Encoding of danger by parabrachial CGRP neurons.

Animals must respond to various threats to survive. Neurons that express calcitonin gene-related peptide in the parabrachial nucleus (CGRPPBN neurons) relay sensory signals that contribute to satiation and pain-induced fear behaviour, but it is unclear how they encode these distinct processes. Here, by recording calcium transients in vivo from individual neurons in mice, we show that most CGRPPBN neurons are activated by noxious cutaneous (shock, heat, itch) and visceral stimuli (lipopolysaccharide). The 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 foods or by an auditory cue that has previously been paired with electrical footshocks. Correspondingly, silencing of CGRPPBN neurons attenuates the expression of food neophobia and conditioned fear responses. Therefore, in addition to transducing primary sensory danger signals, CGRPPBN neurons promote affective-behavioural states that limit harm in response to potential threats.

Prenatal Metal Exposures and Infants' Developmental Outcomes in a Navajo Population.

Early-life exposure to environmental toxicants can have detrimental effects on children's neurodevelopment. In the current study, we employed a causal modeling framework to examine the direct effect of specific maternal prenatal exposures on infants' neurodevelopment in the context of co-occurring metals. Maternal metal exposure and select micronutrients' concentrations were assessed using samples collected at the time of delivery from mothers living across Navajo Nation with community exposure to metal mixtures originating from abandoned uranium mines. Infants' development across five domains was measured at ages 10 to 13 months using the Ages and Stages Questionnaire Inventory (ASQ:I), an early developmental screener. After adjusting for effects of other confounding metals and demographic variables, prenatal exposure to lead, arsenic, antimony, barium, copper, and molybdenum predicted deficits in at least one of the ASQ:I domain scores. Strontium, tungsten, and thallium were positively associated with several aspects of infants' development. Mothers with lower socioeconomic status (SES) had higher lead, cesium, and thallium exposures compared to mothers from high SES backgrounds. These mothers also had infants with lower scores across various developmental domains. The current study has many strengths including its focus on neurodevelopmental outcomes during infancy, an understudied developmental period, and the use of a novel analytical method to control for the effects of co-occurring metals while examining the effect of each metal on neurodevelopmental outcomes. Yet, future examination of how the effects of prenatal exposure on neurodevelopmental outcomes unfold over time while considering all potential interactions among metals and micronutrients is warranted.

Characteristics of Environmental influences on Child Health Outcomes (ECHO) Cohorts Recruited During Pregnancy.

PURPOSE: The objective of this study was to characterize the study designs, recruitment strategies, and other study characteristics among cohorts that initiated during pregnancy as part of the Environmental influences on Child Health Outcomes (ECHO) program. METHODS: ECHO research programs (cohorts) were reviewed. Only those who had or were currently recruiting during pregnancy were surveyed in 2018 about research recruitment strategies (participant incentives, study burden, community collaboration, and cultural adaptations). Data are presented with cohort characteristics (location, inclusion and exclusion criteria, sociodemographics, medical information, behavioral factors, and biospecimens). RESULTS: Forty-seven of the 84 ECHO pediatric cohorts recruited during pregnancy. Findings demonstrate various recruitment strategies, domains of data collection, and biospecimen collection are all characteristics of successful cohorts. CLINICAL IMPLICATIONS: These data that include over 50,000 children from families across the country, many in underserved areas, will be used for research with the potential to lead to profound policy changes. Prenatal conditions such as maternal age, obesity, depression, and drug use can be examined using study data, including biological markers, from pregnancy through childbirth and into childhood and will inform national policies on the role of early life exposures and underlying mechanisms of disease progression.

A tale of two circuits: CCKNTS neuron stimulation controls appetite and induces opposing motivational states by projections to distinct brain regions.

Cholecystokinin (CCK)-expressing neurons within the nucleus of the solitary tract (CCKNTS) of the mouse 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).

Genetically and functionally defined NTS to PBN brain circuits mediating anorexia.

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 (CGRP(PBN)) neurons; however, the molecular identity of the inputs to these neurons was not established. Here we identify cholecystokinin (CCK) and noradrenergic, dopamine ß-hydroxylase (DBH)-expressing NTS neurons as two separate populations that directly excite CGRP(PBN) 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 CGRP(PBN) neurons to promote anorexia.

Parabrachial nucleus (PBn) pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the amygdala: implication for the sensory and behavioral effects of pain.

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.

Regulation of bed nucleus of the stria terminalis PACAP expression by stress and corticosterone.

Single-nucleotide polymorphisms (SNPs) in the genes for pituitary adenylyl cyclase-activating peptide (PACAP) and the PAC1 receptor have been associated with stress-related psychiatric disorders. Although, from recent work, we have argued that stress-induced PACAP expression in the bed nucleus of the stria terminalis (BNST) may mediate stress-related psychopathology, it is unclear whether stress-induced increases in BNST PACAP expression require acute or repeated stressor exposure and whether increased BNST PACAP expression is related to stress-induced increases in circulating glucocorticoids. In the current work, we have used real-time quantitative polymerase chain reaction (qPCR) to assess transcript expression in brain punches from rats after stressor exposure paradigms or corticosterone injection. BNST PACAP and PAC1 receptor transcript expression was increased only after 7 days of repeated stressor exposure; no changes in transcript levels were observed 2 or 24 hours after a single-restraint session. Moreover, repeated corticosterone treatment for 7 days was not sufficient to reliably increase BNST PACAP transcript levels, suggesting that stress-induced elevations in corticosterone may not be the primary drivers of BNST PACAP expression. These results may help clarify the mechanisms and temporal processes that underlie BNST PACAP induction for intervention in stress-related anxiety disorders.

PAC1 receptor antagonism in the bed nucleus of the stria terminalis (BNST) attenuates the endocrine and behavioral consequences of chronic stress.

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.

Excitotoxic lesions of the bed nucleus of the stria terminalis (BNST) attenuate the effects of repeated stress on weight gain: evidence for the recruitment of BNST activity by repeated, but not acute, stress.

Exposure to repeated stress can lead to diverse and widespread behavioral consequences, including reduction in food and water intake and subsequent diminution in weight gain. Many reports have suggested that repeated stress substantially alters the neurochemistry, morphology and physiology of neurons within the bed nucleus of the stria terminalis (BNST). Here we investigate the role of the BNST in mediating the reduced weight gain observed during repeated stress. Rats exposed to a one-week variate stress paradigm exhibited a reduction in weight gain over the course of the 7-day paradigm. Excitotoxic lesions to a subregion of the anterolateral BNST containing the oval nucleus had no effects early in the 7-day paradigm, but significantly attenuated the effects of repeated stress on weight gain by the last day of stress. These data suggest that at least two mechanisms mediate the effects of stress on body weight gain, and that when stressor exposure becomes repeated, the BNST is recruited, worsening the symptoms of stressor exposure.

Pretreatment with nonselective cationic channel inhibitors blunts the PACAP-induced increase in guinea pig cardiac neuron excitability.

Calcium influx is required for the pituitary adenylyl cyclase activating polypeptide (PACAP)-induced increase in guinea pig cardiac neuron excitability, noted as a change from a phasic to multiple action potential firing pattern. Intracellular recordings indicated that pretreatment with the nonselective cationic channel inhibitors, 2-aminoethoxydiphenylborate (2-APB), 1-[ß-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96365), and flufenamic acid (FFA) reduced the 20-nM PACAP-induced excitability increase. Additional experiments tested whether 2-APB, FFA, and SKF 96365 could suppress the increase in excitability by PACAP once it had developed. The increased action potential firing remained following application of 2-APB but was diminished by FFA. SKF 96365 transiently depressed the PACAP-induced excitability increase. A decrease and recovery of action potential amplitude paralleled the excitability shift. Since semiquantitative PCR indicated that cardiac neurons express TRPC subunit transcripts, we hypothesize that PACAP activates calcium-permeable, nonselective cationic channels, which possibly are members of the TRPC family. Our results are consistent with calcium influx being required for the initiation of the PACAP-induced increase in excitability, but suggest that it may not be required to sustain the peptide effect. The present results also demonstrate that nonselective cationic channel inhibitors could have other actions, which might contribute to the inhibition of the PACAP-induced excitability increase.

Roles for pituitary adenylate cyclase-activating peptide (PACAP) expression and signaling in the bed nucleus of the stria terminalis (BNST) in mediating the behavioral consequences of chronic stress.

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 anxiety-like 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 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 and brainstem regions (Walker et al. Eur J Pharmacol 463:199-216, 2003, Prog Neuropsychopharmacol Biol Psychiatry 33(8):1291-1308, 2009; Ulrich-Lai and Herman Nat Rev Neurosci 10:397-409, 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. Behav Neurosci 120:324-336, 2006) and/or when the anxiety-like response is sustained (Walker and Davis Brain Struct Funct 213:29-42, 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. Brain Res 965:290-294, 2003; Pego et al. Eur J Neurosci 27:1503-1516, 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 is substantially upregulated following chronic stress, particularly in the BNST oval nucleus where PACAP-containing neurons closely interact with CRH-containing neurons (Kozicz et al. Brain Res 767:109-119, 1997; Hammack et al. Psychoneuroendocrinology 34:833-843, 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.