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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. 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. 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. 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. 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 endocannabinoid system plays an important role in the organization of panic-like defensive behavior. Threatening situations stimulate brain areas, such as the dorsomedial hypothalamus (DMH). However, there is a lack of studies addressing the role of the DMH endocannabinoid system in panic-like responses. We aimed to verify which mechanisms underlie anandamide-mediated responses in the DMH. To test the hypothesis that the anandamide produces panicolytic-like effects, we treated mice with intra-DMH microinjections of vehicle or increasing doses of anandamide (0.5, 5, or 50 pmol) and then performed confrontation with the South American snake Epicrates cenchria assisi. Intra-DMH anandamide treatment yielded a U-shaped dose-response curve with no effect of the lowest (0.5 pmol) or the highest (50 pmol) dose and significant inhibition of panic-like responses at the intermediate (5 pmol) dose. In addition, this panicolytic-like effect was prevented by pretreatment of the DMH with the CB1 receptor antagonist AM251 (100 pmol). However, pretreatment of the DMH with the TRPV1 receptor antagonist 6-iodo-nordihydrocapsaicin (3 nmol) restored the panicolytic-like effect of the highest dose of anandamide. Immunohistochemistry revealed that CB1 receptors were present primarily on axonal fibers, while TRPV1 receptors were found almost exclusively surrounding the perikarya in DMH. The present results suggest that anandamide exerts a panicolytic-like effect in the DMH by activation of CB1 receptors and that TRPV1 receptors are related to the lack of effect of the highest dose of anandamide.

Background: There is a controversy regarding the key role played by opioid peptide neurotransmission in the modulation of panic-attack-related responses. Aims: Using a prey versus rattlesnakes paradigm, the present work investigated the involvement of the endogenous opioid peptide-mediated system of the inferior colliculus in the modulation of panic attack-related responses. Methods: Wistar rats were pretreated with intracollicular administration of either physiological saline or naloxone at different concentrations and confronted with rattlesnakes ( Crotalus durissus terrificus). The prey versus rattlesnake confrontations were performed in a polygonal arena for snakes. The defensive behaviors displayed by prey (defensive attention, defensive immobility, escape response, flat back approach and startle) were recorded twice: firstly, over a period of 15 min the presence of the predator and a re-exposure was performed 24 h after the confrontation, when animals were exposed to the experimental enclosure without the rattlesnake. Results: The intramesencephalic non-specific blockade of opioid receptors with microinjections of naloxone at higher doses decreased both anxiety- (defensive attention and flat back approach) and panic attack-like (defensive immobility and escape) behaviors, evoked in the presence of rattlesnakes and increased non-defensive responses. During the exposure to the experimental context, there was a decrease in duration of defensive attention. Conclusions: These findings suggest a panicolytic-like effect of endogenous opioid receptors antagonism in the inferior colliculus on innate (panic attack) and conditioned (anticipatory anxiety) fear in rats threatened by rattlesnakes.

The effects of cannabinoids in brain areas expressing cannabinoid receptors, such as hypothalamic nuclei, are not yet well known. Several studies have demonstrated the role of hypothalamic nuclei in the organisation of behavioural responses induced through innate fear and panic attacks. Panic-prone states are experimentally induced in laboratory animals through a reduction in the GABAergic activity. The aim of the present study was to examine panic-like elaborated defensive behaviour evoked by GABAA receptor blockade with bicuculline (BIC) in the dorsomedial division of the ventromedial hypothalamus (VMHdm). We also aimed to characterise the involvement of endocannabinoids and the CB1 cannabinoid receptor in the modulation of elaborated defence behavioural responses organised with the VMHdm. The guide-cannula was stereotaxicaly implanted in VMHdm and the animals were treated with anandamide (AEA) at different doses, and the effective dose was used after the pre-treatment with the CB1 receptor antagonist AM251, followed by GABAA receptor blockade in VMHdm. The results showed that the intra-hypothalamic administration of AEA at an intermediate dose (5 pmol) attenuated defence responses induced through the intra-VMHdm microinjection of bicuculline (40 ng). This effect, however, was inhibited when applied central microinjection of the CB1 receptor antagonist AM251 in the VMHdm. Moreover, AM251 potentiates de non-oriented escape induced by bicuculline, effect blocked by pre-treatment with the TRPV1 channel antagonist 6-I-CPS. These results indicate that AEA modulates the pro-aversive effects of intra-VMHdm-bicuculline treatment, recruiting CB1 cannabinoid receptors and the TRPV1 channel is involved in the AM251-related potentiation of bicuculline effects on non-oriented escape behaviour.

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.

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.

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.

This study investigated the intrinsic connections of a key-structure of the endogenous pain inhibitory system, the pedunculopontine tegmental nucleus (PPTN), in post-ictal antinociceptive process through synaptic inactivation of the PPTN with cobalt chloride. Male Wistar rats (n = 6 or 7 per group), weighing 250–280 g, had the tail-flick baseline recorded and were submitted to a stereotaxic surgery for the introduction of a guide-cannula aiming at the PPTN. After 5 days of postoperative recovery, cobalt chloride (1 mM/0.2 µL) or physiological saline (0.2 µL) were microinjected into the PPTN and after 5 min, the tail-withdrawal latency was measured again at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min after seizures evoked by intraperitoneal injection of pentylenetetrazole (64 mg/kg). The synaptic inactivation of PPTN decreased the post-ictal antinociceptive phenomenon, suggesting the involvement of PPTN intrinsic connections in the modulation of pain, during tonic-clonic seizures. These results showed that the PPTN may be crucially involved in the neural network that organizes the post-ictal analgesia. Synapse 68:369–377, 2014. © 2014 Wiley Periodicals, Inc.

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/0.2 μL), followed by GABAA receptor blockade in the PH. Defensive reactions were analysed for 20 min, and the nociceptive threshold was then measured at 10-min intervals for 60 min. 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.

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.

Objetivo. O estudo objetivou-se em avaliar a participação da via se­rotoninérgica na modulação da dor e descrever a influência dos re­ceptores 5-HT3 na resposta nociceptiva de ratos Wistar submetidos ao teste da formalina. Método. Estudou-se o efeito da administração intraperitoneal do antagonista 5-HT3 ondansetrona sobre o compor­tamento de elevação da pata gerado pela injeção intraplantar de 100μL de formalina a 1%. O número de elevações da pata foi observado pelo período de 60 minutos. Resultados. Ondansetrona (2mg/kg) aumen­tou o comportamento de elevações da pata em relação ao grupo con­trole positivo durante a 1ª fase do teste e provocou uma diminuição deste comportamento durante a 2ª fase. Conclusão. A ondansetrona apresentou efeito frente ao modelo de comportamento de elevação da pata com formalina, sugerindo que a resposta nociceptiva é influencia­da por receptores 5-HT3, em especial em resposta à dor inflamatória.