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In humans, adverse physical and/or psychological traumas in childhood may predispose to developing psychiatric disorders in adulthood, including panic disorder. To model early life adversity in mice, we subjected male and female C57BL/6 J mice to a limited bedding and nesting (LBN) protocol between postnatal days 2-9 and investigated its effect on responsiveness to panicogenic challenges in adulthood. Panic-like escape behaviour was assessed during exposure to a high concentration of CO 2 (20%) or in the beetle mania task (BMT), used to model respiratory and non-respiratory-related types of panic respectively. Neonatal exposure to LBN increased panic-like jumping during the CO 2 challenge in male but not female mice. In an initial pharmacological validation of the BMT as a panic-inducing paradigm, undirected jumping and horizontal escape behaviours were reduced significantly by the panicolytic alprazolam (0.05 and 0.1mg.kg -1 i.p.) whilst tolerance to the close proximity of the aversive robo-beetle increased. The anxiolytic diazepam (1 mg.kg -1  i.p.) reduced only the number of horizontal escape attempts. In both sexes, previous experience of LBN significantly enhanced the number of horizontal escape episodes, indicating a pro-panic phenotype. Directed escape to access a safe ledge on the wall of the test arena, which was seen only in males, was also reduced significantly following LBN. These findings indicate that early life adversity produced by fragmented and unpredictable maternal care promotes a sex-specific increase in susceptibility to panic-like behaviour in adulthood. Whilst non-respiratory-related panic-like behaviour was enhanced in both sexes, females were resilient to respiratory-related challenges., (© 2024 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

CO 2 exposure has been used to investigate the panicogenic response in patients with panic disorder. These patients are more sensitive to CO 2 , and more likely to experience the "false suffocation alarm" which triggers panic attacks. Imbalances in locus coeruleus noradrenergic (LC-NA) neurotransmission are responsible for psychiatric disorders, including panic disorder. These neurons are sensitive to changes in CO 2 /pH. Therefore, we investigated if LC-NA neurons are differentially activated after severe hypercapnia in mice. Further, we evaluated the participation of LC-NA neurons in ventilatory and panic-like escape responses induced by 20% CO 2 in male and female wild type mice and two mouse models of altered LC-NA synthesis. Hypercapnia activates the LC-NA neurons, with males presenting a heightened level of activation. Mutant males lacking or with reduced LC-NA synthesis showed hypoventilation, while animals lacking LC noradrenaline present an increased metabolic rate compared to wild type in normocapnia. When exposed to CO 2 , males lacking LC noradrenaline showed a lower respiratory frequency compared to control animals. On the other hand, females lacking LC noradrenaline presented a higher tidal volume. Nevertheless, no change in ventilation was observed in either sex. CO 2 evoked an active escape response. Mice lacking LC noradrenaline had a blunted jumping response and an increased freezing duration compared to the other groups. They also presented fewer racing episodes compared to wild type animals, but not different from mice with reduced LC noradrenaline. These findings suggest that LC-NA has an important role in ventilatory and panic-like escape responses elicited by CO 2 exposure in mice., Competing Interests: Declaration of competing interest The authors have no competing interests to declare and have no relevant financial or non- financial interests to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bradykinin (BK), a well-studied mediator of physiological and pathological processes in the peripheral system, has garnered less attention regarding its function in the central nervous system, particularly in behavioural regulation. This review delves into the historical progression of research focused on the behavioural effects of BK and other drugs that act via similar mechanisms to provide new insights into the pathophysiology and pharmacotherapy of psychiatric disorders. Evidence from experiments with animal models indicates that BK modulates defensive reactions associated with panic symptoms and the response to acute stressors. The mechanisms are not entirely understood but point to complex interactions with other neurotransmitter systems, such as opioids, and intracellular signalling cascades. By addressing the existing research gaps in this field, we present new proposals for future research endeavours to foster a new era of investigation regarding BK's role in emotional regulation. Implications for psychiatry, chiefly for panic and depressive disorders are also discussed.

Clinical and preclinical studies point towards anxiolytic actions of cannabidiol (CBD), but its effect in panic disorder has been less explored and few studies consider effects in females. We here compared the effect of CBD on the response of male and female rats and mice to a panicogenic challenge; exposure to low O 2 (rats) or high CO 2 (mice) paying attention in females to possible effects of estrous cycle phase. Male and female Sprague-Dawley rats and C57BL/6 J mice were exposed to 7% O 2 for 5 min (rats) or 20% CO 2 (mice) and escape behaviour, which has been associated with panic attacks, was quantified as undirected jumps towards the gas chamber's ceiling. The effect of pretreatment with CBD (1-10 mg kg -1 i.p. in rats or 10-60 mg kg -1 i.p. in mice) was tested. The results showed that low O 2 (rats) or high CO 2 (mice) evoked escape in both sexes. In female rats the response was estrous cycle-sensitive: females in late diestrus made significantly more jumps than females in proestrus. In female mice escape was not influenced by estrous cycle phase and CBD was panicolytic. In female rats CBD attenuated escape behaviour in late diestrus phase but not in proestrus. In male rats and mice CBD had no effect on escape behaviour. Therefore, CBD is panicolytic in female rats and mice but not in males. In rats the effect is estrous cycle-sensitive: rats were most responsive to CBD in late diestrus. In mice higher doses were required to elicit effects and estrous cycle had no effect., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A wealth of evidence associates disruptions of the parent-infant relationship (e.g. childhood parental loss or parental neglect) with the later appearance of panic disorder. In rodents, neonatal maternal separation and maternal deprivation (MD) are reported to increase the expression of anxiety-related defensive responses in adult animals. However, little is known about the long-term consequences of these early-life stressors in animal models of panic. We here investigated the effects of a single 24 h-episode of MD on post-natal day 11 (PND 11) in adult male Wistar rats submitted to two animal models that associate escape expression with panic attacks: the elevated T-maze and exposure to severe hypoxia (7% O 2 ). We also investigated the involvement of serotonin (5-HT) in the observed changes. Although neonatal MD did not affect the behavioral responses measured in the elevated T-maze, it facilitated the expression of escape during hypoxia exposure, indicating a panicogenic-like effect. Pre-test administration of the 5-HT synthesis inhibitor, para-chlorophenylalanine (PCPA; 4 daily injections of 100 mg/kg) facilitated escape attempts in non-deprived animals during the hypoxia challenge, but did not interfere with the expression of this behavior in maternally-deprived rats. The levels of 5-HT1A receptors in key panic- and anxiety-associated areas, the dorsal periaqueductal gray and amygdala, respectively, were not different between previously deprived and non-deprived animals. Plasma corticosterone levels were significantly increased by hypoxia exposure, independently of the animals' previous stress condition or PCPA administration. Therefore, MD on PND 11 predisposes the adult animal to the panic-evoking effects of severe hypoxia, a stimulus also reported to induce panic attacks in humans. The lack of PCPA effect on the pro-escape consequence of MD may be indicative that 5-HT signaling is impaired in the stressed animal., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The antidepressant effect of ketamine has been widely acknowledged and the use of one of its enantiomers, S-ketamine (esketamine), has recently been approved for the clinical management of treatment-resistant depression. As with ketamine, the non-selective opioid receptor-interacting drug buprenorphine is reported to have antidepressant and anxiolytic properties in humans and rodents. Given the fact that antidepressant drugs are also first line treatment for panic disorder, it is surprising that the potential panicolytic effect of these compounds has been scarcely (ketamine), or not yet (buprenorphine) investigated. We here evaluated the effects of ketamine (the racemic mixture), esketamine, and buprenorphine in male Wistar rats submitted to a panicogenic challenge: acute exposure to hypoxia (7% O 2 ). We observed that esketamine (20 mg/kg), but not ketamine, decreased the number of escape attempts made during hypoxia, and this effect could be observed even 7 days after the drug administration. A panicolytic-like effect was also observed with MK801, which like esketamine, antagonizes NMDA glutamate receptors. Buprenorphine (0.3 mg/kg) also impaired hypoxia-induced escape, an effect blocked by the non-selective opioid receptor antagonist naloxone, indicating an interaction with classical ligand sites, such as µ and kappa receptors, but not with nociception/orphanin FQ receptors. Altogether, the results suggest that esketamine and buprenorphine cause rapid-onset panicolytic-like effects, and may be alternatives for treating panic disorder, particularly in patients who are refractory to standard pharmacological treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Anxiety-related diseases are more than twice as common in women than in men, and in women, symptoms may be exacerbated during the late luteal phase of the menstrual cycle. Despite this, most research into the underlying mechanisms, which drives drug development, have been carried out using male animals. In an effort to redress this imbalance, we compared responses of male and female Wistar rats during exposure to two unconditioned threatening stimuli that evoke panic-related defensive behaviours: confrontation with a predator (Bothrops alternatus) and acute exposure to hypoxia (7% O 2 ). Threatened by venomous snake, male and female rats initially displayed defensive attention, risk assessment, and cautious interaction with the snake, progressing to defensive immobility to overt escape. Both males and females displayed higher levels of risk assessment but less interaction with the predator. They also spent more time in the burrow, displaying inhibitory avoidance, and more time engaged in defensive attention, and non-oriented escape behaviour. In females, anxiety-like behaviour was most pronounced in the oestrous and proestrus phases whereas panic-like behaviour was more pronounced during the dioestrus phase, particularly during late dioestrus. Acute hypoxia evoked panic-like behaviour (undirected jumping) in both sexes, but in females, responsiveness in late dioestrus was significantly greater than at other stages of the cycle. The results reveal that females respond in a qualitatively similar manner to males during exposure to naturally occurring threatening stimuli, but the responses of females is oestrous cycle dependent with a significant exacerbation of panic-like behaviour in the late dioestrus phase., (© 2021 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: Acute hypoxia, which is panicogenic in humans, also evokes panic-like behavior in male rats. Panic disorder is more common in women and susceptibility increases during the premenstrual phase of the cycle., Aims: We here investigated for the first time the impact of hypoxia on the expression of panic-like escape behavior by female rats and its relationship with the estrous cycle. We also evaluated functional activation of the midbrain panic circuitry in response to this panicogenic stimulus and whether short-term, low-dose fluoxetine treatment inhibits the hyper-responsiveness of females in late diestrus., Methods: Male and female Sprague Dawley rats were exposed to 7% O 2 . Females in late diestrus were also tested after short-term treatment with fluoxetine (1.75 or 10 mg/kg, i.p.). Brains were harvested and processed for c-Fos and tryptophan hydroxylase immunoreactivity in the periaqueductal gray matter (PAG) and dorsal raphe nucleus (DR)., Results: Acute hypoxia evoked escape in both sexes. Overall, females were more responsive than males and this is clearer in late diestrus phase. In both sexes, hypoxia induced functional activation (c-Fos expression) in non-serotonergic cells in the lateral wings of the DR and dorsomedial PAG, which was greater in late diestrus than proestrus (lowest behavioral response to hypoxia). Increased responding in late diestrus (behavioral and cellular levels) was prevented by 1.75, but not 10 mg/kg fluoxetine., Discussion: The response of female rats to acute hypoxia models panic behavior in women. Low-dose fluoxetine administered in the premenstrual phase deserves further attention for management of panic disorders in women.

Anxiety disorders are more prevalent in women than in men. In women the menstrual cycle introduces another variable; indeed, some conditions e.g., premenstrual syndrome, are menstrual cycle specific. Animal models of fear and anxiety, which form the basis for research into drug treatments, have been developed almost exclusively, using males. There remains a paucity of work using females and the available literature presents a confusing picture. One confound is the estrous cycle in females, which some authors consider, but many do not. Importantly, there are no accepted standardized criteria for defining cycle phase, which is important given the rapidly changing hormonal profile during the 4-day cycle of rodents. Moreover, since many behavioral tests that involve a learning component or that consider extinction of a previously acquired association require several days to complete; the outcome may depend on the phase of the cycle on the days of training as well as on test days. In this article we consider responsiveness of females compared to males in a number of commonly used behavioral tests of anxiety and fear that were developed in male rodents. We conclude that females perform in a qualitatively similar manner to males in most tests although there may be sex and strain differences in sensitivity. Tests based on unconditioned threatening stimuli are significantly influenced by estrous cycle phase with animals displaying increased responsiveness in the late diestrus phase of the cycle (similar to the premenstrual phase in women). Tests that utilize conditioned fear paradigms, which involve a learning component appear to be less impacted by the estrous cycle although sex and cycle-related differences in responding can still be detected. Ethologically-relevant tests appear to have more translational value in females. However, even when sex differences in behavior are not detected, the same outward behavioral response may be mediated by different brain mechanisms. In order to progress basic research in the field of female psychiatry and psychopharmacology, there is a pressing need to validate and standardize experimental protocols for using female animal models of anxiety-related states., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lovick and Zangrossi.)

Changes in 5-HT 1A receptor (5-HT 1A R)-mediated neurotransmission in the hippocampus have been associated with anxiety, depression and in the mode of action of antidepressant drugs. It has been commonly accepted that whereas the dorsal pole of the hippocampus (DH) is involved in cognitive processing, the ventral pole (VH) is associated with emotional regulation. However, to date, only a few studies have directly addressed the role played by VH 5-HT 1A Rs in anxiety and panic processing, and their results are conflicting. Here we report that intra-VH administration of the 5-HT 1A receptor agonist 8-OH-DPAT, the endogenous agonist serotonin (5-HT), or the standard anxiolytic benzodiazepine midazolam impaired the acquisition of inhibitory avoidance in the elevated T-maze (ETM) of male Wistar rats, indicating an anxiolytic effect. Conversely, local injection of the 5-HT 1A R antagonist WAY-100635 caused the opposite effect. These results were equally found in the Vogel conflict test. None of these drugs interfered with locomotor activity in the open-field test, nor did they alter the expression of the escape response in the ETM, a defensive behavior associated with panic. Pre-injection of a sub-effective dose of WAY-100635 in the VH blocked the anxiolytic effect of 5-HT or 8-OH-DPAT in the Vogel test, confirming the involvement of 5-HT 1A R for this behavioral effect. The effect in this test was anxiety-selective as none of the drugs affected water consumption or nociception. In conclusion, our results suggest that 5-HT 1A Rs in the VH play a tonic inhibitory role in anxiety processing. These receptors, however, are not involved in the regulation of panic-related escape behavior., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antidepressant drugs are first-line treatment for panic disorder. Facilitation of 5-HT 1A receptor-mediated neurotransmission in the dorsal periaqueductal gray (dPAG), a key panic-associated area, has been implicated in the panicolytic effect of the selective serotonin reuptake inhibitor fluoxetine. However, it is still unknown whether this mechanism accounts for the antipanic effect of other classes of antidepressants drugs (ADs) and whether the 5-HT interaction with 5-HT 2C receptors in this midbrain area (which increases anxiety) is implicated in the anxiogenic effect caused by short-term treatment with ADs. The results showed that previous injection of the 5-HT 1A receptor antagonist WAY-100635 in the dPAG blocked the panicolytic-like effect caused by chronic systemic administration of the tricyclic AD imipramine in male Wistar rats tested in the elevated T-maze. Neither chronic treatment with imipramine nor fluoxetine changed the expression of 5-HT 1A receptors in the dPAG. Treatment with these ADs also failed to significantly change ERK1/2 (extracellular-signal regulated kinase) phosphorylation level in this midbrain area. Blockade of 5-HT 2C receptors in the dPAG with the 5-HT 2C receptor antagonist SB-242084 did not change the anxiogenic effect caused by a single acute injection of fluoxetine or imipramine in the Vogel conflict test. These results reinforce the view that the facilitation of 5-HT 1A receptor-mediated neurotransmission in the dPAG is a common mechanism involved in the panicolytic effect caused by chronic administration of ADs. On the other hand, the anxiogenic effect observed after short-term treatment with these drugs does not depend on 5-HT 2C receptors located in the dPAG., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Stimulation of serotonergic neurons within the dorsal raphe dorsomedial subnucleus facilitates inhibitory avoidance acquisition in the elevated T-maze. It has been hypothesized that such anxiogenic effect is due to serotonin release in the basolateral nucleus of the amygdala, where facilitation of serotonin 2C receptor-mediated neurotransmission increases anxiety. Besides the dorsal raphe dorsomedial subnucleus, the dorsal raphe caudal subnucleus is recruited by anxiogenic stimulus/situations. However, the behavioral consequences of pharmacological manipulation of this subnucleus are still unknown., Aims: Investigate whether blockade of serotonin 2C receptors in the basolateral nucleus of the amygdala counteracts the anxiogenic effect caused by the stimulation of dorsal raphe dorsomedial subnucleus serotonergic neurons. Evaluate the effects caused by the excitatory amino acid kainic acid or serotonin 1A receptor-modulating drugs in the dorsal raphe caudal subnucleus., Methods: Male Wistar rats were tested in the elevated T-maze and light-dark transition tests after intra-basolateral nucleus of the amygdala injection of the serotonin 2C receptor antagonist SB-242084 (6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride) followed by intra-dorsal raphe dorsomedial subnucleus administration of the serotonin 1A receptor antagonist WAY-100635 (N-[2-[4-2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinil-cyclohexanecarboxamide maleate). In the dorsal raphe caudal subnucleus, animals were injected with kainic acid, WAY-100635 or the serotonin 1A receptor agonist 8-OH-DPAT ((±)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide) and tested in the elevated T-maze., Results: SB-242084 in the basolateral nucleus of the amygdala blocked the anxiogenic effect caused by the injection of WAY-100635 in the dorsal raphe dorsomedial subnucleus. Kainic acid in the dorsal raphe caudal subnucleus increased anxiety, but also impaired escape expression in the elevated T-maze. Neither WAY-100635 nor 8-OH-DPAT in the dorsal raphe caudal subnucleus affected rat's behavior in the elevated T-maze., Conclusion: Serotonin 2C receptors in the basolateral nucleus of the amygdala mediate the anxiogenic effect caused by the stimulation of serotonergic neurons in the dorsal raphe dorsomedial subnucleus. The dorsal raphe caudal subnucleus regulates anxiety- and panic-like behaviors, presumably by a serotonin 1A receptor-independent mechanism.

Introduction and Objectives: Oxytocin (OT) has been widely linked to positive social interactions, and there is great interest in OT as a therapy for a variety of neuropsychiatric conditions. Recent evidence also suggests that OT can play an important role in the mediation of anxiety-associated defensive responses, including a role for serotonin (5-HT) neurotransmission in this action. However, it is presently unknown whether OT additionally regulates the expression of panic-related behaviors, such as escape, by acting in the dorsal periaqueductal gray (dPAG), a key panic-regulating area. This study aimed to investigate the consequence of OT injection in the dPAG on escape expression and whether facilitation of 5-HT neurotransmission in this midbrain area is implicated in this action., Methods: Male Wistar rats were injected with OT in the dPAG and tested for escape expression in the elevated T-maze (ETM) and dPAG electrical stimulation tests. Using the latter test, OT's effect was also investigated after previous intra-dPAG injection of the OT receptor antagonist atosiban, the preferential antagonists of 5-HT 1A and 5-HT 2A receptors, WAY-100635 and ketanserin, respectively, or systemic pretreatment with the 5-HT synthesis inhibitor p-CPA., Results: OT impaired escape expression in the two tests used, suggesting a panicolytic-like effect. In the ETM, the peptide also facilitated inhibitory avoidance acquisition, indicating an anxiogenic effect. Previous administration of atosiban, WAY-100635, ketanserin, or p-CPA counteracted OT's anti-escape effect., Conclusions: OT and 5-HT in the dPAG interact in the regulation of panic- and anxiety-related defensive responses. These findings open new perspectives for the development of novel therapeutic strategies for the treatment of anxiety disorders.

Background: Antidepressants are the first-choice for pharmacological treatment of panic disorder. However, they present disadvantages, such as delayed therapeutic effect, many side effects and a considerable rate of non-responders. These shortcomings prompt the development of new therapeutic strategies. Among these are the adjunctive use of enkephalinase inhibitors, such as opiorphin, which supposedly acts by increasing the availability of brain enkephalins and other endogenous opioids., Aims: We here evaluated whether opiorphin in the dorsal periaqueductal grey matter (dPAG), a key panic-related area, accelerates and/or facilitates the antipanic-like effect of fluoxetine or imipramine. We also verified whether the panicolytic effect of imipramine depends on activation of μ-opioid receptors (MORs)., Methods: Male Wistar rats were submitted to the escape task of the elevated T-maze, an index of panic attack, after treatment with imipramine (3, 7 or 21 days) or fluoxetine (3, 7, 14 or 21 days), combined with an intra-dPAG injection of opiorphin., Results: Opiorphin facilitated and accelerated the panicolytic-like effect caused by imipramine, but not with fluoxetine. The antipanic-like effect caused by chronic imipramine did not depend on MOR activation in the dPAG., Conclusion: Combined treatment of antidepressant drugs with opiorphin for hastening or potentiating the effects of the former compounds may not be generally effective, with the results varying depending on the type/class of these panicolytic drugs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A wealth of evidence indicates that the lateral wings subnucleus of the dorsal raphe nucleus (lwDR) is implicated in the processing of panic-associated stimuli. Escape expression in the elevated T-maze, considered a panic-related defensive behavior, markedly and selectively recruits non-serotonergic cells within this DR subregion and in the dorsal periaqueductal gray (dPAG), another key panic-associated area. However, whether anti-panic drugs may interfere with this pattern of neuronal activation is still unknown. In the present study, the effects of acute (10 mg/kg) or chronic fluoxetine (10 mg/kg/daily/21 days) treatment on the number of serotonergic and non-serotonergic cells induced by escape expression within the rat DR and PAG subnuclei were investigated by immunochemistry. The results showed that chronic, but not acute, treatment with fluoxetine impaired escape expression, indicating a panicolytic-like effect, and markedly decreased the number of non-serotonergic cells that were recruited in the lwDR and dPAG. The same treatment selectively increased the number of serotonergic neurons within the lwDR. Our immunochemistry analyses also revealed that the non-serotonergic cells recruited in the lwDR and dPAG by the escape expression were not nitrergic. Overall, our findings suggest that the anti-panic effect of chronic treatment with fluoxetine is mediated by stimulation of the lwDR-dPAG pathway that controls the expression of panic-associated escape behaviors.

Exposure of rats to an environment with low O 2 levels evokes a panic-like escape behavior and recruits the dorsal periaqueductal gray (dPAG), which is considered to be a key region in the pathophysiology of panic disorder. The neurochemical basis of this response is, however, currently unknown. We here investigated the role played by nitric oxide (NO) within the dPAG in mediation of the escape reaction induced by hypoxia exposure. The results showed that exposure of male Wistar rats to 7% O 2 increased nitrite levels, a NO metabolite, in the dPAG but not in the amygdala or hypothalamus. Nitrite levels in the dPAG were correlated with the number of escape attempts during the hypoxia challenge. Injections of the NO synthesis inhibitor NPA, the NO-scavenger c- PTIO, or the NMDA receptor antagonist AP-7 into the dorsolateral column of the periaqueductal gray (dlPAG) inhibited escape expression during hypoxia, without affecting the rats' locomotion. Intra-dlPAG administration of c-PTIO had no effect on the escape response evoked by the elevated-T maze, a defensive behavior that has also been associated with panic attacks. Altogether, our results suggest that NO plays a critical role in mediation of the panic-like defensive response evoked by exposure to low O 2 concentrations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Although the etiology of panic disorder (PD) remains elusive, accumulating evidence suggests a key role for the dorsal periaqueductal gray matter (dPAG). There is also evidence that this midbrain area is critically involved in mediation of the panicolytic effect of antidepressants, which with high potency benzodiazepines (e.g. alprazolam and clonazepam) are first line treatment for PD. Whether the dPAG is also implicated in the antipanic effect of the latter drugs is, however, still unknown. We here investigated the consequences of blocking GABA A or benzodiazepine receptors within the dPAG, with bicuculline (5 pmol) and flumazenil (80 nmol), respectively, on the panicolytic and anxiolytic effects of alprazolam (4 mg/kg). Microinjection of these antagonists fully blocked the anti-escape effect, considered as a panicolytic-like action, caused by a single systemic injection of alprazolam in male Wistar rats submitted to the elevated T-maze. These antagonists, however, did not affect the anxiolytic effect of the benzodiazepine on inhibitory avoidance acquisition and punished responding, measured in the elevated T-maze and Vogel conflict tests, respectively. Altogether, our findings show the involvement of GABA A /benzodiazepine receptors of the dPAG in the panicolytic, but not the anxiolytic effect caused by alprazolam. They also implicate the dPAG as the fulcrum of the effects of different classes of clinically effective antipanic drugs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The role of 5-HT2C receptors (5-HT2CRs) in the regulation of anxiety has been widely acknowledged. However, conflicting results have been reported on whether stimulation of these receptors increases or decreases anxiety. We here investigated the role of 5-HT2CRs of the dorsal hippocampus (DH) in the mediation of anxiety- or panic-associated defensive behaviors and in the anxiolytic effect of the tricyclic antidepressant imipramine. In the Vogel conflict test, administration of the mixed 5-HT2CR agonist mCPP into the DH of male Wistar rats was anxiogenic, whereas infusions of the more selective agonists MK-212 and RO-600175 were anxiolytic. The 5-HT2CR antagonist SB-242084, on the other hand, was anxiogenic. A sub-effective dose of this antagonist blocked the anxiolytic effect of RO-600175, but not the increase in anxiety observed with mCPP, indicating that the latter effect was not due to 5-HT2CR activation. In full agreement with these findings, MK-212 and RO-600175 in the DH also inhibited inhibitory avoidance acquisition in the elevated T-maze, whereas SB-242084 caused the opposite effect. None of these drugs interfered with escape expression in this test, which has been associated with panic. Chronic administration of imipramine (15 mg/kg, ip, 21 days) caused an anxiolytic effect in the elevated T-maze and light-dark transition tests, which was not blocked by previous infusion of SB-242084 into the DH. Therefore, facilitation of 5-HT2CR-mediated neurotransmission in the DH decreases the expression of anxiety-, but not panic-related defensive behaviors. This mechanism, however, is not involved in the anxiolytic effect caused by imipramine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background:: Serotonin plays an important role in the regulation of anxiety, acting through complex modulatory mechanisms within distinct brain structures. Serotonin can act through complex negative feedback mechanisms controlling the neuronal activity of serotonergic circuits and downstream physiologic and behavioral responses. Administration of serotonin or the serotonin 1A receptor agonist, (±)-8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT), into the prefrontal cortex, inhibits anxiety-like responses. The prelimbic area of the prefrontal cortex regulates serotonergic neurons within the dorsal raphe nucleus and is involved in modulating anxiety-like behavioral responses., Aims:: This study aimed to investigate the serotonergic role within the prelimbic area on anxiety- and panic-related defensive behavioral responses., Methods:: We investigated the effects of serotonin within the prelimbic area on inhibitory avoidance and escape behaviors in the elevated T-maze. We also extended the investigation to serotonin 1A, 2A, and 2C receptors., Results:: Intra-prelimbic area injection of serotonin or 8-OH-DPAT induced anxiolytic effects without affecting escape behaviors. Previous administration of the serotonin 1A receptor antagonist, WAY-100635, into the prelimbic area counteracted the anxiolytic effects of serotonin. Neither the serotonin 2A nor the serotonin 2C receptor preferential agonists, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) and 6-chloro-2-(1-piperazinyl) pyrazine (MK-212), respectively, affected behavioral responses in the elevated T-maze., Conclusion:: Facilitation of serotonergic signaling within the prelimbic area of rats induced an anxiolytic effect in the elevated T-maze test, which was mediated by local serotonin 1A receptors. This inhibition of anxiety-like defensive behavioral responses may be mediated by prelimbic area projections to neural systems controlling anxiety, such as the dorsal raphe nucleus or basolateral amygdala.

Exposure to elevated concentrations of CO 2 or hypoxia has been widely used in psychiatric research as a panic provoking stimulus. However, the use of these respiratory challenges to model panic-like responses in experimental animals has been less straightforward. Little data is available, from behavioral and endocrine perspectives, to support the conclusion that a marked aversive situation, such as that experienced during panic attacks, was evoked in these animals. We here compared the behavioral responses of male CB57BL/6 mice during exposure to 20% CO 2 or 7% O 2 and its consequence on plasma levels of corticosterone. We also evaluated whether clinically-effective panicolytic drugs affect the behavioral responses expressed during CO 2 exposure. The results showed that whereas hypoxia caused a marked reduction in locomotion, inhalation of CO 2 -enriched air evoked an active escape response, characterized by bouts of upward leaps directed to the border of the experimental cage, interpreted as escape attempts. Corticosterone levels were increased 30min after either of the respiratory challenges used, but it was higher in the hypoxia group. Chronic (21days), but not acute, treatment with fluoxetine or imipramine (5, 10 or 15mg/kg) or a single injection of alprazolam (0.025, 0.05 or 0.1mg/kg), but not of the anxiolytic diazepam (0.025, 0.05 or 0.1 and 1mg/kg) reduced the number of escape attempts, indicating a panicolytic-like effect. Altogether, the results suggest that whereas hypoxia increased anxiety, exposure to 20% CO 2 evoked a panic-like state. The latter condition/test protocol seems to be a simple and validated model for studying in mice pathophysiological mechanisms and the screening of novel drugs for panic disorder., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Reported results have shown that the pentapeptide opiorphin inhibits oligopeptidases that degrade brain neuropeptides, and has analgesic and antidepressant effects in experimental animals, without either tolerance or dependency after chronic administration. In a previous study we showed that opiorphin has a panicolytic-like effect in the dorsal periaqueductal gray (dPAG) electrical stimulation test (EST), mediated by the μ-opioid receptor (MOR). This study further analyzes the mechanism of opiorphin panicolytic action, using the EST and drug injection inside the dPAG. The obtained results showed that blockade of the 5-HT 1A receptors with WAY-100635 did not change the escape-impairing effect of opiorphin, and combined injection of sub-effective doses of opiorphin and the 5-HT 1A -agonist 8-OH-DPAT did not have a significant anti-escape effect. In contrast, the anti-escape effect of opiorphin was antagonized by pretreatment with the kinin B2 receptor blocker HOE-140, and association of sub-effective doses of opiorphin and bradykinin caused a significant anti-escape effect. The anti-escape effect of bradykinin was not affected by previous administration of WAY-100635. Therefore, the anti-escape effect of opiorphin in the dPAG seems to be mediated by endogenous bradykinin, acting on kinin B2 receptors, which previous results have shown to interact synergistically with MOR in the dPAG to restrain escape in two animal models of panic. Chemical compounds: Opiorphin (PubChem CID: 25195667); WAY100635 maleate salt (PubChem CID: 11957721); 8-OH-DPAT hydrobromide (PubChem CID: 6917794); Bradykinin (PubChem CID: 439201); HOE-140 (Icatibant) (PubChem CID: 6918173)., (Copyright © 2017 Elsevier Inc. All rights reserved.)

It is known that diabetic (DBT) animals present dysregulation on the serotonergic system in several brain areas associated with anxiety-like responses. The aim of this study was to investigate the involvement of 5-HT1A receptors on dorsal periaqueductal gray (dPAG) in the behavioral response related to panic disorder in type-1 DBT animals. For this, the escape response by electric stimulation (ES) of dPAG in DBT and normoglycemic (NGL) animals was assessed. Both NGL and DBT animals were exposed to an open-field test (OFT) 28 days after DBT confirmation. The current threshold to induce escape behavior in DBT animals was reduced compared with NGL animals. No impairment in locomotor activity was observed when DBT animals were compared with NGL animals. An intra-dPAG injection of the 5-HT1A receptor agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) increased the [INCREMENT] threshold in both DBT and NGL, suggesting a panicolytic-like effect. DBT animals presented a more pronounced panicolytic-like response compared with NGL as a higher [INCREMENT] threshold was observed after 8-OH-DPAT treatment, which could be a consequence of the increased expression of the 5-HT1A receptor in the dPAG from DBT animals. Our results are in line with the proposal that a deficiency in serotonergic modulation of the dPAG is involved in triggering the panic attack and the 5-HT1A receptors might be essential for the panicolytic-like response.

A wealth of evidence has shown that opioid and kinin systems may control proximal defense in the dorsal periaqueductal gray matter (dPAG), a critical panic-associated area. Studies with drugs that interfere with serotonin-mediated neurotransmission suggest that the μ-opioid receptor (MOR) synergistically interacts with the 5-HT 1A receptor in the dPAG to inhibit escape, a panic-related behavior. A similar inhibitory effect has also been reported after local administration of bradykinin (BK), which is blocked by the non-selective opioid receptor antagonist naloxone. The latter evidence, points to an interaction between BK and opioids in the dPAG. We further explored the existence of this interaction through the dPAG electrical stimulation model of panic. We also investigated whether intra-dPAG injection of captopril, an inhibitor of the angiotensin-converting enzyme (ACE) that also degrades BK, causes a panicolytic-like effect. Our results showed that intra-dPAG injection of BK inhibited escape performance in a dose-dependent way, and this panicolytic-like effect was blocked by the BK type 2 receptor (B2R) antagonist HOE-140, and by the selective MOR antagonist CTOP. Conversely, the panicolytic-like effect caused by local administration of the selective MOR agonist DAMGO was antagonized by pre-treatment with either CTOP or HOE-140, indicating cross-antagonism between MOR and B2R. Finally, intra-dPAG injection of captopril also impaired escape in a dose-dependent way, and this panicolytic-like effect was blocked by pretreatment with HOE-140, suggesting mediation by endogenous BK. The panicolytic-like effect of captopril indicates that the use of ACE inhibitors in the clinical management of panic disorder may be worth exploring., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Several studies have shown that serotonin plays a dual role in the modulation of defensive behaviors related to anxiety and panic. A major source of serotonergic projections to limbic structures responsible for this modulation is the dorsal raphe nucleus (DR). Anatomical studies indicate that the prelimbic (PL) cortex sends dense glutamatergic projections to the DR, leading to stimulation or inhibition of serotonin release in structures innervated by the DR. The objective of the present study was to investigate if GABAergic disinhibition of the PL by means of local administration of picrotoxin (PIC), a chloride channel blocker, can affect serotonergic tone and the expression of defensive behaviors related to anxiety and panic. We used the elevated T-maze model and Vogel conflict test to evaluate defensive responses associated with anxiety or panic. The results showed that intra-PL PIC caused an increase in c-Fos activation in serotonergic cells in DR subregions. Furthermore, the intra-PL injection of PIC induced a panicolytic-like effect without affecting behaviors associated with anxiety. Our findings suggest that the PL-DR pathway, through DR serotonergic stimulation, is involved in the control of panic-related behaviors by control of serotonin release in structures that modulate panic responses, such as the dorsal periaqueductal gray.

Panic patients may have abnormalities in serotonergic and opioidergic neurotransmission. The dorsal periaqueductal gray (dPAG) plays an important role in organizing proximal defense, related to panic attacks. The 5-HT 1A receptor (5-HT 1A -R) is involved in regulating escape behavior that is organized in the dPAG. Activation of κ-opioid receptor (KOR) in this region causes anxiogenic effects. In this study, we investigated the involvement of KOR in regulating escape behavior, using systemic and intra-dPAG injection of the KOR antagonist Nor-BNI. As panic models, we used the elevated T-maze (ETM) and the dPAG electrical stimulation test (EST). We also evaluated whether activation of the 5-HT 1A -R or the μ-opioid receptor (MOR) in the dPAG contributes to the Nor-BNI effects. The results showed that systemic administration of Nor-BNI, either subcutaneously (2.0 and 4.0mg/kg) or intraperitoneally (2.0mg/kg), impaired escape in the EST, indicating a panicolytic-like effect. Intra-dPAG injection of this antagonist (6.8nmol) caused the same effect in the EST and in the ETM. Association of ineffective doses of Nor-BNI and the 5-HT 1A -R agonist 8-OH-DPAT caused panicolytic-like effect in these two tests. Previous administration of the 5-HT 1A -R antagonist WAY-100635, but not of the MOR antagonist CTOP, blocked the panicolytic-like effect of Nor-BNI. These results indicate that KOR enhances proximal defense in the dPAG through 5-HT 1A -R modulation, independently of MOR. Because former results indicate that the 5-HT 1A -R is involved in the antipanic action of antidepressants, KOR antagonists may be useful as adjunctive or alternative drug treatment of panic disorder., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The lateral wings subnucleus of the dorsal raphe nucleus (lwDR) has been implicated in the modulation of panic-like behaviors, such as escape. Infusion of non- excitotoxic doses of the excitatory amino acid kainic acid into this subnucleus promptly evokes a vigorous escape response. In addition, rats exposed to panic-inducing situations show an increase in Fos protein expression in neurons within the lwDR. In the present study, we first investigated whether key structures associated with the mediation of escape behavior are recruited after chemical stimulation of the lwDR with kainic acid. We next investigated whether the infusion of the GABA A receptor antagonist bicuculline into the lwDR also evoked escape responses measured both in a circular arena and in the rat elevated T-maze. The effects of bicuculline in the circular arena were compared to those caused by the infusion of this antagonist into the ventrolateral periaqueductal gray (vlPAG), an area in close vicinity to the lwDR. The results showed that kainic acid infusion into the lwDR increased Fos protein immunostaining in brain structures deeply involved in panic-like defensive behaviors, such as the periaqueductal gray and hypothalamus, but not the amygdala. As observed with kainic acid, bicuculline evoked a pronounced escape response in the circular arena when microinjected in the lwDR, but not in the vlPAG. The escape-promoting effect of bicuculline in the lwDR was also evidenced in the elevated T-maze. These findings strength the view that dysfunction in mechanisms controlling escape in the lwDR is critically implicated in the pathophysiology of panic disorder., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The elevated T-maze was developed to test the hypothesis that serotonin plays an opposing role in the regulation of defensive behaviors associated with anxiety and panic. Previous pharmacological exploitation of this test supports the association between inhibitory avoidance acquisition and escape expression with anxiety and fear/panic, respectively. In the present study, we extend the pharmacological validation of this test by investigating the effects of other putative or clinically effective anxiety- and panic-modulating drugs. The results showed that chronic, but not acute injection of the reversible monoamine oxidase-A inhibitor moclobemide (3, 10 and 30mg/kg) inhibited escape expression, indicating a panicolytic-like effect. The same effect was observed after either acute or chronic treatment with alprazolam (1, 2 and 4mg/kg), a high potency benzodiazepine. This drug also impaired inhibitory avoidance acquisition, suggesting an anxiolytic effect. On the other hand, subcutaneous administration of the 5-HT1D/1B receptor agonist sumatriptan (0.1, 0.5 and 2.5μg/kg) facilitated escape performance, indicating a panicogenic-like effect, while treatment with α-para-chlorophenylalanine (p-CPA; 4days i.p injections of 100mg/kg, or a single i.p injection of 300mg/kg), which caused marked 5-HT depletion in the amygdala and striatum, was without effect. Altogether, these results are in full agreement with the clinical effects of these compounds and offer further evidence that the elevated T-maze has broad predictive validity for the effects of anxiety- and panic-modulating drugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The dorsal raphe nucleus (DR), the main source of 5-HT projections to brain areas involved in anxiety regulation, is composed by 5 subnuclei that differ morphologically, functionally and neurochemically. Based on immunohistochemical evidence, it has been proposed that whereas 5-HT cells of the dorsomedial (dmDR) and caudal subnuclei are implicated in the pathophysiology of generalized anxiety disorder (GAD), neurons of the lateral wings (lwDR) are associated with panic disorder (PD). We here tested this hypothesis from a behavioral perspective by investigating the consequences of the non-selective stimulation of neurons within the dmDR and lwDR, or the pharmacological manipulation of 5-HT1A receptors located in these nuclei, of male Wistar rats exposed to the elevated T-maze. This test allows the measurement of both a GAD- (i.e. inhibitory avoidance) and a PD- (i.e. escape) related response in the same animal. Intra-dmDR injection of either the excitatory amino acid kainic acid or the 5-HT1A receptor antagonist WAY-100635 facilitated inhibitory avoidance acquisition, suggesting an anxiogenic effect, and inhibited escape expression, a panicolytic-like effect. Microinjection of the 5-HT1A receptor agonist 8-OH-DPAT caused the opposite effect. Administration of the same drugs into the lwDR only altered escape performance. Whereas kainic acid and 8-OH-DPAT facilitated its expression, WAY-100635 inhibited it. At higher doses, kainic acid administration evoked vigorous escape reactions as measured in an open-field. These findings implicate 5-HT neurons of the dmDR in the regulation of both GAD- and PD-related defensive behaviors. They also support a primary role of the lwDR in the mediation of PD-associated responses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The dorsal raphe nucleus (DRN) is an important brainstem source of 5-hydroxytryptamine (5-HT), and 5-HT plays a key role in the regulation of panic attacks. The aim of the present study was to determine whether 5-HT1A receptor-containing neurons in the medial hypothalamus (MH) receive neural projections from DRN and to then determine the role of this neural substrate in defensive responses. The neurotracer biotinylated dextran amine (BDA) was iontophoretically microinjected into the DRN, and immunohistochemical approaches were then used to identify 5HT1A receptor-labelled neurons in the MH. Moreover, the effects of pre-treatment of the dorsomedial hypothalamus (DMH) with 8-OH-DPAT and WAY-100635, a 5-HT1A receptor agonist and antagonist, respectively, followed by local microinjections of bicuculline, a GABAA receptor antagonist, were investigated. We found that there are many projections from the DRN to the perifornical lateral hypothalamus (PeFLH) but also to DMH and ventromedial (VMH) nuclei, reaching 5HT1A receptor-labelled perikarya. DMH GABAA receptor blockade elicited defensive responses that were followed by antinociception. DMH treatment with 8-OH-DPAT decreased escape responses, which strongly suggests that the 5-HT1A receptor modulates the defensive responses. However, DMH treatment with WAY-100635 failed to alter bicuculline-induced defensive responses, suggesting that 5-HT exerts a phasic influence on 5-HT1A DMH neurons. The activation of the inhibitory 5-HT1A receptor had no effect on antinociception. However, blockade of the 5-HT1A receptor decreased fear-induced antinociception. The present data suggest that the ascending pathways from the DRN to the DMH modulate panic-like defensive behaviours and mediate antinociceptive phenomenon by recruiting 5-HT1A receptor in the MH., (Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.)

Reported evidence indicates that endogenous opioid peptides regulate the expression of escape behavior in rats, a panic-related defensive response, through μ-opioid receptors (MORs) in the dorsal periaqueductal gray (dPAG). These peptides are rapidly catabolized by degrading enzymes, including neutral endopeptidase (NEP) and aminopeptidase N (APN). Opiorphin is a peptide inhibitor of both NEP and APN and potentiates the action of endogenous enkephalins. This study evaluated the effects of intravenous and intra-dPAG administration of opiorphin on escape responses in the elevated T-maze and in a dPAG electrical stimulation test in rats. We also evaluated the involvement of MORs in the effects of opiorphin using the selective MOR antagonist CTOP. A dose of 2.0 mg/kg, i.v., of opiorphin impaired escape performance in both tests. Similar effects were observed with intra-dPAG administration of 5.0 nmol of opiorphin. Local pretreatment with 1.0 nmol CTOP antagonized the anti-escape effects of intra-dPAG opiorphin in both tests, as well as the effect of systemically administered opiorphin (2.0 mg/kg, i.v.) in the electrical stimulation test. These results indicate that opiorphin has an antipanic-like effect that is mediated by MORs in the dPAG. They may open new perspectives for the development of opiorphin analogues with greater bioavailability and physicochemical characteristics in the pursuit of new medications for the treatment of panic disorder., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Previously reported results have shown that the inhibitory effect of fluoxetine on escape behavior, interpreted as a panicolytic-like effect, is blocked by pretreatment with either the opioid receptor antagonist naloxone or the 5-HT1A receptor (5-HT1A-R) antagonist WAY100635 via injection into the dorsal periaqueductal gray matter (dPAG). Additionally, reported evidence indicates that the μ-opioid receptor (MOR) interacts with the 5-HT1A-R in the dPAG. In the present work, pretreatment of the dPAG with the selective MOR blocker CTOP antagonized the anti-escape effect of chronic fluoxetine (10 mg/kg, i.p., daily, for 21 days), as measured in the elevated T-maze (ETM) test, indicating mediation of this effect by the MOR. In addition, the combined administration of sub-effective doses of the selective MOR agonist DAMGO (intra-dPAG) and sub-effective doses of chronic as well as subchronic (7 days) fluoxetine increased avoidance and escape latencies, suggesting that the activation of MORs may facilitate and accelerate the effects of fluoxetine. The current observation that MORs located in the dPAG mediate the anti-escape effect of fluoxetine may open new perspectives for the development of more efficient and fast-acting panic-alleviating drugs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)