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.

Central Autonomic Network Regions and Hypertension: Unveiling Sympathetic Activation and Genetic Therapeutic Perspectives.

INTRODUCTION: Hypertension, a leading cause of death, was investigated in this study to understand the role of specific brain regions in regulating blood pressure. The lateral parabrachial nucleus (LPBN), Kolliker-fuse nucleus (KF), and periductal grey matter (PAG) were examined for their involvement in hypertension. METHODS: Lentiviral vectors were used to alter the activity of these brain regions in hypertensive rats. Over a 75-day period, blood pressure, heart rate, reflex responses, and heart rate variability were measured. RESULTS: Decreasing the activity in the LPBN resulted in a reduced sympathetic outflow, lowering the blood pressure and heart rate. In the KF, the sympathetic activity decreased and chemoreflex variation was attenuated, without affecting the blood pressure. Silencing the PAG had no significant impact on blood pressure or sympathetic tone, but decreased cardiac baroreflex gain. DISCUSSION: These findings highlight the significant role of the LPBN in hypertension-related sympathetic activation. Additionally, LPBN and KF neurons appear to activate mechanisms that control respiration and sympathetic outflow during chemoreceptor activation. CONCLUSIONS: The study provided insights into the contribution of the midbrain and pontine regions to neurogenic hypertension and offers potential avenues for future genetic interventions and developing novel treatment approaches.

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.

Impact of the glutamatergic neurotransmission within the A5 region on the cardiorespiratory response evoked from the midbrain dlPAG.

Stimulation of the dorsolateral periaqueductal grey matter (dlPAG) in rats evokes an active defensive behaviour together with a cardiorespiratory response characterised by tachypnoea, tachycardia and hypertension. The dlPAG neurons involved in these responses are excitatory, presumably glutamatergic, due to the presence of vesicular glutamate transporter VGLUT2 within their axon terminals. Previously, our group described a functional interaction between dlPAG and the pontine A5 region. Accordingly, in the present work, in order to characterize the role of glutamate within this interaction, experiments were carried out in spontaneously breathing anaesthetized rats (sodium pentobarbitone 60 mg/kg i.p., suplemented with 20 mg/kg i.p.). The cardiorespiratory response evoked by electrical stimulation of the dlPAG (1 ms pulses, 20-50 µA, given at 100 Hz, during 5 s) was analysed before and after the microinjection, within the A5 region, of either kynurenic acid (non-specific glutamate receptor antagonist; 5-10 nmol), DAP-5 (NMDA antagonist; 1 pmol), CNQX (non-NMDA antagonist; 1 pmol) or MCPG (metabotropic antagonist; 0,1 nmol). Kynurenic acid decreased the intensity of both the tachypnoea (p < 0,001) and tachycardia (p < 0,001) induced by dl-PAG stimulation. Blockade of no-NMDA receptors reduced the increase of respiratory frequency, heart rate and pressor response to dl-PAG stimulation (p < 0,01, p < 0,001, p < 0,05 respectively). Blockade of either NMDA or metabotropic receptors reduced the dlPAG-evoked tachycardia and pressor response (p < 0,01; p < 0,05 respectively). These results suggest a neuromodulatory role for A5 region via glutamate neurotransmission of the dlPAG-evoked cardiorespiratory response, confirming the role of the ventrolateral pons in the neuronal circuits involved in respiratory and heart rate control.

Positioning the brainstem within the neural network of threat prediction.

In a recent study, Strickland and McDannald dissected the role of brainstem networks in threat prediction. Using probabilistic threat discrimination in rats, the authors demonstrated that brainstem neurons estimate threat probability and generate positive aversive prediction errors after unexpected outcomes. Their findings suggest that, beyond organizing defensive behaviors, brainstem neurons are involved in threat prediction computations.

Translational aspects of deep brain stimulation for chronic pain.

The use of deep brain stimulation (DBS) for the treatment of chronic pain was one of the first applications of this technique in functional neurosurgery. Established brain targets in the clinic include the periaqueductal (PAG)/periventricular gray matter (PVG) and sensory thalamic nuclei. More recently, the anterior cingulum (ACC) and the ventral striatum/anterior limb of the internal capsule (VS/ALIC) have been investigated for the treatment of emotional components of pain. In the clinic, most studies showed a response in 20%-70% of patients. In various applications of DBS, animal models either provided the rationale for the development of clinical trials or were utilized as a tool to study potential mechanisms of stimulation responses. Despite the complex nature of pain and the fact that animal models cannot reliably reflect the subjective nature of this condition, multiple preparations have emerged over the years. Overall, DBS was shown to produce an antinociceptive effect in rodents when delivered to targets known to induce analgesic effects in humans, suggesting a good predictive validity. Compared to the relatively high number of clinical trials in the field, however, the number of animal studies has been somewhat limited. Additional investigation using modern neuroscience techniques could unravel the mechanisms and neurocircuitry involved in the analgesic effects of DBS and help to optimize this therapy.

Role of Enteric Glia as Bridging Element between Gut Inflammation and Visceral Pain Consolidation during Acute Colitis in Rats.

Acute inflammation is particularly relevant in the pathogenesis of visceral hypersensitivity associated with inflammatory bowel diseases. Glia within the enteric nervous system, as well as within the central nervous system, contributes to neuroplasticity during inflammation, but whether enteric glia has the potential to modify visceral sensitivity following colitis is still unknown. This work aimed to investigate the occurrence of changes in the neuron-glial networks controlling visceral perception along the gut-brain axis during colitis, and to assess the effects of peripheral glial manipulation. Enteric glia activity was altered by the poison fluorocitrate (FC; 10 µmol kg-1 i.p.) before inducing colitis in animals (2,4-dinitrobenzenesulfonic acid, DNBS; 30 mg in 0.25 mL EtOH 50%), and visceral sensitivity, colon damage, and glia activation along the pain pathway were studied. FC injection significantly reduced the visceral hyperalgesia, the histological damage, and the immune activation caused by DNBS. Intestinal inflammation is associated with a parallel overexpression of TRPV1 and S100ß along the gut-brain axis (colonic myenteric plexuses, dorsal root ganglion, and periaqueductal grey area). This effect was prevented by FC. Peripheral glia activity modulation emerges as a promising strategy for counteracting visceral pain induced by colitis.

The primary motor cortex electrical and chemical stimulation attenuates the chronic neuropathic pain by activation of the periaqueductal grey matter: The role of NMDA receptors.

BACKGROUND: Motor cortex stimulation (MCS) is proper as a non-pharmacological therapy for patients with chronic and neuropathic pain (NP). AIMS: This work aims to investigate if the MCS in the primary motor cortex (M1) produces analgesia and how the MCS could interfere in the MCS-induced analgesia. Also, to elucidate if the persistent activation of N-methyl-d-aspartic acid receptor (NMDAr) in the periaqueductal grey matter (PAG) can contribute to central sensitisation of the NP. METHODS: Male Wistar rats were submitted to the von Frey test to evaluate the mechanical allodynia after 21 days of chronic constriction injury (CCI) of the sciatic nerve. The MCS was performed with low-frequency (20 µA, 100 Hz) currents during 15 s by a deep brain stimulation (DBS) device. Moreover, the effect of M1-treatment with an NMDAr agonist (at 2, 4, and 8 nmol) was investigated in CCI rats. The PAG dorsomedial column (dmPAG) was pretreated with the NMDAr antagonist LY 235959 (at 8 nmol), followed by MCS. RESULTS: The MCS decreased the mechanical allodynia in rats with chronic NP. The M1-treatment with an NMDA agonist at 2 and 8 nmol reduced the mechanical allodynia in CCI rats. In addition, dmPAG-pretreatment with LY 235959 at 8 nmol attenuated the mechanical allodynia evoked by MCS. CONCLUSION: The M1 cortex glutamatergic system is involved in the modulation of chronic NP. The analgesic effect of MCS may depend on glutamate signaling recruitting NMDAr located on PAG neurons in rodents with chronic NP.

Activation of orexin-1 receptors in the ventrolateral periaqueductal grey matter (vlPAG) modulates pulpal nociception and the induction of substance P in vlPAG and trigeminal nucleus caudalis.

AIM: To characterize the role of orexin-1 receptors (OX1Rs) in ventrolateral periaqueductal grey matter (vlPAG) on modulation of capsaicin-induced pulpal nociception in rats. METHODOLOGY: Sixty-six adult male Wistar rats (2 months old) weighing between 230 and 260 g were used. The animals were cannulated for microinjection of drugs into the vlPAG matter. Pulpalgia was induced by intradental application of capsaicin solution (100 µg) into the incisor teeth of the rats. Ten min prior to capsaicin application, orexin-A (50, 100 and 150 pmol L-1 per rat) was administered. Orexin-A (150 pmol L-1 ) was also co-administrated with SB-334867 (40 nmol L-1 per rat), an OX1Rs antagonist; or bicuculline (1 µg per rat), a GABAA receptors antagonist. Moreover, treatment effects on the release of pro-nociceptive modulator substance P (SP) in vlPAG and trigeminal nucleus caudalis (Vc) of rats were explored using an immunofluorescence technique. One-way analysis of variance was used for the statistical analysis. RESULTS: Orexin-A dose-dependently decreased capsaicin-induced nociceptive behaviour. However, SB-334867 (40 nmol L-1 per rat) pretreatment (P < 0.05), but not bicuculline (1 µg per rat), attenuated the analgesic effect of orexin-A (150 pmol L-1 ). The level of SP was significantly increased in Vc and decreased in vlPAG of capsaicin-treated rats (P < 0.05). Capsaicin-induced changes in SP levels, however, were prohibited by orexin-A treatment (150 pmol L-1 ) (P < 0.05). CONCLUSIONS: Orexin-A administration into the vlPAG was associated with an inhibitory effect on capsaicin-induced pulpal nociception and bidirectional effects on the induction of SP in vlPAG and Vc of rats. Central activation of OX1Rs is a potential therapeutic tool for pulpalgia.

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.

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.

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 9nmol 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 9nmol 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 6nmol 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 9nmol 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.

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.

Effect of electrical vs. chemical deep brain stimulation at midbrain sites on micturition in anaesthetized rats.

AIM: To understand how deep brain stimulation of the midbrain influences control of the urinary bladder. METHODS: In urethane-anaesthetized male rats, saline was infused continuously into the bladder to evoke cycles of filling and voiding. The effect of electrical (0.1-2.0 ms pulses, 5-180 Hz, 0.5-2.5 V) compared to chemical stimulation (microinjection of D,L-homocysteic acid, 50 nL 0.1 M solution) at the same midbrain sites was tested. RESULTS: Electrical stimulation of the periaqueductal grey matter and surrounding midbrain disrupted normal coordinated voiding activity in detrusor and sphincters muscles and suppressed urine output. The effect occurred within seconds was reversible and not secondary to cardiorespiratory changes. Bladder compliance remained unchanged. Chemical stimulation over the same area using microinjection of D,L-homocysteic acid (DLH) to preferentially activate somatodendritic receptors decreased the frequency of micturition but did not disrupt the coordinated pattern of voiding. In contrast, chemical stimulation within the caudal ventrolateral periaqueductal grey, in the area where critical synapses in the micturition reflex pathway are located, increased the frequency of micturition. CONCLUSION: Electrical deep brain stimulation within the midbrain can inhibit reflex micturition. We suggest that the applied stimulus entrained activity in the neural circuitry locally, thereby imposing an unphysiological pattern of activity. In a way similar to the use of electrical signals to 'jam' radio transmission, this may prevent a synchronized pattern of efferent activity being transmitted to the spinal outflows to orchestrate a coordinated voiding response. Further experiments to record neuronal firing in the midbrain during the deep brain stimulation will be necessary to test this hypothesis.

µ- and κ-Opioid receptor activation in the dorsal periaqueductal grey matter differentially modulates panic-like behaviours induced by electrical and chemical stimulation of the inferior colliculus.

It has been shown that electrical stimulation of the mesencephalic tectum (MT) provokes defensive responses in both humans and rodents. During an emotional aversive state, some convergent studies have also demonstrated the existence of a complex interaction between endogenous opioid peptide- and γ-aminobutyric acid (GABA)-containing connections during fear-induced responses. It has been proposed that opioid neurons exert an influence on GABAergic interneurons, which, in turn, exert inhibitory tonic control on the mesencephalic excitatory pathways. Thus, opioid peptides can disinhibit neurons that are tonically inhibited by GABA, therefore, modulating the expression of defensive behavioural reactions. In the present work, we used both electric stimulation and microinjections of the GABAA receptor antagonist bicuculline in the inferior colliculus (IC) of Wistar rats in combination with microinjections of µ- and κ-opioid receptor selective agonists into the dorsal columns of periaqueductal grey matter (dPAG) to evaluate the effects on panic-like behaviours elicited by IC electrical and chemical stimulation. The present results showed that neurochemical lesions of the dPAG caused a significant impairment in the organisation of defensive responses by IC neurons, reducing the duration [t(14)=3.0; p<0.01] of defensive immobility and the duration [t(14)=2.8; p<0.05] and frequency [t(14)=2.5; p<0.05] of escape. Paradoxically, treating the dPAG with the µ-opioid receptor agonist met-enkephalin caused a significant reduction of panic-like behaviours induced by both electrical and chemical stimulation of the IC, increasing the escape behaviour threshold [F(2,23)=13.5; p<0.001] and decreasing the frequency [F(3,36)=11.7; p<0.001] and duration [F(3,36)=11.6; p<0.001] of escape and the duration of defensive immobility [F(3,36)=16.1; p<0.05]. In contrast, treating the dPAG with the κ-opioid receptor agonist salvinorin-A increased the frequency [F(3,36)=12.4; p<0.01] and duration [F(3,34)=16.1; p<0.01] of defensive immobility induced by GABAA receptor blockade in the IC. The present results suggest the existence of a complex neuronal network in the MT in which endogenous opioid peptides and GABAergic pathways interact in the control of fear-related behavioural responses.

Elevation of brain allopregnanolone rather than 5-HT release by short term, low dose fluoxetine treatment prevents the estrous cycle-linked increase in stress sensitivity in female rats.

Withdrawal from long-term dosing with exogenous progesterone precipitates increased anxiety-linked changes in behavior in animal models due to the abrupt decrease in brain concentration of allopregnanolone (ALLO), a neuroactive metabolite of progesterone. We show that a withdrawal-like effect also occurs during the late diestrus phase (LD) of the natural ovarian cycle in rats, when plasma progesterone and ALLO are declining but estrogen secretion maintains a stable low level. This effect at LD was prevented by short-term treatment with low dose fluoxetine. During LD, but not at other stages of the estrous cycle, exposure to anxiogenic stress induced by whole body vibration at 4 Hz for 5 min evoked a significant decrease in tail flick latency (stress-induced hyperalgesia) and a decrease in the number of Fos-positive neurons present in the periaqueductal gray (PAG). The threshold to evoke fear-like behaviors in response to electrical stimulation of the dorsal PAG was lower in the LD phase, indicating an increase in the intrinsic excitability of the PAG circuitry. All these effects were blocked by short-term administration of fluoxetine (2 × 1.75 mg kg(-1) i.p.) during LD. This dosage increased the whole brain concentration of ALLO, as determined using gas chromatography-mass spectrometry, but was without effect on the extracellular concentration of 5-HT in the dorsal PAG, as measured by microdialysis. We suggest that fluoxetine-induced rise in brain ALLO concentration during LD offsets the sharp physiological decline, thus removing the trigger for the development of anxiogenic withdrawal effects.

Cooperative regulation of anxiety and panic-related defensive behaviors in the rat periaqueductal grey matter by 5-HT1A and µ-receptors.

Previous results with the elevated T-maze (ETM) test indicate that the antipanic action of serotonin (5-HT) in the dorsal periaqueductal grey (dPAG) depends on the activation endogenous opioid peptides. The aim of the present work was to investigate the interaction between opioid- and serotonin-mediated neurotransmission in the modulation of defensive responses in rats submitted to the ETM. The obtained results showed that intra-dPAG administration of morphine significantly increased escape latency, a panicolytic-like effect that was blocked by pre-treatment with intra-dPAG injection of either naloxone or the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl] ethyl] -N- 2- pyridinyl-ciclohexanecarboxamide maleate (WAY-100635). In addition, previous administration of naloxone antagonized both the anti-escape and the anti-avoidance (anxiolytic-like) effect of the 5-HT1A agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), but did not affect the anti-escape effect of the 5-HT2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). Moreover, the combination of sub-effective doses of locally administered 5-HT and morphine significantly impaired ETM escape performance. Finally, the µ-antagonist D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN (CTOP) blocked the anti-avoidance as well as the anti-escape effect of 8-OHDPAT, and the association of sub-effective doses of the µ-opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin acetate salt (DAMGO) and of 8-OHDPAT had anti-escape and anti-avoidance effects in the ETM. These results suggest a synergic interaction between the 5-HT1A and the µ-opioid receptor at post-synaptic level on neurons of the dPAG that regulate proximal defense, theoretically related to panic attacks.