Cell-type based analysis of microRNA profiles in the mouse brain
ABSTRACT: MicroRNAs (miRNA) are implicated in brain development and function but the underlying mechanisms have been difficult to study in part due to cellular heterogeneity in neural circuits. To systematically analyze miRNA expression in neurons, we have established a miRNA tagging and affinity purification (miRAP) method that is targeted to cell types through the Cre-loxP binary system in mice. Our studies of the neocortex and cerebellum reveal the expression of a large fraction of known miRNAs with distinct profiles in glutamatergic and GABAergic neurons, and subtypes of GABAergic neurons. We further detected putative novel miRNAs, tissue or cell type-specific strand selection of miRNAs, and miRNA editing. Our method thus will facilitate a systematic analysis of miRNA expression and regulation in specific neuron types in the context of neuronal development, physiology, plasticity, pathology and disease models, and is generally applicable to other cell types and tissues. RNA was extracted from miRNA tagging samples(Ago2 IP or Myc IP), processed and sequenced on Illumina genome analyzer
Project description:We have used RNA immunoprecipitation to identify the set of mRNAs that HIV-1 Tat interacts with in T-cells. We have also performed measurements of relative RNA abundance to determine if Tat binding is associated with an increase in RNA abundance in Tat-expressing T-cells and during HIV infection of primary T-cells. We have also used RNA IP and ChIP-Chip to compare the RNAs with which Tat interacts with to the RNAs that RISC interacts with and the genes associated with pTEF-b.
Project description:The main goal of the experiment was to analize the transcriptome of GABAergic neurons in general and compare their expression profiles among different brain regions Keywords: GABAergic neurons; expression profiling; rare cell Overall design: We used transgenic GAD67-eGFP mice to isolate early postnatal GABAergic neurons by flow cytometric cell sorting from whole mouse brains as well as from the olfactory bulb, the cortex, the striatum, and the cerebellum. Subsequently, microarray analysis was performed from amplified RNA of the isolated cells. Biological replicates: 4 for whole brain, 3 for each brain region. One replicate per array.
Project description:GABAergic interneurons are lost in conditions including epilepsy and CNS injury, but there are few culture models available to study their function. Towards the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally-incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP+ rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2 they exhibit varying patterns of differentiation. Transcriptional profiling and qPCR indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5 and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In co-cultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express BetaIII-tubulin, and staining for synaptophysin and vesicular GABA transporter (VGAT) were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses. The purpose was to compare differentiation patterns of several different immortalized rat neural progenitor clones to identify early stages in differentiation. The cell clones studies were: GE6 (GABAergic neuronal precursor), GE2 (non-neuronal precursor, CTX8 (multipotential precursor), L2.2 (interneuronal precursor), and L2.3 (multipotential precursor). Five rat neural precursor cell clones were compared at three different time points following FGF2 withdrawal, which triggers differentiation. Three sister culture replicates were performed for each cell clone and time point, yielding 45 samples. One microarray failed so we have 44 microarray results in the dataset.
Project description:Certain neuron types fire spontaneously at high rates, an ability that is crucial for their function in brain circuits. The spontaneously active GABAergic neurons of the substantia nigra pars reticulata (SNr), a major output of the basal ganglia, provide tonic inhibition of downstream brain areas. A depolarizing "leak" current supports this firing pattern, but its molecular basis remains poorly understood. To understand how SNr neurons maintain tonic activity, we used single-cell RNA sequencing to determine the transcriptome of individual SNr neurons. We discovered that SNr neurons express the sodium leak current, NaLCN and that SNr neurons lacking NaLCN have impaired spontaneous firing. RNA sequencing profiles from 87 GFP-positive GABAergic SNr neurons and 9 GFP-negative SNr cells were carried out. However only 80 samples that passed initial quality control and that were included in the data processing are represented in this record.
Project description:The preoptic area (POA) of the hypothalamus is known to be crucial for sleep generation, but the spatial intermingling of sleep- and wake-promoting neurons makes it difficult to dissect the sleep control circuit. Here we identified a population of POA sleep-promoting neurons based on their projection target. Using a lentivirus for retrograde labeling with channelrhodopsin-2 (ChR2) followed by optogenetic manipulation and recording, we found that the POA GABAergic neurons projecting to the tuberomammillary nucleus (TMN) are both sleep active and sleep promoting. Cell type- and projection-specific rabies tracing revealed the presynaptic inputs to these neurons, including an amygdala GABAergic input that promotes wakefulness. Using single-cell RNA-seq, we identified several molecular markers for these neurons, and optogenetic activation of the POA neurons labeled by these markers confirmed their sleep-promoting effects. Together, these findings define a group of sleep-promoting neurons functionally, anatomically, and genetically. Overall design: Single-cell RNA-Seq of retrogradely-labeled POA neurons projecting to the tuberomammillary nucleus (TMN).
Project description:A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur prior to any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Neural stem cells derived from mouse embryonic stem cells were differentiated into neurons and FACS purified based on RedStar fluorescence driven by the Tau promoter. Chromatin was prepared from NSCs and neurons (n=1), sonicated to roughly 300bp and immunoprecipitated with antibodies against H3K4me3, H3K27me3, total Histone H3 and total IgG, alongside a 5% input sample. K4/K27 and corresponding input samples were analysed by ChIPSeq
Project description:Six DD class GABAergic neurons are generated in the embryo to synapse with ventral muscles and receive input from cholinergic neurons in the dorsal nerve cord. After hatching and toward the end of the first larval (L1) stage, DD neurons reverse polarity (i.e., synapse with dorsal muscles, receive ventral cholinergic inputs). Expression profiles were generated from DD neurons in the early L1 stage before the initiation of the remodeling program. We used microarray analysis to detect transcripts with potential roles in DD remodeling. We used FACS to isolate ttr-39::mCherry labeled DD GABAergic motor neurons from a synchronized population of L1 larvae and amplified and labeled total RNA to generate Affymetrix Genome Array data. The DD data sets were compared to an expression profile obtained from all cells in a matched population of synchronized L1 larvae. See Spencer et al. PLOS One 9, e112102 (2014).
Project description:During embryonic development, GABAergic interneurons, a main inhibitory component in the cerebral cortex, migrate tangentially from the ganglionic eminence (GE) to cerebral cortex. After reaching the cerebral cortex, they start to extend their neurites for constructing local neuronal circuits around the neonatal stage. Aberrations in migration or neurite outgrowth are implicated in neurological and psychiatric disorders such as epilepsy, schizophrenia and autism. Previous studies revealed that in the early phase of cortical development the neural population migrates tangentially from the GE in the telencephalon and several genes have been characterized as regulators of migration and specification of GABAergic interneurons. However, much less is known about the molecular mechanisms of GABAergic interneurons-specific maturation at later stages of development. Here, we performed genome-wide screening to identify genes related to the later stage by flow cytometry based-microarray (FACS-array) and identified 247 genes expressed in cortical GABAergic interneurons. Among them, Dgkg, a member of diacylglycerol kinase family, was further analyzed. Correlational analysis revealed that Dgkg is dominantly expressed in somatostatin (SST)-expressing GABAergic interneurons. The functional study of Dgkg using GE neurons indicated alteration in neurite outgrowth of GABAergic neurons. This study shows a new functional role for Dgkg in GABAergic interneurons as well as the identification of other candidate genes for their maturation. Overall design: GAD67-EGFP+/- mice (P0) were used for FACS-array analysis. Single cell suspension derived from cerebral cortice were prepared and sorted by fluorescent signal (GFP+ and GFP-). Four biological replicate were used for the comparison of GFP- v.s. GFP+.