Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Neuroligin-3 in dopaminergic circuits promotes behavioural and neurobiological adaptations to chronic morphine exposure.

Chronic opioid exposure causes structural and functional changes in brain circuits, which may contribute to opioid use disorders. Synaptic cell-adhesion molecules are prime candidates for mediating this opioid-evoked plasticity. Neuroligin-3 (NL3) is an X-linked postsynaptic adhesion protein that shapes synaptic function at multiple sites in the mesolimbic dopamine system. We therefore studied how genetic knockout of NL3 alters responses to chronic morphine in male mice. Constitutive NL3 knockout caused a persistent reduction in psychomotor sensitization after chronic morphine exposure and change in the topography of locomotor stimulation produced by morphine. This latter change was recapitulated by conditional genetic deletion of NL3 from cells expressing the Drd1 dopamine receptor, whereas reduced psychomotor sensitization was recapitulated by conditional genetic deletion from dopamine neurons. Without NL3 expression, dopamine neurons in the ventral tegmental area exhibited diminished activation following chronic morphine exposure, by measuring in vivo calcium signals with fibre photometry. This altered pattern of dopamine neuron activity may be driven by aberrant forms of opioid-evoked synaptic plasticity in the absence of NL3: dopamine neurons lacking NL3 showed weaker synaptic inhibition at baseline, which was subsequently strengthened after chronic morphine. In total, our study highlights neurobiological adaptations in dopamine neurons of the ventral tegmental area that correspond with increased behavioural sensitivity to opioids and further suggests that NL3 expression by dopamine neurons provides a molecular substrate for opioid-evoked adaptations in brain function and behaviour.

Differential Patterns of Synaptic Plasticity in the Nucleus Accumbens Caused by Continuous and Interrupted Morphine Exposure.

Opioid exposure and withdrawal both cause adaptations in brain circuits that may contribute to abuse liability. These adaptations vary in magnitude and direction following different patterns of opioid exposure, but few studies have systematically manipulated the pattern of opioid administration while measuring neurobiological impact. In this study, we compared cellular and synaptic adaptations in the nucleus accumbens shell caused by morphine exposure that was either continuous or interrupted by daily bouts of naloxone-precipitated withdrawal. At the behavioral level, continuous morphine administration caused psychomotor tolerance, which was reversed when the continuity of morphine action was interrupted by naloxone-precipitated withdrawal. Using ex vivo slice electrophysiology in female and male mice, we investigated how these patterns of morphine administration altered intrinsic excitability and synaptic plasticity of medium spiny neurons (MSNs) expressing the D1 or D2 dopamine receptor. We found that morphine-evoked adaptations at excitatory synapses were predominately conserved between patterns of administration, but there were divergent effects on inhibitory synapses and the subsequent balance between excitatory and inhibitory synaptic input. Overall, our data suggest that continuous morphine administration produces adaptations that dampen the output of D1-MSNs, which are canonically thought to promote reward-related behaviors. Interruption of otherwise continuous morphine exposure does not dampen D1-MSN functional output to the same extent, which may enhance behavioral responses to subsequent opioid exposure. Our findings support the hypothesis that maintaining continuity of opioid administration could be an effective therapeutic strategy to minimize the vulnerability to opioid use disorders.SIGNIFICANCE STATEMENT Withdrawal plays a key role in the cycle of addiction to opioids like morphine. We studied how repeated cycles of naloxone-precipitated withdrawal from otherwise continuous opioid exposure can change brain function of the nucleus accumbens, which is an important brain region for reward and addiction. Different patterns of opioid exposure caused unique changes in communication between neurons in the nucleus accumbens, and the nature of these changes depended on the type of neuron being studied. The specific changes in communication between neurons caused by repeated cycles of withdrawal may increase vulnerability to opioid use disorders. This highlights the importance of reducing or preventing the experience of withdrawal during opioid treatment.

Characterization and mu opioid receptor sensitivity of neuropeptide Y interneurons in the mouse nucleus accumbens.

Inhibitory interneurons represent less than 5% of neurons within the nucleus accumbens, but are critical for proper microcircuit function within this brain region. In the dorsal striatum, neuropeptide Y is expressed by two interneuron subtypes (low-threshold spiking interneurons and neurogliaform interneurons) that exhibit mu opioid receptor sensitivity in other brain regions. However, few studies have assessed the molecular and physiological properties of neuropeptide Y interneurons within the nucleus accumbens. We used a transgenic reporter mouse to identify and characterize neuropeptide Y interneurons in acute nucleus accumbens brain slices. Nearly all cells exhibited electrophysiological properties of low-threshold spiking interneurons, with almost no neurogliaform interneurons observed among neuropeptide Y interneurons. We corroborated this pattern using fluorescent in situ hybridization, and also identified a high level of mu opioid receptor expression by low-threshold spiking interneurons, which led us to examine the functional consequences of mu opioid receptor activation in these cells using electrophysiology. Mu opioid receptor activation caused a reduction in the rate of spontaneous action potentials in low-threshold spiking interneurons, as well as a decrease in optogenetically-evoked GABA release onto medium spiny neurons. The latter effect was more robust in female versus male mice, and when the postsynaptic medium spiny neuron expressed the Drd1 dopamine receptor. This work is the first to examine the physiological properties of neuropeptide Y interneurons in the nucleus accumbens, and show they may be an important target for mu opioid receptor modulation by endogenous and exogenous opioids.

Angiotensin-converting enzyme gates brain circuit-specific plasticity via an endogenous opioid.

Angiotensin-converting enzyme (ACE) regulates blood pressure by cleaving angiotensin I to produce angiotensin II. In the brain, ACE is especially abundant in striatal tissue, but the function of ACE in striatal circuits remains poorly understood. We found that ACE degrades an unconventional enkephalin heptapeptide, Met-enkephalin-Arg-Phe, in the nucleus accumbens of mice. ACE inhibition enhanced µ-opioid receptor activation by Met-enkephalin-Arg-Phe, causing a cell type-specific long-term depression of glutamate release onto medium spiny projection neurons expressing the Drd1 dopamine receptor. Systemic ACE inhibition was not intrinsically rewarding, but it led to a decrease in conditioned place preference caused by fentanyl administration and an enhancement of reciprocal social interaction. Our results raise the enticing prospect that central ACE inhibition can boost endogenous opioid signaling for clinical benefit while mitigating the risk of addiction.

µ-Opioid Receptor (Oprm1) Copy Number Influences Nucleus Accumbens Microcircuitry and Reciprocal Social Behaviors.

The µ-opioid receptor regulates reward derived from both drug use and natural experiences, including social interaction, through actions in the nucleus accumbens. Here, we studied nucleus accumbens microcircuitry and social behavior in male and female mice with heterozygous genetic knockout of the µ-opioid receptor (Oprm1+/-). This genetic condition models the partial reduction of µ-opioid receptor signaling reported in several neuropsychiatric disorders. We first analyzed inhibitory synapses in the nucleus accumbens, using methods that differentiate between medium spiny neurons (MSNs) expressing the D1 or D2 dopamine receptor. Inhibitory synaptic transmission was increased in D2-MSNs of male mutants, but not female mutants, while the expression of gephyrin mRNA and the density of inhibitory synaptic puncta at the cell body of D2-MSNs was increased in mutants of both sexes. Some of these changes were more robust in Oprm1+/- mutants than Oprm1-/- mutants, demonstrating that partial reductions of µ-opioid signaling can have large effects. At the behavioral level, social conditioned place preference and reciprocal social interaction were diminished in Oprm1+/- and Oprm1-/- mutants of both sexes. Interaction with Oprm1 mutants also altered the social behavior of wild-type test partners. We corroborated this latter result using a social preference task, in which wild-type mice preferred interactions with another typical mouse over Oprm1 mutants. Surprisingly, Oprm1-/- mice preferred interactions with other Oprm1-/- mutants, although these interactions did not produce a conditioned place preference. Our results support a role for partial dysregulation of µ-opioid signaling in social deficits associated with neuropsychiatric conditions.SIGNIFICANCE STATEMENT Activation of the µ-opioid receptor plays a key role in the expression of normal social behaviors. In this study, we examined brain function and social behavior of female and male mice, with either partial or complete genetic deletion of µ-opioid receptor expression. We observed abnormal social behavior following both genetic manipulations, as well as changes in the structure and function of synaptic input to a specific population of neurons in the nucleus accumbens, which is an important brain region for social behavior. Synaptic changes were most robust when µ-opioid receptor expression was only partially lost, indicating that small reductions in µ-opioid receptor signaling can have a large impact on brain function and behavior.

Effect of Aggressive Experience in Female Syrian Hamsters on Glutamate Receptor Expression in the Nucleus Accumbens.

Our social relationships determine our health and well-being. In rodent models, there is now strong support for the rewarding properties of aggressive or assertive behaviors to be critical for the expression and development of adaptive social relationships, buffering from stress and protecting from the development of psychiatric disorders such as depression. However, due to the false belief that aggression is not a part of the normal repertoire of social behaviors displayed by females, almost nothing is known about the neural mechanisms mediating the rewarding properties of aggression in half the population. In the following study, using Syrian hamsters as a well-validated and translational model of female aggression, we investigated the effects of aggressive experience on the expression of markers of postsynaptic structure (PSD-95, Caskin I) and excitatory synaptic transmission (GluA1, GluA2, GluA4, NR2A, NR2B, mGluR1a, and mGluR5) in the nucleus accumbens (NAc), caudate putamen and prefrontal cortex. Aggressive experience resulted in an increase in PSD-95, GluA1 and the dimer form of mGluR5 specifically in the NAc 24 h following aggressive experience. There was also an increase in the dimer form of mGluR1a 1 week following aggressive experience. Aggressive experience also resulted in an increase in the strength of the association between these postsynaptic proteins and glutamate receptors, supporting a common mechanism of action. In addition, 1 week following aggressive experience there was a positive correlation between the monomer of mGluR5 and multiple AMPAR and NMDAR subunits. In conclusion, we provide evidence that aggressive experience in females results in an increase in the expression of postsynaptic density, AMPARs and group I metabotropic glutamate receptors, and an increase in the strength of the association between postsynaptic proteins and glutamate receptors. This suggests that aggressive experience may result in an increase in excitatory synaptic transmission in the NAc, potentially encoding the rewarding and behavioral effects of aggressive interactions.

Interruption of continuous opioid exposure exacerbates drug-evoked adaptations in the mesolimbic dopamine system.

Drug-evoked adaptations in the mesolimbic dopamine system are postulated to drive opioid abuse and addiction. These adaptations vary in magnitude and direction following different patterns of opioid exposure, but few studies have systematically manipulated the pattern of opioid administration while measuring neurobiological and behavioral impact. We exposed male and female mice to morphine for one week, with administration patterns that were either intermittent (daily injections) or continuous (osmotic minipump infusion). We then interrupted continuous morphine exposure with either naloxone-precipitated or spontaneous withdrawal. Continuous morphine exposure caused tolerance to the psychomotor-activating effects of morphine, whereas both intermittent and interrupted morphine exposure caused long-lasting psychomotor sensitization. Given links between locomotor sensitization and mesolimbic dopamine signaling, we used fiber photometry and a genetically encoded dopamine sensor to conduct longitudinal measurements of dopamine dynamics in the nucleus accumbens. Locomotor sensitization caused by interrupted morphine exposure was accompanied by enhanced dopamine signaling in the nucleus accumbens. To further assess downstream consequences on striatal gene expression, we used next-generation RNA sequencing to perform genome-wide transcriptional profiling in the nucleus accumbens and dorsal striatum. The interruption of continuous morphine exposure exacerbated drug-evoked transcriptional changes in both nucleus accumbens and dorsal striatum, dramatically increasing differential gene expression and engaging unique signaling pathways. Our study indicates that opioid-evoked adaptations in brain function and behavior are critically dependent on the pattern of drug administration, and exacerbated by interruption of continuous exposure. Maintaining continuity of chronic opioid administration may, therefore, represent a strategy to minimize iatrogenic effects on brain reward circuits.

Dopamine-Evoked Synaptic Regulation in the Nucleus Accumbens Requires Astrocyte Activity.

Dopamine is involved in physiological processes like learning and memory, motor control and reward, and pathological conditions such as Parkinson's disease and addiction. In contrast to the extensive studies on neurons, astrocyte involvement in dopaminergic signaling remains largely unknown. Using transgenic mice, optogenetics, and pharmacogenetics, we studied the role of astrocytes on the dopaminergic system. We show that in freely behaving mice, astrocytes in the nucleus accumbens (NAc), a key reward center in the brain, respond with Ca2+ elevations to synaptically released dopamine, a phenomenon enhanced by amphetamine. In brain slices, synaptically released dopamine increases astrocyte Ca2+, stimulates ATP/adenosine release, and depresses excitatory synaptic transmission through activation of presynaptic A1 receptors. Amphetamine depresses neurotransmission through stimulation of astrocytes and the consequent A1 receptor activation. Furthermore, astrocytes modulate the acute behavioral psychomotor effects of amphetamine. Therefore, astrocytes mediate the dopamine- and amphetamine-induced synaptic regulation, revealing a novel cellular pathway in the brain reward system.

Nucleus Accumbens Fast-Spiking Interneurons Constrain Impulsive Action.

BACKGROUND: The nucleus accumbens (NAc) controls multiple facets of impulsivity but is a heterogeneous brain region with diverse microcircuitry. Prior literature links impulsive behavior in rodents to gamma-aminobutyric acid signaling in the NAc. Here, we studied the regulation of impulsive behavior by fast-spiking interneurons (FSIs), a strong source of gamma-aminobutyric acid-mediated synaptic inhibition in the NAc. METHODS: Male and female transgenic mice expressing Cre recombinase in FSIs allowed us to identify these sparsely distributed cells in the NAc. We used a 5-choice serial reaction time task to measure both impulsive action and sustained attention. During the 5-choice serial reaction time task, we monitored FSI activity with fiber photometry calcium imaging and manipulated FSI activity with chemogenetic and optogenetic methodology. We used electrophysiology, optogenetics, and fluorescent in situ hybridization to confirm these methods were robust and specific to FSIs. RESULTS: In mice performing the 5-choice serial reaction time task, NAc FSIs showed sustained activity on trials ending with correct responses, but FSI activity declined over time on trials ending with premature responses. The number of premature responses increased significantly after sustained chemogenetic inhibition or temporally delimited optogenetic inhibition of NAc FSIs, without any changes in response latencies or general locomotor activity. CONCLUSIONS: These experiments provide strong evidence that NAc FSIs constrain impulsive actions, most likely through gamma-aminobutyric acid-mediated synaptic inhibition of medium spiny projection neurons. Our findings may provide insight into the pathophysiology of disorders associated with impulsivity and may inform the development of circuit-based therapeutic interventions.

Opioid-Mediated Astrocyte-Neuron Signaling in the Nucleus Accumbens.

Major hallmarks of astrocyte physiology are the elevation of intracellular calcium in response to neurotransmitters and the release of neuroactive substances (gliotransmitters) that modulate neuronal activity. While µ-opioid receptor expression has been identified in astrocytes of the nucleus accumbens, the functional consequences on astrocyte-neuron communication remains largely unknown. The present study has investigated the astrocyte responsiveness to µ-opioid signaling and the regulation of gliotransmission in the nucleus accumbens. Through the combination of calcium imaging and whole-cell patch clamp electrophysiology in brain slices, we have found that µ-opioid receptor activation in astrocytes elevates astrocyte cytoplasmic calcium and stimulates the release of the gliotransmitter glutamate, which evokes slow inward currents through the activation of neuronal N-methyl-D-aspartate (NMDA) receptors. These results indicate the existence of molecular mechanisms underlying opioid-mediated astrocyte-neuron signaling in the nucleus accumbens.

Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.

The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

Autism Spectrum Disorders and Drug Addiction: Common Pathways, Common Molecules, Distinct Disorders?

Autism spectrum disorders (ASDs) and drug addiction do not share substantial comorbidity or obvious similarities in etiology or symptomatology. It is thus surprising that a number of recent studies implicate overlapping neural circuits and molecular signaling pathways in both disorders. The purpose of this review is to highlight this emerging intersection and consider implications for understanding the pathophysiology of these seemingly distinct disorders. One area of overlap involves neural circuits and neuromodulatory systems in the striatum and basal ganglia, which play an established role in addiction and reward but are increasingly implicated in clinical and preclinical studies of ASDs. A second area of overlap relates to molecules like Fragile X mental retardation protein (FMRP) and methyl CpG-binding protein-2 (MECP2), which are best known for their contribution to the pathogenesis of syndromic ASDs, but have recently been shown to regulate behavioral and neurobiological responses to addictive drug exposure. These shared pathways and molecules point to common dimensions of behavioral dysfunction, including the repetition of behavioral patterns and aberrant reward processing. The synthesis of knowledge gained through parallel investigations of ASDs and addiction may inspire the design of new therapeutic interventions to correct common elements of striatal dysfunction.

Input- and Output-Specific Regulation of Serial Order Performance by Corticostriatal Circuits.

The serial ordering of individual movements into sequential patterns is thought to require synaptic plasticity within corticostriatal circuits that route information through the basal ganglia. We used genetically and anatomically targeted manipulations of specific circuit elements in mice to isolate the source and target of a corticostriatal synapse that regulates the performance of a serial order task. This excitatory synapse originates in secondary motor cortex, terminates on direct pathway medium spiny neurons in the dorsolateral striatum, and is strengthened by serial order learning. This experience-dependent and synapse-specific form of plasticity may sculpt the balance of activity in basal ganglia circuits during sequential movements, driving a disparity in striatal output that favors the direct pathway. This disparity is necessary for execution of responses in serial order, even though both direct and indirect pathways are active during movement initiation, suggesting dynamic modulation of corticostriatal circuitry contributes to the choreography of behavioral routines.

Single-Cell mRNA Profiling Reveals Cell-Type-Specific Expression of Neurexin Isoforms.

Neurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell-type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell-type-specific expression patterns of multiple neurexins at the single-cell level and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity.

Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors.

In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology.

Anhedonia requires MC4R-mediated synaptic adaptations in nucleus accumbens.

Chronic stress is a strong diathesis for depression in humans and is used to generate animal models of depression. It commonly leads to several major symptoms of depression, including dysregulated feeding behaviour, anhedonia and behavioural despair. Although hypotheses defining the neural pathophysiology of depression have been proposed, the critical synaptic adaptations in key brain circuits that mediate stress-induced depressive symptoms remain poorly understood. Here we show that chronic stress in mice decreases the strength of excitatory synapses on D1 dopamine receptor-expressing nucleus accumbens medium spiny neurons owing to activation of the melanocortin 4 receptor. Stress-elicited increases in behavioural measurements of anhedonia, but not increases in measurements of behavioural despair, are prevented by blocking these melanocortin 4 receptor-mediated synaptic changes in vivo. These results establish that stress-elicited anhedonia requires a neuropeptide-triggered, cell-type-specific synaptic adaptation in the nucleus accumbens and that distinct circuit adaptations mediate other major symptoms of stress-elicited depression.

Protracted manifestations of acute dependence after a single morphine exposure.

RATIONALE: Acute opiate exposure produces a state of dependence in humans and animals, which is revealed by signs and symptoms of withdrawal precipitated by opioid receptor antagonists. The physiological changes that underlie this state of acute dependence develop rapidly and can persist long after the end of chronic opiate exposure. OBJECTIVES: The purpose of this investigation was to determine the persistence of acute dependence after a single morphine exposure in rodents, focusing on changes in behavior thought to reflect the negative emotional consequences of withdrawal. METHODS: The acoustic startle reflex and conditioned place aversion were measured following naloxone administration at different time points after a single morphine exposure. RESULTS: Naloxone administration produced significant potentiation of acoustic startle-a form of anxiety-like behavior-for at least 80 days after one exposure to morphine. In contrast, naloxone produced a conditioned place aversion 24 h but not 20 days after one morphine exposure. CONCLUSIONS: Together with existing literature, these results suggest acute as well as chronic opiate exposure leave rodents persistently vulnerable to express anxiety-like behavior in response to opioid receptor antagonists or stressful experience. The adaptations in brain function that underlie this protracted state of dependence may provide a foundation for the escalation of withdrawal severity that develops over repeated opiate exposure, and increase the likelihood of progression from casual drug use to compulsive drug abuse.