GABAergic and glutamatergic inputs to the medulla oblongata and locus coeruleus noradrenergic pathways are critical for seizures and postictal antinociception neuromodulation.

We investigated the participation of the nucleus of the tractus solitarius (NTS) in tonic‒clonic seizures and postictal antinociception control mediated by NMDA receptors, the role of NTS GABAergic interneurons and noradrenergic pathways from the locus coeruleus (LC) in these phenomena. The NTS-lateral nucleus reticularis paragigantocellularis (lPGi)-LC pathway was studied by evaluating neural tract tracer deposits in the lPGi. NMDA and GABAergic receptors agonists and antagonists were microinjected into the NTS, followed by pharmacologically induced seizures. The effects of LC neurotoxic lesions caused by DSP-4, followed by NTS-NMDA receptor activation, on both tonic‒clonic seizures and postictal antinociception were also investigated. The NTS is connected to lPGi neurons that send outputs to the LC. Glutamatergic vesicles were found on dendrites and perikarya of GABAergic interneurons in the NTS. Both tonic‒clonic seizures and postictal antinociception are partially dependent on glutamatergic-mediated neurotransmission in the NTS of seizing rats in addition to the integrity of the noradrenergic system since NMDA receptor blockade in the NTS and intrathecal administration of DSP-4 decrease the postictal antinociception. The GABAA receptor activation in the NTS decreases both seizure severity and postictal antinociception. These findings suggest that glutamatergic inputs to NTS-GABAergic interneurons, in addition to ascending and descending noradrenergic pathways from the LC, are critical for the control of both seizures and postictal antinociception.

Panicolytic-like effects of environment enrichment on male mice threatened by Bothrops jararaca lancehead pit vipers.

Environment enrichment (EE) is a well-known eustress model showing beneficial effects in different psychiatric diseases, but its positive properties in panic disorders are not yet established. The confrontation between prey and predator in complex arenas has been validated as a putative panic attack model. The principal aim of this work was to investigate the role of the EE on panic-like defensive responses elicited by mice threatened by venomous snakes. After 6 weeks of exposure either to an enriched or standard environments, 36 male mice were habituated in a complex polygonal arena for snakes containing an artificial burrow and elevated platforms for escape. The animals were confronted by Bothrops jararaca for 5 min, and the following antipredatory responses were recorded: defensive attention, stretched attend posture, flat back approach, prey versus predator interaction, oriented escape behavior, time spent in a safe place, and number of crossings. Mice threatened by snakes displayed several antipredatory reactions as compared to the exploratory behavior of those animals submitted to a nonthreatening situation (toy snake) in the same environment. Notably, EE causes anxiolytic- and panicolytic-like effects significantly decreasing the defensive attention and time spent in safe places and significantly increasing both prey versus predator interaction and exploratory behavior. In conclusion, our data demonstrate that EE can alter the processing of fear modulation regarding both anxiety- and panic-like responses in a dangerous condition, significantly modifying the decision-making defensive strategy.

Alpha1- and Beta-norepinephrinergic receptors of dorsomedial and ventromedial hypothalamic nuclei modulate panic attack-like defensive behaviour elicited by diencephalic GABAergic neurotransmission disinhibition.

Gamma-aminobutyric acid (GABA) disinhibition in medial hypothalamus (MH) nuclei of rats elicits some defensive reactions that are considered panic attack-like behaviours. Recent evidence showed that the norepinephrine-mediated system modulates fear-related defensive behaviours organised by MH neurons at least in part via noradrenergic receptors recruitment on midbrain tegmentum. However, it is unknown whether noradrenergic receptors of the MH also modulate the panic attack-like reactions. The aim of this work was to investigate the distribution of noradrenergic receptors in MH, and the effects of either α1-, α2- or ß-noradrenergic receptors blockade in the MH on defensive behaviours elaborated by hypothalamic nuclei. Defensive behaviours were evaluated after the microinjection of the selective GABAA receptor antagonist bicuculline into the MH that was preceded by microinjection of either WB4101, RX821002, propranolol (α1-, α2- and ß-noradrenergic receptor selective antagonists, respectively), or physiological saline into the MH of male Wistar rats. The α1-, α2- and ß-noradrenergic receptors were found in neuronal perikarya of all MH nuclei, and the α2-noradrenergic receptor were also found on glial cells mainly situated in the ventrolateral division of the ventromedial hypothalamic nucleus. The α1- and ß-noradrenergic receptors blockade in the MH decreased defensive attention and escape reactions elicited by the intra-MH microinjections of bicuculline. These findings suggest that, despite the profuse distributions of α1-, α2- and ß-noradrenergic receptors in the MH, both α1- and ß-noradrenergic receptor- rather than α2-noradrenergic receptor-signalling in MH are critical for the neuromodulation of panic-like behaviour.

The anterior cingulate cortex and its interface with the dorsal periaqueductal grey regulating nitric oxide-mediated panic-like behaviour and defensive antinociception.

The anterior cingulate cortex (ACC) Cg1 (24b) area modulates glutamate-mediated unconditioned fear and antinociception organised by hypothalamus. However, it remains unknown whether 24b area also modulates these latter defensive responses through connections with the dorsal periaqueductal grey matter (dPAG), a midbrain structure implicated in the genesis of innate fear-induced defence. The aim of this work is to examine the correlation between the behavioural effects of intra-ACC microinjections of vehicle, NMDA (1 nmol) or lidocaine (2%) with Fos protein expression and nitrergic activity in the dPAG of male C57BL/6 mice that were threatened by snakes. In addition, the 24b area-dPAG pathways were also characterised by neural tract tracing procedures. Finally, the effect of dPAG pretreatment with the neuronal nitric oxide synthase inhibitor N(omega)-propyl-l-arginine (NPLA; 0.2, 0.4 or 0.8 nmol) 10 min before 24b area treatment with NMDA on behavioural and nociceptive responses of threatened mice was studied. The activation of 24b area N-methyl-d-aspartic acid receptors facilitated escape and freezing rather than risk assessment, and enhanced Fos expression and nitrite levels in dPAG, while lidocaine decreased escape and risk assessment as well as Fos and nitrergic activity in dPAG. In addition, dPAG pretreatment with NPLA suppressed intra-24b NMDA-facilitated panicogenic effects while increased nociception. Infusions of an antegrade neurotracer into 24b area showed axonal fibres surrounding both dorsomedial and dorsolateral PAG perikarya. Neurons were identified in 24b area after deposits of a retrograde neurotracer into dPAG. Our findings suggest that the ACC/24b area modulates innate defensive responses through the recruitment of dPAG nitrergic neurons.

Copaifera langsdorffii Desf. tree oleoresin-induced antinociception recruits µ1- and κ -opioid receptors in the ventrolateral columns of the periaqueductal gray matter.

Popular medicine has been using oleoresin from several species of copaíba tree for the treatment of various diseases and its clinical administration potentially causes antinociception. Electrical stimulation of ventrolateral (vlPAG) and dorsolateral (dlPAG) columns of the periaqueductal gray matter also causes antinociception. The aim this study was to verify the antinociceptive effect of oleoresin extracted from Copaifera langsdorffii tree and to test the hypothesis that oleoresin-induced antinociception is mediated by µ1- and κ-opioid receptors in the vlPAG and dlPAG. Nociceptive thresholds were determined by the tail-flick test in Wistar rats. The copaíba tree oleoresin was administered at different doses (50, 100 and 200 mg/kg) through the gavage technique. After the specification of the most effective dose of copaíba tree oleoresin (200 mg/kg), rats were pretreated with either the µ1-opioid receptor selective antagonist naloxonazine (at 0.05, 0.5 and 5 µg/ 0.2 µl in vlPAG, and 5 µg/ 0.2 µl in dlPAG) or the κ-opioid receptor selective antagonist nor-binaltorphimine (at 1, 3 and 9 nmol/ 0.2 µl in vlPAG, and 9 nmol/ 0.2 µl in dlPAG). The blockade of µ1 and κ opioid receptors of vlPAG decreased the antinociception produced by copaíba tree oleoresin. However, the blockade of these receptors in dlPAG did not alter copaíba tree oleoresin-induced antinociception. These data suggest that vlPAG µ1 and κ opioid receptors are critically recruited in the antinociceptive effect produced by oleoresin extracted from Copaifera langsdorffii.

Unravelling the dorsal periaqueductal grey matter NMDA receptors relevance in the nitric oxide-mediated panic­like behaviour and defensive antinociception organised by the anterior hypothalamus of male mice.

RATIONALE: Previous studies suggested that the dorsal column of the periaqueductal grey matter (dPAG) can be a target of neural pathways from hypothalamic nuclei involved in triggering fear-related defensive responses. In turn, evidence is provided suggesting that microinjection of the nitric oxide (NO) donor SIN-1 into the anterior hypothalamus (AH) of mice evokes panic-like behaviours and fear-induced antinociception. However, it is unknown whether the dPAG of mice mediates these latter defensive responses organised by AH neurons. OBJECTIVES: This study was designed to examine the role of dPAG in mediating SIN-1-evoked fear-induced defensive behavioural and antinociceptive responses organised in the AH of mice. METHODS: First, neural tract tracing was performed to characterise the AH-dPAG pathways. Then, using neuropharmacological approaches, we evaluated the effects of dPAG pretreatment with either the non-selective synaptic blocker cobalt chloride (CoCl2; 1 mM/0.1 µL) or the competitive N-methyl-D-aspartate (NMDA) receptor antagonist LY235959 (0.1 nmol/0.1 µL) on defensive behaviours and antinociception induced by microinjections of SIN-1 in the AH of male C57BL/6 mice. RESULTS: AlexaFluor488-conjugated dextran-labelled axonal fibres from AH neurons were identified in both dorsomedial and dorsolateral PAG columns. Furthermore, we showed that pre-treatment of the dPAG with either CoCl2 or LY235959 inhibited freezing and impaired oriented escape and antinociception induced by infusions of SIN-1 into the AH. CONCLUSIONS: These findings suggest that the panic-like freezing and oriented escape defensive behaviours, and fear-induced antinociception elicited by intra-AH microinjections of SIN-1 depend on the activation of dPAG NMDA receptors.

Neostriatum neuronal TRPV1-signalling mediates striatal anandamide at high concentration facilitatory influence on neostriato-nigral dishinhibitory GABAergic connections.

RATIONALE: Several lines of evidence have demonstrated that the cannabinoid type 1 receptor (CB1) is found in the caudate nucleus and putamen (CPu) in addition to the substantia nigra pars reticulata (SNpr). Here, we investigated the role of endocannabinoid neuromodulation of striato-nigral disinhibitory projections on the activity of nigro-collicular GABAergic pathways that control the expression of unconditioned fear-related behavioural responses elicited by microinjections of the GABAA receptor selective antagonist bicuculline (BIC) in the deep layers of the superior colliculus (dlSC). METHODS: Fluorescent neural tract tracers were deposited in either CPu or in SNpr. Wistar rats received injection of vehicle, anandamide (AEA), either at low (50 pmol) or high (100 pmol) concentrations in CPu followed by bicuculline microinjections in dlSC. RESULTS: Connections between CPu, the SNpr and dlSC were demonstrated. The GABAA receptor blockade in dlSC elicited panic-like behaviour. AEA at the lowest concentration caused a panicolytic-like effect that was antagonised by the CPu pretreatment with AM251 at 100 pmol. AEA at the highest concentration caused a panicogenic-like effect that was antagonised by the CPu pretreatment with 6-iodonordihydrocapsaicin (6-I-CPS) at different concentrations (0.6, 6, 60 nmol). CONCLUSION: These findings suggest that while pre-synaptic CB1-signalling subserves an indirect facilitatory effect of AEA on striato-nigral pathways causing panicolytic-like responses through midbrain tectum enhanced activity, post-synaptic TRPV1-signalling in CPu mediates AEA direct activation of striato-nigral disinhibitory pathways resulting in increasing dlSC neurons activity and a panicogenic-like response. All these actions seem to depend on the interface with the nigro-collicular inhibitory GABAergic pathways.

D2-like receptor activation by intranasal dopamine attenuates fear responses induced by electrical stimulation of the dorsal periaqueductal grey matter, but fails to reduce aversion to pit vipers and T-maze performance.

BACKGROUND: Panic-like reactions elicited by electrical stimulation of the dorsal periaqueductal grey matter (ES-dPAG) seem to be regulated by dopamine (DA). We showed that DA applied intranasally (IN) increased escape-behaviour thresholds induced by ES-dPAG of rats, indicating a panicolytic-like effect. AIMS: We investigated whether IN-DA increases escape-response thresholds induced by ES-dPAG by acting on D2-like receptors, and whether IN-DA affects escape responses elicited by the presence of a potential predator and by open space and height of the elevated T-maze (ETM) as well as motor performance in the open field (OF) test. METHODS: Wistar rats exposed to ES-dPAG were treated with Sulpiride (SUL, 40 mg/kg, D2-like receptor antagonist) previously IN-DA (2 mg/kg). Independent groups of rats treated with IN-DA were submitted to prey versus snake paradigm (PSP), ETM and OF. RESULTS: Anti-aversive effects of the IN-DA were reduced by SUL pretreatment in the ES-dPAG test. IN-DA did not affect the escape number in the PSP nor the escape latencies in the ETM as well as motor performance in the OF. CONCLUSIONS/INTERPRETATION: The IN-DA effects in reducing unconditioned fear responses elicited by ES-dPAG seem to be mediated by D2-like receptors. The lack of effects on panic-related responses in the ETM and PSP may be related to the possibility of avoiding the danger inherent to these models, a defence strategy not available during ES-dPAG. These findings cannot be attributed to motor performance. The decision-making responses to avoid dangerous situations can be orchestrated by supra-mesencephalic structures connected by non-dopaminergic inputs.

Augmented anandamide signalling in the substantia nigra pars reticulata mediates panicolytic-like effects in mice confronted by Crotalus durissus terrificus pit vipers.

RATIONALE: The endocannabinoid modulation of fear and anxiety due to the on-demand synthesis and degradation is supported by a large body of research. Although it has been proposed that anandamide (AEA) in the substantia nigra pars reticulata (SNpr) seems to be important for the organisation of innate fear-related behaviours, a role for endogenous AEA has yet to be clarified. METHODS: Mice were treated with the fatty acid amide hydrolase (FAAH) selective inhibitor URB597 at different concentrations (0.01, 0.1, 1 nmol/0.1 µL) in the SNpr and confronted by rattlesnakes (Crotalus durissus terrificus). The most effective dose of URB597 (1 nmol) was also preceded by microinjections of the CB1 receptor antagonist AM251 (0.1 nmol) into the SNpr, and mice were then confronted by the venomous snake. RESULTS: URB597 (0.1 and 1 nmol) in the SNpr decreased the expression of defensive behaviours such as defensive attention, escape, and time spent inside the burrow of mice confronted by rattlesnakes. Moreover, pretreatment of SNpr with AM251 suppressed these antiaversive effects of URB597 in this midbrain structure. CONCLUSION: Overall, these data clearly indicate that the panicolytic consequences of endogenous AEA enhancement in the SNpr are mediated by CB1 receptor signalling.

Panic-like responses of female Wistar rats confronted by Bothrops alternatus pit vipers, or exposure to acute hypoxia: Effect of oestrous cycle.

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% O2 ). 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.

Nitric oxide-mediated defensive and antinociceptive responses organised at the anterior hypothalamus of mice are modulated by glutamatergic inputs from area 24b of the cingulate cortex.

BACKGROUND: Previous studies suggested that Cg1 area of the cingulate cortex of rats controls glutamate-mediated fear-induced defensive behaviour and antinociception organised at the posterior hypothalamus. In turn, microinjection of the nitric oxide donor SIN-1 into the anterior hypothalamus of mice produced defensive behaviours and fear-induced antinociception. However, it remains unknown whether Cg1 also modulates the latter mechanisms in mice. AIMS: The present study examined the influence of Cg1 on SIN1-evoked fear-induced defensive behaviour and antinociception organised at the anterior hypothalamus of mice. METHODS: The fear-like behavioural and antinociceptive responses to the microinjection of SIN-1 (300 nmol) into the anterior hypothalamus were evaluated after the microinjection of either N-methyl-D-aspartic acid receptor agonist (0.1, 1 and 10 nmol) or physiological saline into the cingulate cortex of C57BL/6 male mice. In addition, neurotracing and immunohistochemistry were used to characterise Cg1-anterior hypothalamus glutamatergic pathways. RESULTS: The data showed that activation of Cg1 N-methyl-D-aspartic acid receptors increased escape while reducing freezing and antinociceptive responses to SIN-1 microinjections into the anterior hypothalamus. Anterograde neural tract tracer co-localised with VGLUT2-labelled fibres suggests these responses are mediated by glutamatergic synapses at the anterior hypothalamus. CONCLUSIONS: In contrast with previous studies showing that Cg1 facilitates both escape and antinociception to chemical stimulation of the posterior hypothalamus in rats, the present data suggest that Cg1 facilitates escape while inhibiting defensive antinociception produced by the microinjection of SIN-1 in the anterior hypothalamus of mice. Accordingly, Cg1 may have opposite effects on antinociceptive responses organised in the anterior and posterior hypothalamus of mice and rats, respectively.

Cannabidiol-induced panicolytic-like effects and fear-induced antinociception impairment: the role of the CB1 receptor in the ventromedial hypothalamus.

RATIONALE: The behavioural effects elicited by chemical constituents of Cannabis sativa, such as cannabidiol (CBD), on the ventromedial hypothalamus (VMH) are not well understood. There is evidence that VMH neurons play a relevant role in the modulation of unconditioned fear-related defensive behavioural reactions displayed by laboratory animals. OBJECTIVES: This study was designed to explore the specific pattern of distribution of the CB1 receptors in the VMH and to investigate the role played by this cannabinoid receptor in the effect of CBD on the control of defensive behaviours and unconditioned fear-induced antinociception. METHODS: A panic attack-like state was triggered in Wistar rats by intra-VMH microinjections of N-methyl-D-aspartate (NMDA). One of three different doses of CBD was microinjected into the VMH prior to local administration of NMDA. In addition, the most effective dose of CBD was used after pre-treatment with the CB1 receptor selective antagonist AM251, followed by NMDA microinjections in the VMH. RESULTS: The morphological procedures demonstrated distribution of labelled CB1 receptors on neuronal perikarya situated in dorsomedial, central and ventrolateral divisions of the VMH. The neuropharmacological approaches showed that both panic attack-like behaviours and unconditioned fear-induced antinociception decreased after intra-hypothalamic microinjections of CBD at the highest dose (100 nmol). These effects, however, were blocked by the administration of the CB1 receptor antagonist AM251 (100 pmol) in the VMH. CONCLUSION: These findings suggest that CBD causes panicolytic-like effects and reduces unconditioned fear-induced antinociception when administered in the VMH, and these effects are mediated by the CB1 receptor-endocannabinoid signalling mechanism in VMH.

The alpha- and beta-noradrenergic receptors blockade in the dorsal raphe nucleus impairs the panic-like response elaborated by medial hypothalamus neurons.

Dorsal raphe nucleus (DRN) neurons are reciprocally connected to the locus coeruleus (LC) and send neural pathways to the medial hypothalamus (MH). The aim of this work was to investigate whether the blockade of α1-, α2- or ß-noradrenergic receptors in the DRN or the inactivation of noradrenergic neurons in the LC modify defensive behaviours organised by MH neurons. For this purpose, Wistar male rats received microinjections of WB4101, RX821002, propranolol (α1-, α2- and ß-noradrenergic receptor antagonists, respectively) or physiological saline in the DRN, followed 10 min later by MH GABAA receptor blockade. Other groups of animals received DSP-4 (a noradrenergic neurotoxin), physiological saline or only a needle insertion (sham group) into the LC, and 5 days later, bicuculline or physiological saline was administered in the MH. In all these cases, after MH treatment, the frequency and duration of defensive responses were recorded over 15 min. An anterograde neural tract tracer was also deposited in the DRN. DRN neurons send pathways to lateral and dorsomedial hypothalamus. Blockade of α1- and ß-noradrenergic receptors in the DRN decreased escape reactions elicited by bicuculline microinjections in the MH. In addition, a significant increase in anxiety-like behaviours was observed after the blockade of α2-noradrenergic receptors in the DRN. LC pretreatment with DSP-4 decreased both anxiety- and panic attack-like behaviours evoked by GABAA receptor blockade in the MH. In summary, the present findings suggest that the norepinephrine-mediated system modulates defensive reactions organised by MH neurons at least in part via noradrenergic receptors recruitment on DRN neurons.

Dorsal raphe nucleus 5-Hydroxytryptamine 2A receptors are critical for the organisation of panic attack-like defensive behaviour and unconditioned fear-induced antinociception elicited by the chemical stimulation of superior colliculus neurons.

Microinjections of N-methyl-d-aspartic acid (NMDA) in the midbrain tectum structures produce panic attack-like defensive behaviours, followed by an antinociceptive response. It has been suggested that fear-related defensive responses organised by brainstem neurons can be modulated by 5-hydroxytryptamine (5-HT). However, there is a shortage of studies showing the role of dorsal raphe nucleus (DRN) 5-HT2A receptors in the modulation of panic-like behaviour and fear-induced antinociception organised by the superior colliculus (SC). The purpose of this study was to investigate the participation of DRN 5-HT2A receptors in the modulation of panic attack-like behaviour and antinociception evoked by intra-SC injections of NMDA. In experiment I, the animals received microinjections of physiological saline or NMDA (6, 9 and 12 nmol) in the deep layers of the SC (dlSC). In experiment II, the most effective dose of NMDA (12 nmol) or vehicle was preceded by microinjections of vehicle or the 5-HT2A receptor selective antagonist R-96544 at different concentrations (0.5, 5 and 10 nM) in the DRN. Both proaversive and antinociceptive effects elicited by intra-dlSC injections of NMDA were attenuated by DRN pretreatment with R-96544. In addition, a morphological analysis showed that 5-HT2A receptors are present in GABAergic interneurons in the DRN. Taken together, these findings suggest that DRN 5-HT2A receptors are critical for the modulation of both panic attack-like defensive behaviour organised by SC neurons and unconditioned fear-induced antinociception. A possible interaction between serotonergic inputs, GABAergic interneurons and serotonergic outputs from the DRN was also considered.

Panicolytic-like effect of µ1-opioid receptor blockade in the inferior colliculus of prey threatened by Crotalus durissus terrificus pit vipers.

BACKGROUND: The endogenous opioid peptide system has been implicated in the neural modulation of fear and anxiety organised by the dorsal midbrain. Furthermore, previous results indicate a fundamental role played by inferior colliculus (IC) opioid mechanisms during the expression of defensive behaviours, but the involvement of the IC µ1-opioid receptor in the modulation of anxiety- and panic attack-related behaviours remains unclear. Using a prey-versus-snake confrontation paradigm, we sought to investigate the effects of µ1-opioid receptor blockade in the IC on the defensive behaviour displayed by rats in a dangerous situation. METHODS: Specific pathogen-free Wistar rats were treated with microinjection of the selective µ1-opioid receptor antagonist naloxonazine into the IC at different concentrations (1.0, 3.0 and 5.0 µg/0.2 µL) and then confronted with rattlesnakes ( Crotalus durissus terrificus). The defensive behavioural repertoire, such as defensive attention, flat back approach (FBA), startle, defensive immobility, escape or active avoidance, displayed by rats either during the confrontations with wild snakes or during re-exposure to the experimental context without the predator was analysed. RESULTS: The blockade of µ1-opioid receptors in the IC decreased the expression of both anxiety-related behaviours (defensive attention, FBA) and panic attack-related responses (startle, defensive immobility and escape) during the confrontation with rattlesnakes. A significant decrease in defensive attention was also recorded during re-exposure of the prey to the experimental apparatus context without the predator. CONCLUSION: Taken together, these results suggest that a decrease in µ1-opioid receptor signalling activity within the IC modulates anxiety- and panic attack-related behaviours in dangerous environments.

5-Hydroxytryptamine 2A receptors of the dorsal raphe nucleus modulate panic-like behaviours and mediate fear-induced antinociception elicited by neuronal activation in the central nucleus of the inferior colliculus.

It has been established that chemical stimulation of the inferior colliculus (IC) of laboratory animals evokes fear-related defensive responses, which are considered panic attack-like behaviours. In addition, there is evidence that defensive reactions provoked by chemical stimulation of midbrain tectum neurons may induce an antinociceptive response. Morphologically, the IC receives projections from other mesencephalic structures, such as the dorsal raphe nucleus (DRN), a region rich in serotonergic neurons that play a critical role in the control of defensive behaviours. Moreover, this monoaminergic brainstem reticular nucleus is suggested to comprise the endogenous pain modulatory system. The aim of the present study was to investigate the role of DRN 5-hydroxytryptamine 2A (5-HT2A) receptors in Wistar rats by local microinjection of R-96544 (a selective antagonist of the 5-HT2A receptor) at doses of 5, 10 or 15 nM on defensive reactions and fear-induced antinociception evoked by chemical stimulation of the central nucleus of the IC with NMDA (6, 9 or 12 nmol). Behavioural responses were analysed for 10 min, and then the nociceptive threshold was measured at 10 min intervals for 70 min. The dose of 12 nmol of NMDA was the most effective in causing panic attack-like defensive behaviours and much higher hypoalgesia. In addition, both effects were attenuated by pretreatment of the DRN with R-96544. These findings suggest the critical participation of DRN 5-HT2A receptors in the modulation of panic attack-like defensive behaviour and unconditioned fear-induced antinociception organised by neurons in the central nucleus of the IC.

The Nitric Oxide Donor SIN-1-Produced Panic-Like Behaviour And Fear-Induced Antinociception Are Modulated By NMDA Receptors In The Anterior Hypothalamus.

BACKGROUND: An excitatory imbalance in the hypothalamus of rodents caused by local chemical stimulation elicits fear-related defensive reactions such as escape and freezing. In addition, these panic attack-like defensive reactions induced by hypothalamic neurons may cause antinociception. However, there is a shortage of studies showing the participation of the anterior hypothalamic nucleus in these adaptive defensive mechanisms. Nitric oxide (NO) donors have been shown to evoke fear-related defensive responses when microinjected into paralimbic and limbic structures, and this excitatory neuromodulation can recruit the glutamatergic system. AIMS: The aim of this work was to investigate the influence of the glutamatergic system in the nitrergic effects on fear-related defensive responses organised by anterior hypothalamic neurons. METHODS: The present study evaluates the effects of the molsidomine active metabolite SIN-1 NO donor administered into the anterior hypothalamus (AH) of mice at different concentrations (75, 150 and 300 nmol/0.1 µL). Then, we investigated the effects of pre-treatment of the AH with AP-7 (an N-methyl-d-aspartate (NMDA) receptor-selective antagonist; 0.02, 0.2 and 2 nmol/0.1 µL) on the behavioural and antinociceptive effects provoked by AH chemical stimulation with SIN-1 microinjections. RESULTS: The 300 nmol dose of SIN-1 was the most effective at causing panic-like defensive behaviours followed by a significant antinociceptive response. In addition, both of these effects were attenuated or inhibited by AH pre-treatment with AP-7. CONCLUSIONS: These findings suggest that the panicogenic and antinociceptive effects evoked by intra-AH microinjections of SIN-1 depend on NMDA receptor activation.

Neurotoxic lesions of the pedunculopontine tegmental nucleus impair the elaboration of postictal antinociception.

Generalised tonic-clonic seizures, generated by abnormal neuronal hyper-activity, cause a significant and long-lasting increase in the nociceptive threshold. The pedunculopontine tegmental nucleus (PPTN) plays a crucial role in the regulation of seizures as well as the modulation of pain, but its role in postictal antinociceptive processes remains unclear. In the present study, we aimed to investigate the involvement of PPTN neurons in the postictal antinociception. Wistar rats had their tail-flick baseline recorded and were injected with ibotenic acid (1.0 µg/0.2 µL) into the PPTN, aiming to promote a local neurotoxic lesion. Five days after the neuronal damage, pentylenetetrazole (PTZ; 64 mg/kg) was intraperitoneally administered to induce tonic-clonic seizures. The tail-withdrawal latency was measured immediately after the seizures (0 min) and subsequently at 10-min intervals until 130 min after the seizures were induced pharmacologically. Ibotenic acid microinjected into the PPTN did not reduce the PTZ-induced seizure duration and severity, but it diminished the postictal antinociception from 0 to 130 min after the end of the PTZ-induced tonic-clonic seizures. These results suggest that the postictal antinociception depends on the PPTN neuronal cells integrity.

The Rodent-versus-wild Snake Paradigm as a Model for Studying Anxiety- and Panic-like Behaviors: Face, Construct and Predictive Validities.

Using an innovative approach to study the neural bases of psychiatric disorders, this study investigated the behavioral, morphological and pharmacological bases of panic attack-induced responses in a prey-versus-coral snake paradigm. Mesocricetus auratus was chronically treated with intraperitoneal administration of the selective serotonin uptake inhibitor paroxetine or the gamma aminobutyric acid (GABA)/benzodiazepine receptor agonist alprazolam at three different doses and were then confronted with a venomous coral snake (Micrurus frontalis, Reptilia, Elapidae). The threatened rodents exhibited defensive attention, flat back approaches, defensive immobility, and escape defensive responses in the presence of the venomous snake, followed by increases in Fos protein in limbic structure neurons. Chronic administration of both paroxetine and alprazolam decreased these responses with morphological correlates between the panicolytic effect of both drugs administered at the highest dose and decreases in Fos protein-immunolabeled perikarya found in the amygdaloid complex, hypothalamus and periaqueductal gray matter columns, which are structures that make up the encephalic aversion system. These findings provide face, construct and predictive validities of this new experimental model of anxiety- and panic attack-like behavioral responses displayed by threatened prey confronted with venomous coral snakes.

Decrease in NMDA receptor-signalling activity in the anterior cingulate cortex diminishes defensive behaviour and unconditioned fear-induced antinociception elicited by GABAergic tonic inhibition impairment in the posterior hypothalamus.

Acute γ-aminobutyric acid (GABA) disinhibition in the posterior hypothalamus (PH) elicits defensive reactions that are considered anxiety- and panic attack-like behaviour, and these defensive reactions are followed by antinociception. Evidence indicates that the PH connects with the medial prefrontal cortex, particularly the anterior cingulate cortex (ACC), which seems to regulate these unconditioned fear-induced defensive responses. However, few studies have shown the participation of cortical regions in the control of behavioural and antinociceptive responses organised by diencephalic structures. It has been suggested that the glutamatergic system can mediate this cortical influence, as excitatory imbalance is believed to play a role in both defensive mechanisms. Thus, the aim of the present study was to investigate the involvement of ACC glutamatergic connections via blockade of local N-methyl-D-aspartate (NMDA) receptors to elaborate panic-like defensive behaviours and unconditioned fear-induced antinociception organised by PH neurons. Wistar rats were treated with microinjections of 0.9% NaCl or LY235959 (a selective NMDA receptor antagonist) in the ACC at different concentrations (2, 4 and 8 nmol/0.2µL), followed by GABAA receptor blockade in the PH. Defensive reactions were analysed for 20min, and the nociceptive threshold was then measured at 10-min intervals for 60min. Pretreatment of the ACC with LY235959 reduced both panic-like defensive behaviour and fear-induced antinociception evoked by PH GABAergic disinhibition. Our findings suggest that ACC NMDA receptor-signalled glutamatergic inputs play a relevant role in the organisation of anxiety- and panic attack-like behaviours and in fear-induced antinociception.