Animals must respond to various threats to survive. Neurons that express calcitonin gene-related peptide (CGRP) in the parabrachial nucleus (PBN) relay sensory signals that contribute to satiation and pain-induced fear behavior, but it is unknown how they encode these distinct processes. By recording calcium transients in vivo from individual CGRPPBN neurons, we reveal that most neurons are activated by noxious cutaneous (shock, heat, itch) and visceral stimuli (lipopolysaccharide). These same neurons are inhibited during feeding, but become activated during satiation, consistent with evidence that CGRPPBN neurons prevent overeating. CGRPPBN neurons are also activated during consumption of novel food or by an auditory cue that was previously paired with electrical foot shocks. Correspondingly, silencing CGRPPBN neurons attenuates expression of food neophobia and conditioned fear responses. Therefore, in addition to transducing primary sensory danger signals, CGRPPBN neurons promote affective-behavioral states that limit harm in response to potential threats.

The central nervous system controls food consumption to maintain metabolic homoeostasis. In response to a meal, visceral signals from the gut activate neurons in the nucleus of the solitary tract (NTS) via the vagus nerve. These NTS neurons then excite brain regions known to mediate feeding behaviour, such as the lateral parabrachial nucleus (PBN). We previously described a neural circuit for appetite suppression involving calcitonin gene-related protein (CGRP)-expressing PBN (CGRPPBN) neurons; however, the molecular identity of the inputs to these neurons was not established. Here we identify cholecystokinin (CCK) and noradrenergic, dopamine β-hydroxylase (DBH)-expressing NTS neurons as two separate populations that directly excite CGRPPBN neurons. When these NTS neurons are activated using optogenetic or chemogenetic methods, food intake decreases and with chronic stimulation mice lose body weight. Our optogenetic results reveal that CCK and DBH neurons in the NTS directly engage CGRPPBN neurons to promote anorexia.

Anxiety disorders are frequently long-lasting and debilitating for more than 40 million American adults. Although stressor exposure plays an important role in the etiology of some anxiety disorders, the mechanisms by which exposure to stressful stimuli alters central circuits that mediate anxietylike emotional behavior are still unknown. Substantial evidence has implicated regions of the central extended amygdala, including the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala (CeA) as critical structures mediating fear-and anxiety-like behavior in both humans and animals. These areas organize coordinated fear-and anxiety-like behavioral responses as well as peripheral stress responding to threats via direct and indirect projections to the paraventricular nucleus of the hypothalamus (PVN) and brainstem regions (Walker et al., 2003;Ulrich-Lai and Herman, 2009;Walker et al., 2009). In particular, the BNST has been argued to mediate these central and peripheral responses when the perceived threat is of long duration (Waddell et al., 2006) and/or when the anxiety-like response is sustained (Walker and Davis, 2008); hence, the BNST may mediate pathological anxiety-like states that result from exposure to chronic stress. Indeed, chronic stress paradigms result in enhanced BNST neuroplasticity that has been associated with pathological anxiety-like states (Vyas et al., 2003;Pego et al., 2008). Here we review evidence that suggests that pituitary adenylate cyclase activating polypeptide (PACAP) and corticotropin-releasing hormone (CRH) work together to modulate BNST function and increase anxiety-like behavior. Moreover, we have shown that BNST PACAP as well as its cognate PAC1 receptor are substantially upregulated following chronic stress, particularly in the BNST oval nucleus where PACAP-containing neurons closely interact with CRH-containing neurons (Kozicz et al., 1997;Hammack et al., 2009). We describe how interactions between PACAP and CRH in the BNST may mediate stress-associated behaviors, including anorexia and anxiety-like behavior. These studies have the potential to define specific mechanisms underlying anxiety disorders, and may provide important therapeutic strategies for stress-and anxiety-management.

Summary Chronic or repeated stressor exposure can induce a number of maladaptive behavioral and physiological consequences and among limbic structures, the bed nucleus of the stria terminalis (BNST) has been implicated in the integration and interpretation of stress responses. Previous work has demonstrated that chronic variate stress (CVS) exposure in rodents increases BNST pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) and PAC1 receptor (Adcyap1r1) transcript expression, and that acute BNST PACAP injections can stimulate anxiety-like behavior. Here we show that chronic stress increases PACAP expression selectively in the oval nucleus of the dorsolateral BNST in patterns distinct from those for corticotropin releasing hormone (CRH). Among receptor subtypes, BNST PACAP signaling through PAC1 receptors not only heightened anxiety responses as measured by different behavioral parameters but also induced anorexic-like behavior to mimic the consequences of stress. Conversely, chronic inhibition of BNST PACAP signaling by continuous infusion with the PAC1 receptor antagonist PACAP(6-38) during the week of CVS attenuated these stress-induced behavioral responses and changes in weight gain. BNST PACAP signaling stimulated the hypothalamic-pituitary-adrenal (HPA) axis and heightened corticosterone release; further, BNST PACAP(6-38) administration blocked corticosterone release in a sensitized stress model. In aggregate with recent associations of PACAP/PAC1 receptor dysregulation with altered stress responses including post-traumatic stress disorder, these data suggest that BNST PACAP/PAC1 receptor signaling mechanisms may coordinate the behavioral and endocrine consequences of stress.

The intricate relationships that associate pain, stress responses and emotional behavior have been well established. Acute stressful situations can decrease nociceptive sensations and conversely, chronic pain can enhance other pain experiences and heighten the emotional and behavioral consequences of stress. Accordingly, chronic pain is comorbid with a number of behavioral disorders including depression, anxiety abnormalities and associated stress-related disorders including post traumatic stress disorder (PTSD). The central nucleus of the amygdala (CeA) represents a convergence of pathways for pain, stress and emotion, and we have identified pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in fiber elements in the lateral capsular division of the CeA (CeLC). The PACAP staining patterns colocalized in part with those for calcitonin gene related peptide (CGRP); anterograde fiber tracing and excitotoxic lesion studies demonstrated that the CeLC PACAP/CGRP immunoreactivities represented sensory fiber projections from the lateral parabrachial nucleus (LPBn) along the spino-parabrachioamygdaloid tract. The same PBn PACAP/CGRP fiber system also projected to the BNST. As in the BNST, CeA PACAP signaling increased anxiety-like behaviors accompanied by weight loss and decreased feeding. But in addition to heightened anxiety-like responses, CeA PACAP signaling also altered nociception as reflected by decreased latency and threshold responses in thermal and mechanical sensitivity tests, respectively. From PACAP expression in major pain pathways, the current observations are novel and suggest that CeA PACAP nociceptive signaling and resulting neuroplasticity via the spino-parabrachioamygdaloid tract may represent mechanisms that associate chronic pain with sensory hypersensitivity, fear memory consolidation and severe behavioral disorders.

Cholecystokinin (CCK)-expressing neurons within the nucleus of the solitary tract (CCKNTS) are responsive to satiety signals and their chemogenetic activation suppresses appetite. Optogenetic activation of CCKNTS axon terminals within either the parabrachial nucleus (PBN) or the paraventricular nucleus of the hypothalamus (PVH) is sufficient to suppress feeding. An interesting dichotomy has been revealed when assessing the motivational valence of these two circuits. Activating CCKNTS cell bodies is aversive as demonstrated by conditioned taste aversion and place-preference assays. Activation of the CCKNTS→PBN pathway is also aversive; however, stimulating the CCKNTS→PVH pathway is appetitive when assayed using a real-time, place-preference task. Thus, these two projections from CCKNTS neurons reduce food intake through opposite motivational states; one pathway signals positive valence (CCKNTS→PVH) and the other signals negative valence (CCKNTS→PBN).

Single nucleotide polymorphisms (SNP) in the genes for pituitary adenylate cyclase-activating peptide (PACAP) and the PAC1 receptor have been associated with several psychiatric disorders whose etiology has been associated with stressor exposure and/or dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. In rats, exposure to repeated variate stress has been shown to increase PACAP and its cognate PAC1 receptor expression in the bed nucleus of the stria terminalis (BNST), a brain region implicated in anxiety and depression-related behaviors as well as the regulation of HPA axis activity. We have argued that changes in BNST PACAP signaling may mediate the changes in emotional behavior and dysregulation of the HPA axis associated with anxiety and mood disorders. The current set of studies was designed to determine whether BNST PACAP infusion leads to activation of the HPA axis as determined by increases in plasma corticosterone. We observed an increase in plasma corticosterone levels 30 minutes following BNST PACAP38 infusion in male and female rats, which was independent of estradiol (E2) treatment in females, and we found that plasma corticosterone levels were increased at both 30 minutes and 60 minutes, but returned to baseline levels 4 hours following the highest dose. PACAP38 infusion into the lateral ventricles immediately above the BNST did not alter plasma corticosterone level, and the increased plasma corticosterone following BNST PACAP was not blocked by BNST corticotropin releasing hormone (CRH) receptor antagonism. These results support others suggesting that BNST PACAP plays a key role in regulating stress responses.

It was recently found that nociceptive sensations (stinging, pricking, or burning) can be evoked by cooling or heating the skin to innocuous temperatures (e.g., 29°, 37°C). Here we show that this lowthreshold thermal nociception (LTN) can be traced to sensitive 'spots' in the skin equivalent to classically defined warm spots and cold spots. Because earlier work had shown that LTN is inhibited by simply touching a thermode to the skin, a spatial search procedure was devised that minimized tactile stimulation by sliding small thermodes (16 mm 2 and 1 mm 2 ) set to 28° or 36°C slowly across the lubricated skin of the forearm. The procedure uncovered three types of temperature-sensitive sites (thermal, bimodal and nociceptive) that contained one or more thermal, nociceptive or (rarely) bimodal spots. Repeated testing indicated that bimodal and nociceptive sites were less stable over time than thermal sites, and that mechanical contact differentially inhibited nociceptive sensations. Intensity ratings collected over a range of temperatures showed that LTN increased monotonically on heat-sensitive sites but not on cold-sensitive sites. These results provide psychophysical evidence that stimulation from primary afferent fibers with thresholds in the range of warm fibers and cold fibers is relayed to the pain pathway. However, the labile nature of LTN implies that these lowthreshold nociceptive inputs are subject to inhibitory controls. The implications of these findings for the roles of putative temperature receptors and nociceptors in innocuous thermoreception and thermal pain are discussed.