Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro

Glutamate is an important regulator of dendrite development that may inhibit, (during ischemic injury), or facilitate (during early development) dendrite growth. Previous studies have reported mainly on the N-methyl-D-aspartate (NMDA) receptor-mediated dendrite growth-promoting effect of glutamate. In this study, we examined how the non-NMDA receptor agonist kainate influenced dendrite growth. E18 mouse cortical neurons were grown for 3 days in vitro and immunolabeled with anti-microtubule-associated protein 2 (MAP2) and anti-neurofilament (NF-H), to identify dendrites and axons, respectively. Exposure of cortical neurons to kainate increased dendrite growth without affecting neuron survival. This effect was dose-dependent, reversible and blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor antagonist NBQX and the low-affinity kainate receptor antagonist NS-102, but not by the AMPA receptor antagonist CFM-2. In addition, the NMDA receptor antagonist MK-801 had no effect on kainate-induced dendrite growth. Immunolabeling and Western blot analysis of kainate receptors using antibodies against the GluR6 and KA2 subunits, demonstrated that the immature cortical neurons used in this study express kainate receptor proteins. These results suggest that kainate-induced non-NMDA receptor activation promotes dendrite growth, and in particular primary dendrite number and length, from immature cortical neurons in vitro, and that kainate receptors may be directly involved in this process. Furthermore, these data support the possibility that like NMDA receptors, kainate receptor activation may also contribute to early neurite growth from cortical neurons in vitro.     

神经元兴奋性毒性对Munc18-1蛋白在大鼠海马神经元和原代培养神经元胞核内分布的影响(英文)

目的 Munc18-1在中枢神经系统递质释放过程中具有重要作用,控制着突触囊泡释放步骤的每一个环节。Munc18-1功能异常与癫痫发病相关。本文主要探讨癫痫是否会引起神经元胞核内Munc18-1定位的改变。方法通过海马内注射海人藻酸建立Sprague-Dawley(SD)大鼠癫痫模型,腹腔注射海人藻酸建立昆明小鼠癫痫模型。分离胎龄18天SD大鼠海马神经元,用Neurobasal培养基培养7天后,用谷氨酸处理3h。用蔗糖密度梯度离心法分离神经元和神经胶质细胞的细胞核组份,通过甲酚紫染色对上述富含细胞核的组份进行形态学鉴别。用免疫组化和免疫电镜分析法确定Munc18-1的细胞核定位。免疫印迹法检测不同细胞组分的Munc18-1蛋白的表达水平。结果免疫组化、免疫电镜以及对神经元细胞核组份的免疫印迹证实了Munc18-1在海马神经元细胞核的分布定位。在海人藻酸诱导癫痫的动物海马内,免疫组化染色显示Munc18-1在海马CA区锥体细胞层、齿状回颗粒细胞层和门区的多型细胞表达减少。同时,免疫印迹分析表明,Munc18-1在海马神经元胞核中的表达水平明显下降。免疫印迹检测显示,原代培养神经元经50μmol/L谷氨酸处理3h...     

Munc18-1 Is Essential for Neuropeptide Secretion in Neurons

Neuropeptide secretion from dense-core vesicles (DCVs) controls many brain functions. Several components of the DCV exocytosis machinery have recently been identified, but the participation of a SEC1/MUNC18 (SM) protein has remained elusive. Here, we tested the ability of the three exocytic SM proteins expressed in the mammalian brain, MUNC18-1/2/3, to support neuropeptide secretion. We quantified DCV exocytosis at a single vesicle resolution on action potential (AP) train-stimulation in mouse CNS neurons (of unknown sex) using pHluorin-tagged and/or mCherry-tagged neuropeptide Y (NPY) or brain-derived neurotrophic factor (BDNF). Conditional inactivation of Munc18-1 abolished all DCV exocytosis. Expression of MUNC18-1, but not MUNC18-2 or MUNC18-3, supported DCV exocytosis in Munc18-1 null neurons. Heterozygous (HZ) inactivation of Munc18-1, as a model for reduced MUNC18-1 expression, impaired DCV exocytosis, especially during the initial phase of train-stimulation, when the release was maximal. These data show that neurons critically and selectively depend on MUNC18-1 for neuropeptide secretion. Impaired neuropeptide secretion may explain aspects of the behavioral and neurodevelopmental phenotypes that were observed in Munc18-1 HZ mice.SIGNIFICANCE STATEMENT Neuropeptide secretion from dense-core vesicles (DCVs) modulates synaptic transmission, sleep, appetite, cognition and mood. However, the mechanisms of DCV exocytosis are poorly characterized. Here, we identify MUNC18-1 as an essential component for neuropeptide secretion from DCVs. Paralogs MUNC18-2 or MUNC18-3 cannot compensate for MUNC18-1. MUNC18-1 is the first protein identified to be essential for both neuropeptide secretion and synaptic transmission. In heterozygous (HZ) Munc18-1 neurons, that have a 50% reduced MUNC18-1expression and model the human STXBP1 syndrome, DCV exocytosis is impaired, especially during the initial phase of train-stimulation, when the release is maximal. These data show that MUNC18-1 is essential for neuropeptide secretion and that impaired neuropeptide secretion on reduced MUNC18-1expression may contribute to the symptoms of STXBP1 syndrome.     

Presynaptic Short-Term Plasticity Persists in the Absence of PKC Phosphorylation of Munc18-1

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.     

Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors

Glutamate is an important regulator of dendrite development; however, during cerebral ischemia, massive glutamate release can lead to neurodegeneration and death. An early consequence of glutamate excitotoxicity is dendrite injury, which often precedes cell death. We examined the effect of glutamate on dendrite growth from embryonic day 18 (E18) mouse cortical neurons grown for 3 days in vitro (DIV) and immunolabeled with anti-microtubule-associated protein (MAP)2 and anti-neurofilament (NF)-H, to identify dendrites and axons, respectively. Cortical neurons exposed to excess extracellular glutamate (100 microM) displayed reduced dendrite growth, which occurred in the absence of cell death. This effect was mimicked by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and blocked by the ionotropic glutamate receptor antagonist kynurenic acid and the NMDA receptor-specific antagonist MK-801. The non-NMDA receptor agonist AMPA, however, did not affect process growth. Neither NMDA nor AMPA influenced neuron survival. Immunolabeling and Western blot analysis of NMDA receptors using antibodies against the NR1 subunit, demonstrated that immature cortical neurons used in this study, express NMDA receptors. These results suggest that excess glutamate decreases dendrite growth through a mechanism resulting from NMDA receptor subclass activation. Furthermore, these data support the possibility that excess glutamate activation of NMDA receptors mediate both cell death in mature neurons and the inhibitory effect of excess glutamate on dendrite growth in immature neurons or in the absence of cell death.     

BMP-7 and excess glutamate: opposing effects on dendrite growth from cerebral cortical neurons in vitro

Glutamate is an important regulator of dendrite development. During cerebral ischemia, however, there is massive release of glutamate reaching millimolar concentrations in the extracellular space. An early consequence of this excess glutamate is reduced dendrite growth. Bone morphogenetic protein-7 (BMP-7) a member of the transforming growth factor-beta (TGF-beta) superfamily has been demonstrated to enhance dendrite output from cerebral cortical and hippocampal neurons in vitro. However, it is not known whether BMP-7can prevent the reduced dendrite growth associated with excess glutamate or enhance dendrite growth after glutamate exposure. Therefore we quantified axon and primary, secondary, and total dendrite growth from embryonic mouse cortical neurons (E18) grown at low density in vitro in a chemically defined medium and exposed to glutamate (1 or 2 mM) for 48 h. Morphology and double immunolabeling (MAP2, NF-H) were used to identify cortical dendrites and axons after 3 DIV. In these short-term cultures, glutamate did not influence neuron survival. The addition of glutamate to cortical neurons, however, significantly attenuated dendrite output. This effect was mimicked by the addition of NMDA but not AMPA agonists and inhibited by the specific NMDA receptor antagonist MK-801. The reduction in dendrite growth mediated by excess glutamate was ameliorated by the administration of 30 or 100 ng/ml of BMP-7. In addition, when administered in a delayed fashion between 1 and 24 h after the initial glutamate exposure, BMP-7 was able to enhance dendrite growth, including primary dendrite number, primary dendrite length, and secondary dendritic branching. These findings demonstrate that BMP-7 can ameliorate reduced dendrite growth from cerebral cortical neurons associated with excess glutamate in vitro and are important because they may help explain why BMP-7 administration is associated with enhanced functional recovery in models of cerebral ischemia.     

Glutamate released spontaneously from astrocytes sets the threshold for synaptic plasticity

Astrocytes exhibit spontaneous calcium oscillations that could induce the release of glutamate as gliotransmitter in rat hippocampal slices. However, it is unknown whether this spontaneous release of astrocytic glutamate may contribute to determining the basal neurotransmitter release probability in central synapses. Using whole‐cell recordings and Ca²⁺ imaging, we investigated the effects of the spontaneous astrocytic activity on neurotransmission and synaptic plasticity at CA3–CA1 hippocampal synapses. We show here that the metabolic gliotoxin fluorocitrate (FC) reduces the amplitude of evoked excitatory postsynaptic currents and increases the paired‐pulse facilitation, mainly due to the reduction of the neurotransmitter release probability and the synaptic potency. FC also decreased intracellular Ca²⁺ signalling and Ca²⁺‐dependent glutamate release from astrocytes. The addition of glutamine rescued the effects of FC over the synaptic potency; however, the probability of neurotransmitter release remained diminished. The blockage of group I metabotropic glutamate receptors mimicked the effects of FC on the frequency of miniature synaptic responses. In the presence of FC, the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N ′,N ′‐tetra‐acetate or group I metabotropic glutamate receptor antagonists, the excitatory postsynaptic current potentiation induced by the spike‐timing‐dependent plasticity protocol was blocked, and it was rescued by delivering a stronger spike‐timing‐dependent plasticity protocol. Taken together, these results suggest that spontaneous glutamate release from astrocytes contributes to setting the basal probability of neurotransmitter release via metabotropic glutamate receptor activation, which could be operating as a gain control mechanism that regulates the threshold of long‐term potentiation. Therefore, endogenous astrocyte activity provides a novel non‐neuronal mechanism that could be critical for transferring information in the central nervous system.     

Glutamate receptors in nucleus accumbens mediate regionally selective increases in cortical acetylcholine release

The basal forebrain cortical cholinergic system (BFCS) is critical for the regulation of attentional information processing. BFCS activity is regulated by several cortical and subcortical structures, including the nucleus accumbens (NAC) and prefrontal cortex (PFC). GABAergic projection neurons from NAC to basal forebrain are modulated by Glu receptors within NAC. We previously reported that intra-NAC perfusions of NMDA or its antagonist CPP stimulate ACh release in PFC. In this experiment we determined whether this trans-synaptic modulation of cortical ACh release is evident in multi-sensory associational areas like the posterior parietal cortex (PPC). Artificial cerebrospinal fluid (aCSF, control), NMDA (250 or 400 muM), or CPP (200 or 400 muM) were perfused into the NAC shell and ACh was measured in the ipsilateral PPC. Amphetamine (2.0 mg/kg, i.p), was systemically administered as a positive control in a fourth session, since it also stimulates cortical ACh release but via mechanisms known to not necessitate transmission within the NAC. Neither NMDA nor CPP increased ACh efflux in the PPC, yet both drugs increased ACh release in PFC, suggesting that NMDA receptor modulation in the NAC increases ACh in the cortex in a regionally-specific manner. Systemic amphetamine administration significantly increased (100-200%) ACh in the PPC, suggesting that levels of ACh in the PPC can be increased following certain pharmacological manipulations. The cortical region-specific modulation of ACh by NAC may underlie the linkage of motivational information with top-down controls of attention as well as guide appropriate motor output following exposure to salient and behaviorally relevant stimuli.     

Chronic caffeine exposure enhances adenosinergic inhibition of cerebral cortical neurons

Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10–70 mg/kg) increased the sensitivity of rat cerebral cortical neurons to the inhibitory action of microiontophoretically applied adenosine. The sensitivity of spontaneously firing rat cerebral cortical neurons in caffeine-treated animals was compared with that of saline-treated controls using the same multiple-barrel micropepettes tested on the same day. Adenosine sensitivity was determined by the I·T₅₀ method. The I·T₅₀ value for 134 neurons in the caffeine-treated rats of 130.77 ± 4.33 (S.E.M.) was significantly (P < 0.001) different to that of 136 neurons in the saline-treated control rats (222.16 ± 6.68), indicating a supersensitivity to adenosine in neurons which had been chronically exposed to caffeine.     

Glutamate Peripherally Administered Exerts Somatostatin-Releasing Action in the Conscious Rat

This study was designed to determine whether glutamate is able to stimulate somatostatin release from in vivo conscious animals when somatostatin release is monitored in unanaesthetized rats stereotaxically implanted with a push-pull cannula in the median eminence. One week after implantation, the median eminence was perfused with artificial cerebrospinal fluid alone or with the addition of either CGS 19755, an N-methyl-D-aspartate (NMDA) receptor antagonist (1(10^?6 M), or glutamate (10^?5 M). The latter (which is able to cross the brain-blood barrier at large doses) was also peripherally administered (1 g/kg ip). Median eminence perfusate samples were collected every 15 min and somatostatin was measured by a sensitive radioimmunoassay. In rats receiving ip glutamate injection, somatostatin release from the median eminence was significantly increased (73.3±10.4 versus 24.8 ± 6.2; P < 0.01; n = 5) when compared to baseline levels measured in the same animals, but no effect was observed when local perfusion of the median eminence with glutamate (10^?4 to 10^?5 M; n = 6) was performed. Glutamate-induced somatostatin release was completely blunted (n = 5) by prior local administration of CGS 19755 (10^?5 M), a potent NMDA-type receptor antagonist able to cross the blood-brain barrier. In contrast, administration of glutamic acid diethylester, a competitive antagonist of non-NMDA receptors, at doses of 10^?4 M (n = 4), was not able to alter this response. Our results are the first in vivo evidence in favour of a neuroendocrine role for glutamate on somatostatin release whose site of action seems to exclude the median eminence.     

Distribution of phosphate-activated glutaminase in the human cerebral cortex

Phosphate-activated glutaminase (PAG), which catalyses conversion of glutamine to glutamate, is a potential marker for glutamatergic, and possibly GABA, neurons in the central nervous system. A polyclonal antibody, raised in rabbits against rat brain PAG, was applied to postmortem human brain tissue to reveal the distribution of PAG in the cerebral cortex. PAG immunoreactivity was observed in pyramidal and non-pyramidal neurons but not in glial cells. In the neocortex, large to medium-sized pyramidal neurons in layers III and V were stained most intensely, while the majority of smaller pyramidal cells were labeled either lightly or moderately. Such modified pyramids as the giant Betz cells, the large pyramidal cells of Meynert, and the solitary cells of Ramón y Cajal were also stained intensely. Fusiform cells in layer VI showed moderate to intense labeling. A number of cortical non-pyramidal neurons of various sizes stained moderately to intensely. These included large basket cells which were identified by their characteristic morphology and size in primary cortical areas. Pyramidal cells in the hippocampal formation as well as basket cells of the stratum oriens stained moderately to intensely. Since pyramidal cells are believed to be glutamatergic and large basket cells GABAergic, these results suggest that PAG plays a role in generating not only transmitter glutamate, but also GABA precursor glutamate.     

Inorganic phosphate enhances phosphonucleotide concentrations in cultured fetal rat cortical neurons

Our laboratory has recently characterized saturable Na⁺-dependent P_i import into cultured fetal rat cortical neurons and shown that a substantial fraction of the P_i so accumulated is incorporated into ATP. We now report that the ATP, NADPH and intracellular free P_i ([P_i]_i) concentrations of cultured fetal rat cortical neurons are dependent on the extracellular P_i concentration ([P_i]_e). [ATP], [NADPH] and [P_i]_i display a hyperbolic dependence upon [P_i]_e, being significantly increased after incubation with [P_i]_e of ≥10 μM, and maximal at ≥500 μM. Increases in both [ATP] and [NADPH] are abolished in the absence of glucose. In the absence of extracellular P_i, both [ATP] and [P_i]_i decline over time. Our data suggest that in cultured fetal rat cortical neurons [P_i]_e has a direct effect on glucose utilization, stimulating both ATP and NADPH synthesis via glycolysis and the pentose phosphate pathway.     

Pentylenetetrazole-induced changes of the single potassium channel in primary cultured cerebral cortical neurons

To elucidate the behavior of the single ionic channels of cerebral cortical neurons during bursting activity, the effects of pentylenetetrazole (PTZ) on a single potassium channel of primary cultured cerebral cortical neurons from mice were examined. All of the examined 10-day-old primary cultured neurons of the cerebral cortex showed clear bursting activity after extracellular application of PTZ using whole-cell patch-clamp recording. Less than half of the examined single potassium channels, both outward and inward, of the 2- and 3-day-old as well as 6–10-day-old primary cultured cerebral cortical neurons showed the bursting-type open-close state by application of PTZ. The PTZ-sensitive single potassium channels were found in the voltage-dependent as well as calcium-activated channels.     

Munc18抗体致痫机制的体外实验研究:诱导海马神经细胞的损伤

目的 前期研究发现将突触前膜胞内蛋白(Munc18)抗体注射到大鼠的海马,大鼠呈痫样发作,故研究此抗体对海马神经细胞的损伤作用.方法 取SD新生鼠海马组织,以此培养的原代海马神经细胞作为靶细胞.抗体在不同的作用时间(20 h和40 h)和稀释倍数下,通过细胞存活率和乳酸脱氢酶(LDH)释放量来测定抗体对神经细胞的毒性;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)测定神经细胞的凋亡.结果 Munc18抗体对神经细胞具有细胞毒性和促凋亡作用,并与抗体的滴度和作用时间呈剂量依赖性.即使在抗体低滴度的情况下(×1 000和×2 000),Munc18抗体仍可诱导凋亡,且与对照组比较有显著性差异(P＜0.01或P＜0.05).结论 Munc18抗体的致痫机制与其诱导神经细胞的损伤作用(可能主要是经凋亡途径)有关.     

Glutamate receptor regulation of rat nucleus accumbens neurons in vivo

Extracellular single cell recording and microiontophoretic techniques were used to characterize the roles of ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs) in glutamate-induced excitation of rat nucleus accumbens (NAc) neurons in vivo. Pulse-ejected glutamate (16–128 nA) induced a current-dependent increase in the firing of quiescent NAc neurons. A stronger excitatory response to α-amino-3-hydroxy-5-methyl-4-iosoxazole-proprionic acid (AMPA) was observed at much lower ejection currents (0.1–6.4 nA). Compared to AMPA and glutamate, N-methyl-D -aspartate (NMDA) induced a much less potent excitation in a narrow current range (1–4 nA) and only when neurons were previously “primed” with other excitatory amino acids (EAAs). Higher ejection currents of all three EAA agonists drove NAc neurons into a state of apparent depolarization block. AMPA-evoked firing was selectively blocked by the AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) whereas NMDA-induced activity was selectively prevented by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (D-AP5). DNQX, but not D-AP5, significantly attenuated glutamate-evoked activity. The mGluR receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-t-ACPD) failed to evoke activity of NAc neurons, but significantly reduced the excitatory effects of other EAAs. This modulatory effect of 1S,3R-t-ACPD was consistently blocked by the selective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3) whereas another mGluR antagonist (RS)-4-carboxy-3-hydroxy phenylglycine (4C3HPG) was inconsistent in this regard. These results indicate that the excitatory effects of glutamate on rat NAc neurons in vivo are primarily mediated by non-NMDA iGluRs and that mGluRs function to dampen excessive glutamate transmission through iGluRs. © 1996 Wiley-Liss, Inc.     

Cocaine causes deficits in radial migration and alters the distribution of glutamate and GABA neurons in the developing rat cerebral cortex

Prenatal cocaine exposure induces cytoarchitectural changes in the embryonic neocortex; however, the biological mechanisms and type of cortical neurons involved in these changes are not known. Previously, we found that neural progenitor proliferation in the neocortical ventricular zone (VZ) is inhibited by cocaine; here, we examine the changes in cortical neurogenesis and migration of glutamate and GABA neurons induced by prenatal cocaine exposure. Pregnant rats received 20 mg/kg of cocaine intraperitoneally twice at an interval of 12 h during three periods of neocortical neurogenesis. Neocortical area and distribution of developing neurons were examined by counting Tuj1+, glutamate+, or GABA+ cells in different areas of the cerebral cortex. Cocaine decreased neocortical area by reducing the size of the Tuj1+ layer, but only when administered during early periods of neocortical neurogenesis. The number of glutamatergic neurons was increased in the VZ but was decreased in the outer cortical laminae. Although the number of GABA+ neurons in the VZ of both the neocortex and ganglionic eminences was unchanged, GABA+ cells decreased in all other neocortical laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. These findings suggest that in utero cocaine exposure disturbs radial migration of neocortical neurons, possibly because of decreased radial glia guiding support through enhanced differentiation of neocortical VZ progenitors. Cocaine interrupts radial migration of both glutamatergic and GABAergic neurons within the neocortex, in addition to the tangential migration of GABAergic neurons from the subcortical telecephalon. This may result in abnormal neocortical cytoarchitecture and concomitant adverse functional effects. Synapse 65:21–34, 2011. © 2010 Wiley‐Liss, Inc.     

Enhanced GABA release in cell-damaging conditions in the adult and developing mouse hippocampus

The release of [³H]GABA from hippocampul slices from adult (3-month-old) and developing (7-day-old) mice was studied in cell-damaging conditions in vitro using a superfusion system. Cell damage was induced by modified superfusion media, including hypoxia, hypoglycemia, ischemia, the presence of free radicals and oxidative stress. The basal release of GABA from the immature and mature hippocampus was generally markedly increased in all cell-damaging conditions. In 7-day-old mice the release was enhanced most in the presence of free radicals, 1.0 mM NaCN and ischemia, whereas in the adults 1.0 mM NaCN provoked the largest release of GABA, followed by ischemia and free radical-containing media. Potassium stimulation (50mM K⁺) was still able to potentiate the release in all cell-damaging conditions in both age groups. It was shown by superfusing the slices in Ca- and Na-free media that ischemia-induced GABA release was Ca-independent, occurring by a reversed operation of Na-dependent cell membrane carriers in both adult and developing hippocampus. Glutamate and its receptor agonists, N-methyl-d-aspartate (NMDA), kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), potentiated GABA release only in the immature hippocampus by a receptor-mediated mechanism. The enhancement by kainate and AMPA receptors also operated under ischemic conditions. The massive amount of GABA released simultaneously with excitatory amino acids in the mature and immature hippocampus may be an important protective mechanism against excitotoxicity, counteracting harmful effects that lead to neuronal death. The GABA release induced by activation of presynaptic glutamate receptors may contribute particularly to the maintenance of homeostasis in the hippocampus upon impending hyper-excitation.     

Neurokinin-receptor-mediated depolarization of cortical neurons elicits an increase in glutamate release at excitatory synapses

Using whole-cell patch-clamp recordings of spontaneous synaptic activity, we have previously shown that activation of neurokinin-1 (NK1) but not NK3 receptors leads to increased GABA release onto principal cells in the rat entorhinal cortex. In the present study, we examine the effect of activation of these receptors on spontaneous excitatory synaptic responses mediated by glutamate. Both neurokinin B (NKB) and the specific NK3 receptor agonist, senktide, increased the spontaneous release of glutamate, and a similar effect was also seen with substance P (SP) and other NK1 receptor agonists. The increased release induced by either SP or senktide was absent in the presence of tetrodotoxin, demonstrating that it was likely to occur via activation of presynaptic excitatory neurons. Current-clamp recordings confirmed that principal neurons were depolarized by both NK3 and NK1 agonists. However, the response to the former but not the latter persisted in tetrodotoxin, allowing us to conclude that NK3 receptor activation provoked glutamate release via recurrent collaterals between principal neurons, whereas the NK1 receptors may be localized to excitatory interneurons. Finally, the increased release induced by senktide, but not SP, was reduced by an antagonist of group III metabotropic glutamate receptors. Thus, glutamate release from recurrent collaterals is facilitated by a presynaptic group III autoreceptor [Evans, D.I.P., Jones, R.S.G. & Woodhall, G.L. (2000) J. Neurophysiol.,83, 2519-2525], whereas the terminals of neurons responsible for the NK1-receptor induced glutamate release may not bear these receptors. These results have implications for control of activity and epileptogenesis in cortical networks.     

Catecholamine release from isolated sensory neurons of cat petrosal ganglia in tissue culture

The petrosal ganglion (PG) is entirely constituted by the perikarya of primary sensory neurons, part of which innervates the carotid body via the carotid sinus nerve (CSN). Application of acetylcholine (ACh) or nicotine (Nic) as well as adenosine 5'-triphosphate (ATP) to the PG in vitro increases the frequency of CSN discharges, an effect that is modified by the concomitant application of dopamine (DA). Since a population of PG neurons expresses tyrosine hydroxylase, and DA is released from the cat carotid body in response to electrical stimulation of C-fibers in the CSN, it is possible that DA may be released from the perikarya of PG neurons. Therefore, we studied whether ACh or Nic, ATP and high KCl could induce DA release from PG neurons in culture. Petrosal ganglia were excised from pentobarbitone-anesthetized adult cats, dissociated and their neurons maintained in culture for 7-21 days. Catecholamine release was measured by amperometry via carbon-fiber microelectrodes. In response to KCl, Nic, ACh or ATP application, about 25% of neurons exhibited electrochemical signals compatible with DA release. This percentage increased to 41% after loading the neurons with exogenous DA. The present results suggest that DA release may be induced from the perikarya of a population of PG neurons.     

Methylphenylpyridium ion (MPP+) enhances glutamate-induced cytotoxicity against dopaminergic neurons in cultured rat mesencephalon

Parkinson's disease is characterized by chronic progression of dopaminergic neuronal death, the mechanism of which is still unknown. Although methyl-4-phenylpyridium ion (MPP+) or MPP+-like substance, that can reduce mitochondrial complex I activity, is supposed to be a causative agent for Parkinson's disease, it is difficult to explain the chronic neuronal degeneration for years. It is important to identify other putative agents capable of causing chronic cell death besides MPP⁺. We hypothesized that treatment with small doses of MPP⁺, not causing severe damage to dopaminergic neurons but merely reducing the activity of mitochondrial complex I, can be a model of Parkinson's disease, and that glutamate can be a putative agent causing chronic neuronal degeneration. Using primary culture of the rat mesencephalon, we investigated glutamate-induced cytotoxicity against dopaminergic and non-dopaminergic neurons with or without the pretreatment with MPP⁺. Brief exposure to glutamate showed similar cytotoxicity against both dopaminergic and non-dopaminergic neurons. An N-methyl-D -aspartate receptor antagonist completely blocked the glutamate-induced cytotoxicity against both dopaminergic and non-dopaminergic neurons. In the dopaminergic neurons, MPP⁺ caused cytotoxicity that was not blocked by co-administration of MK-801. After pretreatment with small doses of MPP⁺, sub-lethal doses of glutamate caused severe cell damage restricted to dopaminergic neurons, suggesting that MPP⁺ potentiates the glutamate-induced cytotoxicity only against dopaminergic neurons. As glutamate is putatively capable of causing cytotoxicity against dopaminergic neurons, the present findings might be important in considering the pathogenesis of dopaminergic neuronal degeneration and a possible therapeutic application of glutamate receptor antagonists in Parkinson's disease. © 1996 Wiley-Liss, Inc.