Renato de Freitas

Serotonergic neural links from the dorsal raphe nucleus modulate defensive behaviours organised by the dorsomedial hypothalamus and the elaboration of fear-induced antinociception via locus coeruleus pathways.

Decrease of γ-aminobutyric acid (GABA)-mediated neurotransmission in the dorsomedial hypothalamus (DMH) evokes instinctive fear-like responses. The aim of the present study was to investigate the involvement of the serotonin (5-HT)- and norepinephrine-mediated pathways of the endogenous pain inhibitory system, including the dorsal raphe nucleus (DRN) and the locus coeruleus (LC), in the defensive responses and antinociceptive processes triggered by the blockade of GABAergic receptors in the DMH. The intra-hypothalamic microinjection of the GABA(A) receptor antagonist bicuculline (40xa0ng/200xa0nL) elicited elaborate defensive behaviours interspersed with exploratory responses. This escape behaviour was followed by significantly increased pain thresholds, a phenomenon known as fear-induced antinociception. Furthermore, at 5 and 14 days after DRN serotonin-containing neurons were damaged using the selective neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), the frequency and duration of alertness and escape behaviour evoked by the GABA(A) receptor blockade in the DMH decreased, as well as fear-induced antinociception. Pre-treatment with the non-selective 5-HT receptor antagonist methysergide, the 5-HT(2A/2C) receptor antagonist ketanserin and the 5-HT(2A) receptor selective antagonist R-96544 in the LC also decreased fear-induced antinociception, without significant changes in the expression of defensive behaviours. These data suggest that the serotonergic neurons of the DRN are directly involved in the organisation of defensive responses as well as in the elaboration of the innate fear-induced antinociception. However, serotonin-mediated inputs from the NDR to the LC modulate only fear-induced antinociception and not the defensive behaviours evoked by GABA(A) receptor blockade in the DMH.

Role of muscarinic and nicotinic cholinergic receptors in an experimental model of epilepsy-induced analgesia

The blockade of GABA-mediated Cl(-) influx with pentylenetetrazol (PTZ) was used in the present work to induce seizures in animals. The neurotransmission in the postictal period has been the focus of many studies, and there is evidence suggesting antinociceptive mechanisms following tonic-clonic seizures in both animals and men. The aim of this work was to study the involvement of acetylcholine in the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg/kg). Analgesia was measured by the tail-flick test in eight albino Wistar rats per group. Convulsions were followed by significant increases in tail-flick latencies (TFLs) at least for 120 min of the postictal period. Peripheral administration of atropine (0.25, 1 and 4 mg/kg) caused a significant dose-dependent decrease in the TFL in seizing animals, as compared to controls. These data were corroborated by peripheral administration of mecamylamine, a nicotinic cholinergic receptor blocker, at the same doses (0.25, 1 and 4 mg/kg) used for the muscarinic cholinergic receptor antagonist. The recruitment of the muscarinic receptor was made 10 min postconvulsions and in subsequent periods of postictal analgesia, whereas the involvement of the nicotinic cholinergic receptor was implicated only after 30 min postseizures. The cholinergic antagonists caused a minimal reduction in body temperature, but did not impair baseline TFL, spontaneous exploration or motor coordination in the rotarod test at the maximal dose of 4 mg/kg. These results indicate that acetylcholine may be involved as a neurotransmitter in postictal analgesia.

Involvement of prelimbic medial prefrontal cortex in panic-like elaborated defensive behaviour and innate fear-induced antinociception elicited by GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei: role of the endocannabinoid CB1 receptor

It has been shown that GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei (DMH and VMH, respectively) induces elaborated defensive behavioural responses accompanied by antinociception, which has been utilized as an experimental model of panic attack. Furthermore, the prelimbic (PL) division of the medial prefrontal cortex (MPFC) has been related to emotional reactions and the processing of nociceptive information. The aim of the present study was to investigate the possible involvement of the PL cortex and the participation of local cannabinoid CB1 receptors in the elaboration of panic-like reactions and in innate fear-induced antinociception. Elaborated fear-induced responses were analysed during a 10-min period in an open-field test arena. Microinjection of the GABAA receptor antagonist bicuculline into the DMH/VMH evoked panic-like behaviour and fear-induced antinociception, which was decreased by microinjection of the non-selective synaptic contact blocker cobalt chloride in the PL cortex. Moreover, microinjection of AM251 (25, 100 or 400xa0pmol), an endocannabinoid CB1 receptor antagonist, into the PL cortex also attenuated the defensive behavioural responses and the antinociception that follows innate fear behaviour elaborated by DMH/VMH. These data suggest that the PL cortex plays an important role in the organization of elaborated forward escape behaviour and that this cortical area is also involved in the elaboration of innate fear-induced antinociception. Additionally, CB1 receptors in the PL cortex modulate both panic-like behaviours and fear-induced antinociception elicited by disinhibition of the DMH/VMH through microinjection of bicuculline.

Antinociception induced by acute oral administration of sweet substance in young and adult rodents: The role of endogenous opioid peptides chemical mediators and μ1-opioid receptors

The present work aimed to investigate the effects of acute sucrose treatment on the perception of painful stimuli. Specifically, we sought to determine the involvement of the endogenous opioid peptide-mediated system as well as the role of the μ(1)-opioid receptor in antinociception organisation induced by acute sucrose intake. Nociception was assessed with the tail-flick test in rats (75, 150 and 250 g) of different ages acutely pre-treated with 500 μL of a sucrose solution (25, 50, 150 and 250 g/L) or tap water. Young and Adult rats (250 g) showed antinociception after treatment with 50 g/L (during 5 min) and 150 g/L and 250 g/L (during 20 min) sucrose solutions. Surprisingly, this antinociception was more consistent in mature adult rodents than in pups. To evaluate the role of opioid systems, mature adult rodents were pre-treated with different doses (0.25, 1 or 4 mg/kg) of the non-selective opioid receptor antagonist naloxone, the selective μ(1)-opioid receptor antagonist naloxonazine or vehicle followed by 250 g/L sucrose solution treatment. Sucrose-induced antinociception was reduced by pre-treatment with both naloxone and naloxonazine. The present findings suggest that sweet substance-induced hypo-analgesia is augmented by increasing sucrose concentrations in young and adult rodents. Acute oral sucrose treatment inhibits pain in laboratory animal by mediating endogenous opioid peptide and μ(1)-opioid receptor actions.

Endogenous opioid peptide-mediated neurotransmission in central and pericentral nuclei of the inferior colliculus recruits μ1-opioid receptor to modulate post-ictal antinociception.

BACKGROUNDnThe aim of the present work was to investigate the involvement of the μ1-endogenous opioid peptide receptor-mediated system in post-ictal antinociception.nnnMETHODSnAntinociceptive responses were determined by the tail-flick test after pre-treatment with the selective μ1-opioid receptor antagonist naloxonazine, peripherally or centrally administered at different doses.nnnRESULTSnPeripheral subchronic (24 h) pre-treatment with naloxonazine antagonised the antinociception elicited by tonic-clonic seizures. Acute (10 min) pre-treatment, however, did not have the same effect. In addition, microinjections of naloxonazine into the central, dorsal cortical and external cortical nuclei of the inferior colliculus antagonised tonic-clonic seizure-induced antinociception. Neither acute (10-min) peripheral pre-treatment with naloxonazine nor subchronic intramesencephalic blockade of μ1-opioid receptors resulted in consistent statistically significant differences in the severity of tonic-clonic seizures shown by Racines index (1972), although the intracollicular specific antagonism of μ1-opioid receptor decreased the duration of seizures.nnnCONCLUSIONnμ1-Opioid receptors and the inferior colliculus have been implicated in several endogenous opioid peptide-mediated responses such as antinociception and convulsion. The present findings suggest the involvement of μ1-opiate receptors of central and pericentral nuclei of the inferior colliculus in the modulation of tonic-clonic seizures and in the organisation of post-ictal antinociception.

Involvement of pre- and post-synaptic serotonergic receptors of dorsal raphe nucleus neural network in the control of the sweet-substance-induced analgesia in adult Rattus norvegicus (Rodentia, Muridae).

In order to investigate the effects of monoaminergic mechanisms of the dorsal raphe nucleus on the elaboration and control of sweet-substance-induced antinociception, male albino Wistar rats weighing 180-200 g received sucrose solution (250 g/L) for 14 days as their only source of liquid. After the chronic consumption of sucrose solution, each animal was pretreated with unilateral microinjection of methiothepin mesylate (5.0 microg/0.2 microL), or methysergide maleate (5.0 microg/0.2 microL) in the dorsal raphe nucleus. Each rat consumed an average of 15.6g sucrose/day. Their tail withdrawal latencies in the tail-flick test were measured immediately before and after this treatment. An analgesia index was calculated from the withdrawal latencies before and after the pharmacological treatment. The blockade of serotonergic receptor in the dorsal raphe nucleus with methysergide after the chronic intake of sucrose decreased the sweet-induced antinociception. However, microinjections of methiothepin in the dorsal raphe nucleus did not cause a similar effect on the tail-flick latencies after the chronic intake of sucrose solution, increasing the sweet-substance-induced analgesia. These results indicate the involvement of serotonin as a neurotransmitter in the sucrose-produced antinociception. Considering that the blockade of pre-synaptic serotonergic receptors of the neural networks of the dorsal raphe nucleus with methiothepin did not decrease the sweet-substance-induced antinociception, and the central blockade of post-synaptic serotonergic receptors decreased the sucrose-induced analgesia, the modulation of the release of serotonin in the neural substrate of the dorsal raphe nucleus seems to be crucial for the organization of this interesting antinociceptive process.

The role of dorsomedial and ventrolateral columns of the periaqueductal gray matter and in situ 5‐HT2A and 5‐HT2C serotonergic receptors in post‐ictal antinociception

The periaqueductal gray matter (PAG) consists in a brainstem structure rich in 5‐hydroxytryptamine (5‐HT) inputs related to the modulation of pain. The involvement of each of the serotonergic receptor subtypes found in PAG columns, such as the dorsomedial (dmPAG) and the ventrolateral (vlPAG) columns, regarding post‐ictal antinociception have not been elucidated. The present work investigated the participation of the dmPAG and vlPAG columns in seizure‐induced antinociception. Specifically, we studied the involvement of serotonergic neurotransmission in these columns on antinociceptive responses that follow tonic‐clonic epileptic reactions induced by pentylenetetrazole (PTZ), an ionophore GABA‐mediated Cl‐ influx antagonist. Microinjections of cobalt chloride (1.0 mM CoCl2/0.2 µL) into the dmPAG and vlPAG caused an intermittent local synaptic inhibition and decreased post‐ictal antinociception that had been recorded at various time points after seizures. Pretreatments of the dmPAG or the vlPAG columns with the nonselective serotonergic receptors antagonist methysergide (5.0 µg/0.2 µL) or intramesencephalic microinjections of ketanserin (5.0 µg/0.2 µL), a serotonergic antagonist with more affinity to 5‐HT2A/2C receptors, decreased tonic‐clonic seizure‐induced antinociception. Both dmPAG and vlPAG treatment with either the 5‐HT2A receptor selective antagonist R‐96544 (10 nM/0.2 µL), or the 5‐HT2C receptors selective antagonist RS‐102221 (0.15 µg/0.2 µL) also decrease post‐ictal antinociception. These findings suggest that serotonergic neurotransmission, which recruits both 5‐HT2A and 5‐HT2C serotonergic receptors in dmPAG and vlPAG columns, plays a critical role in the elaboration of post‐ictal antinociception. Synapse, 68:16–30, 2014.

Paradoxical effect of noradrenaline-mediated neurotransmission in the antinociceptive phenomenon that accompanies tonic–clonic seizures: Role of locus coeruleus neurons and α2- and β-noradrenergic receptors

The postictal state is generally followed by antinociception. It is known that connections between the dorsal raphe nucleus, the periaqueductal gray matter, and the locus coeruleus, an important noradrenergic brainstem nucleus, are involved in the descending control of ascending nociceptive pathways. The aim of the present study was to determine whether noradrenergic mechanisms in the locus coeruleus are involved in postictal antinociception. Yohimbine (an α(2)-receptor antagonist) or propranolol (a β-receptor antagonist) was microinjected unilaterally into the locus coeruleus, followed by intraperitoneal administration of pentylenetetrazole (PTZ), a noncompetitive antagonist that blocks GABA-mediated Cl(-) influx. Although the administration of both yohimbine and propranolol to the locus coeruleus/subcoeruleus area resulted in a significant decrease in tonic or tonic-clonic seizure-induced antinociception, the effect of yohimbine restricted to the locus coeruleus was more distinct compared with that of propranolol, possibly because of the presynaptic localization of α(2)-noradrenergic receptors in locus coeruleus neurons. These effects were related to the modulation of noradrenergic activity in the locus coeruleus. Interestingly, microinjections of noradrenaline into the locus coeruleus also decrease the postictal antinociception. The present results suggest that the mechanism underlying postictal antinociception involves both α(2)- and β-noradrenergic receptors in the locus coeruleus, although the action of noradrenaline on these receptors causes a paradoxical effect, depending on the nature of the local neurotransmission.

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.