Molecular and functional interactions between tumor necrosis factor-alpha receptors and the glutamatergic system in the mouse hippocampus: Implications for seizure susceptibility

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine acting on two distinct receptor subtypes, namely p55 and p75 receptors. TNF-alpha p55 and p75 receptor knockout mice were previously shown to display a decreased or enhanced susceptibility to seizures, respectively, suggesting intrinsic modifications in neuronal excitability. We investigated whether alterations in glutamate system function occur in these naive knockout mice with perturbed cytokine signaling that could explain their different propensity to develop seizures. Using Western blot analysis of hippocampal homogenates, we found that p55^−/− mice have decreased levels of membrane GluR3 and NR1 glutamate receptor subunits while GluR1, GluR2, GluR6/7 and NR2A/B were unchanged as compared to wild-type mice. In p75^−/− mice, GluR2, GluR3, GluR6/7 and NR2A/B glutamate receptor subunits were increased in the hippocampus while GluR1 and NR1 did not change. Extracellular single-cell recordings of the electrical activity of hippocampal neurons were carried out in anesthetized mice by standard electrophysiological techniques. Microiontophoretic application of glutamate increased the basal firing rate of hippocampal neurons in p75^−/− mice versus wild-type mice, and this effect was blocked by 2-amino-5-phosphopentanoic acid and 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione denoting the involvement of N-methyl-d-aspartic acid and AMPA receptors. In p55^−/− mice, hippocampal neurons responses to glutamate were similar to wild-type mice. Spontaneous glutamate release measured by in vivo hippocampal microdialysis was significantly decreased only in p55^−/− mice. No changes were observed in KCl-induced glutamate release in both receptor knockout mice strains versus wild-type mice. These findings highlight specific molecular and functional interactions between p55 and p75 receptor-mediated signaling and the glutamate system. These interactions may be relevant for controlling neuronal excitability in physiological and pathological conditions.     

Repeated administration of imipramine attenuates glutamatergic transmission in rat frontal cortex

The effects of repeated administration of a tricyclic antidepressant, imipramine, lasting 14 days (10 mg/kg p.o., twice daily), were studied ex vivo in rat frontal cortex slices prepared 48 h after last dose of the drug. In slices prepared from imipramine-treated animals the mean frequency, and to a lesser degree the mean amplitude, of spontaneous excitatory postsynaptic currents recorded from layer II/III pyramidal neurons, were decreased. These effects were accompanied by a reduction of the initial slope ratio of pharmacologically isolated N-methyl-d-aspartate to AMPA/kainate receptor-mediated stimulation-evoked excitatory postsynaptic currents. Imipramine treatment also resulted in a decrease of extracellular field potentials evoked in layer II/III by stimulation of underlying sites in layer V. These results indicate that chronic treatment with imipramine results in an attenuation of the release of glutamate and an alteration in the postsynaptic reactivity of ionotropic glutamate receptors in rat cerebral cortex.     

Glutamatergic neurotransmission in the nucleus tractus solitarii: Structural and functional characteristics

Glutamate is the main excitatory transmitter in the central nervous system. As such, it plays a major role in transmitting and processing visceral sensory information within the nucleus tractus solitarii (NTS). Here, we review current knowledge on NTS glutamatergic transmission. We describe the main organizational features of NTS glutamatergic synapses as determined by work performed during the last decade using antibodies against glutamate receptors and transporters proteins. In light of these recent neuronatomical findings, we discuss some functional properties of developing and adult NTS glutamatergic synapses.     

Presynaptic D1 dopamine receptors facilitate glutamatergic neurotransmission in the rat globus pallidus

The effects of D1/5 dopamine agonists on spontaneous excitatory postsynaptic currents (sEPSCs) were studied in neurons of the rat globus pallidus using whole-cell recordings in the presence of TTX and bicuculline. In this condition, CNQX abolished the sEPSCs, indicating that they were solely mediated by AMPA receptors. SKF 38393, a D1-like agonist, increased the frequency but not the amplitude of the sEPSCs, suggesting a presynaptic site of action. The increase in frequency was blocked by SCH 23390, a D1/5 antagonist. Quinpirole, a D2-like agonist, decreased the frequency but did not affect the amplitude of the synaptic currents. SKF 38393 increased the frequency of sEPSCs currents, even in presence of quinpirole, indicating that D1/5- and D2-like receptors independently modulate glutamate release upon a single neuron. The results suggest that the dopaminergic control of the glutamate transmission in the globus pallidus may play a role in processing cortical information in the indirect pathway of the basal ganglia.     

Potentiation of a metabotropic glutamatergic response following NMDA receptor activation in rat hippocampus

Interactions between metabotropic glutamate andN-methyl-D-aspartate (NMDA) receptor-mediated responses were investigated in hippocampal CA3 cells using the single-electrode voltage-clamp method. Bath application (2.5–10 M, 30 s) or iontophoresis of 1-amino-cyclopentyl-trans-1S, 3R-dicarboxylate (ACPD), a selective agonist for metabotropic glutamate receptors, resulted in an inward current associated with a decrease in membrane conductance. Following transient bath application of NMDA (5–10 M, 30–60 s), the ACPD-induced inward current was potentiated for a period of up to 25 min (by 61±8% with bath application, by 32±15% with iontophoresis). Transient application of NMDA did not result in a potentiation of ionotropic RS--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or metabotropic muscarinic responses. ACPD responses were not potentiated following transient AMPA application. Intracellular buffering of calcium with tetrapotassium bis(O-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA) prevented potentiation by NMDA in all cells. Bath application of arachidonic acid did not mimic the NMDA-induced potentiation. These results demonstrate that activation of NMDA receptors can specifically induce a long-lasting potentiation of a metabotropic glutamatergic response in hippocampal CA3 pyramidal cells. The characterization of this interaction may contribute to the elucidation of the physiological significance of metabotropic glutamate receptors.     

Differential regulation of AMPA receptor GluA1 phosphorylation at serine 831 and 845 associated with activation of NMDA receptor subpopulations

AMPA receptors and NMDA receptors are the main subtypes of ionotropic glutamate receptors in the vertebrate central nervous system. Accumulating evidence demonstrates that two serine sites, S831 and S845, on the AMPA receptor GluA1 subunit, are phosphorylation-regulated and profoundly involved in NMDA receptor-dependent synaptic plasticity. On the other hand, recent studies have revealed distinct functional consequences of activating synaptic or extrasynaptic NMDA receptors, or of activating GluN2A- or GluN2B-containing NMDA receptors. Therefore, it is essential to determine how phosphorylation of the GluA1 at S831 and S845 is regulated by NMDA receptor subpopulations. In this study, we demonstrated transiently increased phosphorylation of GluA1 at S831 and persistently decreased phosphorylation of GluA1 at S845 by bath application of NMDA to hippocampal slices from rats. Interestingly, we also found a differential regulation of phosphorylation of GluA1 at S831 and S845 by activation of NMDA receptor subpopulations: the synaptic and/or the GluN2A-containing NMDA receptors were more likely to mediate up-regulation of GluA1 phosphorylation at S831 and down-regulation of GluA1 phosphorylation at S845, while the extrasynaptic NMDA receptors down-regulated GluA1 phosphorylation at S831. Taken together, our results suggest the NMDA receptor subpopulations differentially regulate GluA1 phosphorylation, which may contribute to NMDA receptor-dependent synaptic plasticity.     

Studies of the Roles of NMDA and AMPA Glutamate Receptors in the Mechanism of Corasole Convulsions in Mice

Experiments on mice were performed to study the ability of monocationic and dicationic adamantane and phenylcyclohexyl derivatives to prevent convulsive syndrome induced by i.p. corasole (pentylenetetrazole; 80 mg/kg). Monocationic phenylcyclohexyl derivatives, which are selective blockers of NMDA glutamate receptor channels, along with memantine and MK-801, effectively prevented the appearance of the clonic and tonic components of convulsions at micromolar concentrations. Their dicationic analogs, which block both NMDA and AMPA receptor channels, were ineffective against clonic convulsions, but prevented corasole-induced tonic convulsions at much lower concentrations. The convulsive action of corasole, whose primary target is weakening of the inhibitory action of GABA, appears to be mediated by glutamatergic synaptic transmission. NMDA receptors have a much greater involvement than AMPA receptors in the genesis of clonic convulsions, while AMPA receptor activation appears to be an important component of tonic convulsions.     

Characterization of synaptic transmission in the ventrolateral periaqueductal gray of rat brain slices

Synaptic transmission evoked by focal stimulation in the ventrolateral periaqueductal gray was characterized using the whole-cell recording technique in rat brain slices. At resting membrane potential (-62+/-1 mV), focal stimulation (0.05-0.1 ms, 0.03 Hz) usually evoked a 6-cyano-7-nitroquinoxaline-2, 3-dione-sensitive fast excitatory postsynaptic potential and a DL-2-amino-5-phosphonopentanoic acid-sensitive slow excitatory postsynaptic potential with a bicuculline-sensitive inhibitory postsynaptic potential in between. In the presence of kynurenic acid, bicuculline-sensitive inhibitory postsynaptic currents recorded in the voltage-clamp mode displayed a reversal potential of -68+/-3 mV, resembling GABA(A) receptor-mediated inhibitory postsynaptic currents. However, no GABA(B) receptor-mediated inhibitory postsynaptic current was evoked, even at stronger stimulating intensity. 6-Cyano-7-nitroquinoxaline-2,3-dione-sensitive fast excitatory postsynaptic currents were isolated by DL-2-amino-5-phosphonopentanoic acid plus bicuculline and DL-2-amino-5-phosphonopentanoic acid-sensitive slow fast excitatory postsynaptic currents by bicuculline plus 6-cyano-7-nitroquinoxaline-2,3-dione. Both types of excitatory postsynaptic current reversed at potentials near 0 mV. The I-V curve of slow fast excitatory postsynaptic currents or N-methyl-D-aspartate currents displayed a negative slope at potentials more negative than -30 mV in an Mg(2+)-sensitive manner. The control postsynaptic currents reversed at potentials between -50 and -35 mV, inclined to the reversal potential of GABA(A), but not glutamate, receptor channels. It is concluded that, in the ventrolateral periaqueductal gray, focal stimulation elicits both inhibitory and excitatory transmission, while the former is dominant. The inhibitory transmission is mediated by GABA(A) but not GABA(B) receptors. The excitatory transmission is mediated by glutamate acting on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate as well as N-methyl-D-aspartate receptors.     

Immunocytochemical distribution of ionotropic glutamate receptor subunits in the spinal cord of the rabbit

Several histochemical and physiological studies in the literature suggest that ionotropic glutamate receptors are involved in various sensory and motor control mechanisms at the spinal level. The present immunocytochemical study used three specific antibodies to GluR2,4, GluR5,6,7 and to NMDAR1 to differentiate between the regional distribution of -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and N-methyl- -aspartate (NMDA) subtypes of glutamate receptors throughout the rabbit spinal cord. All of these immunoreactivities were prominent in the superficial dorsal horn and motor column. Each antibody gave rise to regionally specific immunostaining patterns but which were similar at all spinal levels. Numerous small neurons in superficial laminae were immunostained with GluR2,4 antibody while only neuropilar elements were immunostained with the two other antibodies. Cell bodies of the intermediate zone and fibres in the motor column were particularly densely immunostained with GluR5-7. Such an immunostaining pattern, which was particularly abundant with the GluR5-7 antibody, suggests the presence, at the spinal level, of an extensive population of neurons exhibitinh a high density of kainate receptors. Immunostaining with NMDAR1 antibody was less dense in comparison with the two others and especially in the motoneuron area. The present results provide the first immunohistochemical comparison between the respective regional distributions of the three types of ionotropic glutamate receptors in the spinal cord. Their parallel distributions throughout the spinal cord support the concept of a tight functional cooperation between NMDA and non-NMDA receptors which has been extensively described for spinal events.     

GluN2B subunits of the NMDA receptor contribute to the AMPA receptor internalization during long-term depression in the lateral amygdala of juvenile rats

The lateral nucleus of the amygdala (LA) is a critical structure involved in fear conditioning. We recently showed that regulated exocytosis and endocytosis of postsynaptic A-amino-3-hydroxy-5-methylisoxazole-4-propionate subtype of glutamate receptors (AMPARs) are involved in the expression of N-methyl-D-aspartate subtype glutamate receptors (NMDARs) dependent long-term potentiation (LTP) and long-term depression (LTD) in coronal slices of the LA. However, the molecular mechanisms of this effect remain unclear. In the present study, we investigated the role of distinct NMDAR subtypes in the endocytosis of AMPARs during LTD expression at the synapses of the thalamic inputs to the LA neurons. Here we show that the NMDARs antagonist DL-2-amino-5-phosphonovalerate (D-APV) blocked the induction of LTD and thus prevented endocytosis of surface AMPARs, indicating that NMDAR activation enhanced the internalization of AMPARs in LTD expression. Furthermore, the selective blocking of GluN2B-containing NMDARs completely abolished the NMDAR-induced AMPAR endocytosis, whereas preferential inhibition of GluN2A-containing NMDARs did not block the NMDAR-induced AMPAR endocytosis during LTD expression. These results suggest that there exist a preferred NMDAR subtype for AMPAR internalization and activation of GluN2B-containing NMDARs represent the predominate pathway triggered during the early stages of this NMDAR-induced endocytosis of AMPARs during LTD in the thalamic inputs to the LA of juvenile rats.     

The entry of manganese ions into the brain is accelerated by the activation of N-methyl-D-aspartate receptors

Manganese-enhanced magnetic resonance imaging (MEMRI) is receiving increased interest as a valuable tool for monitoring the physiological functions in the animal brain based on the ability of manganese ions to mimic calcium ions entering to excitable cells. Here the possibility that in vivo MEMRI can detect the entry of manganese ions (Mn2+) in the brain of rats behaving without intended stimulation is tested. This hypothesis was a result of the unexpected observation that Mn2+-dependent signal enhancement was dramatically suppressed in ketamine-anesthetized rats compared with other anesthetics, such as urethane, pentobarbital and isoflurane. The effects of noncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonists, ketamine and MK-801, on MEMRI for MnCl2 injected rats were examined. Treatment with MK-801 suppressed the signal enhancement more effectively than with ketamine. NMDAR agonists, glutamate (100 mg/kg) and N-methyl-d-aspartate (NMDA) (35 mg/kg), enhanced the signal intensities on MEMRI, and this signal enhancement was completely antagonized by MK-801. The systemic administration of the competitive NMDAR antagonist, D-2-amino-5-phosphono-pentanoate (D-AP5), which does not cross the blood-brain barrier (BBB), showed no effects on the signal enhancement induced by NMDA and glutamate. A selective AMPA receptor (AMPAR) antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), did not block the signal enhancement. These data indicated that the Mn2+-dependent signal enhancement took place as a result of the activation of glutamatergic neurons through NMDAR, but not through AMPAR in the brain.     

Effects of ionotropic glutamate receptor channel blockers on the development of pentylenetetrazol kindling in mice

Experiments on mice were performed to study the ability of monocationic and dicationic adamantane and phenylcyclohexyl derivatives to prevent the development of kindling induced by i.p. administration of pentylenetetrazol (Corasol, 35 mg/kg). The monocationic phenylcyclohexyl derivative IEM-1921 effectively slowed the development of kindling, this being seen over a wide range of doses (0.0001–0.1 μmol/kg). A monocationic adamantane derivative (memantine), also a selective non-competitive blocker of NMDA receptors, produced a similar effect at doses 100 times higher. The anticonvulsive activity of the dicationic phenylcyclohexyl derivative IEM-1925, which could block both types of glutamate receptors, differed from the activity of the monocationic derivative by having a more complex dose-response relationship. Thus, the development of kindling was suppressed by essentially the same doses as needed for the monocation IEM-1921 (0.001 μmol/kg). However, on reducing the dose by a factor of 10 (0.0001 μmol/kg), IEM-1925 facilitated the development of kindling. This difference in the prophylactic activities of selective NMDA receptor blockers and substances able to block both NMDA and AMPA receptors provides evidence that the mechanism of kindling involves both types of ionotropic glutamate receptor and the effects of compounds depend not only on the ratio of the contributions of these receptors, but also on the kinetic characteristics of the blocking action. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 11, pp. 1241–1251, November, 2005.     

The Ability of New Non-Competitive Glutamate Receptor Blockers to Weaken Motor Disorders in Animals

The ability of mono- and dicationic phenylcyclohexyl derivatives, which are non-competitive glutamate antagonists, to prevent convulsions induced in mice by intragastric NMDA or kainate, to weaken catalepsy induced in rats by haloperidol, and to exert their own influences of movement activity and behavior in animals was studied. The actions of study compounds were compared with those of the known NMDA antagonists memantine and dizocilpine. NMDA-induced convulsions were effectively prevented by both mono- and dications, while only dications were effective against kainate convulsions. Anticataleptic activity was significantly more marked in monocations, which lacked the ability to block non-NMDA receptors. Side effects on motor coordination were less marked with study compounds than with dizocilpine. Thus, the effects of phenylcyclohexyl derivatives in in vivo experimental models correlate with their anti-NMDA and anti-AMPA activity. They can be regarded as potential agents for treating parkinsonism and other motor disorders.     

Insulin-like growth factor 1 (IGF1) and its active peptide (1-3)IGF1 enhance the expression of synaptic markers in neuronal circuits through different cellular mechanisms

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) is neurotrophic and prevents fibril formation of amyloid β protein (Aβ). It is a promising lead compound for the development of therapeutic drugs for Alzheimer's disease. To find even more effective drugs based on the structure of fisetin, we synthesized a series of fisetin analogues lacking the 7-hydroxyl group and compared their effects on Aβ fibril formation determined by the thioflavin T fluorescence assay. 3,3',4'-Trihydroxyflavone and 3',4'-dihydroxyflavone inhibited Aβ fibril formation more potently than fisetin or 3',4',7-trihydroxyflavone, suggesting that the 7-hydroxy group is not necessary for anti-amyloidogenic activity. 3,3',4',5'-Tetrahydroxyflavone and 3',4',5'-trihydroxyflavone inhibited Aβ fibril formation far more potently than 3,3',4'-trihydroxyflavone and 3',4'-dihydroxyflavone, suggesting that 3',4',5'-trihydroxyl group of the B ring is crucial for the anti-amyloidogenic activity of flavonoids. Based on the structure-activity relationship, we synthesized 3,3',4',5,5'-pentahydroxyflavone, and confirmed that this compound is the most potent inhibitor of Aβ fibril formation among fisetin analogues that have been tested. Cytotoxicity assay using rat hippocampal neuron cultures demonstrated that Aβ preincubated with 3,3',4',5,5'-pentahydroxyflavone was significantly less toxic than Aβ preincubated with vehicle. 3,3',4',5,5'-Pentahydroxyflavone could be a new therapeutic drug candidate for the treatment of Alzheimer's disease.     

The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) impairs late reconsolidation of passive avoidance learning in the day-old chick

Both NMDA and non-NMDA receptors participate in the consolidation of passive avoidance learning (PAL) in the day-old chick. NMDA antagonists have also been implicated in reconsolidation processes following reminder-trials. In this study, we examined the effect of administering 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, on reconsolidation following memory reactivation. New HampshirexWhite leghorn cockerels were trained using a modified version of the PAL task. When CNQX was administered 20min following a reminder trial, a retention deficit was detected at 90min, but this had resolved by 24h following the reminder. The parameters of the reconsolidation deficit were similar to those induced by CNQX injections post-training with the exception of their transience. This finding suggests that the action of non-NMDA receptors may perform a similar role in both consolidation and reconsolidation processes.     

Role of presynaptic dopamine receptors in regulation of the glutamatergic neurotransmission in rat neostriatum

In experiments with the use of a push-pull cannula and simultaneous recording of electrical activity at the site of perfusion, the release of L-[3H]glutamic acid from rat neostriatum induced by K+-depolarization (60 mM K+ in perfusate) has been shown to be inhibited by replacing Ca2+ in the perfusion medium by Co2+. In contrast, release of L-[3H]glutamate induced by electrical stimulation of frontal cortex is enhanced by replacement of these cations. Application of dopamine (10(-5)-10(-3) M). apomorphine (10(-4) M) or beta-phenylethylamine (10(-3) M) as well as stimulation of the substantia nigra enhanced the basal release of L-[3H]glutamate. Haloperidol (10(-4) M) completely abolished the effects of apomorphine and beta-phenylethylamine, and partially abolished the effect of dopamine. The enhancement induced by apomorphine is strongly dependent on the presence of Na+ in the perfusion medium. On the other hand, apomorphine (10(-4) M) and beta-phenylethylamine (10(-3) M) inhibited the release of glutamate induced by electrical stimulation of the frontal cortex and that by K+-depolarization (the latter was shown for apomorphine). This inhibition is also abolished by haloperidol. A possible functional role of endogenous dopamine in the regulation of glutamatergic neurotransmission in rat neostriatum is discussed.     

High resolution in situ zymography reveals matrix metalloproteinase activity at glutamatergic synapses

Synaptic plasticity involves remodeling of extracellular matrix. This is mediated, in part, by enzymes of the matrix metalloproteinase (MMP) family, in particular by gelatinase MMP-9. Accordingly, there is a need of developing methods to visualize gelatinolytic activity at the level of individual synapses, especially in the context of neurotransmitters receptors. Here we present a high-resolution fluorescent in situ zymography (ISZ), performed in thin sections of the alcohol-fixed and polyester wax-embedded brain tissue of the rat (Rattus norvegicus), which is superior to the current ISZ protocols. The method allows visualization of structural details up to the resolution-limit of light microscopy, in conjunction with immunofluorescent labeling. We used this technique to visualize and quantify gelatinolytic activity at the synapses in control and seizure-affected rat brain. In particular, we demonstrated, for the first time, frequent colocalization of gelatinase(s) with synaptic N-methyl-D-aspartic acid (NMDA)- and AMPA-type glutamate receptors. We believe that our method represents a valuable tool to study extracellular proteolytic processes at the synapses, it could be used, as well, to investigate proteinase involvement in a range of physiological and pathological phenomena in the nervous system.     

A comparison of paired-pulse facilitation of AMPA and NMDA receptor-mediated excitatory postsynaptic currents in the hippocampus

Paired-pulse facilitation of excitatory synaptic transmission was investigated in the CA1 region of rat hippocampal slices using whole-cell patch-clamp recording. To optimise the measurement of excitatory synaptic transmission, -amino-butyric acid (GABA)-mediated synaptic inhibition was eliminated using both GABA_A and GABA_B antagonists. Pure -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs) were then isolated pharmacologically. Paired-pulse facilitation of either AMPA or NMDA receptor-mediated EPSCs (EPSC_A and EPSC_N, respectively) was investigated using two stimuli of identical strength delivered at intervals of between 25 and 1000 ms. The paired-pulse facilitation profiles of both EPSC_A and EPSC_N were similar. Pairedpulse facilitation of EPSC_A was independent of holding potential. In contrast paired-pulse facilitation of EPSC_N was markedly voltage-dependent; maximum facilitation was recorded at hyperpolarised membrane potentials. At positive membrane potentials there was little or no paired-pulse facilitation and, in most neurones, pairedpulse depression was observed. Voltage-dependence of paired-pulse facilitation of EPSC_N was similar in the presence or nominal absence of Mg²⁺ in the bathing medium, and was unaffected by extensive dialysis of neurones with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA). These data are consistent with a presynaptic locus for paired-pulse facilitation of EPSC_A. However, paired-pulse facilitation of EPSC_N involves postsynaptic factors.     

Role of glutamatergic receptors located in the nucleus raphe magnus on antinociceptive effect of morphine microinjected into the nucleus cuneiformis of rat

Neurons in the nucleus cuneiformis (CnF), located just ventrolateral to the periaqueductal gray, project to medullary nucleus raphe magnus (NRM), which is a key medullary relay for descending pain modulation and is critically involved in opioid-induced analgesia. Previous studies have shown that antinociceptive response of CnF-microinjected morphine can be modulated by the specific subtypes of glutamatergic receptors within the CnF. In this study, we evaluated the role of NMDA and kainate/AMPA receptors that are widely distributed within the NRM on morphine-induced antinociception elicited from the CnF. Hundred and five male Wistar rats weighing 250-300 g were used. Morphine (10, 20 and 40 microg) and NMDA receptor antagonist, MK-801 (10 microg) or kainate/AMPA receptor antagonist, DNQX (0.5 microg) in 0.5 microl saline were stereotaxically microinjected into the CnF and NRM, respectively. The latency of tail-flick response was measured at set intervals (2, 7, 12, 17, 22, 27 min after microinjection) by using an automated tail-flick analgesiometer. The results showed that morphine microinjection into the CnF dose-dependently causes increase in tail-flick latency (TFL). MK-801 microinjected into the NRM, just 1 min before morphine injection into the CnF, significantly attenuated antinociceptive effects of morphine. On the other hand, DNQX microinjected into the NRM, significantly increased TFL after local application of morphine into the CnF. We suggest that morphine related antinociceptive effect elicited from the CnF is mediated, in part, by NMDA receptor at the level of the NRM whereas kainite/AMPA receptor has a net inhibitory influence at the same pathway.