Beyond their immune function, microglia have key homeostatic functions in the brain. It is now well accepted that they control the life of synapses in different contexts: during development, in experience-dependent plasticity processes and pathologies.
The main objective of this proposal is to determine the role of microglia in remodeling nucleus accumbens-(NAc) glutamatergic transmission, through the maturation of newly formed silent synapses. This process underlies critical circuit mechanisms promoting cocaine craving (a risk factor for drug relapse), a feature of cue-associated cocaine memories.
Our central hypothesis, based on the current literature and our preliminary results (see below), is: microglia-neuron interactions dependent on fractalkine/CX3CR1 signaling contribute to the maturation and plasticity of silent glutamatergic synapses in the NAc, which embed critical memory traces that promote cue-induced cocaine craving.
To verify this hypothesis, we will combine behavioral measures, whole-cell patch clamp recordings, biochemical approaches and pharmacological interventions in mice and rats. Our proposal is based on the following 3 main aims: 1) Determine the effects of microglial depletion on glutamatergic transmission in the NAc during forced abstinence from cocaine-induced conditioned place preference (CPP). 2) Determine the role of fractalkine/CX3CR1 signaling on the electrophysiological properties of excitatory synapses in acute slices of NAc after forced abstinence from cocaine-induced CPP. 3) Determine the causal role of fractalkine/CX3CR1 signaling in modulating incubated cocaine craving after forced abstinence from cocaine-self administration (the gold standard procedure employed to study drug addiction-related neurobiological mechanisms).
We believe that our proposal, combining behavioral, electrophysiological and biochemical approaches, will offer a new perspective to address a question of great social and clinical relevance.
The major objective of this proposal is to study the role of microglia in remodeling glutamatergic transmission in the NAc which underlies critical circuit mechanisms promoting cocaine craving and drug relapse after forced abstinence. Our central hypothesis is that CX3CR1 dependent microglia-neuron interaction critically contributes to the maturation and plasticity of silent glutamatergic synapses in the NAc.
If confirmed, these findings will enable novel research in several areas, including sex differences in relapse vulnerability, fractalkine-based treatment strategies, and consequences of cocaine-induced synaptic plasticity for NAc function.
Expected results:
Aim 1. By using a `loss of function strategy, after the completion of this aim, we will have characterized the role of microglia on silent synapse maturation after forced abstinence and its contribution to cocaine CPP retention. We predict that microglia depletion (PLX-induced), by preventing the maturation of silent synapses (indexed by: reduced rectification index, increased % of silent synapses in the minimal stimulation assay, reduced GluA1 subunit and higher levels of GluN2B subunits relative to control) prevents cocaine CPP retention (observed in the preliminary results).
Aim 2. We are aware that CX3CL1/CX3CR1 pathway is not the only one involved in signaling between neurons and microglia. Indeed, there are multiple bidirectional signaling pathways active in different functional contexts[55]. However, understanding the role of fractalkine signaling in cocaine plasticity will be an important step in the comprehension of this phenomenon and will enlarge our understanding on the elusive mechanisms by which the action of microglia on synapses takes place.
Aim 3. After the completion of this study, we will have assessed the efficacy of the CX3CL1 antagonist JMS-17-2 in preventing incubation of cocaine craving. We predict that JMS-17-2, by preventing the maturation of silent synapses, prevents the incubation of cocaine craving. A question of major importance for treating human cocaine users is the extent to which adaptations produced by chronic drug use recover with a given pharmacological-based or behavioral-based therapy during prolonged abstinence. Preclinical cocaine self-administration studies suggest that incubation of cocaine craving intensifies over time and plateau after ~20 days of forced abstinence [5] (and stay high up to 100 days).
Potential pitfalls and contingency plans
In case morphology from microglia in acute slices will be inconclusive, due to resolution and slicing related issues, immunofluorescence will be repeated using perfused mice and anti IBA1 antibody[37].
The lack of expression of Thy-1 on MSN prevents us from using double labeled tomato/GFP mice (Cx3cr1::CreERT2; Rosa26-CAG::LSL-tomato; Thy1::GFP), representing the most validated experimental tool for microglia-neuron contact analysis[26]. For this reason, we will analyze contacts by IBA-1 immunofluorescence in double transgenic mice expressing GFP in MSNs[54].
Potential age-based differences in rats drug metabolism will not affect seeking tests, occurring in drug-free conditions. In case of systemic effects of JMS-17-2, we will inject the drug intracranially. We do not envision other technical concerns given the expertise on the approaches proposed here, routinely used in the laboratory of D. Ragozzino and collaborators.
Innovation of the research
This proposal is innovative, in our opinion, on several counts: (i) The incubation of craving model provides a unique approach that specifically focuses on the propensity to relapse, both of which occur in humans. (ii) At present there are several papers available on the incubation of cocaine craving after forced abstinence, focusing primarily on synaptic plasticity, behavioral pharmacology, neural circuitry and epigenetics. Despite the relevance of neuronal signaling, work to date has not considered a role of microglia on cocaine-induced synaptic adaptations, while is known that microglia play a critical role on the formation and elimination of synapses during development. Our focus on the microglial mechanisms modulating synaptic transmission in the adult brain is unique and is one of the aspects of our research that will open novel future directions in the therapeutic setting. (iii) In addition to the translational relevance, we believe, that cocaine-induced plasticity represents a privileged phenomenon for the study of the mechanisms of action of microglia on synaptic plasticity. In fact, it represents a form of very strong synaptic remodeling associated with the formation and maturation of new synapses in adults with a strong behavioral correlate. (iv) The suggested work combines complex behavior and state-of-the-art electrophysiological and biochemical approaches; the convergence of such approaches to study adult rodents is still relatively rare.