Differential Roles of Oxytocin Receptors in the Prefrontal Cortex and Nucleus Accumbens on Cocaine Self-Administration and Reinstatement of Cued Cocaine Seeking in Male Rats.

BACKGROUND: Little is known about the specific roles of cortical and accumbal oxytocin receptors in drug use disorders. To better understand the importance of the endogenous oxytocin system in cocaine relapse behavior, we developed an adeno-associated viral vector-expressing short hairpin (sh) RNAs to selectively degrade the rat oxytocin receptor (OxyR) mRNA in vivo. METHODS: Male (Sprague-Dawley) rats received bilateral infusions of the shRNA for the oxytocin receptor (shOxyR) or an shRNA control virus into the prefrontal cortex (PFC) or the nucleus accumbens core (NAc). Rats self-administered cocaine on an escalating FR ratio for 14 days, lever responding was extinguished, and rats were tested for cued and cocaine-primed reinstatement of drug seeking. RESULTS: OxyR knockdown in the PFC delayed the acquisition of lever pressing on an fixed ratio 1 schedule of reinforcement. All rats eventually acquired the same level of lever pressing and discrimination, and there were no differences in extinction. OxyR knockdown in the NAc had no effect during acquisition. In both the PFC and NAc, the shOxyR decreased cued reinstatement relative to shRNA control virus but was without effect during drug-primed reinstatement. OxyR knockdown in the PFC increased chamber activity during a social interaction task. CONCLUSIONS: This study provides critical new information about how endogenous OxyRs function to affect drug seeking in response to different precipitators of relapse. The tool developed to knockdown OxyRs in rat could provide important new insights that aid development of oxytocin-based therapeutics to reduce return-to-use episodes in people with substance use disorder and other neuropsychiatric disorders.

The activity-regulated cytoskeleton-associated protein, Arc, functions in the nucleus accumbens shell to limit multiple triggers of cocaine-seeking behaviour.

Use of addictive substances like cocaine produces enduring associations between the drug experience and cues in the drug-taking environment. In individuals with a substance use disorder (SUD) and attempting to remain abstinent, these powerful drug-cue associations can trigger a return to active drug use, but the molecular mechanisms regulating drug-cue associations remain poorly understood. The activity-regulated cytoskeleton-associated protein (Arc) is induced by cocaine in the nucleus accumbens (NAc), an important brain reward region, but Arc's NAc function in SUD-related behaviour remains unclear. We show here that cocaine self-administration (SA) in rats produced a significant upregulation of Arc protein in both the core and shell subregions of the NAc. Subregion-specific Arc reduction (shRNA) in the medial NAc Shell enhanced both context-associated and cue-reinstated cocaine seeking, but without altering the motivation to work for cocaine, the sensitivity to the reinforcing effects of cocaine or the ability of cocaine priming to reinstate drug seeking. In contrast, we observed no effects of Arc knockdown in the NAc core on any aspect of cocaine SA, extinction or reinstated cocaine seeking, suggesting that Arc functions within the medial NAc shell, but not NAc core, to limit the strength of drug-context and drug-cue associations that promote cocaine-seeking behaviour.

It is a complex issue: emerging connections between epigenetic regulators in drug addiction.

Drug use leads to addiction in some individuals, but the underlying brain mechanisms that control the transition from casual drug use to an intractable substance use disorder (SUD) are not well understood. Gene x environment interactions such as the frequency of drug use and the type of substance used likely to promote maladaptive plastic changes in brain regions that are critical for controlling addiction-related behavior. Epigenetics encompasses a broad spectrum of mechanisms important for regulating gene transcription that are not dependent on changes in DNA base pair sequences. This review focuses on the proteins and complexes contributing to epigenetic modifications in the nucleus accumbens (NAc) following drug experience. We discuss in detail the three major mechanisms: histone acetylation and deacetylation, histone methylation, and DNA methylation. We discuss how drug use alters the regulation of the associated proteins regulating these processes and highlight how experimental manipulations of these proteins in the NAc can alter drug-related behaviors. Finally, we discuss the ways that histone modifications and DNA methylation coordinate actions by recruiting large epigenetic enzyme complexes to aid in transcriptional repression. Targeting these multiprotein epigenetic enzyme complexes - and the individual proteins that comprise them - might lead to effective therapeutics to reverse or treat SUDs in patients.

Novel role and regulation of HDAC4 in cocaine-related behaviors.

Epigenetic mechanisms have been proposed to contribute to persistent aspects of addiction-related behaviors. One family of epigenetic molecules that may regulate maladaptive behavioral changes produced by cocaine use are the histone deacetylases (HDACs)-key regulators of chromatin and gene expression. In particular, the class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) respond to changes in neuronal activity by modulating their distribution between the nucleus and cytoplasm-a process controlled in large part by changes in phosphorylation of conserved residues. Cocaine triggers a transient nuclear accumulation of HDAC5 that functions to limit the development of cocaine reward behavior. However, the role and regulation of the close family member, HDAC4, in cocaine behaviors remain largely unknown. In this study, we report that cocaine and cAMP signaling in striatum produced differential phosphorylation and subcellular localization of HDAC4 and HDAC5. Unlike HDAC5, cocaine exposure induced a modest hyperphosphorylation and nuclear export of HDAC4. Genetic deletion of HDAC4 in the nucleus accumbens reduced acute cocaine-produced locomotion, maximum locomotor sensitization and cocaine reward-related behavior. Interestingly, overexpression of an HDAC4 cytoplasm-concentrated mutant (S266E) increased cocaine reward behavior in the cocaine conditioned place preference assay, suggesting that cocaine-induced nuclear export of HDAC4 might function to facilitate the development of cocaine reward behaviors through a role in the cell cytoplasm. Together, our findings suggest that, despite high sequence homology, HDAC4 and HDAC5 are oppositely regulated by cocaine-induced signaling in vivo and have distinct roles in regulating cocaine behaviors.

The activity-regulated cytoskeleton-associated protein (Arc) functions in a cell type- and sex-specific manner in the adult nucleus accumbens to regulate non-contingent cocaine behaviors.

Repeated cocaine use produces adaptations in brain function that contribute to long-lasting behaviors associated with cocaine use disorder (CUD). In rodents, the activity-regulated cytoskeleton-associated protein (Arc) can regulate glutamatergic synaptic transmission, and cocaine regulates Arc expression and subcellular localization in multiple brain regions, including the nucleus accumbens (NAc)-a brain region linked to CUD-related behavior. We show here that repeated, non-contingent cocaine administration in global Arc KO male mice produced a dramatic hypersensitization of cocaine locomotor responses and drug experience-dependent sensitization of conditioned place preference (CPP). In contrast to the global Arc KO mice, viral-mediated reduction of Arc in the adult male, but not female, NAc (shArcNAc) reduced both CPP and cocaine-induced locomotor activity, but without altering basal miniature or evoked glutamatergic synaptic transmission. Interestingly, cell type-specific knockdown of Arc in D1 dopamine receptor-expressing NAc neurons reduced cocaine-induced locomotor sensitization, but not cocaine CPP; whereas, Arc knockdown in D2 dopamine receptor-expressing NAc neurons reduced cocaine CPP, but not cocaine-induced locomotion. Taken together, our findings reveal that global, developmental loss of Arc produces hypersensitized cocaine responses; however, these effects cannot be explained by Arc's function in the adult mouse NAc since Arc is required in a cell type- and sex-specific manner to support cocaine-context associations and locomotor responses.

NPAS4 supports cocaine-conditioned cues in rodents by controlling the cell type-specific activation balance in the nucleus accumbens.

Powerful associations that link drugs of abuse with cues in the drug-paired environment often serve as prepotent relapse triggers. Drug-associated contexts and cues activate ensembles of nucleus accumbens (NAc) neurons, including D1-class medium spiny neurons (MSNs) that typically promote, and D2-class MSNs that typically oppose, drug seeking. We found that in mice, cocaine conditioning upregulated transiently the activity-regulated transcription factor, Neuronal PAS Domain Protein 4 (NPAS4), in a small subset of NAc neurons. The NPAS4+ NAc ensemble was required for cocaine conditioned place preference. We also observed that NPAS4 functions within NAc D2-, but not D1-, MSNs to support cocaine-context associations and cue-induced cocaine, but not sucrose, seeking. Together, our data show that the NPAS4+ ensemble of NAc neurons is essential for cocaine-context associations in mice, and that NPAS4 itself functions in NAc D2-MSNs to support cocaine-context associations by suppressing drug-induced counteradaptations that oppose relapse-related behaviour.

Nicotine trained as a negative feature passes the retardation-of-acquisition and summation tests of a conditioned inhibitor.

Nicotine functions as a negative feature in a Pavlovian discriminated goal-tracking task. Whether withholding of responding to the conditional stimulus (CS) reflects nicotine functioning as a conditioned inhibitor is unknown. Accordingly, the present research sought to determine whether nicotine trained as a negative feature passed the retardation-of-acquisition and summation tests, thus characterizing it as a pharmacological (interoceptive) conditioned inhibitor. In the retardation test, rats received either nicotine (0.4 mg/kg) or chlordiazepoxide (5 mg/kg) negative feature training in which the drug state signaled when a 15-sec light CS would not be paired with sucrose; light was paired with sucrose on intermixed saline sessions. Following acquisition of the discrimination, both groups received nicotine CS training in which sucrose was intermittently available on nicotine but not intermixed saline sessions. Acquisition of conditioned responding to the nicotine CS was slower in the nicotine negative feature group than in the chlordiazepoxide negative feature group. In the summation test, rats were assigned to either the nicotine negative feature group or a pseudoconditioning control. In this control, the light CS was paired with sucrose on half the nicotine and half the saline sessions. Both groups also received excitatory training in which a white noise CS was paired with sucrose. The summation test consisted of presenting the white noise in conjunction with nicotine. Conditioned responding evoked by the white noise was decreased in the negative feature but not the pseudoconditioning group. Combined, the results provide the first evidence that an interoceptive stimulus (nicotine) can become a conditioned inhibitor.

The activity-regulated cytoskeleton-associated protein, Arc/Arg3.1, influences mouse cocaine self-administration.

The activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1), an immediate early gene and synaptic regulator, is upregulated following a single cocaine exposure. However, there is not much known regarding Arc/Arg3.1's potential contribution to addiction-relevant behaviors. Despite known learning and memory deficits in contextual fear and water-maze reversal learning tasks, we find that mice lacking Arc/Arg3.1 perform conditioned place preference and operant conditioning involving positive reinforcers (food and cocaine) with little-to-no impairment. However, following normal saline-extinction, wild type (WT) mice show a classic inverted-U dose-response function, while Arc/Arg3.1 knockout (KO) mice fail to adjust their intake across multiple doses. Importantly, Arc/Arg3.1 KO and WT mice behave comparably on an increasing cost task (FR1-FR3; acquisition dose), providing evidence that both groups find cocaine reinforcing. Differences in individuals that drive variations in use patterns and particularly, drug intake levels, are critical as they influence the likelihood of developing dependence. Our data suggest that Arc/Arg3.1 may contribute to addiction as a regulator of drug-taking vulnerability under different drug availability conditions.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) regulates anxiety- and novelty-related behaviors.

The activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1) regulates glutamatergic synapse plasticity and has been linked to neuropsychiatric illness; however, its role in behaviors associated with mood and anxiety disorders remains unclear. We find that stress upregulates Arc expression in the adult mouse nucleus accumbens (NAc)-a brain region implicated in mood and anxiety behaviors. Global Arc knockout mice have altered AMPAR-subunit surface levels in the adult NAc, and the Arc-deficient mice show reductions in anxiety-like behavior, deficits in social novelty preference, and antidepressive-like behavior. Viral-mediated expression of Arc in the adult NAc of male, global Arc KO mice restores normal levels of anxiety-like behavior in the elevated plus maze (EPM). Consistent with this finding, viral-mediated reduction of Arc in the adult NAc reduces anxiety-like behavior in male, but not female, mice in the EPM. NAc-specific reduction of Arc also produced significant deficits in both object and social novelty preference tasks. Together our findings indicate that Arc is essential for regulating normal mood- and anxiety-related behaviors and novelty discrimination, and that Arc's function within the adult NAc contributes to these behavioral effects.

Nicotine does not produce state-dependent effects on learning in a Pavlovian appetitive goal tracking task with rats.

Past research has shown that when rats received 0.4mg base/kg nicotine paired reliably with intermittent sucrose delivery that anticipatory sucrose-seeking behavior (i.e., goal tracking) was differentially displayed in the nicotine state relative to intermixed saline sessions in which no sucrose was delivered. The present research extended this observation to a lower dose of nicotine (i.e., 0.2mg base/kg) and tested a state-dependent learning account of differential conditioned responding. According to this account, the increase in goal tracking on nicotine sessions reflects a chamber-sucrose association that is only recalled when in the nicotine state. We used a 2x2 factorial design in which rats received sucrose deliveries in one drug state (nicotine or saline) and were then tested in the same state (Nic-->Nic or Sal-->Sal) or a different state (Nic-->Sal or Sal-->Nic) after acquiring the conditioned response. A state-dependency account predicts disruption in conditioned goal tracking for rats that receive a shift in drug state on the test day. This disruption did not occur suggesting that differential control of conditioned responding by nicotine is more likely due to a direct excitatory association between the interoceptive cueing effects of nicotine and the appetitive qualities of sucrose.

HDAC5 and Its Target Gene, Npas4, Function in the Nucleus Accumbens to Regulate Cocaine-Conditioned Behaviors.

Individuals suffering from substance-use disorders develop strong associations between the drug's rewarding effects and environmental cues, creating powerful, enduring triggers for relapse. We found that dephosphorylated, nuclear histone deacetylase 5 (HDAC5) in the nucleus accumbens (NAc) reduced cocaine reward-context associations and relapse-like behaviors in a cocaine self-administration model. We also discovered that HDAC5 associates with an activity-sensitive enhancer of the Npas4 gene and negatively regulates NPAS4 expression. Exposure to cocaine and the test chamber induced rapid and transient NPAS4 expression in a small subpopulation of FOS-positive neurons in the NAc. Conditional deletion of Npas4 in the NAc significantly reduced cocaine conditioned place preference and delayed learning of the drug-reinforced action during cocaine self-administration, without affecting cue-induced reinstatement of drug seeking. These data suggest that HDAC5 and NPAS4 in the NAc are critically involved in reward-relevant learning and memory processes and that nuclear HDAC5 limits reinstatement of drug seeking independent of NPAS4.

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence(2). In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

The presence of cortical neurons in striatal-cortical co-cultures alters the effects of dopamine and BDNF on medium spiny neuron dendritic development.

Medium spiny neurons (MSNs) are the major striatal neuron and receive synaptic input from both glutamatergic and dopaminergic afferents. These synapses are made on MSN dendritic spines, which undergo density and morphology changes in association with numerous disease and experience-dependent states. Despite wide interest in the structure and function of mature MSNs, relatively little is known about MSN development. Furthermore, most in vitro studies of MSN development have been done in simple striatal cultures that lack any type of non-autologous synaptic input, leaving open the question of how MSN development is affected by a complex environment that includes other types of neurons, glia, and accompanying secreted and cell-associated cues. Here we characterize the development of MSNs in striatal-cortical co-culture, including quantitative morphological analysis of dendritic arborization and spine development, describing progressive changes in density and morphology of developing spines. Overall, MSN growth is much more robust in the striatal-cortical co-culture compared to striatal mono-culture. Inclusion of dopamine (DA) in the co-culture further enhances MSN dendritic arborization and spine density, but the effects of DA on dendritic branching are only significant at later times in development. In contrast, exogenous Brain Derived Neurotrophic Factor (BDNF) has only a minimal effect on MSN development in the co-culture, but significantly enhances MSN dendritic arborization in striatal mono-culture. Importantly, inhibition of NMDA receptors in the co-culture significantly enhances the effect of exogenous BDNF, suggesting that the efficacy of BDNF depends on the cellular environment. Combined, these studies identify specific periods of MSN development that may be particularly sensitive to perturbation by external factors and demonstrate the importance of studying MSN development in a complex signaling environment.

Use of Adeno-Associated and Herpes Simplex Viral Vectors for In Vivo Neuronal Expression in Mice.

Adeno-associated viruses and the herpes simplex virus are the two most widely used vectors for the in vivo expression of exogenous genes. Advances in the development of these vectors have enabled remarkable temporal and spatial control of gene expression. This unit provides methods for storing, delivering, and verifying expression of adeno-associated and herpes simplex viruses in the adult mouse brain. It also describes important considerations for experiments using in vivo expression of these viral vectors, including serotype and promoter selection, as well as timing of expression. Additional protocols are provided that describe methods for preliminary experiments to determine the appropriate conditions for in vivo delivery.

Social cognitive bias and neurocognitive deficit in paranoid symptoms: evidence for an interaction effect and changes during treatment.

Persistent paranoid symptoms are best understood as having multiple causal mechanisms. An enhanced multidimensional understanding of paranoia may result from the convergence of two distinct measurement paradigms, experimental psychopathology and social cognitive research. This study investigated the role of neurocognitive deficits and emotion misperception bias as they relate to paranoid symptoms at two different time points in a sample of individuals with severe mental illness (primarily schizophrenia spectrum disorders [N=91]) undergoing intensive psychosocial rehabilitation. Before intensive rehabilitation (but after initial stabilization), paranoid symptoms were related to a tendency to misperceive emotion as disgust. The impact of this social cognitive bias was amplified by perseveration (as measured by the COGLAB Card Sorting Task). Perseverative errors were associated with paranoid symptoms at both time points. After 6 months of treatment, there were significant reductions in paranoid symptoms and perseverative errors but no significant changes in emotion misperception biases. This study is one of few to date to evaluate the contribution of both neurocognitive deficits and social cognitive biases to paranoid symptoms. The results demonstrate how social cognitive biases can interact with neurocognitive deficits in expression of paranoid symptoms, and how these relationships change during treatment.

An embryonic culture system for the investigation of striatal medium spiny neuron dendritic spine development and plasticity.

Dendritic spines of striatal Medium Spiny Neurons (MSNs) receive converging dopaminergic and glutamatergic inputs. These spines undergo experience-dependent structural plasticity following repeated drug administration and during disease states like Huntington's and Parkinson's. Thus, understanding the molecular mechanisms leading to structural plasticity is an important step toward establishing a clear relationship between spine structure and function, and will ultimately contribute to understanding how changes in dendritic spine structure relate to behaviors or diseases. One major difficulty faced when studying MSN development is the lack of a detailed, standardized in vitro model system that produces MSNs with in vivo-like morphologies. For example, unlike cultured pyramidal neurons, MSNs grown in mono-cultures display stunted dendritic arborization and fail to develop a full cohort of mature dendritic spines. Here we report the generation of an embryonic mouse cortical-striatal co-culture that generates high cell yields from a single embryo. Unlike MSNs in striatal mono-culture, MSNs in co-culture develop in vivo-like morphologies and high densities of dendritic spines. Morphological identification of co-cultured MSNs expressing a soluble fluorescent protein can be confirmed by immunochemical detection of DARPP-32 (Dopamine and cyclic AMP regulated phosphoprotein of 32kDa). Additionally, co-cultured MSN spines contain PSD-95 puncta and are apposed to SV2 puncta, indicating the spines express synaptic machinery. Finally, whole-cell recordings of co-cultured MSNs exhibit higher mEPSC frequency compared to mono-cultured MSNs, suggesting that the spines are functionally mature. These studies establish that this co-culture system is suitable for studying the morphological and physiological development and function of MSN dendritic spines.

Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration.

The microtubule-associated protein tau accumulates in Alzheimer's and other fatal dementias, which manifest when forebrain neurons die. Recent advances in understanding these disorders indicate that brain dysfunction precedes neurodegeneration, but the role of tau is unclear. Here, we show that early tau-related deficits develop not from the loss of synapses or neurons, but rather as a result of synaptic abnormalities caused by the accumulation of hyperphosphorylated tau within intact dendritic spines, where it disrupts synaptic function by impairing glutamate receptor trafficking or synaptic anchoring. Mutagenesis of 14 disease-associated serine and threonine amino acid residues to create pseudohyperphosphorylated tau caused tau mislocalization while creation of phosphorylation-deficient tau blocked the mistargeting of tau to dendritic spines. Thus, tau phosphorylation plays a critical role in mediating tau mislocalization and subsequent synaptic impairment. These data establish that the locus of early synaptic malfunction caused by tau resides in dendritic spines.

Nicotine-evoked conditioned responding is dependent on concentration of sucrose unconditioned stimulus.

Previous studies have shown that the interoceptive nicotine conditional stimulus (CS) functions similarly to exteroceptive CSs such as lights or environments. For instance, the appetitive conditioned response (CR) evoked when nicotine is repeatedly paired with sucrose presentations (the unconditioned stimulus; US) is sensitive to changes in training dose (CS salience) and the contiguity between the CS effects and sucrose. The current study was conducted to extend this research by examining the possible role of US intensity in CR acquisition and maintenance. Rats were trained using one of four sucrose concentrations: 0, 4, 16, or 32% (w/v). On nicotine sessions (0.4 mg base/kg), rats received 36 deliveries (4s each) of their assigned concentration intermittently throughout the session; sucrose was withheld on saline sessions. In all groups, an appetitive goal-tracking CR was acquired at a similar rate. However, the asymptotic CR level varied with sucrose concentration. The magnitude of the CR was increased in rats trained with higher sucrose US concentrations. These findings are consistent with previous Pavlovian conditioning research, and extend the conditions under which the nicotine state functions as an interoceptive conditional stimulus.

Interoceptive Pavlovian conditioning with nicotine as the conditional stimulus varies as a function of the number of conditioning trials and unpaired sucrose deliveries.

In rats, the pharmacological (interoceptive) effects of nicotine can serve as a signal (conditional stimulus) in a Pavlovian (classical) conditioning task. In this task, nicotine administration (0.4 mg base/kg, subcutaneous) is typically paired with intermittent access to a liquid sucrose unconditional stimulus; sucrose is withheld on saline sessions. An increase in sucrose receptacle entries (goal tracking) on nicotine sessions indicates conditioning. Given our limited understanding of the functional relationships controlling conditioned responding to a nicotine conditional stimulus, the present research examined nicotine's sensitivity to several manipulations shown to affect the conditioned responding in more widely studied Pavlovian conditioning tasks that use exteroceptive conditional stimuli: number of nicotine conditional stimulus-sucrose unconditional stimulus pairings per session (0, 3, 9, 18, or 36) and the impact of sucrose deliveries in saline sessions. Differential goal tracking developed in fewer sessions and asymptotic conditioned responding magnitude was greater with more nicotine-sucrose pairings. Further, goal tracking was more resistant to extinction (unconditional stimulus withheld) with more conditional-unconditional stimulus pairings during the acquisition phase. The discrimination was not acquired when sucrose presentations (9 or 18) also occurred during saline sessions. Furthermore, expression of the discrimination was disrupted when sucrose was presented in saline sessions; this disruption resulted from goal tracking in saline sessions. These results are consistent with the notion that nicotine-evoked goal tracking results from interoceptive conditioning processes.