Juan Leyva

Characterisation of mGluRs which modulate nociception in the PAG of the mouse

The contribution of metabotropic glutamate receptors (mGluRs) to the modulation of nociception by the periaqueductal gray (PAG) matter was investigated in mice. Intra-PAG microinjection of (IS,3R)-ACPD, an agonist of groups I and II mGluRs, as well as (S)-3,5-DHPG, a selective agonist of group I mGluRs, increased the latency of the nociceptive reaction (NR) in the hot plate test. (RS)-AIDA, an antagonist of group I mGluRs, antagonized the effect of (S)-3,5-DHPG, but changed the effect induced by (1S,3R)-ACPD in that a decrease in the latency for the NR could now be observed. L-CCG-I and L-SOP, which are agonists of groups II and III mGluRs respectively, decreased the latency of the NR. (2S)-alpha-EGlu and (RS)-alpha-MSOP, which are antagonists of groups II and III mGluRs, respectively, antagonized the effect of L-CCG-I and L-SOP. (RS)-AIDA and (RS)-alpha-MSOP alone decreased and increased, respectively, the latency of the NR with the highest doses used. (2S)-alpha-EGlu alone did not change significantly the latency of the NR. Intra-PAG microinjection of LH, an agonist of ionotropic glutamate receptors, induced a dose-dependent analgesia which was blocked by pretreatment with DL-AP5, a selective antagonist of NMDA receptors. No mGluRs antagonists were able to prevent LH-induced analgesia. These results emphasize the possible involvement of mGluRs in the modulation of nociception. It seems that activation of group I mGluRs potentiates, while groups II and III mGluRs decrease, the activity of the PAG for the modulation of nociception.

Pregnenolone sulfate increases the convulsant potency of N-methyl-D-aspartate in mice

The neurosteroid pregnenolone sulfate is known to specifically enhance NMDA-gated currents in spinal cord neurons. The response does not appear to be mediated by the glycine/NMDA modulatory site. Here we found that pregnenolone sulfate significantly increased the convulsant potency of N-methyl-D-aspartate (NMDA), but not of pentylenetetrazol (PTZ). In agreement with previous in vitro reports showing that the glutamergic NMDA receptor is also specifically modulated by steroids, our findings suggest that pregnenolone sulfate selectively activates the NMDA receptors involved in convulsions in the intact animal.

Metabotropic glutamate receptors modulate serotonin release in the rat periaqueductal gray matter

The role of metabotropic (mGluRs) and N-methyl-D-aspartate (NMDA) glutamate receptors on 5-hydroxytryptamine (5-HT) release has been studied in rat periaqueductal gray (PAG) matter by using in vivo microdialysis. (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; 0.5 or 1 mM], a group I/group II mGluRs agonist, increased the dialysate 5-HT concentration. (2S)-α-ethylglutamic acid (EGlu; 1 mM), an antagonist of group II mGluRs, but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), an antagonist of group I mGluRs, antagonized the 1S,3R-ACPD-induced effect. (S)-3,5-dihydroxyphenylglycine (DHPG; 0.5 and 1 mM), an agonist of group I mGluRs, did not modify dialysate 5-HT. (2S, 3S, 4S)-α-(carboxycyclopropyl)-glycine (L-CCG-I; 0.5 and 1 mM), an agonist of group II mGluRs, increased extracellular 5-HT. This effect was antagonized by EGlu. Similarly, L-serine-O-phosphate (L-SOP; 1 and 10 mM), an agonist of group III mGluRs, increased extracellular 5-HT and this effect was antagonized by (RS)-α-methylserine O-phosphate (M-SOP; 1 mM), an antagonist of group III mGluRs. Out of the several N-methyl-D-aspartate concentrations used (NMDA; 10, 50, 100, 500 and 1000 µM) only the 50 µM infusion significantly decreased dialysate 5-HT. The GABAA receptor agonist, bicuculline (30 µM), increased 5-HT release on its own and antagonized the decrease caused by the opiate antagonist, naloxone (2 mM), as well as the increases caused by CCG-I or L-SOP. These data show that stimulation of PAG’s group II/group III mGluRs increases 5-HT release, while stimulation of NMDA glutamate receptors may decrease it. We speculate that glutamate does not modulate 5-HT release in the PAG directly, but via activation of tonically active GABAergic interneurons.

Interactive role of L-glutamate and vasopressin, at the level of the PAG area, for cardiovascular tone and stereotyped behaviour.

The periaqueductal gray (PAG) area may modulate cardiovascular functions and trigger several stereotyped behavioural responses through a mechanism mediated by the interaction of L-glutamate with arginine vasopressin (AVP). Moreover, only the NMDA- but not the non-NMDA-glutamergic subtype receptors might participate in the control of these neurovegetative functions also modifying the homeostasis of the hypothalamic-neurohypophysis system. This latter effect may be due to the tight connections between the PAG area neurons to the more cephalic nuclei within the brainstem.

Metabotropic and ionotropic glutamate receptors mediate opposite effects on periaqueductal gray matter

Microinjections, into the dorso-lateral periaqueductal gray matter, of N-methyl-D-aspartic acid (NMDA, 0.07-7 nmol/rat) significantly (P < 0.01) increased arterial blood pressure in a dose-related manner. Pretreatment, 5 min before NMDA (7 nmol/rat), in the same area with 2-amino-5-phosphonovaleric acid (2-APV, 5 nmol/rat), a selective antagonist of NMDA receptors, significantly (P < 0.01) reduced NMDA-induced arterial hypertension. trans-(+/-)-1-Amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 6-30 nmol/rat), an agonist of metabotropic glutamate receptors (mGlu receptors), significantly (P < 0.01) decreased arterial blood pressure when microinjected into the dorsal-lateral periaqueductal gray matter. Pretreatment, 5 min before t-ACPD (30 nmol/rat), in the same area with L-2-amino-3-phosphono-propionate (L-AP-3, 30 nmol/rat), a putative antagonist of the mGlu receptors, was not able to prevent t-ACPD-induced hypotension. Microinjections of L-AP-3 (30 nmol/rat) induced a hypotension similar to the one obtained with t-ACPD at the dose of 6 nmol/rat. From these data we can suggest that mGlu receptors act inversely to the NMDA receptors in the dorso-lateral periaqueductal gray area and that L-AP-3 is a partial agonist rather than an antagonist of mGlu receptors within the periaqueductal gray area.

Involvement of periaqueductal gray area NMDA receptors in endothelin-induced behavioural effects.

We investigated the behavioural effects induced by endothelin-1 injected into the lateral-caudal periaqueductal gray matter of freely moving rats. Endothelin-1 induced a dose-dependent longitudinal rolling of the body (barrel rolling) which was prevented by D,L-2-amino-5-phosphonovalerate (2-APV), an N-methyl-D-aspartate (NMDA) receptor antagonist, but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist. This effect, not reproducible with NMDA alone, indicates that the activation of the NMDA receptors in the periaqueductal gray area may be a necessary, but not sufficient, step for the triggering of endothelin-1-induced barrel-rolling behaviour.

Effects of the polyamine spermidine on NMDA-induced arterial hypertension in freely moving rats.

We investigated the effect of the polyamine spermidine (SPD) (0.01-1 microgram/rat) on hypertension induced by N-methyl-D-aspartate (NMDA) (0.1 microgram/rat) microinjected into the latero-caudal periaqueductal gray (PAG) area of freely moving rats. Pretreatment with a low dose of SPD (0.01 microgram/rat) significantly increased NMDA-induced hypertension. On the contrary, higher doses of SPD (0.1 and 1 microgram/rat) significantly decreased NMDA-induced cardiovascular changes. SPD alone did not modify arterial blood pressure. Arcaine (1 microgram/rat), a putative antagonist at the polyamine recognition site on NMDA receptors, when microinjected into the PAG area, prevented the negative but not the positive modulatory effects of SPD on the NMDA-induced cardiovascular changes. Pretreatment with SPD did not affect cardiovascular effects induced by quisqualic acid (QUIS), a non-NMDA receptor agonist. These data, in agreement with the in vitro results, suggest that at the level of the PAG area, the polyamines also show multiple actions at NMDA receptors in vivo.

Involvement of opioid receptors in N-Methyl-d-aspartate-induced arterial hypertension in periaqueductal gray matter

Arterial hypertension induced by microinjections of N-methyl-d-aspartate (NMDA) (2 nmol/rat) into the midbrain periaqueductal gray matter was used to assess the involvement of opioid receptors (µ, δ and κ) in modulating pressor periaqueductal gray neurons. Groups (n = 5 – 8) of urethane-anaesthetised rats received, 5 min before NMDA, microinjections of selective opioid receptor antagonists in the periaqueductal gray area and arterial blood pressure was monitored. Pretreatments with naloxone (5 nmol/rat), a non selective ,µ receptor antagonist, or naltrindole hydrochloride (5 nmol/rat), a selective δ receptor antagonist, significantly (P< 0.05) decreased by 31% and 37%, respectively, NMDA-induced hypertension. The latency for the maximum increase of NMDA-induced hypertension was also significantly (P<0.05) increased with naloxone. Pretreatment with nor-binaltorphimine (5 nmol/rat), a selective κ receptor antagonist, only increased the latency of NMDA-induced hypertension. Each opioid antagonist failed per se to alter arterial blood pressure. Microinjection of morphine (13 nmol/rat), a non selective ,µ receptor agonist, significantly decreased (P<0.05) by 57.5% NMDA-induced arterial hypertension and this effect was antagonised by naloxone. Combined pretreatments in the periaqueductal gray area with naloxone and the GABAA antagonist bicuculline (2.5 nmol/rat; 5 min before naloxone) antagonised the effect of naloxone on NMDA induced hypertension. In contrast, bicuculline significantly (P<0.05) potentiated morphine-induced decrease of NMDA hypertension. Combined pretreatments in the periaqueductal gray area with naloxone and the glycine antagonist strychnine (8 nmol/rat; 5 min before naloxone) failed to prevent the effect of naloxone on the NMDA-induced cardiovascular changes.These data suggest that periaqueductal gray vasopressor neurons receive both direct opioid and GABAergic inhibitory inputs. The latter may be, in turn, negatively modulated by opioid fibres mainly through µ and δ subtype receptors.

Evidence that arcaine increases the N-methyl-D-aspartate-induced cardiovascular effects into the periaqueductal gray area of anesthetized rats.

In the present study the influence of arcaine (0.01-1 microgram/rat), an in vitro putative non-competitive antagonist of the NMDA receptors, on cardiovascular changes induced by intracerebral administration of N-methyl-D-aspartate (NMDA) (0.1 microgram/rat) has been evaluated. Both NMDA and arcaine were microinjected into the periaqueductal gray (PAG) area of anesthetized rats. Arcaine did not decrease NMDA-induced arterial hypertension and tachycardia, but, in a dose-related manner, increased the NMDA-induced cardiovascular effects. Moreover, treatment with arcaine was not able per se to modify arterial blood pressure and heart rate basal values. Although updated in vitro reports indicate arcaine as a blocker of the NMDA receptors by an open channel mechanism, our in vivo results, at the level of the PAG area, show this not to be true. Indeed the drug may facilitate NMDA receptor activation.

L-Cysteinesulfinic acid modulates cardiovascular function in the periaqueductal gray area of rat

L-Cysteinesulfinic acid (CSA) involvement in modulating periaqueductal gray (PAG) pressor neurons has been evaluated in the rat. Intra-PAG CSA induced an increase in mean blood pressure partially antagonized by (2S)-alpha-ethylglutamic acid (EGA), a group II metabotropic glutamate receptors (mGluRs) antagonist. Conversely, the NMDA antagonist, DL-AP5, or the mGluRs antagonists, (+)-MCPG, UPF523, or (RS)-alpha-methylserine-O-phosphate (MSOP), were devoid of any activity on the CSA effect. These data show that the excitatory amino acid CSA, probably by stimulating an mGluR, contributes with glutamate in modulating cardiovascular function at the PAG matter.