Etiology, triggers and neurochemical circuits associated with unexpected, expected, and laboratory-induced panic attacks

Panic disorder (PD) is a severe anxiety disorder that is characterized by recurrent panic attacks (PA), which can be unexpected (uPA, i.e., no clear identifiable trigger) or expected (ePA). Panic typically involves an abrupt feeling of catastrophic fear or distress accompanied by physiological symptoms such as palpitations, racing heart, thermal sensations, and sweating. Recurrent uPA and ePA can also lead to agoraphobia, where subjects with PD avoid situations that were associated with PA. Here we will review recent developments in our understanding of PD, which includes discussions on: symptoms and signs associated with uPA and ePAs; Diagnosis of PD and the new DSM-V; biological etiology such as heritability and gene×environment and gene×hormonal development interactions; comparisons between laboratory and naturally occurring uPAs and ePAs; neurochemical systems that are associated with clinical PAs (e.g. gene associations; targets for triggering or treating PAs), adaptive fear and panic response concepts in the context of new NIH RDoc approach; and finally strengths and weaknesses of translational animal models of adaptive and pathological panic states

OREXIN 1 AND 2 RECEPTOR INVOLVEMENT IN CO2 -INDUCED PANIC-ASSOCIATED BEHAVIOR AND AUTONOMIC RESPONSES

BACKGROUND: The neuropeptides orexin A and B play a role in reward and feeding and are critical for arousal. However, it was not initially appreciated that most prepro-orexin synthesizing neurons are almost exclusively concentrated in the perifornical hypothalamus, which when stimulated elicits panic-associated behavior and cardiovascular responses in rodents and self-reported "panic attacks" and "fear of dying" in humans. More recent studies support a role for the orexin system in coordinating an integrative stress response. For instance, orexin neurons are highly reactive to anxiogenic stimuli, are hyperactive in anxiety pathology, and have strong projections to anxiety and panic-associated circuitry. Although the two cognate orexin receptors are colocalized in many brain regions, the orexin 2 receptor (OX2R) most robustly maps to the histaminergic wake-promoting region, while the orexin 1 receptor (OX1R) distribution is more exclusive and dense in anxiety and panic circuitry regions, such as the locus ceruleus. Overall, this suggests that OX1Rs play a critical role in mobilizing anxiety and panic responses. METHODS: Here, we used a CO2 -panic provocation model to screen a dual OX1/2R antagonist (DORA-12) to globally inhibit orexin activity, then a highly selective OX1R antagonist (SORA1, Compound 56) or OX2R antagonist (SORA2, JnJ10397049) to assess OX1R and OX2R involvement. RESULTS: All compounds except the SORA2 attenuated CO2 -induced anxiety-like behaviors, and all but the SORA2 and DORA attenuated CO2 -induced cardiovascular responses. CONCLUSIONS: SORA1s may represent a novel method of treating anxiety disorders, with no apparent sedative effects that were present with a benzodiazepine

Evaluation of Low versus High Volume per Minute Displacement CO₂ Methods of Euthanasia in the Induction and Duration of Panic-Associated Behavior and Physiology

Current recommendations for the use of CO ₂ as a euthanasia agent for rats require the use of gradual fill protocols (such as 10% to 30% volume displacement per minute) in order to render the animal insensible prior to exposure to levels of CO ₂ that are associated with pain. However, exposing rats to CO ₂ , concentrations as low as 7% CO ₂ are reported to cause distress and 10%-20% CO ₂ induces panic-associated behavior and physiology, but loss of consciousness does not occur until CO ₂ concentrations are at least 40%. This suggests that the use of the currently recommended low flow volume per minute displacement rates create a situation where rats are exposed to concentrations of CO ₂ that induce anxiety, panic, and distress for prolonged periods of time. This study first characterized the response of male rats exposed to normoxic 20% CO ₂ for a prolonged period of time as compared to room air controls. It demonstrated that rats exposed to this experimental condition displayed clinical signs consistent with significantly increased panic-associated behavior and physiology during CO ₂ exposure. When atmospheric air was then again delivered, there was a robust increase in respiration rate that coincided with rats moving to the air intake. The rats exposed to CO ₂ also displayed behaviors consistent with increased anxiety in the behavioral testing that followed the exposure. Next, this study assessed the behavioral and physiologic responses of rats that were euthanized with 100% CO ₂ infused at 10%, 30%, or 100% volume per minute displacement rates. Analysis of the concentrations of CO ₂ and oxygen in the euthanasia chamber and the behavioral responses of the rats suggest that the use of the very low flow volume per minute displacement rate (10%) may prolong the duration of panicogenic ranges of ambient CO ₂ , while the use of the higher flow volume per minute displacement rate (100%) increases agitation. Therefore, of the volume displacement per minute rates evaluated, this study suggests that 30% minimizes the potential pain and distress experienced by the animal

Anxiogenic CO2 Stimulus Elicits Exacerbated Hot Flash-like Responses in a Rat Menopause Model and Hot Flashes in Menopausal Women

Objective Since longitudinal studies determined that anxiety is a strong risk factor for hot flashes, we hypothesized that an anxiogenic stimulus that signals air hunger (hypercapnic, normoxic gas) would trigger an exacerbated hot flash-associated increase in tail skin temperature (TST) in a rat ovariectomy (OVEX) model of surgical menopause and hot flashes in symptomatic menopausal women. We also assessed TST responses in OVEX serotonin transporter (SERT)+/− rats that models a common polymorphism that is associated with increased climacteric symptoms in menopausal women and increases in anxiety traits. Methods OVEX and sham-OVEX rats (initial experiment) and wildtype and SERT+/− OVEX rats (subsequent experiment) were exposed to a 5 min infusion of 20%CO2 normoxic gas while measuring TST. Menopausal women were given brief 20% and 35%CO2 challenges, and hot flashes were self-reported and objectively verified. Results Compared to controls, OVEX rats had exacerbated increases in TST, and SERT+/− OVEX rats had prolonged TST increases following CO2. Most women reported mild/moderate hot flashes after CO2 challenges, and the hot flash severity to CO2 was positively correlated with daily hot flash frequency. Conclusions The studies demonstrate that this anxiogenic stimulus is capable of inducing cutaneous vasomotor responses in OVEX rats, and eliciting hot flashes in menopausal women. In rats, the severity of the response was mediated by loss of ovarian function and increased anxiety traits (SERT+/−), and, in women, by daily hot flash frequency. These findings may provide insights into anxiety related triggers and genetic risk factors for hot flashes in thermoneutral environments

Hypothalamic orexin’s role in exacerbated cutaneous vasodilation responses to an anxiogenic stimulus in a surgical menopause model

Distressing symptoms such as hot flashes and sleep disturbances affect over 70% of women approaching menopause for an average of 4-7 years, and recent large cohort studies have shown that anxiety and stress are strongly associated with more severe and persistent hot flashes and can induce hot flashes. Although high estrogen doses alleviate symptoms, extended use increases health risks, and current non-hormonal therapies are marginally better than placebo. The lack of effective non-hormonal treatments is largely due to the limited understanding of the mechanisms that underlie menopausal symptoms. One mechanistic pathway that has not been explored is the wake-promoting orexin neuropeptide system. Orexin is exclusively synthesized in the estrogen receptor rich perifornical hypothalamic region, and has an emerging role in anxiety and thermoregulation. In female rodents, estrogens tonically inhibit expression of orexin, and estrogen replacement normalizes severely elevated central orexin levels in postmenopausal women. Using an ovariectomy menopause model, we demonstrated that an anxiogenic compound elicited exacerbated hot flash-associated increases in tail skin temperature (TST, that is blocked with estrogen), and cellular responses in orexin neurons and efferent targets. Furthermore, systemic administration of centrally active, selective orexin 1 or 2 and dual receptor antagonists attenuated or blocked TST responses, respectively. This included the reformulated Suvorexant, which was recently FDA-approved for treating insomnia. Collectively, our data support the hypothesis that dramatic loss of estrogen tone during menopausal states leads to a hyperactive orexin system that contributes to symptoms such as anxiety, insomnia, and more severe hot flashes. Additionally, orexin receptor antagonists may represent a novel non-hormonal therapy for treating menopausal symptoms, with minimal side effects

Reliability of Striatal [11C]Raclopride Binding in Smokers Wearing Transdermal Nicotine Patches

PURPOSE: In studies where [(11)C]raclopride (RAC) positron emission tomography (PET) is used to assess changes in striatal dopamine, it is important to control for cognitive states, such as drug craving, that could alter dopamine levels. In cigarette smokers, transdermal nicotine patches (TNP) can control nicotine craving, but the effects of nicotine patches on RAC binding are unknown. Thus, we sought to determine the test-retest reliability of RAC binding in the presence of nicotine patches. METHODS: Eleven male smokers were scanned twice with RAC on separate days while wearing TNP. RESULTS: Across the striatum, test-retest variability was 7.63 ± 5.88; percent change in binding potential was 1.11 ± 9.83; and the intraclass correlation coefficient was 0.91 (p < 0.0001). CONCLUSION: Baseline RAC binding is highly reproducible in smokers wearing nicotine patches. This suggests that TNP are an acceptable method for controlling cigarette craving during studies that utilize RAC to examine changes in dopamine

Pharmacological depletion of serotonin in the basolateral amygdala complex reduces anxiety and disrupts fear conditioning

The basolateral and lateral amygdala nuclei complex (BLC) is implicated in a number of emotional responses including conditioned fear and social anxiety. Based on previous studies demonstrating that enhanced serotonin release in the BLC leads to increased anxiety and fear responses, we hypothesized that pharmacologically depleting serotonin in the BLC using 5,7-dihydroxytryptamine (5,7-DHT) injections would lead to diminished anxiety and disrupted fear conditioning. To test this hypothesis, 5,7-DHT(a serotonin-depleting agent) was bilaterally injected into the BLC. Desipramine (a norepinephrine reuptake inhibitor) was systemically administered to prevent non-selective effects on norepinephrine. After 5days, 5-7-DHT-treated rats showed increases in the duration of social interaction (SI) time, suggestive of reduced anxiety-like behavior. We then used a cue-induced fear conditioning protocol with shock as the unconditioned stimulus and tone as the conditioned stimulus for rats pretreated with bilateral 5,7-DHT, or vehicle, injections into the BLC. Compared to vehicle-treated rats, 5,7-DHT rats had reduced acquisition of fear during conditioning (measured by freezing time during tone), also had reduced fear retrieval/recall on subsequent testing days. Ex vivo analyses revealed that 5,7-DHT reduced local 5-HT concentrations in the BLC by ~40% without altering local norepinephrine or dopamine concentrations. These data provide additional support for 5-HT playing a critical role in modulating anxiety-like behavior and fear-associated memories through its actions within the BLC

Differences in IV alcohol-induced dopamine release in the ventral striatum of social drinkers and nontreatment-seeking alcoholics

Background Striatal dopamine (DA) has been implicated in alcohol use disorders, but it is still unclear whether or not alcohol can induce dopamine release in social drinkers. Furthermore, no data exist on dopamine responses to alcohol in dependent drinkers. We sought to characterize the DA responses to alcohol intoxication in moderately large samples of social drinkers (SD) and nontreatment-seeking alcoholics (NTS). Methods Twenty-four SD and twenty-one NTS received two [11C]raclopride (RAC) PET scans; one at rest, and one during an intravenous alcohol infusion, with a prescribed ascent to a target breath alcohol concentration (BrAC), at which it was then “clamped.” The alcohol clamp was started 5 min after scan start, with a linear increase in BrAC over 15 min to the target of 80 mg%, the legal threshold for intoxication. Target BrAC was maintained for 30 min. Voxel-wise binding potential (BPND) was estimated with MRTM2. Results IV EtOH induced significant increases in DA in the right ventral striatum in NTS, but not SD. No decreases in DA were observed in either group. Conclusions Alcohol intoxication results in distinct anatomic profiles of DA responses in SD and NTS, suggesting that in NTS, the striatal DA system may process effects of alcohol intoxication differently than in SD

Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase

Children with Neurofibromatosis type 1 (NF1) are increasingly recognized to have high prevalence of social difficulties and autism spectrum disorders (ASD). We demonstrated selective social learning deficit in mice with deletion of a single Nf1 gene (Nf1+/−), along with greater activation of mitogen activated protein kinase pathway in neurons from amygdala and frontal cortex, structures relevant to social behaviors. The Nf1+/− mice showed aberrant amygdala glutamate/GABA neurotransmissiondeficits in long-term potentiationand specific disruptions in expression of two proteins associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (ADAM22) and heat shock protein 70 (HSP70), respectively. All of these amygdala disruptions were normalized by co-deletion of p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1+/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide novel insights and therapeutic targets for NF1 and ASD patients