Receptors Inhibit Glutamate Release Research Articles

Serotonin 5-HT1A and 5-HT1B receptor-mediated inhibition of glutamatergic transmission onto rat basal forebrain cholinergic neurones.

Cholinergic neurones in the basal forebrain (BF) project into various brain regions and receive excitatory inputs from the cortex and brain stem. These cholinergic neurones receive serotonergic fibres from the dorsal raphe nuclei. This study was aimed to elucidate serotonin (5-HT)-induced modulation of glutamatergic transmission onto rat BF cholinergic neurones identified with Cy3-192IgG. Excitatory postsynaptic currents (EPSCs) were evoked by focal stimulation. Bath application of either 5-HT, the 5-HT1A receptor agonist 8-OH-DPAT (DPAT), or the 5-HT1B receptor agonist CP93129 (CP), inhibited the amplitude of EPSCs. In the presence of both 5-HT1A and 5-HT1B receptor antagonists, the 5-HT-induced effect disappeared. The paired-pulse ratio (PPR) and coefficient of variation (CV) of the EPSCs were increased by CP, whereas DPAT had no effect on PPR or CV. DPAT inhibited the inward currents induced by puff application of l-glutamate, which were unaffected by CP. DPAT suppressed the amplitude of miniature EPSCs (mEPSCs) without affecting their frequency. CP decreased the frequency of mEPSCs in more than half of the neurones examined, whereas the amplitude was unaffected. DPAT or CP alone inhibited the NMDA receptor-mediated currents. 5-HT-induced inhibition of EPSCs was reduced in the presence of ω-agatoxin TK (Aga). Furthermore, CP-induced inhibition of EPSCs was eliminated in the presence of Aga. DPAT-induced inhibition of EPSCs was unchanged in the presence of Aga. These results suggest that activation of 5-HT1A receptors reduces the sensitivity of postsynaptic glutamate receptors to glutamate, whereas presynaptic activation of 5-HT1B receptors inhibits glutamate release by blocking P/Q-type calcium channels. KEY POINTS: We performed a patch-clamp study to investigate serotonin (5-HT)-induced modulation of glutamatergic transmission onto cholinergic neurones in the rat basal forebrain slices. Excitatory postsynaptic currents (EPSCs) were inhibited by 5-HT as well as agonists of 5-HT1A or 5-HT1B receptors. 5-HT-induced inhibition was antagonized by co-application of 5-HT1A and 5-HT1B receptor antagonists. The effects of 5-HT receptor agonists on the paired-pulse ratio, coefficient of variation of EPSCs, inward currents induced by puff application of l-glutamate as well as miniature EPSCs suggest that activation of 5-HT1A receptors decreases the sensitivity of postsynaptic glutamate receptors to glutamate, whereas 5-HT1B receptors presynaptically inhibit glutamate release. The 5-HT1B agonist-induced inhibition was eliminated in the presence of a P/Q-type calcium channel blocker, whereas the 5-HT1A agonist still inhibited the EPSCs even in the presence of the blocker. The present study reveals different pre- and postsynaptic mechanisms underlying 5-HT1A and 5-HT1B receptor-mediated modulation of excitatory transmission.

Effects of M1 and M4 activation on excitatory synaptic transmission in CA1.

Hippocampal networks are particularly susceptible to dysfunction in many neurodegenerative diseases and neuropsychiatric disorders including Alzheimer's disease, Lewy body dementia, and schizophrenia. CA1, a major output region of the hippocampus, receives glutamatergic input from both hippocampal CA3 and entorhinal cortex, via the Schaffer collateral (SC) and temporoammonic (TA) pathways, respectively. SC and TA inputs to CA1 are thought to be differentially involved in the retrieval of previously stored memories versus the encoding of novel information, and switching between these two crucial hippocampal functions is thought to critically depend on acetylcholine (ACh) acting at muscarinic receptors. In this study, we aimed to determine the roles of specific subtypes of muscarinic receptors in mediating the neuromodulatory effects of ACh on glutamatergic synaptic transmission in the SC and TA pathways of CA1. Using selective pharmacological activation of M1 or M4 receptors along with extracellular and intracellular electrophysiology recordings from adult rat hippocampal slices, we demonstrate that activation of M1 receptors increases spontaneous spike rates of neuronal ensembles in CA1 and increases the intrinsic excitability of pyramidal neurons and interneurons. Selective activation of M4 receptors inhibits glutamate release in the SC pathway, while leaving synaptic transmission in the TA pathway comparatively intact. These results suggest specific mechanisms by which M1 and M4 activation may normalize CA1 circuit activity following disruptions of signaling that accompany neurodegenerative dementias or neuropsychiatric disorders. These findings are of particular interest in light of clinical findings that xanomeline, an M1/M4 preferring agonist, was able to improve cognitive and behavioral symptoms in patients with Alzheimer's disease or schizophrenia.

5-Hydroxytryptamine 1A receptors inhibit glutamate release in rat medullary dorsal horn neurons

We examined 5-hydroxytryptamine 1A (5-HT1A) receptor-mediated modulation of glutamatergic transmission in rat medullary dorsal horn neurons using a conventional whole-cell patch clamp technique. 5-HT reversibly and concentration dependently decreased the amplitude of glutamatergic excitatory postsynaptic currents and increased the paired-pulse ratio, indicating that 5-HT acts presynaptically to reduce glutamate release from primary afferents. The 5-HT-induced inhibition of excitatory postsynaptic currents was partially occluded by NAN-190, a 5-HT1A receptor antagonist, and mimicked by 8-OH-DPAT, a 5-HT1A receptor agonist. Our results suggest that presynaptic 5-HT1A receptors inhibit glutamate release from trigeminal primary afferents onto medullary dorsal horn neurons, and thus in addition to other 5-HT1 receptor subtypes, 5-HT1A receptors could be a potential target for treatment of pain from orofacial tissues.

5‐HT1B receptors inhibit glutamate release from primary afferent terminals in rat medullary dorsal horn neurons

Although 5-HT(1B) receptors are expressed in trigeminal sensory neurons, it is still not known whether these receptors can modulate nociceptive transmission from primary afferents onto medullary dorsal horn neurons. Primary afferent-evoked EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices using a conventional whole-cell patch clamp technique under a voltage-clamp condition. CP93129, a selective 5-HT(1B) receptor agonist, reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, CP93129 reduced the frequency of spontaneous miniature EPSCs without affecting the current amplitude. The CP93129-induced inhibition of EPSCs was significantly occluded by GR55562, a 5-HT(1B/1D) receptor antagonist, but not LY310762, a 5-HT(1D) receptor antagonist. Sumatriptan, an anti-migraine drug, also decreased EPSC amplitude, and this effect was partially blocked by either GR55562 or LY310762. On the other hand, primary afferent-evoked EPSCs were mediated by the Ca(2+) influx passing through both presynaptic N-type and P/Q-type Ca(2+) channels. The CP93129-induced inhibition of EPSCs was significantly occluded by ω-conotoxin GVIA, an N-type Ca(2+) channel blocker. The present results suggest that the activation of presynaptic 5-HT(1B) receptors reduces glutamate release from primary afferent terminals onto medullary dorsal horn neurons, and that 5-HT(1B) receptors could be, at the very least, a potential target for the treatment of pain from orofacial tissues.

A1 receptors inhibit glutamate release in rat medullary dorsal horn neurons

We have investigated the adenosine-mediated presynaptic inhibition of primary afferent-evoked glutamate release in rat substantia gelatinosa neurons of the trigeminal subnucleus caudalis using a conventional whole-cell patch clamp technique. Adenosine reversibly and concentration dependently decreased the amplitude of glutamatergic excitatory postsynaptic currents and increased the paired-pulse ratio, indicating that adenosine acts presynaptically to reduce glutamate release from primary afferents. The adenosine-induced inhibition of excitatory postsynaptic currents was occluded by a selective A₁ receptor antagonist, DPCPX, and was mimicked by a selective A₁ receptor agonist CPA. The results suggest that presynaptic A₁ receptors decrease action potential-dependent glutamate release from trigeminal primary afferents onto medullary dorsal horn neurons, and thus adenosine A₁ receptors could be a potential target for the treatment of pain of orofacial tissues.

The GABAB1aIsoform Mediates Heterosynaptic Depression at Hippocampal Mossy Fiber Synapses

GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed that GABA(B(1a,2)) receptors are more abundant than GABA(B(1b,2)) receptors at glutamatergic terminals. Accordingly, it was found that GABA(B(1a,2)) receptors are more efficient than GABA(B(1b,2)) receptors in inhibiting glutamate release when maximally activated by exogenous application of the agonist baclofen. Here, we used a combination of genetic, ultrastructural and electrophysiological approaches to analyze to what extent GABA(B(1a,2)) and GABA(B(1b,2)) receptors inhibit glutamate release in response to physiological activation. We first show that at hippocampal mossy fiber (MF)-CA3 pyramidal neuron synapses more GABA(B1a) than GABA(B1b) protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations >or=1 microm, both GABA(B(1a,2)) and GABA(B(1b,2)) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABA(B(1a,2)) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABA(B(1a,2)) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABA(B(1a,2)) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance.

Pre‐synaptic GABAB receptors inhibit glutamate release through GIRK channels in rat cerebral cortex

Neuronal G protein-gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post-synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA(B) receptors. In this study, we show for the first time that GABA(B) receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA(B) receptors reduces glutamate release and the Ca(2+) influx mediated by N-type Ca(2+) channels in a mode insensitive to the GIRK channel blocker tertiapin-Q and consistent with direct inhibition of this voltage-gated Ca(2+) channel. However, by means of weak stimulation protocols, we reveal that GABA(B) receptors also reduce glutamate release mediated by P/Q-type Ca(2+) channels, and that these responses are reversed by the GIRK channel blocker tertiapin-Q. Consistent with the functional interaction between GABA(B) receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre-synaptic boutons of asymmetric synapses co-express GABA(B) receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post-synaptic level, also occurs at glutamatergic nerve terminals.

The inhibition of release by mGlu7 receptors is independent of the Ca 2+ channel type but associated to GABA B and adenosine A 1 receptors

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca 2+ channels and adenylyl cyclase. Through Ca 2+ imaging and immunocytochemistry, we have recently shown that adenosine A 1, GABA B and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca 2+ channels by analysing this process in nerve terminals from mice lacking the α 1B subunit (Ca v 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca 2+ channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABA B and adenosine A 1. Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A 1, GABA B and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca 2+ channel type but rather, it is associated to functional mGlu7 receptors.

Enhanced activity of GABA receptors inhibits glutamate release induced by focal cerebral ischemia in rat striatum

Cerebral ischemia causes an excess release of glutamate, which can injure neurons. The striatum is one of the important regions vulnerable to hypoxia and ischemia. Using push-pull perfusion technique, we investigated the regulatory role of gamma-aminobutyric acid (GABA) and its receptors in modifying the amount of glutamate in rat striatum with ischemia. Perfusion with exogenous GABA (1 mM) inhibited cerebral ischemia-induced glutamate release by as much as 47%. We further characterized relative roles of subtype receptors of GABA on glutamate release by using pharmacological tools. While baclofen (500 microM), a GABA(B) receptor agonist, suppressed ischemia-induced glutamate release by 52%, GABA(B) receptor antagonist saclofen (500 microM) failed to produce a significant increase of glutamate release. The GABA(A) receptor agonist muscimol (500 microM) also reduced by 38% the release of glutamate induced by cerebral ischemia but the GABA(A) receptor antagonist bicuculline (500 microM) had very little effect. The present study demonstrates that the excessive release of glutamate or the overly activated glutamate receptor, triggered by cerebral ischemia, can be down-regulated by exogenous GABA or by increased activity of GABA receptors, especially the presynaptic GABA(B) receptors, which might be one of the important mechanisms to protect against striatum neuronal damage from over stimulation by excessive glutamate during ischemia.

Group II metabotropic glutamate receptors inhibit glutamate release at thalamocortical synapses in the developing somatosensory cortex

Thalamocortical synapses provide a strong glutamatergic excitation to cortical neurons that is critical for processing sensory information. Unit recordings in vivo indicate that metabotropic glutamate receptors (mGluRs) reduce the effect of thalamocortical input on cortical circuits. However, it is not known whether this reduction is due to a reduction in glutamate release from thalamocortical terminals or from a decrease in cortical neuron excitability. To directly determine whether mGluRs act as autoreceptors on thalamocortical terminals, we examined the effect of mGluR agonists on thalamocortical synapses in slices. Thalamocortical excitatory postsynaptic currents (EPSCs) were recorded in layer IV cortical neurons in developing mouse brain slices. The activation of group II mGluRs with (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG IV) reduced thalamocortical EPSCs in both excitatory and inhibitory neurons, while the stimulation of group I or group III mGluRs had no effect on thalamocortical EPSCs. Consistent with a reduction in glutamate release, DCG IV increased the paired pulse ratio and the coefficient of variation of the EPSCs. The reduction induced by DCG IV was reversed by the group II mGluR antagonist, LY341495, and mimicked by another selective group II agonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC). The mGluR2 subtype appears to mediate the reduction of thalamocortical EPSCs, since the selective mGluR3 agonist, N-acetylaspartylglutamate (NAAG), had no effect on the EPSCs. Consistent with this, we showed that mGluR2 is expressed in the barrels. Furthermore, blocking group II mGluRs with LY341495 reduced the synaptic depression induced by a short stimulus train, indicating that synaptically released glutamate activates these receptors. These results indicate that group II mGluRs modulate thalamocortical processing by inhibiting glutamate release from thalamocortical synapses. This inhibition provides a feedback mechanism for preventing excessive excitation of cortical neurons that could play a role in the plasticity and refinement of thalamocortical connections during this early developmental period.

Nalfurafine, the kappa opioid agonist, inhibits icilin-induced wet-dog shakes in rats and antagonizes glutamate release in the dorsal striatum

Icilin, a cooling compound, produces vigorous wet-dog shakes in rats. We have reported previously that icilin-induced wet-dog shakes are blocked by the kappa opioid receptor agonists, nalfurafine and U50,488H, and that icilin evokes a dose- and time-dependent increase in glutamate within the dorsal striatum. Since activation of kappa opioid receptors inhibits glutamate release intrastriatally, we targeted glutamate release within the dorsal striatum using nalfurafine and examined the role of the dorsal striatum in icilin-induced wet-dog shakes, more specifically, the effect that icilin-evoked intrastriatal glutamate release has on the overt stimulant behavior. We report that nalfurafine (0.04 mg/kg) inhibits icilin (0.50 mg/kg)-induced wet-dog shakes and that this inhibition is reversed by intrastriatal perfusion of the kappa opioid receptor antagonist, norbinaltorphimine (100 nM). Furthermore,we antagonized icilin-evoked glutamate release with nalfurafine (0.04 mg/kg), and reversed inhibition of glutamate release with intrastriatal norbinaltorphimine (100 nM). These findings support a central component in the behavioral response to icilin and suggest that activation of kappa opioid receptors antagonizes icilin-induced wet-dog shakes in rats by inhibiting glutamate release within the dorsal striatum.

Chronic intake of caffeine during gestation down regulates metabotropic glutamate receptors in maternal and fetal rat heart

Caffeine is the most widely consumed substance in the world which antagonizes adenosine effects. Adenosine acting through A(1) receptors inhibits glutamate release which binds to metabotropic glutamate receptors (mGluRs). Recently, we have shown that maternal caffeine intake during gestation causes down-regulation of A(1) and metabotropic glutamate receptors in the brain of both rat mothers and fetuses. In the present work we provide evidence that caffeine also affects receptors in hearts, causing a decrease in mGluRs from both maternal and fetal hearts. A decrease in G(q/11) and PLC beta(1) proteins level was also observed in both tissues. However, phospholipase C activity was only affected in fetal heart, being significantly decreased. These results suggest an in vivo cross-talk mechanism between adenosine and glutamate receptors in peripheral tissues. Therefore, special attention should be paid to caffeine ingestion during gestation.

Activation of neuropeptide Y Y1 receptors inhibits glutamate release through reduction of voltage-dependent Ca 2+ entry in the rat cerebral cortex nerve terminals: Suppression of this inhibitory effect by the protein kinase C-dependent facilitatory pathway

Neuropeptide Y (NPY) is known to regulate the presynaptic glutamate release and neuronal responses to excitatory neurotransmission. The aim of this study was to investigate the effect of NPY on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes). NPY inhibited the Ca 2+-dependent glutamate release evoked by 4-aminopyridine, and this inhibitory effect was mediated via NPY Y1 receptors, because it was mimicked by the specific NPY Y1 receptor agonist [Leu 31 Pro 34] NPY and blocked by the NPY Y1 receptor antagonist GR 231118. The inhibitory action of NPY was not due to it decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca 2+ influx, because NPY did not alter the 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential or ionomycin and hypertonic solution-induced glutamate release. Examination of the effect of NPY on the cytosolic [Ca 2+] revealed that the inhibition of glutamate release could be attributed to a reduction in voltage-dependent Ca 2+ influx. Consistent with this, the NPY-mediated inhibition of glutamate release was completely abolished in synaptosomes pretreated with N- and P/Q-type Ca 2+ channel blocker, ω-conotoxin MVIIC. Moreover, NPY-mediated inhibition of 4-aminopyridine-evoked glutamate release was insensitive to KT 5720 and Ro32–0432 but was suppressed when protein kinase C was stimulated with phorbol ester. Together, these results suggest that NPY acting predominantly on NPY Y1 receptors inhibits glutamate release from rat cerebrocortical synaptosomes, likely by a mechanism involving direct coupling of receptors to N- and P/Q-type Ca 2+ channels, and this coupling is subject to regulation by protein kinase C-dependent pathway. This implies that selective ligand for NPY receptors may be of value for treatment of conditions characterized by excessive glutamate release in the cerebral cortex.

Overexpression of NPY and Y2 receptors in epileptic brain tissue: an endogenous neuroprotective mechanism in temporal lobe epilepsy?

Recurrent epileptic seizures in the rat enhance the expression of neuropeptide Y (NPY) and its mRNA in various brain areas including the hippocampus, cerebral cortex and the amygdala. In the hippocampus, the most prominent expression of NPY is observed in mossy fibers and in GABAergic interneurons. At the same time, expression of Y2 receptors is also increased whereas Y1 receptors are reduced. Similar changes in Y1 and Y2 receptors were observed in the hippocampus of patients with temporal lobe epilepsy (TLE). In contrast to the rat, NPY expression is not enhanced in mossy fibers in TLE. In the same tissue, surviving NPY interneurons show marked axonal sprouting into areas innervated by mossy fibers (dentate hilus, stratum lucidum, inner molecular layer of the dentate gyrus). Stimulation of presynaptic Y2 receptors inhibits glutamate release, and exert an anticonvulsant action in experimental models. Y1 receptors mediate a weak excitatory component of NPY action. These findings suggest that changes in the NPY system induced by seizures represent an endogenous adaptive mechanism aimed at counteracting hyperexcitability underlying epileptic activity. This concept is strongly supported by evidence that genetically modified rats overexpressing the NPY gene are less susceptible to seizures while deletion of NPY or Y2 receptor genes results in increased susceptibility to seizures.

Presynaptic kynurenate-sensitive receptors inhibit glutamate release.

Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and alpha7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30-100 nM) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3-10 nM), a selective alpha7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nM), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mM)-induced release of transmitter or previously taken-up [3H]-D-aspartate. This inhibition was not modified by glycine, N-methyl-D-aspartate or subtype-selective kainate receptor agents, while CNQX or DNQX (10 microM), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]-kainate (high and low affinity) or [3H]-AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1-100 nM) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate-induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.