Specific Aims
The three specific aims of this project have not been modified from those stated in the original application. The aims of the project are:
- To measure ACh in NAc, PFC, hippocampus and amygdala during methamphetamine (MA) self-administration in B6D2F1 mice. Comparisons of ACh responses in self-administration animals will be made with mice that receive equal amounts of MA passively. Experiments in this aim were planned to extend over the length of the project.
- To determine the effect(s) of MA on the induction of transcription factors and choline transporters in cholinergic cells and target nuclei. This aim was planned to begin in the third year of the project.
- To measure the ability of stress to induce reinstatement of MA-seeking behavior after extinction and the ability of cholinergic drugs to block reinstatement. This project was planned to run in the fourth and fifth years of the project.
Studies and Results
The overall goal of this component project is to understand the neurochemical systems that participate in the development of MA self-administration and relapse. The focus is on cholinergic pathways that innervate dopamine regions and may be important for controlling the amount of MA mice will self-administer. Here we report results from two of the three aims and important new data on the ACh circuit that is affected by MA treatment.
Studies to address Specific Aim 1:
We have conducted MA self-administration sessions in conjunction with real-time measurements of ACh in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) using microdialysis. In the animals tested to date, we found that ACh levels in the mPFC varied positively and synchronously with the amount of MA self-administered (Figure 1). In contrast, ACh levels in the NAc were not affected by MA. ACh responses from animals that received passive MA are currently being analyzed.
Studies to address Specific Aim 2:
As planned, we have obtained brains from a cohort of 4 mice that self-administered MA and are now running the next cohort through the self-administration procedure. When all the brains have been collected, we will begin processing the tissue for ITF measures in Dr. Ryabinin's lab.
Studies to address Specific Aim 3:
In another experiment we found that
the metabolic stressor, 2-deoxyglucose (2-DG) effectively reinstated extinguished MA-seeking behavior. This was the first demonstration that a metabolic stress resulted in reinstatement of drug seeking. We will use this model in the next experiments to determine if cholinergic agents affect stress-induced reinstatement.
Additional Studies. We examined the circuit by which MA affects ACh and DA in the mesolimbic system. We administered systemic and intra-tegmental MA and measured ACh and DA in VTA of mice. We found that MA acted directly in the VTA to induce a strong and short-lived increase in somatodendritic DA release but acted in an area upstream from the VTA to produce a prolonged intra-VTA increase in ACh. We are now preparing these results for publication.
Significance
Methamphetamine abuse has developed into a significant health-care concern in the United States. Recent advances in drug abuse research have suggested that one reason MA has such a high abuse potential is its ability to inappropriately activate (or, in some cases, suppress) the activity of neurotransmitters.
In particular, neurochemical systems that interact with dopamine (DA) are potentially important components in both the development of addiction and the expression of compulsive behaviors. One such modulatory system appears to be cholinergic pathways that interact with DA systems at all levels of the reward circuit.
In the experiments in this component, we have developed animal models of MA self-administration and relapse that are suitable for examining the role of cholinergic systems in MA reward and addiction-like behavior.
Plans
Specific Aim 1:
Microdialysis experiments for ACh in NAc and mPFC, amygdala and hippocampus in MA self-administration mice are continuing. We have concentrated first on ACh measures in the mesolimbic terminals (NAc and mPFC) and will be expanding the analysis to cholinergic systems in the hippocampus and amygdala. Measurements of ACh in mice that receive passive MA are proceeding in parallel with the self-administration tests. We anticipate submitting a research report on the effect of MA self-administration on mesolimbic ACh systems within the next 3 months.
Specific Aim 2:
As planned, in the next year of the project we will begin analyzing the effect(s) of both active and passive MA exposure on the induction of transcription factors, c-Fos, Egr1 and FosB as markers of neuronal activity. We have collected brains from 4 mice that self-administered a criterion amount of >20µg of MA ICV per session. All of the tissue must be analyzed together, so we will begin this analysis as soon as we have collected the remainder of the active and passive groups.
Specific Aim 3:
The manuscript describing reinstatement of MA-seeking behavior induced by 2-DG is being re-submitted for review. We will use this mild-stress model to test the effects of the nicotinic receptor antagonist mecamylamine on 2-DG reinstatement. Since we did not find a significant change in ACh response to MA in the NAc, but we did see a pronounced response in mPFC, we will target the mPFC for our first tests of reinstatement.
Component Interactions:
Mice selectively bred for MA-drinking from Dr. Tamara Phillips' group (Component 6) have been reserved for us to test for acquisition and maintenance of systemic self-administration behavior. These animals will be sufficiently old for catheterization in 4-5 weeks. We plan to test 6 animals from each of the high- and low-MA drinking lines. There will be no changes in the research involving vertebrate animals, i.e. B6D2F1 mice.
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