Δ9 -Tetrahydrocannabinol self-administration induces cell type-specific adaptations in the nucleus accumbens core.

Drugs of abuse induce cell type-specific adaptations in D1- and D2-medium spiny neurons (MSNs) in the nucleus accumbens core (NAcore) that can bias signalling towards D1-MSNs and enhance relapse vulnerability. Whether Δ9 -tetrahydrocannabinol (THC) use initiates similar neuroadaptations is unknown. D1- and D2-Cre transgenic rats were transfected with Cre-dependent reporters and trained to self-administer THC + cannabidiol (THC + CBD). After extinction training spine morphology, glutamate transmission, CB1R function and cFOS expression were quantified. We found that extinction from THC + CBD induced a loss of large spine heads in D1- but not D2-MSNs and commensurate reductions in glutamate synaptic transmission. Also, presynaptic CB1R function was impaired selectively at glutamatergic synapses on D1-MSNs, which augmented the capacity to potentiate glutamate transmission. Using cFOS expression as an activity marker, we found no change after extinction but increased cFOS expression in D1-MSNs after cue-induced drug seeking. Contrasting D1-MSNs, CB1R function and glutamate synaptic transmission on D2-MSN synapses were unaffected by THC + CBD use. However, cFOS expression was decreased in D2-MSNs of THC + CBD-extinguished rats and was restored after drug seeking. Thus, CB1R adaptations in D1-MSNs partially predicted neuronal activity changes, posing pathway specific modulation of eCB signalling in D1-MSNs as a potential treatment avenue for cannabis use disorder (CUD).

Relapse-Associated Transient Synaptic Potentiation Requires Integrin-Mediated Activation of Focal Adhesion Kinase and Cofilin in D1-Expressing Neurons.

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced drug seeking in rodent models correlates with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses in the nucleus accumbens core (NAcore). Matrix metalloproteinases (MMPs) are inducible endopeptidases that degrade extracellular matrix (ECM) proteins, and reveal tripeptide Arginine-Glycine-Aspartate (RGD) domains that bind and signal through integrins. Integrins are heterodimeric receptors composed of αß subunits, and a primary signaling kinase is focal adhesion kinase (FAK). We previously showed that MMP activation is necessary for and potentiates cued reinstatement of cocaine seeking, and MMP-induced catalysis stimulates ß3-integrins to induce t-SP. Here, we determined whether ß3-integrin signaling through FAK and cofilin (actin depolymerization factor) is necessary to promote synaptic growth during t-SP. Using a small molecule inhibitor to prevent FAK activation, we blocked cued-induced cocaine reinstatement and increased spine head diameter (dh). Immunohistochemistry on NAcore labeled spines with ChR2-EYFP virus, showed increased immunoreactivity of phosphorylation of FAK (p-FAK) and p-cofilin in dendrites of reinstated animals compared with extinguished and yoked saline, and the p-FAK and cofilin depended on ß3-integrin signaling. Next, male and female transgenic rats were used to selectively label D1 or D2 neurons with ChR2-mCherry. We found that p-FAK was increased during drug seeking in both D1 and D2-medium spiny neurons (MSNs), but increased p-cofilin was observed only in D1-MSNs. These data indicate that ß3-integrin, FAK and cofilin constitute a signaling pathway downstream of MMP activation that is involved in promoting the transient synaptic enlargement in D1-MSNs induced during reinstated cocaine by drug-paired cues.SIGNIFICANCE STATEMENT Drug-associated cues precipitate relapse, which is correlated with transient synaptic enlargement in the accumbens core. We showed that cocaine cue-induced synaptic enlargement depends on matrix metalloprotease signaling in the extracellular matrix (ECM) through ß3-integrin to activate focal adhesion kinase (FAK) and phosphorylate the actin binding protein cofilin. The nucleus accumbens core (NAcore) contains two predominate neuronal subtypes selectively expressing either D1-dopamine or D2-dopamine receptors. We used transgenic rats to study each cell type and found that cue-induced signaling through cofilin phosphorylation occurred only in D1-expressing neurons. Thus, cocaine-paired cues initiate cocaine reinstatement and synaptic enlargement through a signaling cascade selectively in D1-expressing neurons requiring ECM stimulation of ß3-integrin-mediated phosphorylation of FAK (p-FAK) and cofilin.

The loss of NMDAR-dependent LTD following cannabinoid self-administration is restored by positive allosteric modulation of CB1 receptors.

Glutamatergic plasticity in the nucleus accumbens core (NAcore) is a key neuronal process in appetitive learning and contributes to pathologies such as drug addiction. Understanding how this plasticity factors into cannabis addiction and relapse has been hampered by the lack of a rodent model of cannabis self-administration. We used intravenous self-administration of two constituents of cannabis, Δ9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) to examine how contingent cannabis use and cue-induced cannabinoid-seeking alters glutamatergic neurotransmission and synaptic plasticity in NAcore. NMDA receptor (NMDAR)-dependent long-term depression (LTD) in the NAcore was lost after cannabinoid, but not sucrose self-administration. Surprisingly, when rats underwent cue-induced cannabinoid seeking, LTD was restored. Loss of LTD was accompanied by desensitization of cannabinoid receptor 1 (CB1R). CB1R are positioned to regulate synaptic plasticity by being expressed on glutamatergic terminals and negatively regulating presynaptic excitability and glutamate release. Supporting this possibility, LTD was restored by promoting CB1R signaling with the CB1 positive allosteric modulator GAT211. These data implicate NAcore CB1R as critical regulators of metaplasticity induced by cannabis self-administration and the cues predicting cannabis availability.

Drug Refraining and Seeking Potentiate Synapses on Distinct Populations of Accumbens Medium Spiny Neurons.

Cocaine-associated cues and contexts can precipitate drug seeking in humans and in experimental animals. Glutamatergic synapses in the core subcompartment of the nucleus accumbens (NAcore) undergo transient potentiation in response to presenting drug-associated cues. The NAcore contains two populations of medium spiny neurons (MSNs) that differentially express D1 or D2 dopamine receptors. By recording the ratio of AMPA and NMDA glutamate receptor currents (AMPA/NMDA ratio) from MSNs in NAcore tissue slices, we endeavored to understand which subpopulation of MSNs was undergoing transient potentiation. Transgenic female and male mice differentially expressing fluorescent reporters in D1 or D2 MSNs were withdrawn for 2-3 weeks after being trained to self-administer cocaine. In some mice, discrete cocaine-conditioned cues were isolated from the drug-associated context via extinction training, which causes rodents to refrain from drug seeking in the extinguished context. By measuring AMPA/NMDA ratios in the drug context with or without contextual or discrete cues, and with or without extinction training, we made the following three discoveries: (1) mice refraining from cocaine seeking in the extinguished context showed selective elevation in AMPA/NMDA ratios in D2 MSNs; (2) without extinction training, the drug-associated context selectively increased AMPA/NMDA ratios in D1 MSNs; (3) mice undergoing cue-induced cocaine seeking after extinction training in the drug-associated context showed AMPA/NMDA ratio increases in both D1 and D2 MSNs. These findings reveal that the NAcore codes drug seeking through transient potentiation of D1 MSNs, and that refraining from cocaine seeking in an extinguished context is coded through transient potentiation of D2 MSNs.SIGNIFICANCE STATEMENT Relapse is a primary symptom of addiction that can involve competition between the desire to use drugs and the desire to refrain from using drugs. Drug-associated cues induce relapse, which is correlated with transiently potentiated glutamatergic synapses in the nucleus accumbens core. We determined which of two cell populations in the accumbens core, D1-expressing or D2-expressing neurons, undergo transient synaptic potentiation. After being trained to self-administer cocaine, mice underwent withdrawal, some with and others without extinguishing responding in the drug-associated context. Extinguished mice showed transient potentiation in D2-expressing neurons in the extinguished environment, and all mice engaged in context-induced or cue-induced drug seeking showed transient potentiation of D1-expressing neurons. A simple binary engram in accumbens for seeking drugs and refraining from drugs offers opportunities for cell-specific therapies.

Loss of Plasticity in the D2-Accumbens Pallidal Pathway Promotes Cocaine Seeking.

Distinct populations of D1- and D2-dopamine receptor-expressing medium spiny neurons (D1-/D2-MSNs) comprise the nucleus accumbens, and activity in D1-MSNs promotes, whereas activity in D2-MSNs inhibits, motivated behaviors. We used chemogenetics to extend D1-/D2-MSN cell specific regulation to cue-reinstated cocaine seeking in a mouse model of self-administration and relapse, and found that either increasing activity in D1-MSNs or decreasing activity in D2-MSNs augmented cue-induced reinstatement. Both D1- and D2-MSNs provide substantial GABAergic innervation to the ventral pallidum, and chemogenetic inhibition of ventral pallidal neurons blocked the augmented reinstatement elicited by chemogenetic regulation of either D1- or D2-MSNs. Because D1- and D2-MSNs innervate overlapping populations of ventral pallidal neurons, we next used optogenetics to examine whether changes in synaptic plasticity in D1- versus D2-MSN GABAergic synapses in the ventral pallidum could explain the differential regulation of VP activity. In mice trained to self-administer cocaine, GABAergic LTD was abolished in D2-, but not in D1-MSN synapses. A µ opioid receptor antagonist restored GABA currents in D2-, but not D1-MSN synapses of cocaine-trained mice, indicating that increased enkephalin tone on presynaptic µ opioid receptors was responsible for occluding the LTD. These results identify a behavioral function for D1-MSN innervation of the ventral pallidum, and suggest that losing LTDGABA in D2-MSN, but not D1-MSN input to ventral pallidum may promote cue-induced reinstatement of cocaine-seeking. SIGNIFICANCE STATEMENT: More than 90% of ventral striatum is composed of two cell types, those expressing dopamine D1 or D2 receptors, which exert opposing roles on motivated behavior. Both cell types send GABAergic projections to the ventral pallidum and were found to differentially promote cue-induced reinstatement of cocaine seeking via the ventral pallidum. Furthermore, after cocaine self-administration, synaptic plasticity was selectively lost in D2, but not D1 inputs to the ventral pallidum. The selective impairment in D2 afferents may promote the influence of D1 inputs to drive relapse to cocaine seeking.

Accumbens nNOS Interneurons Regulate Cocaine Relapse.

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced relapse is correlated with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses on medium spiny neurons (MSNs) in the nucleus accumbens core (NAcore) and requires spillover of glutamate from prefrontal cortical afferents. We used a rodent self-administration/reinstatement model of relapse to show that cue-induced t-SP and reinstated cocaine seeking result from glutamate spillover, initiating a metabotropic glutamate receptor 5 (mGluR5)-dependent increase in nitric oxide (NO) production. Pharmacological stimulation of mGluR5 in NAcore recapitulated cue-induced reinstatement in the absence of drug-associated cues. Using NO-sensitive electrodes, mGluR5 activation by glutamate was shown to stimulate NO production that depended on activation of neuronal nitric oxide synthase (nNOS). nNOS is expressed in ∼1% of NAcore neurons. Using a transgene strategy to express and stimulate designer receptors that mimicked mGluR5 signaling through Gq in nNOS interneurons, we recapitulated cue-induced reinstatement in the absence of cues. Conversely, using a transgenic caspase strategy, the intensity of cue-induced reinstatement was correlated with the extent of selective elimination of nNOS interneurons. The induction of t-SP during cued reinstatement depends on activating matrix metalloproteinases (MMPs) and selective chemogenetic stimulation of nNOS interneurons recapitulated MMP activation and t-SP induction (increase in AMPA currents in MSNs). These data demonstrate critical involvement of a sparse population of nNOS-expressing interneurons in cue-induced cocaine seeking, revealing a bottleneck in brain processing of drug-associated cues where therapeutic interventions could be effective in treating drug addiction. SIGNIFICANCE STATEMENT: Relapse to cocaine use in a rat model is associated with transient increases in synaptic strength at prefrontal cortex synapses in the nucleus accumbens. We demonstrate the sequence of events that mediates synaptic potentiation and reinstated cocaine seeking induced by cocaine-conditioned cues. Activation of prefrontal inputs to the accumbens by cues initiates spillover of synaptic glutamate, which stimulates metabotropic glutamate receptor 5 (mGluR5) on a small population of interneurons (∼1%) expressing neuronal nitric oxide synthase. Stimulating these glutamate receptors increases nitric oxide (NO) production, which stimulates matrix metalloprotease-2 (MMP-2) and MMP-9 activity in the extracellular space. Manipulating the interaction between mGluR5, NO production, or MMP-2 and MMP-9 pharmacologically or genetically is sufficient to recapitulate transient synaptic potentiation and reinstate cocaine seeking.

Metaplasticity at the addicted tetrapartite synapse: A common denominator of drug induced adaptations and potential treatment target for addiction.

In light of the current worldwide addiction epidemic, the need for successful therapies is more urgent than ever. Although we made substantial progress in our basic understanding of addiction, reliable therapies are lacking. Since 40-60% of patients treated for substance use disorder return to active substance use within a year following treatment discharge, alleviating the vulnerability to relapse is regarded as the most promising avenue for addiction therapy. Preclinical addiction research often focuses on maladaptive synaptic plasticity within the reward pathway. However, drug induced neuroadaptations do not only lead to a strengthening of distinct drug associated cues and drug conditioned behaviors, but also seem to increase plasticity thresholds for environmental stimuli that are not associated with the drug. This form of higher order plasticity, or synaptic metaplasticity, is not expressed as a change in the efficacy of synaptic transmission but as a change in the direction or degree of plasticity induced by a distinct stimulation pattern. Experimental addiction research has demonstrated metaplasticity after exposure to multiple classes of addictive drugs. In this review we will focus on the concept of synaptic metaplasticity in the context of preclinical addiction research. We will take a closer look at the tetrapartite glutamatergic synapse and outline forms of metaplasticity that have been described at the addicted synapse. Finally we will discuss the different potential avenues for pharmacotherapies that target glutamatergic synaptic plasticity and metaplasticity. Here we will argue that aberrant metaplasticity renders the reward seeking circuitry more rigid and hence less able to adapt to changing environmental contingencies. An understanding of the molecular mechanisms that underlie this metaplasticity is crucial for the development of new strategies for addiction therapy. The correction of drug-induced metaplasticity could be used to support behavioral and pharmacotherapies for the treatment of addiction.

In Science Journals.

Highlights from the Science family of journals.

In Science Journals.

Highlights from the Science family of journals.

In Science Journals.

Highlights from the Science family of journals.

In Science Journals.

Highlights from the Science family of journals.

Differential Modulation of GABAergic and Glutamatergic Neurons in the Ventral Pallidum by GABA and Neuropeptides.

The ventral pallidum (VP) is an integral locus in the reward circuitry and a major target of GABAergic innervation of both D1-medium spiny neurons (MSNs) and D2-MSNs from the nucleus accumbens. The VP contains populations of GABAergic [VPGABA, GAD2(+), or VGluT(-)] and glutamatergic [VPGlutamate, GAD2(-), or VGluT(+)] cells that facilitate positive reinforcement and behavioral avoidance, respectively. MSN efferents to the VP exert opponent control over behavioral reinforcement with activation of D1-MSN afferents promoting and D2-MSN afferents inhibiting reward seeking. How this afferent-specific and cell type-specific control of reward seeking is integrated remains largely unknown. In addition to GABA, D1-MSNs corelease substance P to stimulate neurokinin 1 receptors (NK1Rs) and D2-MSNs corelease enkephalin to activate µ-opioid receptors (MORs) and δ-opioid receptors. These neuropeptides act in the VP to alter appetitive behavior and reward seeking. Using a combination of optogenetics and patch-clamp electrophysiology in mice, we found that GAD2(-) cells receive weaker GABA input from D1-MSN, but GAD2(+) cells receive comparable GABAergic input from both afferent types. Pharmacological activation of MORs induced an equally strong presynaptic inhibition of GABA and glutamate transmission on both cell types. Interestingly, MOR activation hyperpolarized VPGABA but not VGluT(+). NK1R activation inhibited glutamatergic transmission only on VGluT(+) cells. Our results indicate that the afferent-specific release of GABA and neuropeptides from D1-MSNs and D2-MSNs can differentially influence VP neuronal subtypes.

In Science Journals.

Highlights from the Science family of journals.

In Science Journals.

Highlights from the Science family of journals.

In Science Journals.

Highlights from the Science family of journals.

Neuronal precision and the limits for acoustic signal recognition in a small neuronal network.

Recognition of acoustic signals may be impeded by two factors: extrinsic noise, which degrades sounds before they arrive at the receiver's ears, and intrinsic neuronal noise, which reveals itself in the trial-to-trial variability of the responses to identical sounds. Here we analyzed how these two noise sources affect the recognition of acoustic signals from potential mates in grasshoppers. By progressively corrupting the envelope of a female song, we determined the critical degradation level at which males failed to recognize a courtship call in behavioral experiments. Using the same stimuli, we recorded intracellularly from auditory neurons at three different processing levels, and quantified the corresponding changes in spike train patterns by a spike train metric, which assigns a distance between spike trains. Unexpectedly, for most neurons, intrinsic variability accounted for the main part of the metric distance between spike trains, even at the strongest degradation levels. At consecutive levels of processing, intrinsic variability increased, while the sensitivity to external noise decreased. We followed two approaches to determine critical degradation levels from spike train dissimilarities, and compared the results with the limits of signal recognition measured in behaving animals.

Understanding the Munchies.

In this issue of Neuron, Thoeni et al. (2020) demonstrate that both food restriction and a high-fat diet cause an endocannabinoid-dependent inhibition of D1 medium spiny neuron terminals in the lateral hypothalamus that promotes overeating.

Endocannabinoid LTD in Accumbal D1 Neurons Mediates Reward-Seeking Behavior.

The nucleus accumbens (NAc) plays a key role in drug-related behavior and natural reward learning. Synaptic plasticity in dopamine D1 and D2 receptor medium spiny neurons (MSNs) of the NAc and the endogenous cannabinoid (eCB) system have been implicated in reward seeking. However, the precise molecular and physiological basis of reward-seeking behavior remains unknown. We found that the specific deletion of metabotropic glutamate receptor 5 (mGluR5) in D1-expressing MSNs (D1miRmGluR5 mice) abolishes eCB-mediated long-term depression (LTD) and prevents the expression of drug (cocaine and ethanol), natural reward (saccharin), and brain-stimulation-seeking behavior. In vivo enhancement of 2-arachidonoylglycerol (2-AG) eCB signaling within the NAc core restores both eCB-LTD and reward-seeking behavior in D1miRmGluR5 mice. The data suggest a model where the eCB and glutamatergic systems of the NAc act in concert to mediate reward-seeking responses.

Visual perception of motion in a hunting spider.

Like most other spiders Cupiennius salei (Keyserling 1877) has two different eye types, one pair of principal eyes and three pairs of secondary eyes. The principal eyes have two eye muscles each, which allow movement of the retina and are mainly used for the discrimination of stationary objects. The secondary eyes without such eye muscles are supposed to detect moving objects. Masking experiments were used to analyse the role of these two eye types in motion detection. In a white arena the animals were stimulated with short sequences of moving black bars. The principal eyes move involuntarily when objects are moving within the visual field of an ipsilateral secondary eye. The eye muscle activity of the principal eyes was recorded using single channel telemetry, and activity changes were taken as an indicator for the perception of motion. Masking the principal eyes with black paint and presenting a moving visual stimulus did not modify the induced muscle activity, whereas masking the secondary eyes eliminated the increase in eye muscle activity. This suggests that the secondary eyes are responsible for movement detection. We conclude that the animals are able to detect moving targets visually only with the secondary eyes. The principal eyes, by contrast, do not seem to be involved in the detection of moving targets.

Extracellular Matrix Signaling Through ß3 Integrin Mediates Cocaine Cue-Induced Transient Synaptic Plasticity and Relapse.

BACKGROUND: Cue-induced relapse to drug use is a primary symptom of cocaine addiction. Cue-induced transient excitatory synaptic potentiation (t-SP) induced in the nucleus accumbens mediates cued cocaine seeking in rat models of relapse. Cue-induced t-SP depends on extracellular signaling by matrix metalloproteases (MMPs), but it is unknown how this catalytic activity communicates with nucleus accumbens neurons to induce t-SP and cocaine seeking. METHODS: Male Sprague Dawley rats (N = 125) were trained to self-administer cocaine, after which self-administration was extinguished and then reinstated by cocaine-conditioned cues. We used a morpholino antisense strategy to knock down the ß1 or ß3 integrin subunits or inhibitors to prevent phosphorylation of the integrin signaling kinases focal adhesion kinase (FAK) or integrin-linked kinase. We quantified protein changes with immunoblotting and t-SP by measuring dendritic spine morphology and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/N-methyl-D-aspartate glutamate currents. Integrin signaling was stimulated by microinjecting an MMP activator or integrin peptide ligand into the accumbens. RESULTS: Knockdown of ß3 integrin or FAK inhibitor, but not ß1 integrin or integrin-linked kinase inhibitor, prevented cue-induced cocaine seeking but not sucrose seeking. ß3 integrin knockdown prevented t-SP as measured by preventing the cue-induced increases in both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/N-methyl-D-aspartate glutamate ratio and spine head diameter. Activating MMP gelatinases with tissue plasminogen activator potentiated cue-induced reinstatement, which was prevented by ß3 integrin knockdown and FAK inhibition. Stimulating integrin receptors with the RGD ligand liberated by MMP gelatinase activity also potentiated cued cocaine seeking. CONCLUSIONS: Activation of MMP gelatinase in the extracellular space is necessary for and potentiates cued cocaine seeking. This extracellular catalysis stimulates ß3 integrins and activates FAK to induce t-SP and promote cue-induced cocaine seeking.