Quisqualate Research Articles

Metabotropic glutamate receptor type 1alpha and tubulin assemble into dynamic interacting complexes.

Metabotropic glutamate receptors (mGlu receptors) are coupled to G-protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. Very recently, we identified tubulin as an interacting partner of the mGlu(1alpha) receptor in rat brain. Using BHK-570 cells permanently expressing the receptor we have shown that this interaction occurs predominantly with soluble tubulin, following its translocation to the plasma membrane. In addition, treatment of the cells with the agonist quisqualic acid induce tubulin depolymerization and its translocation to the plasma membrane. Immunofluorescence detection of both the receptor and tubulin in agonist-treated cells reveals a disruption of the microtubule network and an increased clustering of the receptor. Collectively these data demonstrate that the mGlu(1alpha) receptor interacts with soluble tubulin and that this association can take place at the plasma membrane.

Spinal and supraspinal changes in opioid mRNA expression are related to the onset of pain behaviors following excitotoxic spinal cord injury

Excitotoxic spinal cord injury (SCI) causes anatomic, physiologic and molecular changes within the spinal cord and brain. Intraspinal injection of quisqualic acid (QUIS) produces an excitotoxic injury that leads to the onset of behavioral syndromes, believed to be related to the clinical condition of chronic pain. The opioid system, classically involved in the suppression of pain transmission, has been associated with the onset of pain-related behaviors and changes in spinal opioid peptide expression have been demonstrated in various models of SCI and chronic pain. Recently, changes in opioid peptide expression have been demonstrated in both spinal and supraspinal areas following excitotoxic SCI. Therefore, the purpose of this study was to examine changes in opioid peptide gene expression as they relate to the onset of pain behaviors following excitotoxic SCI. Male, Long–Evans rats were given an intraspinal injection of 1.2 μl of 125 mM QUIS and allowed to survive for 10 days, a duration sufficient for the development of pain-related behaviors. Animals were assessed daily for the presence of excessive grooming behavior, i.e. self-directed biting and scratching resulting in damage to superficial and deeper layers of the skin. Animals were also tested for thermal hypersensitivity using a cold plate apparatus on days 5, 7, and 10 following QUIS injection. After sacrifice, quantitative in situ hybridization was performed on regions of the spinal cord surrounding the lesion site as well as whole brain sections through various levels of the thalamus and cortex. Spinal preproenkephalin (PPE) and preprodynorphin (PPD) expression was significantly increased in animals that developed excessive grooming behaviors vs. those that did not. For PPE, this difference was seen bilaterally, in areas of cord caudal to the site of injury. For PPD, this difference was seen only ipsilateral to the site of injection, rostral to the site of injury. In addition, PPE expression in the anterior cingulate cortex and PPD expression in the contralateral parietal cortex were significantly higher in grooming vs. non-grooming animals. These results support previous conclusions that both spinal and supraspinal regulation of endogenous opioid peptide expression plays a role in the response to or onset of post-SCI pain. These results also suggest that the opioid peptides are regulated independently and serve different functions in response to SCI.

Physiological and pharmacological characteristics of quisqualic acid-induced K(+)-current response in the ganglion cells of Aplysia.

The extracellular application of either quisqualic acid (QA) or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces an outward current in identified neurons of Aplysia ganglion under voltage clamp. The time course of the QA-induced response is significantly slower than that induced by FMRFamide. The reversal potential for both responses was -92 mV and was shifted 17 mV in a positive direction for a twofold increase in the extracellular K(+) concentration. The QA-induced response was markedly depressed in the presence of Ba(2+), a blocker of inward rectifier K(+)-channel, whereas TEA, a Ca(2+)-activated K(+)-channel (BK(Ca)) blocker, or 4-AP, a transient K(+) (A)-channel blocker, had no effect on the response. The QA-induced K(+)-current was significantly suppressed by CNQX and GYKI52466, antagonists of non-NMDA receptors. However, the application of either kainate or AMPA, agonists for non-NMDA receptors, produced no type of response in the same neurons. The QA-induced K(+)-current response was not depressed at all by an intracellular injection of either guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) or guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS), but the FMRFamide-induced response was markedly blocked by both GDP-betaS and GTP-gammaS in the same cell. Furthermore, the QA- and FMRFamide-induced K(+)-current responses were both decreased markedly when the temperature was lowered to 15 degrees C, from 23 degrees C. These results suggested that the QA-induced K(+)-current response is produced by an activation of a novel type of QA-receptor and that this response is not produced by an activation of the G protein.

NO-Flurbiprofen Attenuates Excitotoxin-Induced Brain Inflammation, and Releases Nitric Oxide in the Brain

Brain inflammation underlies the pathogenesis of Alzheimer's disease (AD) and nonsteroidal anti-inflammatory drug therapy may delay the onset of AD. We investigated, in vivo, the effects of NO-flurbiprofen on brain inflammation in rats injected with quisqualic acid into the nucleus basalis and on the release of nitric oxide from the drug in naive rat brains. We showed that the excitotoxin-induced microglia reaction, the expression of inducible nitric oxide synthase-positive cells and the production of interleukin-1beta and prostaglandin-E2 in the injected area were attenuated by the NO-flurbiprofen (15 mg/kg, p.o.) treatment. An oral administration of NO-flurbiprofen (25, 50 and 100 mg/kg) to naive rats was followed by significant increases in cortical nitrite levels. This drug may have important therapeutic implications for the treatment of AD.

Protective effect of excitatory amino acids on cold-restraint stress-induced gastric ulcers in mice: role of cyclic nucleotides.

Previous studies have shown that excitatory amino acids (EAAs) and their receptors may play important roles in the mammalian enteric system. In this study, we investigated whether EEAs, including L-glutamate (L-Glu) and subtypes N-methyl-D-aspartate (NMDA), kainic acid (KA), and quisqualic acid (QA), reduce cyclic AMP (cAMP) levels and play a role in protecting gastric lesions in cold-restraint stress (CRS) mice. First, we found that dose-dependent administration of four selected EAAs significantly attenuated the increase of cAMP content and exhibited a protective effect on the development of gastric lesions induced by CRS. Second, CRS treatment exhibited a decrease of cGMP content and an increase of cAMP content with marked time-dependent changes, and a high cAMP/cGMP ratio in mice gastric mucosa. Third, pretreatment with 0.25 microg/kg or 0.5 microg/kg dibutyryl cGMP (db-cGMP) exhibited protective effects on CRS-induced gastric lesions, with preventive ratios of 24.61% and 35.32%, respectively. Moreover, db-cGMP at 0.5 microgg/kg significantly attenuated the increase in both cAMP content and the cAMP/cGMP ratio in CRS-treated gastric mucosa. In contrast, db-cAMP exhibited no protective effect, and significantly decreased cGMP content and increased the cAMP/cGMP ratio. These results suggest that EAAs significantly reduce CRS-induced gastric ulcers in mice. The possible mechanism of the antiulcer activity of EAAs may be related to a decrease in the cAMP content in the gastric mucosa of mice. In addition, an increase of the cAMP/cGMP ratio significantly involved in CRS-induced gastric ulcer formation in mice.

Another aspect of anticipation in schizophrenia: Suicide attempts in successive generations

The role of the ERK1/2 signal transduction pathway and related transcription factors in the regulation of gene expression and pain behavior following excitotoxic spinal cord injury (SCI) was examined. Specifically, phosphorylation of ERK1/2, activation of transcription factors NF-kB, ELK-1, and CREB, and gene expression of the neurokinin-1 receptor and NMDA receptor subunits NR1 and NR-2A were investigated. Excitotoxic injury was produced by intraspinal injection of quisqualic acid (QUIS) in male Sprague–Dawley rats. Western blots were used to evaluate phosphorylation and activation of ERK1/2 and transcription factors using phospho-specific or total antibodies. Real-time PCR was used to evaluate gene expression of NK-1R, NR-1, and NR-2A. Assessment of excessive grooming behavior was used to evaluate the presence of spontaneous pain behavior. Excitotoxic spinal injury resulted in: (1) increased phosphorylation of ERK1/2; (2) increased activation of NF-kB and phosphorylation of ELK-1; and (3) increased gene expression for the NK-1 receptor and NR1 and NR-2A subunits of the NMDA receptor. Blockade of the ERK cascade with the MEK inhibitor PD98059 inhibited phosphorylation of ELK-1, activation of NF-kB and gene expression of NR1, NR-2A and NK-1R, and prevented the development of excessive grooming behavior. The results have shown that excitotoxic spinal injury leads to the injury-induced activation of the ERK→ELK-1 and NF-kB signaling cascades and transcriptional regulation of receptors important in the development of chronic pain. Blockade of this intracellular kinase cascade prevented the onset of injury-induced pain behavior.

Cognitive effects of neurotoxic lesions of the nucleus basalis magnocellularis in rats: differential roles for corticopetal versus amygdalopetal projections.

The cholinergic hypothesis states that cholinergic neurons of the basal forebrain nucleus basalis magnocellularis (nbm) that project to cortical and amygdalar targets play an important role in memory. Biochemical studies have shown that these target areas are differentially sensitive to different excitotoxins (e.g., ibotenate vs. quisqualate). This observation might explain the finding from many behavioural studies of memory that different excitotoxins affect memory differentially even though they produce about the same level of depletion of cholinergic markers in the cortex and similar cortical electrophysiological effects. Thus, the magnitude of mnemonic impairment might be related to the extent of damage to cholinergic projections to the amygdala more than to the extent of damage to corticopetal cholinergic projections. This explanation might similarly apply to the observation that the immunotoxin 192 IgG-saporin produces mild effects on memory when injected into the nbm. This is because it damages cholinergic neurons projecting to the cortex but not those projecting to the amygdala. Studies comparing the effects on memory of ibotenic acid vs. quisqualic acid lesions of the nbm are reviewed as are studies of the mnemonic effects of 192 IgG-saporin. Results support the cholinergic hypothesis and suggest that amygdalopetal cholinergic neurons of the nbm play an important role in the control of memory.

The effects of nucleus basalis magnocellularis lesions in Long-Evans hooded rats on two learning set formation tasks, delayed matching-to-sample learning, and open-field activity.

Rats with quisqualic acid lesions of the nucleus basalis magnocellularis (nBM) and control rats were compared in discrimination reversal learning set (DRLS) and olfactory discrimination learning set (ODLS) tasks, a delayed matching-to-sample task (DMTS), and open-field activity. Evidence of learning set formation was seen in control rats but not in nBM-lesioned rats in both the DRLS and ODLS tasks. Better-than-chance performances were seen for both groups in DMTS, indicating no impairment after nBM lesions. There were no group differences in open-field activity. These findings suggest that the nBM is important for higher cognitive processing such as "learning to learn" and thus is important for a complex form of reference memory. In addition, perseverational, working memory, and attentional deficits could not explain learning set impairment after nBM lesions.

Characterization with [ 3H]quisqualate of group I metabotropic glutamate receptor subtype in rat central and peripheral excitable tissues

Radioligand binding studies were performed to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes using [ 3H]quisqualic acid (QA) synthesized in our laboratory as a radioligand. In the presence of ionotropic glutamate receptor (iGluR) agonists, including N-methyl- d-aspartic (NMDA), DL-α-amino-3-hydroxy-5-methylisoxasole-4-propionic (AMPA) and kainic acids (KA), at concentrations maximally effective in displacing each receptor binding, the agonists for group I mGluR subtype (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( trans-ACPD) and ( S)-3,5-dihydroxyphenylglycine (( S)-3,5-DHPG) more potently displaced [ 3H]QA binding in a concentration-dependent manner than their absence. The addition of these three iGluR agonists did not significantly affect potencies of (2 S,2′ R,3′ R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) and l-(+)-2-amino-4-phosphonobutyric acid (L-AP4) to displace [ 3H]QA binding. Scatchard analysis revealed that [ 3H]QA binding consisted of a single component with a maximal number of binding sites ( B max) of 431.6 fmol/mg protein and a dissociation constant ( K d) of 50.9 nM, in the presence of the three iGluR agonists. [ 3H]QA binding was markedly inhibited by GTP and its analogues; but not by GDP, GMP and ATP, under these conditions. Inhibition by GTP was seen in all central structures examined, but [ 3H]QA binding was not detectable in peripheral tissues, such as pituitary and adrenal glands. Neither reverses transcription polymerase chain reaction nor immunoblotting analysis demonstrated the expression of mGluR1 and mGluR5 subunits in the aforementioned two peripheral tissues. These results suggest that [ 3H]QA indeed labels group I mGluR subtype functionally coupled to GTP binding protein in rat brain synaptic membranes under the experimental conditions employed. Group I mGluR subtype seems to be selectively distributed in central structures but not in pituitary and adrenal glands.

Chlormethiazole inhibits epileptiform activity by potentiating GABA A receptor function

Chlormethiazole has sedative, hypnotic, anticonvulsant and neuroprotective properties. Using in vitro grease-gap recordings, we show that it inhibits epileptiform activity in neocortical slices superfused with Mg 2+-free medium (IC 50∼200 μM). At an antiepileptic concentration (300 μM), chlormethiazole potentiated the action of exogenously applied GABA (1 mM) but did not affect responses to the glutamate receptor agonists N-methyl- d-aspartate (10 μM) or l-quisqualic acid (3 μM). The GABA A receptor antagonist N-methyl-bicuculline (50 μM) reduced chlormethiazole’s potency to inhibit the epileptiform activity. These results indicate that chlormethiazole’s anticonvulsant action is likely mediated by potentiating GABA Aergic inhibition rather than by antagonising glutamatergic excitation.

Direct radiolabeling by [ 3H]quisqualic acid of group I metabotropic glutamate receptor in rat brain synaptic membranes

[ 3H]Quisqualic acid (QA) was synthesized and used to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes in the presence of three different ionotropic glutamate receptor agonists at respective saturating concentrations. Of several mGluR agonists tested, group I agonists were more potent in displacing [ 3H]QA binding than group II and group III agonists in the presence of the three ionotropic agonists. [ 3H]QA binding was markedly inhibited by guanine nucleotide analogues in a concentration-dependent manner at a concentration range of 10 nM to 1 mM. Scatchard analysis revealed that [ 3H]QA binding consisted of a single component with a K d of 50.9±5.3 nM and a B max of 431.6±18.3 fmol/mg protein. These results suggest that [ 3H]QA indeed labels group I mGluR functionally coupled to GTP binding protein in rat brain synaptic membranes when determined under the experimental conditions employed.

Atonia-related regions in the rodent pons and medulla.

Electrical stimulation of circumscribed areas of the pontine and medullary reticular formation inhibits muscle tone in cats. In this report, we present an analysis of the anatomical distribution of atonia-inducing stimulation sites in the brain stem of the rat. Muscle atonia could be elicited by electrical stimulation of the nuclei reticularis pontis oralis and caudalis in the pons as well as the nuclei gigantocellularis, gigantocellularis alpha, gigantocellularis ventralis, and paragigantocellularis dorsalis in the medulla of decerebrate rats. This inhibitory effect on muscle tone was a function of the intensity and frequency of the electrical stimulation. Average latencies of muscle-tone suppressions elicited by electrical stimulation of the pontine reticular formation were 11.02 +/- 2.54 and 20.49 +/- 3.39 (SD) ms in the neck and in the hindlimb muscles, respectively. Following medullary stimulation, these latencies were 11.29 +/- 2.44 ms in the neck and 18.87 +/- 2. 64 ms in the hindlimb muscles. Microinjection of N-methyl-D-aspartate (NMDA, 7 mM/0.1 microliter) agonists into the pontine and medullary inhibitory sites produced muscle-tone facilitation, whereas quisqualate (10 mM/0.1 microliter) injection induced an inhibition of muscle tone. NMDA-induced muscle tone change had a latency of 31.8 +/- 35.3 s from the pons and 10.5 +/- 0. 7 s from the medulla and a duration of 146.7 +/- 95.2 s from the pons and 55.5 +/- 40.4 s from the medulla. The latency of quisqualate (QU)-induced reduction of neck muscle tone was 30.1 +/- 37.9 s after pontine and 39.5 +/- 21.8 s after medullary injection. The duration of muscle-tone suppression induced by QU injection into the pons and medulla was 111.5 +/- 119.2 and 169.2 +/- 145.3 s. Smaller rats (8 wk old) had a higher percentage of sites producing muscle-tone inhibition than larger rats (16 wk old), indicating an age-related change in the function of brain stem inhibitory systems. The anatomical distribution of atonia-related sites in the rat has both similarities and differences with the distribution found in the cat, which can be explained by the distinct anatomical organization of the brain stem in these two species.

Basal forebrain nitric oxide synthase (NOS)-containing neurons project to microvessels and NOS neurons in the rat neocortex: cellular basis for cortical blood flow regulation.

Stimulation of basal forebrain neurons results in local increases in cortical cerebral blood flow that are dependent upon cholinergic and nitrergic mechanisms. In the present study, we investigated the possibility that basal forebrain nitric oxide synthase (NOS)-containing neurons project to microvessels and NOS interneurons in the rat cerebral cortex. We performed quisqualic (QUIS) acid lesions of the basal forebrain and evaluated their effects on cortical NOS immunostained nerve terminals, with emphasis on those associated with microvessels and NOS interneurons, both at the light and/or electron microscopic levels. The results show that basal forebrain NOS neurons provide about one third of the overall cortical NOS innervation. Further, the data indicate that basalocortical NOS fibres establish privileged associations with microvessels and NOS neurons, as respective denervations of 60 and 45% were observed following lesion. At the electron microscopic level, most perivascular NOS neuronal elements corresponded to nerve terminals and a majority ( approximately 25%) of these were located in the immediate vicinity of the blood vessels, similar to the perivascular distribution reported previously for classic neurotransmitters/neuromediators. NOS terminals abutting on cortical NOS neurons were primarily nonjunctional. Altogether, these results raise the possibility that not only cholinergic but also nitrergic basal forebrain neurons are involved in the flow response observed following stimulation of the basal forebrain. Further, they suggest interactions between basalocortical and intracortical NOS neurons. We conclude that these interactions are involved in the spatial and temporal regulation of cortical perfusion following basal forebrain activation, and that they may become dysfunctional in pathologies such as Alzheimer's disease which affects both the basal forebrain and the cortical NOS neurons.

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 μl, 125 mM). After one, three, five, seven or 10 days, 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.

Metabotropic glutamate autoreceptors of the mGlu 5 subtype positively modulate neuronal glutamate release in the rat forebrain in vitro

In the present study we have examined the role of presynaptic group I metabotropic glutamate (mGlu) receptors in the control of neuronal glutamate release using rat forebrain slices pre-loaded with [ 3H]D-aspartate. We have also addressed the question of which group I mGlu receptor subtype, mGlu 1 or mGlu 5, mediates the facilitatory response observed by the use of a range of established and some more novel agonists and antagonists showing selectivity for these receptors. The electrically-stimulated release of pre-loaded [ 3H]D-aspartate from rat forebrain slices was markedly potentiated by the potent group I mGlu receptor agonist, L-quisqualic acid (L-QUIS), in a concentration-dependent manner (EC 50 17.31 μM). This response was inhibited by the mGlu receptor antagonists (S)-MCPG (100 μM) and (RS)-MTPG (100 μM) but not by the AMPA-type ionotropic glutamate receptor antagonist, NBQX (100 μM). The selective group I mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine ((S)-DHPG) also enhanced electrically-stimulated efflux of label, although responses diminished with high (10–100 μM) concentrations of the agonist. Maximum responses were fully restored when (S)-DHPG (10 μM) was applied in the presence of the proposed mGlu 5 receptor desensitization inhibitor, cyclothiazide (10 μM). The positive modulatory response to (S)-DHPG (1 μM) was powerfully inhibited by (S)-MCPG (IC 50 0.08 μM) but was resistant to the mGlu 1 receptor antagonists, (RS)-AIDA (1–500 μM), CPCCOEt (0.1–100 μM) and (+)-2-methyl-4-carboxyphenylglycine (LY367385) (0.1–10 μM). The recently developed, selective mGlu 5 receptor agonist (RS)-2-chloro-5-hydroxyphenylglycine ((RS)-CHPG) enhanced electrically-stimulated [ 3H]D-aspartate efflux from rat forebrain slices with a similar concentration-response profile to that of (S)-DHPG. Responses to this receptor subtype-selective agonist were also blocked by (S)-MCPG (IC 50 1.13 μM) but were unaffected by (RS)-AIDA (500 μM), CPCCOEt (100 μM) or LY367385 (10 μM). These results indicate that the positive modulation of neuronal glutamate release seen in the rat forebrain in the presence of group I mGlu receptor agonists is mediated by presynaptically located mGlu 5 glutamate autoreceptors. The pharmacological profile of these receptors appears to be distinct from that of postsynaptic mGlu receptors. Novel antagonists acting at these presynaptic receptors may provide new drugs for the experimental therapy of a range of acute or chronic neurodegenerative disorders.

The role of Arg 78 in the metabotropic glutamate receptor mGlu 1 for agonist binding and selectivity

The metabotropic glutamate receptors belong to family C of the G-protein coupled receptor superfamily. These receptors all possess large extracellular amino terminal domains, where agonist binding takes place. We have previously constructed a molecular model of the amino terminal domain of the mGlu 1 receptor based on a weak amino acid sequence similarity with a family of bacterial periplasmic binding proteins (PBPs). The residues Ser 165 and Thr 188 were demonstrated to be involved in agonist binding to the receptor. Here, we report that mutation of Arg 78 in the mGlu 1b receptor to leucine or glutamate completely knocks out [ 3H]quisqualic acid binding to the receptor. The constructed mutants, R78L and R78E, have also been characterized in a inositol phosphate assay. Here, the potency of ( S)-glutamic acid and ( S)-quisqualic acid was reduced 1000- and 100-fold, respectively, on R78L compared to the wild type (WT) receptor. ( S)-Quisqualic acid was as potent on mutant R78E as it was on R78L, whereas ( S)-glutamic acid was unable to activate R78E significantly at concentrations up to 10 mM. In conclusion, Arg 78 appears to be essential for agonist binding to the mGlu 1 receptor, most likely, through the formation of an ionic bond between its positively charged side chain and the distal acid group of the agonists. Furthermore, the different impact of the two mutations on ( S)-glutamic acid and ( S)-quisqualic acid potencies strongly indicates that while Arg 78 appears to be a common site of interaction for the agonists, the Group I subtype selectivity of ( S)-quisqualic acid is probably determined by other residues in the amino terminal domain.

Brain inflammatory reaction in an animal model of neuronal degeneration and its modulation by an anti-inflammatory drug: implication in Alzheimer's disease.

Brain inflammatory processes underlie the pathogenesis of Alzheimer's disease, and nonsteroidal anti-inflammatory drugs have a protective effect in the disease. The aim of this study was to characterize in vivo in the rat brain the inflammatory reaction in response to excitotoxic insult and to investigate the efficacy of nimesulide treatment. Quisqualic acid was injected into the right nucleus basalis of rats. The excitotoxin induced cholinergic degeneration, an intense glial reaction and the production of inflammatory mediators. Three hours after injection, a five-fold elevation in the concentration of interleukin-1beta in the injected area was observed. This elevation was reduced by 50% by nimesulide (10 mg/kg, i.m.) pretreatment. Electron microscope examination and immunocytochemical staining revealed an intense activation of microglia and astrocytes at both 24 h and 7 days after injection. Cyclooxygenase-2-immunoreactivity was induced in the blood vessels of the injected hemisphere in perivascular microglial and endothelial cells 24 h after injection. Seven days postinjection, a cyclooxygenase-2-positive signal was induced in the parenchymal microglia and large amounts of prostaglandin-E2 were measured in the injected area. Twenty-four hours and 7 days after injection, many inducible nitric oxide synthase-positive cells and a high level of nitrite were detected at the injection site. Seven days of nimesulide (10 mg/kg/day, i.m.) treatment strongly attenuated the microglial reaction, reduced the number of inducible nitric oxide synthase-positive cells and completely abolished the increase in prostaglandin-E2 formation. These data provide valuable support in vivo for the potential efficacy of cyclooxygenase-2 inhibitors in Alzheimer's disease therapy.

Contribution of glutamate receptors to spontaneous and stimulus-evoked discharge in afferent fibers innervating hair cells of the Xenopus lateral line organ

The relative contributions of NMDA ( N-methyl- D-aspartate) and non-NMDA glutamate receptors to spontaneous and stimulus-evoked transmission at the hair cell/afferent fiber synapse were determined in the Xenopus laevis lateral line organ. The non-NMDA receptor antagonist, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), reversibly reduced both spontaneous and stimulus-evoked discharge rate with an EC 50 of 0.5 μM. NMDA receptor antagonism with the combination of chlorokynurenic acid (100 μM) and elevated magnesium (1.1 mM), or elevated magnesium alone, blocked responses to NMDA without significantly altering spontaneous or stimulus-evoked discharge rate or the responses to kainate. All non-NMDA receptor agonists tested increased discharge rate at low concentrations and, at higher concentrations, increased, then suppressed discharge rate. The EC 50s were: domoic acid (2.4 μM)<quisqualic acid (6 μM)<kainic acid (18 μM)<AMPA (82 μM)⪡glutamate (1150 μM). NMDA and ibotenic acid also produced an increase in discharge followed by a suppression, but the suppressive phase of the response predominated and maximum increases in discharge rates were low compared to effects of the non-NMDA agonists. The EC 50s were: NMDA (148 μM)<ibotenic acid (463 μM). The EC 50 for the suppression of afferent discharge that followed the initial excitatory effect was similar to the EC 50 for excitation. Perfusion with active concentrations of kainate, AMPA, or NMDA did not alter the threshold for electrical stimulation of these nerve fibers. We conclude that most of the postsynaptic signal normally seen in afferent fibers is mediated by non-NMDA receptors.

GABA neurons provide a rich input to microvessels but not nitric oxide neurons in the rat cerebral cortex: A means for direct regulation of local cerebral blood flow

Basal forebrain neurons project to microvessels and the somata of nitric oxide (NO) synthase-containing neurons in the cerebral cortex, and their stimulation results in increases in cortical perfusion. gamma-Aminobutyric acid (GABA) is the second major neurotransmitter synthesized by these neurons and it has also been reported to modify cerebromicrovascular tone. We thus investigated by light and electron microscopy the association of GABA neurons (labeled for glutamic acid decarboxylase [GAD]) with cortical microvessels and/or NO neurons (identified by nicotinamide adenine dinucleotide [NADPH-D] histochemistry) within the frontoparietal and perirhinal cerebral cortex in the rat. On thick and semithin sections, a high density of GAD puncta was observed, several surrounded intracortical blood vessels and neuronal perikarya. In contrast, NADPH-D cell somata and proximal dendrites were only occasionally contacted by GAD nerve terminals. Perivascular and perisomatic GAD appositions were identified at the ultrastructural level as large (0.44-0.50 microm(2)) neuronal varicosities located in the immediate vicinity of, or being directly apposed to, vessels or unstained neuronal cell bodies. In both cortical areas, perivascular GAD terminals were located at about 1 microm from the vessels and were seen to frequently establish junctional contacts (synaptic frequency of 25-40% in single thin sections) with adjacent neuronal but not vascular elements. Ibotenic or quisqualic acid lesion of the substantia innominata did not significantly affect the density of cortical and perivascular GAD terminals, suggesting that they mostly originated locally in the cortex. These results suggest that GABA terminals can interact directly with the microvascular bed and that the somata and proximal dendrites of NO neurons are not a major target for cortical GABA neurotransmission. However, based on the colocalization of GABA and NADPH-D in a subset of cortical neurons, we suggest that these interneurons could be implicated in the cortical vascular response elicited by stimulation of basal forebrain neurons.

Long-term evolution of local, proximal and remote astrocyte responses after diverse nucleus basalis lesioning (an experimental Alzheimer model): GFAP immunocytochemical study

A study on long-term astrocytic responses (from 1 day to 20 months after lesioning in 4-month-old rats, and from 1 day to 6 months in 20-month-old rats) to diverse unilateral damage of the nucleus basalis (nbM) by injection of 40 nmol of ibotenic acid, or 50 or 100 nmols of quisqualic acid was performed using a histochemical method (immunoreactivity against the glial fibrillary acidic protein GFAP). Glial reactivity (i.e., isolated or clustered hypertrophic and/or hyper-reactive astrocytes) was evaluated in several ipsilateral and contralateral brain regions: the ‘local response’ within the damaged nbM region; the ‘proximal response’ (a new concept proposed by us) in the non-damaged structures neighbouring the nbM; and the ‘remote response’ in the ipsilateral brain cortex and in the contralateral cortex and nbM. In 4-month-old animals, the remote cortical glial responses, independent of the involution of cortical cholinergic activity and randomly located in layers I–V of motor and somatosensory cortical regions, were similar in appearance over a long period (13–20 months), with the highest reactivity 45 days after lesioning. The proximal response lasted from 1 day to 13 months and afterwards tended to disappear. Contralateral reactivity and ipsilateral cortical scars were observed. The local (nbM) glial response was maintained throughout the period studied. Subsets of astrocytes of different reactivities were observed, most of their elements being highly intermeshed. In 20-month-old animals, nbM lesions produced less positive, but similar, glial reactive patterns. This glial reactivity was superposed onto the glial reactivity of old age. All these results are discussed. The maintenance of reactive astrocytes many months after lesioning suggests the existence of cellular factors other than those produced by damaged nbM neurons. Taking into account the role of glial cells under pathological conditions, it is possible that these reactive astrocytes in humans could promote neurodegenerative processes, such as amyloid plaque formation and neurodegeneration (Alzheimer’s disease). Along this line, nbM cholinergic involution could then originate cortical involution through induced reactive astrocytosis.