慢性应激对大鼠海马锥体细胞形态结构的效应

为研究慢性应激相关精神障碍的发病机制,采用尼氏（Nissl）染色法、高尔基（Golgi）镀染法和透射电镜技术,探讨慢性应激对大鼠海马CA1、CA3区锥体细胞形态结构的效应.结果显示应激组大鼠海马CA1区锥体细胞形态结构较对照组无明显变化.应激组海马CA3区锥体细胞数（35.14±3.85）较对照组(38.74±3.54)显著减少（P＜0.05）;顶树突的总长度（155.67 μm±33.32 μm）较对照组（195.63 μm±34.61 μm）显著缩短（P＜0.05）;应激组大鼠海马CA3区锥体细胞出现超微结构的改变,包括细胞固缩、体积缩小、核膜皱缩、线粒体变性和粗面内质网模糊不清.这提示海马CA3区锥体细胞形态结构的改变,可能是慢性应激相关精神障碍的病理生理基础.     

Calcium Channels in the Nuclear Envelope of Pyramidal Neurons of the Rat Hippocampus

As is known, an increase in the concentration of Са²⁺ in the nuclei of nerve cells leads to activation of genes responsible for the formation of long-lasting postsynaptic changes; mechanisms of memory and learning are based on such changes. The pathways necessary for the entry of calcium into the nuclei of hippocampal pyramidal neurons remained unstudied. Using a patch-clamp technique, we studied what types of calcium channels exist in the membranes of isolated nuclei of pyramidal neurons of the hippocampal СА1 area. In the inner nuclear membrane of these cells, we, for the first time, found inositol trisphosphate receptors (IP₃Rs) activated by inositol trisphosphate applied in the concentration of ≥0.1 μM. The conductivity of single channels of such receptors was, on average, 366 pS; these channels were permeable for both monovalent and bivalent cations. Our data indicate that the nuclear envelope of pyramidal neurons of the hippocampal СА1 area can play the role of the calcium store from which Са²⁺ enter the cell nucleus directly. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 288–292, July–August, 2008.     

An Ibotenate-Selective Metabotropic Glutamate Receptor: Mediates Protein Phosphorylation in Cultured Hippocampal Pyramidal Neurons

Abstract: Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca²⁺ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120-and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and ionotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca²⁺ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca²⁺ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca²⁺ but not in 50 nM extracellular Ca²⁺. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphates in both normal and low extracellular Ca²⁺. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca²⁺. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50–60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml),-suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation. These results suggest that the stimulation of phosphorylation in pyramidal neurons by glutamate occurs predominantly through the activation of an ibotenate-selective metabotropic glutamate receptor.     

海马区锥体神经元轴突高频电刺激对胞体的影响

目的 深部脑刺激（deep brain stimulation,DBS）利用持续的电脉冲高频刺激（high-frequency stimulation,HFS）调控神经元的活动，可望用于治疗更多脑疾病。为了深入了解HFS的作用机制，促进DBS的发展，本文研究轴突HFS在引起轴突阻滞期间神经元胞体的改变。方法 在麻醉大鼠海马CA1区的锥体神经元轴突上施加脉冲频率为100 Hz的1 min逆向高频刺激（antidromic high-frequency stimulation,A-HFS）。为了研究胞体的响应，利用线性垂直排列的多通道微电极阵列，记录刺激位点上游CA1区锥体神经元胞体附近各结构分层上的诱发电位，包括A-HFS脉冲诱发的逆向群峰电位（antidromic population spike,APS）以及A-HFS期间施加的顺向测试脉冲诱发的顺向群峰电位（orthodromic population spike,OPS），并计算诱发电位的电流源密度（current-source density,CSD），用于分析A-HFS期间锥体神经元胞体附近动作电位的生成和传导。结果 锥体神经...     

弱激光对大鼠海马神经元钠通道特性的影响

利用波长670nm、功率5mW的半导体激光器照射急性分离的大鼠海马CA3区锥体神经元,应用全细胞膜片钳技术研究其电压门控Na 通道的特性.实验发现:弱激光作用5min时,Na 通道激活电位和峰值电位开始向负电位方向移动,7min激光作用达稳定;激光照射对Na 通道电流峰值无影响,对照组和激光照射组峰值电流密度分别为(-383.51±26.93)pA/pF和(-368.36±33.14)pA/pF(n=8,P>0.05);激光作用降低了Na 通道的激活阈值电位和峰值电位,对照组通道电流在-40mV激活,-30mV达峰值,激光照射组通道电流在-60mV激活,-40mV达峰值;激光照射改变了Na 通道半数激活电压和斜率因子,对照组和激光照射组的半数激活电压分别为(-42.091±1.537)mV和(-54.971±1.846)mV(n=8,P<0.01),斜率因子分别为(1.529±0.667)mV和(2.634±0.519)mV(n=8,P<0.05).结果表明,弱激光照射海马神经元可改变Na 通道的激活特性,从而影响动作电位的去激化过程,进而会引起神经元细胞生理功能发生变化.     

乌拉坦对大鼠海马CA1区锥体神经元自发放电的抑制作用

为了深入研究麻醉药乌拉坦对大鼠海马CA1锥体神经元自发放电的作用及其机制,分析了10 mmol/L乌拉坦对自发放电、电压门控钠通道、电压门控钾通道的作用．从自发放电信号中计算了放电频率、提取了峰峰间隔序列(ISI)并利用样品熵和去趋势波动法对ISI进行了非线性分析．结果表明,乌拉坦不仅抑制了自发放电的频率,而且降低了自发放电ISI序列的复杂度并弱化了其长时程相关性．离子通道研究结果表明,乌拉坦显著地抑制了钠通道电流(I_Na),对延迟整流钾通道电流(I_K)和瞬时外向钾通道电流(I_A)虽然也有抑制作用但无统计学意义．由于乌拉坦不影响突触传递,因此它可能通过抑制I_Na使自发放电的阈值升高而降低放电频率,同时,由于参与的通道数量或活性降低而使得ISI的复杂度下降,长时程相关性弱化．     

成年大鼠海马CA1区锥体神经元外向整流氯离子单通道特性

采用膜片钳内面向外式技术,在急性分离成年大鼠海马CAl区锥体细胞上记录到了外向整流氯离子通道（outwardly rectifying chloride channel,ORCC）.长时间去极化（≥60 mV）刺激后,在30%的游离膜片上记录到有外向整流特性的单通道氯电流,膜电位在－60 mV到0 mV之间的单通道电导为(16.58±1.54) pS（n=10）,而在0 mV到+60 mV之间电导为（40.92±3.17） pS.通道开放概率有明显的电压依赖性(膜电位－60 mV时,P_o=0.44±0.12;膜电位为+60 mV时,P_o=0.86±0.06, n=10).在对称Cl^－浓度（150 mmol/L）时,通道翻转电位为(－4.17±1.84) mV.当溶液中部分NaCl被葡萄糖酸钠替代后,翻转电位为：(－34.23±4.86) mV (［Cl^－］_i/［Cl^－］_o=(30 mmol/L)/(150 mmol/L)),接近氯离子通道的理论值,这表明通道具有氯离子选择性.浴槽液中分别加入氯通道阻断剂DIDS和SITS可以使+40 mV的通道开放概率从(0.83±0.06)和(0.86±0.06)分别降低到(0.12±0.05)和(0.13±0.04)（n=5）,冲洗后可使开放概率基本恢复.上述研究结果显示,在成年大鼠海马CA1神经元上存在外向整流氯离子通道.     

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II by Extracellular Calcium in Cultured Hippocampal Pyramidal Neurons

Abstract: The ability of various stimuli to convert Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) into a Ca²⁺-independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca²⁺] ([Ca²⁺]_o) (～50 nM) and then shifted to normal [Ca²⁺]_o (～1.26 mM) by addition of CaCl₂ (referred to as “Ca²⁺ stimulation”). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30–50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K⁺] or treatment with glutamate or a Ca²⁺ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca²⁺-stimulated increase in CaMKII autonomy coincided with a two- to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca²⁺ stimulation, ³²P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr²⁸⁶Thr²⁸⁷ of the α/β subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine-containing peptides showed a significant increase. The activation of CaMKII by Ca²⁺ stimulation was accompanied by only a small rise in intracellular [Ca²⁺]. Inhibitor studies showed that Na⁺-dependent action potentials and Ca²⁺ influx through glutamate receptors or voltage-sensitive Ca²⁺ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn²⁺, Mg²⁺, Co²⁺, or Gd³⁺. Although the mechanism of Ca²⁺ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca²⁺ selectivity of the response suggest a direct and specific relationship between [Ca²⁺]_o and the state of activation of the kinase in intact neurons.     

琥珀酸对大鼠海马CA1区神经元电压依赖性钙电流的影响

目的：观察不同浓度的琥珀酸对大鼠海马CA1区神经元电压依赖性钙通道（voltage—dependent calcium channels,VDCC）电流的作用,初步探讨琥珀酸对神经元保护的电生理学基础。方法：采用传统全细胞膜片钳技术和制霉菌素（nystatin）穿孔膜片钳技术观察琥珀酸对海马CA1区神经元VDCC电流的影响。结果：不同浓度的琥珀酸（10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1）在海马CA1区对低电压激活（low—voltage activated,LVA）钙通道电流未见任何影响,而对高电压激活（high—voltage activated,HVA）钙通道电流的抑制呈浓度依赖性。对照组HVA钙电流为580．05±17．32pA,分别给予10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1。的琥珀酸后,HVA钙电流依次为563．74±16．65,517．99±15．24,444．66±13．26,405．32±19．11,269．03±9．96和86．41±3．25pA,同对照组相比差异有统计学意义（n=8,P〈0．01）。结论：琥珀酸能浓度依赖性地抑制HVA钙电流,而对LVA钙电流无影响。由此推测琥珀酸可能通过抑制HVA钙电流减少Ca^2＋内流而影响海马CA1区神经元的兴奋性,从而抑制癫痫的形成,其脑保护作用可能与此有关。     

不同强度工频磁场对神经元延迟整流钾通道特性的影响

工频磁场是人类生活中接触最多的一类磁场,其引起的生物效应与人类健康的关系备受关注．本文选用1 mT、5 mT及10 mT工频磁场照射急性分离的小鼠皮层神经元(15 min),应用全细胞膜片钳技术离线记录通道电流,研究了工频磁场对神经元延迟整流钾通道特性的影响．结果显示,1 mT、5 mT及10 mT 3个强度的工频磁场对I_k均有抑制作用,但随着去极化电压的增加,发现1 mT和5 mT工频磁场的抑制率几乎不变,抑制率分别为(30 ± 4.2)%和(20 ± 2.2)%,而10 mT工频磁场的抑制率增加,最大抑制率为43.4%．另外,1 mT和5 mT工频磁场影响了延迟整流钾通道的激活特性,通道的半数激活电压变大,斜率因子不变．而10 mT工频磁场对通道的激活特性没有影响,半数激活电压和斜率因子均不改变．研究表明,工频磁场可能影响了细胞膜上离子通道蛋白质的结构和功能,并且不同强度工频磁场对通道的影响不同,存在强度窗口效应．     

Elevation of Basal Protein Kinase C Activity Increases Ethanol Sensitivity of GABAA Receptors in Rat Hippocampal CA1 Pyramidal Neurons

Abstract: The ability of ethanol to enhance GABA_A receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA_A inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11–15°C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA_A IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31–33°C. In addition, the increase in ethanol sensitivity of GABA_A IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA_A receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA_A receptors in the mammalian brain.     

Coriaria Lactone Increased the Intracellular Level of Calcium through the Voltage-gated Calcium Channels in Rat Hippocampal Neurons

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca²⁺]_i) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca²⁺]_i from 118 ± 21 to 440 ± 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL’s epileptogenic process. Q. Zhang and X. Lai contributed equally to this article.     

氧化还原对成年大鼠海马CA1区锥体神经元外向整流氯通道的调控作用

采用膜片钳内面向外式记录技术,研究急性分离成年大鼠海马CAl区锥体神经元外向整流氯离子通道的氧化还原调控。发现细胞内侧给予氧化剂DTNB(5,5'-dithiobis-2-nitrobenzoic acid),可显著减弱氯通道的活动,IC50值为(28.05±2.42)μmol/L;还原剂DTT(dithiothreitol)对氯通道没有明显影响,但可逆转DTNB引起的抑制效应。说明DTNB不改变通道电导,其引起的通道活动减弱是由氯通道关闭时间延长而开放时间缩短所致。研究还发现,另一对氧化型和还原型谷胱甘肽具有与DTNB和DTT同样的效应。本研究结果显示,成年大鼠海马CA1区锥体神经元外向整流氯通道可以被细胞内氧化还原剂所调控。     

银杏叶提取物对过氧化氢刺激下的血管内皮细胞的影响

目的:探讨银杏叶提取物对血管内皮细胞的保护作用及可能的保护机制。方法:进行血管内皮细胞培养。用过氧化氢(H2O2)处理血管内皮细胞建立细胞凋亡模型。将细胞分为四组:空白对照组、H2O2处理组、单独银杏叶提取物处理组、银杏叶提取物预处理组(提前2h给药后H2O2处理)。进行MTT检测细胞的相对活力、RT-PCR检测目的基因CHOP的表达、Western Blot分析目的蛋白CHOP的表达等。结果:与对照处理组比较,H2O2处理组细胞凋亡率、CHOPmRNA相对表达及CHOP蛋白表达量明显升高。与H2O2处理组比较,银杏叶提取物预处理组细胞凋亡指数、CHOPmRNA相对表达及CHOP蛋白表达量明显降低(P0.05)。结论:作为一种清除自由基、抗氧化、抗衰老药物,银杏叶提取物可能通过调节CHOP蛋白选择性地抑制过度的内质网应激来保护血管内皮细胞。     

Effect of cannabinoids on glycine-activated currents in pyramidal neurons of the rat hippocampus

Using electrophysiological techniques (a patch-clamp technique in the whole-cell configuration and intracellular perfusion of neurons), we studied the effect of cannabinoids on the characteristics of glycine-activated currents in freshly isolated pyramidal neurons of the rat hippocampus. We found that endocannabinoids (anandamide and 2-arachidonoyl glycerol), as well as a synthetic cannabinoid, WIN 55,212-2, when applied in physiological concentrations, decreased the amplitude of glycine-activated currents. The agents under study accelerated the kinetics of activation and desensitization of glycine-induced Cl⁻ currents. The characteristics of the currents recovered after washout from cannabinoids. Changes in the kinetics of desensitization of glycine-activated currents depended noticeably on the holding potential; at positive potentials the sensitivity to cannabinoids was higher. These effects of cannabinoids were also observed in the presence of antagonists of CB1/CB3 receptors and an inhibitor of G proteins, GDPβS. These data indicate that under our experimental conditions cannabinoids exerted direct effects on glycine receptors. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 15–21, January–February, 2007.     

海南捕鸟蛛毒素-IV对大鼠背根神经节细胞钠通道的抑制影响(英文)

海南捕鸟蛛毒素 IV(HNTX IV)是从中国捕鸟蛛Seleconosmiahainana粗毒中分离得到的一种肽类神经毒素 ,在成年大鼠背根神经节 (DRG)细胞上观察了该毒素对电压门控钠通道的影响。在全细胞膜片钳条件下 ,HNTX IV能明显抑制哺乳动物神经性河豚毒敏感型 (TTX S)钠电流 ,但不影响河豚毒不敏感型 (TTX R)钠电流。HNTX IV对DRG细胞TTX S钠电流的抑制作用具有浓度依从性 ,其有效半抑制浓度 (IC50 )为 44 .6nmol/L。该毒素不影响DRG钠电流的激活与失活时间特征 ,但能导致钠通道的半数稳态失活电压向超极化方向漂移约 10 .1mV。结果表明HNTX IV是一种新型的蜘蛛毒素 ,其影响电压门控钠通道的机制可能有别于那些结合于通道位点 3来延缓钠电流失活时间特征的蜘蛛毒素如δ 澳洲漏斗网蛛毒素、μ 美洲漏斗网蛛毒素I VI等。     

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.     

高效氯氰菊酯对大鼠海马CA3区神经元电压门控钾通道的影响

利用全细胞膜片钳技术,在急性分离的新生大鼠海马CA3区锥体细胞上研究高效氯氰菊酯的两种组分高顺氯氰菊酯和高反氯氰菊酯对瞬时外向钾电流（transient outward potassiumcurrent,IA）和延迟整流钾电流（delayed rectifier potassiumcurrent,Ik）的影响。高顺氯氰菊酯使IA增大,而高反氯氰菊酯则使IA减小。高顺和高反氯氰菊酯均使IA激活曲线左移,反式结构还可促进IA的失活。高顺和高反氯氰菊酯均使IK减小,并使其激活曲线左移,而对IK的失活过程无影响,高反氯氰菊酯可使IK失活后恢复过程延长。结果表明,瞬时外向钾通道和延迟整流钾通道同样是高效氯氰菊酯的作用靶点,这可能是高效氯氰菊酯对哺乳动物产生毒性作用的原因之一。     

Cyclic AMP Regulates the Expression of Neurokinin1 Receptors by Neonatal Rat Spinal Neurons in Culture

Neurokinin1 (NK1) receptors are up-regulated in the spinal cord during peripheral inflammation, but the biochemical mediators regulating this change have not been resolved. The promoter region of the gene encoding the NK1 receptor contains a cyclic AMP (cAMP)-responsive element. Therefore, we used primary cultures of neonatal rat spinal cord to test whether increasing intracellular cAMP can increase expression of NK1 receptors. Treatment with dibutyryl-cAMP (dbcAMP) resulted in a time-dependent increase in 125I-Bolton-Hunter-substance P (BHSP) binding in the cultures; treatment with dibutyryl-cyclic GMP did not. Treatment with forskolin plus 3-isobutyl-1-methylxanthine mimicked the increase in binding, providing further evidence for the involvement of cAMP in this effect. Scatchard analyses indicated that the increase in BHSP binding was due to an increase in binding capacity. The cAMP-induced increase in BHSP binding was preceded by an increase in levels of mRNA for NK1 receptor and was attenuated by pretreatment with cycloheximide. These data indicate that the cAMP-induced increase in binding was due to increased synthesis of NK1 receptors. Comparison of substance P (SP)-induced production of inositol phosphates between cultures pretreated with dbcAMP and controls suggested that increased expression of NK1 receptors did not result in increased generation of second messenger by NK1 receptor activation. Together, these data indicate that a persistent increase in intracellular cAMP increases expression of NK1 receptors. Because NK1 receptor activation contributes to increased excitability of spinal neurons, the increased expression of NK1 receptors may be important in maintaining responsiveness of spinal neurons to SP in central mechanisms underlying hyperalgesia.     

Activation of Mitogen-Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Abstract: Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca²⁺ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with ³²P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c- raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca²⁺-dependent pathways and that the activation of Raf-1 is not involved.