To address the specific questions mentioned above, we have outline five projects and a brief experimental design summary.? ? Project 1: How does chronic cocaine treatment affect synaptic function and morphology? ? Wild-type and transgenic mice will receive passive cocaine/saline administration (IP) daily for 5-30 days. This will be followed by behaivoral testing: locomotor activity and place preference tested every day with injection. We will then prepare acute brain slices / brain slice cultures and perform in vitro procedures? Different techniques and in vitro procedures will be applied to determine the changes that take place in the brain of addicted animals. We will investigate:? - Morphological changes: such as alterations in spine numbers, size, motility and dendritic branching.? - Changes in synaptic strength, synaptic plasticity and modulation.? - Biochemical changes in expression of synaptic proteins such as glutamate AMPA receptors, NMDA receptors, dopamine receptors and signaling molecules.? - Changes in intracellular calcium signaling in spines and dendrites? ? Project 2: Animals that display high impulsivity behavior show increased vulnerability to become compulsive drug seekers when chronically exposed to cocaine (Belin et al., 2008 Science 320:1352-1355). We will compare the synaptic function and the structure of neurons in different brain regions of mice that display high and low impulsivity. ? Wild-type and transgenic mice will undergo behavioral testing (5 choice serial reaction time task) daily for 30-40 days followed by in vitro procedures. Different techniques will be used to identify neuronal markers highly impulsive animals. We will investigate:? - Morphology: spine numbers, size, motility and dendritic branching.? - Biochemistry: expression levels of synaptic proteins and signaling molecules.? - Calcium signaling in spines and dendrites? - Synaptic strength and plasticity? ? ? Project 3: Only a fraction of cocaine users become compulsive drug-takers. What is different in the brain of subjects exposed and addicted in comparison to those exposed but non-addicted?? Is there a difference in synaptic function and morphology between mice that display addictive-like behaviors and mice that do not after they all have been chronically exposed to cocaine? ? Wild-type mice and transgenic mice will receive cocaine/saline self-administration for 30 days followed by behavioral testing to determine addictive score after 1, 10 or 30 days of last exposure. We will then perform in vitro procedures on these mice. Electrophysiological, morphological and biochemical analysis will be performed as described in project 1 and 2.? ? ? Project 4: What is the effect of voluntary ethanol exposure on neuronal function and morphology in specific brain regions? What is different in the brain of animals that actively seek ethanol and those that avoid it after the chronic treatment? ? Wild-type and transgenic mice will undergo voluntary ethanol intake for 15-30 days followed by behavioral testing to determine degree of dependence. In vitro procedures will then be performed on brain tissue from these mice. Electrophysiological, morphological and biochemical analysis will be performed as described above.
