Opioid peptide messenger RNA expression is increased at spinal and supraspinal levels following excitotoxic spinal cord injury

Spinal cord injury in rats is known to cause anatomical, physiological and molecular changes within the spinal cord. These changes may account for behavioral syndromes that appear following spinal cord injury, syndromes believed to be related to the clinical condition of chronic pain. Intraspinal injection of quisqualic acid produces an excitotoxic injury with pathological characteristics similar to those associated with ischemic and traumatic spinal cord injury. In addition, recent studies have demonstrated changes in blood flow, neuronal excitability and gene expression in the brain following excitotoxic injury, indicating that behavioral changes may result from modification of neuronal substrates at supraspinal levels of the neuraxis. Because changes in spinal opioid peptide expression have been demonstrated in models of traumatic spinal cord injury and chronic pain, the present study investigated messenger RNA expression of the opioid peptides, preproenkephalin and preprodynorphin, at spinal and supraspinal levels following excitotoxic spinal cord injury. Male, Long-Evans rats were given three intraspinal injections of quisqualic acid (total 1.2 microl, 125mM). After one, three, five, seven or 10days, animals were killed and quantitative in situ hybridization performed on regions of the spinal cord surrounding the lesion site, as well as whole-brain sections through various levels of the thalamus. Preproenkephalin and preprodynorphin expression was increased in spinal cord areas adjacent to the site of quisqualic injection and in cortical regions associated with nociceptive function, preproenkephalin in the cingulate cortex and preprodynorphin in the parietal cortex, both ipsilaterally and contralaterally at various time-points following injury.These results further our knowledge of the secondary events that occur following spinal cord injury, specifically implicating supraspinal opioid systems in the CNS response to spinal cord injury.     

Expression of c-fos mRNA is increased and related to dynorphin mRNA expression following excitotoxic spinal cord injury in the rat.

Previous studies have demonstrated that excitotoxic spinal cord injury (SCI) created by the intraspinal injection of quisqualic acid (QUIS) is capable of inducing opioid peptide gene expression within the spinal cord and cortex. The opioids are classically involved in the suppression of pain transmission but specifically, dynorphin, has been implicated in the secondary pathophysiologic response to SCI. Activation of the immediate early gene, c-fos, has been implicated in the induction of preprodynorphin (PPD) gene expression and therefore, may be an important intermediate step in the generation of the opioid response to SCI. The purpose of this study was to investigate whether intraspinal QUIS injection induces c-fos expression within the spinal cord. Male, Long-Evans, adult rats (n=5) received an intraspinal injection of 1.2 microl of 125 mM QUIS directed at spinal segments T12-L2. Four hours post-injection brain and spinal cord tissues were removed and processed for in situ hybridization. Integrated density of c-fos and PPD mRNA expression was increased in the spinal dorsal horn following QUIS injection as compared to sham-injected animals. This indicates that SCI rapidly induces c-fos and PPD expression and suggests that c-fos plays a role in the induction of PPD expression.     

Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by trans-1-aminocyclopentane-1,3-dicarboxylic acid.

In freshly isolated spinal dorsal horn (DH) neurons (laminae I-IV) of the young rat the effects of 25-100 microM of (+/-)-trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), 1S,3R-ACPD and 1R,3S-ACPD, a metabotropic glutamate receptor (mGluR) agonist, on inward currents induced by glutamate (Glu), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate were studied under whole-cell voltage-clamp conditions. When the cells were clamped to -60 mV, the racemic mixture and both stereo isomers of trans-ACPD increase the responses elicited by Glu, AMPA, and NMDA, but little those of kainate. In addition, quisqualate (10-50 microM), in the presence of CNQX (5-20 microM) or NBQX (5 microM), potentiated NMDA-induced currents. The enhancing effect lasted 10-75 min, depending upon both dose and length of application. In a smaller proportion of dorsal horn neurons, the enhancing effect was preceded by a transient depression of the responses to Glu, AMPA, and NMDA. 2-Amino-3-phosphonopropionic acid (L-AP3), a putative antagonist of mGluR exerted little effect on responses to AMPA itself, but reduced or prevented the enhancing effect of 1S,3R-ACPD. It is concluded that activation of a metabotropic glutamate receptor by trans-ACPD, and its two enantiomers, may mediate the enhancement of AMPA and NMDA responses in acutely isolated rat spinal dorsal horn neurons. These results are consistent with the possibility that the activation of metabotropic glutamate receptor may contribute to the regulation of the strength of excitatory amino-mediated primary afferent neurotransmission, including nociception.     

Evaluation of the activity of a novel metabotropic glutamate receptor antagonist (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) in the in vitro neonatal spinal cord and in an in vivo pain model

The cyclobutylglycine (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1alpha and 5alpha, with IC50 values of 1.0+/-0.4 microM and 1.6+/-1.4 microM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent Kd values were estimated to be 0.3 microM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 microM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root-ventral root potential elicited at 8 x threshold was depressed by LY393053 with IC50 values of 9.0+/-0.7 microM and 12.7+/-1.7 microM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1-10 mg/kg intraperitoneally. An ED50 value of 6.0 mg/kg was obtained in this test. By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.     

Morphine induction of c-fos expression in the rat forebrain through glutamatergic mechanisms: role of non-n-methyl-D-aspartate receptors

Acute injection of morphine induces expression of the immediate-early genes c-Fos and JunB in several forebrain regions of the rat, in part through an N-methyl-D-aspartate (NMDA) receptor-dependent mechanism. Because membrane depolarization through (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors is believed to be necessary for full activation of NMDA receptors, we determined the role of AMPA receptors in morphine-induced c-Fos expression. Rats were given the AMPA receptor antagonist GYKI-52466 (12.9 mg/kg, i.p.) 15 min before morphine (10 mg/kg, s.c.), or the AMPA receptor enhancer CX516 (30 mg/kg, i.p.) 5 min after morphine. The c-Fos response was attenuated by the antagonist and augmented by the enhancer. Using double immunocytochemistry, we found that morphine induced c-Fos in neurons containing the GluR2/3, but not the GluR1 and rarely the GluR4, subunits of the AMPA receptor. Double immunocytochemistry for mu opioid receptor and c-Fos showed that c-Fos expression was mainly absent in the patch compartment of the striatum, which is enriched in mu opioid receptors.The glutamatergic synapse often contains metabotropic receptors as well as ionotropic receptors. Type I metabotropic glutamate receptors are coupled to activation of protein kinase C, which has also been shown to mediate the immediate-early gene response to morphine. To determine if activation of metabotropic glutamate receptors is involved in rapid effects of morphine on the brain, rats were given the type I metabotropic glutamate receptor antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.2 mg/kg, i.p.) or vehicle 30 min before morphine treatment. Pretreatment with AIDA completely blocked morphine-induced c-Fos expression in the caudate-putamen.Taken together, these results demonstrate involvement of both AMPA and type I metabotropic glutamate receptors in the acute effects of morphine on the forebrain, supporting an important role for glutamatergic neurotransmission mediated by non-NMDA glutamate receptors in morphine's actions.     

Group I metabotropic glutamate receptor inhibition selectively blocks a prolonged Ca(2+) elevation associated with age-dependent excitotoxicity

It has been recognized for some years that a prolonged Ca(2+) elevation that is predictive of impending cell death develops in cultured neurons following excitotoxic insult. In addition, neurons exhibit enhanced sensitivity to excitotoxic insult with increasing age in culture. However, little is known about the processes that selectively regulate the post-insult Ca(2+) elevation and therefore, it remains unclear whether it is associated specifically with age-dependent toxicity.Here, we tested the hypothesis that a group I metabotropic glutamate receptor antagonist selectively modulates the prolonged Ca(2+) elevation in direct association with its protective effects against excitotoxicity. Rat hippocampal cultures of two ages (8-9 and 21-28 days in vitro) were exposed to a 5-min glutamate insult (400 microM in younger and 10 microM in older cultures) sufficient to kill >50% of the neurons, and were treated with vehicle or the specific group I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), throughout and following the insult. Neuronal survival was quantified 24 h after insult. In parallel studies, neurons of similar age in culture were imaged ratiometrically with a confocal microscope during and for 60 min after the glutamate insult. A large post-insult Ca(2+) elevation was present in older but not most younger neurons. The N-methyl-D-aspartate receptor antagonist, MK-801, blocked the Ca(2+) elevation both during and following the insult. In contrast, AIDA blocked only the post-insult prolonged Ca(2+) elevation in older neurons. Moreover, AIDA was neuroprotective in older but not younger cultures.From these results we suggest that the post-insult Ca(2+) elevation is regulated differently from the Ca(2+) elevation during glutamate insult and is modulated by group I metabotropic glutamate receptors. Further, the prolonged Ca(2+) elevation appears to be directly linked to an age-dependent component of vulnerability.     

The striopallidal pathway is involved in antiparkinsonian-like effects of the blockade of group I metabotropic glutamate receptors in rats

The aim of the study was to examine the influence of the blockade of group I metabotropic glutamate receptors (mGluRs) on the haloperidol-induced catalepsy and proenkephalin mRNA expression in the rat striatum. Bilateral, intrastriatal injection of AIDA ((RS)-1-aminoindan-1,5-dicarboxylic acid, 3-15 microg/0.5 microl), a selective antagonist of group I mGluRs, inhibited catalepsy induced by haloperidol (0.5 mg/kg i.p.). Repeated intrastriatal AIDA administrations (3 x 15 microg/0.5 microl, 3 h apart) counteracted the haloperidol-induced (3 x 1.5 mg/kg s.c., 3 h apart) increase in the proenkephalin mRNA expression in that structure. The present study indicates that the blockade of the striatal group I mGluRs may inhibit parkinsonian akinesia by normalizing the function of the striopallidal pathway.     

Inhibitory effects of Group I metabotropic glutamate receptors antagonists on the expression of NMDA receptor NR1 subunit in morphine tolerant rats

N-Methyl-d-aspartate receptor (NMDAR) and Group I metabotropic glutamate receptors (mGluRs) are involved in the process of morphine tolerance. Previous studies have shown that Group I mGluRs can modulate NMDAR functions in the central nervous system. The aim of the present study was to examine the influence of Group I mGluRs antagonists on the expression of NMDA receptor NR1 subunit (NR1) in the rat spinal cord. Morphine tolerance was induced in rats by repeated administration of 10 μg morphine (intrathecal, i.t.) twice a day for 7 consecutive days. Tail flick test was used to assess the effect of Group I mGluRs antagonist, AIDA ((RS)-1-Aminoindan-1,5 dicarboxylic acid) or mGluR5 antagonist, MPEP (2-methyl-6-(phenylethynyl)pyridine) on morphine antinociceptive tolerance. The expression of NR1 was measured by immunofluorescence and Western blot. Behavioral tests revealed that both AIDA and MPEP attenuated the development of morphine tolerance. The expression of NR1 was upregulated in the dorsal horn of spinal cord after chronic morphine treatment. AIDA or MPEP co-administered with morphine attenuated morphine induced upregulation of NR1. These findings suggest that the development of morphine tolerance partly prevented by Group I mGluRs antagonists may due to its inhibitory effect on the expression of NR1 subunit.     

An approach to experimental synaptic pathology using green fluorescent protein-transgenic mice and gene knockout mice to show mitochondrial permeability transition pore-driven excitotoxicity in interneurons and motoneurons

Researchers used transgenic mice expressing enhanced-green fluorescent protein (eGFP) driven by either the glycine transporter-2 gene promoter to specifically visualize glycinergic interneurons or the homeobox-9 (Hb9) gene promoter to visualize motoneurons for assessing their vulnerabilities to excitotoxins in vivo. Stereotaxic excitotoxic lesions were made in adult male and female mouse lumbar spinal cord with the specific N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA) and the non-NMDA ion channel glutamate receptor agonist kainic acid (KA). QA and KA induced large-scale degeneration of glycinergic interneurons in spinal cord. Glycinergic interneurons were more sensitive than motoneurons to NMDA receptor-mediated and non-NMDA glutamate receptor-mediated excitotoxicity. Outcome after spinal cord excitotoxicity was gender-dependent, with males showing greater sensitivity than females. Excitotoxic degeneration of spinal interneurons resembled apoptosis, while motoneuron degeneration appeared non-apoptotic. Perikaryal mitochondrial accumulation was antecedent to both NMDA and non-NMDA receptor-mediated excitotoxic stimulation of interneurons and motoneurons. Genetic ablation of cyclophilin D, a regulator of the mitochondrial permeability transition pore (mPTP), protected both interneurons and motoneurons from excitotoxicity. The results demonstrate in adult mouse spinal cord that glycinergic interneurons are more sensitive than motoneurons to excitotoxicity that stimulates mitochondrial accumulation, and that the mPTP has pro-death functions mediating apoptotic and non-apoptotic neuronal degeneration in vivo.     

The mRNA expression of AMPA type glutamate receptors in the primary motor cortex of patients with amyotrophic lateral sclerosis: an in situ hybridization study

The pathogenetic mechanisms leading to progressive neurodegeneration in amyotrophic lateral sclerosis (ALS) have not been fully elucidated. One possible factor responsible for the selective motor neuron loss in the motor cortex, brain stem and spinal cord is glutamate-induced excitotoxicity particularly mediated via alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) type glutamate receptors. Data about the expression pattern of AMPA receptors in the primary motor cortex are lacking so far. The pharmacological and physiological properties of AMPA receptors are defined by the heteromeric composition of the four different receptor subunits. Different expression patterns of these subunits at motor neurons may provide a molecular basis for increased vulnerability to excitotoxic damage. Using in situ hybridization histochemistry we did not detect any significant differences in the distribution of AMPA receptor mRNA in the motor cortex of ALS patients compared to controls.     

The excitation of frog motoneurones in vitro by the glutamate analogue, DI-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and the effect of amino acid antagonists

Intracellular recordings were made from motoneurones of the frog spinal cord in vitro and the excitatory effects of the glutamate analogue DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) examined. AMPA (25-100 microM) depolarized motoneurones with an increase in input conductance and an increase in spontaneous cell firing. The time-course of the AMPA response was prolonged compared to L-glutamate. D-Aminophosphonovalerate (1 microM) and D-aminoadipate (100 microM) did not antagonize AMPA-induced excitations while depressing L-glutamate responses. Glutamate diethylester up to 1 mM was not found to be a useful amino acid antagonist since it did not significantly reduce the depolarizations to quisqualate, L-glutamate and AMPA. The results are compatible with an action of AMPA on receptors distinct from those to N-methyl-D-aspartate and shared by quisqualate.     

Excitatory amino acid receptors on isolated retinal ganglion cells from the goldfish.

1. Whole-cell currents activated by the excitatory amino acids L-glutamic acid (glutamate, Glu), L-aspartic acid (Asp), and their analogues N-methyl-D-aspartate (NMDA), kainic acid (KA), quisqualic acid (QA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were recorded from ganglion cells enzymatically dissociated from goldfish retina and grown in culture. All agonists induced detectable whole-cell responses in the majority of cells cultured from 2 to 72 h. 2. Currents activated by each of the agonists were selective for cations (Na+) over anions (Cl-). The responses to Glu, NMDA, and Asp were each potentiated when 1 microM glycine was coapplied. Extracellular Mg2+ blocked completely the response to NMDA plus glycine in cells held at negative potentials, but the block was relieved when cells were more depolarized. 3. Dose-response measurements revealed a rank order of sensitivity to the Glu analogues in the presence of 1 microM glycine and zero extracellular Mg2+; QA greater than AMPA greater than NMDA greater than KA. Cells were not responsive to APB (L-2-aminophosphonobutyric acid). 4. Kynurenic acid (Ky) produced a noncompetitive block of the NMDA response in the presence of 1 microM glycine with a Ki of 40 microM. Responses to KA, QA, and AMPA were blocked competitively by Ky with Kis of 72, 148, and 656 microM, respectively. QA and AMPA competitively blocked the response to KA with Kis of 114 microM and 1 mM, respectively. NMDA single channels had a mainstate slope conductance of 27-30 pS and two subconductance levels of 5 and 24 pS at 13 degrees C in symmetrical Na+. 5. Whole-cell responses to QA and AMPA were highly correlated, suggesting that QA and AMPA activated the same receptor or class of receptors; whereas, responses to QA and KA were not well correlated, suggesting that these agonists at least in part activated separate receptor populations.     

Differential activation and survival of basal forebrain neurons following infusions of excitatory amino acids: studies with the immediate early gene c-fos

These experiments investigated, by studying patterns of c-fos expression, the distribution of neurons activated or destroyed by the infusion into the basal forebrain of various excitatory amino acids at toxic and subtoxic doses. The results of experiment 1 showed that N-methyl-d-aspartic acid (NMDA), quisqualic acid and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) differentially increased the expression of c-fos in magnocellular cholinergic nucleus basalis, dorsal and ventral pallidal neurons. AMPA was the most, and NMDA the least, effective in inducing FOS in nucleus basalis magnocellularis (nbM) neurons, with quisqualic acid having an intermediate effect, whereas the reverse was true in terms of the induction of FOS in pallidal neurons. In experiment 2, it was demonstrated that, in animals with ibotenic acid-induced lesions of the basal forebrain that were targetted on the nbM, virtually no pallidal neurons could be visualized that expressed FOS following AMPA-induced excitation of the dorsal and ventral striatum. By contrast, in animals with AMPA-induced lesions of the nbM, excitation of the striatum was followed by the expression of FOS in many dorsal and ventral pallidal neurons. Thus, infusions of AMPA into the basal forebrain appears preferentially to activate or destroy, depending on the concentration infused, cholinergic nbM neurons, whereas ibotenic acid or NMDA preferentially destroys or activates neurons of the dorsal and ventral pallidum. These results provide novel and complementary information regarding the organization of the basal forebrain and allow a clearer understanding of the different behavioural consequences of NMDA agonist-induced and non-NMDA agonist-induced excitotoxic lesions of this area.     

Inhibition of glutamate uptake in the spinal cord induces hyperalgesia and increased responses of spinal dorsal horn neurons to peripheral afferent stimulation

Glutamate is a primary excitatory neurotransmitter in the mammalian CNS. Glutamate released from presynaptic neurons is cleared from the synaptic cleft passively by diffusion and actively by glutamate transporters. In this study, the role of glutamate transporters in sensory processing in the spinal cord has been investigated in behavioral, in vivo and in vitro experiments. Intrathecal application of a non-selective glutamate transport inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (10 microl of 100 microM solution) induced hypersensitivity to peripheral mechanical and thermal stimuli. Topical application of L-trans-pyrrolidine-2,4-dicarboxylic acid (100 microM) onto the dorsal surface of the L3-L6 spinal cord increased spontaneous activities, innocuous and noxious stimulus-evoked responses and after-discharges of wide dynamic range neurons in the L4-5 spinal segments. Whole cell recordings made from superficial dorsal horn neurons in an isolated whole spinal cord from newborn rats (2-3 weeks old) revealed that bath-applied L-trans-pyrrolidine-2,4-dicarboxylic acid (100 microM) produced partial membrane depolarization, increased spontaneous action potentials with decreased neuronal membrane resistance and time constant, but without significant changes of capacitance. Finally, the amplitude and duration of primary afferent evoked-excitatory postsynaptic currents recorded from neurons in the substantia gelatinosa in the spinal slices from young adult rats (6-8 weeks old) were increased in the presence of L-trans-pyrrolidine-2,4-dicarboxylic acid (100 microM). This study indicates that glutamate transporters regulate baseline excitability and responses of dorsal horn neurons to peripheral stimulation, and suggests that dysfunction of glutamate transporters may contribute to certain types of pathological pain.     

Dopamine-opiate interaction in the regulation of neostriatal and pallidal neuronal activity as assessed by opioid precursor peptides and glutamate decarboxylase messenger RNA expression.

Neostriatal GABAergic neurons projecting to the globus pallidus synthesize the opioid peptide enkephalin, while those innervating the substantia nigra pars reticulata and the entopeduncular nucleus synthesize dynorphin. The differential control exerted by dopamine on the activity of these two efferent projections concerns also the biosynthesis of these opioid peptides. Using in situ hybridization histochemistry, we investigated the role of opioid co-transmission in the regulation of neostriatal and pallidal activity. The expression of the messenger RNAs encoding glutamate decarboxylase-the biosynthetic enzyme of GABA-and the precursor peptides of enkephalin (preproenkephalin) and dynorphin (preprodynorphin) were measured in rats after a sustained blockade of opioid receptors by naloxone (s.c. implanted osmotic minipump, eight days, 3 mg/kg per h), and/or a subchronic blockade of D2 dopamine receptors by haloperidol (one week, 1.25 mg/kg s.c. twice a day). The density of mu opioid receptors in the neostriatum and globus pallidus was determined by autoradiography. Naloxone treatment resulted in a strong up-regulation of neostriatal and pallidal mu opioid receptors that was not affected by the concurrent administration of haloperidol. Haloperidol alone produced a moderate down-regulation of neostriatal and pallidal micro opioid receptors. Haloperidol strongly stimulated the expression of neostriatal preproenkephalin and preprodynorphin messenger RNAs. This effect was partially attenuated by naloxone, which alone produced moderate increases in preproenkephalin and preprodynorphin messenger RNA levels. In the neostriatum, naloxone did not affect either basal or haloperidol-stimulated glutamate decarboxylase messenger RNA expression. A strong reduction of glutamate decarboxylase messenger RNA expression was detected over pallidal neurons following either naloxone or haloperidol treatment, but concurrent administration of the two antagonists did not result in a further decrease. The amplitude of the variations of mu opioid receptor density and of preproenkephalin and preprodynorphin messenger RNA levels suggests that the regulation of neostriatal and pallidal micro opioid receptors is more susceptible to a direct opioid antagonism, while the biosynthesis of opioid peptides in the neostriatum is more dependent on the dopaminergic transmission. The down-regulation of mu opioid receptors following haloperidol represents probably an adaptive change to increased enkephalin biosynthesis and release. The haloperidol-induced increase in neostriatal preprodynorphin messenger RNA expression might result from an indirect, intermittent stimulation of neostriatal D1 receptors. The haloperidol-induced decrease of pallidal glutamate decarboxylase messenger RNA expression suggests, in keeping with the current functional model of the basal ganglia, that the activation of the striatopallidal projection produced by the interruption of neostriatal dopaminergic transmission reduces the GABAergic output of the globus pallidus. The reduction of pallidal glutamate decarboxylase messenger RNA expression following opioid receptor blockade indicates an indirect, excitatory influence of enkephalin upon globus pallidus neurons and, consequently, a functional antagonism between the two neuroactive substances (GABA and enkephalin) of the striatopallidal projection in the control of globus pallidus output. Through this antagonism enkephalin could partly attenuate the GABA-mediated effects of a dopaminergic denervation on pallidal neuronal activity.     

Stimulation of Na-K-2Cl cotransporter in neurons by activation of Non-NMDA ionotropic receptor and group-I mGluRs

In a previous study, we found that Na(+)-K(+)-2Cl(-) cotransporter in immature cortical neurons was stimulated by activation of the ionotropic N-methyl-D-aspartate (NMDA) glutamate receptor in a Ca(2+)-dependent manner. In this report, we investigated whether the Na(+)-K(+)-2Cl(-) cotransporter in immature cortical neurons is stimulated by non-NMDA glutamate receptor-mediated signaling pathways. Expression of the Na(+)-K(+)-2Cl(-) cotransporter and metabotropic glutamate receptors (mGluR1 and 5) was detected in cortical neurons via immunoblotting and immunofluorescence staining. Significant stimulation of cotransporter activity was observed in the presence of both trans-(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) (10 microM), a metabotropic glutamate receptor (mGluR) agonist, and (RS)-3,5-dihydroxyphenylglycine (DHPG) (20 microM), a selective group-I mGluR agonist. Both trans-ACPD and DHPG-mediated effects on the cotransporter were eradicated by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM, a Ca(2+) chelator. In addition, DHPG-induced stimulation of the cotransporter activity was inhibited in the presence of mGluRs antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (1 mM) and also with selective mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (100 microM). A DHPG-induced rise in intracellular Ca(2+) in cortical neurons was detected with Fura-2. Moreover, DHPG-mediated stimulation of the cotransporter was abolished by inhibition of Ca(2+)/CaM kinase II. Interestingly, the cotransporter activity was increased by activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These results suggest that the Na(+)-K(+)-2Cl(-) cotransporter in immature cortical neurons is stimulated by group-I mGluR- and AMPA-mediated signal transduction pathways. The effects are dependent on a rise of intracellular Ca(2+).     

Involvement of mu- and delta-opioid receptors in the antinociceptive effects induced by AMPA receptor antagonist in the spinal cord of rats

The present study was performed to explore the involvement of opioid receptors in the antinociception induced by a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist in rats. The hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation was assessed by hot plate test and the Randall Selitto Test. Intrathecal injection of 20 nmol of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX) disodium, a competitive AMPA receptor antagonist, increased significantly the HWLs to both thermal and mechanical stimulation in rats. The increased HWLs induced by NBQX were dose-dependently attenuated by the opioid receptor antagonist naloxone, while naloxone itself had no marked influences on the HWL of rats. Furthermore, the increased HWLs induced by NBQX were inhibited by the mu-opioid antagonist beta-funaltrexamine (beta-FNA) or the delta-opioid antagonist naltrindole, but not by the kappa-opioid antagonist nor-binaltorphimine (nor-BNI). The results suggest that mu- and delta-opioid receptors, not kappa-opioid receptor, are involved in the antinociception induced by AMPA antagonist in the spinal cord of rats.     

Involvement of ionotropic glutamate receptors in low frequency electroacupuncture analgesia in rats

The present study was conducted to determine whether blockage of both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid/kainate (AMPA/KA) receptors influences the induction of low frequency electroacupuncture (EA) analgesia. Although neither intrathecal injection of NMDA antagonist D-2-amino-5-phosphonopentanoic acid (D-AP-5) or AMPA/KA antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonami-de (NBQX) disodium alone had an effect on analgesia, spinal application of D-AP-5 and NBQX disodium significantly prevented analgesia induced by 2 Hz EA. The intrathecal injection of the excitatory amino acid NMDA produced analgesia for several minutes after intrathecal injection, as did EA stimulation. These results suggest that ionotropic glutamate receptors may be involved in the induction of 2 Hz EA analgesia.     

Olfactory nerve stimulation-evoked mGluR1 slow potentials, oscillations, and calcium signaling in mouse olfactory bulb mitral cells

Fast synaptic transmission between olfactory receptor neurons and mitral cells (MCs) is mediated through AMPA and NMDA ionotropic glutamate receptors. MCs also express high levels of metabotropic glutamate receptor 1 (mGluR1) whose functional significance is less understood. Here we characterized a slow mGluR1-mediated potential that was evoked by high-frequency (100-Hz) olfactory nerve (ON) stimulation in the presence of NBQX and D-APV, blockers of ionotropic glutamate receptors, and that was associated with a local Ca2+ transient in the MC dendritic tuft. High-frequency ON stimulation in the presence of NBQX and D-APV also evoked a slow, nearly 2-Hz oscillation of MC membrane potential that was abolished by the mGluR1 antagonist LY367385 (50 microM). Both mGluR slow potential and slow oscillation persisted in the presence of gabazine (10 microM), a GABA(A) receptor antagonist, and intracellular QX-314 (10 mM), a Na+ channel blocker. In contrast to a slow mGluR1 potential in cerebellar Purkinje neurons, the MC mGluR1 potential was not depressed by SKF96365 (< or =250 microM) and thus is likely not mediated by TRPC1 cation channels, nor was it potentiated by an elevation of intracellular Ca2+ level. Imaging with the Na+ indicator SBFI revealed a Na+ transient in the MC dendrite accompanying the mGluR1 slow potential. We conclude that the MC mGluR1 potential triggered by glutamate released from the ON supports oscillations and synchronizations of MCs associated within one glomerulus.     

A metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, attenuates immediate early gene mRNA expression following traumatic injury in cultured rat cortical glial cells

The effects of three glutamate receptor antagonists, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801) for the N-methyl-D-aspartate receptor, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f] quinoxaline-7-sulfonamide (NBQX) for the alpha-amino-3-hydroxy-5methyl-4-isoxazole propionate /kinate receptor and (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) for the metabotropic receptor, on c-fos and c-jun mRNA expression were investigated in cultured cortical glial cells following traumatic scratch injury. Expression of the two genes along the edges of wounds detected by in situ hybridization was not affected by MK-801 and NBQX. However, 100 and 500 microM of MCPG remarkably reduced the hybridization signals for both c-fos and c-jun mRNAs. The present results suggest that group I metabotropic glutamate receptors might have some association with immediate early gene induction after in vitro traumatic injury in glial cells.