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.

Sex differences in cognitive flexibility are driven by the estrous cycle and stress-dependent.

Stress is associated with psychiatric disorders such as post-traumatic stress disorder, major depressive disorder, anxiety disorders, and panic disorders. Women are more likely to be diagnosed with these stress-related psychiatric disorders than men. A key phenotype in stress-related psychiatric disorders is impairment in cognitive flexibility, which is the ability to develop new strategies to respond to different patterns in the environment. Because gonadal hormones can contribute to sex differences in response to stress, it is important to consider where females are in their cycle when exposed to stress and cognitive flexibility testing. Moreover, identifying neural correlates involved in cognitive flexibility could not only build our understanding of the biological mechanisms behind this crucial skill but also leads to more targeted treatments for psychiatric disorders. Although previous studies have separately examined sex differences in cognitive flexibility, stress effects on cognitive flexibility, and the effect of gonadal hormones on cognitive flexibility, many of the findings were inconsistent, and the role of the estrous cycle in stress-induced impacts on cognitive flexibility is still unknown. This study explored potential sex differences in cognitive flexibility using an operant strategy shifting-paradigm after either control conditions or restraint stress in freely cycling female and male rats (with estrous cycle tracking in the female rats). In addition, we examined potential neural correlates for any sex differences observed. In short, we found that stress impaired certain aspects of cognitive flexibility and that there were sex differences in cognitive flexibility that were driven by the estrous cycle. Specifically, stress increased latency to first press and trials to criterion in particular tasks. The female rats demonstrated more omissions and perseverative errors than the male rats; the sex differences were mostly driven by proestrus female rats. Interestingly, the number of orexinergic neurons was higher in proestrus female rats than in the male rats under control conditions. Moreover, orexin neural count was positively correlated with number of perseverative errors made in cognitive flexibility testing. In sum, there are sex differences in cognitive flexibility that are driven by the estrous cycle and are stress-dependent, and orexin neurons may underlie some of the sex differences observed.

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.

Stress, coping, resilience, and sleep during the COVID-19 pandemic: A representative survey study of US adults.

INTRODUCTION: The COVID-19 pandemic is a global health emergency resulting in widespread death and substantial disruption to daily life. Previous research has shown that novel disease outbreaks are associated with high stress levels and sleep impairments that lead to neuropsychiatric consequences. Therefore, it is vital to study both stress and protective factors such as coping and resilience that may hinder or help sleep quality during the COVID-19 pandemic. Further, as gender disparities exist in sleep quality, it is important to understand the relationship between pandemic-related stress, coping strategies, resilience, and sleep in bothgenders during the COVID-19 pandemic. METHODS: Our study examined how gender, stress, coping, and resilience were associated with sleep cross-sectionally during the COVID-19 pandemic in a representative sample of US adults (N = 393). RESULTS: Consistent with many recent studies, we found that worsened sleep quality in women compared to men persisted during the COVID-19 pandemic. Interestingly, pandemic-related stress was not significantly associated with sleep quality, but pandemicrelated coping was associated with sleep independent of robust controls and trait resilience. CONCLUSIONS: Greater primary control engagement coping was associated with better sleep quality, while involuntary engagement coping was associated with poor sleep quality. Future research should extend the findings with actigraphy and explore ways to enhance beneficial coping and sleep health during pandemics.

Sex differences in stress-induced sleep deficits.

Sleep disruptions are hallmarks in the pathophysiology of several stress-related disorders, including Major Depressive Disorder (MDD) and Post-Traumatic Stress Disorder (PTSD), both known to disproportionately affect female populations. Although previous studies have attempted to investigate disordered sleep in women, few studies have explored and compared how repeated stress affects sleep in both sexes in either human or animal models. We have previously shown that male rats exhibit behavioral and neuroendocrine habituation to 5 days of repeated restraint, whereas females do not; additional days of stress exposure are required to observe habituation in females. This study examined sex differences in sleep measures prior to, during, and after repeated restraint stress in adult male and female rats. Our data reveal that repeated stress increased time spent awake and decreased slow-wave sleep (SWS) and REM sleep (REMS) in females, and these effects persisted over 2 days of recovery. In contrast, the effects of stress on males were transient. These insomnia-like symptoms were accompanied by a greater number of exaggerated motor responses to waking from REMS in females, a phenotype similar to trauma-related nightmares. In sum, these data demonstrate that repeated stress produces disruptions in sleep that persist days after the stress is terminated in female rats. These disruptions in sleep produced by 5 days of repeated restraint may be due to their lack of habituation.

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.

Assessment of Stress Effects on Cognitive Flexibility using an Operant Strategy Shifting Paradigm.

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and duration of the stressor; 2) type of cognitive function under study; and 3) timing of the stressor in relation to learning or executing the cognitive task. Furthermore, sex differences among the effects of stress on cognitive function have been widely documented. Described here is an adaptation of an automated operant strategy shifting paradigm to assess how variations in stress affect cognitive flexibility in male and female Sprague Dawley rats. Specifically, restraint stress is used before or after training in this operant-based task to examine how stress affects cognitive performance in both sexes. Particular brain areas associated with each task in this automated paradigm have been well-established (i.e., the medial prefrontal cortex and orbitofrontal cortex). This allows for targeted manipulations during the experiment or the assessment of particular genes and proteins in these regions upon completion of the paradigm. This paradigm also allows for the detection of different types of performance errors that occur after stress, each of which has defined neural substrates. Also identified are distinct sex differences in perseverative errors after a repeated restraint stress paradigm. The use of these techniques in a preclinical model may reveal how stress affects the brain and impairs cognition in psychiatric disorders, such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD), which display marked sex differences in prevalence.

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.

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.

Methylphenidate Enhances Early-Stage Sensory Processing and Rodent Performance of a Visual Signal Detection Task.

Methylphenidate (MPH) is used clinically to treat attention-deficit/hyperactivity disorder (ADHD) and off-label as a performance-enhancing agent in healthy individuals. MPH enhances catecholamine transmission via blockade of norepinephrine (NE) and dopamine (DA) reuptake transporters. However, it is not clear how this action affects neural circuits performing cognitive and sensorimotor functions driving performance enhancement. The dorsal lateral geniculate nucleus (dLGN) is the primary thalamic relay for visual information from the retina to the cortex and is densely innervated by NE-containing fibers from the locus coeruleus (LC), a pathway known to modulate state-dependent sensory processing. Here, MPH was evaluated for its potential to alter stimulus-driven sensory responses and behavioral outcomes during performance of a visual signal detection task. MPH enhanced activity within individual neurons, ensembles of neurons, and visually-evoked potentials (VEPs) in response to task light cues, while increasing coherence within theta and beta oscillatory frequency bands. MPH also improved reaction times to make correct responses, indicating more efficient behavioral performance. Improvements in reaction speed were highly correlated with faster VEP latencies. Finally, immunostaining revealed that catecholamine innervation of the dLGN is solely noradrenergic. This work suggests that MPH, acting via noradrenergic mechanisms, can substantially affect early-stage sensory signal processing and subsequent behavioral outcomes.