Yonatan M. Kupchik

Optogenetic inhibition of cocaine seeking in rats

Inhibitory optogenetics was used to examine the roles of the prelimbic cortex (PL), the nucleus accumbens core (NAcore) and the PL projections to the NAcore in the reinstatement of cocaine seeking. Rats were microinjected into the PL or NAcore with an adeno‐associated virus containing halorhodopsin or archaerhodopsin. After 12 days of cocaine self‐administration, followed by extinction training, animals underwent reinstatement testing along with the presence/absence of optically induced inhibition via laser light. Bilateral optical inhibition of the PL, NAcore or the PL fibers in the NAcore inhibited the reinstatement of cocaine seeking.

Coding the direct/indirect pathways by D1 and D2 receptors is not valid for accumbens projections

It is widely accepted that D1 dopamine receptor–expressing striatal neurons convey their information directly to the output nuclei of the basal ganglia, whereas D2-expressing neurons do so indirectly via pallidal neurons. Combining optogenetics and electrophysiology, we found that this architecture does not apply to mouse nucleus accumbens projections to the ventral pallidum. Thus, current thinking attributing D1 and D2 selectivity to accumbens projections akin to dorsal striatal pathways needs to be reconsidered.

Reinstatement of nicotine seeking is mediated by glutamatergic plasticity

Nicotine abuse and addiction is a major health liability. Nicotine, an active alkaloid in tobacco, is self-administered by animals and produces cellular adaptations in brain regions associated with drug reward, such as the nucleus accumbens. However, it is unknown whether, akin to illicit drugs of abuse such as cocaine or heroin, the adaptations endure and contribute to the propensity to relapse after discontinuing nicotine use. Using a rat model of cue-induced relapse, we made morphological and electrophysiological measures of synaptic plasticity, as well as quantified glutamate overflow, in the accumbens after 2 wk of withdrawal with extinction training. We found an enduring basal increase in dendritic spine head diameter and in the ratio of AMPA to NMDA currents in accumbens spiny neurons compared with yoked saline animals at 2 wk after the last nicotine self-administration session. This synaptic potentiation was associated with an increase in both AMPA (GluA1) and NMDA (GluN2A and GluN2B) receptor subunits, and a reduction in the glutamate transporter-1 (GLT-1). When nicotine seeking was reinstated by presentation of conditioned cues, there were parallel increases in behavioral responding, extracellular glutamate, and further increases in dendritic spine head diameter and ratio of AMPA to NMDA currents within 15 min. These findings suggest that targeting glutamate transmission might inhibit cue-induced nicotine seeking. In support of this hypothesis, we found that pharmacological inhibition of GluN2A with 3-Chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulfonamide (TCN-201) or GluN2B with ifenprodil abolished reinstated nicotine seeking. These results indicate that up-regulated GluN2A, GluN2B, and rapid synaptic potentiation in the accumbens contribute to cue-induced relapse to nicotine use.

Relapse induced by cues predicting cocaine depends on rapid, transient synaptic potentiation.

Cocaine addiction is characterized by long-lasting vulnerability to relapse arising because neutral environmental stimuli become associated with drug use and then act as cues that induce relapse. It is not known how cues elicit cocaine seeking, and why cocaine seeking is more difficult to regulate than seeking a natural reward. We found that cocaine-associated cues initiate cocaine seeking by inducing a rapid, transient increase in dendritic spine size and synaptic strength in the nucleus accumbens. These changes required neural activity in the prefrontal cortex. This is not the case when identical cues were associated with obtaining sucrose, which did not elicit changes in spine size or synaptic strength. The marked cue-induced synaptic changes in the accumbens were correlated with the intensity of cocaine, but not sucrose seeking, and may explain the difficulty addicts experience in managing relapse to cocaine use.

The effect of N-acetylcysteine in the nucleus accumbens on neurotransmission and relapse to cocaine

BACKGROUND Relapse to cocaine seeking has been linked with low glutamate in the nucleus accumbens core (NAcore) causing potentiation of synaptic glutamate transmission from prefrontal cortex (PFC) afferents. Systemic N-acetylcysteine (NAC) has been shown to restore glutamate homeostasis, reduce relapse to cocaine seeking, and depotentiate PFC-NAcore synapses. Here, we examine the effects of NAC applied directly to the NAcore on relapse and neurotransmission in PFC-NAcore synapses, as well as the involvement of the metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5). METHODS Rats were trained to self-administer cocaine for 2 weeks and following extinction received either intra-accumbens NAC or systemic NAC 30 or 120 minutes, respectively, before inducing reinstatement with a conditioned cue or a combined cue and cocaine injection. We also recorded postsynaptic currents using in vitro whole cell recordings in acute slices and measured cystine and glutamate uptake in primary glial cultures. RESULTS NAC microinjection into the NAcore inhibited the reinstatement of cocaine seeking. In slices, a low concentration of NAC reduced the amplitude of evoked glutamatergic synaptic currents in the NAcore in an mGluR2/3-dependent manner, while high doses of NAC increased amplitude in an mGluR5-dependent manner. Both effects depended on NAC uptake through cysteine transporters and activity of the cysteine/glutamate exchanger. Finally, we showed that by blocking mGluR5 the inhibition of cocaine seeking by NAC was potentiated. CONCLUSIONS The effect of NAC on relapse to cocaine seeking depends on the balance between stimulating mGluR2/3 and mGluR5 in the NAcore, and the efficacy of NAC can be improved by simultaneously inhibiting mGluR5.

Optogenetic Evidence That Pallidal Projections, Not Nigral Projections, from the Nucleus Accumbens Core Are Necessary for Reinstating Cocaine Seeking

The core subcompartment of the nucleus accumbens (NAcore) contributes significantly to behavioral responses following motivationally relevant stimuli, including drug-induced, stress-induced, and cue-induced reinstatement of cocaine seeking. Projections from NAcore that could carry information necessary to initiate reinstated cocaine seeking include outputs via the indirect pathway to the dorsolateral subcompartment of the ventral pallidum (dlVP) and through the direct pathway to the medial substantia nigra (SN). Here we used an optogenetic strategy to determine whether the dlVP or nigral projections from the NAcore are necessary for cocaine seeking initiated by a cocaine and conditioned cue combination in rats extinguished from cocaine self-administration. Rats were pretreated in the NAcore with an adeno-associated virus expressing the inhibitory opsin archaerhodopsin, and fiber-optic cannulae were implanted above the indirect pathway axon terminal field in the dlVP, or the direct pathway terminal field in the SN. Inhibiting the indirect pathway to the dlVP, but not the direct pathway to the SN, prevented cocaine-plus-cue-induced reinstatement. We also examined projections back to the NAcore from the ventral tegmental area (VTA) and dlVP. Inhibiting the dlVP to NAcore projection did not alter, while inhibiting VTA afferents abolished reinstated cocaine seeking. Localization of green fluorescent protein reporter expression and whole-cell patch electrophysiology were used to verify opsin expression. These data reveal a circuit involving activation of VTA inputs to the NAcore and NAcore projections through the indirect pathway to the dlVP as critical for cocaine-plus-cue-induced reinstatement of cocaine seeking.

Synaptic plasticity mediating cocaine relapse requires matrix metalloproteinases

Relapse to cocaine use necessitates remodeling excitatory synapses in the nucleus accumbens and synaptic reorganization requires matrix metalloproteinase (MMP) degradation of the extracellular matrix proteins. We found enduring increases in MMP-2 activity in rats after withdrawal from self-administered cocaine and transient increases in MMP-9 during cue-induced cocaine relapse. Cue-induced heroin and nicotine relapse increased MMP activity, and increased MMP activity was required for both cocaine relapse and relapse-associated synaptic plasticity.

Addiction-like Synaptic Impairments in Diet-Induced Obesity

BACKGROUND There is increasing evidence that the pathological overeating underlying some forms of obesity is compulsive in nature and therefore contains elements of an addictive disorder. However, direct physiological evidence linking obesity to synaptic plasticity akin to that occurring in addiction is lacking. We sought to establish whether the propensity to diet-induced obesity (DIO) is associated with addictive-like behavior, as well as synaptic impairments in the nucleus accumbens core considered hallmarks of addiction. METHODS Sprague Dawley rats were allowed free access to a palatable diet for 8 weeks then separated by weight gain into DIO-prone and DIO-resistant subgroups. Access to palatable food was then restricted to daily operant self-administration sessions using fixed ratio 1, 3, and 5 and progressive ratio schedules. Subsequently, nucleus accumbens brain slices were prepared, and we tested for changes in the ratio between α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate currents and the ability to exhibit long-term depression. RESULTS We found that propensity to develop DIO is linked to deficits in the ability to induce long-term depression in the nucleus accumbens, as well as increased potentiation at these synapses as measured by AMPA/N-methyl-D-aspartate currents. Consistent with these impairments, we observed addictive-like behavior in DIO-prone rats, including 1) heightened motivation for palatable food; 2) excessive intake; and 3) increased food seeking when food was unavailable. CONCLUSIONS Our results show overlap between the propensity for DIO and the synaptic changes associated with facets of addictive behavior, supporting partial coincident neurological underpinnings for compulsive overeating and drug addiction.

A novel fast mechanism for GPCR-mediated signal transduction—control of neurotransmitter release

In addition to calcium influx, charge movement in the G protein–coupled M2-muscarinic receptor is required for the control of acetylcholine release.

Cocaine Dysregulates Opioid Gating of GABA Neurotransmission in the Ventral Pallidum

The ventral pallidum (VP) is a target of dense nucleus accumbens projections. Many of these projections coexpress GABA and the neuropeptide enkephalin, a δ and μ opioid receptor (MOR) ligand. Of these two, the MOR in the VP is known to be involved in reward-related behaviors, such as hedonic responses to palatable food, alcohol intake, and reinstatement of cocaine seeking. Stimulating MORs in the VP decreases extracellular GABA, indicating that the effects of MORs in the VP on cocaine seeking are via modulating GABA neurotransmission. Here, we use whole-cell patch-clamp on a rat model of withdrawal from cocaine self-administration to test the hypothesis that MORs presynaptically regulate GABA transmission in the VP and that cocaine withdrawal changes the interaction between MORs and GABA. We found that in cocaine-extinguished rats pharmacological activation of MORs no longer presynaptically inhibited GABA release, whereas blocking the MORs disinhibited GABA release. Moreover, MOR-dependent long-term depression of GABA neurotransmission in the VP was lost in cocaine-extinguished rats. Last, GABA neurotransmission was found to be tonically suppressed in cocaine-extinguished rats. These substantial synaptic changes indicated that cocaine was increasing tone on MOR receptors. Accordingly, increasing endogenous tone by blocking the enzymatic degradation of enkephalin inhibited GABA neurotransmission in yoked saline rats but not in cocaine-extinguished rats. In conclusion, our results indicate that following withdrawal from cocaine self-administration enkephalin levels in the VP are elevated and the opioid modulation of GABA neurotransmission is impaired. This may contribute to the difficulties withdrawn addicts experience when trying to resist relapse.