Unknown,Transcriptomics,Genomics,Proteomics

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Translational profiling of hypothalamic and midbrain neurons that project to the nucleus accumbens.

ABSTRACT: Neuroanatomical methods enable high-resolution mapping of neural circuitry, but do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of a novel approach for molecularly profiling projective neurons. We show that ribosomes can be labeled with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from cells expressing GFP. We generated a transgenic mouse encoding a nanobody-ribosomal protein fusion (Syn-NBL10) and used a retrograde virus (CAV) encoding GFP to immunoprecipitate ribosomes from projection neurons. This enabled us to profile neurons projecting to the nucleus accumbens. The current method provides a new means for profiling neurons based on their projections. Translating mRNAs immunoprecipitated from neurons projecting to the nucleus accumbens. Each Input and IP sample corrspond to a pooled group of 6 mice.

ORGANISM(S): Mus musculus

SUBMITTER: Jeffrey Friedman

PROVIDER: E-GEOD-55800 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Molecular profiling of neurons based on connectivity.

Ekstrand Mats I MI Nectow Alexander R AR Knight Zachary A ZA Latcha Kaamashri N KN Pomeranz Lisa E LE Friedman Jeffrey M JM

Cell 20140501 5

The complexity and cellular heterogeneity of neural circuitry presents a major challenge to understanding the role of discrete neural populations in controlling behavior. While neuroanatomical methods enable high-resolution mapping of neural circuitry, these approaches do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of an approach for molecularly profiling projective neurons. We show that ribosomes can be tagged with a camelid n ...[more]

PMID: 24855954

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Project description:Here, we identify persistent and substantial variation in ethanol drinking behavior within an inbred mouse strain and utilize this model to identify gene networks influencing such non-genetic variation in ethanol intake. C57BL/6NCrl mice showed persistent inter-individual variation of ethanol intake in a two-bottle choice paradigm over a three week period, ranging from less than 1 g/kg to over 14 g/kg ethanol in an 18h interval. Whole genome microarray expression analysis in nucleus accumbens, prefrontal cortex and ventral tegmental area of individual animals identified gene expression patterns correlated with ethanol intake. Results included several gene networks previously implicated in ethanol behaviors, such as glutamate signaling, BDNF and genes involved in synaptic vesicle function. Additionally, genes functioning in epigenetic chromatin or DNA modifications such as acetylation and/or methylation also had expression patterns correlated with ethanol intake. Our results thus implicate specific brain regional gene networks, including chromatin modification factors, as potentially important mechanisms underlying individual variation in ethanol intake. Voluntary two-bottle choice drinking was performed as described previously (Khisti et al. 2006). Briefly, two bottles containing 10%(w/v) ethanol (Aaper Alcohol and Chemical Co. Shelbyville, KY) or tap water were placed into the home cage at the beginning of the dark cycle. Tube position was varied every two days (L, L, R, R). Drinking sessions lasted 18 hours/day followed by 6 hours access to water only. Mice had four consecutive drinking sessions followed by four days of abstinence repeated four times to give 16 total drinking sessions. Three brain regions were harvested 6 days after the last drinking session: prefrontal cortex (PFC), nucleus accumbens (NAc) and ventral tegmental area (VTA) as previously described (Kerns et al. 2005). Labeled cRNA from individual animals (n=19) was hybridized to a single microarray for each brain region (n=58 total microarrays).

Project description:Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a novel mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine, and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine. Mice were treated for 7 days with daily injections of cocaine (20 mg/kg) and sacrificed 24 hrs later. Chromatin from bilateral punches of NAc was immunoprecipitated with an antibody against MEF2A as described previously with minor modifications (Renthal et al., 2007). Chromatin was sonicated to an average of ~500 bp and immunoprepitated with antibody against MEF2A (Santa Cruz, sc-313) or an IgG control (Upstate/Millipore). Antibody-bound chromatin was precipitated using Protein A beads from Upstate (06-157), which were washed with low salt, high salt, and LiCl buffers to remove non-specific DNA binding. Eluted chromatin was reverse-crosslinked at 65oC in the presence of proteinase K and EDTA. DNA was purified by chloroform extraction/ethanol precipitation and the enrichment of specific promoters was amplified by ligation-mediated PCR for genome-wide analysis (Sikder et al., 2006). Amplified DNA was then labeled with Cy3 (input-enriched) or Cy5 (MEF2-enriched) and hybridized to Nimblegen (Madison, WI) MM8 mouse promoter arrays. Bilateral nucleus accumbens from eight mice were pooled for microarray analysis.

Dataset Information

Translational profiling of hypothalamic and midbrain neurons that project to the nucleus accumbens.

Publications

Molecular profiling of neurons based on connectivity.

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