMARY Even before integrating into existing circuitry, adult-born neurons express receptors for neurotransmitters, but the intercellular mechanisms and their impact on neurogenesis remain largely unexplored. Here, we show that neuroblasts born in the postnatal subventricular zone (SVZ) acquire NMDA receptors (NMDARs) during their migration to the olfactory bulb. Along their route, neuroblasts are ensheathed by astrocyte-like cells expressing vesicular glutamate release machinery. Increasing calcium in these specialized astrocytes induced NMDAR-activity in neuroblasts and blocking astrocytic vesicular release eliminated spontaneous NMDAR-activity. Single-cell knockout of NMDARs using neonatal electroporation resulted in neuroblast apoptosis at the time of NMDAR acquisition. This cumulated in a 40% loss of neuroblasts along their migratory route demonstrating that NMDAR acquisition is critical for neuroblast survival, prior to entering a synaptic network. In addition, our findings suggest an unexpected mechanism where SVZ astrocytes use glutamate signaling through NMDARs to control the number of adult-born neurons reaching their final destination.

Activity-induced remodeling of neuronal circuits is critical for memory formation. This process relies in part on transcription, but neither the rate of activity nor baseline transcription is equal across neuronal cell types. In this study, we isolated mouse hippocampal populations with different activity levels and used single nucleus RNA-seq to compare their transcriptional responses to activation. One hour after novel environment exposure, sparsely active dentate granule (DG) neurons had a much stronger transcriptional response compared to more highly active CA1 pyramidal cells and vasoactive intestinal polypeptide (VIP) interneurons. Activity continued to impact transcription in DG neurons up to 5 h, with increased heterogeneity. By re-exposing the mice to the same environment, we identified a unique transcriptional signature that selects DG neurons for reactivation upon re-exposure to the same environment. These results link transcriptional heterogeneity to functional heterogeneity and identify a transcriptional correlate of memory encoding in individual DG neurons.

In the postnatal neurogenic niche, two populations of astrocyte-like cells (B cells) persist, one acting as neural progenitor cells (NPCs, B1 cells) and one forming a structural boundary between the neurogenic niche and the striatum (B2 cells, niche astrocytes). Despite being viewed as two distinct entities, we found that B1 and B2 cells express the gap junction protein connexin 43 and display functional coupling involving 50-60 cells. Using neonatal electroporation to label slowly cycling radial glia-derived B1 cells, which send a basal process onto blood vessels, we further confirmed dye coupling between NPCs. To assess the functionality of the coupling, we used calcium imaging in a preparation preserving the 3-dimensional architecture of the SVZ. Intercellular calcium waves were observed among B cells. These waves travelled bidirectionally between B1 and B2 cells and propagated on blood vessels. Inter-B cell calcium waves were absent in the presence of a gap junction blocker but persisted with purinergic receptor blockers. These findings show that privileged microdomains of communication networks exist among NPCs and niche astrocytes. Such functional coupling between these two cell types suggests that niche astrocytes do not exert a mere structural role, but may play an active role in shaping the behavior of NPCs.