NAAG peptidase inhibitor increases dialysate NAAG and reduces glutamate, aspartate and GABA levels in the dorsal hippocampus following fluid percussion injury in the rat

Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI.     

Regional Heterogeneity of Nicotine Effects on Neurotransmitters in Rat Brains <Emphasis Type="Italic">in vivo</Emphasis> at Low Doses

In our recent studies on nicotine-induced changes in neurotransmitters in brain areas associated with cognitive function using a nicotine dose of 0.5 mg/kg administered subcutaneously to conscious freely moving rats, we found changes in dopamine, norepinephrine, and serotonin, and their metabolites, in the areas examined. For the present report we examined changes in these neurotransmitters following administration of lower nicotine doses, to test regional differences in nicotine response and possible threshold levels for some effects of nicotine. The doses used were 0.15 mg/kg and 0.03 mg/kg nicotine administered subcutaneously. Nicotine levels in the brain reached peak values in less than 10 min and decreased with a half-life of about 60 min (0.15 mg/kg) or 30 min (0.03 mg/kg) to values below detection limits (1 ng/g), by the later time points of the 0.03 mg/kg experiments. Nicotine-induced dopamine (DA) increase (and increase in DA metabolites) and decrease in 5-HT levels at 0.15 mg/kg were significant in the cortex, less so in the hippocampus. Norepinephrine (NE) increase at 0.15 mg/kg was much less significant than found previously at 0.5 mg/kg. At a low nicotine dose (0.03 mg/kg), the significant changes observed were a decrease in 5-HT in the hippocampus and small increases of DA and NE in the prefrontal cortex and of NE in the medial temporal cortex. In the nucleus accumbens DA, NE, and 5-HT and their metabolites in the ventral tegmental area, mostly DA and metabolites were increased. We conclude that in areas of cognitive function nicotine-induced DA changes are more concentration dependent than changes in NE or 5-HT, and that there are regional differences in neurotransmitter changes induced by nicotine, with NE changes detectable only in the cortex and 5-HT changes only in the hippocampus at a low nicotine dose, indicating significant regional variation in sensitivity to nicotine-induced neurotransmitter changes in brain areas associated with cognitive function. The decrease in 5-HT shows that nicotine also has indirect effects caused by neurotransmitters released by nicotine. The effects of low nicotine dose are more significant in areas of reward function, indicating differences in sensitivity between cognitive and reward functions.     

In vivo modulation of extracellular hippocampal glutamate and GABA levels and limbic seizures by group I and II metabotropic glutamate receptor ligands

The effects of several metabotropic receptor (mGluR) ligands on baseline hippocampal glutamate and GABA overflow in conscious rats and the modulation of limbic seizure activity by these ligands were investigated. Intrahippocampal mGluR group I agonist perfusion via a microdialysis probe [1 mm (R,S)-3,5-dihydroxyphenylglycine] induced seizures and concomitant augmentations in amino acid dialysate levels. The mGlu1a receptor antagonist LY367385 (1 mm) decreased baseline glutamate but not GABA concentrations, suggesting that mGlu1a receptors, which regulate hippocampal glutamate levels, are tonically activated by endogenous glutamate. This decrease in glutamate may contribute to the reported LY367385-mediated anticonvulsant effect. The mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (50 mg/kg) also clearly abolished pilocarpine-induced seizures. Agonist-mediated actions at mGlu2/3 receptors by LY379268 (100 microm, 10 mg/kg intraperitoneally) decreased basal hippocampal GABA but not glutamate levels. This may partly explain the increased excitation following systemic LY379268 administration and the lack of complete anticonvulsant protection within our epilepsy model with the mGlu2/3 receptor agonist. Group II selective mGluR receptor blockade with LY341495 (1-10 microm) did not alter the rats' behaviour or hippocampal amino acid levels. These data provide a neurochemical basis for the full anticonvulsant effects of mGlu1a and mGlu5 antagonists and the partial effects observed with mGlu2/3 agonists in vivo.     

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.     

Locally Administered Low Nicotine-Induced Neurotransmitter Changes in Areas of Cognitive Function

The present study examined the effect of a low-dose of nicotine; below that one expects to be achieved from a single cigarette, on brain regional heterogeneity and sensitivity of catecholaminergic responses. 1 μM nicotine was infused into six brain areas via a microdialysis probe: the dorsal and ventral hippocampus, the medial temporal and prefrontal cortex, the basolateral amygdala, and the ventral tegmental area (VTA). The nicotine concentration in the brain tissue near the probe site was approximately 0.1 μM. Nicotine-induced increases and decreases could be noted in dopamine (DA), norepinephrine (NE), and serotonin (5HT) levels. In particular, DA and 5HT decreased in both hippocampal areas, while NE increased in the dorsal and decreased in the ventral hippocampus. In the cortical areas, DA and NE increased and 5HT was not significantly altered. In the amygdala all three neurotransmitters increased and in the VTA, all three decreased. Many of the nicotine-induced changes in neurotransmitter concentrations were reversed in the presence of atropine. Where nicotine induced decreases in DA and 5HT in the VTA, increases were observed in the presence of atropine. A similar reversal was seen with NE in the VTA and ventral hippocampus. In contrast, the increases in DA observed in the cortex and amygdala and the increases in NE observed in the cortex, amygdala and dorsal hippocampus were inhibited by the presence of atropine. 5HT was also significantly decreased in the amygdala and both cortical areas in the presence of atropine, where nicotine alone had no significant effect. We conclude, that at low doses, nicotine significantly alters the release of DA, NE, and 5HT – in some areas increasing, in others decreasing endogenous neurotransmitter levels. This data, in conjunction with previous experiments, indicates that the effects of nicotine are regionally heterogeneous and arise from both direct and indirect actions on various receptors and neurotransmitter systems and nicotine’s effects at low doses differ from that at higher doses. The changes in effects in the presence of atropine suggest that muscarinic acetylcholine receptors play a major role in nicotine’s actions on neurotransmitter systems.     

Plasticity of glutamatergic control of striatal acetylcholine release in experimental parkinsonism: opposite changes at group-II metabotropic and NMDA receptors

To investigate whether adaptive changes of glutamatergic transmission underlie dysfunction of the cholinergic system in experimental parkinsonism, the effects of group-II metabotropic glutamate and NMDA receptor ligands on acetylcholine release was studied in striatal slices and synaptosomes obtained from naive rats, 6-hydroxydopamine hemi-lesioned rats and 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa (L-DOPA) plus benserazide (non-dyskinetic). Group-II metabotropic glutamate receptor agonists LY354740, DCG-IV and L-CCG-I inhibited the electrically-evoked endogenous acetylcholine release from slices, while NMDA facilitated it. LY354740 also inhibited K+-evoked acetylcholine release from synaptosomes. LY354740-induced inhibition was prevented by the group-II metabotropic glutamate receptor antagonist LY341495. In hemi-parkinsonian rats, sensitivity towards LY354740 was reduced while that to NMDA was enhanced in the lesioned (denervated) compared with unlesioned striatum. Moreover, dizocilpine inhibited acetylcholine release in the lesioned compared with unlesioned striatum. Chronic treatment with L-DOPA normalized sensitivity towards glutamatergic agonists. We conclude that striatal dopamine denervation results in plastic changes at group-II metabotropic glutamate and NMDA receptors that may shift glutamatergic control of acetylcholine release towards facilitation. From a clinical perspective, L-DOPA and NMDA antagonists appear effective in counteracting overactivity of striatal cholinergic interneurones associated with Parkinson's disease.     

Synthesis and structure-activity relationship studies of novel 2-diarylethyl substituted (2-carboxycycloprop-1-yl)glycines as high-affinity group II metabotropic glutamate receptor ligands

The major excitatory neurotransmitter in the central nervous system, (S)-glutamic acid , activates both ionotropic and metabotropic excitatory amino acid receptors. Its importance in connection to neurological and psychiatric disorders has directed great attention to the development of compounds that modulate the effects of this endogenous ligand. Whereas L-carboxycyclopropylglycine (L-CCG-1) is a potent agonist at, primarily, group II metabotropic glutamate receptors, alkylation of at the alpha-carbon notoriously result in group II mGluR antagonists, of which the most potent compound described so far, LY341495, displays IC(50) values of 23 and 10 nM at the group II receptor subtypes mGlu2 and mGlu3, respectively. In this study we synthesized a series of structural analogues of in which the xanthyl moiety is replaced by two substituted-phenyl groups. The pharmacological characterization shows that these novel compounds have very high affinity for group II mGluRs when tested as their racemates. The most potent analogues demonstrate K(i) values in the range of 5-12 nM, being thus comparable to LY341495.     

N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABAAα6 Subunit in Cerebellar Granule Cells

Abstract: We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 µM) results in the transient increase in content of GABA_Aα6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABA_B receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in α6 levels induced by 30 µM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional α6-containing GABA_A receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA_Aα6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA_A receptor subunit composition.     

Receptors in the Ventral Tegmental Area Mediating Nicotine-Induced Dopamine Release in the Nucleus Accumbens

Nicotine or cocaine, when administered intravenously, induces an increase of extracellular dopamine in the nucleus accumbens. The nicotine-mediated increase was shown to occur at least in part through increase of the activity of dopamine neurons in the ventral tegmental area. As part of our continuing studies of the mechanisms of nicotine effects in the brain, in particular, effects on reward and cognitive mechanisms, in the present study we examined the role of various receptors in the ventral tegmental area in nicotine and cocaine reward. We assayed inhibition of the increase of dopamine in the nucleus accumbens induced by intravenous nicotine or cocaine administration by antagonists administered into the ventral tegmental area. Nicotine-induced increase of accumbal dopamine release was inhibited by intrategmental nicotinic (mecamylamine), muscarinic (atropine), dopaminergic (D₁: SCH 23390, D₂: eticlopride), and NMDA glutamatergic (MK 801) and GABA_B (saclofen) antagonists, but not by AMPA-kainate (CNQX, GYKI-52466) antagonists under our experimental circumstances. The intravenous cocaine-induced increase of dopamine in the nucleus accumbens was inhibited by muscarinic (atropine), dopamine 2 (eticlopride), and GABA_B (saclofen) antagonists but not by antagonists to nicotinic (mecamylamine), dopamine D₁ (SCH 23390), glutamate (MK 801), or AMPA-kainate (CNQX, GYKI-52466) receptors. Antagonists administered in the ventral tegmental area in the present study had somewhat different effects when they were previously administered intravenously. When administered intravenously atropine did not inhibit cocaine effects. The inhibition by atropine may be indirect, since this compound, when administered intrategmentally, decreased basal dopamine levels in the accumbens. The findings indicate that a number of receptors in the ventral tegmental area mediate nicotine-induced dopamine changes in the nucleus accumbens, a major component of the nicotine reward mechanism. Some, but not all, of these receptors in the ventral tegmental area also seem to participate in the reward mechanism of cocaine. The importance of local receptors in the ventral tegmental area was further indicated by the increase in accumbal dopamine levels after intrategmental administration of nicotine or also cocaine.     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996     

GABA<Subscript>B</Subscript> Receptors in Neuroendocrine Regulation

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA_A and GABA_C (ionotropic receptors) and GABA_B (metabotropic receptor). Here we reviewed the participation of GABA_B receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA_B1 knock-out mice, that lack functional GABA_B receptors. Our general conclusion indicates that GABA_B receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA_B receptor activation, as demonstrated in the rat and also in the GABA_B1 knock-out mouse. In addition, hypothalamic and pituitary GABA_B receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA_B receptors depends on physiological conditions, being age and sex critical factors. These results indicate that patients receiving GABA_B agonists/antagonists should be monitored for possible endocrine side effects.     

Nicotine-Induced Changes in Neurotransmitter Levels in Brain Areas Associated with Cognitive Function

Nicotine, one of the most widespread drugs of abuse, has long been shown to impact areas of the brain involved in addiction and reward. Recent research, however, has begun to explore the positive effects that nicotine may have on learning and memory. The mechanisms by which nicotine interacts with areas of cognitive function are relatively unknown. Therefore, this paper is part of an ongoing study to evaluate regional effects of nicotine enhancement of cognitive function. Nicotine-induced changes in the levels of three neurotransmitters, dopamine (DA), serotonin (5-HT), norepinepherine (NE), their metabolites, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), and their precursor, L-DOPA, were evaluated in the ventral and dorsal hippocampus (VH and DH), prefrontal and medial temporal cortex (PFC and MTC), and the ventral tegmental area (VTA) using in vivo microdialysis in awake, freely moving, male Sprague-Dawley rats. The animals were treated with acute nicotine (0.5 mg/kg, s.c.) halfway through the 300-min experimental period. The reuptake blockers, desipramine (100 microM) and fluoxetine (30 microM), were given to increase the levels of NE and 5-HT so that they could be detected. Overall, a nicotine-induced DA increase was found in some areas, and this increase was potentiated by desipramine and fluoxetine. The two DA metabolites, HVA and DOPAC, increased in all the areas throughout the experiments, both with and without the inhibitors, indicating a rapid metabolism of the released DA. The increase in these metabolites was greater than the increase in DA. 5-HT was increased in the DH, MTC, and VTA in the presence of fluoxetine; its metabolite, 5-HIAA, was increased in the presence and absence of fluoxetine. Except in the VTA, NE levels increased to a similar extent with desipramine and fluoxetine. Overall, nicotine appeared to increase the release and turnover of these three neurotransmitters, which was indicated by significant increases in their metabolites. Furthermore, DA, and especially HVA and DOPAC, increased for the 150 min following nicotine administration; 5-HT and NE changes were shorter in duration. As gas chromatography experiments showed that nicotine levels in the brain decreased by 75% after 150 min, this may indicate that DA is more susceptible to lower levels of nicotine than 5-HT or NE. In conclusion, acute nicotine administration caused alterations in the levels of DA, 5-HT, and NE, and in the metabolism of DA and 5-HT, in brain areas that are involved in cognitive processes.     

NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR

Phencyclidine (PCP) administration elicits positive and negative symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochemical effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. We synthesized and characterized a urea-based NAAG analogue, ZJ43. This novel compound is a potent inhibitor of enzymes, glutamate carboxypeptidase II (K(i) = 0.8 nM) and III (K(i) = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 microM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Additionally, LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.     

Regulation of Akt and Wnt signaling by the group II metabotropic glutamate receptor antagonist LY341495 and agonist LY379268

Metabotropic glutamate receptors 2/3 (mGlu(2/3)) have been implicated in schizophrenia and as a novel treatment target for schizophrenia. The current study examined whether mGlu(2/3) regulates Akt (protein kinase B) and Wnt (Wingless/Int-1) signaling, two cascades associated with schizophrenia and modified by antipsychotics. Western blotting revealed increases in phosphorylated Akt (pAkt) and phosphorylated glycogen synthase kinase-3 (pGSK-3) following acute and repeated treatment of LY379268 (mGlu(2/3) agonist), whereas increases in dishevelled-2 (Dvl-2), dishevelled-3 (Dvl-3), GSK-3 and β-catenin were only observed following repeated treatment. LY341495 (mGlu(2/3) antagonist) induced the opposite response compared with LY379268. Co-immunoprecipitation experiments showed an association between the mGlu(2/3) complex and Dvl-2 providing a possible mechanism to explain how the mGlu(2/3) can mediate changes in Wnt signaling. However, there was no association between the mGlu(2/3) complex and Akt suggesting that changes in Akt signaling following LY341495 and LY379268 treatments may not be directly mediated by the mGlu(2/3) . Finally, an increase in locomotor activity induced by LY341495 treatment correlated with increased pAkt and pGSK-3 levels and was attenuated by the administration of the GSK-3 inhibitor, SB216763. Overall, the results suggest that mGlu(2/3) regulates Akt and Wnt signaling and LY379268 treatment has overlapping effects with D(2) dopamine receptor antagonists (antipsychotic drugs).     

Differential negative coupling of type 3 metabotropic glutamate receptor to cyclic GMP levels in neurons and astrocytes

Metabotropic receptors may couple to different G proteins in different cells or perhaps even in different regions of the same cell. To date, direct studies of group II and group III metabotropic glutamate receptors' (mGluRs) relationships to second messenger cascades have reported negative coupling of these receptors to cyclic AMP (cAMP) levels in neurons, astrocytes and transfected cells. In the present study, we found that the peptide neurotransmitter N-acetylaspartylglutamate (NAAG), an mGluR3-selective agonist, decreased sodium nitroprusside (SNP)-stimulated cyclic GMP (cGMP) levels in cerebellar granule cells and cerebellar astrocytes. The mGluR3 and group II agonists FN6 and LY354740 had similar effects on cGMP levels. The mGluR3 and group II antagonists beta-NAAG and LY341495 blocked these actions. Treatment with pertussis toxin inhibited the effects of NAAG on SNP-stimulated cGMP levels in rat cerebellar astrocytes but not in cerebellar neurons. These data support the conclusion that mGluR3 is also coupled to cGMP levels and that this mGluR3-induced reduction of cGMP levels is mediated by different G proteins in cerebellar astrocytes and neurons. We previously reported that this receptor is coupled to a cAMP cascade via a pertussis toxin-sensitive G protein in cerebellar neurons, astrocytes and transfected cells. Taken together with the present data, we propose that mGluR3 is coupled to two different G proteins in granule cell neurons. These data greatly expand knowledge of the range of second messenger cascades induced by mGluR3, and have implications for clinical conditions affected by NAAG and other group II mGluR agonists.     

Inhibition of NAALADase by 2‐PMPA attenuates cocaine‐induced relapse in rats: a NAAG‐mGluR2/3‐mediated mechanism

Pharmacological activation of group II metabotropic glutamate receptors (mGluR2/3) inhibits cocaine self‐administration and reinstatement of drug‐seeking behavior, suggesting a possible use of mGluR2/3 agonists in the treatment of cocaine dependence. In this study, we investigated whether elevation of the endogenous mGluR2/3 ligand N‐acetyl‐aspartatylglutamate (NAAG) levels by the N‐acetylated‐alpha‐linked‐acidic dipeptidase inhibitor 2‐(phosphonomethyl)pentanedioic acid (2‐PMPA) attenuates cocaine self‐administration and cocaine‐induced reinstatement of drug seeking. N‐acetylated‐alpha‐linked‐acidic dipeptidase is a NAAG degradation enzyme that hydrolyzes NAAG to N‐acetylaspartate and glutamate. Systemic administration of 2‐PMPA (10‐100 mg/kg, i.p.) inhibited intravenous self‐administration maintained by low unit doses of cocaine and cocaine (but not sucrose)‐induced reinstatement of drug‐seeking behavior. Microinjections of 2‐PMPA (3–5 μg/side) or NAAG (3–5 μg/side) into the nucleus accumbens (NAc), but not into the dorsal striatum, also inhibited cocaine‐induced reinstatement, an effect that was blocked by intra‐NAc injection of LY341495, a selective mGluR2/3 antagonist. In vivo microdialysis demonstrated that 2‐PMPA (10‐100 mg/kg, i.p.) produced a dose‐dependent reduction in both extracellular dopamine (DA) and glutamate, an effect that was also blocked by LY341495. Finally, pre‐treatment with 2‐PMPA partially attenuated cocaine‐enhanced extracellular NAc DA, while completely blocking cocaine‐enhanced extracellular NAc glutamate in rats during reinstatement testing. Intra‐NAc perfusion of LY341495 blocked 2‐PMPA‐induced reductions in cocaine‐enhanced extracellular NAc glutamate, but not DA. These findings suggest that 2‐PMPA is effective in attenuating cocaine‐induced reinstatement of drug‐seeking behavior, likely by attenuating cocaine‐induced increases in NAc DA and glutamate via pre‐synaptic mGluR2/3s.     

Pharmacological blockade of mGlu2/3 metabotropic glutamate receptors reduces cell proliferation in cultured human glioma cells

Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT-PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group-II mGlu receptors. In two selected cultures (MZC-12 and FCN-9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nm, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)-alpha-ethylglutamate. The anti-proliferative effect of LY341495 was confirmed by measuring [methyl-3H]-thymidine incorporation in cultures arrested in G0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen-activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl-3H]-thymidine incorporation, were partially reduced by co-addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group-II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo.     

Nicotine-Stimulated Release of [3H]Norepinephrine from Fetal Rat Locus Coeruleus Cells in Culture

Abstract: Acute nicotine administration stimulated [³H]norepinephrine ([³H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC₅₀ of 0.9 µ M , and was abolished by removal of calcium from, or addition of tetrodotoxin (500 n M ) to, the assay buffer. Other nicotinic receptor agonists stimulated [³H]NE release, with the rank order of potency being (±)-epibatidine > (−)-nicotine > 1,1-dimethyl-4-phenylpiperazinium (DMPP). Whereas (−)-nicotine and (±)-epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (−)-cytisine had no agonist activity. Nicotine-stimulated release of [³H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro-β-erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an α4β2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA- and nicotine-stimulated [³H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK-801 blockade of nicotine-induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [³H]NE from LC cells and does not act indirectly via activation of glutamate release.     

The potent, selective mGlu2/3 receptor agonist LY379268 increases extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid in the medial prefrontal cortex of the freely moving rat

Previous work has shown that the potent, selective metabotropic glutamate mGlu2/3 receptor agonist LY379268 acts like the atypical antipsychotic clozapine in behavioral assays. To investigate further the potential antipsychotic actions of this agent, we examined the effects of LY379268 using microdialysis in awake, freely moving rats, on extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in rat medial prefrontal cortex. Systemic LY379268 increased extracellular levels of dopamine, DOPAC, HVA, and 5-HIAA in a dose-dependent, somewhat delayed manner. LY379268 (3 mg/kg s.c. ) increased levels of dopamine, DOPAC, HVA, and 5-HIAA to 168, 170, 169, and 151% of basal, respectively. Clozapine (10 mg/kg) also increased dopamine, DOPAC, and HVA levels, with increases of 255, 262, and 173%, respectively, but was without effect on extracellular 5-HIAA levels by 3 mg/kg LY379268 were reversed by the selective mGlu2/3 receptor antagonist LY341495 (1 mg/kg). Furthermore, LY379268 (3 mg/kg)-evoked increases in DOPAC and HVA were partially blocked and the increase in 5-HIAA was completely blocked by local application of 3 microM tetrodotoxin. Therefore, we have demonstrated that mGlu2/3 receptor agonists activate dopaminergic and serotonergic brain pathways previously associated with the action of atypical antipsychotics such as clozapine and other psychiatric agents.     

Microglial neurotransmitter receptors trigger superoxide production in microglia; consequences for microglial-neuronal interactions

Microglia express three isoforms of the NADPH oxidase, Nox1, Nox2 and Nox4, with the potential to produce superoxide (O(2) ˙(-) ). Microglia also express neurotransmitter receptors, which can modulate microglial responses. In this study, microglial activity of Nox1, Nox2 and Nox4 in primary rat cultured microglia or the rodent BV2 cell line were altered by microglial neurotransmitter receptor modulation. Glutamate, GABA or ATP triggered microglial O(2) ˙(-) production via Nox activation. Nox activation was elicited by agonists of metabotropic mGlu3 receptors and by group III receptors, by GABA(A) but not GABA(B) receptors, and by purinergic P2X(7) or P2Y(2/4) receptors but not P2Y(1) receptors, and inhibited by metabotropic glutamate receptor 5 antagonists. The neurotransmitters also modulated Nox mRNA expression and NADPH activity. The activation of Nox by BzATP or GABA promoted a neuroprotective phenotype whilst the activation of Nox by glutamate promoted a neurotoxic phenotype. Taken together, these data indicate that microglial neurotransmitter receptors can signal via Nox to promote neuroprotection or neurotoxicity. This has implications for the subsequent neurotoxic profile of microglia when neurotransmitter levels may become skewed in neurodegeneration.