Synapses In Nucleus Accumbens Research Articles

Focused Ultrasound Combined With Microbubbles Attenuate Symptoms in Heroin-Addicted Mice

ObjectiveTo explore the efficacy and mechanisms of stimulating the nucleus accumbens (NAc) in heroin-addicted mice using focused ultrasound and microbubbles (MBs). MethodsThe conditioned place preference (CPP) method was employed to establish a heroin-addicted mice model. Mice were randomized into control (C), heroin (H), heroin + ultrasound (H + U) and H + U + MBs. Ultrasound (2 MHz fundamental frequency, 1.34 MPa peak-negative pressure, 1 MHz pulse repetition frequency, 5% duty cycle, 15 min/d, over 2 d) was applied to stimulate the NAc in the latter 2 groups. Whereas H + U + MBs received an injection of sulfur hexafluoride MBs during the stimulation. Subsequently, CPP scores, open-field test (OFT), and elevated plus-maze test (EPMT) were conducted to assess behavioral changes in addiction memory, anxiety and exercise status. HE staining was performed to detect pathological structures. Neurotransmitters such as dopamine (DA), serotonin (5-HT) and glutamate (Glu) were detected using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Transmission electron microscopy (TEM) was used to observe ultrastructural changes of synapses in NAc. Immunohistochemistry (IHC) was utilized to detect Cleaved Caspase-3 in the NAc region. Western blotting (WB) was used to detect the protein expression of Cleaved Caspase-3, Bax and Bcl-2 in NAc. ResultsHE staining showed small patches of erythrocyte exudation were observed in the NAc and adjacent areas in H + U + MBs. The CPP scores of H + U + MBs were lower (p < 0.05) than H. After ultrasound treatment, all indices of the OFT and EPMT in H + U + MBs were significantly higher than H (p < 0.05). UPLC-MS/MS revealed that the levels of DA, 5-HT and Glu in H + U + MBs were lower than H (p < 0.01). TEM showed decrease the number of synapses (p < 0.05), and noticeable swelling of mitochondria, membrane damage, as well as damage to the cristae. Further detection by IHC and WB showed that the pro-apoptotic proteins Cleaved Caspase-3 and Bax increased and Bcl-2 decreased as anti-apoptotic proteins after ultrasound combined with MBs (p < 0.05). ConclusionFocused ultrasound combined with MBs stimulate the NAc can weaken the addictive memory and improve anxiety of heroin-related mice. The mechanical effect of ultrasound combined with the cavitation effect may be a potential treatment for addiction.

Sex-Specific Mechanisms Underlie Long-Term Potentiation at Hippocampus→Medium Spiny Neuron Synapses in the Medial Shell of the Nucleus Accumbens.

Sex differences have complicated our understanding of the neurobiological basis of many behaviors that are key for survival. As such, continued elucidation of the similarities and differences between sexes is necessary to gain insight into brain function and vulnerability. The connection between the hippocampus (Hipp) and nucleus accumbens (NAc) is a crucial site where modulation of neuronal activity mediates reward-related behavior. Our previous work demonstrated that long-term potentiation (LTP) of Hipp→NAc synapses is rewarding, and mice can establish learned associations between LTP of these synapses and the contextual environment in which LTP occurred. Here, we investigated sex differences in the mechanisms underlying Hipp→NAc LTP using whole-cell electrophysiology and pharmacology. We observed similarities in basal synaptic strength between males and females and found that LTP occurs postsynaptically with similar magnitudes in both sexes. However, key sex differences emerged as LTP in males required NMDA receptors (NMDAR), whereas LTP in females utilized an NMDAR-independent mechanism involving L-type voltage-gated Ca2+ channels (VGCCs) and estrogen receptor α (ERα). We also uncovered sex-similar features as LTP in both sexes depended on CaMKII activity and occurred independently of dopamine-1 receptor (D1R) activation. Our results have elucidated sex-specific molecular mechanisms for LTP in an integral pathway that mediates reward-related behaviors, emphasizing the importance of considering sex as a variable in mechanistic studies. Continued characterization of sex-specific mechanisms underlying plasticity will offer novel insight into the neurophysiological basis of behavior, with significant implications for understanding how diverse processes mediate behavior and contribute to vulnerability to developing psychiatric disorders.

Microglia-mediated calcium-permeable AMPAR accumulation in the nucleus accumbens drives hyperlocomotion during cocaine withdrawal

During withdrawal from cocaine, calcium permeable-AMPA receptors (CP-AMPAR) progressively accumulate in nucleus accumbens (NAc) synapses, a phenomenon linked to behavioral sensitization and drug-seeking. Recently, it has been suggested that neuroimmune alterations might promote aberrant changes in synaptic plasticity, thus contributing to substance abuse-related behaviors. Here, we investigated the role of microglia in NAc neuroadaptations after withdrawal from cocaine-induced conditioned place preference (CPP). We depleted microglia using PLX5622-supplemented diet during cocaine withdrawal, and after the place preference test, we measured dendritic spine density and the presence of CP-AMPAR in the NAc shell. Microglia depletion prevented cocaine-induced changes in dendritic spines and CP-AMPAR accumulation. Furthermore, microglia depletion prevented conditioned hyperlocomotion without affecting drug-context associative memory. Microglia displayed fewer number of branches, resulting in a reduced arborization area and microglia control domain at late withdrawal. Our results suggest that microglia are necessary for the synaptic adaptations in NAc synapses during cocaine withdrawal and therefore represent a promising therapeutic target for relapse prevention.

Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

Positive Allosteric Modulation of mGlu1 Reverses Cocaine-Induced Behavioral and Synaptic Plasticity Through the Integrated Stress Response and Oligophrenin-1

BackgroundCue-induced cocaine craving progressively intensifies (incubates) during abstinence from cocaine self-administration. Expression of incubated cocaine craving depends on elevated calcium-permeable AMPA receptors (CP-AMPARs) on medium spiny neurons in the nucleus accumbens (NAc) core. After incubation has occurred, stimulation of NAc metabotropic glutamate 1 (mGlu1) receptors or systemic administration of mGlu1 positive allosteric modulators removes CP-AMPARs from NAc synapses via dynamin-dependent internalization (mGlu1 long-term depression [LTD]) and thereby reduces incubated cocaine craving. Because mGlu1 positive allosteric modulators are potential therapeutics for cocaine craving, it is important to further define the mechanism triggering this mGlu1-LTD. MethodsMale and female rats self-administered saline or cocaine (10 days) using a long access regimen (6 h/day). Following ≥40 days of abstinence, we assessed the ability of an mGlu1 positive allosteric modulator to inhibit expression of incubated craving and remove CP-AMPARs from NAc synapses under control conditions, after blocking the integrated stress response (ISR), or after knocking down oligophrenin-1, a mediator of the ISR that can promote AMPAR endocytosis. AMPAR transmission in NAc medium spiny neurons was assessed with ex vivo slice recordings. ResultsmGlu1 stimulation reduced cue-induced craving and removed synaptic CP-AMPARs. When the ISR was blocked prior to mGlu1 stimulation, there was no reduction in cue-induced craving, nor were CP-AMPARs removed from the synapse. Further, selective knockdown of oligophrenin-1 blocked mGlu1-LTD. ConclusionsOur results indicate that mGlu1-LTD in the NAc and consequently the reduction of cue-induced seeking occur through activation of the ISR, which induces translation of oligophrenin-1. We also demonstrate CP-AMPAR accumulation and mGlu1 reversal in female rats, as previously shown in male rats.

Dopaminylation in Psychostimulant Use Disorder Protects Against Psychostimulant Seeking Behavior by Normalizing Nucleus Accumbens (NAc) Dopamine Expression.

Repeated cocaine administration changes histone acetylation and methylation on Lys residues and Deoxyribonucleic acid (DNA) within the nucleus accumbens (NAc). Recently Nestler's group explored histone Arg (R) methylation in reward processing models. Damez-Werno et al. (2016) reported that during human investigations and animal self-administration experiments, the histone mark protein-R-methyltransferase-6 (PRMT6) and asymmetric dimethylation of R2 on histone H3 (H3R2me2a) decreased in the rodent and cocaine-dependent human NAc. Overexpression of PRMT6 in D2-MSNs in all NAc neurons increased cocaine seeking, whereas PRMT6 overexpression in D1-MSNs protects against cocaine-seeking. The hypothesis is that dopaminylation (H3R2me2a binding) occurs in psychostimulant use disorder (PSU), and the binding inhibitor Srcin1, like the major DRD2 A2 allelic polymorphism, protects against psychostimulant seeking behavior by normalizing nucleus accumbens (NAc) dopamine expression. Numerous publications confirmed the association between the DRD2 Taq A1 allele (30-40 lower D2 receptor numbers) and severe cocaine dependence. Lepack et al. (2020) found that acute cocaine increases dopamine in NAc synapses, and results in histone H3 glutamine 5 dopaminylation (H3Q5dop) and consequent inhibition of D2 expression. The inhibition increases with chronic cocaine use and accompanies cocaine withdrawal. They also found that the Src kinase signaling inhibitor 1 (Srcin1 or p140CAP) during cocaine withdrawal reduced H3R2me2a binding. Consequently, this inhibited dopaminylation induced a "homeostatic brake." The decrease in Src signaling in NAc D2-MSNs, (like the DRD2 Taq A2 allele, a well-known genetic mechanism protective against SUD) normalizes the NAc dopamine expression and decreases cocaine reward and motivation to self-administer cocaine. The Srcin1 may be an important therapeutic target.

Heroin Cue–Evoked Astrocytic Structural Plasticity at Nucleus Accumbens Synapses Inhibits Heroin Seeking

BackgroundOpioid addiction is a critical medical and societal problem characterized by vulnerability to relapse. Glutamatergic synapses in the nucleus accumbens regulate the motivation to relapse to opioid use, and downregulation of glutamate transporters on astroglial processes adjacent to accumbens synapses contributes to heroin seeking induced by cues. However, it is not known how astroglial processes themselves respond to heroin cues or if changes in astroglial morphology are necessary for heroin seeking. MethodsMale Sprague Dawley rats (n = 62) were trained to self-administer heroin or sucrose and were reinstated by heroin-conditioned or sucrose-conditioned cues. Astroglial proximity to accumbens synapses was estimated using a confocal-based strategy, and the association between digitally isolated astroglia and the presynaptic marker synapsin I was quantified. To determine the functional consequence of astroglial morphological plasticity on cued heroin seeking, a morpholino antisense strategy was used to knock down expression of the actin binding protein ezrin, which is expressed almost exclusively in peripheral astroglial processes in the adult rat brain. ResultsAfter heroin extinction, there was an enduring reduction in synaptic proximity by astroglia. Synaptic proximity was restored during 15 minutes of cued heroin seeking but returned to extinction levels by 120 minutes. Extinction from sucrose self-administration and reinstated sucrose seeking induced no changes in astroglial synaptic association. Ezrin knockdown reduced astroglial association with synapses and potentiated cued heroin seeking. ConclusionsCue-induced heroin seeking transiently increased synaptic proximity of accumbens astrocytes. Surprisingly, the reassociation of astroglia with synapses was compensatory, and preventing cue-induced morphological plasticity in astrocytes potentiated heroin seeking.

Emergence of Endocytosis-Dependent mGlu1 LTD at Nucleus Accumbens Synapses After Withdrawal From Cocaine Self-Administration.

Extended-access cocaine self-administration induces a progressive intensification of cue-induced drug craving during withdrawal termed “incubation of cocaine craving”. Rats evaluated after >1 month of withdrawal (when incubation of craving is robust) display alterations in excitatory synapses onto medium spiny neurons (MSNs) of the nucleus accumbens (NAc), including elevated levels of Ca2+-permeable AMPA receptors (CP-AMPAR) and a transition from group I metabotropic glutamate receptor (mGluR) mGlu5- to mGlu1-mediated synaptic depression. It is important to further characterize the emergent form of mGlu1-mediated synaptic depression because it has been demonstrated that mGlu1 stimulation, by normalizing CP-AMPAR transmission, reduces cue-induced cocaine craving. In the present study, we conducted whole-cell patch-clamp recordings in NAc core MSNs, comparing rats that underwent >35 days of withdrawal from cocaine self-administration to control rats that had self-administered saline. Bath application of the nonselective group I mGluR agonist dihydroxyphenylglycine (DHPG) produced a transient mGlu5-mediated synaptic depression in saline controls, whereas a persistent mGlu1-mediated synaptic depression emerged in cocaine rats. This form of long-term depression (LTD) was abolished by the inclusion of dynamin inhibitory peptide (DIP) in the recording electrode, indicating that it is mediated by removal of CP-AMPARs through a dynamin-dependent endocytosis mechanism. We further showed that CP-AMPAR endocytosis is normally coupled to the PICK1-mediated insertion of Ca2+-impermeable AMPARs (CI-AMPAR). Interestingly, this coupling is not obligatory because disruption of PICK1-mediated CI-AMPAR insertion with pep2-EVKI spared mGlu1-mediated CP-AMPAR endocytosis. Collectively, these results reveal similarities but also differences from mGlu1-LTD observed in other brain regions, and further our understanding of a form of plasticity that may be targeted to reduce cue-induced craving for cocaine and methamphetamine.

Persistent pain alters AMPA receptor subunit levels in the nucleus accumbens.

BackgroundA variety of pain conditions have been found to be associated with depressed mood in clinical studies. Depression-like behaviors have also been described in animal models of persistent or chronic pain. In rodent chronic neuropathic pain models, elevated levels of GluA1 subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the nucleus accumbens (NAc) have been found to inhibit depressive symptoms. However, the effect of reversible post-surgical pain or inflammatory pain on affective behaviors such as depression has not been well characterized in animal models. Neither is it known what time frame is required to elicit AMPA receptor subunit changes in the NAc in various pain conditions.ResultsIn this study, we compared behavioral and biochemical changes in three pain models: the paw incision (PI) model for post-incisional pain, the Complete Freund’s Adjuvant (CFA) model for persistent but reversible inflammatory pain, and the spared nerve injury (SNI) model for chronic postoperative neuropathic pain. In all three models, rats developed depressive symptoms that were concurrent with the presentation of sensory allodynia. GluA1 levels at the synapses of the NAc, however, differed in these three models. The level of GluA1 subunits of AMPA-type receptors at NAc synapses was not altered in the PI model. GluA1 levels were elevated in the CFA model after a period (7 d) of persistent pain, leading to the formation of GluA2-lacking AMPA receptors. As pain symptoms began to resolve, however, GluA1 levels returned to baseline. Meanwhile, in the SNI model, in which pain persisted beyond 14 days, GluA1 levels began to rise after pain became persistent and remained elevated. In addition, we found that blocking GluA2-lacking AMPA receptors in the NAc further decreased the depressive symptoms only in persistent pain models.ConclusionOur study shows that while both short-term and persistent pain can trigger depression-like behaviors, GluA1 upregulation in the NAc likely represents a unique adaptive response to minimize depressive symptoms in persistent pain states.

α2δ-1 signaling in nucleus accumbens is necessary for cocaine-induced relapse.

Relapse to cocaine seeking is associated with potentiated excitatory synapses in nucleus accumbens. α2δ-1 is an auxiliary subunit of voltage-gated calcium channels that affects calcium-channel trafficking and kinetics, initiates extracellular signaling cascades, and promotes excitatory synaptogenesis. Previous data demonstrate that repeated exposure to alcohol, nicotine, methamphetamine, and morphine upregulates α2δ-1 in reward-related brain regions, but it was unclear whether this alteration generalized to cocaine. Here, we show that α2δ-1 protein was increased in nucleus accumbens after cocaine self-administration and extinction compared with saline controls. Furthermore, the endogenous ligand thrombospondin-1, responsible for the synaptogenic properties of the α2δ-1 receptor, was likewise elevated. Using whole-cell patch-clamp recordings of EPSCs in nucleus accumbens, we demonstrated that gabapentin, a specific α2δ-1 antagonist, preferentially reduced the amplitude and increased the paired-pulse ratio of EPSCs evoked by electrical stimulation in slices from cocaine-experienced rats compared with controls. In vivo, gabapentin microinjected in the nucleus accumbens core attenuated cocaine-primed but not cue-induced reinstatement. Importantly, gabapentin's effects on drug seeking were not due to a general depression of spontaneous or cocaine-induced locomotor activity. Moreover, gabapentin had no effect on reinstatement of sucrose seeking. These data indicate that α2δ-1 contributes specifically to cocaine-reinstated drug seeking, and identifies this protein as a target for the development of cocaine relapse medications. These results also inform ongoing discussion in the literature regarding efficacy of gabapentin as a candidate addiction therapy.

A Protein Synthesis-Dependent Mechanism Sustains Calcium-Permeable AMPA Receptor Transmission in Nucleus Accumbens Synapses during Withdrawal from Cocaine Self-Administration

Extended-access cocaine self-administration results in withdrawal-dependent incubation of cocaine craving. Rats evaluated after ∼1 month of withdrawal from such regimens ("incubated rats") exhibit changes in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) that include accumulation of Ca(2+)-permeable AMPA receptors (CP-AMPARs) and a switch in group I metabotropic glutamate receptor (mGluR)-mediated suppression of synaptic transmission from mGluR5-dependent to mGluR1-dependent. To determine the role of protein synthesis in mediating these adaptations, we conducted whole-cell patch-clamp recordings in NAc core MSNs of "incubated rats" in the presence of translational inhibitors (anisomycin, cycloheximide, rapamycin) or the transcriptional inhibitor actinomycin-D. The contribution of CP-AMPARs to synaptic transmission was determined by the rectification index and the sensitivity to the CP-AMPAR antagonist 1-naphthyl acetyl spermine. We found that CP-AMPAR-mediated transmission in the NAc of "incubated rats" was reduced to levels comparable to those found in saline control rats when brain slices were treated with translational inhibitors, whereas actinomycin-D had no effect. We also investigated the effect of protein translation inhibitors on the switch of mGluR function in MSNs of "incubated rats" using the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine in combination with either an mGluR1 (LY367385) or an mGluR5 (3-[(2-methyl-4-thiazolyl)ethynyl]pyridine) antagonist. Data revealed that inhibition of protein translation eliminated the mGluR1-mediated inhibition and restored the mGluR5 responsiveness to a state functionally similar to that of saline control rats. Together, these results suggest that aberrant regulation of local protein synthesis contributes to the maintenance of adaptations accrued at NAc MSN synapses during incubation of cocaine craving.

Maturation of silent synapses in amygdala-accumbens projection contributes to incubation of cocaine craving

In rat models of drug relapse and craving, cue-induced cocaine seeking progressively increases after drug withdrawal. This ‘incubation of cocaine craving’ is partially mediated by time-dependent adaptations at glutamatergic synapses in nucleus accumbens. However, the circuit-level adaptations mediating this plasticity remain elusive. Here we studied silent synapses—often regarded as immature synapses that express stable NMDA receptors with AMPA receptors either absent or labile—in basolateral amygdala-to-accumbens projection in incubation of cocaine craving. Silent synapses were detected within this projection during early withdrawal from cocaine. As the withdrawal period progressed, these silent synapses became ‘unsilenced’, a process involving synaptic insertion of calcium-permeable AMPA receptors (CP-AMPARs). In vivo optogenetic stimulation-induced downregulation of CP-AMPARs at amygdala-to-NAc synapses, which re-silenced some of the previously silent synapses after prolonged withdrawal, decreased cocaine incubation. Our finding indicates that silent synapse-based reorganization of the amygdala-to-accumbens projection is critical for persistent cocaine craving and relapse after withdrawal.

Differential effects of natural rewards and pain on vesicular glutamate transporter expression in the nucleus accumbens

BackgroundPain and natural rewards such as food elicit different behavioral effects. Both pain and rewards, however, have been shown to alter synaptic activities in the nucleus accumbens (NAc), a key component of the brain reward system. Mechanisms by which external stimuli regulate plasticity at NAc synapses are largely unexplored. Medium spiny neurons (MSNs) from the NAc receive excitatory glutamatergic inputs and modulatory dopaminergic and cholinergic inputs from a variety of cortical and subcortical structures. Glutamate inputs to the NAc arise primarily from prefrontal cortex, thalamus, amygdala, and hippocampus, and different glutamate projections provide distinct synaptic and ultimately behavioral functions. The family of vesicular glutamate transporters (VGLUTs 1–3) plays a key role in the uploading of glutamate into synaptic vesicles. VGLUT1-3 isoforms have distinct expression patterns in the brain, but the effects of external stimuli on their expression patterns have not been studied.ResultsIn this study, we use a sucrose self-administration paradigm for natural rewards, and spared nerve injury (SNI) model for chronic pain. We examine the levels of VGLUTs (1–3) in synaptoneurosomes of the NAc in these two behavioral models. We find that chronic pain leads to a decrease of VGLUT1, likely reflecting decreased projections from the cortex. Pain also decreases VGLUT3 levels, likely representing a decrease in projections from GABAergic, serotonergic, and/or cholinergic interneurons. In contrast, chronic consumption of sucrose increases VGLUT3 in the NAc, possibly reflecting an increase from these interneuron projections.ConclusionOur study shows that natural rewards and pain have distinct effects on the VGLUT expression pattern in the NAc, indicating that glutamate inputs to the NAc are differentially modulated by rewards and pain.

Calcium-permeable AMPA receptors in the VTA and nucleus accumbens after cocaine exposure: when, how, and why?

In animal models of drug addiction, cocaine exposure has been shown to increase levels of calcium-permeable AMPA receptors (CP-AMPARs) in two brain regions that are critical for motivation and reward—the ventral tegmental area (VTA) and the nucleus accumbens (NAc). This review compares CP-AMPAR plasticity in the two brain regions and addresses its functional significance. In VTA dopamine neurons, cocaine exposure results in synaptic insertion of high conductance CP-AMPARs in exchange for lower conductance calcium-impermeable AMPARs (CI-AMPARs). This plasticity is rapid in onset (hours), GluA2-dependent, and can be observed with a single cocaine injection. Whereas it is short-lived after experimenter-administered cocaine, it persists for months after cocaine self-administration. In addition to strengthening synapses and altering Ca2+ signaling, CP-AMPAR insertion alters subsequent induction of plasticity at VTA synapses. However, CP-AMPAR insertion is unlikely to mediate the increased DA cell activity that occurs during early withdrawal from cocaine exposure. Metabotropic glutamate receptor 1 (mGluR1) exerts a negative influence on CP-AMPAR accumulation in the VTA. Acutely, mGluR1 stimulation elicits a form of LTD resulting from CP-AMPAR removal and CI-AMPAR insertion. In medium spiny neurons (MSNs) of the NAc, extended access cocaine self-administration is required to increase CP-AMPAR levels. This is first detected after approximately a month of withdrawal and then persists. Once present in NAc synapses, CP-AMPARs mediate the expression of incubation of cue-induced cocaine craving. The mechanism of their accumulation may be GluA1-dependent, which differs from that observed in the VTA. However, similar to VTA, mGluR1 stimulation removes CP-AMPARs from MSN synapses. Loss of mGluR1 tone during cocaine withdrawal may contribute to CP-AMPAR accumulation in the NAc. Thus, results in both brain regions point to the possibility of using positive modulators of mGluR1 as treatments for cocaine addiction.

Group I mGluR Activation Reverses Cocaine-Induced Accumulation of Calcium-Permeable AMPA Receptors in Nucleus Accumbens Synapses via a Protein Kinase C-Dependent Mechanism

Following prolonged withdrawal from extended access cocaine self-administration in adult rats, high conductance Ca2+ -ermeable AMPA receptors (CP-AMPARs) accumulate in nucleus accumbens (NAc) synapses and mediate the expression of "incubated" cue-induced cocaine craving. Using patch-clamp recordings from NAc slices prepared after extended access cocaine self-administration and >45 d of withdrawal, we found that group I metabotropic glutamate receptor (mGluR) stimulation using 3,5-dihydroxyphenylglycine (DHPG; 50 μm) rapidly eliminates the postsynaptic CP-AMPAR contribution to NAc synaptic transmission. This is accompanied by facilitation of Ca2+ -impermeable AMPAR (CI-AMPAR)-mediated transmission, suggesting that DHPG may promote an exchange between CP-AMPARs and CI-AMPARs. In saline controls, DHPG also reduced excitatory transmission but this occurred through a CB1 receptor-dependent presynaptic mechanism rather than an effect on postsynaptic AMPARs. Blockade of CB1 receptors had no significant effect on the alterations in AMPAR transmission produced by DHPG in the cocaine group. Interestingly, the effect of DHPG in the cocaine group was mediated by mGluR1 whereas its effect in the saline group was mediated by mGluR5. These results indicate that regulation of synaptic transmission in the NAc is profoundly altered after extended access cocaine self-administration and prolonged withdrawal. Furthermore, they suggest that activation of mGluR1 may represent a potential strategy for reducing cue-induced cocaine craving in abstinent cocaine addicts.

Calcium-Permeable AMPA Receptors Are Present in Nucleus Accumbens Synapses after Prolonged Withdrawal from Cocaine Self-Administration But Not Experimenter-Administered Cocaine

Repeated noncontingent cocaine injections, which lead to behavioral sensitization, increase AMPA receptor (AMPAR) transmission in the rodent nucleus accumbens (NAc) in a withdrawal-dependent manner. On withdrawal days (WD) 10-21, this is attributable to upregulation of GluA1A2-containing AMPARs. However, synaptic incorporation of GluA2-lacking/Ca(2+)-permeable AMPARs (CP-AMPARs) was observed after longer withdrawal (WD35) from repeated noncontingent cocaine injections in young mice (Mameli et al., 2009). CP-AMPARs had previously been observed in NAc synapses only after prolonged (WD30-WD47) withdrawal from extended-access cocaine self-administration. Our goal was to determine whether rats receiving repeated noncontingent cocaine injections during adulthood similarly exhibit CP-AMPARs in the NAc after prolonged withdrawal. For comparison, we began by evaluating CP-AMPARs on WD35-WD49 after extended-access cocaine self-administration. Confirming our previous results, whole-cell recordings revealed inwardly rectifying AMPAR EPSCs, a hallmark of CP-AMPARs. This was observed in both core and shell. Next, we conducted the same analysis in adult rats treated with eight daily noncontingent cocaine injections and recorded on WD35-WD49. AMPAR EPSCs in core and shell did not show inward rectification and were insensitive to 1-naphthylacetylspermine (a selective antagonist of CP-AMPARs). Locomotor sensitization could still be demonstrated after this long withdrawal period, although the upregulation of GluA1A2-containing AMPARs observed at earlier withdrawal times was no longer detected. In conclusion, in adult rats, accumulation of synaptic CP-AMPARs in the NAc occurs after prolonged withdrawal from extended-access cocaine self-administration but not after prolonged withdrawal from noncontingent cocaine injections.

Synaptic Adaptations in the Nucleus Accumbens Caused by Experiences Linked to Relapse

Excitatory synaptic transmission in the nucleus accumbens (NAc) regulates the reinstatement of drug seeking, an animal model of relapse in human drug addicts. However, the functional adaptations at NAc synapses that mediate reinstatement are not clearly understood. We assessed the behavioral responses of mice to cocaine administration by measuring locomotor stimulation and the acquisition, extinction, and reinstatement of conditioned place preference. Synaptic function was then examined by preparing acute brain slices and performing whole cell voltage-clamp recordings from individual medium spiny neurons in the NAc shell. We find that reduced excitatory synaptic strength in the NAc shell is a common functional adaptation induced by multiple experiences known to cause reinstatement, including stress and drug re-exposure. The same synaptic adaptation is observed shortly after reinstatement of conditioned place preference by a cocaine priming injection. This common synaptic modification associated with stress, drug re-exposure, and reinstatement defines a potential synaptic gateway to relapse.

Environmental novelty causes stress-like adaptations at nucleus accumbens synapses: Implications for studying addiction-related plasticity

Exposure to abused drugs and stressful experience, two factors that promote the development of addiction, also modify synaptic function in the mesolimbic dopamine system. Here, we show that exposure to a novel environment produces functional synaptic adaptations in the nucleus accumbens (NAc) that mirror the effect of conventional forms of stress. We find an enhancement of excitatory synaptic strength in the NAc shell one day after exposure to a novel environment for 60 min – an effect not observed in NAc core. This effect disappeared following repeated exposure to the same environment, but then reappeared if mice are returned to the same environment 10–14 days later. There were no interactions between the effects of environmental novelty and a single exposure to cocaine (15 mg/kg), with no effect of the latter on synaptic strength in NAc shell. These results have important implications for designing studies of NAc synapses in the context of behavioral analysis, and expand our understanding of how different forms of stress modify NAc synaptic function. This article is part of a Special Issue entitled ‘Synaptic Plasticity and Addiction’.

Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca 2+-permeable AMPA receptors during the incubation of cocaine craving

Cue-induced cocaine seeking intensifies or incubates after withdrawal from extended access cocaine self-administration, a phenomenon termed incubation of cocaine craving. The expression of incubated craving is mediated by Ca 2+-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). Thus, CP-AMPARs are a potential target for therapeutic intervention, making it important to understand mechanisms that govern their accumulation. Here we used subcellular fractionation and biotinylation of NAc tissue to examine the abundance and distribution of AMPAR subunits, and GluA1 phosphorylation, in the incubation model. We also studied two transmembrane AMPA receptor regulatory proteins (TARPs), γ-2 and γ-4. Our results, together with earlier findings, suggest that some of the new CP-AMPARs are synaptic. These are probably associated with γ-2, but they are loosely tethered to the PSD. Levels of GluA1 phosphorylated at serine 845 (pS845 GluA1) were significantly increased in biotinylated tissue and in an extrasynaptic membrane-enriched fraction. These results suggest that increased synaptic levels of CP-AMPARs may result in part from an increase in pS845 GluA1 in extrasynaptic membranes, given that S845 phosphorylation primes GluA1-containing AMPARs for synaptic insertion and extrasynaptic AMPARs supply the synapse. Some of the new extrasynaptic CP-AMPARs are likely associated with γ-4, rather than γ-2. The maintenance of CP-AMPARs in NAc synapses during withdrawal is accompanied by activation of CaMKII and ERK2 but not CaMKI. Overall, AMPAR plasticity in the incubation model shares some features with better described forms of synaptic plasticity, although the timing of the phenomenon and the persistence of related neuroadaptations are significantly different. This article is part of a Special Issue entitled ‘Synaptic Plasticity and Addiction’.

Intermittent morphine treatment induces a long-lasting increase in cholinergic modulation of GABAergic synapses in nucleus accumbens of adult rats

Repeated exposure to drugs of abuse causes persistent behavioral sensitization and associated adaptations of striatal neurotransmission, which is thought to play an important role in certain aspects of drug addiction. Microdialysis and neurochemical studies suggest that intermittent morphine treatment may lead to a long-term increase in both ACh and dopaminergic neurotransmission in the nucleus accumbens (NAc). This implies that both cholinergic modulation of GABA synapses and their sensitivity to dopaminergic transmission might be changed, ultimately leading to a modified NAc output. Here we investigate to what extent cholinergic modulation and sensitivity to amphetamine, causing endogenous dopamine efflux, of GABAergic transmission in the nucleus accumbens are affected 3 weeks after a period of daily morphine injections in adult rats. To this end, we recorded medium spiny neurons using whole cell voltage clamp and monitored the frequency and amplitude of spontaneous GABAergic synaptic currents. We observed that the effect of nicotine on the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was suppressed in rats pretreated with morphine, whereas the effects of mecamylamine and tetrodotoxin (TTX) were increased. These results indicate that the probability of GABA release was increased and that this effect resulted from an upregulation of the endogenous activation of presynaptic nicotinic receptors. In addition, we observed an increased sensitivity to in vitro application of amphetamine. This suggests that the long-term increase in dopaminergic transmission caused by the morphine treatment affects GABA synapses in the NAc. Hence, there may be two parallel synaptic mechanisms by which drugs of abuse may affect processing and integration of NAc inputs.