A major problem in treating cocaine addiction is the high likelihood of relapse, even after months of abstinence. The persistence of relapse vulnerability suggests that it is maintained by long-lasting adaptations in the brain. There is an urgent need for therapeutic strategies to combat this problem. To this end, we have studied a rat model in which cue-induced cocaine craving progressively intensifies (incubates) over the first 2 months of withdrawal from extended access cocaine self-administration (SA). We showed previously that enhanced craving after ~1.5 months of withdrawal is mediated by Ca2+-permeable AMPARs (CP-AMPARs) that accumulate in nucleus accumbens (NAc) synapses. Thus, detailed information exists on the time-dependency of AMPAR and behavioral plasticity during incubation. In contrast, no information exists about plasticity of spines or NMDAR transmission in this model. The vast majority of studies on these latter endpoints have been performed after non-contingent cocaine regimens which are not directly useful for assessing cocaine craving, and most have examined only one withdrawal time. The objective of our proposal is to evaluate spine density and morphology, and NMDAR transmission at the single spine level, at 5 withdrawal times (15, 25, 35, 60 and 180 days) after discontinuing cocaine SA or saline SA (a control condition). Our central hypothesis is that there are increases in silent synapses early in withdrawal corresponding to an increased number of immature spines;later in withdrawal, silent synapses dissipate, the number of immature spines normalizes, and an increase in mature spines occurs coincident with CP-AMPAR insertion. This hypothesis will be tested by pursuing 3 Aims: 1) Characterize dendritic spine density and morphology in NAc neurons during the incubation of cocaine craving. Single NAc neurons will be filled with Lucifer yellow, imaged with confocal microscopy, and analyzed with NeuronStudio. 2) Investigate silent synapse formation and the contribution of NR2B-containing NMDARs to silent synapses during the incubation of cocaine craving. Silent synapses, particularly those expressing NR2B subunits, are preferentially primed for long term plasticity and represent a potential substrate fo AMPAR insertion. Patch clamp recordings, using minimal stimulation and coefficient of variation analyses, will assess the number of silent synapses;pharmacological approaches will assess the contribution of NR2B. 3) Determine Ca2+ signaling dynamics mediated by NMDARs and the relative proportion mediated by NR2B- containing NMDARs in NAc spines during the incubation of cocaine craving. To evaluate functional changes in NMDARs, NMDAR-mediated Ca2+ influx will be measured in single spines using patch clamp electrophysiology, 2-photon Ca2+ imaging, and flash photolysis of caged glutamate. Overall, by comparing the time-course of changes in cocaine craving, spine plasticity, silent synapse formation, and measures of Ca2+ influx, this exploratory proposal will generate necessary data that will enable us to formulate hypotheses about causality and ultimately develop strategies for reversing craving-related changes in synaptic function.
In abstinent cocaine users, environmental cues previously associated with drug use are powerful triggers for relapse. The proposed research is relevant to NIH's mission of reducing the burden of illness because it explores novel cellular mechanisms that potentially contribute to enhanced cue-induced cocaine craving during withdrawal. Understanding such mechanisms will accelerate the search for effective therapeutic strategies for cocaine addiction.