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Tissue
Physiopathology
Type
Name

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User name	FranÃ§ois Tronche
Email	francois.tronche@gmail.com
Phone / Fax number	+33 1 44 27 60 0 / +33 1 44 27 61 5
Location	INSERM URMS 952 , CNRS UMR 7224, UniversitÃ© Pierre et Marie Curie (Physiopathologie des Maladies du SystÃ¨me Nerveux Central) - 7-9 quai Saint Bernard, Bat A, B - 75252 Paris, France

Scientific description

The etiology of addiction is multifaceted and implicates interactions between life experience and genetic background. Stress and stress-induced glucocorticoid secretion are determinant to facilitate addiction. We previously showed that genetic inactivation of glucocorticoid receptor (GR) gene in dopaminoceptive neurons impaired cocaine seeking. Here we examined the contribution of GR to the responses to opiates, a pharmacologically-distinct class of abused substances. We showed that inactivating GR within dopaminoceptive neurons substantially reduced cocaine-induced conditioned place preference and the expression of locomotor sensitization. In contrast, behavioral responses to morphine remained unchanged when GR was absent from these neurons or from dopamine cells. The dopaminoceptive mutation engendered alterations of striatal genes expression implicated in glutamatergic transmission and plasticity. Impaired molecular reactivity to cocaine was conspicuous as we observed a deficit in activation of extracellular-signal regulated kinase 1/2 and expression of the key immediate early genes c-Fos and Zif268 in mutants. Furthermore, cocaine-elicited extracellular accumbal dopamine release was markedly diminished. Mirroring the lack of effect on morphineâ€™s behaviors, the dopaminoceptive mutation did not modify neither molecular nor neurochemical morphineâ€™s responses. The combined behavioural and molecular approaches identified a subset of striatal neurons in which GR differently underlies psychostimulant- and opiate-induced maladaptive changes contributing to addiction.

Technical description

Tissue collection and total RNA isolation and labelling

One week before dissection, animals (GRD1Cre and controls littermates) are isolated in single cages with tap water and food ad libitum. Mice are sacrificed between 10 and 11 a.m. and the striata are quickly removed and put in RNA later solution (Qiagen) on ice. Collected striata are disrupted in Qiazol reagent (Qiagen) using ultra turax (IKA-Werke??). Total RNA preparation was performed using â€œRNeasy lipid mini kitâ€ (Qiagen). RNA quality and concentration were both determined by absorbance at 260 and 280 nm and by analysis using agilent bioanalyzer (Agilent). The obtained RNA presented a RIN between 8.3 and 9.1. Sample labelling and purification were done using â€œNanoAmp RT-IVTâ€ kit (Applied Biosystems). Briefly, first and second strand cDNA synthesis was performed from 0.5 Âµg of each sample. Then, in vitro transcription was achieved. During this step, DIG labelled-UTP is incorporated to obtain labelled cRNA. 


Microarray Hybridization, Quality control and Analysis

IVT products were then fragmented for hybridization to Applied Biosystem Mouse Genome Survey Microarray v0.2. Arrays were hybridized for 16 h at 55Â°C, washed, stained with ant-DIG antibodies using the â€œChemioluminescence Detection kitâ€ protocol (Applied Biosystems) and scanned in AB1700 platform.  Primary image and data analysis was performed as described in (Wilhelm et al. 2008) using Applied Biosystems software in version 1.1.1. The resulting transcriptome profile data distributions (Noth et al. 2006b)  were then compared to a large reference set of 500 unrelated and, biologically speaking, heterogeneous, previously acquired profiles. A similarity index calculated individually for each experiment towards the reference group was used as a quality control measure. Mean Pearson correlation coefficients for all possible inter- and intra-group combinations were determined by calculating the average correlation as a function of the individual probe by probe correlations. Weighted mean expression profiles were calculated using the inverse of the coefficient of variation (CV) as a weight with the sum of the weights being equal to unity (previously discussed in Firley et al. 2008). Subtraction profiles were calculated either between the average mutant and control profiles or in a everyone-against-everyone scheme using a mixed-distribution ANOVA (see also Wilhelm et al. 2008) to reveal statistically significant (p<0.05) changes in gene expression. Hierarchical bi-clustering was performed using average Euclidean distances calculated from the individual experiments. Correlation between qPCR and microarray data was determined using Pearson correlation coefficients.



Gene ontology analysis

Gene Ontology (GO) and KEGG annotations were analyzed using the Panther Protein Classification System (http://www.pantherdb.org) to identify functional annotations that were significantly enriched in the different gene sets when compared to the whole set of genes present on the ABI microarray. Note that a given gene can be assigned to different pathways or biological processes. P-values are determined using a binominal distribution and a null hypothesis of a random set of genes with identical size.

References

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/20554270

Glucocorticoid receptors in dopaminoceptive neurons, key for cocaine, are dispensable for molecular and behavioral morphine responses.

Barik J, Parnaudeau S, Saint Amaux AL, Guiard BP, Golib Dzib JF, Bocquet O, Bailly A, Benecke A, Tronche F.

Biol Psychiatry. 2010 Aug 1;68(3):231-9.