Luiz Luciano Falconi-Sobrinho

CB1 cannabinoid receptor-mediated anandamide signalling reduces the defensive behaviour evoked through GABAA receptor blockade in the dorsomedial division of the ventromedial hypothalamus

&NA; 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. HighlightsAdministration of bicuculline intra‐VMHdm induced panic‐like responses.Anandamide attenuated defence responses induced by bicuculline.Anandamide modulates the pro‐aversive effects of bicuculline, recruiting CB1 receptors.AM251 potentiated the non‐oriented escape induced by bicuculline.AM251‐induced potentiation of bicuculline effect is related to TRPV1 vanilloid receptor activity.

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

&NA; 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. HighlightsLidocaine in ACC decreased the proaversive effect and antinociception evoked by bicuculline in PH.NBQX in PH attenuated anxiety‐ and panic‐like defensive reaction and antinociception.LY235959 in PH attenuated panic‐like defensive reactions and antinociception.ACC modulates defensive behaviour and antinociception organised by PH neurons.Glutamatergic efferent pathways from the ACC to the PH modulate fear‐induced defensive responses.

Neuroethological validation of an experimental apparatus to evaluate oriented and non-oriented escape behaviours: Comparison between the polygonal arena with a burrow and the circular enclosure of an open-field test.

Inhibition of GABAergic neural inputs to dorsal columns of the periaqueductal grey matter (dPAG), posterior (PH) and dorsomedial (DMH) hypothalamic nuclei elicits distinct types of escape behavioural reactions. To differentiate between the variety and intensity of panic-related behaviours, the pattern of defensive behaviours evoked by blockade of GABAA receptors in the DMH, PH and dPAG were compared in a circular open-field test and in a recently designed polygonal arena. In the circular open-field, the defensive behaviours induced by microinjection of bicuculline into DMH and PH were characterised by defensive alertness behaviour and vertical jumps preceded by rearing exploratory behaviour. On the other hand, explosive escape responses interspersed with horizontal jumps and freezing were observed after the blockade of GABAA receptors on dPAG neurons. In the polygonal arena apparatus, the escape response produced by GABAergic inhibition of DMH and PH neurons was directed towards the burrow. In contrast, the blockade of GABAA receptors in dPAG evoked non-oriented escape behaviour characterised by vigorous running and horizontal jumps in the arena. Our findings support the hypothesis that the hypothalamic nuclei organise oriented escape behavioural responses whereas non-oriented escape is elaborated by dPAG neurons. Additionally, the polygonal arena with a burrow made it easy to discriminate and characterise these two different patterns of escape behavioural responses. In this sense, the polygonal arena with a burrow can be considered a good methodological tool to discriminate between these two different patterns of escape behavioural responses and is very useful as a new experimental animal model of panic attacks.

Connexions between the dorsomedial division of the ventromedial hypothalamus and the dorsal periaqueductal grey matter are critical in the elaboration of hypothalamically mediated panic-like behaviour

&NA; The electrical and chemical stimulation of the dorsal periaqueductal grey matter (dPAG) elicits panic‐like explosive escape behaviour. Although neurons of the ventromedial hypothalamus (VMH) seem to organise oriented escape behaviour, when stimulated with excitatory amino acids at higher doses, non‐oriented/explosive escape reactions can also be displayed. The aim of this work was to examine the importance of reciprocal projections between the VMH and the dPAG for the organisation of this panic‐like behaviour. The chemical stimulation of the VMH with 9 nmol of N‐methyl‐D‐aspartic acid (NMDA) elicited oriented and non‐oriented escape behaviours. The pretreatment of the dPAG with a non‐selective blocker of synaptic contacts, cobalt chloride (CoCl2), followed by stimulation of the dorsomedial part of the ventromedial hypothalamus (dmVMH) with 9 nmol of NMDA, abolished the non‐oriented/explosive escape and freezing responses elicited by the stimulation of the dmVMH. Nonetheless, the rats still showed oriented escape to the burrow. On the other hand, when the blockade of the dmVMH with CoCl2 was followed by stimulation of the dPAG with 6 nmol of NMDA, no effect was observed either on the non‐oriented/explosive escape or on the freezing behaviour organised by the dPAG. Furthermore, Fos protein‐labelled neurons were observed in the dPAG after the stimulation of the dmVMH with 9 nmol of NMDA. Additionally, when the anterograde neurotracer biotinylated dextran amine (BDA) was deposited in the dmVMH subsequent stimulation of the dmVMH produced BDA‐labelled neural fibres with terminal boutons surrounding Fos‐labelled neurons in the dPAG, suggesting synaptic contacts between dmVMH and dPAG neurons for eliciting panic‐like behavioural responses. The current data suggest that the dPAG is the key structure that organises non‐oriented/explosive escape reactions associated with panic attack‐like behaviours. Graphical abstract The chemical stimulation of the dorsomedial division of the ventromedial hypothalamic nucleus (dmVMH) with NMDA at 9 nmol elicits panic‐like behavioural responses with increase of Fos protein in neuronal nuclei of the dorsal periaqueductal grey matter (dPAG). Fos‐immunolabelled neurons in dPAG are surrounded by dmVMH‐dPAG neuronal pathways terminal buttons. The inactivation of synaptic contacts in dPAG with CoCl2 abolishes the panic‐like behaviour elicited by dmVMH neuronal activation with 9 nmol of NMDA. Figure. No caption available. HIGHLIGHTSdmVMH neurons can trigger panic‐like escape but does not act independently.dPAG neurons mainly produce freezing and explosive escape independently.Projections from dmVMH to dPAG play an important role in the elaboration of defence.

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

HighlightsThe electrical stimulation of dlSC and dlPAG neurons elicits fear‐induced antinociception.Gi/PGi&agr; 5‐HT2A/2C receptors modulate fear‐induced antinociception evoked by dlSC activation.Gi/PGi&agr; 5‐HT2A/2C receptors modulate fear‐induced antinociception evoked by dlPAG activation.NRM 5‐HT2A/2C receptors modulate fear‐induced antinociception evoked by dlSC activation.NRM 5‐HT2A/2C receptors modulate fear‐induced antinociception evoked by dlPAG activation. ABSTRACT 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 &agr; reticular nuclei (Gi/PGi&agr;) 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 5.0 &mgr;g/0.2 &mgr;L) either in Gi/PGi&agr; or in NRM. The blockade of 5‐HT2 receptors in both Gi/PGi&agr; 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/PGi&agr; complex and NRM are critically recruited in pain modulation during the panic‐like emotional behaviour.

Intrinsic connections within the pedunculopontine tegmental nucleus are critical to the elaboration of post-ictal antinociception.

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.

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.

Nicotinic and muscarinic cholinergic receptors are recruited by acetylcholine-mediated neurotransmission within the locus coeruleus during the organisation of post-ictal antinociception.

Post-ictal antinociception is characterised by an increase in the nociceptive threshold that accompanies tonic and tonic-clonic seizures (TCS). The locus coeruleus (LC) receives profuse cholinergic inputs from the pedunculopontine tegmental nucleus. Different concentrations (1μg, 3μg and 5μg/0.2μL) of the muscarinic cholinergic receptor antagonist atropine and the nicotinic cholinergic receptor antagonist mecamylamine were microinjected into the LC of Wistar rats to investigate the role of cholinergic mechanisms in the severity of TCS and the post-ictal antinociceptive response. Five minutes later, TCS were induced by systemic administration of pentylenetetrazole (PTZ) (64mg/kg). Seizures were recorded inside the open field apparatus for an average of 10min. Immediately after seizures, the nociceptive threshold was recorded for 130min using the tail-flick test. Pre-treatment of the LC with 1μg, 3μg and 5μg/0.2μL concentrations of both atropine and mecamylamine did not cause a significant effect on seizure severity. However, the same treatments decreased the post-ictal antinociceptive phenomenon. In addition, mecamylamine caused an earlier decrease in the post-ictal antinociception compared to atropine. These results suggest that muscarinic and mainly nicotinic cholinergic receptors of the LC are recruited to organise tonic-clonic seizure-induced antinociception.

Dorsal raphe nucleus acetylcholine-mediated neurotransmission modulates post-ictal antinociception: The role of muscarinic and nicotinic cholinergic receptors

The dorsal raphe nucleus (DRN) is a key structure of the endogenous pain inhibitory system. Although the DRN is rich in serotoninergic neurons, cholinergic neurons are also found in that nucleus. Both ictal and inter-ictal states are followed by post-ictal analgesia. The present study investigated the role of cholinergic mechanisms in postictal antinociceptive processes using microinjections of atropine and mecamylamine, muscarinic and nicotinic cholinergic receptor antagonists, respectively, in the DRN of rats. Intraperitoneal injection of pentylenetetrazole (PTZ) (at 64mg/kg) caused tonic and tonic-clonic seizures. The convulsive motor reactions were followed by an increase in pain thresholds, a phenomenon known as post-ictal analgesia. Pre-treatment of the DRN with atropine or mecamylamine at 1µg, 3µg and 5µg/0.2µL decreased the post-ictal antinociceptive phenomenon. The present results showed that the post-ictal analgesia was mediated by muscarinic and nicotinic cholinergic receptors in the DRN, a structure crucially involved in the neural network that organises post-ictal hypoalgesia.