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The nucleus accumbens shell (NAcSh) is a key brain region where environmental cues acquire incentive salience to reinforce motivated behaviors. Principal medium spiny neurons (MSNs) in the NAcSh receive extensive glutamatergic projections from limbic regions, among which, the ventral hippocampus (vH) transmits information enriched in contextual cues, and the basolateral amygdala (BLA) encodes real-time arousing states. The vH and BLA project convergently to NAcSh MSNs, both activated in a time-locked manner on a cue-conditioned motivational action. In brain slices prepared from male and female mice, we show that co-activation of the two projections induces long-term potentiation (LTP) at vH-to-NAcSh synapses without affecting BLA-to-NAcSh synapses, revealing a heterosynaptic mechanism through which BLA signals persistently increase the temporally contingent vH-to-NAcSh transmission. Furthermore, this LTP is more prominent in dopamine D1 receptor-expressing (D1) MSNs than D2 MSNs and can be prevented by inhibition of either D1 receptors or dopaminergic terminals in NAcSh. This heterosynaptic LTP may provide a dopamine-guided mechanism through which vH-encoded cue inputs that are contingent to BLA activation acquire increased circuit representation to reinforce behavior. SIGNIFICANCE STATEMENT In motivated behaviors, environmental cues associated with arousing stimuli acquire increased incentive salience, processes mediated in part by the nucleus accumbens (NAc). NAc principal neurons receive glutamatergic projections from the ventral hippocampus (vH) and basolateral amygdala (BLA), which transmit information encoding contextual cues and affective states, respectively. Our results show that co-activation of the two projections induces long-term potentiation (LTP) at vH-to-NAc synapses without affecting BLA-to-NAc synapses, revealing a heterosynaptic mechanism through which BLA signals potentiate the temporally contingent vH-to-NAc transmission. Furthermore, this LTP is prevented by inhibition of either D1 receptors or dopaminergic axons. This heterosynaptic LTP may provide a dopamine-guided mechanism through which vH-encoded cue inputs that are contingent to BLA activation acquire increased circuit representation to reinforce behavior.

Background Synaptogenesis is essential in forming new neurocircuits during development, and this is mediated in part by astrocyte-released thrombospondins (TSPs) and activation of their neuronal receptor, α2δ-1. Here, we show that this developmental synaptogenic mechanism is utilized during cocaine experience to induce spinogenesis and the generation of AMPA receptor–silent glutamatergic synapses in the adult nucleus accumbens shell (NAcSh). Methods Using multidisciplinary approaches including astrocyte Ca2+ imaging, genetic mouse lines, viral-mediated gene transfer, and operant behavioral procedures, we monitor the response of NAcSh astrocytes to cocaine administration and examine the role of astrocytic TSP–α2δ-1 signaling in cocaine-induced silent synapse generation as well as the behavioral impact of astrocyte-mediated synaptogenesis and silent synapse generation. Results Cocaine administration acutely increases Ca2+ events in NAcSh astrocytes, while decreasing astrocytic Ca2+ blocks cocaine-induced generation of silent synapses. Furthermore, knockout of TSP2, or pharmacological inhibition or viral-mediated knockdown of α2δ-1, prevents cocaine-induced generation of silent synapses. Moreover, disrupting TSP2–α2δ-1–mediated spinogenesis and synapse generation in NAcSh decreases cue-induced cocaine seeking after withdrawal from cocaine self-administration and cue-induced reinstatement of cocaine seeking after drug extinction. Conclusions These results establish that silent synapses are generated by an astrocyte-mediated synaptogenic mechanism in response to cocaine experience and embed critical cue-associated memory traces that promote cocaine relapse.

Pain experience can change the central processing of nociceptive inputs, resulting in persistent allodynia and hyperalgesia. However, the underlying circuit mechanisms remain underexplored. Here, we focus on pain-induced remodeling of the projection from the mediodorsal thalamus (MD) to the anterior cingulate cortex (ACC), a projection that relays spinal nociceptive input for central processing. Using optogenetics combined with slice electrophysiology, we detected in male mice that 7 days of chronic constriction injury (CCI; achieved by loose ligation of the sciatic nerve) generated AMPA receptor (AMPAR)-silent glutamatergic synapses within the contralateral MD-to-ACC projection. AMPAR-silent synapses are typically GluN2B-enriched nascent glutamatergic synapses that mediate the initial formation of neural circuits during early development. During development, some silent synapses mature and become "unsilenced" by recruiting and stabilizing AMPARs, consolidating and strengthening the newly formed circuits. Consistent with these synaptogenic features, pain-induced generation of silent synapses was accompanied by increased densities of immature dendritic spines in ACC neurons and increased synaptic weight of GluN2B-containing NMDA receptors (NMDARs) in the MD-to-ACC projection. After prolonged (∼30 days) CCI, injury-generated silent synapses declined to low levels, which likely resulted from a synaptic maturation process that strengthens AMPAR-mediated MD-to-ACC transmission. Consistent with this hypothesis, viral-mediated knockdown of GluN2B in ACC neurons, which prevented pain-induced generation of silent synapses and silent synapse-mediated strengthening of MD-to-ACC projection after prolonged CCI, prevented the development of allodynia. Taken together, our results depict a silent synapse-mediated mechanism through which key supraspinal neural circuits that regulate pain sensitivity are remodeled to induce allodynia and hyperalgesia.

Cocaine-associated memories are persistent, but, on retrieval, become temporarily destabilized and vulnerable to disruptions, followed by reconsolidation. To explore the synaptic underpinnings for these memory dynamics, we studied AMPA receptor (AMPAR)-silent excitatory synapses, which are generated in the nucleus accumbens by cocaine self-administration, and subsequently mature after prolonged withdrawal by recruiting AMPARs, echoing acquisition and consolidation of cocaine memories. We show that, on memory retrieval after prolonged withdrawal, the matured silent synapses become AMPAR-silent again, followed by re-maturation ~6 h later, defining the onset and termination of a destabilization window of cocaine memories. These synaptic dynamics are timed by Rac1, with decreased and increased Rac1 activities opening and closing, respectively, the silent synapse-mediated destabilization window. Preventing silent synapse re-maturation within the destabilization window decreases cue-induced cocaine seeking. Thus, cocaine-generated silent synapses constitute a discrete synaptic ensemble dictating the dynamics of cocaine-associated memories and can be targeted for memory disruption. Cocaine-generated silent synapses dictate the encoding, consolidation, retrieval-induced destabilization and reconsolidation of cocaine memories, and these syapses can be targeted to reduce drug seeking and relapse.

Frequent relapse prevents the successful treatment of substance use disorders and is triggered in part by retrieval of drug-associated memories. Drug-conditioned behaviours in rodents are reinstated upon drug memory retrieval following re-exposure to cues previously associated with the drug, or the drug itself. Therapies based on mechanistic insights from rodent studies have focused on amnesic procedures of cue-drug associations but with so far limited success. Conversely, more recent studies propose that inhibiting drug memory retrieval offers improved anti-relapse efficacy. However, mechanisms of memory retrieval are poorly understood. Here, we used a conditioned place preference (CPP) procedure in mice to investigate the cellular and molecular underpinnings of drug-induced memory retrieval. After extinction training of CPP, Ca

Cocaine experience generates AMPA receptor (AMPAR)-silent synapses in the nucleus accumbens (NAc), which are thought to be new synaptic contacts enriched in GluN2B-containing NMDA receptors (NMDARs). After drug withdrawal, some of these synapses mature by recruiting AMPARs, strengthening the newly established synaptic transmission. Silent synapse generation and maturation are two consecutive cellular steps through which NAc circuits are profoundly remodeled to promote cue-induced cocaine seeking after drug withdrawal. However, the basic cellular processes that mediate these two critical steps remains underexplored. Using a combination of electrophysiology, viral-mediated gene transfer, and confocal imaging in male rats as well as knock-in (KI) mice of both sexes, our current study characterized the dynamic roles played by AMPARs and NMDARs in generation and maturation of silent synapses on NAc medium spiny neurons after cocaine self-administration and withdrawal. We report that cocaine-induced generation of silent synapses not only required synaptic insertion of GluN2B-containing NMDARs, but also, counterintuitively, involved insertion of AMPARs, which subsequently internalized, resulting in the AMPAR-silent state on withdrawal day 1. Furthermore, GluN2B NMDARs functioned to maintain these cocaine-generated synapses in the AMPAR-silent state during drug withdrawal, until they were replaced by nonGluN2B NMDARs, a switch that allowed AMPAR recruitment and maturation of silent synapses. These results reveal dynamic interactions between AMPARs and NMDARs during the generation and maturation of silent synapses after cocaine experience and provide a mechanistic basis through which new synaptic contacts and possibly new neural network patterns created by these synapses can be manipulated for therapeutic benefit.SIGNIFICANCE STATEMENTStudies over the past decade reveal a critical role of AMPA receptor-silent, NMDA receptor-containing synapses in forming cocaine-related memories that drive cocaine relapse. However, it remains incompletely understood how AMPA and NMDA receptors traffic at these synapses during their generation and maturation. The current study characterizes a two-step AMPA receptor trafficking cascade that contributes to the generation of silent synapses in response to cocaine experience, and a two-step NMDA receptor trafficking cascade that contributes to the maturation of these synapses after cocaine withdrawal. These results depict a highly regulated cellular procedure through which nascent glutamatergic synapses are generated in the adult brain after drug experience and provide significant insight into the roles of glutamate receptors in synapse formation and maturation.

Significance During critical periods, restricted time windows in early brain development, cortical neural circuits get refined in an experience-dependent manner. Postsynaptic density protein-95 (PSD-95)-dependent maturation of silent glutamatergic synapses onto principal neurons is required for the termination of the critical period for ocular dominance plasticity (ODP) in the primary visual cortex (V1) of mice. Using chronic in vivo two-photon imaging of dendritic spine dynamics in V1 of awake mice, here we show that adult PSD-95-deficient neurons display increased spine elimination after a brief monocular deprivation, a structural hallmark of juvenile ODP. Thus, adult PSD-95-deficient neurons display both functional and structural hallmarks of critical period plasticity so that PSD-95-dependent silent synapse maturation and stabilization is correlated with critical period closure., Critical periods (CPs) are time windows of heightened brain plasticity during which experience refines synaptic connections to achieve mature functionality. At glutamatergic synapses on dendritic spines of principal cortical neurons, the maturation is largely governed by postsynaptic density protein-95 (PSD-95)-dependent synaptic incorporation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors into nascent AMPA-receptor silent synapses. Consequently, in mouse primary visual cortex (V1), impaired silent synapse maturation in PSD-95-deficient neurons prevents the closure of the CP for juvenile ocular dominance plasticity (jODP). A structural hallmark of jODP is increased spine elimination, induced by brief monocular deprivation (MD). However, it is unknown whether impaired silent synapse maturation facilitates spine elimination and also preserves juvenile structural plasticity. Using two-photon microscopy, we assessed spine dynamics in apical dendrites of layer 2/3 pyramidal neurons (PNs) in binocular V1 during ODP in awake adult mice. Under basal conditions, spine formation and elimination ratios were similar between PSD-95 knockout (KO) and wild-type (WT) mice. However, a brief MD affected spine dynamics only in KO mice, where MD doubled spine elimination, primarily affecting newly formed spines, and caused a net reduction in spine density similar to what has been observed during jODP in WT mice. A similar increase in spine elimination after MD occurred if PSD-95 was knocked down in single PNs of layer 2/3. Thus, structural plasticity is dictated cell autonomously by PSD-95 in vivo in awake mice. Loss of PSD-95 preserves hallmark features of spine dynamics in jODP into adulthood, revealing a functional link of PSD-95 for experience-dependent synapse maturation and stabilization during CPs.

Synaptic incorporation and removal of AMPA receptors is highly regulated to modulate the strength of synaptic transmission for long-term synaptic plasticity during brain development and associative learning. PSD-93α2 and PSD-95α, two paralogs of the DLG-MAGUK protein family of signaling scaffolds govern the synaptic incorporation and stabilization of AMPA receptors opposingly, with PSD-95α promoting and PSD-93α2 inhibiting it. The associated signaling mechanisms that control the synaptic incorporation and stabilization remain elusive. Here, we used domain swapping between the antagonizing signaling scaffolds to identify the protein motifs responsible for enhancing synaptic AMPA receptors and the associated signaling protein. We narrowed down multiple motifs in the N-terminal domain that are principally responsible for governing the enhancement by Src. Specific activation and inhibiting peptides revealed continuous activity of Src. Together, the results depict a mutual dependence of Src and PSD-95α in enhancing and maintaining synaptic AMPA receptors.

Critical periods are postnatal, restricted time windows of heightened plasticity in cortical neural networks, during which experience refines principal neuron wiring configurations. Here, we propose a model with two distinct types of synapses, innate synapses that establish rudimentary networks with innate function, and gestalt synapses that govern the experience-dependent refinement process. Nascent gestalt synapses are constantly formed as AMPA receptor-silent synapses which are the substrates for critical period plasticity. Experience drives the unsilencing and stabilization of gestalt synapses, as well as synapse pruning. This maturation process changes synapse patterning and consequently the functional architecture of cortical excitatory networks. Ocular dominance plasticity (ODP) in the primary visual cortex (V1) is an established experimental model for cortical plasticity. While converging evidence indicates that the start of the critical period for ODP is marked by the maturation of local inhibitory circuits, recent results support our model that critical periods end through the progressive maturation of gestalt synapses. The cooperative yet opposing function of two postsynaptic signaling scaffolds of excitatory synapses, PSD-93 and PSD-95, governs the maturation of gestalt synapses. Without those proteins, networks do not progress far beyond their innate functionality, resulting in rather impaired perception. While cortical networks remain malleable throughout life, the cellular mechanisms and the scope of critical period and adult plasticity differ. Critical period ODP is initiated with the depression of deprived eye responses in V1, whereas adult ODP is characterized by an initial increase in non-deprived eye responses. Our model proposes the gestalt synapse-based mechanism for critical period ODP, and also predicts a different mechanism for adult ODP based on the sparsity of nascent gestalt synapses at that age. Under our model, early life experience shapes the boundaries (the gestalt) for network function, both for its optimal performance as well as for its pathological state. Thus, reintroducing nascent gestalt synapses as plasticity substrates into adults may improve the network gestalt to facilitate functional recovery.

The nucleus accumbens shell (NAcSh) regulates emotional and motivational responses, a function mediated, in part, by integrating and prioritizing extensive glutamatergic projections from limbic and paralimbic brain regions. Each of these inputs is thought to encode unique aspects of emotional and motivational arousal. The projections do not operate alone, but rather are often activated simultaneously during motivated behaviors, during which they can interact and coordinate in shaping behavioral output. To understand the anatomic and physiological bases underlying these interprojection interactions, the current study in mice of both sexes focused on how the basolateral amygdala projection (BLAp) to the NAcSh regulates, and is regulated by, projections from the medial prefrontal cortex (mPFCp) and paraventricular nucleus of the thalamus (PVTp). Using a dual-color SynaptoTag technique combined with a backfilling spine imaging strategy, we found that all three afferent projections primarily targeted the secondary dendrites of NAcSh medium spiny neurons, forming putative synapses. We detected a low percentage of BLAp contacts closely adjacent to mPFCp or PVTp presumed synapses, and, on some rare occasions, the BLAp formed heterosynaptic interactions with mPFCp or PVTp profiles or appeared to contact the same spines. Using dual-rhodopsin optogenetics, we detected signs of dendritic summation of BLAp with PVTp and mPFCp inputs. Furthermore, high-frequency activation of BLAp synchronous with the PVTp or mPFCp resulted in a transient enhancement of the PVTp, but not mPFCp, transmission. These results provide anatomic and functional indices that the BLAp interacts with the mPFCp and PVTp for informational processing within the NAcSh. SIGNIFICANCE STATEMENT The nucleus accumbens regulates emotional and motivational responses by integrating extensive glutamatergic projections, but the anatomic and physiological bases on which these projections integrate and interact remain underexplored. Here, we used dual-color synaptic markers combined with backfilling of nucleus accumbens medium spiny neurons to reveal some unique anatomic alignments of presumed synapses from the basolateral amygdala, medial prefrontal cortex, and paraventricular nucleus of thalamus. We also used dual-rhodopsin optogenetics in brain slices, which reveal a nonlinear interaction between some, but not all, projections. These results provide compelling anatomic and physiological mechanisms through which different glutamatergic projections to the nucleus accumbens, and possibly different aspects of emotional and motivational arousal, interact with each other for final behavioral output.

Synaptogenesis is essential in forming new neurocircuits during development, and this is mediated in part by astrocyte-released thrombospondins (TSPs) and activation of their neuronal receptor, α2δ-1. Here, we show that this developmental synaptogenic mechanism is utilized during cocaine experience to induce spinogenesis and the generation of AMPA receptor-silent glutamatergic synapses in the adult nucleus accumbens (NAc). Specifically, cocaine administration activates NAc astrocytes, and preventing this activation blocks cocaine-induced generation of silent synapses. Furthermore, knockout of TSP2, or pharmacological inhibition or viral-mediated knockdown of α2δ-1, prevents cocaine-induced generation of silent synapses. Moreover, disrupting TSP2-α2δ-1-mediated spinogenesis and silent synapse generation in the NAc occludes cue-induced cocaine seeking after withdrawal from cocaine self-administration and cue-induced reinstatement of cocaine seeking after drug extinction. These results establish that silent synapses are generated by an astrocyte-mediated synaptogenic mechanism in response to cocaine experience and embed critical cue-associated memory traces that promote cocaine relapse.

The ventral tegmental area (VTA) projection to the nucleus accumbens shell (NAcSh) regulates NAcSh-mediated motivated behaviors in part by modulating the glutamatergic inputs. This modulation is likely to be mediated by multiple substances released from VTA axons, whose phenotypic diversity is illustrated here by ultrastructural examination. Furthermore, we show in mouse brain slices that a brief optogenetic stimulation of VTA-to-NAc projection induced a transient inhibition of excitatory postsynaptic currents (EPSCs) in NAcSh principal medium spiny neurons (MSNs). This inhibition was not accompanied by detectable alterations in presynaptic release properties of electrically-evoked EPSCs, suggesting a postsynaptic mechanism. The VTA projection to the NAcSh releases dopamine, GABA and glutamate, and induces the release of other neuronal substrates that are capable of regulating synaptic transmission. However, pharmacological inhibition of dopamine D1 or D2 receptors, GABAA or GABAB receptors, NMDA receptors, P2Y1 ATP receptors, metabotropic glutamate receptor 5, and TRP channels did not prevent this short-term inhibition. These results suggest that an unknown mechanism mediates this form of short-term plasticity induced by the VTA-to-NAc projection.

In human drug users, cue-induced drug craving progressively intensifies after drug abstinence, promoting drug relapse. This time-dependent progression of drug craving is recapitulated in rodent models, in which rats exhibit progressive intensification of cue-induced drug seeking after withdrawal from drug self-administration, a phenomenon termed incubation of drug craving. Although recent results suggest that functional alterations of the nucleus accumbens (NAc) contribute to incubation of drug craving, it remains poorly understood how NAc function evolves after drug withdrawal to progressively intensify drug seeking. The functional output of NAc relies on how the membrane excitability of its principal medium spiny neurons (MSNs) translates excitatory synaptic inputs into action potential firing. Here, we report a synapse-membrane homeostatic crosstalk (SMHC) in male rats, through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the intrinsic membrane excitability of NAc MSNs, and vice versa. After short-term withdrawal from cocaine self-administration, despite no actual change in the AMPA receptor-mediated excitatory synaptic strength, GluN2B NMDA receptors, the SMHC sensors of synaptic strength, are upregulated. This may create false SMHC signals, leading to a decrease in the membrane excitability of NAc MSNs. The decreased membrane excitability subsequently induces another round of SMHC, leading to synaptic accumulation of calcium-permeable AMPA receptors and upregulation of excitatory synaptic strength after long-term withdrawal from cocaine. Disrupting SMHC-based dysregulation cascades after cocaine exposure prevents incubation of cocaine craving. Thus, cocaine triggers cascades of SMHC-based dysregulation in NAc MSNs, promoting incubated cocaine seeking after drug withdrawal.SIGNIFICANCE STATEMENTHere, we report a bidirectional homeostatic plasticity between the excitatory synaptic input and membrane excitability of nucleus accumbens (NAc) medium spiny neurons (MSNs), through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the membrane excitability, and vice versa. Cocaine self-administration creates a false homeostatic signal that engages this synapse-membrane homeostatic crosstalk mechanism, and produces cascades of alterations in excitatory synapses and membrane properties of NAc MSNs after withdrawal from cocaine. Experimentally preventing this homeostatic dysregulation cascade prevents the progressive intensification of cocaine seeking after drug withdrawal. These results provide a novel mechanism through which drug-induced homeostatic dysregulation cascades progressively alter the functional output of NAc MSNs and promote drug relapse.

The nucleus accumbens (NAc) gates motivated behaviors through the functional output of principle medium spiny neurons (MSNs), whereas dysfunctional output of NAc MSNs contributes to a variety of psychiatric disorders. Fast-spiking interneurons (FSIs) are sparsely distributed throughout the NAc, forming local feedforward inhibitory circuits. It remains elusive how FSI-based feedforward circuits regulate the output of NAc MSNs. Here, we investigated a distinct subpopulation of NAc FSIs that express the cannabinoid receptor type-1 (CB1). Using a combination of paired electrophysiological recordings and pharmacological approaches, we characterized and compared feedforward inhibition of NAc MSNs from CB1+ FSIs and lateral inhibition from recurrent MSN collaterals. We observed that CB1+ FSIs exerted robust inhibitory control over a large percentage of nearby MSNs in contrast to local MSN collaterals that provided only sparse and weak inhibitory input to their neighboring MSNs. Furthermore, CB1+ FSI-mediated feedforward inhibition was preferentially suppressed by endocannabinoid (eCB) signaling, whereas MSN-mediated lateral inhibition was unaffected. Finally, we demonstrated that CB1+ FSI synapses onto MSNs are capable of undergoing experience-dependent long-term depression in a voltage- and eCB-dependent manner. These findings demonstrated that CB1+ FSIs are a major source of local inhibitory control of MSNs and a critical component of the feedforward inhibitory circuits regulating the output of the NAc.

Abnormalities in synaptic inhibition play a critical role in psychiatric disorders, and accordingly, it is essential to understand the molecular mechanisms linking components of the inhibitory postsynapse to psychiatrically relevant neural circuits and behaviors. Here we study the role of IgSF9b, an adhesion protein that has been associated with affective disorders, in the amygdala anxiety circuitry. We show that deletion of IgSF9b normalizes anxiety-related behaviors and neural processing in mice lacking the synapse organizer Neuroligin-2 (Nlgn2), which was proposed to complex with IgSF9b. This normalization occurs through differential effects of Nlgn2 and IgSF9b at inhibitory synapses in the basal and centromedial amygdala (CeM), respectively. Moreover, deletion of IgSF9b in the CeM of adult Nlgn2 knockout mice has a prominent anxiolytic effect. Our data place IgSF9b as a key regulator of inhibition in the amygdala and indicate that IgSF9b-expressing synapses in the CeM may represent a target for anxiolytic therapies., IgSF9b is a synaptic adhesion protein that has been linked to psychiatric disorders. Here the authors show that deletion of IgSF9b regulates anxiety-like behaviour in mice by increasing inhibitory synaptic transmission in the centromedial amygdala.

Depression is a complex, heterogeneous disorder and a leading contributor to the global burden of disease. Most previous research has focused on individual brain regions and genes contributing to depression. However, emerging evidence in humans and animal models suggests that dysregulated circuit function and gene expression across multiple brain regions drive depressive phenotypes. Here, we performed RNA sequencing on four brain regions from control animals and those susceptible or resilient to chronic social defeat stress at multiple time points. We employed an integrative network biology approach to identify transcriptional networks and key driver genes that regulate susceptibility to depressive-like symptoms. Further, we validated in vivo several key drivers and their associated transcriptional networks that regulate depression susceptibility and confirmed their functional significance at the levels of gene transcription, synaptic regulation, and behavior. Our study reveals novel transcriptional networks that control stress susceptibility and offers fundamentally new leads for antidepressant drug discovery.

The basolateral amygdala (BLA) sends excitatory projections to the nucleus accumbens (NAc) and regulates motivated behaviors partially by activating NAc medium spiny neurons (MSNs). Here, we characterized a feedforward inhibition circuit, through which BLA-evoked activation of NAc shell (NAcSh) MSNs was fine-tuned by GABAergic monosynaptic innervation from adjacent fast-spiking interneurons (FSIs). Specifically, BLA-to-NAcSh projections predominantly innervated NAcSh FSIs compared with MSNs and triggered action potentials in FSIs preceding BLA-mediated activation of MSNs. Due to these anatomical and temporal properties, activation of the BLA-to-NAcSh projection resulted in a rapid FSI-mediated inhibition of MSNs, timing-contingently dictating BLA-evoked activation of MSNs. Cocaine self-administration selectively and persistently up-regulated the presynaptic release probability of BLA-to-FSI synapses, entailing enhanced FSI-mediated feedforward inhibition of MSNs upon BLA activation. Experimentally enhancing the BLA-to-FSI transmission in vivo expedited the acquisition of cocaine self-administration. These results reveal a previously unidentified role of an FSI-embedded circuit in regulating NAc-based drug seeking and taking.

A transient but prominent increase in the level of “silent synapses”—a signature of immature glutamatergic synapses that contain only NMDA receptors without stably expressed AMPA receptors—has been identified in the nucleus accumbens (NAc) following exposure to cocaine. As the NAc is a critical forebrain region implicated in forming addiction-associated behaviors, the initial discoveries have raised speculations about whether and how these drug-induced synapses mature and potentially contribute to addiction-related behaviors. Here, we summarize recent progress in recognizing the pathway-specific regulations of silent synapse maturation, and its diverse impacts on behavior. We provide an update of the guiding hypothesis—the “neural rejuvenation hypothesis”—with recently emerged evidence of silent synapses in cocaine craving and relapse.

The postsynaptic density proteins 95 (PSD95) and 93 (PSD93) belong to a family of scaffolding proteins, the membrane-associated guanylate kinases (MAGUKs), which are highly enriched in synapses and responsible for organizing the numerous protein complexes required for synaptic development and plasticity. Genetic studies have associated MAGUKs with diseases like autism and schizophrenia, but knockout mice show severe, complex defects with difficult-to-interpret behavioral abnormalities due to major motor dysfunction which is atypical for psychiatric phenotypes. Therefore, rather than studying loss-of-function mutants, we comprehensively investigated the behavioral consequences of reduced PSD95 expression, using heterozygous PSD95 knockout mice (PSD95+/-). Specifically, we asked whether heterozygous PSD95 deficient mice would exhibit alterations in the processing of social stimuli and social behavior. Additionally, we investigated whether PSD95 and PSD93 would reveal any indication of functional or biological redundancy. Therefore, homozygous and heterozygous PSD93 deficient mice were examined in a similar behavioral battery as PSD95 mutants. We found robust hypersocial behavior in the dyadic interaction test in both PSD95+/- males and females. Additionally, male PSD95+/- mice exhibited higher levels of aggression and territoriality, while female PSD95+/- mice showed increased vocalization upon exposure to an anesthetized female mouse. Both male and female PSD95+/- mice revealed mild hypoactivity in the open field but no obvious motor deficit. Regarding PSD93 mutants, homozygous (but not heterozygous) knockout mice displayed prominent hypersocial behavior comparable to that observed in PSD95+/- mice, despite a more severe motor phenotype, which precluded several behavioral tests or their interpretation. Considering that PSD95 and PSD93 reduction provoke strikingly similar behavioral consequences, we explored a potential substitution effect and found increased PSD93 protein expression in hippocampal synaptic enrichment preparations of PSD95+/- mice. These data suggest that both PSD95 and PSD93 are involved in processing of social stimuli and control of social behavior. This important role may be partly assured by functional/behavioral and biological/biochemical redundancy.

Exposure to cocaine generates silent synapses in the nucleus accumbens (NAc), whose eventual unsilencing/maturation by recruitment of calcium‐permeable AMPA‐type glutamate receptors (CP‐AMPARs) after drug withdrawal results in profound remodeling of NAc neuro‐circuits. Silent synapse‐based NAc remodeling was shown to be critical for several drug‐induced behaviors, but its role in acquisition and retention of the association between drug rewarding effects and drug‐associated contexts has remained unclear. Here, we find that the postsynaptic proteins PSD‐93, PSD‐95, and SAP102 differentially regulate excitatory synapse properties in the NAc. Mice deficient for either of these scaffold proteins exhibit distinct maturation patterns of silent synapses and thus provided instructive animal models to examine the role of NAc silent synapse maturation in cocaine‐conditioned place preference (CPP). Wild‐type and knockout mice alike all acquired cocaine‐CPP and exhibited increased levels of silent synapses after drug‐context conditioning. However, the mice differed in CPP retention and CP‐AMPAR incorporation. Collectively, our results indicate that CP‐AMPAR‐mediated maturation of silent synapses in the NAc is a signature of drug–context association, but this maturation is not required for establishing or retaining cocaine‐CPP. ![][1] Various cocaine‐induced behaviors are linked to generation and remodeling of silent synapses in the nucleus accumbens. Postsynaptic signaling scaffold proteins PSD‐93, PSD‐95, and SAP102 differentially affect the properties of such excitatory synapses as well as the acquisition and retention of drug reward/context association in mice. [1]: /embed/graphic-1.gif

Ocular dominance (OD) plasticity in mouse primary visual cortex (V1) declines during postnatal development and is absent beyond postnatal day 110 if mice are raised in standard cages (SCs). An enriched environment (EE) promotes OD plasticity in adult rats. Here, we explored cellular mechanisms of EE in mouse V1 and the therapeutic potential of EE to prevent impairments of plasticity after a cortical stroke. Using in vivo optical imaging, we observed that monocular deprivation in adult EE mice (i) caused a very strong OD plasticity previously only observed in 4-wk-old animals, (ii) restored already lost OD plasticity in adult SC-raised mice, and (iii) preserved OD plasticity after a stroke in the primary somatosensory cortex. Using patch-clamp electrophysiology in vitro, we also show that (iv) local inhibition was significantly reduced in V1 slices of adult EE mice and (v) the GABA/AMPA ratio was like that in 4-wk-old SC-raised animals. These observations were corroborated by in vivo analyses showing that diazepam treatment significantly reduced the OD shift of EE mice after monocular deprivation. Taken together, EE extended the sensitive phase for OD plasticity into late adulthood, rejuvenated V1 after 4 mo of SC-rearing, and protected adult mice from stroke-induced impairments of cortical plasticity. The EE effect was mediated most likely by preserving low juvenile levels of inhibition into adulthood, which potentially promoted adaptive changes in cortical circuits.

In rat models of drug relapse and craving, cue-induced cocaine seeking progressively increases after withdrawal from the drug. This 'incubation of cocaine craving' is partially mediated by time-dependent adaptations at glutamatergic synapses in nucleus accumbens (NAc). However, the circuit-level adaptations mediating this plasticity remain elusive. We studied silent synapses, often regarded as immature synapses that express stable NMDA receptors with AMPA receptors being either absent or labile, in the projection from the basolateral amygdala to the NAc in incubation of cocaine craving. Silent synapses were detected in this projection during early withdrawal from cocaine. As the withdrawal period progressed, these silent synapses became unsilenced, a process that involved 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 incubation of cocaine craving. Our findings indicate that silent synapse-based reorganization of the amygdala-to-NAc projection is critical for persistent cocaine craving and relapse after withdrawal.

Synaptic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) make up the backbone of the brain reward pathway, a neural circuit that mediates behavioural responses elicited by natural rewards as well as by cocaine and other drugs of abuse. In addition to the well-known modulatory dopaminergic projection, the VTA also provides fast excitatory and inhibitory synaptic input to the NAc, directly regulating NAc medium spiny neurons (MSNs). However, the cellular nature of VTA-to-NAc fast synaptic transmission and its roles in drug-induced adaptations are not well understood. Using viral-mediated in vivo expression of channelrhodopsin 2, the present study dissected fast excitatory and inhibitory synaptic transmission from the VTA to NAc MSNs in rats. Our results suggest that, following repeated exposure to cocaine (15 mg kg(-1) day(-1) × 5 days, i.p., 1 or 21 day withdrawal), a presynaptic enhancement of excitatory transmission and suppression of inhibitory transmission occurred at different withdrawal time points at VTA-to-NAc core synapses. In contrast, no postsynaptic alterations were detected at either type of synapse. These results suggest that changes in VTA-to-NAc fast excitatory and inhibitory synaptic transmissions may contribute to cocaine-induced alteration of the brain reward circuitry.

Medium spiny neurons (MSNs) within the nucleus accumbens shell (NAc) function to gate and prioritize emotional/motivational arousals for behavioral output. The neuronal output NAc MSNs is mainly determined by the integration of membrane excitability and excitatory/inhibitory synaptic inputs. Whereas cocaine-induced alterations at excitatory synapses and membrane excitability have been extensively examined, the overall functional output of NAc MSNs following cocaine exposure still poorly defined because little is known about whether inhibitory synaptic input to these neurons is affected by cocaine. Here, our results demonstrate multidimensional alterations at inhibitory synapses in NAc neurons following cocaine self-administration in rats. Specifically, the amplitude of miniature (m) inhibitory postsynaptic currents (IPSCs) was decreased after 21-d withdrawal from 5-d cocaine self-administration. Upon re-exposure to cocaine after 21-day withdrawal, whereas the amplitude of mIPSCs remained down-regulated, the frequency became significantly higher. Furthermore, the reversal potential of IPSCs, which was not significantly altered during withdrawal, became more hyperpolarized upon cocaine re-exposure. Moreover, the relative weight of excitatory and inhibitory inputs to NAc MSNs was significantly decreased after 1-d cocaine withdrawal, increased after 21-d withdrawal, and returned to the basal level upon cocaine re-exposure after 21-d withdrawal. These results, taken together with previous results showing cocaine-induced adaptations at excitatory synapses and intrinsic membrane excitability of NAc MSNs, may provide a relatively thorough picture of the functional state of NAc MSNs following cocaine exposure.

The nucleus accumbens (NAc) regulates motivated behavior by, in part, processing excitatory synaptic projections from several brain regions. Among these regions, the prefrontal cortex (PFC) and basolateral amygdala, convey executive control and affective states, respectively. Whereas glutamatergic synaptic transmission within the NAc has been recognized as a primary cellular target for cocaine and other drugs of abuse to induce addiction-related pathophysiological motivational states, the understanding has been thus far limited to drug-induced postsynaptic alterations. It remains elusive whether exposure to cocaine or other drugs of abuse influences presynaptic functions of these excitatory projections, and if so, in which projection pathways. Using optogenetic methods combined with biophysical assays, we demonstrate that the presynaptic release probability (Pr) of the PFC-to-NAc synapses was enhanced after short-term withdrawal (1 d) and long-term (45 d) withdrawal from either noncontingent (i.p. injection) or contingent (self-administration) exposure to cocaine. After long-term withdrawal of contingent drug exposure, the Pr was higher compared with i.p. injected rats. In contrast, within the basolateral amygdala afferents, presynaptic Pr was not significantly altered in any of these experimental conditions. Thus, cocaine-induced procedure- and pathway-specific presynaptic enhancement of excitatory synaptic transmission in the NAc. These results, together with previous findings of cocaine-induced postsynaptic enhancement, suggest an increased PFC-to-NAc shell glutamatergic synaptic transmission after withdrawal from exposure to cocaine. This presynaptic alteration may interact with other cocaine-induced cellular adaptations to shift the functional output of NAc neurons, contributing to the addictive emotional and motivational state.

Impairments in social skills are central to mental disease, and developing tools for their assessment in mouse models is essential. Here we present the SocioBox, a new behavioral paradigm to measure social recognition memory. Using this paradigm, we show that male wildtype mice of different strains can readily identify an unfamiliar mouse among 5 newly acquainted animals. In contrast, female mice exhibit lower locomotor activity during social exploration in the SocioBox compared to males and do not seem to discriminate between acquainted and unfamiliar mice, likely reflecting inherent differences in gender-specific territorial tasks. In addition to a simple quantification of social interaction time of mice grounded on predefined spatial zones (zone-based method), we developed a set of unbiased, data-driven analysis tools based on heat map representations and characterized by greater sensitivity. First proof-of-principle that the SocioBox allows diagnosis of social recognition memory deficits is provided using male PSD-95 heterozygous knockout mice, a mouse model related to psychiatric pathophysiology.

Exposure to cocaine induces addiction-associated behaviors partially through remodeling neurocircuits in the nucleus accumbens (NAc). The paraventricular nucleus of thalamus (PVT), which projects to the NAc monosynaptically, is activated by cocaine exposure and has been implicated in several cocaine-induced emotional and motivational states. Here we show that disrupting synaptic transmission of select PVT neurons with tetanus toxin activated via retrograde trans-synaptic transport of cre from NAc efferents decreased cocaine self-administration in rats. This projection underwent complex adaptations after self-administration of cocaine (0.75 mg/kg/infusion; 2 h/d × 5 d, 1d overnight training). Specifically, 1d after cocaine self-administration, we observed increased levels of AMPA receptor (AMPAR)-silent glutamatergic synapses in this projection, accompanied by a decreased ratio of AMPAR-to-NMDA receptor (NMDAR)-mediated EPSCs. Furthermore, the decay kinetics of NMDAR EPSCs was significantly prolonged, suggesting insertion of new GluN2B-containing NMDARs to PVT-to-NAc synapses. After 45-d withdrawal, silent synapses within this projection returned to the basal levels, accompanied by a return of the AMPAR/NMDAR ratio and NMDAR decay kinetics to the basal levels. In amygdala and infralimbic prefrontal cortical projections to the NAc, a portion of cocaine-generated silent synapses becomes unsilenced by recruiting calcium-permeable AMPARs (CP-AMPARs) after drug withdrawal. However, the sensitivity of PVT-to-NAc synapses to CP-AMPAR-selective antagonists was not changed after withdrawal, suggesting that CP-AMPAR trafficking is not involved in the evolution of cocaine-generated silent synapses within this projection. Meanwhile, the release probability of PVT-to-NAc synapses was increased after short- and long-term cocaine withdrawal. These results reveal complex and profound alterations at PVT-to-NAc synapses after cocaine exposure and withdrawal.

Environmental enrichment (EE) has long been postulated as a behavioral treatment for drug addiction based on its preventive effects in animal models: rodents experiencing prior EE exhibit increased resistance to establishing drug taking and seeking. However, the therapeutic effects of EE, namely, the effects of EE when applied after drug exposure, are often marginal and transient. Using incubation of cue-induced cocaine craving, a rat relapse model depicting progressive intensification of cocaine seeking after withdrawal from cocaine self-administration, our present study reveals that after cocaine withdrawal, in vivo circuit-specific long-term depression (LTD) unmasks the therapeutic power of EE to achieve long-lasting anti-relapse effects. Specifically, our previous results show that cocaine self-administration generates AMPA receptor (AMPAR)-silent excitatory synapses within the basolateral amygdala (BLA) to nucleus accumbens (NAc) projection, and maturation of these silent synapses via recruiting calcium-permeable (CP) AMPARs contributes to incubation of cocaine craving. Here, we show that after cocaine withdrawal and maturation of silent synapses, the BLA-to-NAc projection became highly resistant to EE. However, optogenetic LTD applied to this projection in vivo transiently re-silenced these silent synapses by removing CP-AMPARs. During this transient window, application of EE resulted in the insertion of nonCP-AMPARs, thereby remodeling the "incubated" BLA-to-NAc projection. Consequently, incubation of cocaine craving was decreased persistently. These results reveal a mechanistic basis through which the persistent anti-relapse effects of EE can be unleashed after drug withdrawal.

Exposures to cocaine and morphine produce similar adaptations in nucleus accumbens (NAc)-based behaviors, yet produce very different adaptations at NAc excitatory synapses. In an effort to explain this paradox, we found that both drugs induced NMDA receptor-containing, AMPA receptor-silent excitatory synapses, albeit in distinct cell types through opposing cellular mechanisms. Cocaine selectively induced silent synapses in D1-type neurons, likely via a synaptogenesis process, whereas morphine induced silent synapses in D2-type neurons via internalization of AMPA receptors from pre-existing synapses. After drug withdrawal, cocaine-generated silent synapses became 'unsilenced' by recruiting AMPA receptors to strengthen excitatory inputs to D1-type neurons, whereas morphine-generated silent synapses were likely eliminated to weaken excitatory inputs to D2-type neurons. Thus, these cell type-specific, opposing mechanisms produced the same net shift of the balance between excitatory inputs to D1- and D2-type NAc neurons, which may underlie certain common alterations in NAc-based behaviors induced by both classes of drugs.

Spike timing-dependent synaptic plasticity (STDP) serves as a key cellular correlate of associative learning, which is facilitated by elevated attentional and emotional states involving activation of adrenergic signaling. At cellular levels, adrenergic signaling increases dendrite excitability, but the underlying mechanisms remain elusive. Here we show that activation of β2-adrenoceptors promoted STD long-term synaptic potentiation at mouse hippocampal excitatory synapses by inactivating dendritic Kv1.1-containing potassium channels, which increased dendrite excitability and facilitated dendritic propagation of postsynaptic depolarization, potentially improving co-incidental activation of pre- and postsynaptic terminals. We further demonstrate that adrenergic modulation of Kv1.1 was mediated by the signaling scaffold SAP97, which, through direct protein-protein interactions, escorts β2-signaling to remove Kv1.1 from the dendrite surface. These results reveal a mechanism through which the postsynaptic signaling scaffolds bridge the aroused brain state to promote induction of synaptic plasticity and potentially to enhance spike timing and memory encoding.

The N-methyl-D-aspartate (NMDA) receptor (NMDAR)-stimulated autophosphorylation of calmodulin-dependent kinase IIα at Thr286 may regulate many aspects of neuroplasticity. Here, we show that low NMDA concentration (20 μM) up-regulated Thr286 phosphorylation, and high concentration (100 μM) caused dephosphorylation. We next modulated the strength of NMDAR activation by manipulating NMDAR 2A subunit (NR2A) and NMDAR 2B subunit (NR2B), which represent the major NMDAR subtypes in forebrain regions. Pharmacological inhibition and molecular knockdown of NR2A or NR2B blocked 20 μM NMDA-induced phosphorylation. Conversely, over-expression of NR2A or NR2B enhanced phosphorylation by 20 μM NMDA. The 100 μM NMDA-induced dephosphorylation was suppressed by inhibition or knockdown of NR2A or NR2B, and enhanced by over-expression of NR2A or NR2B. Compared to NR2A, NR2B showed a higher impact on the NMDA-stimulated bi-directional regulation of Thr286 phosphorylation. We further found that activation of NR2A and NR2B by 100 μM NMDA-induced dephosphorylation through protein phosphatases (PP) that are inhibited by high concentration okadaic acid (1 μM), but not by PP2A and PP2B inhibitors. This novel function of NMDAR in dynamic regulation of calmodulin-dependent kinase IIα activity provides new evidence to support the current understanding that, depending on the degree of activation, NMDAR may lead to different and even opposing effects on intracellular signaling.

The nucleus accumbens shell (NAc) is a key brain region mediating emotional and motivational learning. In rodent models, dynamic alterations have been observed in synaptic N-Methyl-D-aspartic acid receptors (NMDARs) within the NAc following incentive stimuli, and some of these alterations are critical for acquiring new emotional/motivational states. NMDARs are prominent molecular devices for controlling neural plasticity and memory formation. Although synaptic NMDARs are predominately located postsynaptically, recent evidence suggests that they may also exist at presynaptic terminals and reshape excitatory synaptic transmission by regulating presynaptic glutamate release. However, it remains unknown whether presynaptic NMDARs exist in the NAc to contribute to emotional and motivational learning. In an attempt to identify presynaptically-located NMDARs in the NAc, the present study utilizes slice electrophysiological recording combined with pharmacological and genetic tools to examine the physiological role of the putative presynaptic NMDARs in rats. Our results show that application of glycine, the glycine-site agonist of NMDARs, potentiated presynaptic release of glutamate at excitatory synapses on NAc neurons, whereas application of 5,7-dichlorokynurenic acid (DCKA) or 7-chlorokynurenic acid, the glycine-site antagonists of NMDARs, produced the opposite effect. However, these seemingly presynaptic NMDAR-mediated effects could not be prevented by application of D-APV, the glutamate-site NMDAR antagonist, and were still present in the mice in which NMDAR NR1 or NR3 subunits were genetically deleted. Thus, rather than suggesting the existence of presynaptic NMDARs, our results support the idea that an unidentified type of glycine-activated substrate may account for the presynaptic effects appearing to be mediated by NMDARs.

Background Dysregulation of excitatory synaptic input to nucleus accumbens (NAc) medium spiny neurons (MSNs) underlies a key pathophysiology of drug addiction and addiction-associated emotional and motivational alterations. Dynorphin peptides, which exhibit higher affinity to κ type opioid receptors, are upregulated within the NAc upon exposure to cocaine administration, and the increased dynorphin-signaling in the NAc has been critically implicated in negative mood observed in cocaine- or stress-exposed animals. Despite such apparent behavioral significance of the NAc dynorphins, the understanding of how dynorphins regulate excitatory synaptic transmission in the NAc remains incomplete. Methods We used electrophysiological recording in brain slices to examine the effects of dynorphins on excitatory synaptic transmission in the NAc. Results We focused on two key dynorphins, dynorphin A and B. Our current results show that dynorphin A and B differentially regulated excitatory postsynaptic currents (EPSCs) in NAc MSNs. Whereas perfusions of both dynorphin A and B to NAc slices decreased EPSCs in MSNs, the effect of dynorphin A but not dynorphin B was completely reversed by the κ receptor-selective antagonist nor-binaltorphimine. These results implicate κ receptor-independent mechanisms in dynorphin B-mediated synaptic effects in the NAc. Furthermore, repeated exposure to cocaine (15 mg/kg/day via intraperitoneal injection for 5 days, with 1, 2, or 14 days withdrawal) completely abolished dynorphin A-mediated modulation of EPSCs in NAc MSNs, whereas the effect of dynorphin B remained largely unchanged. Conclusions Given the quantitatively higher abundance of dynorphin B in the NAc, our present results suggest that the dynorphin B-mediated, κ receptor-independent pathways predominate in the overall effect of dynorphins in cocaine-pretreated animals and potentially in cocaine-induced alterations in mood.

The recently introduced term 'optogenetics' describes a variety of techniques for expressing genes in nerve cells that render them responsive to light. This approach makes use of light-sensitive channel proteins that can be used to manipulate neuronal function. Using genetic strategies, these channel proteins can be expressed in neurons defined by a common genetic identity, which can then be selectively activated or silenced through illumination. In this minireview, we shall describe the basic principles of such manipulative optogenetic approaches in neuroscience and summarize how these tools are being exploited to investigate neuronal circuits and behavior.

Drug-induced malfunction of nucleus accumbens (NAc) neurons underlies a key pathophysiology of drug addiction. Drug-induced changes in intrinsic membrane excitability of NAc neurons are thought to be critical for producing behavioral alterations. Previous studies demonstrate that, after short-term (2 d) or long-term (21 d) withdrawal from noncontingent cocaine injection, the intrinsic membrane excitability of NAc shell (NAcSh) neurons is decreased, and decreased membrane excitability of NAcSh neurons increases the acute locomotor response to cocaine. However, animals exhibit distinct cellular and behavioral alterations at different stages of cocaine exposure, suggesting that the decreased membrane excitability of NAc neurons may not be a persistent change. Here, we demonstrate that the membrane excitability of NAcSh neurons is differentially regulated depending on whether cocaine is administered contingently or noncontingently. Specifically, the membrane excitability of NAcSh medium spiny neurons (MSNs) was decreased at 2 d after withdrawal from either 5 d intraperitoneal injections (15 mg/kg) or cocaine self-administration (SA). At 21 d of withdrawal, the membrane excitability of NAcSh MSNs, which remained low in intraperitoneally pretreated rats, returned to a normal level in SA-pretreated rats. Furthermore, after a reexposure to cocaine after long-term withdrawal, the membrane excitability of NAcSh MSNs instantly returned to a normal level in intraperitoneally pretreated rats. Conversely, in SA-pretreated rats, the reexposure elevated the membrane excitability of NAcSh MSMs beyond the normal level. These results suggest that the dynamic alterations in membrane excitability of NAcSh MSNs, together with the dynamic changes in synaptic input, contribute differentially to the behavioral consequences of contingent and noncontingent cocaine administration.

The endocytosis of AMPA receptors underlies several forms of synaptic plasticity. Here, the authors show that PSD-95, via its binding to AKAP150, is important for NMDAR-triggered endocytosis of synaptic AMPARS. The endocytosis of AMPA receptors (AMPARs) underlies several forms of synaptic plasticity, including NMDA receptor (NMDAR)-dependent long-term depression (LTD), but the molecular mechanisms responsible for this trafficking remain unknown. We found that PSD-95, a major postsynaptic density protein, is important for NMDAR-triggered endocytosis of synaptic AMPARs in rat neuron cultures because of its binding to A kinase–anchoring protein 150 (AKAP150), a scaffold for specific protein kinases and phosphatases. Knockdown of PSD-95 with shRNA blocked NMDAR-triggered, but not constitutive or mGluR-triggered, endocytosis of AMPARs. Deletion of PSD-95's Src homology 3 and guanylate kinase–like domains, as well as a point mutation (L460P), both of which inhibit binding of PSD-95 to AKAP150, also blocked NMDAR-triggered AMPAR endocytosis. Furthermore, expression of a mutant AKAP150 that does not bind calcineurin inhibited this NMDAR-triggered trafficking event. Our results suggest that PSD-95's interaction with AKAP150 is critical for NMDAR-triggered AMPAR endocytosis and LTD, possibly because these scaffolds position calcineurin in the appropriate subsynaptic domain.

During critical periods, all cortical neural circuits are refined to optimize their functional properties. The prevailing notion is that the balance between excitation and inhibition determines the onset and closure of critical periods. In contrast, we show that maturation of silent glutamatergic synapses onto principal neurons was sufficient to govern the duration of the critical period for ocular dominance plasticity in the visual cortex of mice. Specifically, postsynaptic density protein-95 (PSD-95) was absolutely required for experience-dependent maturation of silent synapses, and its absence before the onset of critical periods resulted in lifelong juvenile ocular dominance plasticity. Loss of PSD-95 in the visual cortex after the closure of the critical period reinstated silent synapses, resulting in reopening of juvenile-like ocular dominance plasticity. Additionally, silent synapse-based ocular dominance plasticity was largely independent of the inhibitory tone, whose developmental maturation was independent of PSD-95. Moreover, glutamatergic synaptic transmission onto parvalbumin-positive interneurons was unaltered in PSD-95 KO mice. These findings reveal not only that PSD-95-dependent silent synapse maturation in visual cortical principal neurons terminates the critical period for ocular dominance plasticity but also indicate that, in general, once silent synapses are consolidated in any neural circuit, initial experience-dependent functional optimization and critical periods end.

PSD-95 and SAP97 are scaffolding proteins that have been implicated in regulating AMPA receptor incorporation and function at synapses. Gain- and loss-of-function approaches, however, have generated conflicting results. To minimize adaptations during development and potential dominant-negative effects of overexpression, we have combined silencing of endogenous PSD-95 in mature neurons with heterologous expression of specific SAP97 or PSD-95 isoforms. We find that both PSD-95 and SAP97 contain alternative N termini expressing either double cysteines that normally are palmitoylated (alpha-isoforms) or an L27 domain (beta-isoforms). Whereas alpha-isoforms of PSD-95 and SAP97 influence AMPA receptor-mediated synaptic strength independent of activity, the effects of beta-isoforms are regulated by activity in a CaMKII-dependent manner. Importantly, the synaptic effects of the beta-isoforms are masked by the endogenous alpha-isoform of PSD-95. These results demonstrate that the different N termini of the predominant endogenous forms of PSD-95 (alpha-isoform) and SAP97 (beta-isoform) govern their role in regulating synaptic function.

Presynaptic vesicle trafficking and priming are important steps in regulating synaptic transmission and plasticity. The four closely related small GTP-binding proteins Rab3A, Rab3B, Rab3C, and Rab3D are believed to be important for these steps. In mice, the complete absence of all Rab3s leads to perinatal lethality accompanied by a 30% reduction of probability of Ca2+-triggered synaptic release. This study examines the role of Rab3 during Ca2+-triggered release in more detail and identifies its impact on short-term plasticity. Using patch-clamp electrophysiology of autaptic neuronal cultures from Rab3-deficient mouse hippocampus, we show that excitatory Rab3-deficient neurons display unique time- and frequency-dependent short-term plasticity characteristics in response to spike trains. Analysis of vesicle release and repriming kinetics as well as Ca2+sensitivity of release indicate that Rab3 acts on a subset of primed, fusion competent vesicles. They lower the amount of Ca2+required for action potential-triggered release, which leads to a boosting of release probability, but their action also introduces a significant delay in the supply of these modified vesicles. As a result, Rab3-induced modifications to primed vesicles causes a transient increase in the transduction efficacy of synaptic action potential trains and optimizes the encoding of synaptic information at an intermediate spike frequency range.

Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.

In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca(2+)-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca(2+)-triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Luthi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca(2+)-independent exocytosis that correlated with the inhibition of regulated Ca(2+)-triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knock-out studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.

Quick, Quick, Slow The slow muscles of postural stability and the fast muscles of running and jumping are driven by motor neurons that are differentiated by fast and slow biophysical properties. By retrograde labeling of mouse and chick muscle fibers, Müller et al. (p. 1264 ) characterized the developmental distinctions between fast and slow motor neurons. A transmembrane protein, when over- or underexpressed, was discovered to drive specification of the motor neurons and a downstream effector specified some, but not all, of the biophysical attributes.