Eugenol reduces acute pain in mice by modulating the glutamatergic and tumor necrosis factor alpha (TNF-α) pathways.

Eugenol is utilized together with zinc oxide in odontological clinical for the cementation of temporary prostheses and the temporary restoration of teeth and cavities. This work explored the antinociceptive effects of the eugenol in different models of acute pain in mice and investigated its possible modulation of the inhibitory (opioid) and excitatory (glutamatergic and pro-inflammatory cytokines) pathways of nociceptive signaling. The administration of eugenol (3-300 mg/kg, p.o., 60 min or i.p., 30 min) inhibited 82 ± 10% and 90 ± 6% of the acetic acid-induced nociception, with ID50 values of 51.3 and 50.2 mg/kg, respectively. In the glutamate test, eugenol (0.3-100 mg/kg, i.p.) reduced the response behavior by 62 ± 5% with an ID50 of 5.6 mg/kg. In addition, the antinociceptive effect of eugenol (10 mg/kg, i.p.) in the glutamate test was prevented by the i.p. treatment for mice with naloxone. The pretreatment of mice with eugenol (10 mg/kg, i.p.) was able to inhibit the nociception induced by the intrathecal (i.t.) injection of glutamate (37 ± 9%), kainic (acid kainite) (41 ± 12%), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (55 ± 5%), and substance P (SP) (39 ± 8%). Furthermore, eugenol (10 mg/kg, i.p.) also inhibited biting induced by tumor necrosis factor alpha (TNF-α, 65 ± 8%). These results extend our current knowledge of eugenol and confirm that it promotes significant antinociception against different mouse models of acute pain. The mechanism of action appears to involve the modulation of the opioid system and glutamatergic receptors (i.e., kainate and AMPA), and the inhibition of TNF-α. Thus, eugenol could represent an important compound in the treatment for acute pain.

Blockade of the NMDA and AMPA/kainate receptors in the dorsal raphe nucleus prevents the 5-HT3 receptor agonist m-chlorophenylbiguanide-induced suppression of REM sleep in the rat.

The effects of the selective 5-HT(3) receptor agonist m-chlorophenylbiguanide (m-CPBG), and of the NMDA (N-methyl-D-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate)/kainate antagonists AP-5 [(±)-2-amino-5-phosphono-pentanoic acid] and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), respectively, were studied in adult male Wistar rats implanted for chronic sleep recordings. The compounds were microinjected directly into the dorsal raphe nucleus (DRN) during the light period of the 12-h light/12-h dark cycle. Infusion of m-CPBG (2 and 4mM) into the DRN induced a significant reduction of rapid-eye-movement sleep (REMS) and of the number of REM periods. Local infusion of AP-5 (0.5-1 mM) and CNQX (2 mM) significantly increased slow wave sleep (SWS). Pretreatment with AP-5 (0.5 mM) or CNQX (0.5 mM) antagonized the m-CPBG-induced suppression of REMS. It is proposed that the reduction of REMS after microinjection of m-CPBG into de DRN is related to the activation of glutamatergic interneurons that express the 5-HT(3) receptor and make synaptic contacts with serotonergic cells. The resultant increase of serotonin release at postsynaptic sites involved in the induction of REMS would provoke the suppression of the behavioral state. Our findings provide, in addition, new details concerning the pharmacology of DRN serotonergic neurons in the rat that may become relevant to the development of drugs for enhancing cortical and subcortical serotonergic neurotransmission.

Involvement of AMPA/kainate-excitotoxicity in MK801-induced neuronal death in the retrosplenial cortex.

MK801 is a prototypical non-competitive NMDA receptor-antagonist that induces behavioural changes and reversible toxicity at low doses, while at higher doses triggers neuronal death that mainly affects the retrosplenial cortex (RSC) and to a lesser extent other structures such as the posterolateral cortical amygdaloid nucleus (PLCo). The mechanism of MK801-induced neurodegeneration remains poorly understood. In this study we analysed the participation of GABA-ergic and glutamatergic neurotransmission in MK801-induced neuronal death. We used a single i.p. injection of MK801 (2.5 mg/kg) that induced moderate neuronal death in the RSC and PLCo of female rats, and combined this treatment with the i.p., i.c.v., or intra-RSC infusion of drugs that are selective agonists or antagonists of the GABA-ergic or glutamatergic neurotransmission. We found that neuronal death in the RSC, but not the PLCo, was significantly reduced by the i.p. injection of thiopental, and the i.c.v. application of muscimol, both GABA-A agonists. MK801-toxicity in RSC was abrogated by intra-RSC infusion of muscimol, but the GABA antagonist picrotoxin had no effect. HPLC-analysis showed that levels of glutamate, but not GABA, in the RSC decreased after i.p. treatment with MK801. Intra-RSC infusion of MK801 did not enhance toxicity triggered by the i.p. injection of MK801, indicating that toxicity is not due to direct blockade of NMDA receptors in RSC neurons. MK801-toxicity in the RSC was abrogated by i.c.v. and intra-RSC infusions of the AMPA/kainate antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Interestingly, i.c.v. application of neither muscimol or DNQX inhibited MK801-toxicity in the PLCo, suggesting that the mechanism of neuronal death in the RSC and the PLCo might be different. 1-naphthylacetyl spermine trihydrochloride (NASPM), which blocks Ca2+ permeable AMPA/kainate receptors, also reduced MK801-induced toxicity in the RSC. Intra-RSC infusion of AMPA or kainic acid alone promoted death of RSC neurons and was reminiscent of the degeneration induced by the i.p. treatment with MK801. Collectively, these experiments provide evidence for an AMPA/kainate-dependent mechanism of excitotoxicity in the death of RSC neurons after i.p. treatment with MK801.

Aniracetam and DNQX affect the acquisition of rapid tolerance to ethanol in mice.

Several studies have emphasized the role of learning in the development of rapid tolerance and have shown that glutamate-mediated neurotransmission plays an important role in this phenomenon. Since the AMPA/kainate receptor system is directly involved in plasticity mechanisms, the influence of this receptor system on rapid tolerance induced by ethanol was studied using the rotarod. In the first experiment, mice were pretreated with aniracetam, an agonist of AMPA/kainate receptors, 30 min before ethanol (2.75 g/kg; IP) treatment, and tested on the rotarod. After 24 h, the groups were tested on the rotarod under ethanol treatment. Aniracetam facilitated the acquisition of rapid tolerance to ethanol. In the second experiment, mice received DNQX, a competitive antagonist of the AMPA receptor, 30 min before ethanol treatment (3 g/kg) and submitted to the rotarod. This dose of ethanol produced tolerance per se. Groups were tested under ethanol treatment (1.75 g/kg) after 24 h. DNQX blocked rapid tolerance to ethanol. Using a similar protocol, the third experiment showed that DNQX blocked the aniracetam-induced facilitation of rapid tolerance to ethanol. Our results show that aniracetam facilitates whereas DNQX blocks ethanol tolerance, suggesting that the non-NMDA receptors are involved in this phenomenon.

The blockade of AMPA-kainate and NMDA receptors in the dorsal periaqueductal gray reduces the effects of diazepam withdrawal in rats.

It is well established that the most persistent sign of withdrawal from chronic benzodiazepine use in humans is anxiety. In contrast to other types of drugs of abuse, the emergence of this anxiety does not seem to be linked directly to alterations in the levels of dopamine in the mesolimbic system. Some studies have proposed that fear-like behaviors elicited by benzodiazepine withdrawal could be the result either of alterations in the sensitivity of GABAA receptors or in the neuronal hyperexcitability that results from neuroadaptative responses to chronic treatment, probably mediated by glutamate. The increased fear-like behaviors induced by benzodiazepine withdrawal are similar to the defense reaction displayed by animals exposed to dangerous situations or submitted to electrical or chemical stimulation of the dorsal periaqueductal gray (dPAG), a key structure of the brain aversive system. However, the involvement of the dPAG in drug abuse has been investigated only in the context of the physical effects of drug dependence. Thus, in this study we investigated the effects of injections into the dPAG of the glutamic acid diethyl ester (GDEE) and 2-amino-7-phosphonoheptanoate (AP-7) (AMPA-kainate and NMDA receptors antagonists, respectively) on fear-like behaviors promoted by benzodiazepine withdrawal in rats submitted to aversive events (foot-shocks) immediately before chronic diazepam administration in a conditioning place-preference paradigm, using a light-dark box. Our results showed that inhibition of the glutamatergic neurotransmission in the dPAG reduces the consequence of the diazepam withdrawal in rats, implicating the excitatory amino acids of the dPAG in the modulation of the aversive state induced by benzodiazepine drugs withdrawal.

Ionotropic glutamate-receptor antagonists inhibit the aversive effects of nitric oxide donor injected into the dorsolateral periaqueductal gray of rats.

RATIONALE: Nitric oxide (NO) is a gas neurotransmitter that may facilitate glutamate release in the central nervous system. NO donors or glutamate agonists injected into the dorsolateral periaqueductal grey (dlPAG) induce flight behaviour. OBJECTIVES: To test the hypothesis that the defensive reactions induced by an NO donor in the dlPAG would be attenuated by pretreatment with AMPA/kainate or NMDA glutamate receptor antagonists. METHODS: Male Wistar rats with cannulae aimed at the dlPAG received vehicle, AP7 (a NMDA receptor antagonist, 2 nmol) or NBQX (an AMPA/kainite receptor antagonist, 100 nmol) injection 10 min before the administration of SIN-1 (an NO donor, 300 nmol). Immediately after the last injection, their behavior was observed in an open arena during 10 min. RESULTS: SIN-1 induced flight reactions characterized by running and jumps. Pretreatment with AP7 or NBQX completely prevented the effects of SIN-1. CONCLUSION: The results suggest that the aversive reactions induced by an NO donor in the dlPAG depend on ionotropic glutamate receptor activation.

Bradycardic responses to microinjection of N-methyl-D-aspartate into the nucleus tractus solitarius are inhibited by local activation of 5-HT(3) receptors.

Previous reports have described that glutamate ionotropic receptors in the nucleus tractus solitarius (NTS) are involved in the reflex control of heart rate, and that such a control can be inhibited by NTS-5-HT(3) receptor stimulation. In the present study, we examined in urethane anaesthetized rats the effects of intra-NTS microinjection of 1-(m-chlorophenyl)-biguanide (CPBG), a potent and selective 5-HT(3) receptor agonist, on the cardiovascular responses to local administration of glutamate ionotropic receptor agonists. Intra-NTS microinjection of CPBG reduced the atropine-sensitive bradycardia elicited by local microinjection of NMDA without affecting the cardiovascular responses to intra-NTS microinjections of AMPA or kainic acid. The reduction by CPBG of the NMDA-evoked cardiac response was blocked by prior intra-NTS microinjection of granisetron, a 5-HT(3) receptor antagonist, as well as bicuculline, a GABA(A) receptor antagonist. These results suggest that the stimulation of NTS 5-HT(3) receptors specifically reduces, via a GABA-dependent mechanism, the cardiac response to local NMDA administration.