mGluR Agonist DHPG Research Articles

ArcKR expression modifies synaptic plasticity following epileptic activity: Differential effects with invitro and invivo seizure-induction protocols.

Pathological forms of neural activity, such as epileptic seizures, modify the expression pattern of multiple proteins, leading to persistent changes in brain function. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which is critically involved in protein-synthesis-dependent synaptic plasticity underlying learning and memory. In the present study, we have investigated how the expression of ArcKR, a form of Arc in which the ubiquitination sites have been mutated, resulting in slowed Arc degradation, modifies group I metabotropic glutamate receptor-mediated long-term depression (G1-mGluR-LTD) following seizures. We used a knock-in mice line that express ArcKR and two hyperexcitation models: an invitro model, where hippocampal slices were exposed to zero Mg2+, 6 mM K+; and an invivo model, where kainic acid was injected unilaterally into the hippocampus. In both models, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices in response to Schaffer collateral stimulation and G1-mGluR-LTD was induced chemically with the group 1 mGluR agonist DHPG. In the invitro model, ArcKR expression enhanced the effects of seizure activity and increased the magnitude of G1-mGluR LTD, an effect that could be blocked with the mGluR5 antagonist MTEP. In the invivo model, fEPSPs were significantly smaller in slices from ArcKR mice and were less contaminated by population spikes. In this model, the amount of G1-mGluR-LTD was significantly less in epileptic slices from ArcKR mice as compared to wildtype (WT) mice. We have shown that expression of ArcKR, a form of Arc in which degradation is reduced, significantly modulates the magnitude of G1-mGluR-LTD following epileptic seizures. However, the effect of ArcKR on LTD depends on the epileptic model used, with enhancement of LTD in an invitro model and a reduction in the kainate mouse model.

Group III Metabotropic Glutamate Receptors Regulate Excitability of Hypothalamic Presympathetic Neurons and Sympathetic Output in Hypertension

Enhanced glutamatergic input to the hypothalamic paraventricular nucleus (PVN) contributes to the development of hypertension. Group III metabotropic glutamate receptors (mGluRs) are expressed in the PVN. In the present study, we determined the role of group III mGluRs in the control of the excitability of spinally projecting PVN neurons and sympathetic outflow in hypertension. We found that stimulation of group III mGluRs with L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) consistently inhibited the firing activity of PVN neurons in normotensive WKY rats. However, L‐AP4 inhibited 37%, but excited 48%, of spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs). Both the inhibitory and excitatory effects of L‐AP4 on the firing activity were abolished by the group III mGluR antagonist CPPG. Inclusion of GDP‐β‐S in the intracellular recording solution eliminated the excitatory effect of L‐AP4 on the firing activity of PVN neurons in SHRs, but it did not significantly change the inhibitory effect of L‐AP4 on PVN neurons in WKY rats or SHRs. L‐AP4 significantly reduced the frequency of spontaneous excitatory postsynaptic currents of PVN neurons in both WKY rats and SHRs. Interestingly, pretreatment of brain slices with the group I mGluR agonist DHPG converted the L‐AP4 effect on PVN neurons from inhibitory to excitatory in WKY rats. Also, L‐AP4 consistently inhibited PVN neurons in SHRs after treatment with the group I mGluR antagonist MPEP. In addition, microinjection of L‐AP4 into the PVN dose‐dependently decreased arterial Bp and lumbar sympathetic nerve activity (LSNA) in WKY rats and SHRs. Microinjection of L‐AP4 in the presence of MPEP produced a more significant reduction in arterial BP and LSNA than did with microinjection of L‐AP4 alone in SHRs. These findings suggest that group III mGluRs in the PVN regulate the synaptic plasticity and excitability of PVN presympathetic neurons and sympathetic vasomotor activity in hypertension. The signaling of postsynaptic group III mGluRs is influenced by increased group I mGluR activity in PVN presympathetic neurons in hypertension. Supported by NIH grants HL131161 and HL142133.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Soluble Guanylate Cyclase As the Key Enzyme in the Modulating Effect of NO on Metabotropic Glutamate Receptors

The synaptic plasticity of the afferent synapse of the vestibular apparatus is defined by the dynamic interaction of ionotropic and metabotropic glutamate receptors and the modulators of synaptic transmission. It was shown that nitric oxide modulates iGluR responses. In this paper, the effect of NO on the function of the afferent synapse mGluR was investigated. Inhibitor of nitric oxide synthase lowered the level of background activity but increased the amplitude of the responses of groups I and II mGluR agonist ACPD. Donor NO SNAP increased the level of background activity. Short-term perfusion of the synaptic region with low concentrations of SNAP led to a decrease in the amplitude of the answers of mGluR agonists ACPD and DHPG. The inhibitory effect of the NO donor was eliminated under blockade of soluble guanylate cyclase with a specific inhibitor ODQ. A prolonged application of NO did not cause a statistically significant change in the amplitude of the ACPD response. However, SNAP at concentrations of 10 and 100 μM increased the amplitude of the mGluR agonist responses 30 and 15 minutes, respectively, after termination of the NO donor exposure. The obtained data show the multidirectional effect of NO on the function of mGluR and testify to the existence of a complex modulating mechanism of the afferent flow from vestibular organs to the central nervous system.

Endocannabinoids control vesicle release mode at midbrain periaqueductal grey inhibitory synapses.

The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA. The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices. Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular. Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode. Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release. These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition. The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress-induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)-3,5-dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca2+ , and when release probability was reduced by lowering Ca2+ . However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca2+ . Lowering external Ca2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca2+ . The low affinity GABAA receptor antagonist TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca2+ . Together these findings indicate that the normal mode of GABA release is multivesicular within the PAG, and that DHPG and lowering external Ca2+ switch this to a univesicular mode. The effects of DHPG were mediated by mGlu5 receptor engagement of the retrograde endocannabinoid system. Blockade of endocannabinoid breakdown produced a similar shift in the mode of release. We conclude that endocannabinoids control both the mode and the probability of GABA release within the PAG.

MGluR1 and mGluR5 Synergistically Control Cholinergic Synaptic Transmission in the Thalamic Reticular Nucleus.

Acetylcholine (ACh) signaling is involved in a wide range of processes, including arousal, attention, and learning. An increasing number of studies indicate that cholinergic control of these functions is highly deterministic, mediated by synaptic afferents that generate reliable and precise responses in postsynaptic neurons. However, mechanisms that govern plastic changes of cholinergic synaptic strength are poorly understood, even though they are likely critical in shaping the impact of cholinergic inputs on neuronal networks. We have recently shown that in the thalamic reticular nucleus (TRN), synaptic release of ACh generates excitatory-inhibitory biphasic postsynaptic responses, mediated by the activation of α4β2 nicotinic (nAChRs) and M2 muscarinic receptors (mAChRs), respectively. Here, using voltage-clamp recordings from TRN neurons in thalamocortical slices of mice, we demonstrate that the activation of Group I metabotropic glutamate receptors (mGluRs) by ambient or synaptically released glutamate evokes transient increases of nicotinic EPSCs. Additionally, we find that the selective Group I mGluR agonist DHPG [(S)-3,5-dihydroxyphenylglycine] evokes long-term potentiation of nicotinic EPSCs (mGluR-nLTP), dependent on increases in postsynaptic Ca(2+) concentration and the activation of phospholipase C. Both the induction and the maintenance of mGluR-nLTP require synergistic activation of mGluR1 and mGluR5. Together, our results show that postsynaptic Group I mGluRs are critically involved in the regulation of cholinergic synaptic strength on different time scales, suggesting that cholinergic control of local thalamic circuits is highly context-dependent and regulated by the overall levels of glutamatergic afferent activity. Cholinergic signaling controls information processing and plasticity in neuronal circuits, but the mechanisms underlying the regulation of cholinergic synaptic strength on different time scales are unknown. Here we identify mGluR1 and mGluR5 as key elements in the dynamic regulation of cholinergic synaptic inputs onto neurons of the TRN. Our findings highlight potential mechanisms that regulate cholinergic signaling in the mammalian brain.

MGluR-mediated calcium signalling in the thalamic reticular nucleus

The thalamic reticular nucleus (TRN) plays a major role in modulating the transfer of information from the thalamus to the cortex. GABAergic inhibition via the TRN is differentially regulated by metabotropic glutamate receptors (mGluRs) and the effect of mGluRs on the membrane potential, on ion channels, and on the plasticity of electrical coupling of TRN neurons has been studied previously. Although mGluRs are generally known to trigger Ca2+ transients, mGluR-mediated Ca2+-transients in TRN neurons have not yet been investigated. In this study, we show that mGluRs can trigger Ca2+-transients in TRN neurons, that these transients depend on intracellular Ca2+-stores, and are mediated by IP3 receptors. Ca2+ transients caused by the group I mGluR agonist DHPG elicit a current that is sensitive to flufenamic acid and has a reversal potential around −40mV. Our results add mGluR-mediated Ca2+-signalling in the TRN to the state-dependent modulators of the thalamocortical system.

Sex Differences in Molecular Signaling at Inhibitory Synapses in the Hippocampus.

Many brain disorders vary between the sexes, yet the degree to which this variation arises from differential experience versus intrinsic biological sex differences is unclear. In this study, we demonstrate intrinsic sex differences in molecular regulation of a key neuromodulatory system, the endocannabinoid system, in the hippocampus. Endocannabinoids are involved in diverse aspects of physiology and behavior that involve the hippocampus, including cognitive and motivational state, responses to stress, and neurological disorders such as epilepsy. Our finding that molecular regulation of the endocannabinoid system differs between the sexes suggests mechanisms through which experiences or therapeutics that engage endocannabinoids could affect males and females differently.

MGluR5 protect astrocytes from ischemic damage in postnatal CNS white matter

Astrocytes perform essential neuron-supporting functions in the central nervous system (CNS) and their disruption has devastating effects on neuronal integrity in multiple neuropathologies. Although astrocytes are considered resistant to most pathological insults, ischemia can result in astrocyte injury and astrocytes in postnatal white matter are particularly vulnerable. Metabotropic glutamate receptors (mGluR) are neuroprotective in ischemia and are widely expressed by astrocytes throughout CNS grey matter, but their potential cytoprotective role in astrocytes had not been determined. Here, we identify functional expression of group I mGluR in white matter astrocytes and demonstrate their activation protects astrocytes from ischemic damage in the postnatal mouse optic nerve. Optic nerve astrocytes are shown to express mGluR5 using immunolabelling of sections and explant cultures from transgenic reporter mice in which GFAP drives expression of EGFP. In addition, using Fluo-4 calcium imaging in isolated intact optic nerves, we show that the group I/II mGluR agonist ACPD and the specific group I mGluR agonist DHPG evoke glial Ca2+ signals that were significantly inhibited by the group I mGluR antagonist AIDA. A key finding is that activation of group I mGluR protects astrocytes against oxygen-glucose deprivation (OGD) in situ, in isolated intact optic nerves from GFAP-EGFP mice. This study identifies a role for group I mGluR in protecting astrocytes against ischemia in postnatal white matter and suggests this may be a strategy for limiting damage in neuropathologies involving excitotoxity.

Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations

The activation of Group I metabotropic glutamate receptors (GI mGluRs) in the hippocampus results in the appearance of persistent bursts of synchronised neuronal activity. In response to other stimuli, such activity is known to cause the release of the purines ATP and its neuroactive metabolite, adenosine. We have thus investigated the potential release and role of the purines during GI mGluR-induced oscillations in rat hippocampal areas CA3 and CA1 using pharmacological techniques and microelectrode biosensors for ATP and adenosine. The GI mGluR agonist DHPG induced both persistent oscillations in neuronal activity and the release of adenosine in areas CA1 and CA3. In contrast, the DHPG-induced release of ATP was only observed in area CA3. Whilst adenosine acting at adenosine A1 receptors suppressed DHPG-induced burst activity, the activation of mGlu5 and P2Y1 ATP receptors were necessary for the induction of DHPG-induced oscillations. Selective inhibition of pannexin-1 hemichannels with a low concentration of carbenoxolone (10 μM) or probenecid (1 mM) did not affect adenosine release in area CA3, but prevented both ATP release in area CA3 and DHPG-induced bursting. These data reveal key aspects of GI mGluR-dependent neuronal activity that are subject to bidirectional regulation by ATP and adenosine in the initiation and pacing of burst firing, respectively, and which have implications for the role of GI mGluRs in seizure activity and neurodevelopmental disorders.

Group I metabotropic glutamate receptors: a potential target for regulation of proliferation and differentiation of an immortalized human neural stem cell line.

Human neural stem cells (NSCs) from the developing embryo or the subventricular zone of the adult brain can potentially elicit brain repair after injury or disease, either via endogenous cell proliferation or by cell transplantation. Profound knowledge of the diverse signals affecting these cells is, however, needed to realize their therapeutic potential. Glutamate and group I metabotropic glutamate receptors (mGluRs) affect proliferation and survival of rodent NSCs both during embryonic and post-natal development. To investigate the role of group I mGluRs (mGluR1 and mGluR5) on human NSCs, we differentiated an immortalized, forebrain-derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists, MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were present on the cells. Addition of glutamate to the growth medium significantly increased cell proliferation and reduced cell death, resulting in increased cell numbers. In the presence of glutamate, selective activation of group I mGluRs reduced gliogenesis, whereas selective inhibition of group I mGluRs reduced neurogenesis. Our results substantiate the importance of glutamate signalling in the regulation of human NSCs and may as such be applied to promote proliferation and neuronal differentiation.

Inositol 1,4,5-Triphosphate Drives Glutamatergic and Cholinergic Inhibition Selectively in Spiny Projection Neurons in the Striatum

The striatum is critically involved in the selection of appropriate actions in a constantly changing environment. The spiking activity of striatal spiny projection neurons (SPNs), driven by extrinsic glutamatergic inputs, is shaped by local GABAergic and cholinergic networks. For example, it is well established that different types of GABAergic interneurons, activated by extrinsic glutamatergic and local cholinergic inputs, mediate powerful feedforward inhibition of SPN activity. In this study, using mouse striatal slices, we show that glutamatergic and cholinergic inputs exert direct inhibitory regulation of SPN activity via activation of metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors. While pressure ejection of the group I mGluR (mGluR1/5) agonist DHPG [(S)-3,5-dihydroxyphenylglycine] equally engages both mGluR1 and mGluR5 subtypes, the mGluR-dependent component of IPSCs elicited by intrastriatal electrical stimulation is almost exclusively mediated by the mGluR1 subtype. Ca(2+) release from intracellular stores specifically through inositol 1,4,5-triphospahte receptors (IP(3)Rs) and not ryanodine receptors (RyRs) mediates this form of inhibition by gating two types of Ca(2+)-activated K(+) channels (i.e., small-conductance SK channels and large-conductance BK channels). Conversely, spike-evoked Ca(2+) influx triggers Ca(2+) release solely through RyRs to generate SK-dependent slow afterhyperpolarizations, demonstrating functional segregation of IP(3)Rs and RyRs. Finally, IP(3)-induced Ca(2+) release is uniquely observed in SPNs and not in different types of interneurons in the striatum. These results demonstrate that IP(3)-mediated activation of SK and BK channels provides a robust mechanism for glutamatergic and cholinergic inputs to selectively suppress striatal output neuron activity.

Metabotropic Glutamate Receptor I (mGluR1) Antagonism Impairs Cocaine-Induced Conditioned Place Preference via Inhibition of Protein Synthesis

Antagonism of group I metabotropic glutamate receptors (mGluR1 and mGluR5) reduces behavioral effects of drugs of abuse, including cocaine. However, the underlying mechanisms remain poorly understood. Activation of mGluR5 increases protein synthesis at synapses. Although mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome, it remains unknown whether group I mGluR-mediated protein synthesis is involved in any behavioral effects of drugs of abuse. We report that group I mGluR agonist DHPG induced more pronounced initial depression of inhibitory postsynaptic currents (IPSCs) followed by modest long-term depression (I-LTD) in dopamine neurons of rat ventral tegmental area (VTA) through the activation of mGluR1. The early component of DHPG-induced depression of IPSCs was mediated by the cannabinoid CB1 receptors, while DHPG-induced I-LTD was dependent on protein synthesis. Western blotting analysis indicates that mGluR1 was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. We also show that cocaine conditioning activated translation machinery in the VTA via an mGluR1-dependent mechanism. Furthermore, intra-VTA microinjections of mGluR1 antagonist JNJ16259685 and protein synthesis inhibitor cycloheximide significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP) and activation of translation elongation factors. Taken together, these results suggest that mGluR1 antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine.

Astrocyte GRK2 as a novel regulator of glutamate transport and brain damage

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates cellular signaling via desensitization of GPCRs and by direct interaction with intracellular signaling molecules. We recently described that ischemic brain injury decreases cerebral GRK2 levels. Here we studied the effect of astrocyte GRK2-deficiency on neonatal brain damage in vivo. As astrocytes protect neurons by taking up glutamate via plasma-membrane transporters, we also studied the effect of GRK2 on the localization of the GLutamate ASpartate Transporter (GLAST).Brain damage induced by hypoxia–ischemia was significantly reduced in GFAP-GRK2+/− mice, which have a 60% reduction in astrocyte GRK2 compared to GFAP-WT littermates. In addition, GRK2-deficient astrocytes have higher plasma-membrane levels of GLAST and an increased capacity to take up glutamate in vitro.In search for the mechanism by which GRK2 regulates GLAST expression, we observed increased GFAP levels in GRK2-deficient astrocytes. GFAP and the cytoskeletal protein ezrin are known regulators of GLAST localization. In line with this evidence, GRK2-deficiency reduced phosphorylation of the GRK2 substrate ezrin and enforced plasma-membrane GLAST association after stimulation with the group I mGluR-agonist DHPG. When ezrin was silenced, the enhanced plasma-membrane GLAST association in DHPG-exposed GRK2-deficient astrocytes was prevented.In conclusion, we identified a novel role of astrocyte GRK2 in regulating plasma-membrane GLAST localization via an ezrin-dependent route. We demonstrate that the 60% reduction in astrocyte GRK2 protein level that is observed in GFAP-GRK2+/− mice is sufficient to significantly reduce neonatal ischemic brain damage. These findings underline the critical role of GRK2 regulation in astrocytes for dampening the extent of brain damage after ischemia.

Role of group 1 metabotropic glutamate receptors (mGluR) in the hypothalamic paraventricular nucleus (PVN) in sympathetic and blood pressure control during water deprivation

Compared to WKY rats, sympathetic nerve activity (SNA) and elevated arterial pressure (AP) in spontaneous hypertensive rats (SHR) is supported by enhanced glutamatergic drive of group 1 mGluR (mGluR1 & mGluR5) in the PVN. Whether enhanced group 1 mGluR activity is a genetically determined feature of sympathetic control unique to SHR or is a mechanism of PVN activation common to other hypertensive states is undetermined. To investigate this, euhydrated (EU) and 48 h water deprived (WD) rats were studied, as dehydration also acutely increases AP by increasing glutamatergic drive of the PVN. In conscious EU rats, injection of the group 1 mGluR agonist DHPG (2 nmol in 200 nl) increased AP (n=3, p<0.05). This effect was attenuated by prior PVN injection of the mGluR1 antagonist LY‐367385 (4 nmol in 200 nl; n=3, p<0.05), but not the mGluR5 antagonist MPEP (4 nmol in 200 nl; n=2). Injections of LY and MPEP alone were each without affect in EU (n=2) and WD rats (n=3). Similar results were obtained in EU (n=3) and WD (n=2) rats under anesthesia in which renal SNA was also unaffected by blockade of PVN group 1 mGluR. Thus, whereas activation of PVN group 1 mGluR is sympathoexcitatory and contributes to sympathetic support of ongoing AP in SHR, the latter appears not to be a common mechanism of PVN driven support of SNA or elevated AP, as no tonic role for PVN group 1 mGluR activity was observed either in EU or 48 h WD rats. HL102310 and HL088052 (GMT)

Differential regulation of voltage-gated Ca 2+ currents and metabotropic glutamate receptor activity by measles virus infection in rat cortical neurons

Measles virus (MV) infection may lead to severe chronic CNS disease processes, including MV-induced encephalitis. Because the intracellular Ca 2+ concentration ([Ca 2+] i) is a major determinant of the (patho-)physiological state in all cells we asked whether important Ca 2+ conducting pathways are affected by MV infection in cultured cortical rat neurons. Patch-clamp measurements revealed a decrease in voltage-gated Ca 2+ currents during MV-infection, while voltage-gated K + currents and NMDA-evoked currents were unaffected. Calcium-imaging experiments using 50 mM extracellular KCl showed reduced [Ca 2+] i increases in MV-infected neurons, confirming a decreased activity of voltage-gated Ca 2+ channels. In contrast, the group-I metabotropic glutamate receptor (mGluR) agonist DHPG evoked changes in [Ca 2+] i that were increased in MV-infected cells. Our results show that MV infection conversely regulates Ca 2+ signals induced by group-I mGluRs and by voltage-gated Ca 2+ channels, suggesting that these physiological impairments may contribute to an altered function of cortical neurons during MV-induced encephalitis.

A novel non‐CB1/TRPV1 endocannabinoid‐mediated mechanism depresses excitatory synapses on hippocampal CA1 interneurons

Endocannabinoids (eCBs) mediate various forms of synaptic plasticity at excitatory and inhibitory synapses in the brain. The eCB anandamide binds to several receptors including the transient receptor potential vanilloid 1 (TRPV1) and cannabinoid receptor 1 (CB1). We recently identified that TRPV1 is required for long-term depression at excitatory synapses on CA1 hippocampal stratum radiatum interneurons. Here we performed whole-cell patch clamp recordings from CA1 stratum radiatum interneurons in rat brain slices to investigate the effect of the eCB anandamide on excitatory synapses as well as the involvement of Group I metabotropic glutamate receptors (mGluRs), which have been reported to produce eCBs endogenously. Application of the nonhydrolysable anandamide analog R-methanandamide depressed excitatory transmission to CA1 stratum radiatum interneurons by ∼50%. The Group I mGluR agonist DHPG also depressed excitatory glutamatergic transmission onto interneurons to a similar degree, and this depression was blocked by the mGluR5 antagonist MPEP (10 μM) but not by the mGluR1 antagonist CPCCOEt (50 μM). Interestingly, however, neither DHPG-mediated nor R-methanandamide-mediated depression was blocked by the TRPV1 antagonist capsazepine (10 μM), the CB1 antagonist AM-251 (2 μM) or a combination of both, suggesting the presence of a novel eCB receptor or anandamide target at excitatory hippocampal synapses. DHPG also occluded R-methanandamide depression, suggesting the possibility that the two drugs elicit synaptic depression via a shared signaling mechanism. Collectively, this study illustrates a novel CB1/TRPV1-independent eCB pathway present in the hippocampus that mediates depression at excitatory synapses on CA1 stratum radiatum interneurons.

Increased Group I Metabotropic Glutamate Receptor Activity in Paraventricular Nucleus Contributes to Elevated Sympathetic Vasomotor Tone in Hypertension

Enhanced glutamatergic input in the paraventricular nucleus (PVN) contributes to elevated sympathetic outflow in hypertension. We determined the role of group I metabotropic glutamate receptors (mGluRs) in the PVN in the control of sympathetic outflow in spontaneously hypertensive rats (SHR). Lumbar sympathetic nerve activity (LSNA), arterial blood pressure (Bp), and heart rate (HR) were recorded from anesthetized SHR and WKY rats. Bilateral microinjection of MPEP (1–10 nmol), a mGluR5 antagonist, or LY367385 (10–100 nmol), a mGluR1 antagonist, into the PVN dose‐dependently decreased LSNA and Bp in SHR but not in WKY. MPEP‐induced decreases in LSNA and Bp were greater than that produced by LY367385 in SHR. Furthermore, microinjection of group I mGluR agonist DHPG (10–100 nmol) caused dose‐dependent increase in LSNA and Bp in both WKY and SHR. DHPG‐induced responses were significantly attenuated by MPEP or LY367385 and abolished by a combination of MPEP and LY367385 in both WKY and SHR. In addition, prior injection of NMDA receptor antagonist AP5 (1.4 nmol) into the PVN decreased LSNA and Bp in SHR and attenuated DHPG‐induced sympathoexcitation in both WKY and SHR. Thus, our study suggests that elevated sympathetic outflow is maintained by activation of group I mGluR receptors in the PVN in SHR. Increased group I mGluR receptor activity contributes to increased glutamatergic input via NMDA receptor in hypertension.

Elongation Factor 2 and Fragile X Mental Retardation Protein Control the Dynamic Translation of Arc/Arg3.1 Essential for mGluR-LTD

Group I metabotropic glutamate receptors (mGluR) induce long-term depression (LTD) that requires protein synthesis. Here, we demonstrate that Arc/Arg3.1 is translationally induced within 5 min of mGluR activation, and this response is essential for mGluR-dependent LTD. The increase in Arc/Arg3.1 translation requires eEF2K, a Ca(2+)/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eEF2. Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis but simultaneously increases Arc/Arg3.1 translation. Genetic deletion of eEF2K results in a selective deficit of rapid mGluR-dependent Arc/Arg3.1 translation and mGluR-LTD. This rapid translational mechanism is disrupted in the fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis but does require Arc/Arg3.1. We propose a model in which eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc/Arg3.1 mRNA in dendrites that is critical for synapse-specific LTD.

A ‘group pacemaker’ mechanism for respiratory rhythm generation

Central pattern generator (CPG) networks emit neural rhythms that drive behaviours such as breathing, locomotion and mastication. Understanding their mechanisms of rhythmogenesis is a longstanding problem. One contemporary debate focuses on whether pacemaker neurons that encapsulate network activity in their intrinsic autorhythmicity or emergent network properties are the fundamental basis for CPG function. Pacemakerdriven mechanisms are intuitive and straightforward – examples in the CNS of many animals from invertebrates to mammals abound – but emergent properties can be abstruse and ill-defined. In this issue of The Journal of Physiology, Mironov (2008) presents a breakthrough analysis of emergent network properties – and emphasizes their importance – using the respiratory oscillator in the pre-Botzinger complex (preBotC) of the ventral medulla in mammals as a model system (Smith et al. 1991; Feldman & Del Negro, 2006). The major proposal for emergent network properties in the preBotC – dubbed the group-pacemaker hypothesis – posited that periodic inspiratory bursts originated due to intrinsic currents, which are ordinarily latent and unavailable, except when evoked synaptically in the context of network function (Rekling & Feldman, 1998). Ca2+-activated non-specific cation current (ICAN) has been recognized as a predominant inspiratory burst-generating current coupled to metabotropic glutamate receptors (mGluRs) (Pace et al. 2007) that could satisfy the group-pacemaker mechanism. Mironov now validates the group-pacemaker hypothesis; he shows the subcellular machinery for synaptically evoking ICAN, and then manipulates this machinery to perturb and/or abolish respiratory rhythms in vitro. Performing on-cell recordings in the preBotC while monitoring inspiratory motor output from the hypoglossal (XII) nerve, Mironov recorded ion channel activity prior to – and during – the inspiratory burst. TRPM4, and its close relative TRPM5 (subtypes of the transient receptor potential, i.e. TRP, family) are monovalent cation channels activated by intracellular Ca2+. Both subtypes were previously suggested to constitute ICAN (Crowder et al. 2007). Mironov establishes that the underlying channels in the soma are TRPM4 by testing their sensitivity to Ca2+ and phosphatidylinositol 4,5-bisphosphate (PIP2) as well as blockade by flufenamate (FFA) and Gd3+. At the systems level, bath-applied Gd3+ stopped the respiratory rhythm within several minutes, which suggests that TRPM4-mediated ICAN is essential for rhythmogenesis, while avoiding the pharmacological caveats associated with FFA. To examine the role of ICAN in networks, Mironov performs two-photon imaging combined with on-cell patch recordings in pairs of connected preBotC neurons. Synaptic excitation gives rise to propagating Ca2+ waves in dendrites, which then evoke TRPM4 in the soma (Fig. 1A). During endogenous network activity these same propagating Ca2+ waves cause vigorous TRPM4 activity in the prelude to, and during, the inspiratory burst. Additionally, the mGluR agonist DHPG, which modulates respiratory frequency and augments TRPM4 activity, here is shown to accelerate the propagation speed of the Ca2+ wave. Interestingly, local application of thapsigargin to deplete the Ca2+ stores halts the wave in mid-dendrite, and occludes the inspiratory burst altogether. These observations suggest that group 1 mGluRs contribute to inspiratory bursts via inositol 1,4,5-trisphosphate (IP3) and Ca2+-induced Ca2+ release, and may influence respiratory frequency by regulating the speed of Ca2+ wave propagation. Figure 1 The cellular mechanisms underlying a ‘group pacemaker’ in the respiratory CPG contained in the preBotC To test this novel idea regarding frequency modulation Mironov turns to simulations. He creates Ca2+ release compartments coupled to soma-like compartments with ICAN, which he couples in a ring of alternating compartment types (Fig. 1B). This ring system generates coherent rhythmicity only when propagation speed is held near ∼70 μm s−1, matching the measured value. The range of propagation speeds that support coherent rhythmicity can be extended when ring oscillators are sparsely coupled to one another, roughly representing distinct clusters of rhythmic neurons observed in the preBotC (Hartelt et al. 2008). This arrangement may be advantageous to synchronize preBotC neurons, which may be sparsely connected (Rekling et al. 2000), and by ‘clamping’ the frequency of the network rhythm on the basis of wave propagation speed. This innovative new idea suggests that frequency modulation may not necessarily depend on regulating somatic ion channels to hyper- or depolarize baseline membrane potential, but could be tuned by metabotropic receptors that modify dendritic cable properties or Ca2+ release mechanisms and influence the speed of Ca2+ waves. Mironov's analysis establishes a viable model for emergent network properties in a CPG. Post-synaptic ion channels become active in the context of network function; bursts of channel activity depend on metabotropic receptors and intracellular signalling. While some uncertainties remain, i.e. regarding the specific role of ionotropic AMPA receptors (AMPARs) and whether TRPM4 is expressed in dendrites, this demonstration of a group pacemaker in the preBotC should cause us to re-examine the underlying mechanism in other important CPGs (e.g. locomotion, swimming and oral-motor activity) as well as rhythmic brain networks in general.

Effects of subtype-selective group I mGluR antagonists on synchronous activity induced by 4-aminopyridine/CGP 55845 in adult guinea pig hippocampal slices

Co-application of the convulsant 4-aminopyridine (4-AP) and the GABA B receptor antagonist CGP 55845 to adult guinea pig hippocampal slices elicits giant GABA-mediated postsynaptic potentials (GPSPs) and epileptiform discharges. Here we tested the effects of the group I metabotropic glutamate receptor (mGluR) subtype-selective antagonists LY 367385 (mGlu1, 100 μM), MPEP (mGlu5, 10 μM), and MTEP (mGlu5, 500 nM) on this synchronous activity. Electrophysiological field recordings were performed in the CA3 region of hippocampal slices from adult guinea pigs. The mGlu5 receptor antagonists increased GPSP rate, but the mGlu1 receptor antagonist did not. This ability of mGlu5 receptor antagonists to increase the rate of GPSPs indicates that enough endogenous glutamate is released under these conditions to activate group I mGluR; nevertheless, co-application of a mGlu1 receptor antagonist (LY 367385 or JNJ 16259685) and MPEP did not decrease pre-existing epileptiform activity. Furthermore, co-application of LY 367385 and MPEP did not prevent the emergence of epileptiform activity. When ionotropic glutamate receptor (iGluR) antagonists were present, neither MPEP nor the group I mGluR agonist DHPG changed GPSP rate, suggesting that pyramidal cell-to-interneuron iGluR-mediated synaptic connections are involved in the rate change mechanism. In contrast to the lack of effect of group I mGluR antagonists on epileptiform activity in the 4-AP/CGP 55845 model, group I mGluR antagonists blocked the emergence of longer epileptiform events and decreased the overall amount of synchronous activity in the GABA A antagonist/4-AP model. In conclusion, in the 4-AP/CGP 55845 model, enough glutamate was released to activate group I mGluRs and affect GPSP rate via mGlu5 receptors; however, this group I mGluR activation was not required for the generation of the epileptiform activity.