piperidines and 6-methyl-2-(phenylethynyl)pyridine

Although there are numerous strategies to counteract the death of dopaminergic neurons in Parkinson's disease (PD), there are currently no treatments that delay or prevent the disease course, indicating that early protective treatments are needed. Targeting axonal degeneration, a key initiating event in PD, is required to develop novel therapies; however, its underlying molecular mechanisms are not fully understood. Here, we studied axonal degeneration induced by 6-hydroxydopamine (6-OHDA) in vitro and in vivo. We found that metabotropic glutamate receptor 5 (mGluR5) expression increased during 6-OHDA-induced axonal degeneration in primary neurons and that blockade of mGluR5 by its antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and 3-[(2-methyl-1, 3-thiazol-4-yl) ethynyl]-pyridine (MTEP) almost completely attenuated the degenerative process in vitro. Furthermore, a rapid increase in intra-axonal calcium levels following 6-OHDA treatment was visualized using a calcium-sensitive fluorescence probe and a calcium chelator prevented the axonal degenerative process induced by 6-OHDA in vitro, whereas application of the mGluR5 antagonist MPEP partially attenuated the increase in intra-axonal calcium. The screening of calcium targets revealed that calpain activation and an increase in phosphorylated extracellular signal-regulated kinase (p-ERK) were calcium dependent during 6-OHDA-induced axonal degeneration in vitro. Consistent with these in vitro findings, blockade of mGluR5 with MPEP attenuated the degeneration of dopaminergic axons induced by 6-OHDA injection into the striatum prior to soma death in the early stage of PD in an in vivo animal model. In addition, MPEP inhibited the increase in mGluR5 expression levels, calpain activation and the elevation of p-ERK in the striatum triggered by 6-OHDA injection in vivo. Taken together, these data identify an mGluR5-calcium-dependent cascade that causes axonal degeneration, and suggest that mGluR5 antagonists could provide effective therapy to prevent the disease process of PD.

Topics: Animals; Axons; Calcium Signaling; Cells, Cultured; Excitatory Amino Acid Antagonists; Extracellular Signal-Regulated MAP Kinases; Female; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Piperidines; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Thiazoles

2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike fenobam. This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions.

Topics: Adenosine Triphosphate; Animals; Cell Hypoxia; Disease Models, Animal; Hepatocytes; Imidazoles; Liver; Mice; Mitochondria, Liver; Piperidines; Pyridines; Rats; Receptor, Metabotropic Glutamate 5; Reperfusion Injury; Thiazoles; Tumor Necrosis Factor-alpha

Hippocampal synaptic plasticity and learning are regulated by metabotropic glutamate receptors (mGlu) and particularly by mGlu5. In the hippocampus, synaptic plasticity is tightly linked to neuronal network oscillations in theta (5-10 Hz) and gamma (∼30-100 Hz) frequency ranges, and specific changes in theta and gamma spectral power can predict for the success of patterned afferent stimulation in inducing robust long-term potentiation (LTP). In this study, we hypothesized that activation of mGlu5 mediates tetanisation-driven changes in network oscillations and thereby determines the longevity of LTP. To explore this, we applied high-frequency stimulation (HFS) to the perforant path input to the dentate gyrus (DG), in the presence of the negative allosteric modulator, 2-methyl-6-(phenylethynyl)pyridine (MPEP), or the positive allosteric modulator (S)-(4-fluorophenyl)-[3-(3-(3-(4-fluorophenyl)-[1,2,4]oxadiazol-5-yl)-piperidin-1-yl)]methanone (ADX47273). In freely behaving rats, administration of MPEP resulted in a significant impairment, whereas treatment with ADX47273 led to a significant enhancement, of LTP (>24 h) compared to vehicle-treated controls. Allosteric potentiation of mGlu5 also resulted in a significantly greater increase of the spectral power of theta and gamma oscillations within the period of 300 s after HFS, as compared to MPEP-treated animals or controls. Our findings show that the regulation of hippocampal LTP by mGlu5 is associated with modulation of network oscillatory activity in the period shortly after LTP induction. Taken together, these data demonstrate that changes in the spectral contents of local field activity that occur in response to patterned afferent stimulation require activation of mGlu5 and may be instrumental for the successful expression of persistent LTP. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Topics: Allosteric Regulation; Animals; Excitatory Amino Acid Antagonists; Gamma Rhythm; Hippocampus; Male; Neuronal Plasticity; Oxadiazoles; Piperidines; Pyridines; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Theta Rhythm

Several forms of long-term depression (LTD) of glutamatergic synaptic transmission have been identified in the dorsal striatum and in the nucleus accumbens (NAc). Such experience-dependent synaptic plasticity might play important roles in reward-related learning. The GABAA receptor agonist muscimol was recently found to trigger a long-lasting depression of glutamatergic synaptic transmission in the NAc of adolescent mice, but the mechanisms that underlie this novel form of LTD were not studied. Here we examined the effect of muscimol applied in the perfusion solution on the amplitude of field excitatory postsynaptic potentials/population spikes (fEPSP/PSs) in mouse brain slices. We found that muscimol depressed the fEPSP/PS in the NAc of adolescent mice but not adult mice, through both postsynaptic and presynaptic mechanisms. Indeed, muscimol altered the fEPSP/PS paired-pulse ratio, depolarized the membrane of projection neurons, and decreased the frequency, but not amplitude, of spontaneous excitatory postsynaptic currents in the NAc of adolescent mice. The LTD induced by muscimol likely involved endocannabinoids, metabotropic glutamate receptors (mGluRs), but not TRPV1 receptors. Muscimol-LTD was occluded by prior induction of LTD through low-frequency stimulation (LFS) of the slice, demonstrating a common pathway in the induction of LFS-LTD and muscimol-LTD. We also found that muscimol induced a form of LTD in the dorsolateral striatum of adult but not adolescent mice. This LTD was mediated by endocannabinoids but did not involve mGluRs or TRPV1 receptors. These results identify a novel form of synaptic plasticity, and its mechanisms of induction, which is age and region dependent. These findings may contribute to a better understanding of the increased susceptibility of the adolescent brain to long-term synaptic changes in regions associated with reward mechanisms.

Topics: Animals; Corpus Striatum; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA-A Receptor Agonists; Long-Term Synaptic Depression; Male; Mice, Inbred C57BL; Muscimol; Piperidines; Pyrazoles; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, GABA-A; TRPV Cation Channels

Pre-clinical studies suggest that negative allosteric modulators (NAMs) of the metabotropic glutamate receptor subtype 5 (mGluR5), including 2-methyl-6-(phenylethynyl)pyridine (MPEP), 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and fenobam are highly effective in attenuating drug-taking and drug-seeking behaviors. However, both MPEP and MTEP have no translational potential for use in humans because of their off-target effects and short half-lives. Here, we report that 3-fluoro-5-[(6-methylpyridin-2-yl)ethynyl]benzonitrile (MFZ 10-7), a novel mGluR5 NAM, is more potent and selective than MPEP, MTEP and fenobam in both in vitro binding and functional assays. Similar to MTEP, intraperitoneal administration of MFZ 10-7 inhibited intravenous cocaine self-administration, cocaine-induced reinstatement of drug-seeking behavior and cocaine-associated cue-induced cocaine-seeking behavior in rats. Although MFZ 10-7 and MTEP lowered the rate of oral sucrose self-administration, they did not alter total sucrose intake. Further, MFZ 10-7 appeared to be more potent than MTEP in inducing downward shifts in the cocaine dose-response curve, but less effective than MTEP in attenuating sucrose-induced reinstatement of sucrose-seeking behavior. MFZ 10-7 and MTEP had no effect on basal locomotor behavior. These findings not only provide additional evidence supporting an important role for mGluR5 in cocaine reward and addiction, but also introduce a new tool for both in vitro and in vivo investigations with which to further characterize this role.

Topics: Allosteric Regulation; Analysis of Variance; Animals; Binding, Competitive; Cocaine; Cues; Dopamine Uptake Inhibitors; Dose-Response Relationship, Drug; Drug-Seeking Behavior; Enzyme-Linked Immunosorbent Assay; Excitatory Amino Acid Antagonists; HEK293 Cells; Humans; Imidazoles; In Vitro Techniques; Infusions, Intravenous; Inhibitory Concentration 50; Male; Motor Activity; Nitriles; Piperidines; Pyridines; Random Allocation; Rats; Receptor, Metabotropic Glutamate 5; Reinforcement Schedule; Reward; Secondary Prevention; Self Administration; Sucrose; Thiazoles

Metabotropic glutamate 5 (mGlu5) receptor antagonists reduce L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias (LID) in Parkinson's disease (PD). The aim of this study was to investigate the long-term effect of the prototypal mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) on glutamate receptors known to be involved in the development of LID in the de novo chronic treatment of monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP monkeys were treated for one month with L-DOPA and developed dyskinesias while those treated with L-DOPA and MPEP (10 mg/kg) developed significantly less. Normal control and saline-treated MPTP monkeys were also included. All MPTP monkeys were extensively and similarly denervated. The basal ganglia [(3)H]ABP688 specific binding (mGlu5 receptors) was elevated in L-DOPA-treated MPTP monkeys compared to controls but not in those treated with L-DOPA and MPEP; dyskinesia scores of these monkeys correlated positively with their [(3)H]ABP688 specific binding. Striatal density (B(max)) of [(3)H]ABP688 specific binding increased in L-DOPA-treated MPTP monkeys compared to other groups and affinity (Kd) remained unchanged. Striatal mGlu5 receptor mRNA remained unchanged following treatments. Elevated basal ganglia specific binding of [(3)H]Ro 25-6981 (NMDA NR1/NR2B receptors), [(3)H]Ro 48-8587 (AMPA receptors) but not [(3)H]CGP-39653 (NMDA NR1/NR2A receptors) was observed only in L-DOPA-treated MPTP monkeys; dyskinesias scores correlated with binding. By contrast, basal ganglia [(3)H]LY341495 specific binding (mGlu2/3 receptors) decreased in L-DOPA-treated MPTP monkeys compared to controls, saline and L-DOPA + MPEP treated MPTP monkeys; dyskinesias scores correlated negatively with this binding. Hence, chronic MPEP treatment reduces the development of LID and is associated with a normalization of glutamate neurotransmission.

Topics: 2-Amino-5-phosphonovalerate; Amino Acids; Animals; Basal Ganglia; Corpus Striatum; Dyskinesia, Drug-Induced; Female; Imidazoles; Levodopa; Macaca fascicularis; Oximes; Parkinsonian Disorders; Phenols; Piperidines; Pyridines; Quinazolines; Radioligand Assay; Receptor, Metabotropic Glutamate 5; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tritium; Xanthenes

This study analyzed the effect of intra-ventrolateral periaqueductal grey (VL PAG) cannabinoid receptor (CB) stimulation on pain responses and rostral ventromedial medulla (RVM) neural activity in the chronic constriction injury (CCI) model of neuropathic pain in rats. Interaction between CB1 and metabotropic glutamate 1 and 5 (mGlu(1)/mGlu(5)) receptors was also investigated together with the expression of the CB1 receptor associated Gαi3 and cannabinoid receptor interacting 1a (CRIP 1a) proteins and the endocannabinoid synthesising and hydrolysing enzymes. In rats not subjected to CCI-induced pain, intra-VL PAG (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) (2-4-8 nmol), a CB receptor agonist, increased the tail flick latency and changed the ongoing activity of RVM OFF and the tail flick-related activity of the ON and OFF cells, accordingly. These effects were prevented by SR141716A and MPEP, selective CB(1) and mGlu(5) receptor antagonists, respectively, though not by CPCCOEt, a selective mGlu(1) receptor antagonist. A higher dose up to 16 nmol of WIN 55,212-2 was necessary to increase tail flick latency and change ON and OFF cell activity in CCI rats. Consistently, CCI rats showed a decrease in the expression of CB(1) receptors, NAPE-PLD, Gαi3 and CRIP 1a proteins;the expression of diacylglycerol lipase A (DAGLA) was increased while fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) did not change. As in control rats, MPEP and SR141716A also blocked WIN 55,212-2- induced effects in CCI rats. These data demonstrate a down regulation of the endocannabinoid system and a functional interaction between mGlu(5) and CB(1) receptors for cannabinoid-mediated effect in the PAG-RVM pain circuitry in neuropathic pain inflicted rats.

Topics: Analgesics; Animals; Benzoxazines; Disease Models, Animal; Drug Interactions; Excitatory Amino Acid Antagonists; Male; Medulla Oblongata; Morpholines; Naphthalenes; Neuralgia; Periaqueductal Gray; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant

Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB(1) cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-β isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α colocalized with mGlu(5) at postsynaptic sites of the dlPAG, whereas CB(1) was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-α, but not DGL-β, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.

Topics: Analgesia; Analysis of Variance; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cyclohexanones; Dose-Response Relationship, Drug; Electroconvulsive Therapy; Endocannabinoids; Excitatory Amino Acid Antagonists; Glycerides; Lipoprotein Lipase; Male; Methoxyhydroxyphenylglycol; Mice; Microscopy, Immunoelectron; Pain; Periaqueductal Gray; Piperidines; Protease Inhibitors; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant; RNA, Messenger; RNA, Small Interfering; Synapses; Tandem Mass Spectrometry

Soluble amyloid beta (Aβ) oligomers are widely accepted to be neurotoxic and lead to the memory loss and neuronal death observed in Alzheimer's disease (AD). Ample evidence suggests that impairment in glutamatergic signalling is associated with AD pathology. In particular, Aβ(1-42) is thought to affect N-methyl-d-aspartate (NMDA) receptor function and abolish the induction of long-term potentiation (LTP), which is regarded to be a phenomenon relevant to memory formation. The involvement of glutamatergic signalling in the pathology of AD is underscored by the therapeutic success of memantine, an uncompetitive NMDA receptor antagonist, used to treat patients with moderate to severe AD. In this study we show that Aβ(1-42) oligomers applied to acute murine hippocampal slices prevented, in a concentration-dependent manner, the development of CA1-LTP after tetanic stimulation of the Schaffer collaterals with a half maximal inhibitory concentration of around 2 nM (before oligomerization). The highest concentration of Aβ(1-42) oligomers (50 nM before oligomerization) completely blocked LTP (105 ± 1% potentiation versus 141 ± 3% in control) whereas scrambled Aβ(1-42) (50 nM) was without effect (144 ± 10% potentiation). Pre-incubation with memantine (1 μM) restored LTP in the presence of Aβ(1-42) (50 nM; 135 ± 5% potentiation). NMDA receptors containing the NR2B subunit have been proposed to play a particularly important role in excitotoxicity, functioning as extracellular "death receptors". The metabotropic glutamate receptor 5 (mGluR5) is mechanistically coupled to postsynaptic NMDA receptors. As such, allosteric sites on both receptors offer alternative means to modulate NMDA receptor function. We therefore tested low concentrations (each 300 nM) of allosteric antagonists of NR2B (Ro 25-6981, [R-(R∗,S∗)]-α-(4-Hydroxyphenyl)-β-methyl-4(phenylmethyl)-1-piperidine propanol hydrochloride) and mGluR5 receptors (MPEP, 2-methyl-6-(phenylethynyl)-pyridine). Both compounds restored LTP in the presence of Aβ(1-42) oligomers (50 nM, fEPSPs were potentiated to 129 ± 13% and 133 ± 7% respectively). Finally, we demonstrated that slices from mice heterozygous for NR2B receptor) in the forebrain are not susceptible to the toxic effects of Aβ(1-42) oligomers but express normal LTP (138 ± 6%). These experiments demonstrate that glutamate receptor antagonists delivered at concentrations which still allow physiological activities in vitro, are able to prevent Aβ(1-42) oligomer-indu

Topics: Amyloid beta-Peptides; Animals; Electric Stimulation; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Long-Term Potentiation; Male; Memantine; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptide Fragments; Phenols; Piperidines; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Synapses

Astrocytes show a complex structural and physiological interplay with neurons and respond to neuronal activation in vitro and in vivo with intracellular calcium elevations. These calcium changes enable astrocytes to modulate synaptic transmission and plasticity through various mechanisms. However, the response pattern of astrocytes to single neuronal depolarization events still remains unresolved. This information is critical for fully understanding the coordinated network of neuron-glial signaling in the brain. To address this, we developed a system to map astrocyte calcium responses along apical dendrites of CA1 pyramidal neurons in hippocampal slices using single-neuron stimulation with channelrhodopsin-2. This technique allowed selective neuronal depolarization without invasive manipulations known to alter calcium levels in astrocytes. Light-evoked neuronal depolarization was elicited and calcium events in surrounding astrocytes were monitored using the calcium-sensitive dye Calcium Orange. Stimulation of single neurons caused calcium responses in populations of astrocytes along the apical axis of CA1 cell dendrites. Calcium responses included single events that were synchronized with neuronal stimulation and poststimulus changes in calcium event frequency, both of which were modulated by glutamatergic and purinergic signaling. Individual astrocytes near CA1 cells showed low ability to respond to repeated neuronal depolarization events. However, the response of the surrounding astrocyte population was remarkably accurate. Interestingly, the reliability of responses was graded with respect to astrocyte location along the CA1 cell dendrite, with astrocytes residing in the primary dendrite subregion being most responsive. This study provides a new perspective on the dynamic response property of astrocyte ensembles to neuronal activity.

Topics: Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Aspartic Acid; Astrocytes; Bacterial Proteins; Benzoates; Benzoxazines; Calcium; Calcium Channel Blockers; Carbenoxolone; Channelrhodopsins; Electric Stimulation; Excitatory Amino Acid Antagonists; Female; Glial Fibrillary Acidic Protein; Glycine; Green Fluorescent Proteins; Hippocampus; In Vitro Techniques; Luminescent Proteins; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Models, Biological; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Peptide Fragments; Phosphopyruvate Hydratase; Photic Stimulation; Piperidines; Pyrazoles; Pyridines; Sodium Channel Blockers; Statistics, Nonparametric; Tetanus Toxin; Tetrodotoxin; Transduction, Genetic

The frequency and duration of glutamatergic inputs to the striatum are strong determinants of the net effect of retrograde endocannabinoid (eCB) signaling, and key factors in determining if long-term depression (LTD) has a net disinhibitory or inhibitory action in striatum. Low to moderate frequency stimulation in the dorsolateral striatum elevates eCB levels to an extent that primarily depresses transmitter release at inhibitory synapses, leading to a long-lasting disinhibition (DLL) of synaptic output. The aim of this study was to further characterize the basic features of endocannabinoid-mediated DLL of striatal output induced by moderate frequency stimulation (5 Hz, 60 s). DLL was inhibited in slices treated with the group 1 metabotropic glutamate receptor (mGluR) antagonists MPEP (40 μM) and CPCCOEt (40 μM), the dopamine D2 receptor antagonist sulpiride (5 μM), the L-type calcium channel blocker nifedipine (20 μM), the nicotinic receptor antagonist mecamylamine (10 μM), the muscarinic agonist oxotremorine sesquifumarate (10 μM), and strychnine (0.1 μM). Strychnine did not block DLL induced by WIN55,212-2 (250 nM), showing that glycine receptor-mediated modulation of eCB signaling occurs upstream from CB(1)R activation. Scopolamine (10 μM) restored DLL in strychnine-treated slices, suggesting that inhibition of glycine receptors on cholinergic interneurons could modulate eCB signaling by enhancing muscarinic receptor activation and reducing the opening of L-type calcium channels in response to depolarization. These data suggests that similar activation points are required for stimulation-induced DLL as for LTD at excitatory striatal synapses, and that cholinergic interneurons are key modulators of stimulation-induced eCB signaling in the striatum.

Topics: Animals; Animals, Newborn; Benzazepines; Bicuculline; Biophysics; Cannabinoid Receptor Modulators; Cholinergic Agents; Chromones; Corpus Striatum; Dopamine Antagonists; Electric Stimulation; Endocannabinoids; Excitatory Amino Acid Antagonists; GABA-A Receptor Antagonists; In Vitro Techniques; Interneurons; Long-Term Potentiation; Models, Neurological; Neural Inhibition; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Wistar; Signal Transduction; Sulpiride; Valine

The reason why neurons synthesize more than one endocannabinoid (eCB) and how this is involved in the regulation of synaptic plasticity in a single neuron is not known. We found that 2-arachidonoylglycerol (2-AG) and anandamide mediate different forms of plasticity in the extended amygdala of rats. Dendritic L-type Ca(2+) channels and the subsequent release of 2-AG acting on presynaptic CB1 receptors triggered retrograde short-term depression. Long-term depression was mediated by postsynaptic mGluR5-dependent release of anandamide acting on postsynaptic TRPV1 receptors. In contrast, 2-AG/CB1R-mediated retrograde signaling mediated both forms of plasticity in the striatum. These data illustrate how the eCB system can function as a polymodal signal integrator to allow the diversification of synaptic plasticity in a single neuron.

Topics: Animals; Arachidonic Acids; Biophysics; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cannabinoid Receptor Modulators; Chromones; Cyclohexanones; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glycerides; In Vitro Techniques; Long-Term Synaptic Depression; Neurons; Nimodipine; Patch-Clamp Techniques; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Septal Nuclei; Signal Transduction; Synapses; Time Factors; TRPV Cation Channels

The NMDA (N-methyl-D-aspartate)-receptor is fundamentally involved in cognitive functions. Recent studies demonstrated a functional interaction between the metabotropic glutamate receptor 5 (mGlu(5) receptor) and the NMDA-receptor in neurons. In rat hippocampal slices, it was shown that activation of mGlu(5) receptor by a positive modulator in the presence of a subthreshold agonist concentration potentiated NMDA-receptor mediated currents and phosphorylation of intracellular signalling proteins. In the present study, we investigated the functional interaction of mGlu(5) receptor and NMDA-receptor by the selective mGlu(5) receptor positive modulator ADX-47273 in-vitro and in-vivo. In rat primary neurons, this compound potentiated Ca(2+) mobilization in the presence of a subthreshold concentration of the mGluR(1/5) agonist DHPG (0.3 microM) with an EC(50) of 0.28+/-0.05 microM. NMDA-induced Ca(2+)-mobilization in primary neurons could be potentiated when neurons were pre-stimulated with 1 microM ADX-47273 in the presence of 0.3 microM DHPG. The specific mGlu(5) receptor antagonist MPEP and the Src-family kinase inhibitor PP2 blocked this potentiation demonstrating the functional interaction of the NMDA-receptor and mGlu(5) receptor in neurons. Furthermore, ADX-47273 elicited an enhancement of NMDA-receptor dependent long-term potentiation in rat hippocampal slices that could be reversed by MPEP. After intraperitoneal administration to rats, ADX-47273 showed a dose-dependent reduction of NMDA-receptor antagonist (ketamine) induced hyperlocomotion, supporting the mechanistic interaction of the NMDA-receptor and mGlu(5) receptor in-vivo. In conclusion, these findings further support the idea of a functional interaction between the mGlu(5) receptor and NMDA-receptor, which may provide a pharmacological strategy for addressing CNS diseases with cognitive impairments linked to NMDA-receptor hypofunction.

Topics: Allosteric Regulation; Animals; Calcium; Cognition; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Hippocampus; Injections, Intraperitoneal; Male; Methoxyhydroxyphenylglycol; Neurons; Oxadiazoles; Phosphorylation; Piperidines; Pyridines; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; src-Family Kinases

Endocannabinoids and their receptor CB1 play key roles in brain function. Astrocytes express CB1Rs that are activated by endocannabinoids released by neurons. However, the consequences of the endocannabinoid-mediated neuron-astrocyte signaling on synaptic transmission are unknown. We show that endocannabinoids released by hippocampal pyramidal neurons increase the probability of transmitter release at CA3-CA1 synapses. This synaptic potentiation is due to CB1R-induced Ca(2+) elevations in astrocytes, which stimulate the release of glutamate that activates presynaptic metabotropic glutamate receptors. While endocannabinoids induce synaptic depression in the stimulated neuron by direct activation of presynaptic CB1Rs, they indirectly lead to synaptic potentiation in relatively more distant neurons by activation of CB1Rs in astrocytes. Hence, astrocyte calcium signal evoked by endogenous stimuli (neuron-released endocannabinoids) modulates synaptic transmission. Therefore, astrocytes respond to endocannabinoids that then potentiate synaptic transmission, indicating that astrocytes are actively involved in brain physiology.

Topics: Animals; Animals, Newborn; Astrocytes; Benzoates; Biophysics; Calcium; Cannabinoid Receptor Modulators; Chelating Agents; Drug Interactions; Egtazic Acid; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glycine; Hippocampus; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Patch-Clamp Techniques; Photolysis; Piperidines; Pyramidal Cells; Pyrazoles; Pyridines; Receptor, Cannabinoid, CB1; Resorcinols; Synaptic Transmission; Thapsigargin

Glutamatergic transmission is one of the main components of the stress response; nevertheless, its role in the emotional stress sequelae is not known. Here, we investigated whether interactions between group I metabotropic glutamate receptors (metabotropic glutamate receptor 1 and metabotropic glutamate receptor 5 [mGluR5]) and Homer proteins mediate the delayed and persistent enhancement of fear induced by acute stress.. Antagonists and inverse agonists of metabotropic glutamate receptor 1 and mGluR5 were injected into the hippocampus after immobilization stress and before contextual fear conditioning. Metabotropic glutamate receptor 5 was displaced from constitutive Homer scaffolds by viral transfection of Homer1a or injection of Tat decoy peptides. The effects of these manipulations on stress-enhanced fear were determined.. We show that stress induces interactions between hippocampal mGluR5 and Homer1a; causes a sustained, ligand-independent mGluR5 activity; and enhances contextual fear. Consistent with this mechanism, enhancement of fear was abolished by delayed poststress application of inverse agonists, but not antagonists, of mGluR5. The effect of stress was mimicked by virally transfected Homer1a or injection of Tat-metabotropic glutamate receptor C-tail decoy peptides into the hippocampus.. Constitutive activation of mGluR5 is identified as a principal hippocampal mechanism underlying the delayed stress effects on emotion and memory. Inverse agonists, but not antagonists, of mGluR5 are therefore proposed as a preventive treatment option for acute and posttraumatic stress disorders.

Topics: Analysis of Variance; Animals; Association Learning; Blotting, Western; Carrier Proteins; Cells, Cultured; Conditioning, Psychological; Excitatory Amino Acid Antagonists; Fear; Hippocampus; Homer Scaffolding Proteins; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Neurons; Piperidines; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Restraint, Physical; Stress, Physiological; Stress, Psychological; Thiazoles

Blockade of the cannabinoid CB1 receptors (CB1R) has been shown to reduce psychostimulant-induced hyperactivity, an effect that we sought to further characterize here. The CB1R antagonist SR141716A dose-dependently decreased d-amphetamine-induced hyperactivity.Also, d-amphetamine-induced hyperlocomotion was reduced in CB1R knockout (KO) mice. However, CB1R KO and wild-type mice showed a similar d-amphetamine-induced increase in nucleus accumbens DA release. Hence, we investigated whether CB1R antagonism/invalidation reduces d-amphetamine-induced hyperlocomotion through a mechanism involving changes in glutamatergic neurotransmission. Blockade of metabotropic-glutamate-receptors-5 (mGluR5)with MPEP, but not blockade of N-methyl-D-aspartate-receptors (NMDA) with MK-801,restored to a great extent the blunted d-amphetamine-induced hyperlocomotion seen after CB1R antagonism/invalidation. Thus, hyporesponsiveness to the psychostimulant effects of d-amphetamine as a result of CB1R antagonism/invalidation is not due to an ensuing decrease in d-amphetamine-induced DA release in the nucleus accumbens, but rather due to a hyperglutamatergic state and facilitation of glutamatergic neurotransmission at the mGlu5, but not NMDA, receptors.

Topics: Amphetamine; Analysis of Variance; Animals; Behavior, Animal; Dizocilpine Maleate; Dopamine; Dopamine Uptake Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Hyperkinesis; Mice; Mice, Inbred C57BL; Mice, Knockout; Microdialysis; Motor Activity; Nucleus Accumbens; Piperidines; Pyrazoles; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant

Neurotensin modulates pain via its actions within descending analgesic pathways which include brain regions such as the midbrain periaqueductal grey (PAG). The aim of this study was to examine the cellular actions of neurotensin on PAG neurons. Whole cell patch clamp recordings were made from rat midbrain PAG slices in vitro to examine the postsynaptic effects of neurotensin and its effects on GABA(A) mediated inhibitory postsynaptic currents (IPSCs). Neurotensin (100-300 nM) produced an inward current in subpopulations of opioid sensitive and insensitive PAG neurons which did not reverse over membrane potentials between -50 and -130 mV. The neurotensin induced current was abolished by the NTS1 and NTS1/2 antagonists SR48692 (300 nM) and SR142948A (300 nM). Neurotensin also produced a reduction in the amplitude of evoked IPSCs, but had no effect on the rate and amplitude of TTX-resistant miniature IPSCs. The neurotensin induced inhibition of evoked IPSCs was reduced by the mGluR5 antagonist MPEP (5microM) and abolished by the cannabinoid CB(1) receptor antagonist AM251 (3 microM). These results suggest that neurotensin produces direct neuronal depolarisation via NTS1 receptors and inhibits GABAergic synaptic transmission within the PAG. The inhibition of synaptic transmission is mediated by neuronal excitation and action potential dependent release of glutamate, leading to mGluR5 mediated production of endocannabinoids which activate presynaptic CB(1) receptors. Thus, neurotensin has cellular actions within the PAG which are consistent with both algesic and analgesic activity, some of which are mediated via the endocannabinoid system.

Topics: Adamantane; Animals; Cannabinoid Receptor Modulators; Endocannabinoids; Excitatory Amino Acid Antagonists; Female; gamma-Aminobutyric Acid; Imidazoles; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Male; Miniature Postsynaptic Potentials; Neural Inhibition; Neurons; Neurotensin; Pain; Periaqueductal Gray; Piperidines; Presynaptic Terminals; Pyrazoles; Pyridines; Quinolines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Receptors, Neurotensin; Synaptic Transmission; Tetrodotoxin; Time Factors

Endocannabinoid (eCB) signaling mediates short-term and long-term synaptic depression (LTD) in many brain areas. In the ventral tegmental area (VTA) and striatum, D(2) dopamine receptors cooperate with group I metabotropic glutamate receptors (mGluRs) to induce eCB-mediated LTD of glutamatergic excitatory and GABAergic inhibitory (I-LTD) synaptic transmission. Because D(2) receptors and group I mGluR agonists are capable of inducing the release of eCBs, the predominant hypothesis is that the cooperation between these receptors to induce eCB-mediated synaptic depression results from the combined activation of type I cannabinoid (CB(1)) receptors by the eCBs. By determining the downstream effectors for D(2) receptor and group I mGluR activation in VTA dopamine neurons, we show that group I mGluR activation contributes to I-LTD induction by enhancing eCB release and CB(1) receptor activation. However, D(2) receptor activation does not enhance CB(1) receptor activation, but facilitates I-LTD induction via direct inhibition of cAMP-dependent protein kinase A (PKA) signaling. We further demonstrate that cAMP/PKA signaling pathway is the downstream effector for CB(1) receptors and is required for eCB-mediated I-LTD induction. Our results suggest that D(2) receptors and CB(1) receptors target the same downstream effector cAMP/PKA signaling pathway to induce I-LTD and D(2) receptor activation facilitates eCB-mediated I-LTD in dopamine neurons not by enhancing CB(1) receptor activation, but by enhancing its downstream effects.

Topics: Analysis of Variance; Animals; Animals, Newborn; Benzoxazines; Chromones; Cocaine; Cyclic AMP-Dependent Protein Kinases; Dopamine; Dopamine Agents; Dopamine D2 Receptor Antagonists; Drug Combinations; Egtazic Acid; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; gamma-Aminobutyric Acid; In Vitro Techniques; Long-Term Synaptic Depression; Male; Mesencephalon; Mice; Mice, Knockout; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Dopamine D2

Endocannabinoids released from the postsynaptic neuronal membrane can activate presynaptic CB1 receptors and inhibit neurotransmitter release. In hippocampal slices, depolarization of the CA1 pyramidal neurons elicits an endocannabinoid-mediated inhibition of gamma-aminobutyric acid release known as depolarization-induced suppression of inhibition (DSI). Using the highly reduced neuron/synaptic bouton preparation from the CA1 region of hippocampus, we have begun to examine endocannabinoid-dependent short-term depression (STD) of inhibitory synaptic transmission under well-controlled physiological and pharmacological conditions in an environment free of other cells. Application of the CB1 synthetic agonist WIN55212-2 and endogenous cannabinoids 2-AG and anandamide produced a decrease in spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude, indicating the presence of CB1 receptors at synapses in this preparation. Endocannabinoid-dependent STD is different from DSI found in hippocampal slices and the neuron/bouton preparation from basolateral amygdala (BLA) since depolarization alone was not sufficient to induce suppression of sIPSCs. However, concurrent application of the metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) and postsynaptic depolarization resulted in a transient (30-50 s) decrease in sIPSC frequency and amplitude. Application of DHPG alone had no effect on sIPSCs. The depolarization/DHPG-induced STD was blocked by the CB1 antagonist SR141716A and the mGluR5 antagonist MPEP and was sensitive to intracellular calcium concentration. Comparing the present findings with earlier work in hippocampal slices and BLA, it appears that endocannabinoid release is less robust in isolated hippocampal neurons.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Drug Interactions; Endocannabinoids; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glycerides; Hippocampus; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Methoxyhydroxyphenylglycol; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant; Synapses; Synapsins

Learning and memory are thought to involve activity-dependent changes in synaptic efficacy such as long-term potentiation (LTP) and long-term depression (LTD). Recent studies have indicated that endocannabinoid-dependent modulation of inhibitory transmission facilitates induction of hippocampal LTP and that endocannabinoids play a key role in certain forms of LTD. Here, we show that repetitive low-frequency synaptic stimulation (LFS) produces persistent up-regulation of endocannabinoid signaling at hippocampal CA1 GABAergic synapses. This LFS also produces LTD of inhibitory synapses and facilitates LTP at excitatory, glutamatergic synapses. These endocannabinoid-mediated plastic changes could contribute to information storage within the brain.

Topics: Animals; Cannabinoid Receptor Modulators; Dose-Response Relationship, Radiation; Electric Stimulation; Endocannabinoids; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Neural Inhibition; Neuronal Plasticity; Neurons; Patch-Clamp Techniques; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Rimonabant; Synapses; Time Factors

The metabotropic glutamate receptor subtype 5 (mGluR5) has been reported to be implicated in various neurological disorders in the central nervous system. To investigate physiological and pathological functions of mGluR5, noninvasive imaging in a living body with PET technology and an mGluR5-specific radiotracer is urgently needed. Here, we report the synthesis of 3-[(18)F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([(18)F]FPEB) through a convenient thermal reaction as a highly specific PET radiotracer for mGluR5. The precursor and standard compounds were prepared by a coupling reaction catalyzed by palladium. Radiosynthesis of [(18)F]FPEB was performed using nitro as a leaving group replaced by [(18)F]fluoride under conventional heating condition. Biodistribution, metabolite, and microPET studies were performed using Sprague-Dawley rats. Upto 30 mCi of [(18)F]FPEB was obtained with a radiochemical yield of 5% and a specific activity of 1900 +/- 200 mCi/mumol at the end of syntheses. Biodistribution showed rapid clearance from the blood pool and fast and steady accumulation of radioactivity into the brain. Metabolite studies indicated that only 22% of [(18)F]FPEB remained in the blood system 10 min after administration, and that a metabolite existed which was much more polar than the parent tracer. MicroPET studies demonstrated that [(18)F]FPEB accumulated specifically in mGluR5-rich regions of the brain such as striatum and hippocampus, and that blockade with 2-methyl-6-(2-phenylethynyl)pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) substantially reduced the activity uptake in these regions. Selectivity was investigated by blockage with 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-caroxamide (YM-298198), a specific antagonist for mGluR1. [(18)F]FPEB was prepared conveniently and showed high specificity and selectivity toward mGluR5. It possesses the potential to be used in human studies to evaluate mGluR5 functions in various neurological disorders.

Topics: Animals; Benzimidazoles; Brain; Evaluation Studies as Topic; Excitatory Amino Acid Antagonists; Male; Nitriles; Piperidines; Positron-Emission Tomography; Pyridines; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Reaction Time; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Thiazoles; Tissue Distribution; Whole Body Imaging

This study was undertaken to analyze the involvement of periaqueductal gray (PAG) cannabinoid or group I metabotropic glutamate receptors in the formalin-induced changes on the rostral ventromedial medulla (RVM) ON- and OFF-cells activities. S.c. injection of formalin into the hind paw produced a transient decrease (4-6 min) followed by a longer increase (25-35 min) in tail flick latencies. Formalin also increased basal activity in RVM ON-cells (42+/-7%) and decreased it in OFF-cells (35+/-4%). Intra-PAG microinjection of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) (2 nmol/rat), a cannabinoid receptor agonist, prevented the formalin-induced changes in RVM cell activities. Higher dosages of WIN 55,212-2 (4-8 nmol/rat) increased the tail flick latencies, delayed the tail flick-related onset to ON-cell burst, and decreased the duration of OFF-cell pause. Furthermore, WIN 55,212-2 at a dosage of 8 nmol/rat decreased RVM ON-cell (57+/-7%) and increased OFF-cell ongoing activities (26+/-4%). These effects were prevented by N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide SR141716A, (1 pmol/rat), a CB1 cannabinoid receptor antagonist, or by 2-methyl-6-(phenylethynyl)pyridine (MPEP 20 nmol/rat), a selective mGlu5 glutamate receptor antagonist. T7-(hydroxyimino) cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCOOE/50 nmol/rat) and (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385, 20 nmol/rat), selective mGlu1 glutamate receptor antagonists, were ineffective in preventing the WIN-induced effects. This study suggests that s.c. injection of formalin modifies RVM neuronal activities and this effect is prevented by PAG cannabinoid receptor stimulation. Moreover, the physiological stimulation of PAG mGlu5, but not mGlu1 glutamate receptors, seems to be required for the cannabinoid-mediated effect.

Topics: Action Potentials; Analgesics; Animals; Benzoates; Benzoxazines; Chromones; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Formaldehyde; Glycine; Injections, Subcutaneous; Male; Medulla Oblongata; Morpholines; Naphthalenes; Neurons; Pain Measurement; Periaqueductal Gray; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Wistar; Reaction Time; Receptor, Cannabinoid, CB1; Receptors, Metabotropic Glutamate; Rimonabant

Group I metabotropic glutamate (mGlu) receptors (i.e. mGlu1 and mGlu5) coupled to phospholipase C have been widely investigated for their possible role in excitotoxic and post-ischemic neuronal death. Recently, phospholipase C has been shown to directly stimulate the activity of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair that has been proposed to play a key role in necrotic cell death. In this study, we investigated whether the stimulation of group I mGlu receptors leads to an increase in PARP activity, as detected by flow cytometry, immunodot blot and immunocytochemistry, both in baby hamster kidney cells transfected with mGlu1a or mGlu5a receptors and in cultured cortical cells. Our results show that the group I mGlu receptor agonist DHPG elicited a significant increase in PARP activity that was completely abolished by the administration of the mGlu1 antagonist 3-MATIDA and partially prevented, in cortical neurons, by the mGlu5 antagonist MPEP. To evaluate whether this pathway is involved in post-ischemic neuronal death, we used a sublethal model of oxygen-glucose deprivation in mixed cortical cell cultures. DHPG exacerbated neuronal death, and this effect was significantly prevented by the application of the PARP inhibitor DPQ. This novel pathway may contribute to the effects of mGlu1 receptors in the mechanisms leading to post-ischemic neuronal death.

Topics: Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Cerebral Cortex; Cricetinae; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Gene Expression Regulation, Enzymologic; Glial Fibrillary Acidic Protein; Glucose; Hydrogen Peroxide; Hypoxia; Isoquinolines; Methoxyhydroxyphenylglycol; Mice; Neuroglia; Neurons; Piperidines; Poly(ADP-ribose) Polymerases; Pyridines; Receptors, Metabotropic Glutamate; Thiophenes; Time Factors; Transfection

Methamphetamine (METH) is a strongly addictive psychostimulant that dramatically affects the central nervous system (CNS). On the other hand, protein kinase C (PKC) plays a major role in cellular regulatory and signalling processes that involve protein phosphorylation. The purpose of this study was to investigate the role of neuronal and astrocytic PKC in changes in the central glutamatergic system induced by METH. We show here that in vitro treatment with METH caused the phosphorylation of both neuronal and astrocytic PKC and the activation of astrocytes in cortical neuron/glia co-cultures. Treatment of cortical neuron/glia co-cultures with either the PKC activator phorbol 12,13-dibutyrate (PDBu) or glutamate also caused the PKC-dependent activation of astrocytes. The PKC inhibitor chelerythrine suppressed the Ca2+ responses to glutamate in both cortical neurons and astrocytes. Moreover, a low concentration of PDBu significantly enhanced the Ca2+ responses to glutamate, but not to dopamine, in both cortical neurons and astrocytes. Notably, treatment with METH also enhanced the Ca2+ responses to glutamate in cortical neurons. The activation of astrocytes induced by METH was also reversed by co-treatment with glutamate receptor antagonists (ifenprodil, DNQX or MPEP) in cortical neuron/glia co-cultures. In the conditioned place preference paradigm, intracerebroventricular administration of glutamate receptor antagonists (ifenprodil, DNQX or MPEP) attenuated the METH-induced rewarding effect. These findings provide evidence that the changes in PKC-dependent neuronal and astrocytic glutamatergic transmission induced by METH may, at least in part, contribute to the development of psychological dependence on METH.

Topics: Amphetamine-Related Disorders; Animals; Astrocytes; Calcium; Cell Communication; Central Nervous System Stimulants; Coculture Techniques; Conditioning, Operant; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; Immunohistochemistry; Male; Methamphetamine; Mice; Mice, Inbred ICR; Microscopy, Confocal; Neuroglia; Neurons; Piperidines; Pregnancy; Protein Kinase C; Pyridines; Quinoxalines; Receptors, Glutamate; Synaptic Transmission

The stress-induced hyperthermia test is a paradigm developed several years ago to model the expression of autonomic hyperactivity in anxiety. Whereas in the classical stress-induced hyperthermia, cohort removal was used, in a recently described modification of the stress-induced hyperthermia model singly housed mice rather than groups of mice were used. The modification of this model can be summarized as follows: rectal temperature is recorded in singly housed animals at two consecutive time-points (T1 and T2) which are interspaced by a defined time-interval (15 min). Since the value at the second temperature-recording exceeds the value of the initial measure it is the difference between these two core-temperatures which reflects stress-induced hyperthermia. In the present study, the stress-induced hyperthermia paradigm, in its modified design, was evaluated in OF1/IC mice. By comparing the effect of various compounds in both the modified as well as the classical (cohort removal) stress-induced hyperthermia paradigm, a very high correlation was found for the pharmacological sensitivity of the two paradigms. Furthermore, it was demonstrated that other anxiolytics, all known to be active in the classical stress-induced hyperthermia paradigm, such as the benzodiazepines chlordiazepoxide (0.3, 1, 3, 10 mg/kg, p.o.), diazepam (0.1, 0.3, 1, 3 mg/kg, p.o.), clobazam (5 or 10 mg/kg, p.o.) and oxazepam (5 or 10 mg/kg, p.o.) as well as the non-benzodiazepines buspirone (7.5 or 15 mg/kg, p.o.) and ethanol (15% or 30%, 10 ml/kg, p.o.), showed a marked reduction in stress-induced hyperthermia in the modified design. New candidate anxiolytics, i.e. the metabotropic glutamate (mGlu) receptor group 2 agonist LY314582 (1 or 10 mg/kg, p.o.; racemic mixture of LY354740 ((2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid), the metabotropic glutamate 5 receptor antagonist MPEP (1, 7.5, 15 or 30 mg/kg, p.o.; 2-methyl-6-(phenylethynyl)pyridine) and the neurokinin 1 (NK1) receptor antagonist NKP608 (0.01 or 0.1 mg/kg, p.o.; quinoline-4-carboxylic acid [trans-(2R,4S)-1-(3,5-bis-trifluoromethyl-benzoyl)-2-(4-chloro-benzyl)-piperidin-4-yl]-amide) also reduced stress-induced hyperthermia in the modified paradigm clearly indicating anxiolytic-like activity for these compounds. Finally, the effects of the classical benzodiazepine chlordiazepoxide (10 mg/kg, p.o.), in parallel with its effect on stress-induced hyperthermia, were also investigated for its effect on plasma c

Topics: Adrenocorticotropic Hormone; Analysis of Variance; Animals; Anti-Anxiety Agents; Benzodiazepines; Body Temperature; Bridged Bicyclo Compounds; Buspirone; Chlordiazepoxide; Clobazam; Corticosterone; Diazepam; Ethanol; Hyperthermia, Induced; Male; Mice; Mice, Inbred ICR; Oxazepam; Piperidines; Pyridines; Quinolines; Reproducibility of Results; Stress, Physiological

This study investigates the effect of microinjections of capsaicin in the periaqueductal grey matter of rats on nociceptive behaviour and the possible interactions with NMDA and mGlu receptors. Intra-periaqueductal grey microinjection of capsaicin (1-3-6 nmol/rat) increased the latency of the nociceptive reaction in the plantar test. This effect was prevented by pretreatment with capsazepine (6 nmol/rat), which had no effect per se on the latency of the nociceptive reaction. 7-(Hydroxyimino)cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 50 nmol/rat) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP, 50 nmol/rat), antagonists of mGlu(1) and mGlu(5) receptors, respectively, completely blocked the effect of capsaicin. Similarly, pretreatment with DL-2-Amino-5-phosphonovaleric acid (DL-AP5, 5 nmol/rat) and riluzole (4 nmol/rat), an NMDA receptor antagonist and a voltage-dependent Na(+) channels blocker which inhibits glutamate release, respectively, completely antagonized the effect of capsaicin. However, pretreatment with (2S)-alpha-Ethylglutamic acid (30 nmol/rat) and (RS)-alpha-Methylserine-O-phosphate (MSOP, 30 nmol/rat), antagonists of group II and group III mGlu receptors, respectively, had no effects on capsaicin-induced analgesia. Similarly, pretreatment with N-(piperidin-1-yl)-5-(4-chlophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 5 pmol/rat), a selective cannabinoid CB(1) receptor antagonist, did not affect the capsaicin-induced antinociception. In conclusion, this study shows that capsaicin might produce antinociception at the periaqueductal grey level by increasing glutamate release, which activates postsynaptic group I mGlu and NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; Animals; Capsaicin; Chromones; Excitatory Amino Acid Antagonists; Male; Pain; Periaqueductal Gray; Phosphoserine; Piperidines; Protein Binding; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptors, Drug; Receptors, Glutamate; Riluzole; Rimonabant; Time Factors