Context-specific sensitization is due to a learned association between drug and stimuli in the administration environment. We had previously shown that this learned association is encoded by a pattern of sparsely distributed neurons called a neuronal ensemble. Until now, scientists have studied molecular neuroadaptations in brain homogenates without differentiating between activated neuronal ensembles and surrounding non-activated neurons. This likely obscures the changes seen only in the activated cells. Therefore, we developed a novel method for purifying activated neurons from rat striatal neuronal ensembles and assessing their unique set of cocaine-induced molecular neuroadaptations.
We used c-fos-lacZ transgenic rats to identify cocaine-activated neuronal ensembles in striatum. Electrophysiological activation of these neurons induces β-galactosidase protein that can be labeled with a fluorescent antibody against β-galactosidase. We then separated these fluorescently labeled activated neurons from the majority of non-activated neurons using Fluorescence Activated Cell Sorting (FACS).
Compared to non-activated neurons from the same cocaine-treated rats or all neurons from saline-treated rats, microarray analysis and quantitative PCR showed that activated neurons from cocaine-treated rats had much higher expression levels of many immediate early genes including arc, fos, fosB, and nr4a3. Because several of these genes are transcription factors, they can influence the expression of many downstream genes, and thus produce a vastly different gene expression profile in the activated neurons. The activated β-galactosidase-expressing neurons also had increased prodynorphin mRNA (a marker of D1-type neurons) and decreased dopamine D2 receptor mRNA; thus, the majority of neurons activated by cocaine are of the D1-type. Finally, the β-galactosidase-positive neurons likely have attenuated p38 MAPK signaling, because they expressed lower levels of the kinase that activates p38 (map2k6) and higher levels of the phosphatase that deactivates p38 (dusp1 or mkp1). Since p38 has been demonstrated to be involved in long term depression and to inhibit long term potentiation, these neurons may be primed for altered plasticity.
We have shown that the neurons mediating context-specific locomotor sensitization to cocaine have a broadly different gene expression profile than the surrounding non-activated neurons. This represents the first multi-gene analysis of a neuronal ensemble encoding a learned behavior.
|Advisor:||Hope, Bruce T.|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 72/10, Dissertation Abstracts International|
|Subjects:||Molecular biology, Neurosciences|
|Keywords:||Cocaine, Fluorescence activated cell sorting, Locomotor sensitization, Neuroadaptations, Neuronal ensembles|
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