Periaqueductal gray matter modulates the hypercapnic ventilatory response

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anxiogenic-like effect induced by TRPV1 receptor activation within the dorsal periaqueductal gray matter in mice

Pharmacological manipulation of TRPV1 (Transient Receptor Potential Vanilloid type-1) receptors has been emerging as a novel target in the investigation of anxiety states. Here, we attempt to show the role played by the TRPV1 receptors within the dorsal periaqueductal gray matter (dPAG), a midbrain structure strongly involved in the modulation of anxiety. Anxiety was assessed by recording spatiotemporal [percent open arm entries (%OE) and percent open arm time (%OT)] and ethological [e.g., head dipping (HD), stretched-attend postures (SAP)] measures in mice exposed to the elevated plus-maze (EPM). Mice received an intra-dPAG injection of the TRPV1 agonist capsaicin (0, 0.01, 0.1 or 1.0. nmol/0.2. μL; Experiment 1) or antagonist capsazepine (0, 10, 30 or 60. nmol/0.2. μL; Experiment 2), or combined injections of capsazepine (30. nmol) and capsaicin (1.0. nmol) (Experiment 3), and were exposed to the EPM to record spatiotemporal and ethological measures. While capsaicin produced an anxiogenic-like effect (it reduced %OE and %OT and frequency of SAP and HD in the open arms), capsazepine did not change any behavior in the EPM. However, when injected before capsaicin (1.0. nmol), intra-dPAG capsazepine (30. nmol-a dose devoid of intrinsic effects) antagonized completely the anxiogenic-like effect of the TRPV1 agonist. These results suggest that the anxiogenic-like effect produced by capsaicin is primarily due to TRPV1 activation within the dPAG in mice, but that dPAG TRPV1 receptors do not exert a tonic control over defensive behavior in mice exposed to the EPM. © 2013 Elsevier B.V.

Periaqueductal gray matter modulates the hypercapnic ventilatory response

The periaqueductal gray (PAG) is a midbrain structure directly involved in the modulation of defensive behaviors. It has direct projections to several central nuclei that are involved in cardiorespiratory control. Although PAG stimulation is known to elicit respiratory responses, the role of the PAG in the CO2-drive to breathe is still unknown. The present study assessed the effect of chemical lesion of the dorsolateral and dorsomedial and ventrolateral/lateral PAG (dlPAG, dmPAG, and vPAG, respectively) on cardiorespiratory and thermal responses to hypercapnia. Ibotenic acid (IBO) or vehicle (PBS, Sham group) was injected into the dlPAG, dmPAG, or vPAG of male Wistar rats. Rats with lesions outside the dlPAG, dmPAG, or vPAG were considered as negative controls (NC). Pulmonary ventilation (Ve), mean arterial pressure (MAP), heart rate (HR), and body temperature (Tb) were measured in unanesthetized rats during normocapnia and hypercapnic exposure (5, 15, 30 min, 7 % CO2). IBO lesioning of the dlPAG/dmPAG caused 31 % and 26.5 % reductions of the respiratory response to CO2 (1,094.3 +/- 115 mL/kg/min) compared with Sham (1,589.5 +/- 88.1 mL/kg/min) and NC groups (1,488.2 +/- 47.7 mL/kg/min), respectively. IBO lesioning of the vPAG caused 26.6 % and 21 % reductions of CO2 hyperpnea (1,215.3 +/- 108.6 mL/kg/min) compared with Sham (1,657.3 +/- 173.9 mL/kg/min) and NC groups (1,537.6 +/- 59.3). Basal Ve, MAP, HR, and Tb were not affected by dlPAG, dmPAG, or vPAG lesioning. The results suggest that dlPAG, dmPAG, and vPAG modulate hypercapnic ventilatory responses in rats but do not affect MAP, HR, or Tb regulation in resting conditions or during hypercapnia.

GLUTAMATERGIC MECHANISMS OF THE DORSAL PERIAQUEDUCTAL GRAY MATTER MODULATE THE EXPRESSION OF CONDITIONED FREEZING AND FEAR-POTENTIATED STARTLE

It is well known that excitatory amino acids induce unconditioned fear responses when locally injected into the dorsal periaqueductal gray matter (dPAG). However, there are only few studies about the involvement of excitatory amino acids mediation in dPAG in the expression of conditioned fear. The present series of experiments evaluates the participation of AMPA/Kainate and NMDA glutamatergic receptors of dPAG in the expression of conditioned fear, assessed by the fear-potentiated startle (FPS) and conditioned freezing responses. Wistar rats were subjected to fear conditioning to light. Twenty-four hours later, they received intra-dPAG injections of kainic acid or NMDA (AMPA/Kainate and NMDA agonists) and 1,2,3,4-Tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium salt hydrate (NBQX) or D(-)-2-Amino-7-phosphonoheptanoic acid (APT) (AMPA/Kainate and NMDA antagonists) and were submitted to the FPS test. Conditioned freezing response was simultaneously measured. Effects of drug treatment on motor activity were evaluated in the open-field test. Intra-dPAG injections of glutamatergic agonists enhanced conditioned freezing and promoted pro-aversive effects in the FPS. Lower doses of the agonists had no effect or enhanced FPS whereas higher doses disrupted FPS, indicating a non-monotonic relationship between fear and FPS. The antagonist NBQX had no significant effects while AP7 decreased conditioned freezing but did not affect FPS. Both antagonists reduced the effects of the agonists. The obtained results cannot be attributed to motor deficits. The results suggest an important role of the AMPA/Kainate and NMDA mechanisms of the dPAG in the expression of conditioned freezing and FPS. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Involvement of the midbrain periaqueductal gray 5-HT1A receptors in social conflict induced analgesia in mice

Recent results from our laboratory have shown that 30-bites social conflict in mice produces a high-intensity, short-term analgesia which is attenuated by systemically injected 5-HT1A receptor agonists, such as BAY R 1531 (6-methoxy-4-(di-n-propylamino)-1,3,4,5-tetrahydrobenz(c,d)indole hydrochloride) and gepirone. The present study investigated the effects of these drugs, as well as the 5-HT1A receptor antagonist WAY 100135 (N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide) injected into the midbrain periaqueductal gray matter of mice on 30-bites analgesia. Four to five days after guide-cannula implantation, each mouse received microinjection of gepirone (30 nmol/0.2 mu l), BAY R 1531 (10 nmol/0.2 mu l), WAY 100135 (10 nmol/0.2 mu l), saline (0.9% NaCl) or vehicle (saline + 4% Tween 80) 5 min before either an aggressive (30 bites) or a non-aggressive interaction. Nociception was assessed by the tail-flick test made before as well as 1, 5, 10 and 20 min after social interaction. The full 5-HT1A receptor agonist BAY R 1531 blocked, whereas, WAY 100135 and gepirone intensified 30-bites analgesia, Neither non-aggressive interaction, per se, nor the three compounds given after this type of social interaction significantly changed nociception. These results indicate that 5-HT1A receptors in the periaqueductal gray inhibit analgesia induced by social conflict in mice. (C) 1998 Elsevier B.V. B.V.

Concurrent nociceptive stimulation impairs the anxiolytic effect of midazolam injected into the periaqueductal gray in mice

This study investigated whether the opportunity to avoid or escape the open arms of an elevated plus-maze (EPM) affects the antinociceptive response observed when mice are subjected to open arm confinement. Furthermore, in order to better characterize the relationship between emotion and antinociception in the EPM, we examined the behavioral effects of midazolam injection into the midbrain periaqueductal gray matter (PAG). As our main aim was to evaluate the relevance of different levels of approach-avoid conflict (i.e. The presence of open and closed arms) to maze-induced antinociception, mice were exposed to one of three types of EPM-a standard EPM (sEPM), an open EPM (oEPM: four open arms) or, as a control condition, an enclosed EPM (eEPM: four enclosed arms). Nociception was assessed using the formalin test. Twenty minutes after formalin injection (50 mu l, 2.5% formalin) into the dorsal right hind paw, mice received an intra-PAG injection of saline or midazolam (10-20 nmol). Five minutes later, they were individually exposed to one of the mazes for 10 min (25-35 min after formalin injection). Videotapes of the test sessions were scored for a variety of behavioral measures including time spent licking the formalin-injected paw. To examine whether the effects of midazolam on anxiety-like behavior may have been influenced by concurrent nociceptive stimulation (i.e. formalin pretreatment), naive mice were submitted to a similar procedure to that described above for the sEPM test but without formalin pretreatment. Results showed that mice exposed to the oEPM spent significantly less time licking the injected paw compared to groups exposed to either the sEPM or eEPM. Although exposure to the sEPM induced anxiety-like behaviors (i.e. open arm avoidance), it did not result in antinociception. Intra-PAG infusions of midazolam failed to block oEPM-induced antinociception or to alter sEPM-induced anxiety in mice that had received formalin injection. However, under normal test conditions (i.e. in the absence of formalin-induced nociceptive stimulation), intra-PAG midazolam produced clear anti-anxiety effects in mice exposed to the sEPM. Findings are discussed in terms of different emotional states induced by the oEPM and sEPM and the influence of concurrent nociceptive stimulation on the anti-anxiety effect of intra-PAG midazolam. (c) 2005 Elsevier B.V. All rights reserved.

Activation of 5-HT2C receptors in the dorsal periaqueductal gray increases antinociception in mice exposed to the elevated plus-maze

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Role of nitric oxide in the periaqueductal gray in defensive behavior in mice: influence of prior local N-methyl-D-aspartate receptor activation and aversive condition

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of TRPV1 channels of the dorsal periaqueductal gray in the modulation of nociception and open elevated plus maze-induced antinociception in mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Activation of 5-HT2C receptors in the dorsal periaqueductal gray increases antinociception in mice exposed to the elevated plus-maze

Several findings have pointed to the role of the dorsal periaqueductal gray (dPAG) serotonin 5-HT1A and 5-HT2(A-C) receptor subtypes in the modulation of defensive behavior in animals exposed to the elevated plus-maze (EPM). Besides displaying anxiety-like behavior, rodents also exhibit antinociception in the EPM. This study investigated the effects of intra-dPAG injections of 5-HT1A and 5-HT2B/2C receptor ligands on EPM-induced antinociception in mice. Male Swiss mice received 0.1 mu l intra-dPAG injections of vehicle, 5.6 and 10 nmol of 8-OHDPAT, a 5-HT1A receptor agonist (Experiment 1), or 0.01, 0.03 and 0.1 nmol of mCPP, a 5-HT2B/2C receptor agonist (Experiment 2). Five minutes later, each mouse received an intraperitoneal injection of 0.6% acetic acid (0.1 ml/10 g body weight; nociceptive stimulus) and was individually confined in the open (OA) or enclosed (EA) arms of the EPM for 5 min, during which the number of abdominal writhes induced by the acetic acid was recorded. While intra-dPAG injection of 8-OHDPAT did not change open-arm antinociception (OAR). mCPP (0.01 nmol) enhanced it. Combined injections of ketanserin (10 nmol/0.1 mu l), a 5-HT2A/2C receptor antagonist, and 0.01 nmol of mCPP (Experiment 3), selectively and completely blocked the OAR enhancement induced by mCPP. Although intra-dPAG injection of mCPP (0.01 nmol) also produced antinociception in EA-confined mice (Experiment 2), this effect was not confirmed in Experiment 3. Moreover, no other compound changed the nociceptive response in EA-confined animals. These results suggest that the 5-HT2C receptors located within the PAG play a role in this type of environmentally induced pain inhibition in mice. (c) 2012 Elsevier B.V. All rights reserved.

New perspective on the pathophysiology of panic: merging serotonin and opioids in the periaqueductal gray

Panic disorder patients are vulnerable to recurrent panic attacks. Two neurochemical hypotheses have been proposed to explain this susceptibility. The first assumes that panic patients have deficient serotonergic inhibition of neurons localized in the dorsal periaqueductal gray matter of the midbrain that organize defensive reactions to cope with proximal threats and of sympathomotor control areas of the rostral ventrolateral medulla that generate most of the neurovegetative symptoms of the panic attack. The second suggests that endogenous opioids buffer normal subjects from the behavioral and physiological manifestations of the panic attack, and their deficit brings about heightened suffocation sensitivity and separation anxiety in panic patients, making them more vulnerable to panic attacks. Experimental results obtained in rats performing one-way escape in the elevated T-maze, an animal model of panic, indicate that the inhibitory action of serotonin on defense is connected with activation of endogenous opioids in the periaqueductal gray. This allows reconciliation of the serotonergic and opioidergic hypotheses of panic pathophysiology, the periaqueductal gray being the fulcrum of serotonin-opioid interaction.

Dorsal periaqueductal gray stimulation facilitates anxiety-, but not panic-related, defensive responses in rats tested in the elevated T-maze

The escape response to electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) has been associated with panic attacks. In order to explore the validity of the DPAG stimulation model for the study of panic disorder, we determined if the aversive consequences of the electrical or chemical stimulation of this midbrain area can be detected subsequently in the elevated T-maze. This animal model, derived from the elevated plus-maze, permits the measurement in the same rat of a generalized anxiety- and a panic-related defensive response, i.e., inhibitory avoidance and escape, respectively. Facilitation of inhibitory avoidance, suggesting an anxiogenic effect, was detected in male Wistar rats (200-220 g) tested in the elevated T-maze 30 min after DPAG electrical stimulation (current generated by a sine-wave stimulator, frequency at 60 Hz) or after local microinjection of the GABA A receptor antagonist bicuculline (5 pmol). Previous electrical (5, 15, 30 min, or 24 h before testing) or chemical stimulation of this midbrain area did not affect escape performance in the elevated T-maze or locomotion in an open-field. No change in the two behavioral tasks measured by the elevated T-maze was observed after repetitive (3 trials) electrical stimulation of the DPAG. The results indicate that activation of the DPAG caused a short-lived, but selective, increase in defensive behaviors associated with generalized anxiety.

Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator

The midbrain periaqueductal gray (PAG) is part of the brain system involved in active defense reactions to threatening stimuli. Glutamate N-methyl-d-aspartate (NMDA) receptor activation within the dorsal column of the PAG (dPAG) leads to autonomic and behavioral responses characterized as the fear reaction. Nitric oxide (NO) has been proposed to be a mediator of the aversive action of glutamate, since the activation of NMDA receptors in the brain increases NO synthesis. We investigated the effects of intra-dPAG infusions of NMDA on defensive behaviors in mice pretreated with a neuronal nitric oxide synthase (nNOS) inhibitor [N omega-propyl-l-arginine (NPLA)], in the same midbrain site, during a confrontation with a predator in the rat exposure test (RET). Male Swiss mice received intra-dPAG injections of NPLA (0.1 or 0.4 nmol/0.1 mu l), and 10 min later, they were infused with NMDA (0.04 nmol/0.1 mu l) into the dPAG. After 10 min, each mouse was placed in the RET. NMDA treatment enhanced avoidance behavior from the predator and markedly increased freezing behavior. These proaversive effects of NMDA were prevented by prior injection of NPLA. Furthermore, defensive behaviors (e.g., avoidance, risk assessment, freezing) were consistently reduced by the highest dose of NPLA alone, suggesting an intrinsic effect of nitric oxide on defensive behavior in mice exposed to the RET. These findings suggest a potential role of glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice during a confrontation with a predator in the RET.

Contrasting effects of nitric oxide and corticotropin-releasing factor within the dorsal periaqueductal gray on defensive behavior and nociception in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of the NMDA receptor/Nitric Oxide pathway in the dorsolateral periaqueductal gray causes anxiolytic-like effects in rats submitted to the Vogel conflict test

Abstract Background Several studies had demonstrated the involvement of the dorsolateral portion of periaqueductal grey matter (dlPAG) in defensive responses. This region contains a significant number of neurons containing the enzyme nitric oxide synthase (NOS) and previous studies showed that non-selective NOS inhibition or glutamate NMDA-receptor antagonism in the dlPAG caused anxiolytic-like effects in the elevated plus maze. Methods In the present study we verified if the NMDA/NO pathway in the dlPAG would also involve in the behavioral suppression observed in rats submitted to the Vogel conflict test. In addition, the involvement of this pathway was investigated by using a selective nNOS inhibitor, Nω-propyl-L-arginine (N-Propyl, 0.08 nmol/200 nL), a NO scavenger, carboxy-PTIO (c-PTIO, 2 nmol/200 nL) and a specific NMDA receptor antagonist, LY235959 (4 nmol/200 nL). Results Intra-dlPAG microinjection of these drugs increased the number of punished licks without changing the number of unpunished licks or nociceptive threshold, as measure by the tail flick test. Conclusion The results indicate that activation of NMDA receptors and increased production of NO in the dlPAG are involved in the anxiety behavior displayed by rats in the VCT.