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This protocol describes how to sequence the transcriptome from a single nucleus. It is particularly suited to cell types that are difficult to isolate as intact whole cells, such as neurons. 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.

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. The molecular dynamics associated with neuronal activation patterns in vivo are unclear. Lacar et al . perform single-nuclei RNA-sequencing of hippocampal neurons from mice exposed to a novel environment, and identify large-scale transcriptome changes in individual neurons associated with the experience.

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. Single nuclei RNA-seq has been used to characterize transcriptional signature of environment-related activity in cells of the dentate gyrus. Here the authors use this approach to show that whether a neuron will be reactivated in response to re-exposure to a previous environment can be predicted by its transcriptional signature.

The postnatal subventricular zone (SVZ) contains proliferating neural progenitor cells in close proximity to blood vessels. Insults and drug treatments acutely stimulate cell proliferation in the SVZ, which was assessed by labeling cells entering S phase. Although G1-to-S progression is metabolically demanding on a minute-to-hour time scale, it remains unknown whether increased SVZ cell proliferation is accompanied by a local hemodynamic response. This neurovascular coupling provides energy substrates to active neuronal assemblies. Transcardial dye perfusion revealed the presence of capillaries throughout the SVZ that constrict upon applications of the thromboxane A(2) receptor agonist U-46119 in acute brain slice preparations. We then monitored in vivo blood flow using laser Doppler flowmetry via a microprobe located either in the SVZ or a mature network. U-46119 injections into the lateral ventricle decreased blood flow in the SVZ and the striatum, which are near the ventricle. A 1-hour ventricular injection of epidermal and basic fibroblast growth factor (EGF and bFGF) significantly increased the percentage of Sox2 transcription factor-positive cells in S phase 1.5 hours post-injection. This increase was accompanied by a sustained rise in blood flow in the SVZ but not in the striatum. Direct growth factor injections into the cortex did not alter local blood flow, ruling out direct effects on capillaries. These findings suggest that an acute increase in the number of G1-to-S cycling SVZ cells is accompanied by neurometabolic-vascular coupling, which may provide energy and nutrient for cell cycle progression.

The subventricular zone (SVZ) is one of two regions where neurogenesis persists in the postnatal brain. The SVZ, located along the lateral ventricle, is the largest neurogenic zone in the brain that contains multiple cell populations including astrocyte-like cells and neuroblasts. Neuroblasts migrate in chains to the olfactory bulb where they differentiate into interneurons. Here, we discuss the experimental approaches to record the electrophysiology of these cells and image their migration and calcium activity in acute slices. Although these techniques were in place for studying glial cells and neurons in mature networks, the SVZ raises new challenges due to the unique properties of SVZ cells, the cellular diversity, and the architecture of the region. We emphasize different methods, such as the use of transgenic mice and in vivo electroporation that permit identification of the different SVZ cell populations for patch clamp recording or imaging. Electroporation also permits genetic labeling of cells using fluorescent reporter mice and modification of the system using either RNA interference technology or floxed mice. In this review, we aim to provide conceptual and technical details of the approaches to perform electrophysiological and imaging studies of SVZ cells.

Nature Communications 7: Article number: 11022 (2016); Published 19 April 2016; Updated 17 March 2017 An incorrect version of Supplementary Data 1, in which normalized counts were analysed instead of raw counts, resulting in a smaller number of differentially expressed genes, was inadvertently published with this article.

Nature Communications 7: Article number:11022 (2016); Published 19 April 2016; Updated 14 June 2016 In the original version of this Article, the middle names or initials of the authors Suguna Rani Krishnaswami, Jerika J. Barron, Martijn J.E. Kelder, Sarah L. Parylak, Apuã C.M. Paquola and Jennifer A. Erwin were omitted from the author information.

Most neuroimaging studies linking regional brain volumes with cognition correct for total intracranial volume (ICV), but methods used for this correction differ across studies. It is unknown whether different ICV correction methods yield consistent results. Using a brain‐wide association approach in the MRI substudy of UK Biobank (N = 41,964; mean age = 64.5 years), we used regression models to estimate the associations of 58 regional brain volumetric measures with eight cognitive outcomes, comparing no correction and four ICV correction approaches. Approaches evaluated included: no correction; dividing regional volumes by ICV (proportional approach); including ICV as a covariate in the regression (adjustment approach); and regressing the regional volumes against ICV in different normative samples and using calculated residuals to determine associations (residual approach). We used Spearman‐rank correlations and two consistency measures to quantify the extent to which associations were inconsistent across ICV correction approaches for each possible brain region and cognitive outcome pair across 2320 regression models. When the association between brain volume and cognitive performance was close to null, all approaches produced similar estimates close to the null. When associations between a regional volume and cognitive test were not null, the adjustment and residual approaches typically produced similar estimates, but these estimates were inconsistent with results from the crude and proportional approaches. For example, when using the crude approach, an increase of 0.114 (95% confidence interval [CI]: 0.103–0.125) in fluid intelligence was associated with each unit increase in hippocampal volume. However, when using the adjustment approach, the increase was 0.055 (95% CI: 0.043–0.068), while the proportional approach showed a decrease of −0.025 (95% CI: −0.035 to −0.014). Different commonly used methods to correct for ICV yielded inconsistent results. The proportional method diverges notably from other methods and results were sometimes biologically implausible. A simple regression adjustment for ICV produced biologically plausible associations. Different commonly used methods to correct for intracranial volume (ICV) yielded inconsistent results, representing significant challenges for reproducibility in neuroimaging research. The proportional method diverges notably from other methods and results were sometimes biologically implausible. A simple regression adjustment for ICV produced biologically plausible associations.

The neurotransmitter GABA exerts a strong negative influence on the production of adult-born olfactory bulb interneurons via tightly regulated, non-synaptic GABAergic signaling. After discussing some findings on GABAergic signaling in the neurogenic subventricular zone (SVZ), we provide data suggesting ambient GABA clearance via two GABA transporter subtypes and further support for a non-vesicular mechanism of GABA release from neuroblasts. While GABA works in cooperation with the neurotransmitter glutamate during embryonic cortical development, the role of glutamate in adult forebrain neurogenesis remains obscure. Only one of the eight metabotropic glutamate receptors (mGluRs), mGluR5, has been reported to tonically increase the number of proliferative SVZ cells in vivo, suggesting a local source of glutamate in the SVZ. We show here that glutamate antibodies strongly label subventricular zone (SVZ) astrocytes, some of which are stem cells. We also show that some SVZ neuroblasts express one of the ionotropic glutamate receptors, AMPA/kainate receptors, earlier than previously thought. Collectively, these findings suggest that neuroblast-to-astrocyte GABAergic signaling may cooperate with astrocyte-to-neuroblast glutamatergic signaling to provide strong homeostatic control on the production of adult-born olfactory bulb interneurons.