Matthew C. Hearing

Repeated N-Acetylcysteine Administration Alters Plasticity-Dependent Effects of Cocaine

Cocaine produces a persistent reduction in cystine–glutamate exchange via system xc− in the nucleus accumbens that may contribute to pathological glutamate signaling linked to addiction. System xc− influences glutamate neurotransmission by maintaining basal, extracellular glutamate in the nucleus accumbens, which, in turn, shapes synaptic activity by stimulating group II metabotropic glutamate autoreceptors. In the present study, we tested the hypothesis that a long-term reduction in system xc− activity is part of the plasticity produced by repeated cocaine that results in the establishment of compulsive drug seeking. To test this, the cysteine prodrug N-acetylcysteine was administered before daily cocaine to determine the impact of increased cystine–glutamate exchange on the development of plasticity-dependent cocaine seeking. Although N-acetylcysteine administered before cocaine did not alter the acute effects of cocaine on self-administration or locomotor activity, it prevented behaviors produced by repeated cocaine including escalation of drug intake, behavioral sensitization, and cocaine-primed reinstatement. Because sensitization or reinstatement was not evident even 2–3 weeks after the last injection of N-acetylcysteine, we examined whether N-acetylcysteine administered before daily cocaine also prevented the persistent reduction in system xc− activity produced by repeated cocaine. Interestingly, N-acetylcysteine pretreatment prevented cocaine-induced changes in [35S]cystine transport via system xc−, basal glutamate, and cocaine-evoked glutamate in the nucleus accumbens when assessed at least 3 weeks after the last N-acetylcysteine pretreatment. These findings indicate that N-acetylcysteine selectively alters plasticity-dependent behaviors and that normal system xc− activity prevents pathological changes in extracellular glutamate that may be necessary for compulsive drug seeking.

Glutamate Transporters Regulate Extrasynaptic NMDA Receptor Modulation of Kv2.1 Potassium Channels

Delayed-rectifier Kv2.1 potassium channels regulate somatodendritic excitability during periods of repetitive, high-frequency activity. Recent evidence suggests that Kv2.1 channel modulation is linked to glutamatergic neurotransmission. Because NMDA-type glutamate receptors are critical regulators of synaptic plasticity, we investigated NMDA receptor modulation of Kv2.1 channels in rodent hippocampus and cortex. Bath application of NMDA potently unclustered and dephosphorylated Kv2.1 and produced a hyperpolarizing shift in voltage-dependent activation of voltage-sensitive potassium currents (IK). In contrast, driving synaptic activity in Mg2+-free media to hyperactivate synaptic NMDA receptors had no effect on Kv2.1 channels, and moderate pentylenetetrazole-induced seizure activity in adult mice did not dephosphorylate hippocampal Kv2.1 channels. Selective activation of extrasynaptic NMDA receptors unclustered and dephosphorylated Kv2.1 channels and produced a hyperpolarizing shift in neuronal IK. In addition, inhibition of glutamate uptake rapidly activated NMDA receptors and dephosphorylated Kv2.1 channels. These observations demonstrate that regulation of intrinsic neuronal activity by Kv2.1 is coupled to extrasynaptic but not synaptic NMDA receptors. These data support a novel mechanism for glutamate transporters in regulation of neuronal excitability and plasticity through extrasynaptic NMDA receptor modulation of Kv2.1 channels.

Repeated Cocaine Weakens GABAB-Girk Signaling in Layer 5/6 Pyramidal Neurons in the Prelimbic Cortex

Repeated cocaine exposure triggers adaptations in layer 5/6 glutamatergic neurons in the medial prefrontal cortex (mPFC) that promote behavioral sensitization and drug-seeking behavior. While suppression of metabotropic inhibitory signaling has been implicated in these behaviors, underlying mechanisms are unknown. Here, we show that Girk/K(IR)3 channels mediate most of the GABA(B) receptor (GABA(B)R)-dependent inhibition of layer 5/6 pyramidal neurons in the mPFC and that repeated cocaine suppresses this pathway. This adaptation was selective for GABA(B)R-dependent Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex (PrLC) and involved a D₁/₅ dopamine receptor- and phosphorylation-dependent internalization of GABA(B)R and Girk channels. Persistent suppression of Girk signaling in layer 5/6 of the dorsal mPFC enhanced cocaine-induced locomotor activity and occluded behavioral sensitization. Thus, the cocaine-induced suppression of GABA(B)R-Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex appears to represent an early adaptation critical for promoting addiction-related behavior.

Acute Cocaine Exposure Weakens GABAB Receptor-Dependent G-Protein-Gated Inwardly Rectifying K+ Signaling in Dopamine Neurons of the Ventral Tegmental Area

Enhanced glutamatergic neurotransmission in dopamine (DA) neurons of the ventral tegmental area (VTA), triggered by a single cocaine injection, represents an early adaptation linked to the more enduring effects of abused drugs that characterize addiction. Here, we examined the impact of in vivo cocaine exposure on metabotropic inhibitory signaling involving G-protein-gated inwardly rectifying K+ (Girk) channels in VTA DA neurons. Somatodendritic Girk currents evoked by the GABAB receptor (GABABR) agonist baclofen were diminished in a dose-dependent manner in mice given a single cocaine injection. This adaptation persisted for 3–4 d, was specific for DA neurons of the VTA, and occurred in parallel with an increase in spontaneous glutamatergic neurotransmission. No additional suppression of GABABR–Girk signaling was observed following repeated cocaine administration. While total Girk2 and GABABR1 mRNA and protein levels were unaltered by cocaine exposure in VTA DA neurons, the cocaine-induced decrease in GABABR–Girk signaling correlated with a reduction in Girk2-containing channels at the plasma membrane in VTA DA neurons. Systemic pretreatment with sulpiride, but not SCH23390 (7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol), prevented the cocaine-induced suppression of GABABR–Girk signaling, implicating D2/3 DA receptor activation in this adaptation. The acute cocaine-induced weakening of somatodendritic Girk signaling complements the previously demonstrated cocaine-induced strengthening of glutamatergic neurotransmission, likely contributing to enhanced output of VTA DA neurons during the early stages of addiction.

Reversal of morphine-induced cell-type–specific synaptic plasticity in the nucleus accumbens shell blocks reinstatement

Significance The recent rise in opioid addiction has made development of new treatments a public health priority. The effort has been impeded by a distinct lack of understanding how opioid-induced alterations in synaptic transmission and cellular plasticity within reward brain regions, such as the nucleus accumbens (NAc), drive addiction behavior. We examined whether repeated morphine induces differential alterations in synaptic strength and transmission in subpopulations of NAc neurons, those expressing dopamine D1 or D2 receptors, that play opposing roles in addiction behavior. Morphine enhanced synaptic strength and transmission at D1 medium spiny neuron (MSN) synapses and reduced signaling in D2-MSN. Reversal of this plasticity with in vivo optogenetics or the antibiotic ceftriaxone disrupted the rewarding properties of morphine, providing a targetable molecular mechanism for future pharmacotherapies. Drug-evoked plasticity at excitatory synapses on medium spiny neurons (MSNs) of the nucleus accumbens (NAc) drives behavioral adaptations in addiction. MSNs expressing dopamine D1 (D1R-MSN) vs. D2 receptors (D2R-MSN) can exert antagonistic effects in drug-related behaviors, and display distinct alterations in glutamate signaling following repeated exposure to psychostimulants; however, little is known of cell-type–specific plasticity induced by opiates. Here, we find that repeated morphine potentiates excitatory transmission and increases GluA2-lacking AMPA receptor expression in D1R-MSNs, while reducing signaling in D2-MSNs following 10–14 d of forced abstinence. In vivo reversal of this pathophysiology with optogenetic stimulation of infralimbic cortex-accumbens shell (ILC-NAc shell) inputs or treatment with the antibiotic, ceftriaxone, blocked reinstatement of morphine-evoked conditioned place preference. These findings confirm the presence of overlapping and distinct plasticity produced by classes of abused drugs within subpopulations of MSNs that may provide targetable molecular mechanisms for future pharmacotherapies.

Differential GABAB-Receptor-Mediated Effects in Perisomatic- and Dendrite-Targeting Parvalbumin Interneurons

Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABAB receptors (GABABRs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABABRs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABABRs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABABR-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABABR activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABAB IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABABRs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABABRs may shift the balance between perisomatic and dendritic inhibition.

Suppression of activity-regulated cytoskeleton-associated gene expression in the dorsal striatum attenuates extinction of cocaine-seeking

The caudate putamen (CPu) has been implicated in habit learning and neuroadaptive changes that mediate the compulsive nature of drug-seeking following chronic cocaine self-administration. Re-exposure to an operant chamber previously associated with cocaine, but not yoked-saline, increases activity-regulated cytoskeleton-associated (Arc) gene mRNA expression within the dorsolateral (dl) CPu following prolonged abstinence. In this study, we tested the hypothesis that antisense gene knockdown of Arc within the dlCPu would alter cocaine-seeking. Initial studies showed that a single infusion of Arc antisense oligodeoxynucleotide (ODN) into the dlCPu significantly attenuated the induction of Arc mRNA and Arc protein by a single cocaine exposure (20 mg/kg i.p.) compared to scrambled-ODN-infused controls. In cocaine self-administering rats, infusion of Arc antisense ODN into the dlCPu 3 h prior to a test of context-driven drug-seeking significantly attenuated Arc protein induction, but failed to alter responding during testing, suggesting striatal Arc does not facilitate context-induced drug-seeking following prolonged abstinence. However, Arc antisense ODN infusion blunted the decrease in responding during subsequent 1-h extinction tests 24 and 48 h later. Following re-exposure to a cocaine-paired context, surface expression of the AMPA-type glutamate receptor GluR1 was significantly reduced whereas GluR2 was significantly increased in the dlCPu, independent of Arc antisense ODN infusion. Together, these findings indicate an important role for Arc in neuroadaptations within brain regions responsible for drug-seeking after abstinence and direct attention to changes occurring within striatal circuitry that are necessary to break down the habitual behaviour that leads to relapse.

Mechanisms underlying the activation of G-protein–gated inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297

Significance Many neurotransmitters dampen excitability in the heart and brain by activating G-protein–gated inwardly rectifying K+ (GIRK) channels. The lack of selective pharmacological tools for GIRK channels has hindered investigations into their physiological and pathophysiological relevance. Here, we examined the mechanisms underlying the activation of GIRK channels by ML297, the prototypical member of a new family of small molecule GIRK channel modulators. ML297 activates GIRK channels via a unique mechanism that requires two amino acids specific to the GIRK1 subunit. In addition, ML297 reduces anxiety-related behavior in mice, in a GIRK1-dependent manner, without triggering sedation or addiction-related behavior. Thus, ML297 is a new tool for probing the therapeutic potential of GIRK channel modulation, which may benefit individuals with anxiety-related disorders. ML297 was recently identified as a potent and selective small molecule agonist of G-protein–gated inwardly rectifying K+ (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP2), but is otherwise distinct from receptor-induced, G-protein–dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.

GIRK channels modulate opioid-induced motor activity in a cell type- and subunit-dependent manner

G-protein-gated inwardly rectifying K+ (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.

Cocaine-induced adaptations in metabotropic inhibitory signaling in the mesocorticolimbic system.

Abstract The addictive properties of psychostimulants such as cocaine are rooted in their ability to activate the mesocorticolimbic dopamine (DA) system. This system consists primarily of dopaminergic projections arising from the ventral tegmental area (VTA) and projecting to the limbic and cortical brain regions, such as the nucleus accumbens (NAc) and prefrontal cortex (PFC). While the basic anatomy and functional relevance of the mesocorticolimbic DA system is relatively well-established, a key challenge remaining in addiction research is to understand where and how molecular adaptations and corresponding changes in function of this system facilitate a pathological desire to seek and take drugs. Several lines of evidence indicate that inhibitory signaling, particularly signaling mediated by the Gi/o class of heterotrimeric GTP-binding proteins (G proteins), plays a key role in the acute and persistent effects of drugs of abuse. Moreover, recent evidence argues that these signaling pathways are targets of drug-induced adaptations. In this review we discuss inhibitory signaling pathways involving DA and the inhibitory neurotransmitter GABA in two brain regions – the VTA and PFC – that are central to the effects of acute and repeated cocaine exposure and represent sites of adaptations linked to addiction-related behaviors including sensitization, craving, and relapse.