Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.     

Presynaptic kainate receptors in the hippocampus: slowly emerging from obscurity.

Kainate receptor agonists depress transmitter release at several synapses in the hippocampus. Distinct mechanisms appear to underlie this phenomenon at different synapses. Recently, it has emerged that presynaptic kainate receptors can also potentiate the release of both GABA and glutamate and that axonal kainate receptors can trigger ectopic action potentials in interneurons. Because synaptically released glutamate mimics many of the actions of exogenous agonists, presynaptic kainate receptors potentially play an extensive role in hippocampal signaling.     

Release of arachidonic acid by NMDA-receptor activation in the rat hippocampus

In hippocampal slices arachidonic acid released after NMDA post-synaptic receptor activation is thought to act as a retrograde trans-synaptic messenger which facilitates the pre-synaptic release of L-glutamate to be involved in the expression of long-term synaptic potentiation (LTP). We measured the mass amount of arachidonic acid released from hippocampal slices incubated under conditions which maintain the electrophysiological responsiveness of the slice. Melittin released arachidonic, oleic and docosahexaenoic acids by phospholipase A₂ activation but not palmitic or stearic acids. Of greater interestl-glutamate, N-methyl-d-aspartate and incubation conditions known to induce LTP selectively and rapidly increased the release of archidonic acid in amounts over basal levels of 200–300 ng/mg protein. This is the first direct determination of the mass amount of arachidonic acid released following NMDA receptor activation in the hippocampus.Special issue dedicated to Dr. Louis Sokoloff.     

Presynaptic glycine receptors facilitate spontaneous glutamate release onto hilar neurons in the rat hippocampus

Although glycine receptors are found in most areas of the brain, including the hippocampus, their functional significance remains largely unknown. In the present study, we have investigated the role of presynaptic glycine receptors on excitatory nerve terminals in spontaneous glutamatergic transmission. Spontaneous EPSCs (sEPSCs) were recorded in mechanically dissociated rat dentate hilar neurons attached with native presynaptic nerve terminals using a conventional whole-cell patch recording technique under voltage-clamp conditions. Exogenously applied glycine or taurine significantly increased the frequency of sEPSCs in a concentration-dependent manner. This facilitatory effect of glycine was blocked by 1 μM strychnine, a specific glycine receptor antagonist, but was not affected by 30 μM picrotoxin. In addition, Zn²⁺ (10 μM) potentiated the glycine action on sEPSC frequency. Pharmacological data suggested that the activation of presynaptic glycine receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. Bumetanide (10 μM), a specific Na-K-2C co-transporter blocker, gradually attenuated the glycine-induced sEPSC facilitation, suggesting that the depolarizing action of presynaptic glycine receptors was due to a higher intraterminal Cl⁻ concentration. The present results suggest that presynaptic glycine receptors on excitatory nerve terminals might play an important role in the excitability of the dentate gyrus-hilus-CA3 network in physiological and/or pathological conditions.     

Modification by arachidonic acid of extracellular adenosine metabolism and neuromodulatory action in the rat hippocampus

Adenosine and arachidonate (AA) fulfil opposite modulatory roles, arachidonate facilitating and adenosine inhibiting cellular responses. To understand if there is an inter-play between these two neuromodulatory systems, we investigated the effect of AA on extracellular adenosine metabolism in hippocampal nerve terminals. AA (30 microm) facilitated by 67% adenosine evoked release and by 45% ATP evoked release. These effects were not significantly modified upon blockade of lipooxygenase or cyclooxygenase and were attenuated (52-61%) by the protein kinase C inhibitor, chelerythrine (6 microm). The ecto-5'-nucleotidase inhibitor, alpha,beta-methylene ADP (100 microm), caused a larger inhibition (54%) of adenosine release in the presence of AA (30 microm) compared with control (37% inhibition) indicating that the AA-induced extracellular adenosine accumulation is mostly originated from an increased release and extracellular catabolism of ATP. This AA-induced extracellular adenosine accumulation is further potentiated by an AA-induced decrease (48%) of adenosine transporters capacity. AA (30 microm) increased by 36-42% the tonic inhibition by endogenous extracellular adenosine of adenosine A(1) receptors in the modulation of acetylcholine release and of CA1 hippocampal synaptic transmission in hippocampal slices. These results indicate that AA increases tonic adenosine modulation as a possible feedback loop to limit AA facilitation of neuronal excitability.     

Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals

One of the pathways implicated in a fine-tuning control of synaptic transmission is activation of the receptors located at the presynaptic terminal. Here we investigated the intracellular events in rat brain cortical and hippocampal nerve terminals occurring under the activation of presynaptic glutamate receptors by exogenous glutamate and specific agonists of ionotropic receptors, NMDA and kainate. Involvement of synaptic vesicles in exocytotic process was assessed using [³H]GABA and pH-sensitive fluorescent dye acridine orange (AO). Glutamate as well as NMDA and kainate were revealed to induce [³H]GABA release that was not blocked by NO-711, a selective blocker of GABA transporters. AO-loaded nerve terminals responded to glutamate application by the development of a two-phase process. The first phase, a fluorescence transient completed in ∼1 min, was similar to the response to high K⁺. It was highly sensitive to extracellular Ca²⁺ and was decreased in the presence of the NMDA receptor antagonist, MK-801. The second phase, a long-lasting process, was absolutely dependent on extracellular Na⁺ and attenuated in the presence of CNQX, the kainate receptor antagonist. NMDA as well as kainate per se caused a rapid and abrupt neurosecretory process confirming that both glutamate receptors, NMDA and kainate, are involved in the control of neurotransmitter release. It could be suggested that at least two types ionotropic receptor are attributed to glutamate-induced two-phase process, which appears to reflect a rapid synchronous and a more prolonged asynchronous vesicle fusion.     

Presynaptic effects of anandamide and WIN55,212-2 on glutamatergic nerve endings isolated from rat hippocampus

We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEA's enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA on glutamatergic nerve terminals and demonstrate that the effects of endogenous and synthetic cannabinoids are not always identical.     

Activation of GluR6-containing kainate receptors induces ubiquitin-dependent Bcl-2 degradation via denitrosylation in the rat hippocampus after kainate treatment

We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.     

Role of excitatory amino acid receptors in synaptic transmission in area CA1 of rat hippocampus

The new antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), which blocks responses to kainate and quisqualate, has been used in conjunction with D-2-amino-5-phosphonovalerate (APV), which blocks selectively responses to N-methyl-D-aspartate (NMDA), to determine the role of excitatory amino acid receptors in synaptic transmission. An excitatory postsynaptic potential (EPSP)-inhibitory postsynaptic potential (IPSP) sequence was evoked in CA1 neurons by stimulation of the Schaffer collateral-commissural pathway in rat hippocampal slices. CNQX (10 microM) substantially reduced the EPSP without having any effect on input resistance or membrane potential. The IPSP was also reduced provided that the stimulating electrode was place approximately 1 mm from the recording electrode. The EPSP that remained in the presence of CNQX had characteristics of an NMDA receptor-mediated potential; it had a slow timecourse, summated at high frequencies, was blocked reversibly by APV, increased greatly in size in Mg2+-free medium, and showed an anomalous voltage dependence in Mg2+-containing medium. In the presence of CNQX, an APV-sensitive polysynaptic GABAergic IPSP could be evoked, indicating that NMDA receptors can mediate suprathreshold EPSPS in inhibitory interneurons. It is suggested that either NMDA or non-NMDA receptors can, under different circumstances, mediate the synaptic excitation of pyramidal neurons and inhibitory interneurons in area CA1 of the hippocampus.     

Presynaptic kainate receptors that enhance the release of GABA on CA1 hippocampal interneurons

We report that kainate receptors are present on presynaptic GABAergic terminals contacting interneurons and that their activation increases GABA release. Application of kainate increased the frequency of miniature inhibitory postsynaptic currents recorded in CA1 interneurons. Local applications of glutamate but not of AMPA or NMDA also increased GABA quantal release. Application of kainate as well as synaptically released glutamate reduced the number of failures of GABAergic neurotransmission between interneurons. Thus, activation of presynaptic kainate receptors increases the probability of GABA release at interneuron-interneuron synapses. Glutamate may selectively control the communication between interneurons by increasing their mutual inhibition.     

Effect of arachidonic acid on [3H]d-aspartate outflow in the rat hippocampus

The aim of this study was to investigate the effect of arachidonic acid on [³H]d-aspartate outflow in rat hippocampus synaptosomes and slices. Arachidonic acid 1) increased basal outflow of [³H]d-aspartate in both synaptosomes and slices, and 2) increased K⁺-evoked overflow in slices but not in synaptosomes. The latter effect was dependent (at least in part) on arachidonic acid metabolism, most likely mediated by lipo-oxygenase metabolites and free radical production. It was prevented by nordihydroguaiaretic acid but not by indomethacin, and was significantly reduced by free radical scavengers (superoxide-desmutase and catalase). This effect was dependent upon stimulation since it could not be observed after a continuous perfusion of arachidonic acid in the absence of stimulation. Furthermore, it was long-lasting since a 30 min perfusion of arachidonic acid was sufficient to exert a significant effect on a stimulation following termination of the application.     

Up-modulation of phorbol dibutyrate receptors by carbachol and arachidonic acid in rat prostatic epithelial cells

Phorbol dibutyrate (PDBu) binding to rat prostatic epithelial cells has been measured as an indirect determination of protein kinase C in this cell system. Analysis of [³H]PDBu binding using competitive displacement demonstrated a single class of PDBu receptors with a Kd=141 nM and a binding capacity of 4.8 pmol PDBu bound/mg cell protein. Raising cytosolic Ca²⁺ levels by redistribution of intracellular Ca²⁺ after cell treatment with carbachol or arachidonic acid (which also affects the bulk biophysical properties of the cell membrane) resulted in up-regulation of the available number of PDBu receptors. These results appear to be a consequence of PKC translocation from the cytosolic compartment to the plasma membrane after a cytosolic Ca²⁺ increase, confirming previous results in other cell systems.     

Thrombin-induced transfer of arachidonic acid in human platelets is not inhibited by trifluoperazine

Human platelets have been shown to contain a Ca++- and CoA-independent transacylase enzyme that catalyzes the transfer of arachidonic acid from phosphatidylcholine (PC) to lysoplasmenylethanolamine. It has been suggested that this route may represent a major source for released arachidonic acid in stimulated platelets. In this study, we have shown using arachidonic-labelled human platelets that the thrombin-induced activation of a transacylase reaction was not affected by concentrations of trifluoperazine (TFP) (15 micrograms/2 X 10(9) cells) which abolished the accumulation of free [3H]arachidonic acid in the presence of the cyclooxygenase/lipoxygenase inhibitor BW755C. TFP, at this concentration failed to block the hydrolysis of phosphatidylcholine (PC) completely and had no effect on the increased radioactivity seen in total phosphatidylethanolamine (PE) (160% of control after 4 min of incubation). These results suggest that the transacylase pathway activated in response to thrombin is not likely dependent on calcium. As TFP blocks effectively both the accumulation of free [3H]arachidonic acid and the mass of arachidonic acid without affecting the transfer of this fatty acid from PC to PE in thrombin-stimulated human platelets, it is very unlikely that the transacylation pathway represents a major source of release arachidonic acid. Based on these findings, we conclude that the above pathway may be primarily involved in the turnover of plasmenylethanolamine lipids in stimulated human platelets.     

The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS-cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.     

Glutamate-induced cobalt uptake elicited by kainate receptors in rat taste bud cells

Glutamate-induced cobalt uptake reveals non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) in rat taste bud cells. However, it is not known which type of non-NMDA glutamate receptors is involved. We used a cobalt staining technique combined with pharmacological tests for kainate or alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors and/or immunohistochemistry against subunits of GluRs to examine the presence of non-NMDA receptors in rat foliate tastebud cells. Cobalt uptake into taste cells was elicited by treating taste buds with glutamate, kainate or SYM 2081, a kainate receptor agonist. Treating taste buds with AMPA or fluorowillardiine did not stimulate significant cobalt uptake. Moreover, 6-cyano-7-nitro-quinoxaline-2, 3-dione significantly reduced cobalt staining elicited by glutamate or kainate receptor agonists, but SYM 2206, an AMPA receptor antagonist, did not. Immunohistochemistry against subunits of GluRs reveals GluR6 and KA1-like immunoreactivity. Moreover, most glutamate-induced cobalt-stained cells showed GluR6 and KA1-like immunoreactivity. These results suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs.     

High-affinity kainate and domoate receptors in rat brain.

Mammalian brain expresses receptors which bind the potent neurotoxins, kainate and domoate, with high affinity, and which form a subclass of ionotropic glutamate receptors. A new member of these receptors, expressed in both adult and embryonic CNS is compared in its ligand binding properties to its closely sequence-related homologs.     

Chronic clozapine reduces rat brain arachidonic acid metabolism by reducing plasma arachidonic acid availability

Chronic administration of mood stabilizers to rats down‐regulates the brain arachidonic acid (AA) cascade. This down‐regulation may explain their efficacy against bipolar disorder (BD), in which brain AA cascade markers are elevated. The atypical antipsychotics, olanzapine (OLZ) and clozapine (CLZ), also act against BD. When given to rats, both reduce brain cyclooxygenase activity and prostaglandin E₂ concentration; OLZ also reduces rat plasma unesterified and esterified AA concentrations, and AA incorporation and turnover in brain phospholipid. To test whether CLZ produces similar changes, we used our in vivo fatty acid method in rats given 10 mg/kg/day i.p. CLZ, or vehicle, for 30 days; or 1 day after CLZ washout. [1‐¹⁴C]AA was infused intravenously for 5 min, arterial plasma was collected and high‐energy microwaved brain was analyzed. CLZ increased incorporation coefficients and rates J_in,i of plasma unesterified AA into brain phospholipids i, while decreasing plasma unesterified but not esterified AA. These effects disappeared after washout. Thus, CLZ and OLZ similarly down‐regulated kinetics and cyclooxygenase expression of the brain AA cascade, likely by reducing plasma unesterified AA availability. Atypical antipsychotics and mood stabilizers may be therapeutic in BD by down‐regulating, indirectly or directly respectively, the elevated brain AA cascade of that disease.     

Trimethyltin-induced loss of NMDA and kainate receptors in the rat brain

Summary Adult rats exposed acutely to trimethyltin (TMT) manifest a number of behavioral alterations, in conjunction with neuronal degeneration in the limbic system. In the present study, changes in³H-TCP binding to N-methyl-D-aspartate (NMDA) receptors and³H-kainic acid (KA) binding to kainate receptors were studied by autoradiographic methods following TMT exposure (8 mg/kg, i.p.) in adult Sprague Dawley rats. No significant alterations were found at 4 hours after exposure. An extensive loss of³H-TCP and³H-KA binding was seen in the hilar region of the CA3 field at 2 and 12 weeks after TMT exposure. Also, the³H-TCP binding was decreased in piriform cortex and in striatum. Thus, TMT exposure leads to a major and regional selective loss of NMDA and kainate receptors in the limbic system, alterations that may be involved in the neuropathology and behavioral sequelae of TMT toxicity.Abbreviations TMT trimethyltin - NMDA N-methyl-D-aspartate - KA Kainic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine