CB1 receptor signalling mediates cannabidiol-induced panicolytic-like effects and defensive antinociception impairment in mice threatened by Bothrops jararaca lancehead pit vipers.

BACKGROUND: Cannabis sativa-derived substances such as cannabidiol (CBD) have attracted increasing clinical interest and consist in a new perspective for treating some neurological and psychiatric diseases. AIMS: The aim of this work was to investigate the effect of acute treatment with CBD on panic-like defensive responses displayed by mice threatened by the venomous snake Bothrops jararaca. METHODS: Mice were habituated in the enriched polygonal arena for snake panic test. After recording the baseline responses of the tail-flick test, the prey were pretreated with intraperitoneal (i.p.) administrations of the endocannabinoid type 1 receptor (CB1) antagonist AM251 (selective cannabinoid 1 receptor antagonist with an IC50 of 8 nM) at different doses, which were followed after 10 min by i.p. treatment with CBD (3 mg/kg). Thirty minutes after treatment with CBD, mice were subjected to confrontations by B. jararaca for 5 min, and the following defensive responses were recorded: risk assessment, oriented escape behaviour, inhibitory avoidance and prey-versus-snake interactions. Immediately after the escape behaviour was exhibited, the tail-flick latencies were recorded every 5 min for 30 min. OUTCOMES: Mice threatened by snakes displayed several anti-predatory defensive and innate fear-induced antinociception responses in comparison to the control. CBD significantly decreased the risk assessment and escape responses, with a consequent decrease in defensive antinociception. The CBD panicolytic effect was reversed by i.p. treatment with AM251. CONCLUSIONS: These findings suggest that the anti-aversive effect of CBD depends at least in part on the recruitment of CB1 receptors.

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.

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.

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.

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.

Opioid neurotransmission modulates defensive behavior and fear-induced antinociception in dangerous environments.

The effects of endogenous opioid peptide antagonists on panic-related responses are controversial. Using elevated mazes and a prey-versus-predator paradigm, we investigated the involvement of the endogenous opioid peptide-mediated system in the modulation of anxiety- and panic attack-induced responses and innate fear-induced antinociception in the present work. Wistar rats were intraperitoneally pretreated with either physiological saline or naloxone at different doses and were subjected to either the elevated plus- or T-maze test or confronted by Crotalus durissus terrificus. The defensive behaviors of the rats were recorded in the presence of the predator and at 24h after the confrontation, when the animals were placed in the experimental enclosure without the rattlesnake. The peripheral non-specific blockade of opioid receptors had a clear anxiolytic-like effect on the rats subjected to the elevated plus-maze but not on those subjected to the elevated T-maze; however, a clear panicolytic-like effect was observed, i.e., the defensive behaviors decreased, and the prey-versus-predator interaction responses evoked by the presence of the rattlesnakes increased. A similar effect was noted when the rats were exposed to the experimental context in the absence of the venomous snake. After completing all tests, the naloxone-treated groups exhibited less anxiety/fear-induced antinociception than the control group, as measured by the tail-flick test. These findings demonstrate the anxiolytic and panicolytic-like effects of opioid receptor blockade. In addition, the fearlessness behavior displayed by preys treated with naloxone at higher doses enhanced the defensive behavioral responses of venomous snakes.

Unravelling cortico-hypothalamic pathways regulating unconditioned fear-induced antinociception and defensive behaviours.

The medial prefrontal cortex can influence unconditioned fear-induced defensive mechanisms organised by diencephalic neurons that are under tonic GABAergic inhibition. The posterior hypothalamus (PH) is involved with anxiety- and panic attack-like responses. To understand this cortical mediation, our study characterised anterior cingulate cortex (ACC)-PH pathways and investigated the effect of ACC local inactivation with lidocaine. We also investigated the involvement of PH ionotropic glutamate receptors in the defensive behaviours and fear-induced antinociception by microinjecting NBQX (an AMPA/kainate receptor antagonist) and LY235959 (a NMDA receptor antagonist) into the PH. ACC pretreatment with lidocaine decreased the proaversive effect and antinociception evoked by GABAA receptor blockade in the PH, which suggests that there may be descending excitatory pathways from this cortical region to the PH. Microinjections of both NBQX and LY235959 into the PH also attenuated defensive and antinociceptive responses. This suggests that the blockade of AMPA/kainate and NMDA receptors reduces the activity of glutamatergic efferent pathways. Both inputs from the ACC to the PH and glutamatergic hypothalamic short links disinhibited by intra-hypothalamic GABAA receptors blockade are potentially implicated. Microinjection of a bidirectional neurotracer in the PH showed a Cg1-PH pathway and PH neuronal reciprocal connections with the periaqueductal grey matter. Microinjections of an antegrade neurotracer into the Cg1 showed axonal fibres and glutamatergic vesicle-immunoreactive terminal boutons surrounding both mediorostral-lateroposterior thalamic nucleus and PH neuronal perikarya. These data suggest a critical role played by ACC-PH glutamatergic pathways and AMPA/kainate and NMDA receptors in the panic attack-like reactions and antinociception organised by PH neurons.

5-Hydroxytryptamine2A/2C receptors of nucleus raphe magnus and gigantocellularis/paragigantocellularis pars α reticular nuclei modulate the unconditioned fear-induced antinociception evoked by electrical stimulation of deep layers of the superior colliculus and dorsal periaqueductal grey matter.

The electrical stimulation of the dorsolateral columns of the periaquedutal grey matter (dlPAG) or deep layers of the superior colliculus (dlSC) evokes defensive behaviours followed by an antinociceptive response. Monoaminergic brainstem reticular nuclei are suggested to comprise the endogenous pain modulatory system. The aim of the present work was to investigate the role played by 5-HT2 subfamily of serotonergic receptors of the nucleus raphe magnus (NRM) and the gigantocellularis/paragigantocellularis pars α reticular nuclei (Gi/PGiα) in the elaboration of instinctive fear-induced antinociception elicited by electrical stimulation of dlPAG or of dlSC. The nociceptive thresholds were measured by the tail-flick test in Wistar rats. The 5-HT2A/2C-serotonergic receptors antagonist ritanserin was microinjected at different concentrations (0.05, 0.5 and 5.0µg/0.2µL) either in Gi/PGiα or in NRM. The blockade of 5-HT2 receptors in both Gi/PGiα and NRM decreased the innate fear-induced antinociception elicited by electrical stimulation of the dlSC or the dlPAG. These findings indicate that serotonin is involved in the hypo-algesia induced by unconditioned fear-induced behavioural responses and the 5-HT2A/2C-serotonergic receptor subfamily in neurons situated in the Gi/PGiα complex and NRM are critically recruited in pain modulation during the panic-like emotional behaviour.

Dissociation between the panicolytic effect of cannabidiol microinjected into the substantia nigra, pars reticulata, and fear-induced antinociception elicited by bicuculline administration in deep layers of the superior colliculus: The role of CB1-cannabinoid receptor in the ventral mesencephalon.

Many studies suggest that the substantia nigra, pars reticulata (SNpr), a tegmental mesencephalic structure rich in γ-aminobutyric acid (GABA)- and cannabinoid receptor-containing neurons, is involved in the complex control of defensive responses through the neostriatum-nigral disinhibitory and nigro-tectal inhibitory GABAergic pathways during imminently dangerous situations. The aim of the present work was to investigate the role played by CB1-cannabinoid receptor of GABAergic pathways terminal boutons in the SNpr or of SNpr-endocannabinoid receptor-containing interneurons on the effect of intra-nigral microinjections of cannabidiol in the activity of nigro-tectal inhibitory pathways. GABAA receptor blockade in the deep layers of the superior colliculus (dlSC) elicited vigorous defensive behaviour. This explosive escape behaviour was followed by significant antinociception. Cannabidiol microinjection into the SNpr had a clear anti-aversive effect, decreasing the duration of defensive alertness, the frequency and duration of defensive immobility, and the frequency and duration of explosive escape behaviour, expressed by running and jumps, elicited by transitory GABAergic dysfunction in dlSC. However, the innate fear induced-antinociception was not significantly changed. The blockade of CB1 endocannabinoid receptor in the SNpr decreased the anti-aversive effect of canabidiol based on the frequency and duration of defensive immobility, the frequency of escape expressed by running, and both the frequency and duration of escape expressed by jumps. These findings suggest a CB1 mediated endocannabinoid signalling in cannabidiol modulation of panic-like defensive behaviour, but not of innate fear-induced antinociception evoked by GABAA receptor blockade with bicuculline microinjection into the superior colliculus, with a putative activity in nigro-collicular GABAergic pathways.

NMDA and AMPA/kainate glutamatergic receptors in the prelimbic medial prefrontal cortex modulate the elaborated defensive behavior and innate fear-induced antinociception elicited by GABAA receptor blockade in the medial hypothalamus.

The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) and amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptors of the prelimbic (PL) division of the medial prefrontal cortex (MPFC) on the panic attack-like reactions evoked by γ-aminobutyric acid-A receptor blockade in the medial hypothalamus (MH). Rats were pretreated with NaCl 0.9%, LY235959 (NMDA receptor antagonist), and NBQX (AMPA/kainate receptor antagonist) in the PL at 3 different concentrations. Ten minutes later, the MH was treated with bicuculline, and the defensive responses were recorded for 10 min. The antagonism of NMDA receptors in the PL decreased the frequency and duration of all defensive behaviors evoked by the stimulation of the MH and reduced the innate fear-induced antinociception. However, the pretreatment of the PL cortex with NBQX was able to decrease only part of defensive responses and innate fear-induced antinociception. The present findings suggest that the NMDA-glutamatergic system of the PL is critically involved in panic-like responses and innate fear-induced antinociception and those AMPA/kainate receptors are also recruited during the elaboration of fear-induced antinociception and in panic attack-related response. The activation of the glutamatergic neurotransmission of PL division of the MPFC during the elaboration of oriented behavioral reactions elicited by the chemical stimulation of the MH recruits mainly NMDA receptors in comparison with AMPA/kainate receptors.

Panic-like defensive behavior but not fear-induced antinociception is differently organized by dorsomedial and posterior hypothalamic nuclei of Rattus norvegicus (Rodentia, Muridae)

The hypothalamus is a forebrain structure critically involved in the organization of defensive responses to aversive stimuli. Gamma-aminobutyric acid (GABA)ergic dysfunction in dorsomedial and posterior hypothalamic nuclei is implicated in the origin of panic-like defensive behavior, as well as in pain modulation. The present study was conducted to test the difference between these two hypothalamic nuclei regarding defensive and antinociceptive mechanisms. Thus, the GABA A antagonist bicuculline (40 ng/0.2 µL) or saline (0.9% NaCl) was microinjected into the dorsomedial or posterior hypothalamus in independent groups. Innate fear-induced responses characterized by defensive attention, defensive immobility and elaborate escape behavior were evoked by hypothalamic blockade of GABA A receptors. Fear-induced defensive behavior organized by the posterior hypothalamus was more intense than that organized by dorsomedial hypothalamic nuclei. Escape behavior elicited by GABA A receptor blockade in both the dorsomedial and posterior hypothalamus was followed by an increase in nociceptive threshold. Interestingly, there was no difference in the intensity or in the duration of fear-induced antinociception shown by each hypothalamic division presently investigated. The present study showed that GABAergic dysfunction in nuclei of both the dorsomedial and posterior hypothalamus elicit panic attack-like defensive responses followed by fear-induced antinociception, although the innate fear-induced behavior originates differently in the posterior hypothalamus in comparison to the activity of medial hypothalamic subdivisions.