Orexin-1 receptor signalling in the prelimbic cortex and ventral tegmental area regulates cue-induced reinstatement of ethanol-seeking in iP rats.

Orexins (hypocretins) are hypothalamic neuropeptides that innervate the entire neuraxis, including the prelimbic cortex and ventral tegmental area and have been implicated in ethanol-seeking behaviour. The present study aimed to use the orexin-1 (OX1 ) receptor antagonist SB-334867 to examine the role of prelimbic cortex and ventral tegmental area OX1 receptors in cue-induced reinstatement of ethanol-seeking. Ethanol-preferring rats (iP) rats were trained to self-administer ethanol (10 percent v/v, FR3) or sucrose (0.2-1 percent w/v, FR3) in the presence of reward-associated cues before being implanted with indwelling guide cannulae. Rats then underwent extinction training for 11 days. On test days, rats were given a microinjection of vehicle or SB-334867 (3 µg/side) and presented with reward-associated cues to precipitate reinstatement. Results show SB-334867 infused into the prelimbic cortex attenuated cue-induced reinstatement of ethanol-seeking, but not sucrose-seeking. OX1 antagonism in the ventral tegmental area also attenuated cue-induced reinstatement of ethanol-seeking. These findings suggest that OX1 receptors located in the prelimbic cortex and ventral tegmental area are part of a circuit driving cue-mediated ethanol-seeking behaviour.

Central orexin (hypocretin) 2 receptor antagonism reduces ethanol self-administration, but not cue-conditioned ethanol-seeking, in ethanol-preferring rats.

Orexins are hypothalamic neuropeptides which bind to two G-protein-coupled receptors, orexin-1 (OX(1)R) and orexin-2 (OX(2)R) receptor. While a role for OX(1)R has been established in both ethanol reinforcement and ethanol-seeking behaviour, the role of OX(2)R in these behaviours is relatively less-studied. The aim of this study was to determine the role of central OX(2)R in ethanol-taking and ethanol-seeking behaviour. Indiana ethanol-preferring rats were trained to self-administer ethanol (10% w/v) or sucrose (0.7­1% w/v) in the presence of reward-associated cues before being implanted with indwelling guide cannulae. The selective OX(2)R antagonist TCS-OX2-29 was administered i.c.v. to assess its effect on operant self-administration and cue-induced reinstatement following extinction. Following i.c.v. injection TCS-OX2-29 reduced self-administration of ethanol, but not sucrose. Despite reducing ethanol self-administration, TCS-OX2-29 had no impact on cue-induced reinstatement of ethanol seeking. To determine where in the brain OX(2)R were acting to modulate ethanol self-administration, TCS-OX2-29 was microinjected into either the shell or core of the nucleus accumbens (NAc). Intra-NAc core, but not shell, infusions of TCS-OX2-29 decreased responding for ethanol. Importantly, the doses of TCS-OX2-029 used were non-sedating. Collectively, these findings implicate OX(2)R in the NAc in mediating the reinforcing effects of ethanol. This effect appears to be drug-specific as antagonism of central OX(2)R had no impact on sucrose self-administration. Thus, OX(2)R in addition to OX(1)R may represent a potential therapeutic target for the treatment of ethanol-use disorders. However, unlike OX(1)R, no impact of OX(2)R antagonism was observed on cue-induced reinstatement, suggesting a more prominent role for OX(2)R in ethanol self-administration compared to cue-conditioned ethanol-seeking.

Metabotropic glutamate group II receptor activation in the ventrolateral dorsal striatum suppresses incentive motivation for cocaine in rats.

RATIONALE: After a history of intermittent cocaine intake, rats develop patterns of drug use characteristic of substance use disorder. The dorsal striatum is involved in the increased pursuit of cocaine after intermittent drug self-administration experience. Within the dorsal striatum, chronic cocaine use changes metabotropic glutamate type II receptor (mGlu2/3) density and function. OBJECTIVES: We examined the extent to which activity at Glu2/3 receptors mediates responding for cocaine after intermittent cocaine use. METHODS: Male (n = 11) and female (n = 10) Wistar rats self-administered 0.25 mg/kg/infusion cocaine during 10 daily intermittent access (IntA) sessions (5 min ON/25 min OFF, for 5 h/session). We then examined the effects of microinjections of the mGlu2/3 receptor agonist LY379268 (0, 1, and 3 µg/hemisphere) into the ventrolateral part of the dorsal striatum on cocaine self-administration under a progressive ratio schedule of reinforcement. RESULTS: Across 10 IntA sessions, the sexes showed similar levels of cocaine intake. In females only, locomotion significantly increased over sessions, suggesting that female rats developed psychomotor sensitization to self-administered cocaine. After 10 IntA sessions, intra-dorsal striatum LY379268 significantly reduced breakpoints achieved for cocaine, active lever presses, and cocaine infusions earned under progressive ratio. LY379268 had no effects on locomotion or inactive lever presses, indicating no motor effects. CONCLUSIONS: These results suggest that mGlu2/3 receptor activation in the ventrolateral dorsal striatum suppresses incentive motivation for cocaine, and this holds promise for new treatments to manage substance use disorder.

Effects of ketamine optical isomers, fluoxetine and naloxone on timing in differential reinforcement of low-rate response (DRL) 72-s task in rats.

(S)-ketamine-induced rapid-acting antidepressant effects have revolutionized the pharmacotherapy of major depression; however, this medication also produces psychotomimetic effects such as timing distortion. While (R)-ketamine produces fewer dissociative effects, its antidepressant actions are less studied. Depression is associated with time overestimation (i.e., subjectively, time passes slowly). Our recent report suggests that while (S)-ketamine induces an opposite effect, i.e., time underestimation, the (R)-isomer does not affect timing. It has been suggested that opioid receptors are involved in the antidepressant effect of ketamine. In the present study we tested (R)- and (S)-ketamine, and fluoxetine as a positive control in the differential-reinforcement-of-low-rate (DRL) 72-s schedule of reinforcement in male rats following naloxone pretreatment. DRL classic metrics as well as peak deviation analyses served to determine antidepressant-like actions and those associated with timing. We report antidepressant-like effects of (S)-ketamine (30-60 mg/kg) that resemble fluoxetine's (2.5-10 mg/kg), as both compounds increased reinforcement rate and peak location (suggesting increased performance), reduced premature responses (suggesting time underestimation) and decreased Weber's fraction (suggesting increased timing precision). (R)-ketamine (30, but not 60 mg/kg) increased only the reinforcement rate and peak location but did not affect timing. Only fluoxetine decreased burst responses, suggesting decreased impulsivity. Naloxone pretreatment did not block ketamine enantiomers' actions, but unexpectedly, increased fluoxetine' performance. Thus, while all three medications produced antidepressant-like effects in DRL 72-s, fluoxetine- and (S)- but not (R)- ketamine-induced time underestimation (the subject experiences the time as passing quickly). The potentiation of DRL performance of fluoxetine by naloxone was unexpected and warrants clinical studies.

Effects of ketamine optical isomers, psilocybin, psilocin and norpsilocin on time estimation and cognition in rats.

RATIONALE: Ketamine and psilocybin belong to the rapid-acting antidepressants but they also produce psychotomimetic effects including timing distortion. It is currently debatable whether these are essential for their therapeutic actions. As depressed patients report that the "time is dragging," we hypothesized that ketamine and psilocybin-like compounds may produce an opposite effect, i.e., time underestimation, purportedly contributing to their therapeutic properties. OBJECTIVES: Timing was tested following administration of (R)- and (S)-ketamine, and psilocybin, psilocin, and norpsilocin in the discrete-trial temporal discrimination task (TDT) in male rats. Timing related to premature responses, and cognitive and unspecific effects of compounds were tested in the 5-choice serial reaction time task (5-CSRTT) in the standard 1-s, and "easier" 2-s stimulus duration conditions, as well as in the vITI variant promoting impulsive responses. RESULTS: (S)-ketamine (15 but not 3.75 or 7.5 mg/kg) shifted psychometric curve to the right in TDT and reduced premature responses in 5-CSRTT, suggesting expected time underestimation, but it also decreased the accuracy of temporal discrimination and increased response and reward latencies, decreased correct responses, and increased incorrect responses. While (R)-ketamine did not affect timing and produced no unspecific actions, it reduced incorrect responses in TDT and increased accuracy in 5-CSRTT, suggesting pro-cognitive effects. Psilocin and psilocybin produced mainly unspecific effects in both tasks, while norpsilocin showed no effects. CONCLUSIONS: Time underestimation produced by (S)-ketamine could be associated with its antidepressant effects; however, it was accompanied with severe behavioral disruption. We also hypothesize that behavioral disruption produced by psychedelics objectively reflects their psychotomimetic-like actions.

Effects of dopamine receptor antagonism and amphetamine-induced psychomotor sensitization on sign- and goal-tracking after extended training.

The dopamine system is important for incentive salience attribution, where motivational value is assigned to conditioned cues that predict appetitive reinforcers. However, the role of dopamine in this process may change with extended training. We tested the effects of dopamine D1-like and D2-like receptor antagonism on the expression of sign-tracking and goal-tracking conditioned responses following extended Pavlovian conditioned approach (PCA) training. We also tested if amphetamine-induced psychomotor sensitization accelerates the enhanced acquisition of sign-tracking that is observed with extended training. In experiment 1, 24 male Long-Evans rats received 20 PCA sessions in which one lever (CS+, 10 s) predicted 0.2 ml sucrose (10 %, w/v) delivery and the other lever (CS-) did not. SCH-23390 (D1-like antagonist) or eticlopride (D2-like antagonist) were administered before non-reinforced behavioural tests at doses of 0, 0.01, and 0.1 mg/kg (s.c.). In experiment 2, rats received vehicle or 2 mg/kg amphetamine (i.p.) for 7 days (n = 12/group). Ten days later, they received 16 PCA training sessions. Both doses of SCH-23390 reduced sign- and goal-tracking, but also reduced locomotor behaviour. A low dose of eticlopride (0.01 mg/kg) selectively reduced goal-tracking, without affecting sign-tracking or locomotor behaviour. Amphetamine produced psychomotor sensitization, and this did not affect the acquisition of sign- or goal-tracking. Following extended PCA training, dopamine D2-like receptor activity is required for the expression of goal-tracking but not sign-tracking. Psychomotor sensitization to amphetamine did not impact incentive salience attribution; however, more selective manipulations of the dopamine system may be needed.

Metabotropic group II glutamate receptors in the basolateral amygdala mediate cue-triggered increases in incentive motivation.

RATIONALE: Reward-associated cues can trigger incentive motivation for reward and invigorate reward-seeking behaviour via Pavlovian-to-instrumental transfer (PIT). Glutamate signaling within the basolateral amygdala (BLA) modulates cue-triggered increases in incentive motivation. However, the role of BLA metabotropic group II glutamate (mGlu2/3) receptors is largely unknown. OBJECTIVES: In Experiment 1, we characterized cue-triggered increases in incentive motivation for water reward using the PIT paradigm. In Experiment 2, we assessed the influence of intra-BLA microinjections of the mGlu2/3 receptor agonist LY379268 on this effect. METHODS: Water-restricted male Sprague-Dawley rats learned to press a lever for water. Separately, they learned to associate one of two auditory cues with free water. On test days, rats could lever press under extinction conditions (no water), with intermittent, non-contingent CS+ and CS- presentations. In Experiment 1, rats were tested under baseline conditions. In Experiment 2, rats received intra-BLA microinjections of LY379268 (0, 3 and 6 [Formula: see text]g/hemisphere) before testing. RESULTS: Across experiments, CS+, but not CS-, presentations increased water-associated lever pressing during testing, even though responding was reinforced neither by water nor the CS+. Intra-BLA LY379268 abolished both CS+ potentiated pressing on the water-associated lever and CS+ evoked conditioned approach to the site of water delivery. LY379268 did not influence locomotion or instrumental and Pavlovian response rates during intervals between CS presentations or during the CS-, indicating no motor effects. CONCLUSIONS: mGlu2/3 receptor activity in the BLA mediates cue-triggered potentiation of incentive motivation for reward, suppressing both cue-induced increases in instrumental pursuit of the reward and anticipatory approach behaviour.

Comparing ABA, AAB, and ABC Renewal of Appetitive Pavlovian Conditioned Responding in Alcohol- and Sucrose-Trained Male Rats.

Conditioned responding can be renewed by re-exposure to the conditioning context following extinction in a different context (ABA renewal) or by removal from the extinction context (AAB or ABC renewal). ABA renewal is robust in Pavlovian and operant conditioning paradigms. However, fewer studies have investigated AAB and ABC renewal of appetitive conditioning, and those that did predominantly used operant conditioning tasks. Renewal has theoretical relevance for extinction and for exposure-based treatments for substance use disorders that aim to extinguish reactivity to drug-predictive cues. We therefore investigated ABA, AAB, and ABC renewal of Pavlovian conditioned responding to cues that predicted either alcohol or sucrose. Male, Long-Evans rats (Charles River) were exposed to either 15% ethanol (Study 1: "alcohol") or 10% sucrose (Study 2: "sucrose") in their home cages. Next, they were trained to discriminate between two auditory stimuli (white noise and clicker; 10 s) in conditioning chambers equipped with distinct olfactory, visual, and tactile contextual stimuli (context A). One conditioned stimulus (CS+) was paired with fluid delivery (0.2 ml/CS+; 3.2 ml/session; alcohol or sucrose in separate experiments), and the second CS (CS-) was not. In all sessions (conditioning, extinction, and test), each CS was presented 16 times/session on a variable-time 67-s schedule, and entries into the fluid port were recorded. CS+ port entries were then extinguished by withholding fluid delivery either in context A or in a second, different context (context B). Next, we assessed ABA, AAB, and ABC renewal in the absence of fluid delivery. During extinction, CS+ port entries were initially elevated in context A relative to context B. ABA renewal of CS+ port entries occurred in both alcohol- and sucrose-trained rats. ABC renewal approached statistical significance when data from both experiments were combined. No AAB renewal was observed, and, in fact, alcohol-trained rats showed AAB suppression. These results corroborate the reliability of ABA renewal and suggest that ABC renewal is a modest effect that may require greater statistical power to detect. From a treatment perspective, the lack of AAB renewal suggests that exposure-based treatments for substance use disorders might benefit from implementation in real-world, drug-use contexts.

Distinct cognitive and discriminative stimulus effects of ketamine enantiomers in rats.

Although (S)-ketamine was approved for use in treatment-resistant depression in 2019, new preclinical findings suggest that (R)-ketamine might produce better efficacy and tolerability relative to (S)-ketamine. Here we evaluated the effects of (R)-, (S)-, and (R,S)-ketamine on executive functions as measured in the attentional set shifting task (ASST) and on their discriminative stimulus effects in rats. Earlier data demonstrated that cognitive flexibility is compromised by (R,S)-ketamine, but the effects of enantiomers in rats are unknown. Separate cohorts of rats were tested in ASST and trained to discriminate either (R,S)-ketamine, (S)-ketamine, or (R)-ketamine (all at 10 mg/kg) from saline; in order to maintain the discrimination, a higher (R)-ketamine dose (17.5 mg/kg) was subsequently instituted. In ASST, all three forms increased the trials to criterion measure at reversal learning and extra-dimensional set-shifting phases. However, in contrast to (R)- and (S)-ketamine, (R,S)-ketamine prolonged the mean time to complete a single trial during early stages, suggesting increased reaction time, and/or unspecific side-effects related to motor or motivational impairments. In the drug discriminations, all rats acquired their respective discriminations between drug and saline. In (R,S)-ketamine-trained rats, (R)-ketamine and (S)-ketamine only partially substituted for the training dose of (R,S)-ketamine. Further, (R)-ketamine did not fully substitute in rats trained to (S)-ketamine. The data suggest more serious cognitive deficits produced by (R,S)-ketamine than its enantiomers. Furthermore, (R,S)-ketamine and its isomers share overlapping but not isomorphic discriminative stimulus effects predicting distinct subjective responses to (R)- vs. (S)-ketamine in humans.

Context and topography determine the role of basolateral amygdala metabotropic glutamate receptor 5 in appetitive Pavlovian responding.

Preclinical data have shown that the excitatory metabotropic Gαq-coupled glutamate receptor, mGluR5, has a role in substance abuse and relapse. However, little is known about the contribution of mGluR5 to the expression of conditioned responding elicited by appetitive Pavlovian cues. We investigated this question in rats that were trained to associate a discrete, auditory conditioned stimulus (CS) with a fructose-glucose solution (5.5% fructose/4.5% glucose; "sugar"). In subsequent tests for the expression of conditioned responding without sugar delivery, CS-elicited fluid port entries were elevated in a context associated with sugar, relative to an equally familiar, neutral context. Inhibiting mGluR5 via systemic injections of a negative allosteric modulator (MTEP; 5 mg/kg) reduced CS port entries in both the sugar context and neutral context. Targeting MTEP microinjections (3 µg/side; 0.3 µl/min) to the nucleus accumbens (Acb) core had no effect on CS port entries at test, whereas the same manipulation in the basolateral amygdala (BLA) produced effects that were topographically dependent. Specifically, microinjecting MTEP in the posterior BLA had no effect on behavior, whereas inhibiting mGluR5 in the anterior BLA enhanced the contextual discrimination of CS port entries. These data are the first to show a role of mGluR5 in the context-dependent expression of appetitive Pavlovian conditioned responding, with a topographically defined arrangement of mGluR5 in the BLA being particularly important for context-based responding to a discrete, appetitive cue.

Justifiability and Animal Research in Health: Can Democratisation Help Resolve Difficulties?

Current animal research ethics frameworks emphasise consequentialist ethics through cost-benefit or harm-benefit analysis. However, these ethical frameworks along with institutional animal ethics approval processes cannot satisfactorily decide when a given potential benefit is outweighed by costs to animals. The consequentialist calculus should, theoretically, provide for situations where research into a disease or disorder is no longer ethical, but this is difficult to determine objectively. Public support for animal research is also falling as demand for healthcare is rising. Democratisation of animal research could help resolve these tensions through facilitating ethical health consumerism or giving the public greater input into deciding the diseases and disorders where animal research is justified. Labelling drugs to disclose animal use and providing a plain-language summary of the role of animals may help promote public understanding and would respect the ethical beliefs of objectors to animal research. National animal ethics committees could weigh the competing ethical, scientific, and public interests to provide a transparent mandate for animal research to occur when it is justifiable and acceptable. Democratic processes can impose ethical limits and provide mandates for acceptable research while facilitating a regulatory and scientific transition towards medical advances that require fewer animals.

Palatable food self-administration and reinstatement are not affected by dual orexin receptor antagonism.

The orexins are widely regarded potential therapeutic targets for a range of disorders of appetitive motivation, including obesity. The motivational activator theory, the first coherent account of the orexin system's role in appetitive motivation, predicts that orexin release motivates appetitive behaviour when the reinforcer is highly salient, available under a high unit-cost or when reward seeking is cue-driven. The present study tested the effect of intracerebroventricular (i.c.v.) administration of the highly potent and commercially available dual orexin receptor antagonist, TCS 1102, on self-administration and reinstatement of palatable food seeking in hungry and sated rats. TCS 1102 was also tested on FR1, FR5, FR10 and PR schedules. Orexin neuron activation was measured by c-Fos/orexin-A immunohistochemistry after cue-induced reinstatement, an extinction test, or a home-cage control. No effect of i.c.v. TCS 1102 was observed on self-administration at any fixed or progressive ratio schedule of reinforcement or reinstatement in hungry or sated rats. Although there was robust recruitment of orexin neurons during behavioural testing conditions, there was no specific activation of these neurons during cue-induced reinstatement when compared to extinction testing conditions. These results suggest that orexin antagonism may not be a useful therapeutic target for obesity as it does not appear to regulate food-seeking, and that the conditions determining orexin involvement as a motivational activator may be less clear than currently understood.

The dual orexin receptor antagonist TCS1102 does not affect reinstatement of nicotine-seeking.

The orexin/hypocretin system is important for appetitive motivation towards multiple drugs of abuse, including nicotine. Both OX1 and OX2 receptors individually have been shown to influence nicotine self-administration and reinstatement. Due to the increasing clinical use of dual orexin receptor antagonists in the treatment of disorders such as insomnia, we examined whether a dual orexin receptor antagonist may also be effective in reducing nicotine seeking. We tested the effect of intracerebroventricular (i.c.v.) administration of the potent and selective dual orexin receptor antagonist TCS1102 on orexin-A-induced food self-administration, nicotine self-administration and reinstatement of nicotine-seeking in rats. Our results show that 30 µg of TCS1102 i.c.v. abolishes orexin-A-induced increases in food self-administration but does not reduce nicotine self-administration. Neither i.c.v. 10 µg nor 30 µg of TCS1102 reduced compound reinstatement after short-term (15 days) self-administration nicotine, but 30 µg transiently reduced cue/nicotine compound reinstatement after chronic self-administration (29 days). These results indicate that TCS1102 has no substantial effect on motivation for nicotine seeking following chronic self-administration and no effect after shorter periods of intake. Orexin receptor antagonists may therefore have little clinical utility against nicotine addiction.

Orexin/hypocretin based pharmacotherapies for the treatment of addiction: DORA or SORA?

Addiction is a chronic relapsing disorder which presents a significant global health burden and unmet medical need. The orexin/hypocretin system is an attractive potential therapeutic target as demonstrated by the successful clinical trials of antagonist medications like Suvorexant for insomnia. It is composed of two neuropeptides, orexin-A and orexin-B and two excitatory and promiscuous G-protein coupled receptors, OX1 and OX2. Orexins are known to have a variety of functions, most notably in regulating arousal, appetite and reward. The orexins have been shown to have a role in mediating the effects of several drugs of abuse, such as cocaine, morphine and alcohol via projections to key brain regions such as the ventral tegmental area, nucleus accumbens and prefrontal cortex. However, it has not yet been demonstrated whether the dual orexin receptor antagonists (DORAs) under development for insomnia are ideal drugs for the treatment of addiction. The question of whether to use a DORA or single orexin receptor antagonist (SORA) for the treatment of addiction is a key question that will need to be answered in order to maximize the clinical utility of orexin receptor antagonists. This review will examine the role of the orexin/hypocretin system in addiction, orexin-based pharmacotherapies under development and factors affecting the selection of one or both orexin receptors as drug targets for the treatment of addiction.