John W. Muschamp

Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area

Significance Hypocretin (orexin) and dynorphin are neuromodulators that play an important role in regulating affect and motivation. Orexin is critical for reward and is implicated in drug seeking, whereas dynorphin mediates negative mood and is implicated in depressive-like states. Considering these opposing effects, reports that both peptides are expressed in the same neurons and are coreleased are counterintuitive. Here, we demonstrate that orexin and dynorphin are coexpressed within the same synaptic vesicles and that this colocalization has a profound influence on reward, drug taking, and impulsive-like behavior. The fact that orexin occludes the depressive-like antireward effects of dynorphin significantly changes how we view the functional role of orexin in the brain. Hypocretin (orexin) and dynorphin are neuropeptides with opposing actions on motivated behavior. Orexin is implicated in states of arousal and reward, whereas dynorphin is implicated in depressive-like states. We show that, despite their opposing actions, these peptides are packaged in the same synaptic vesicles within the hypothalamus. Disruption of orexin function blunts the rewarding effects of lateral hypothalamic (LH) stimulation, eliminates cocaine-induced impulsivity, and reduces cocaine self-administration. Concomitant disruption of dynorphin function reverses these behavioral changes. We also show that orexin and dynorphin have opposing actions on excitability of ventral tegmental area (VTA) dopamine neurons, a prominent target of orexin-containing neurons, and that intra-VTA orexin antagonism causes decreases in cocaine self-administration and LH self-stimulation that are reversed by dynorphin antagonism. Our findings identify a unique cellular process by which orexin can occlude the reward threshold-elevating effects of coreleased dynorphin and thereby act in a permissive fashion to facilitate reward.

Kappa Opioid Receptor Signaling in the Basolateral Amygdala Regulates Conditioned Fear and Anxiety in Rats

BACKGROUND The kappa opioid receptor (KOR) system contributes to the prodepressive and aversive consequences of stress and is implicated in the facilitation of conditioned fear and anxiety in rodents. Here, we sought to identify neural circuits that mediate KOR system effects on fear and anxiety in rats. METHODS We assessed whether fear conditioning induces plasticity in KOR or dynorphin (the endogenous KOR ligand) messenger RNA (mRNA) expression in the basolateral (BLA) and central (CeA) nuclei of the amygdala, hippocampus, or striatum. We then assessed whether microinfusions of the KOR antagonist JDTic (0-10 μg/side) into the BLA or CeA affect the expression of conditioned fear or anxiety. Finally, we examined whether fear extinction induces plasticity in KOR mRNA expression that relates to the quality of fear extinction. RESULTS Fear conditioning upregulated KOR mRNA in the BLA by 65% and downregulated it in the striatum by 22%, without affecting KOR levels in the CeA or hippocampus, or dynorphin levels in any region. KOR antagonism in either the BLA or CeA decreased conditioned fear in the fear-potentiated startle paradigm, whereas KOR antagonism in the BLA, but not the CeA, produced anxiolytic-like effects in the elevated plus maze. Effective fear extinction was associated with a 67% reduction in KOR mRNA in the BLA. CONCLUSIONS These findings suggest that fear conditioning and extinction dynamically regulate KOR expression in the BLA and provide evidence that the BLA and CeA are important neural substrates mediating the anxiolytic-like effects of KOR antagonists in models of fear and anxiety.

Effects of striatal ΔFosB overexpression and ketamine on social defeat stress-induced anhedonia in mice

BACKGROUND Chronic social defeat stress (CSDS) produces persistent behavioral adaptations in mice. In many behavioral assays, it can be difficult to determine if these adaptations reflect core signs of depression. We designed studies to characterize the effects of CSDS on sensitivity to reward because anhedonia (reduced sensitivity to reward) is a defining characteristic of depressive disorders in humans. We also examined the effects of striatal ΔFosB overexpression and the N-methyl-D-aspartate receptor antagonist ketamine, both of which promote resilience, on CSDS-induced alterations in reward function and social interaction. METHODS Intracranial self-stimulation (ICSS) was used to quantify CSDS-induced changes in reward function. Mice were implanted with lateral hypothalamic electrodes, and ICSS thresholds were measured after each of 10 daily CSDS sessions and during a 5-day recovery period. We also examined if acute intraperitoneal administration of ketamine (2.5-20 mg/kg) reverses CSDS-induced effects on reward or, in separate mice, social interaction. RESULTS ICSS thresholds were increased by CSDS, indicating decreases in the rewarding impact of lateral hypothalamic stimulation (anhedonia). This effect was attenuated in mice overexpressing ∆FosB in striatum, consistent with pro-resilient actions of this transcription factor. High, but not low, doses of ketamine administered after completion of the CSDS regimen attenuated social avoidance in defeated mice, although this effect was transient. Ketamine did not block CSDS-induced anhedonia in the ICSS test. CONCLUSIONS This study found that CSDS triggers persistent anhedonia and confirms that ΔFosB overexpression produces stress resilience. The findings of this study also indicate that acute administration of ketamine fails to attenuate CSDS-induced anhedonia despite reducing other depression-related behavioral abnormalities.

Activation of CREB in the Nucleus Accumbens Shell Produces Anhedonia and Resistance to Extinction of Fear in Rats

Stress triggers psychiatric conditions including depressive and anxiety disorders. The mechanisms by which stress produces persistent changes in behavior are not fully understood. Here we show in rats that stress (footshock) activates the transcription factor cAMP response element binding protein (CREB) within the nucleus accumbens shell (NAS), a brain area involved in encoding reward and aversion. To examine the behavioral significance of altered CREB function in the NAS, we used viral vectors to elevate or disrupt CREB in this region. Elevated CREB produced increases in intracranial self-stimulation thresholds, a depressive-like sign reflecting anhedonia (decreased sensitivity to reward), whereas disruption of CREB function by expression of a dominant-negative CREB had the opposite effect. To determine whether neuroadaptations that produce anhedonia subsequently affect vulnerability to stress-induced behavioral adaptations, we subjected rats with altered CREB function in the NAS to fear conditioning. Although neither elevation nor disruption of CREB function altered the development of conditioned fear, elevation of CREB impaired extinction of conditioned fear. To mimic downstream effects of CREB activation on expression of the opioid peptide dynorphin, we microinjected the κ-opioid receptor (KOR) agonist U50,488 directly into the NAS. KOR stimulation produced anhedonia but had no effect on expression or extinction of conditioned fear. These findings demonstrate that activation of CREB in the NAS produces multiple behavioral signs (anhedonia, impaired extinction) characteristic of experience-dependent psychiatric conditions such as posttraumatic stress disorder. Although CREB activation is a common trigger, expression of these individual signs appears to involve divergent downstream mechanisms.

Sex Differences in Sensitivity to the Depressive-like Effects of the Kappa Opioid Receptor Agonist U-50488 in Rats

BACKGROUND Dynorphin, an endogenous ligand at kappa opioid receptors (KORs), produces depressive-like effects and contributes to addictive behavior in male nonhuman primates and rodents. Although comorbidity of depression and addiction is greater in women than men, the role of KORs in female motivated behavior is unknown. METHODS In adult Sprague-Dawley rats, we used intracranial self-stimulation to measure effects of the KOR agonist (±)-trans-U-50488 methanesulfonate salt (U-50488) (.0-10.0 mg/kg) on brain stimulation reward in gonadally intact and castrated males and in females at estrous cycle stages associated with low and high estrogen levels. Pharmacokinetic studies of U-50488 in plasma and brain were conducted. Immunohistochemistry was used to identify sex-dependent expression of U-50488-induced c-Fos in brain. RESULTS U-50488 dose-dependently increased the frequency of stimulation (threshold) required to maintain intracranial self-stimulation responding in male and female rats, a depressive-like effect. However, females were significantly less sensitive than males to the threshold-increasing effects of U-50488, independent of estrous cycle stage in females or gonadectomy in males. Although initial plasma concentrations of U-50488 were higher in females, there were no sex differences in brain concentrations. Sex differences in U-50488-induced c-Fos activation were observed in corticotropin releasing factor-containing neurons of the paraventricular nucleus of the hypothalamus and primarily in non-corticotropin releasing factor-containing neurons of the bed nucleus of the stria terminalis. CONCLUSIONS These data suggest that the role of KORs in motivated behavior of rats is sex-dependent, which has important ramifications for the study and treatment of mood-related disorders, including depression and drug addiction in people.

Differential Roles of GABA A Receptor Subtypes in Benzodiazepine-Induced Enhancement of Brain-Stimulation Reward

Benzodiazepines such as diazepam are widely prescribed as anxiolytics and sleep aids. Continued use of benzodiazepines, however, can lead to addiction in vulnerable individuals. Here, we investigate the neural mechanisms of the behavioral effects of benzodiazepines using the intracranial self-stimulation (ICSS) test, a procedure with which the reward-enhancing effects of these drugs can be measured. Benzodiazepines bind nonselectively to several different GABAA receptor subtypes. To elucidate the α subunit(s) responsible for the reward-enhancing effects of benzodiazepines, we examined mice carrying a histidine-to-arginine point mutation in the α1, α2, or α3 subunit, which renders the targeted subunit nonresponsive to diazepam, other benzodiazepines and zolpidem. In wild-type and α1-point-mutated mice, diazepam caused a dose-dependent reduction in ICSS thresholds (reflecting a reward-enhancing effect) that is comparable to the reduction observed following cocaine administration. This effect was abolished in α2- and α3-point-mutant mice, suggesting that these subunits are necessary for the reward-enhancing action of diazepam. α2 Subunits appear to be particularly important, since diazepam increased ICSS thresholds (reflecting an aversive-like effect) in α2-point-mutant animals. Zolpidem, an α1-preferring benzodiazepine-site agonist, had no reward-enhancing effects in any genotype. Our findings implicate α2 and α3 subunit containing GABAA receptors as key mediators of the reward-related effects of benzodiazepines. This finding has important implications for the development of new medications that retain the therapeutic effects of benzodiazepines but lack abuse liability.

Roles of Nucleus Accumbens CREB and Dynorphin in Dysregulation of Motivation

Psychostimulants such as amphetamine and cocaine are believed to produce dependence by causing rapid, supraphysiological elevations in synaptic dopamine (DA) within the nucleus accumbens (NAc) (Volkow et al. 2009, Neuropharmacology 56: 3-8). These changes in forebrain DA transmission are similar to those evoked by natural reinforcers (Louilot et al. 1991, Brain Res 553: 313-317; Roitman et al. 2004, J Neurosci 24: 1265-1271), but are of greater magnitude and longer duration. Repeated drug exposure causes compensatory neuroadaptations in neurons of the NAc, some of which may modulate excess DA in a homeostatic fashion. One such adaptation is the activation of the transcription factor CREB (cAMP response element-binding protein) within neurons of the NAc. Although elevated levels of transcriptionally active CREB appear to attenuate DA transmission by increasing expression of the endogenous κ opioid receptor (KOR) ligand dynorphin, increased dynorphin transmission may ultimately have undesirable effects that contribute to drug withdrawal states as well as comorbid psychiatric illnesses such as depression. This state may prompt a return to drug use to mitigate the adverse effects of withdrawal. This article summarizes our current understanding of how CREB and dynorphin contribute to the dysregulation of motivation and describes novel therapeutic strategies that derive from preclinical research in this area.

Suvorexant, an orexin/hypocretin receptor antagonist, attenuates motivational and hedonic properties of cocaine

Orexins (‘hypocretins’) are peptides produced by neurons of the hypothalamus that project to structures implicated in reward and emotion processing. Converging evidence demonstrates functional roles of orexin signaling in arousal, sleep/wakefulness and motivated behaviors for natural and drug rewards. Suvorexant, a dual orexin receptor antagonist, recently received approval from the US Food and Drug Administration to treat insomnia. In Experiment 1, rats self‐administered cocaine under a progressive‐ratio schedule of reinforcement and the effects of suvorexant on motivation to self‐administer cocaine were measured. In Experiment 2, the effects of suvorexant on cocaine reward were assessed by using a place conditioning paradigm, and 50‐kHz ultrasonic vocalizations were also recorded to track changes in hedonic reactivity to cocaine. To rule out potentially confounding effects of suvorexant‐induced somnolence, locomotor activity was also measured. In Experiment 3, the effects of suvorexant on cocaine‐evoked elevations in ventral striatal dopamine were examined. Data reveal that suvorexant (i) reduced the number of cocaine infusions earned during progressive‐ratio self‐administration; (ii) attenuated initial positive hedonic reactivity to cocaine and prevented cocaine place preference; (iii) did not affect cocaine‐induced hyperlocomotion and (iv) reduced cocaine‐induced elevations in extracellular ventral striatal dopamine. The present study examined the therapeutic potential of suvorexant in rodent models of cocaine use disorder. These results contribute toward a growing literature supporting therapeutic roles of orexin receptor antagonists in treating substance use disorders.

Effects of Suvorexant, a Dual Orexin/Hypocretin Receptor Antagonist, on Impulsive Behavior Associated with Cocaine

Hypothalamic hypocretin (orexin) peptides mediate arousal, attention, and reward processing. Fibers containing orexins project to brain structures that govern motivated behavior, including the ventral tegmental area (VTA). A number of psychiatric conditions, including attention deficit hyperactivity disorder (ADHD) and substance use disorders, are characterized by deficits in impulse control, however the relationship between orexin and impulsive behavior is incompletely characterized. The effects of systemic or centrally administered orexin receptor (OXR) antagonists on measures of impulsive-like behavior in rats were evaluated using the five-choice serial reaction time task (5-CSRTT) and delay discounting procedures. These paradigms were also used to test the capacity of OXR antagonists to attenuate acute cocaine-evoked impulsivity. Finally, immunohistochemistry and calcium imaging were used to assess potential cellular mechanisms by which OXR blockade may influence motor impulsivity. Suvorexant, a dual (OX1/2R) orexin receptor antagonist, reduced cocaine-evoked premature responses in 5-CSRTT when administered systemically or directly into VTA. Neither suvorexant nor OX1R- or OX2R-selective compounds (SB334867 or TCS-OX2-29, respectively) altered delay discounting. Finally, suvorexant did not alter Fos-immunoreactivity within tyrosine hydroxylase-immunolabeled neurons of VTA, but did attenuate cocaine- and orexin-induced increases in calcium transient amplitude within neurons of VTA. Results from the present studies suggest potential therapeutic utility of OXR antagonists in reducing psychostimulant-induced motor impulsivity. These findings also support the view that orexin transmission is closely involved in executive function in normal and pathological conditions.

Comparing rewarding and reinforcing properties between ‘bath salt’ 3,4-methylenedioxypyrovalerone (MDPV) and cocaine using ultrasonic vocalizations in rats

Abuse of synthetic psychostimulants like synthetic cathinones has risen in recent years. 3,4‐Methylenedioxypyrovalerone (MDPV) is one such synthetic cathinone that demonstrates a mechanism of action similar to cocaine. Compared to cocaine, MDPV is more potent at blocking dopamine and norepinephrine reuptake and is readily self‐administered by rodents. The present study compared the rewarding and reinforcing properties of MDPV and cocaine using systemic injection dose‐response and self‐administration models. Fifty kilohertz ultrasonic vocalizations (USVs) were recorded as an index of positive affect throughout experiments. In Experiment 1, MDPV and cocaine dose‐dependently elicited 50‐kHz USVs upon systemic injection, but MDPV increased USVs at greater rates and with greater persistence relative to cocaine. In Experiment 2, latency to begin MDPV self‐administration was shorter than latency to begin cocaine self‐administration, and self‐administered MDPV elicited greater and more persistent rates of 50‐kHz USVs versus cocaine. MDPV‐elicited 50‐kHz USVs were sustained over the course of drug load‐up whereas cocaine‐elicited USVs waned following initial infusions. Notably, we observed a robust presence of context‐elicited 50‐kHz USVs from both MDPV and cocaine self‐administering rats. Collectively, these data suggest that MDPV has powerfully rewarding and reinforcing effects relative to cocaine at one‐tenth doses. Consistent with prior work, we additionally interpret these data in supporting that MDPV has significant abuse risk based on its potency and subjectively positive effects. Future studies will be needed to better refine therapeutic strategies targeted at reducing the rewarding effects of cathinone analogs in efforts to ultimately reduce abuse liability.