Effect of the Non-NMDA Receptor Antagonist GYKI 52466 on the Microdialysate and Tissue Concentrations of Amino Acids Following Transient Forebrain Ischaemia

Abstract: The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and γ-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 μM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/ kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.     

A novel anti-epileptic agent, perampanel, selectively inhibits AMPA receptor-mediated synaptic transmission in the hippocampus

Perampanel is a non-competitive AMPA receptor antagonist that is under development as an anti-epileptic therapy. Although it is known to reduce calcium flux mediated by AMPA receptors in cultured cortical neurons, there are no studies of its selectivity in synaptic transmission in more intact systems. In the present study using hippocampal slices, perampanel (0.01-10μM) has been tested on pharmacologically isolated synaptic responses mediated by AMPA, NMDA or kainate receptors. Perampanel reduced AMPA receptor-mediated excitatory postsynaptic field potentials (f-EPSPs) with an IC(50) of 0.23μM and a full block at 3μM. This compares with an IC(50) of 7.8μM for GYKI52466 on these responses. By contrast, perampanel at 10μM had no effect on responses mediated by NMDA or kainate receptors, which were completely blocked by 30μM D-AP5 and 10μM NBQX respectively. The concentrations of perampanel required to reduce AMPA receptor-mediated responses are not dissimilar to those in plasma following anti-convulsant doses and are consistent with AMPA receptor antagonism being its primary mode of action.     

The non-competitive AMPA receptor antagonist (GYKI 52466) blocks quisqualate-induced acetylcholine release from the rat hippocampus and striatum: an in vivo microdialysis study

The effects of the non-N-methyl-D-aspartate (NMDA) agonist quisqualate (QUIS) and selective AMPA/kainate receptor antagonist 1-(aminophenyl)-methyl-7, 8-methyilendioxy-5H-2,3-benzodiazepine (GYKI 52466) on the release of acetylcholine (ACh) from the hippocampus and striatum of freely moving rats were studied by transversal microdialysis. Acetylcholine level in the dialisate was measured by the high performance liquid chromatography (HPLC) method with an electrochemical detector. The QUIS (100 microM) perfused through the striatum induced an increase of extracellular ACh level (250%) which lasted for over 1h and gradually returned to basal values. Local perfusion of GYKI 52466 (10-100 microM) to the striatum did not change the basal release of ACh. GYKI 52466 (10 microM) administered together with QUIS (100 microM) in he striatum antagonized the stimulant effect of QUIS on the ACh release.Local administration of the QUIS (100 microM) through the microdialysis fiber implanted in the hippocampus, caused a long lasting increase of extracellular hippocampal ACh level (360%) which was reversed when the drug was withdrawn from the perfusion solution. The stimulant effect of QUIS was antagonized by concomitant perfusion of GYKI (10 microM). No effect was seen on the basal ACh release when GYKI (10-100 microM) was perfused through the hippocampus.Local perfusion with tetrodotoxin (1 microM) decrease the basal release of ACh and prevented the QUIS-induced increase of ACh both in the hippocampus and striatum.Our in vivo neurochemical results indicate that hippocampal and striatal cholinergic systems are regulated by non-NMDA (probably AMPA) glutamatergic receptors located in the hippocampus and striatum.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

The generation of action potentials in the terminals of the Schaffer collaterals during long-term potentiation in the CA1 region of the hippocampus]

Experiments were performed in rat hippocampal slices. Activity of individual CA3 pyramidal neurons and field potentials in the CA1 areas were recorded extracellularly. The collision technique was applied to detect the antidromic origin of the background action potentials in the somata of CA3 neurons. Threshold stimulation of terminals of the Schaffer collaterals in the stratum radiatum of the CA1 area was applied to study their excitability during the CA1 long-term potentiation. During the long-term potentiation, antidromic action potentials appeared in the somata of the CA3 neurons. The obtained evidence suggests that the synaptic potentiation is accompanied by an enhancement of axon terminal excitability resulting in generation of the action potentials.     

Effects of MK-801 and CNQX on various neurotoxic responses induced by kainic acid in mice

Effects of MK-801 (a NMDA receptor blocker) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker) on several neurotoxic responses induced by kainic acid (KA) were examined in ICR mice. In a lethality test, intracerebroventricular (i.c.v.) pretreatment of MK-801 (1 microg), but not CNQX (0.5 microg), attenuated the time to lethality induced by KA (0.5 microg) administered i.c.v. In the memory test (a passive avoidance test), MK-801, but not CNQX, prevented the memory loss induced by KA (0.1 microg). The damage induced by KA (0.1 microg) administered i.c.v. in the hippocampus was markedly concentrated in the CA3 pyramidal neurons. Both MK-801 and CNQX blocked the pyramidal cell death in CA3 hippocampal region induced by KA. In the immunocytochemical study, KA dramatically increased the phosphorylated ERK (p-ERK) and decreased the phosphorylated CREB (p-CREB) in the hippocmapus. Both MK-801 and CNQX attenuated, in part, the increased p-ERK and the decreased p-CREB induced by KA. In addition, both MK-801 and CNQX partially reduced the increased c-Fos and c-Jun protein expression in hippocampus induced by KA. Our results suggest that both NMDA and non-NMDA receptors are involved in supraspinally administered KA-induced pyramidal cell death in CA3 region of hippocampus in the mouse and the p-ERK and the dephosphorylation of CREB protein may play an important role in CA3 region cell death of the hippocampus induced by KA administered supraspinally. Furthermore, c-Fos and c-Jun proteins may serve as third messengers responsible for CA3 pyramidal cell death induced by supraspinally administered KA.     

The anticonvulsive effect of non-NMDA antagonist GYKI 52466 on 3-aminopyridine-induced primary and secondary cortical ictal activity in rat

The effect of GYKI 52466, a selective, non-competitive antagonist of the AMPA glutamate receptor subtype was investigated on the development, expression and propagation of 3-aminopyridine-induced cortical ictal activity, both in the primary and secondary focus. In one group of animals GYKI 52466 was administered intraperitoneally, 20 minutes prior to the local application of the convulsant the surface of the cortex of anaesthetized rats. Control animals were injected by physiological solution. Different parameters of electrographic ictal discharges were measured under the influence of the antagonist and compared to control values. The results demonstrate that GYKI 52466 exerts anticonvulsive effects on both the induction and the expression of primary and secondary electrical ictal activity, by delaying the onset of the first ictal period, shortening the duration of ictal activity and decreasing the amplitudes of epileptiform discharges. However, the seizure propagation was not influenced significantly. It is suggested that the initiation, maintenance and the propagation of spontaneous seizures may be controlled by separate mechanisms and that changes can occur in one of the procedures without parallel changes in others. The observations of the present study extend those reported previously by others, namely that activation of non-NMDA receptors is significantly involved in the initiation and maintenance of cortical epileptiform activity.     

Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons

ABSTRACT: BACKGROUND: Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain. RESULTS: Glutamate robustly increased [Ca2+]i in 14.4 +/- 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3). 75% of cells that responded to glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals. CONCLUSIONS: Bulk-loading fura-2AM into spinal cord slices is a successful means for determining Ca2+ responses in adult dorsal horn neurons. Glutamate-evoked Ca2+ signals in adult dorsal horn neurons are mediated predominantly by AMPA channels and are potentiated by peripheral neuropathic injury.     

Mechanism of inhibition of the GluA2 AMPA receptor channel opening: consequences of adding an N-3 methylcarbamoyl group to the diazepine ring of 2,3-benzodiazepine derivatives

2,3-Benzodiazepine derivatives are AMPA receptor inhibitors, and they are potential drugs for treating some neurological diseases caused by excessive activity of AMPA receptors. Using a laser-pulse photolysis and rapid solution flow techniques, we characterized the mechanism of action of a 2,3-benzodiazepine derivative, termed BDZ-f, by measuring its inhibitory effect on the channel-opening and channel-closing rate constants as well as the whole-cell current amplitude of the homomeric GluA2Q AMPA receptor channels. We also investigated whether BDZ-f competes with GYKI 52466 for binding to the same site on GluA2Q(flip). GYKI 52466 is the prototypic 2,3-benzodiazepine compound, and BDZ-f is the N-3 methylcarbamoyl derivative. We found that BDZ-f is a noncompetitive inhibitor with a slight preference for the closed-channel state of both the flip and the flop variants of GluA2Q. Similar to other 2,3-benzodiazepine compounds that we have previously characterized, BDZ-f inhibits GluA2Q(flip) by forming an initial, loose intermediate that is partially conducting; however, this intermediate rapidly isomerizes into a tighter, fully inhibitory receptor-inhibitor complex. BDZ-f binds to the same noncompetitive site as GYKI 52466 does. Together, our results show that the addition of an N-3 methylcarbamoyl group to the diazepine ring with the azomethine feature (i.e., GYKI 52466) is what makes BDZ-f more potent and more selective toward the closed-channel conformation than the original GYKI 52466. Our results have useful implications for the structure-activity relationship of the 2,3-benzodiazepine series.     

Modulatory effects of oligodendrocytes on the conduction velocity of action potentials along axons in the alveus of the rat hippocampal CA1 region

Like neurons and astrocytes, oligodendrocytes have a variety of neurotransmitter receptors and ion channels. However, except for facilitating the rapid conduction of action potentials by forming myelin and buffering extracellular K(+), little is known about the direct involvement of oligodendrocytes in neuronal activities. To investigate their physiological roles, we focused on oligodendrocytes in the alveus of the rat hippocampal CA1 region. These cells were found to respond to exogenously applied glutamate by depolarization through N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors. Electrical stimulation of the border between the alveus and stratum oriens evoked inward currents through several routes involving glutamate receptors and inward rectifier K(+) channels. Moreover, electrical stimulation resembling in vivo activity evoked long-lasting depolarization. To examine the modulatory effects of oligodendrocytes on neuronal activities, we performed dual, whole-cell recording on CA1 pyramidal neurons and oligodendrocytes. Direct depolarization of oligodendrocytes shortened the latencies of action potentials evoked by antidromic stimulation. These results indicate that oligodendrocytes increase the conduction velocity of action potentials by a mechanism additional to saltatory conduction, and that they have active roles in information processing in the brain.     

Excitability of the soma in central nervous system neurons

The ability of the soma of a spinal dorsal horn neuron, a spinal ventral horn neuron (presumably a motoneuron), and a hippocampal pyramidal neuron to generate action potentials was studied using patch-clamp recordings from rat spinal cord slices, the "entire soma isolation" method, and computer simulations. By comparing original recordings from an isolated soma of a dorsal horn neuron with simulated responses, it was shown that computer models can be adequate for the study of somatic excitability. The modeled somata of both spinal neurons were unable to generate action potentials, showing only passive and local responses to current injections. A four- to eightfold increase in the original density of Na(+) channels was necessary to make the modeled somata of both spinal neurons excitable. In contrast to spinal neurons, the modeled soma of the hippocampal pyramidal neuron generated spikes with an overshoot of +9 mV. It is concluded that the somata of spinal neurons cannot generate action potentials and seem to resist their propagation from the axon to dendrites. In contrast, the soma of the hippocampal pyramidal neuron is able to generate spikes. It cannot initiate action potentials in the intact neurons, but it can support their back-propagation from the axon initial segment to dendrites.     

Phencyclidine actions measured intracellularly in hippocampal CA1 neurons

The electrophysiological effects of phencyclidine (PCP) were measured intracellularly in guinea pig hippocampal CA1 neurons in vitro. At all doses tested (0.2 microM - 10 mM), PCP increased the width of action potentials (APs). Doses of 10 microM and higher were associated with decreased action potential amplitude. PCP decreased inhibitory postsynaptic potentials and excitatory postsynaptic potentials but did not alter responses to focally applied GABA. At the lowest dose (0.2 microM), PCP decreased the input resistance (Rin), while at all other doses Rin was increased. PCP decreased post-spike train afterhyperpolarizations at low and medium doses. PCP effects persisted in low calcium medium and also in medium containing 10(-6) M tetrodotoxin. It is concluded that in these central neurons, PCP primarily blocks potassium conductances at all doses and, at anesthetic doses, depresses sodium-dependent spikes.     

应用多电极记录技术研究海马切片癫痫样放电的起始点和传播方向

弄清癫痫样放电的起始位置和传播方向对研究癫痫机制及其临床治疗有重要意义．为了解决这一问题,应用微电极阵列对低镁人工脑脊液诱导的Sprague-Dawley (SD)大鼠海马切片的癫痫样放电进行记录．分别用癫痫样放电的两种成分：场电位和多单元信号来确定癫痫样放电的起始位置和传播方向．首先计算并比较了海马切片锥体细胞层位置电极记录的癫痫样放电场电位的起始时间,由起始时间的先后关系确定癫痫样放电在锥体细胞层的起始位置和传播方向．然后用整个切片上记录的癫痫样放电的多单元信号动作电位序列进行互相关分析,进一步确定了癫痫样放电在整个海马切片内的起始位置和传播方向．结果显示,CA3区的癫痫样放电具有比CA1区更高的幅度和更长的持续时间,表明CA3区有更高的兴奋性．对于记录到的同步癫痫样放电,CA3b区场电位和多单元信号均比CA3c和CA1区出现更早,起始位置和其随后位置之间的传播延　时与二者之间的距离成正相关．因此,在低镁模型的大鼠海马切片中,癫痫样放电起始于CA3b区并分别向CA3c和CA1区传播．     

Barbiturate effects on hippocampal excitatory synaptic responses are selective and pathway specific

Barbiturate actions on excitatory synaptic responses in CA 1 and dentate regions of hippocampal slices were studied to determine whether different effects occur on anatomically distinct synaptic pathways. Pentobarbital facilitated transmission between stratum radiatum inputs and CA 1 neurons at low concentrations (0.02-0.08 mM) and produced postsynaptic depression at higher concentrations. Only depression was observed for stratum oriens inputs to CA 1 and perforant path inputs to dentate granulae neurons. The (+) isomer of pentobarbital was approximately four times more potent than the (-) isomer of racemic mixture. Phenobarbital (0.04-0.12 mM) produced only depression of synaptic responses in CA 1 and dentate pathways. Comparison of effect on field excitatory postsynaptic potentials and population spike responses indicated that the barbiturates act at selective and pathway-specific sites. The results provide further evidence for specific cellular and membrane recognition sites for barbiturate action.     

Cav1.2 calcium channels modulate the spiking pattern of hippocampal pyramidal cells

Ca(v)1.2 L-type calcium channels support hippocampal synaptic plasticity, likely by facilitating dendritic Ca2+ influx evoked by action potentials (AP) back-propagated from the soma. Ca2+ influx into hippocampal neurons during somatic APs is sufficient to activate signalling pathways associated with late phase LTP. Thus, mechanisms controlling AP firing of hippocampal neurons are of major functional relevance. We examined the excitability of CA1 pyramidal cells using somatic current-clamp recordings in brain slices from control type mice and mice with the Ca(v)1.2 gene inactivated in principal hippocampal neurons. Lack of the Ca(v)1.2 protein did not affect either affect basic characteristics, such as resting membrane potential and input resistance, or parameters of single action potentials (AP) induced by 5 ms depolarising current pulses. However, CA1 hippocampal neurons from control and mutant mice differed in their patterns of AP firing during 500 ms depolarising current pulses: threshold voltage for repetitive firing was shifted significantly by about 5 mV to more depolarised potentials in the mutant mice (p<0.01), and the latency until firing of the first AP was prolonged (73.2+/-6.6 ms versus 48.1+/- 7.8 ms in control; p<0.05). CA1 pyramidal cells from the mutant mice also showed a lowered initial spiking frequency within an AP train. In control cells, isradipine had matching effects, while BayK 8644 facilitated spiking. Our data demonstrate that Ca(v)1.2 channels are involved in regulating the intrinsic excitability of CA1 pyramidal neurons. This cellular mechanism may contribute to the known function of Ca(v)1.2 channels in supporting synaptic plasticity and memory.     

Effects of hypergravic fields on serotonergic neuromodulation in the rat hippocampus

The effects of 7 day exposure to 2G fields on serotonergic modulation at two synapses on a hippocampal pathway were examined by recording dentate gyrus and CA1 pyramidal cell layer electrical activity. Serotonin decreased the amplitude of the population spike (synchronous action potentials in hundreds of neurons) in both the dentate gyrus and CA1 regions of rats exposed to 2G fields for 7 days. The inhibition, averaging 26 +/- 4% (mean +/- SEM) in the dentate gyrus and 80 +/- 5% in the CA1 region, was not significantly different from inhibitory responses observed in 1G controls. The 5-HT1A agonist 8-OH-DPAT mimicked this inhibition in the dentate and CA1 regions of 1G rats. 8-OH-DPAT responses were not affected by exposure to 2G fields. We conclude that the hippocampus contains surplus 5-HT receptors so that decreases in receptor density reported in receptor binding studies do not result in a decrease in modulatory capability. A model to account for the physiological pathway that relates gravitational field strength to 5-HT receptor density without changing the effectiveness of 5-HT neuromodulation is discussed.     

Effects of extracellular calcium on low frequency induced potentiation and habituation in the in vitro hippocampal slice preparation

The electrophysiological characteristics of frequency potentiation and habituation were investigated in two afferent systems of the in vitro hippocampal slice preparation. Low frequency stimulation (1 Hz) of the Schaffer collateral - commissural (Sch-comm) fibers results in a short-term potentiation of the amplitude and rate of rise of the EPSP and population spike responses recorded in the CA1 region. In contrast, 1-Hz stimulation of the perforant path (PP) evokes a short-term, habituation-like depression of the dentate granule cell EPSP and population spike. An inverse relationship was observed between stimulus intensity and the magnitude of frequency potentiation or habituation. Changes in afferent fiber volleys or general excitability of postsynaptic membranes did not contribute significantly to the development of either of these forms of short-term plasticity. Perfusion with a medium containing a high calcium - low magnesium concentration (4 mM Ca+2 and 1 mM Mg+2) produced a differential effect on CA1 and dentate evoked potentials. Following a 20-min exposure to this medium, the amplitude of CA1 potentials was increased while dentate responses were decreased. Frequency potentiation of CA1 responses and habituation of dentate responses were depressed or eliminated by the high calcium medium. The opposing influence of extracellular calcium on CA1 and dentate evoked potentials indicates a fundamental difference in the process of transmitter release in these systems, a characteristic that may contribute to the production of frequency potentiation and habituation.     

Selective activation of field CA1-CA2 by perforant neurons discharging in response to hippocampal efferent volleys

A long lasting evoked response, generated in the hippocampal field CA1-CA2 following postsynaptic sequential discharge of hippocampal pyramids and entorhinal neurons, has been analyzed by multiple simultaneous surface and depth recordings in the guinea pig dorsal hippocampal region. Results obtained suggest that it can be associated with postsynaptic excitatory potentials evoked in the distal portion of the apical dendrites of field CA1-CA2 pyramids by perforant neurons selectively discharged by impulses of hippocampal origin.     

Temporal expression of hippocampal lysophosphatidic acid receptors and their roles in kainic acid-induced neurotoxicity

In this investigation, the role of hippocampal lysophosphatidic acid (LPA) receptors in the regulation of kainic acid (KA)-induced neurotoxicity was investigated. KA (0.07 μg) intracerebroventricular (i.c.v.) administration increased hippocampal Lpar1, 2, 3, and 5 mRNA levels. In the immunohistochemical study, alteration of LPA₁ or LPA₃ immunoreactivity was different depending on the hippocampal regions, such as CA1, CA2, CA3, and dentate gyrus. In addition, the i.c.v. pretreatment with LPA₁ and LPA₃ antagonists, such as VPC12249 (0.05 μg) and VPC32183 (0.05 μg) attenuated KA-induced neuronal cell death in the hippocampal CA3 region. However, the i.c.v. 18:1 LPA (0.05 μg) pretreatment aggravated KA-induced neuronal cell death in the hippocampal CA3 region. Our results suggest that LPA receptors, such as LPA₁ and LPA₃ activation might play an important role in the regulation of KA-induced neuronal cell death in the hippocampal CA3 region.     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.