Metabotropic Glutamate Receptor Antagonist Research Articles

The mGluR5 antagonist fenobam induces analgesic conditioned place preference in mice with spared nerve injury.

Antagonists of metabotropic glutamate receptors (mGluRs) have the potential to act as analgesic drugs that may help alleviate chronic pain. This study was done to look at the possible rewarding properties of the mGluR5 antagonist, fenobam, in a cognitive assay. Analgesic conditioned place preference (aCPP) was used to examine the effects of fenobam (30 mg/kg) and the prototypical mGluR5 antagonist, MPEP, and these effects were compared to those of a drug with known analgesic properties, morphine (10 mg/kg). In each experiment, one group of mice received spared nerve injury (SNI) surgery to model chronic pain; the other group received a control sham surgery. Both fenobam and MPEP induced preference in the SNI mice, such that SNI mice spent significantly more time in the mGluR5 antagonist-paired chamber compared to a vehicle-paired chamber. No such preference developed for sham mice. Morphine induced preference in male and female mice in both the SNI and sham groups. The results showed that fenobam and MPEP likely reduced on-going distress in the SNI mice, causing them to prefer the chamber paired with the drug compared to the vehicle-paired chamber. Since sham animals did not prefer the drug-paired chamber, these data demonstrate that mGluR5 antagonism is non-rewarding in the absence of pain-like injury.

Requirement of AMPA receptor stimulation for the sustained antidepressant activity of ketamine and LY341495 during the forced swim test in rats

Ketamine, a non-competitive N-methyl-d-aspartate receptor antagonist, and group II metabotropic glutamate (mGlu2/3) receptor antagonists produce antidepressant effects in animal models of depression, which last for at least 24h, through the transient increase in glutamate release, leading to activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor. Both ketamine and an mGlu2/3 receptor antagonist reportedly increase the expression of GluR1, an AMPA receptor subunit, within 24h, which may account for the sustained enhancement of excitatory synaptic transmission following ketamine administration. However, whether the sustained increase in AMPA receptor-mediated synaptic transmission is associated with the antidepressant effects of ketamine and mGlu2/3 receptor antagonists has not yet been investigated.In the present study, to address this question, we tested whether AMPA receptor stimulation at 24h after a single injection of ketamine or an mGlu2/3 receptor antagonist, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY341495) was necessary for the antidepressant effect of these compounds using a forced swim test in rats.A single injection of ketamine or LY341495 at 24h before the test significantly decreased the immobility time. An AMPA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), administered 30min prior to the test significantly and dose-dependently reversed the antidepressant effects of ketamine and LY341495, while NBQX itself had no effect on the immobility time.Our findings suggest that AMPA receptor stimulation at 24h after a single injection of ketamine or LY341495 is required to produce the anti-immobility effects of these compounds. Moreover, the present results provide additional evidence that an mGlu2/3 receptor antagonist may share some of neural mechanisms with ketamine to exert antidepressant effects.

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs.

Reversal of activity‐mediated spine dynamics and learning impairment in a mouse model of Fragile X syndrome

Fragile X syndrome (FXS) is characterized by intellectual disability and autistic traits, and results from the silencing of the FMR1 gene coding for a protein implicated in the regulation of protein synthesis at synapses. The lack of functional Fragile X mental retardation protein has been proposed to result in an excessive signaling of synaptic metabotropic glutamate receptors, leading to alterations of synapse maturation and plasticity. It remains, however, unclear how mechanisms of activity-dependent spine dynamics are affected in Fmr knockout (Fmr1-KO) mice and whether they can be reversed. Here we used a repetitive imaging approach in hippocampal slice cultures to investigate properties of structural plasticity and their modulation by signaling pathways. We found that basal spine turnover was significantly reduced in Fmr1-KO mice, but markedly enhanced by activity. Additionally, activity-mediated spine stabilization was lost in Fmr1-KO mice. Application of the metabotropic glutamate receptor antagonist α-Methyl-4-carboxyphenylglycine (MCPG) enhanced basal turnover, improved spine stability, but failed to reinstate activity-mediated spine stabilization. In contrast, enhancing phosphoinositide-3 kinase (PI3K) signaling, a pathway implicated in various aspects of synaptic plasticity, reversed both basal turnover and activity-mediated spine stabilization. It also restored defective long-term potentiation mechanisms in slices and improved reversal learning in Fmr1-KO mice. These results suggest that modulation of PI3K signaling could contribute to improve the cognitive deficits associated with FXS.

Properties of 4 Hz stimulation‐induced parallel fiber–Purkinje cell presynaptic long‐term plasticity in mouse cerebellar cortex in vivo

Cerebellar parallel fiber-Purkinje cell (PF-PC) long-term synaptic plasticity is important for the formation and stability of cerebellar neuronal circuits, and provides substrates for motor learning and memory. We previously reported both presynaptic long-term potentiation (LTP) and long-term depression (LTD) in cerebellar PF-PC synapses in vitro. However, the expression and mechanisms of cerebellar PF-PC synaptic plasticity in the cerebellar cortex in vivo are poorly understood. In the present study, we studied the properties of 4Hz stimulation-induced PF-PC presynaptic long-term plasticity using in vivo the whole-cell patch-clamp recording technique and pharmacological methods in urethane-anesthetised mice. Our results demonstrated that 4Hz PF stimulation induced presynaptic LTD of PF-PC synaptic transmission in the intact cerebellar cortex in living mice. The PF-PC presynaptic LTD was attenuated by either the N-methyl-D-aspartate receptor antagonist, D-aminophosphonovaleric acid, or the group 1 metabotropic glutamate receptor antagonist, JNJ16259685, and was abolished by combined D-aminophosphonovaleric acid and JNJ16259685, but enhanced by inhibition of nitric oxide synthase. Blockade of cannabinoid type 1 receptor activity abolished the PF-PC LTD and revealed a presynaptic PF-PC LTP. These data indicate that both endocannabinoids and nitric oxide synthase are involved in the 4Hz stimulation-induced PF-PC presynaptic plasticity, but the endocannabinoid-dependent PF-PC presynaptic LTD masked the nitric oxide-mediated PF-PC presynaptic LTP in the cerebellar cortex in urethane-anesthetised mice.

Activation of the mTOR signaling pathway in the antidepressant-like activity of the mGlu5 antagonist MTEP and the mGlu7 agonist AMN082 in the FST in rats

Clinical studies have demonstrated rapid and long-lasting antidepressant effects of ketamine in depressive patients. It has been proposed that these effects are related to changes in synaptogenesis in the mechanism involving mammalian target of rapamycin (mTOR) activation. Similar mechanisms have been proposed for a group II metabotropic glutamate (mGlu) receptor antagonist, LY341495. We aimed to investigate whether other mGlu receptor ligands that produce antidepressant-like effects, namely, the mGlu5 antagonist MTEP and the mGlu7 agonist AMN082, induce the activation of mTOR signaling in the prefrontal cortex (PFC) in rats. AMN082 administered 60 min before the test increased the levels of pmTOR and pp70S6K, and the mTORC1 antagonist rapamycin reversed AMN082-induced changes in the forced swim test (FST) in rats. Furthermore, AMN082 administered 23 h before the decapitation of the rats increased the levels of synapsin I and GluR1, although it did not produce any effect in the FST at the same time point. However, MTEP induced a rapid but unsustained antidepressant-like effect, which was not related to the activation of the mTOR cascade. Finally, the antidepressant-like effects of MTEP or AMN082 were not antagonized by NBQX. In summary, the antidepressant-like activity of MTEP did not depend on the activation of mTOR signaling. However, we observed a unique feature of the mechanism of AMN082. The drug stimulated the mTOR signaling pathway and synaptic protein levels (like ketamine), while it did not induce a sustained antidepressant effect and its action was not directly dependent on AMPA receptor activation (as in classic antidepressants (ADs)).

NMDA receptor-dependent metaplasticity by high-frequency magnetic stimulation.

High-frequency magnetic stimulation (HFMS) can elicit N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal cell synapses. Here, we investigated the priming effect of HFMS on the subsequent magnitude of electrically induced LTP in the CA1 region of rat hippocampal slices using field excitatory postsynaptic potential (fEPSP) recordings. In control slices, electrical high-frequency conditioning stimulation (CS) could reliably induce LTP. In contrast, the same CS protocol resulted in long-term depression when HFMS was delivered to the slice 30 min prior to the electrical stimulation. HFMS-priming was diminished when applied in the presence of the metabotropic glutamate receptor antagonists (RS)-α-methylserine-O-phosphate (MSOP) and (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Moreover, when HFMS was delivered in the presence of the NMDA receptor-antagonist D-2-amino-5-phosphonovalerate (50 µM), CS-induced electrical LTP was again as high as under control conditions in slices without priming. These results demonstrate that HFMS significantly reduced the propensity of subsequent electrical LTP and show that both metabotropic glutamate and NMDA receptor activation were involved in this form of HFMS-induced metaplasticity.

Non-dopaminergic Treatments for Motor Control in Parkinson’s Disease

The pathological processes underlying Parkinson's disease (PD) involve more than dopamine cell loss within the midbrain. These non-dopaminergic neurotransmitters include noradrenergic, serotonergic, glutamatergic, and cholinergic systems within cortical, brainstem and basal ganglia regions. Several non-dopaminergic treatments are now in clinical use to treat motor symptoms of PD, or are being evaluated as potential therapies. Agents for symptomatic monotherapy and as adjunct to dopaminergic therapies for motor symptoms include adenosine A2A antagonists and the mixed monoamine-B inhibitor (MAO-BI) and glutamate release agent safinamide. The largest area of potential use for non-dopaminergic drugs is as add-on therapy for motor fluctuations. Thus adenosine A2A antagonists, safinamide, and the antiepileptic agent zonisamide can extend the duration of action of levodopa. To reduce levodopa-induced dyskinesia, drugs that target overactive glutamatergic neurotransmission can be used, and include the non-selective N-methyl D-aspartate antagonist amantadine. More recently, selective metabotropic glutamate receptor (mGluR₅) antagonists are being evaluated in phase II randomized controlled trials. Serotonergic agents acting as 5-HT2A/2C antagonists, such as the atypical antipsychotic clozapine, may also reduce dyskinesia. 5-HT1A agonists theoretically can reduce dyskinesia, but in practice, may also worsen PD motor symptoms, and so clinical applicability has not yet been shown. Noradrenergic α2A antagonism using fipamezole can potentially reduce dyskinesia. Several non-dopaminergic agents have also been investigated to reduce non-levodopa-responsive motor symptoms such as gait and tremor. Thus the cholinesterase inhibitor donepezil showed mild benefit in gait, while the predominantly noradrenergic re-uptake inhibitor methylphenidate had conflicting results in advanced PD subjects. Tremor in PD may respond to muscarinic M4 cholinergic antagonists (anticholinergics), but tolerability is often poor. Alternatives include β-adrenergic antagonists such as propranolol. Other options include 5-HT2A antagonists, and drugs that have mixed binding properties involving serotonin and acetylcholine, such as clozapine and the antidepressant mirtazapine, can be effective in reducing PD tremor. Many other non-dopaminergic agents are in preclinical and phase I/II early stages of study, and the reader is directed to recent reviews. While levodopa remains the most effective agent to treat motor symptoms in PD, the overall approach to using non-dopaminergic drugs in PD is to reduce reliance on levodopa and to target non-levodopa-responsive symptoms.

Neuroprotective Effects of NMDA and Group I Metabotropic Glutamate Receptor Antagonists Against Neurodegeneration Induced by Homocysteine in Rat Hippocampus: In Vivo Study

Homocysteine (Hcy), a neurotoxic amino acid, is a risk factor for neurodegenerative diseases. Previous in vitro studies have demonstrated that group I metabotropic glutamate receptors along with N-methyl-D-aspartic acid (NMDA) receptors participate in acute and chronic aspects of Hcy-induced neuronal damage. In the present study, we examined whether the same mechanism may be involved in homocysteine neurotoxicity in vivo. Memantine, MPEP, and LY367385 were used as NMDA, mGlu5, and mGlu1 antagonists, respectively. Repeated i.c.v injection of Hcy was performed for three consecutive days. Neuronal loss in different zones of the hippocampus was assessed by Nissl, Fluoro-Jade B, and TUNEL staining. Neuronal degeneration was observed in both types of apoptosis and necrosis. All glutamate receptor antagonists, even when given alone, provided some degree of neuroprotection. The degree of protection was dependent on the area of the hippocampus. While memantine was more potent against Hcy-induced apoptosis, the potency of mGluR antagonists in neuronal protection against apoptosis and necrosis was almost equal. No more protection was observed when all three antagonists were used simultaneously. It seems that Fluoro-Jade could be a useful marker of apoptotic cell death. Taken together, results demonstrate that, in vivo, Hcy neurotoxicity is mediated mainly by the NMDA receptors and group I mGluRs.

Effects of ketamine and LY341495 on the depressive-like behavior of repeated corticosterone-injected rats

In the present study, to further validate repeated corticosterone (CORT)-treated rats as a treatment-resistant depression (TRD) model, we first examined the effect of ketamine, which is known to be effective for the treatment of TRD, on the depressive-like behavior of CORT-treated rats. In this model, ketamine significantly reduced the increased immobility time of CORT-treated rats during the forced swim test (FST), indicating that its efficacy against TRD could be detected using this model. We next examined the effect of LY341495, a group ΙΙ metabotropic glutamate (mGlu2/3) receptor antagonist, in this model to evaluate its potential for the alleviation of TRD. LY341495, similar to ketamine, attenuated the increased immobility time of CORT-treated rats during the FST. Therefore, these results suggest that mGlu2/3 receptor antagonists might be effective for patients with depression, including TRD.

Ionotropic but not metabotropic glutamatergic receptors in the Locus Coeruleus modulate the hypercapnic ventilatory response in unanesthetized rats

Central chemoreception is an important mechanism by which changes in tissue CO2/H+ regulate breathing, and the Locus coeruleus (LC) is one of many putative central chemoreceptor sites. Here, we studied the contribution of LC glutamatergic receptors on ventilatory, cardiovascular and thermal responses to hypercapnia. To this end, we measured pulmonary ventilation (VE), body temperatures (Tb), mean arterial pressure (MAP) and heart rate (fH) of male Wistar rats before and after microinjection of Kynurenic Acid (KY, an ionotropic glutamate receptors antagonist, 10 mM) or α‐methyl‐4‐carboxyphenylglycine (MCPG, a metabotropic glutamate receptor antagonist, 10 ηM) unilateraly into the LC, followed by 60 minutes of air or hypercapnia exposure (7% CO2). Ventilatory response to hypercapnia was higher in animals treated with KY intra‐LC (1899 ± 83 mL kg−1 min−1) compared to the control group (1141 ± 64 mL kg−1 min−1; P < 0.01). However, the MCPG treatment within the LC had no effect on the hypercapnia‐induced hyperpnea. The cardiovascular and thermal control were not affected by hypercapnia or by the injection of KY and MCPG in the LC. These data suggest that the glutamate acting on ionotropic but not metabotropic receptors in the LC exerts an inibitory modulation of hypercapnia‐induced hyperpnea.Financial support: FAPESP, CNPq, PROPe.

Ionotropic but not metabotropic glutamatergic receptors in the locus coeruleus modulate the hypercapnic ventilatory response in unanaesthetized rats

Central chemoreceptors are important to detect changes of CO2/H(+), and the Locus coeruleus (LC) is one of the many putative central chemoreceptor sites. Here, we studied the contribution of LC glutamatergic receptors on ventilatory, cardiovascular and thermal responses to hypercapnia. To this end, we determined pulmonary ventilation (V(E)), body temperatures (T(b)), mean arterial pressure (MAP) and heart rate (HR) of male Wistar rats before and after unilateral microinjection of kynurenic acid (KY, an ionotropic glutamate receptor antagonist, 10 nmol/0.1 μL) or α-methyl-4-carboxyphenylglycine (MCPG, a metabotropic glutamate receptor antagonist, 10 nmol/0.1 μL) into the LC, followed by 60 min of air breathing or hypercapnia exposure (7% CO2). Ventilatory response to hypercapnia was higher in animals treated with KY intra-LC (1918.7 ± 275.4) compared with the control group (1057.8 ± 213.9, P < 0.01). However, the MCPG treatment within the LC had no effect on the hypercapnia-induced hyperpnea. The cardiovascular and thermal controls were not affected by hypercapnia or by the injection of KY and MCPG in the LC. These data suggest that glutamate acting on ionotropic, but not metabotropic, receptors in the LC exerts an inhibitory modulation of hypercapnia-induced hyperpnea.

Role of BDNF/TrkB signaling in antidepressant-like effects of a group II metabotropic glutamate receptor antagonist in animal models of depression

We previously revealed that the activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor and mammalian target of rapamycin signaling contributed to the antidepressant-like effects of group II metabotropic glutamate (mGlu2/3) receptor antagonists, suggesting that the signaling pathway may be similar to the molecular mechanisms underlying the antidepressant-like action of ketamine, a noncompetitive N-methyl-d-aspartate receptor antagonist that exertes rapid and sustained antidepressant effects in patients with depressive disorder. Although brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) signaling reportedly participates in the antidepressant-like effects of ketamine, the involvement of BDNF/TrkB signaling in the action of mGlu2/3 receptor antagonists has not been investigated. We therefore examined whether the activation of BDNF/TrkB signaling is required for the antidepressant-like effects of LY341495, an mGlu2/3 receptor antagonist, in animal models of depression such as the tail suspension test (TST) and the novelty-suppressed feeding test (NSFT). The administration of LY341495 at 30min prior to the test exerted antidepressant-like effects (acute effects) lasting for at least 24h (sustained effects) when evaluated using the TST and NSFT. Pretreatment with K252a, a TrkB tyrosine kinase inhibitor, blocked the sustained, but not the acute, effects of LY341495. These results suggest that BDNF/TrkB signaling may be involved in the sustained antidepressant-like effects of LY341495, as observed for ketamine treatment.

Cell type-specific regulation of inhibition via cannabinoid type 1 receptors in rat neocortex

Endogenous cannabinoid type 1 (CB1) receptors demonstrate a cell type-specific expression and are potent modulators of synaptic transmission within the central nervous system. We aimed to investigate whether two classes of multipolar interneuron in the neocortex displayed a form of short-term synaptic plasticity, depolarization-induced suppression of inhibition (DSI), and whether the DSI was mediated by a common receptor. Paired whole cell recordings combined with biocytin labeling were performed between pyramidal cells and either multipolar adapting or multipolar nonadapting interneurons in layers II-IV of male Wistar rat (postnatal day 17-22) somatosensory cortex. Inhibitory postsynaptic potentials elicited by multipolar adapting interneurons were sensitive to DSI, which was blocked by the CB1 receptor antagonist AM-251 (8 μM), indicating that the suppression of inhibition was mediated by CB1 receptors. Two subpopulations of multipolar nonadapting interneuron-to-pyramidal cell connections were discovered on the basis of their susceptibility to DSI. Whereas 50% were insensitive to DSI, the remaining half were sensitive to DSI, which could not be prevented by AM-251. DSI at these connections was also insensitive to the group I (mGluRIa) and III metabotropic glutamate receptor antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (100 μM) and (RS)-α-cyclopropyl-4-phosphonophenylglycine (100 μM) and the group III agonist l-2-amino-4-phosphonobutanoate (50 μM). However, multipolar nonadapting interneuron-to-pyramidal cell connections were sensitive to the endocannabinoid anandamide (9 μM), mimicking the effects of DSI, which also could not be prevented by AM-251, implying a CB1 receptor-independent suppression of inhibition. These results reveal an interneuron type-specific modulation of synaptic transmission via CB receptors in the neocortex.

Peripheral nerve injury produces a sustained shift in the balance between glutamate release and uptake in the dorsal horn of the spinal cord

Peripheral nerve injury provokes heightened excitability of primary sensory afferents including nociceptors, and elicits ectopic activity in lesioned and neighboring intact nerve fibers. The major transmitter released by sensory afferents in the superficial dorsal horn of the spinal cord is glutamate. Glutamate is critically involved in nociceptive signaling and the development of neuropathic pain. We recorded miniature excitatory postsynaptic currents (mEPSCs) from neurons in lamina II of the rat dorsal horn to assess spontaneous synaptic activity after spared nerve injury (SNI), a model of chronic neuropathic pain. Following SNI, the frequency of mEPSCs doubled, indicating heightened glutamate release from primary afferents or spinal interneurons. Consistent with this finding, glutamate concentrations in the cerebrospinal fluid were elevated at 1 and 4weeks after SNI. Transmitter uptake was insufficient to prevent the rise in extracellular glutamate as the expression of glutamate transporters remained unchanged or decreased. 2-Methyl-6-(phenylethynyl)pyridine hydrochloride, an antagonist of metabotropic glutamate receptor 5 (mGluR5), reduced the frequency of mEPSCs to its preinjury level, suggesting a positive feedback mechanism that involves facilitation of transmitter release by mGluR5 activation in the presence of high extracellular glutamate. Treatment with the β-lactam antibiotic ceftriaxone increased the expression of glutamate transporter 1 (Glt1) in the dorsal horn after SNI, raised transmitter uptake, and lowered extracellular glutamate. Improving glutamate clearance prevented the facilitation of transmitter release by mGluR5 and attenuated neuropathic pain-like behavior. Balancing glutamate release and uptake after nerve injury should be an important target in the management of chronic neuropathic pain.

Clinical studies with the metabotropic glutamate receptor antagonist AFQ056voglurant in adults and adolescents with fragile X syndrome

Clinical studies with the metabotropic glutamate receptor antagonist AFQ056voglurant in adults and adolescents with fragile X syndrome

Parabrachial complex glutamate receptors modulate the cardiorespiratory response evoked from hypothalamic defense area

To characterize the possible role of glutamate in the interaction between Hypothalamic Defense Area (HDA) and Parabrachial complex (PBc) nuclei, cardiorespiratory changes were analyzed in response to electrical stimulation of the HDA (1 ms pulses, 30-50 μA given at 100 Hz for 5s) before and after the microinjection of the nonspecific glutamate receptor antagonist kynurenic acid (50 nl, 5 nmol), NMDA receptor antagonist MK-801 (50 nl, 50 nmol), non-NMDA receptor antagonist CNQX (50 nl, 50 nmol) or metabotropic glutamate receptor antagonist MCPG (50 nl, 5 nmol) within the PBc. HDA stimulation evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (p<0.001) due to a decrease in expiratory time (p<0.01). The respiratory response was accompanied by a pressor (p<0.001) and a tachycardic response (p<0.001). Kynurenic acid within the lateral parabrachial region (lPB) abolished the tachycardia (p<0.001) and decreased the magnitude of blood pressure response (p<0.001) to HDA stimulation. Similarly, the magnitude of the tachycardia and the pressor response was decreased after the microinjection of MK-801 (p<0.01 and p<0.001, respectively) and CNQX (p<0.05 in both cases) into the lPB. Kynurenic acid microinjection in this region produced an inhibition of the tachypnea (p<0.001) to HDA stimulation but the respiratory response persisted unchanged after MK-801 or CNQX microinjection into the lPB. Kynurenic acid within the medial parabrachial region (mPB) abolished the tachycardia (p<0.01) and decreased the magnitude of the pressor response (p<0.001) to HDA stimulation. MK-801 and CNQX microinjection in this region decreased the magnitude of the tachycardia (p<0.05, in both cases) and pressor response (p<0.05, in both cases). The respiratory response evoked by HDA stimulation was not changed after the microinjection of kynurenic acid, MK-801 or CNQX within the mPB. No changes were observed in the cardiorespiratory response evoked to HDA stimulation after MCPG microinjection within lPB and mPB. These results indicate that glutamate PBc receptors are involved in the cardiorespiratory response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

NMDA Receptor-Dependent Synaptic Activation of TRPC Channels in Olfactory Bulb Granule Cells

Canonical transient receptor potential (TRPC) channels are widely expressed throughout the nervous system including the olfactory bulb where their function is largely unknown. Here, we describe their contribution to central synaptic processing at the reciprocal mitral and tufted cell-granule cell microcircuit, the most abundant synapse of the mammalian olfactory bulb. Suprathreshold activation of the synapse causes sodium action potentials in mouse granule cells and a subsequent long-lasting depolarization (LLD) linked to a global dendritic postsynaptic calcium signal recorded with two-photon laser-scanning microscopy. These signals are not observed after action potentials evoked by current injection in the same cells. The LLD persists in the presence of group I metabotropic glutamate receptor antagonists but is entirely absent from granule cells deficient for the NMDA receptor subunit NR1. Moreover, both depolarization and Ca²⁺ rise are sensitive to the blockade of NMDA receptors. The LLD and the accompanying Ca²⁺ rise are also absent in granule cells from mice deficient for both TRPC channel subtypes 1 and 4, whereas the deletion of either TRPC1 or TRPC4 results in only a partial reduction of the LLD. Recordings from mitral cells in the absence of both subunits reveal a reduction of asynchronous neurotransmitter release from the granule cells during recurrent inhibition. We conclude that TRPC1 and TRPC4 can be activated downstream of NMDA receptor activation and contribute to slow synaptic transmission in the olfactory bulb, including the calcium dynamics required for asynchronous release from the granule cell spine.

SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABAA receptor channels. In a recent study, we showed that Zn2+ regulates GABAA reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn2+ initiates this process by activating the Gq-coupled metabotropic Zn2+ receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca2+ indicator and exposing cells to Zn2+, which triggered consistent Ca2+ responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn2+ treatment under these conditions did not increase the intracellular concentrations of Zn2+ itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH4+ influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn2+ pretreatment induced a Ca2+-dependent increase in KCC2 activity. The effects of Zn2+ on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39−/− mice, suggesting that Zn2+ acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn2+-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn2+. These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn2+ stimulation of mZnR/GPR39.

Investigation of the antidyskinetic site of action of metabotropic and ionotropic glutamate receptor antagonists. Intracerebral infusions in 6-hydroxydopamine-lesioned rats with levodopa-induced dyskinesia

Long-term levodopa replacement therapy in Parkinson's disease is confounded by abnormal involuntary movements, known as levodopa induced dyskinesia (LID). Dysfunctional glutamatergic neurotransmission has been implicated in the pathogenesis of LID making metabotropic and ionotropic glutamate receptors attractive novel therapeutic targets. The objective of the present study was to investigate the antidyskinetic site of action of different glutamate receptor antagonists in the brain. For that purpose, metabotropic glutamate subtype 5 (3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride, MTEP), NMDA NR2B selective ((aR,bS)-a-(4-Hydroxyphenyl)-b-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate, Ro 25-6981) and AMPA (2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, NBQX) receptor antagonists or saline were administered by intracerebral infusion in the caudate–putamen (CPu), the substantia nigra zona reticulata (SNr) or the subthalamic nucleus (STN) of 6-hydroxydopamine-lesioned rats exhibiting LID. Dyskinesia was assessed with the modified version of the rat Abnormal Involuntary Movements scale (AIMS). Ro 25-6981 and to a lesser extent NBQX improved dyskinesia (82% and 19% reduction in AIM score respectively) after infusion in the caudate–putamen. None of the three drugs managed to noticeably reduce AIM score after infusion in the SNr. MTEP was the only drug that produced a reduction in AIM score (48%) when infused in STN. In conclusion, while the striatum proved important in the antidyskinetic action of NMDA and AMPA receptor antagonists, the results of this study highlight also the importance of the metabotropic glutamate receptors that reside in the STN as therapeutic targets in the treatment of LID.