Restoration of Intrinsic Inhibition in the PFC to Prevent Relapse to Cocaine Seeking

Reward learning involves burst firing of midbrain dopamine neurons in the Ventral Tegmental Area (VTA). The resulting dopamine release from VTA terminals instructs regions like the prefrontal cortex (PFC) about rewards and reward-related cues. Glutamatergic PFC neurons initiate motivated behaviors via innervation of the nucleus accumbens. Chronic use of drugs of abuse such as cocaine disrupts this natural reward pathway, leading to enduring cellular adaptations in dopaminergic signaling that contribute to relapse vulnerability. However, how dopamine modulates activity in this circuitry is not known. We posit that dopamine release from VTA terminals gates intrinsic inhibition in the PFC via a dopamine D1 receptor mediated reduction in potassium channel function. By blocking dopamine reuptake in the cortex, cocaine elevates dopamine signaling at these receptors, increasing D1 receptor activation and the subsequent activation of intracellular signaling cascades. We propose that disruptions in these mechanisms following chronic cocaine use contribute to addiction pathology, resulting in long–lasting reductions in intrinsic inhibition that contribute to drug-seeking in response to cues. We test this hypothesis using the extinction–reinstatement rodent model of cocaine addiction. This in vivo protocol resembles the human condition, in that exposure to drug-associated cues induces drug-seeking behavior. In combination with in situ electrophysiology, chemogenetics, optogenetics, and retrograde tracing, we show that activation of VTA terminals reduces intrinsic inhibition in accumbens core-projecting prelimbic (PL) PFC cells. Operant cocaine self-administration renders PL cells hypersensitive to depolarization via elevated D1 receptor signaling, resulting in calcium store dependent desensitization of inhibitory Kv7 potassium channels and an enduring reduction in intrinsic inhibition. The deficit in Kv7 function and intrinsic inhibition is overcome by pharmacological stabilization of Kv7 channels with retigabine, which when microinjected into the PL reduces cue-induced reinstatement of cocaine-seeking. These studies underscore the significance of dopamine modulation of intrinsic inhibition in accumbens-projecting PL neurons as a mediator of relapse to drug seeking, and offer Kv7 as a potential novel drug target for cocaine addiction

Dopamine terminals from the ventral tegmental area gate intrinsic inhibition in the prefrontal cortex.

Spike frequency adaptation (SFA or accommodation) and calcium-activated potassium channels that underlie after-hyperpolarization potentials (AHP) regulate repetitive firing of neurons. Precisely how neuromodulators such as dopamine from the ventral tegmental area (VTA) regulate SFA and AHP (together referred to as intrinsic inhibition) in the prefrontal cortex (PFC) remains unclear. Using whole cell electrophysiology, we measured intrinsic inhibition in prelimbic (PL) layer 5 pyramidal cells of male adult rats. Results demonstrate that bath application of dopamine reduced intrinsic inhibition (EC50: 25.0 μmol/L). This dopamine action was facilitated by coapplication of cocaine (1 μmol/L), a blocker of dopamine reuptake. To evaluate VTA dopamine terminals in PFC slices, we transfected VTA dopamine cells of TH::Cre rats in vivo with Cre-dependent AAVs to express channelrhodopsin-2 (ChR2) or designer receptors exclusively activated by designer drugs (DREADDS). In PFC slices from these animals, stimulation of VTA terminals with either blue light to activate ChR2 or bath application of clozapine-N-oxide (CNO) to activate Gq-DREADDs produced a similar reduction in intrinsic inhibition in PL neurons. Electrophysiological recordings from cells expressing retrograde fluorescent tracers showed that this plasticity occurs in PL neurons projecting to the accumbens core. Collectively, these data highlight an ability of VTA terminals to gate intrinsic inhibition in the PFC, and under appropriate circumstances, enhance PL neuronal firing. These cellular actions of dopamine may be important for dopamine-dependent behaviors involving cocaine and cue-reward associations within cortical-striatal circuits

Dynamic CRMP2 Regulation of CaV2.2 in the Prefrontal Cortex Contributes to the Reinstatement of Cocaine Seeking

Cocaine addiction remains a major health concern with limited effective treatment options. A better understanding of mechanisms underlying relapse may help inform the development of new pharmacotherapies. Emerging evidence suggests that collapsin response mediator protein 2 (CRMP2) regulates presynaptic excitatory neurotransmission and contributes to pathological changes during diseases, such as neuropathic pain and substance use disorders. We examined the role of CRMP2 and its interactions with a known binding partner, CaV2.2, in cocaine-seeking behavior. We employed the rodent self-administration model of relapse to drug seeking and focused on the prefrontal cortex (PFC) for its well-established role in reinstatement behaviors. Our results indicated that repeated cocaine self-administration resulted in a dynamic and persistent alteration in the PFC expression of CRMP2 and its binding partner, the CaV2.2 (N-type) voltage-gated calcium channel. Following cocaine self-administration and extinction training, the expression of both CRMP2 and CaV2.2 was reduced relative to yoked saline controls. By contrast, cued reinstatement potentiated CRMP2 expression and increased CaV2.2 expression above extinction levels. Lastly, we utilized the recently developed peptide myr-TAT-CBD3 to disrupt the interaction between CRMP2 and CaV2.2 in vivo. We assessed the reinstatement behavior after infusing this peptide directly into the medial PFC and found that it decreased cue-induced reinstatement of cocaine seeking. Taken together, these data suggest that neuroadaptations in the CRMP2/CaV2.2 signaling cascade in the PFC can facilitate drug-seeking behavior. Targeting such interactions has implications for the treatment of cocaine relapse behavior.12 month embargo; published online: 29 July 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Restoration of Kv7 Channel-Mediated Inhibition Reduces Cued-Reinstatement of Cocaine Seeking

Cocaine addicts display increased sensitivity to drug-associated cues, due in part to changes in the prelimbic prefrontal cortex (PL-PFC). The cellular mechanisms underlying cue-induced reinstatement of cocaine seeking remain unknown. Reinforcement learning for addictive drugs may produce persistent maladaptations in intrinsic excitability within sparse subsets of PFC pyramidal neurons. Using a model of relapse in male rats, we sampled >600 neurons to examine spike frequency adaptation (SFA) and after hyperpolarizations (AHPs), two systems that attenuate low-frequency inputs to regulate neuronal synchronization. We observed that training to self-administer cocaine or nondrug (sucrose) reinforcers decreased SFA and AHPs in a subpopulation of PL-PFC neurons. Only with cocaine did the resulting hyperexcitability persist through extinction training and increase during reinstatement. In neurons with intact SFA, dopamine enhanced excitability by inhibiting Kv7 potassium channels that mediate SFA. However, dopamine effects were occluded in neurons from cocaine-experienced rats, where SFA and AHPs were reduced. Pharmacological stabilization of Kv7 channels with retigabine restored SFA and Kv7 channel function in neuroadapted cells. When microinjected bilaterally into the PL-PFC 10 min before reinstatement testing, retigabine reduced cue-induced reinstatement of cocaine seeking. Last, using cFos-GFP transgenic rats, we found that the loss of SFA correlated with the expression of cFos-GFP following both extinction and re-exposure to drug-associated cues. Together, these data suggest that cocaine self-administration desensitizes inhibitory Kv7 channels in a subpopulation of PL-PFC neurons. This subpopulation of neurons may represent a persistent neural ensemble responsible for driving drug seeking in response to cues.National Institute on Drug Abuse [F31-DA-036989, T32-DA-007288, R01-DA-027664, R01-NS-098772, R01-DA-042852, P50-DA-015369, R01-DA-033342A]6 month embargo; published online: 25 April 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

Ventral tegmental area (VTA) dopamine (DA) neurons perform diverse functions in motivation and cognition, but their precise roles in addiction-related behaviors are still debated. Here, we targeted VTA DA neurons for bidirectional chemogenetic modulation during specific tests of cocaine reinforcement, demand, and relapse-related behaviors in male rats, querying the roles of DA neuron inhibitory and excitatory G-protein signaling in these processes. Designer receptor stimulation of Gq signaling, but not Gs signaling, in DA neurons enhanced cocaine seeking via functionally distinct projections to forebrain limbic regions. In contrast, engaging inhibitory Gi/o signaling in DA neurons blunted the reinforcing and priming effects of cocaine, reduced stress-potentiated reinstatement, and altered behavioral strategies for cocaine seeking and taking. Results demonstrate that DA neurons play several distinct roles in cocaine seeking, depending on behavioral context, G-protein-signaling cascades, and DA neuron efferent targets, highlighting their multifaceted roles in addiction.SIGNIFICANCE STATEMENT G-protein-coupled receptors are crucial modulators of ventral tegmental area (VTA) dopamine neuron activity, but how this metabotropic signaling impacts the complex roles of dopamine in reward and addiction is poorly understood. Here, we bidirectionally modulate dopamine neuron G-protein signaling with DREADDs (designer receptors exclusively activated by designer drugs) during a variety of cocaine-seeking behaviors, revealing nuanced, pathway-specific roles in cocaine reward, effortful seeking, and relapse-like behaviors. Gq and Gs stimulation activated dopamine neurons, but only Gq stimulation robustly enhanced cocaine seeking. Gi/o inhibitory signaling reduced some, but not all, types of cocaine seeking. Results show that VTA dopamine neurons modulate numerous distinct aspects of cocaine addiction- and relapse-related behaviors, and point to potential new approaches for intervening in these processes to treat addiction

Acute reorganization of postsynaptic GABAA receptors reveals the functional impact of molecular nanoarchitecture at inhibitory synapses

Summary: Neurotransmitter receptors partition into nanometer-scale subdomains within the postsynaptic membrane that are precisely aligned with presynaptic neurotransmitter release sites. While spatial coordination between pre- and postsynaptic elements is observed at both excitatory and inhibitory synapses, the functional significance of this molecular architecture has been challenging to evaluate experimentally. Here we utilized an optogenetic clustering approach to acutely alter the nanoscale organization of the postsynaptic inhibitory scaffold gephyrin while monitoring synaptic function. Gephyrin clustering rapidly enlarged postsynaptic area, laterally displacing GABAA receptors from their normally precise apposition with presynaptic active zones. Receptor displacement was accompanied by decreased synaptic GABAA receptor currents even though presynaptic release probability and the overall abundance and function of synaptic GABAA receptors remained unperturbed. Thus, acutely repositioning neurotransmitter receptors within the postsynaptic membrane profoundly influences synaptic efficacy, establishing the functional importance of precision pre-/postsynaptic molecular coordination at inhibitory synapses