Pituitary adenylate cyclase-activating polypeptide (PACAP) in the paraventricular nucleus of the thalamus: Influence on binge-type eating in male and female mice.

Binge eating disorder, characterized by the overconsumption of food in a discrete time period, is the most common eating disorder in the United States, but its neurological basis is not fully understood. The paraventricular nucleus of the thalamus (PVT) is a limbic brain region implicated in eating, and the anorexigenic neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is densely expressed in the PVT. This study sought to examine the possible involvement of PACAP in the PVT in binge-type eating. First, a model of binge-type eating was established in mice. Male and female C57BL/6J mice were given limited access to Milk Chocolate Ensure Plus® or had access only to chow and water. Under this model, while males and females both engaged in binge-type eating with Ensure, females engaged in this behavior to a greater degree than males. Next, the role of PACAP in the PVT was defined in relation to binge-type eating. Using quantitative real-time PCR, females were found to have higher baseline levels of PVT PACAP mRNA than males, but only males showed an increase in levels of PACAP after a history of binge-type eating, and only males showed a reduction in levels of PACAP immediately prior to a binge session. Using chemogenetics in PACAP-Cre transgenic mice on a C57BL/6J background, activation of PVT PACAP+ cells with a Cre-dependent Gq-DREADD was found to reduce binge-type eating, significantly in male but not female mice. These results indicate that PVT PACAP is involved in binge-type eating in a sex-dependent manner, with a decrease in PVT PACAP levels preceding binge-type eating in male mice, and enhanced PVT PACAP+ cell activity suppressing binge-type eating in male mice. Together, these results suggest that the PACAP system could be targeted in specific patient populations to help treat binge eating disorder.

Kappa-opioid receptor stimulation in the nucleus accumbens shell and ethanol drinking: Differential effects by rostro-caudal location and level of drinking.

Although the kappa-opioid receptor (KOR) and its endogenous ligand, dynorphin, are believed to be involved in ethanol drinking, evidence on the direction of their effects has been mixed. The nucleus accumbens (NAc) shell densely expresses KORs, but previous studies have not found KOR activation to influence ethanol drinking. Using microinjections into the NAc shell of male and female Long-Evans rats that drank under the intermittent-access procedure, we found that the KOR agonist, U50,488, had no effect on ethanol drinking when injected into the middle NAc shell, but that it promoted intake in males and high-drinking females in the caudal NAc shell and high-drinking females in the rostral shell, and decreased intake in males and low-drinking females in the rostral shell. Conversely, injection of the KOR antagonist, nor-binaltorphimine, stimulated ethanol drinking in low-drinking females when injected into the rostral NAc shell and decreased drinking in high-drinking females when injected into the caudal NAc shell. These effects of KOR activity were substance-specific, as U50,488 did not affect sucrose intake. Using quantitative real-time PCR, we found that baseline gene expression of the KOR was higher in the rostral compared to caudal NAc shell, but that this was upregulated in the rostral shell with a history of ethanol drinking. Our findings have important clinical implications, demonstrating that KOR stimulation in the NAc shell can affect ethanol drinking, but that this depends on NAc subregion, subject sex, and ethanol intake level, and suggesting that this may be due to differences in KOR expression.

Sex-related differences in endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) in the thalamic paraventricular nucleus: Implications for addiction neuroscience.

Males and females exhibit differences in motivated and affective behavior; however, the neural substrates underlying these differences remain poorly understood. In the paraventricular nucleus of the thalamus (PVT), sex-related differences in neuronal activity have been identified in response to motivated behavior tasks and affective challenges. Within the PVT, the neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is highly expressed and is also involved in motivated and affective behavior. The purpose of this study was to compare the expression of PACAP mRNA and peptide in the PVT of males and females. Analysis with quantitative real-time PCR in mice revealed that females had significantly higher levels of PACAP mRNA than males in the whole PVT, but no differences in the neuropeptides enkephalin or corticotropin releasing factor (CRF) in this brain region. While in rats, females demonstrated a trend for greater gene expression than males in the anterior/middle and middle/posterior PVT, they again showed no differences in enkephalin or CRF. Analysis with immunofluorescent histochemistry revealed that female mice had significantly more PACAP-containing cells than males as a function of area throughout the PVT, and that female rats had significantly more PACAP-27 and PACAP-38-containing cells than males, both as a percentage of total cells and as a function of PVT area. For PACAP-27, this specifically occurred in the anterior PVT, and for PACAP-38, it occurred throughout the anterior, middle, and posterior PVT. These results suggest that sex-related differences in PVT PACAP may underly some of the established sex-related differences in motivated and affective behavior.

Inactivation of the thalamic paraventricular nucleus promotes place preference and sucrose seeking in male rats.

RATIONALE: The experience of reward entails both positive affect and motivation. While the brain regions responsible for these distinct aspects of reward are dissociable from each other, the paraventricular nucleus of the thalamus (PVT) may play a role in both. OBJECTIVES: To investigate the role of the PVT in both affect and motivation, and to identify neuropeptides that might mediate these effects. METHODS: Male rats were tested for conditioned place preference following temporary inactivation of the anterior or posterior PVT with local injections of the GABAB and GABAA agonists, baclofen + muscimol. They were tested for sucrose seeking under a fixed ratio 3 (FR3) schedule of reinforcement and after extinction, following injection into the posterior PVT of baclofen + muscimol or saline vehicle. Finally, quantitative real-time PCR was used to examine local neuropeptide gene expression following injection into the posterior PVT of baclofen + muscimol or saline vehicle. RESULTS: Conditioned place preference was induced by temporary inactivation of the posterior but not anterior PVT. While sucrose seeking under an FR3 schedule of reinforcement was unaffected by inactivation of the posterior PVT, reinstatement of sucrose seeking was promoted by posterior PVT inactivation. Local gene expression of pituitary adenylate cyclase-activating polypeptide (PACAP), but not enkephalin or neurotensin, was reduced following inactivation of the posterior PVT. CONCLUSIONS: Temporary inactivation of the posterior PVT affects both affect and motivation as well as local gene expression of PACAP. These results suggest that the posterior PVT is one brain region that may participate in both major aspects of reward.

Sex-related differences in pattern of ethanol drinking under the intermittent-access model and its impact on exploratory and anxiety-like behavior in Long-Evans rats.

BACKGROUND: While men in the United States consume more alcohol than women, rates of drinking are converging. Nevertheless, females remain underrepresented in preclinical alcohol research. Here, we examined rats' sex-related differences in patterns of ethanol (EtOH) drinking and the effects of this drinking on exploratory and anxiety-like behavior. METHODS: Adult male and female Long-Evans rats were given 20% ethanol under the intermittent-access two-bottle-choice paradigm. Their intake was measured daily for the first 7 weeks. During the eighth week, intake was measured over the 24 h of daily access. During the ninth week, they, along with EtOH-naive controls, were tested prior to daily access in a novel chamber, light-dark box, and hole board apparatus. During the tenth week, blood ethanol concentration (BEC) was assessed after 30 to 40 min of access. RESULTS: Females overall demonstrated higher ethanol intake and preference across all access weeks than males, although only half of females drank significantly more than males. Across 24 h of daily access, both sexes had their highest intake in the first 30 min and their lowest in the middle of the light phase of the light/dark cycle. Despite their greater ethanol intake, females did not show significantly different BECs than males. In behavioral tests, females showed less vertical time in a novel activity chamber, more movement between chambers in a light-dark box, and more nose pokes in a hole-board apparatus than males. While a history of ethanol drinking led to a trend for lower vertical time in the activity chamber and greater chamber entries in the light-dark box, the effects were not sex-dependent. CONCLUSIONS: These results suggest that female and male rats could both be tested for acute effects of ethanol after 30 min of daily access, but that nuanced considerations are needed in the design of these experiments and the interpretation of their findings.

Effects of pituitary adenylate cyclase-activating polypeptide isoforms in nucleus accumbens subregions on ethanol drinking.

While limited research has implicated the neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), in problematic alcohol use, the brain regions and isoforms involved in this effect remain to be determined. One region that has been found both to exhibit PACAP binding and, separately, to be involved in ethanol drinking is the nucleus accumbens (NAc). Thus, this study sought to characterize the effect of the PACAP isoforms in the NAc on ethanol drinking under the intermittent-access two-bottle-choice paradigm, in male and female Long-Evans rats. With microinjection into the medial NAc shell, PACAP-27 but not PACAP-38 was found to dose-dependently reduce binge-like ethanol drinking. In contrast, the PACAP receptor antagonist, PACAP (6-27), but not PACAP (6-38), enhanced ethanol drinking. This effect of PACAP was substance specific, as neither isoform in the NAc shell affected binge-like sucrose drinking. It was also anatomically specific, as PACAP-38 rather than PACAP-27 suppressed ethanol drinking when injected into the NAc core, and PACAP-27 instead enhanced drinking when injected into the caudal third of the medial NAc shell. Finally, while PACAP-38 in the NAc shell affected stress-related exploratory behavior, reducing time spent in the light chamber of a light-dark box, PACAP-27 did not significantly affect behavior in a light-dark box or open field. Together, these results, showing that PACAP-27 in the NAc shell attenuates binge-like ethanol drinking without affecting select stress-related behaviors, suggest that compounds related to this PACAP isoform should be investigated as potential novel therapeutics for the treatment of alcohol use disorder.

Kappa-opioid receptor-dependent changes in dopamine and anxiety-like or approach-avoidance behavior occur differentially across the nucleus accumbens shell rostro-caudal axis.

Neural circuit engagement within the nucleus accumbens (NAc) shell is implicated in the regulation of both negative and positive affect. Classically, the dynorphin/kappa opioid receptor (KOR) system in the NAc was believed to promote aversion, while dopamine was viewed as interacting with reward behavior, and KOR activation was known to inhibit dopamine release. Recently, however, both the KOR and dopamine systems have, separately, been shown to have differential effects across the rostro-caudal axis of the NAc shell on hedonic responses. Whether or not this is due to interactions between KORs and dopamine, and if it extends to anxiety-like or approach-avoidance behaviors, remains to be determined. In this study, we examined in rats the relationship between the KOR and dopamine systems in both the rostral and caudal NAc shell using ex vivo fast scan cyclic voltammetry and the impact of KOR activation on affective behavior using exploration-based tasks. We report here that activation of KORs in the caudal NAc shell significantly inhibits dopamine release, stimulates rearing behavior in a novel environment, increases anxiety-like or avoidance behavior, and reduces locomotor activity. In contrast, activation of KORs in the rostral NAc shell inhibits dopamine release to a lesser extent and instead reduces anxiety-like behavior or increases approach behavior. Taken together, these results indicate that there is heterogeneity across the rostro-caudal axis of the NAc shell in the effects of KOR stimulation on affective behaviors, and they suggest that this might be due to differences in KOR control over dopamine release.

Expression and Distribution of Neuropeptide-Expressing Cells Throughout the Rodent Paraventricular Nucleus of the Thalamus.

The paraventricular nucleus of the thalamus (PVT) has been shown to make significant contributions to affective and motivated behavior, but a comprehensive description of the neurochemicals expressed in the cells of this brain region has never been presented. While the PVT is believed to be composed of projection neurons that primarily use as their neurotransmitter the excitatory amino acid, glutamate, several neuropeptides have also been described in this brain region. In this review article, we combine published literature with our observations from the Allen Brain Atlas to describe in detail the expression and distribution of neuropeptides in cells throughout the mouse and rat PVT, with a special focus on neuropeptides known to be involved in behavior. Several themes emerge from this investigation. First, while the majority of neuropeptides are expressed across the antero-posterior axis of the PVT, they generally exist in a gradient, in which expression is most dense but not exclusive in either the anterior or posterior PVT, although other neuropeptides display somewhat more equal expression in the anterior and posterior PVT but have reduced expression in the middle PVT. Second, we find overall that neuropeptides involved in arousal are more highly expressed in the anterior PVT, those involved in depression-like behavior are more highly expressed in the posterior PVT, and those involved in reward are more highly expressed in the medial PVT, while those involved in the intake of food and drugs of abuse are distributed throughout the PVT. Third, the pattern and content of neuropeptide expression in mice and rats appear not to be identical, and many neuropeptides found in the mouse PVT have not yet been demonstrated in the rat. Thus, while significantly more work is required to uncover the expression patterns and specific roles of individual neuropeptides in the PVT, the evidence thus far supports the existence of a diverse yet highly organized system of neuropeptides in this nucleus. Determined in part by their location within the PVT and their network of projections, the function of the neuropeptides in this system likely involves intricate coordination to influence both affective and motivated behavior.

Pleiotropic pituitary adenylate cyclase-activating polypeptide (PACAP): Novel insights into the role of PACAP in eating and drug intake.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was discovered thirty years ago, but its role in eating and drug use disorders has only recently begun to be investigated. The present review develops the hypothesis that, although PACAP normally functions to tightly regulate intake, inhibiting it through negative feedback, this relationship can become dysregulated with the development of dependence, such that PACAP instead acts through positive feedback to promote excessive intake. We propose that repeated exposure to palatable food and drugs of abuse can alter the downstream responses of specific populations of neurons to stimulation by PACAP, leading to the perpetuation of the addiction cycle. Thus, this review will first describe published literature on homeostatic food intake, which shows that PACAP suppresses food intake, while its levels are themselves increased by overfeeding. Next, it will present literature on palatable food, cocaine, alcohol, and nicotine, which overall demonstrates that PACAP in specific limbic brain regions can promote their seeking and intake and itself is stimulated by their intake. Then, it will present literature on affective behavior, which shows that chronic stress increases levels of PACAP, which then promotes anxiety and depression, factors that can trigger substance seeking. Finally, the review will address mechanisms through which chronic substance exposure may dysregulate the PACAP system, proposing that it alters expression of PACAP receptor splice variants. While many questions remain to be addressed, the current evidence suggests that PACAP could be a viable medication target for the treatment of binge eating and drug and alcohol use disorders.

Short- and long-access palatable food self-administration results in different phenotypes of binge-type eating.

Binge eating disorder (BED), the most common eating disorder in the United States, is characterized by binge-type eating and is associated with higher body mass index and greater motivation for food. This disorder tends to first appear in late adolescence or young adulthood and is more common in women than men. While some animal models of BED have recapitulated both the overeating and the excessive body weight / fat of BED, very few have examined the motivational aspects of this disorder or utilized young females as subjects. In the present study, female Long-Evans rats, starting in late adolescence, were trained in operant chambers to self-administer the highly palatable Milk Chocolate Ensure Plus®, in 30-minute ("short access") or 6-hour ("long access") sessions, 5 days per week, over 6.5 weeks. For comparison, other subjects were provided with Ensure ad libitum or maintained on chow and water only. Both short and long access to Ensure led rats to develop binge-type eating, measured as greater 30-minute caloric intake than rats with ad libitum or chow access and as increasing 30-minute intake across weeks. Compared to those with short access, rats with long access demonstrated moderately increased motivation for Ensure (measured by progressive ratio testing) and, compared to those with only chow access, they eventually showed significant hyperphagia on Ensure access days and hypophagia on non-access days. Rats with long access also showed greater body weight/fat than those maintained on chow. These findings suggest that, while both short and long operant access to Ensure causes young female rats to meet the definition of binge-type eating, they lead to different phenotypes of this behavior, with long access promoting the development of a greater number of features found in clinical BED. Ultimately, both models may be useful in future studies aimed at identifying the neurobiological basis of binge eating.

Neurotensin in the posterior thalamic paraventricular nucleus: inhibitor of pharmacologically relevant ethanol drinking.

Individuals prone to ethanol overconsumption may have preexisting neurochemical disturbances that contribute to their vulnerability. This study examined the paraventricular nucleus of the thalamus (PVT), a limbic structure recently shown to participate in ethanol intake. To identify individuals prone to ethanol overconsumption, we tested Long-Evans rats in behavioral paradigms and found high levels of vertical time (rearing behavior) in a novel activity chamber to be a consistent predictor of subsequent excessive 20 percent ethanol drinking under the intermittent access model. Examining neurochemicals in the PVT, we found before ethanol exposure that prone rats with high rearing, compared with non-prone rats, had significantly lower levels of neurotensin (NTS) mRNA and peptide in the posterior (pPVT) but not anterior (aPVT) subregion of the PVT. Our additional finding that ethanol intake has no significant impact on either rearing or NTS levels indicates that these measures, which are different in prone rats before ethanol consumption, remain stable after ethanol consumption. The possibility that NTS directly controls ethanol drinking is supported by our finding that NTS administration specifically suppresses ethanol drinking when injected into the pPVT but not aPVT, with this effect occurring exclusively in higher drinkers that presumably have lower endogenous levels of NTS. Further, an NTS antagonist in the pPVT augments intake in lower drinkers with presumably more endogenous NTS, while NTS in the pPVT inhibits novelty-induced rearing that predicts excessive drinking. Together, these results provide strong evidence that low endogenous levels of NTS in the pPVT contribute to an increased propensity toward excessive ethanol drinking.

Pituitary Adenylate Cyclase-Activating Polypeptide-27 (PACAP-27) in the Thalamic Paraventricular Nucleus Is Stimulated by Ethanol Drinking.

BACKGROUND: The paraventricular nucleus of the thalamus (PVT) is a limbic brain structure that affects ethanol (EtOH) drinking, but the neurochemicals transcribed in this nucleus that may participate in this behavior have yet to be fully characterized. The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is known to be transcribed in other limbic areas and to be involved in many of the same behaviors as the PVT itself, possibly including EtOH drinking. It exists in 2 isoforms, PACAP-38 and PACAP-27, with the former expressed at higher levels in most brain regions. The purpose of this study was to characterize PACAP in the PVT and to assess its response to EtOH drinking. METHODS: First, EtOH-naïve, Sprague Dawley rats were examined using quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry, to characterize PACAP mRNA and peptide throughout the rostrocaudal axis of the PVT. Next, EtOH-naïve, vGLUT2-GFP transgenic mice were examined using immunohistochemistry, to identify the neurochemical phenotype of the PACAPergic cells in the PVT. Finally, Long Evans rats were trained to drink 20% EtOH under the intermittent-access paradigm and then examined with PCR and immunohistochemistry, to determine the effects of EtOH on endogenous PACAP in the PVT. RESULTS: Gene expression of PACAP was detected across the entire PVT, denser in the posterior than the anterior portion of this nucleus. The protein isoform, PACAP-27, was present in a high percentage of cell bodies in the PVT, again particularly in the posterior portion, while PACAP-38 was instead dense in fibers. All PACAP-27+ cells colabeled with glutamate, which itself was identified in the majority of PVT cells. EtOH drinking led to an increase in PACAP gene expression and in levels of PACAP-27 in individual cells of the PVT. CONCLUSIONS: This study characterizes the PVT neuropeptide, PACAP, and its understudied protein isoform, PACAP-27, and demonstrates that it is involved in pharmacologically relevant EtOH drinking. This indicates that PACAP-27 should be further investigated for its possible role in EtOH drinking.