5-HT_(1A)和5-HT_2受体功能与觉醒、睡眠成分关系的研究

目的　观察 5 HT1A受体激动剂 8 OH DPAT和HT2受体拮抗剂利坦色林 (ritanserin)对大鼠清醒和睡眠成分的影响 ,给予 5 HT1A受体激动剂和剥夺REM睡眠后皮层 5 HT2 受体结合能力的变化 ,进一步分析两种 5 HT受体亚型之间的关系以及 5 HT2 受体在睡眠中的作用。方法　利用大鼠睡眠自动分析系统定量分析清醒和睡眠成分的变化 ,注射PCPA制作剥夺睡眠的大鼠模型 ,水上平台法制作剥夺REM睡眠的大鼠模型 ,利用放射配体结合实验研究配体与受体结合力的变化。结果　 8 OH DPAT小剂量 (0 0 1mg·kg-1,sc)可以增加深睡眠和浅睡眠 ,减少觉醒 ;大剂量(0 375mg·kg-1,sc)则增加觉醒 ,减少全部睡眠成分。 5 HT2 受体拮抗剂ritanserin可以明显增加深睡眠 ,减少觉醒和REM睡眠。而 8 OH DPAT低剂量与ritanserin联合用药则使深浅睡眠明显增加 ,清醒和REM睡眠明显减少 ,具有协同作用。对于PCPA化使 3种睡眠成分均明显减少 ,觉醒比例明显增加的大鼠 ,ritanserin仅使浅睡眠增加其它成分不变。用 [3 H] ritanserin放射性配基结合显示 ,剥夺大鼠REM睡眠后皮层 5 HT2 受体Bmax值明显增加但Kd 值无变化 ;给予 5 HT1A激动剂 8 OH DPAT后皮层 5 HT2 受体Kd 和Bmax均降低。结论　 5 HT1A受体和 5 HT2 受体与睡眠有密切关系     

5-HT_(1A)和5-HT_2受体功能与觉醒、睡眠成分关系的研究

目的　观察 5 HT1A受体激动剂 8 OH DPAT和HT2受体拮抗剂利坦色林 (ritanserin)对大鼠清醒和睡眠成分的影响 ,给予 5 HT1A受体激动剂和剥夺REM睡眠后皮层 5 HT2 受体结合能力的变化 ,进一步分析两种 5 HT受体亚型之间的关系以及 5 HT2 受体在睡眠中的作用。方法　利用大鼠睡眠自动分析系统定量分析清醒和睡眠成分的变化 ,注射PCPA制作剥夺睡眠的大鼠模型 ,水上平台法制作剥夺REM睡眠的大鼠模型 ,利用放射配体结合实验研究配体与受体结合力的变化。结果　 8 OH DPAT小剂量 (0 0 1mg·kg-1,sc)可以增加深睡眠和浅睡眠 ,减少觉醒 ;大剂量(0 375mg·kg-1,sc)则增加觉醒 ,减少全部睡眠成分。 5 HT2 受体拮抗剂ritanserin可以明显增加深睡眠 ,减少觉醒和REM睡眠。而 8 OH DPAT低剂量与ritanserin联合用药则使深浅睡眠明显增加 ,清醒和REM睡眠明显减少 ,具有协同作用。对于PCPA化使 3种睡眠成分均明显减少 ,觉醒比例明显增加的大鼠 ,ritanserin仅使浅睡眠增加其它成分不变。用 [3 H] ritanserin放射性配基结合显示 ,剥夺大鼠REM睡眠后皮层 5 HT2 受体Bmax值明显增加但Kd 值无变化 ;给予 5 HT1A激动剂 8 OH DPAT后皮层 5 HT2 受体Kd 和Bmax均降低。结论　 5 HT1A受体和 5 HT2 受体与睡眠有密切关系     

半胱氨酰白三烯受体2通过介导小胶质细胞激活诱导缺血性神经元损伤

目的探索半胱氨酰白三烯(CysLT)受体在大鼠皮层神经元缺氧缺糖损伤中的调节作用及作用机制。方法培养大鼠原代皮层神经元或大鼠原代皮层混合培养细胞,以缺糖缺氧(OGD)模拟脑缺血损伤,以MTT还原法/LDH释放检测确定神经元存活/损伤,以免疫组化做神经元(抗MAP2)、星形胶质细胞(抗GFAP)、小胶质细胞(抗Iba1)特异性染色,观察各细胞数量变化,以PI/Hoechst染色确定细胞凋亡/坏死。以受体激动剂LTD4,NMLTC4,CysLT1拮抗剂孟鲁司特、CysLT2拮抗剂HAMI3379/CysLT1-siRNA及CysLT2-shRNA为药理学干预/基因干扰工具。结果 LTD4(0.1～1000 nmol·L-1),NMLTC4(0.1～1000 nnmol·L-1)不能诱导单纯神经元损伤;CysLT1-siRNA及CysLT2-shRNA不能减轻OGD诱导的神经元损伤;LTD4(1～1000 nnmol·L-1),NMLTC4(0.1～1000 nnmol·L-1)能诱导皮层混合细胞损伤;HAMI3379(0.001～1μmol·L-1)和CysLT2-shRNA能减轻OGD,LTD4或NMLTC4诱导的皮层混合细胞损伤及其诱导的小胶质细胞激活,CysLT1-siRNA无此作用。结论 CysLT2受体可能参与OGD诱导神经元损伤的调节,CysLT2拮抗剂可通过减轻小胶质细胞激活而减轻OGD诱导的神经元损伤。     

羟丁酸钠对大鼠原代培养皮层神经元缺氧复氧损伤的保护作用与GABAA受体的关系

目的 :探讨羟丁酸钠 (SO)对大鼠皮层神经元缺氧复氧损伤的保护作用与GABAA 受体的关系。方法 :采用原代培养大鼠皮层神经元建立缺氧复氧损伤模型 ,观察细胞的形态学 ,测定其LDH漏出率、MDA含量、SOD、GPx活力。结果 :缺氧复氧可引起神经元损伤 ,LDH漏出率增加 ,MDA含量升高 (P <0 .0 1) ,SOD、GPx活力降低 (P <0 .0 1)。SO能显著减少损伤神经元的LDH漏出率及MDA的生成 (P <0 .0 1) ,升高SOD、GPx(P <0 .0 1)的活力 ,这种作用可被GABAA 受体阻断剂seurinine减弱 (P <0 .0 1)。结论 :SO对缺氧复氧神经元损伤的保护作用可能与其激动GABAA 受体有关。     

塞络通胶囊对大鼠多发性脑梗死恢复期Glu和GABA合成以及NMDA受体亚型表达的影响

观察塞络通胶囊(塞络通)对大鼠多发性脑梗死恢复期脑组织中神经递质谷氨酸(Glu)和γ-氨基丁酸(GABA)含量以及NMDA受体亚型NR1、NR2A和NR2B表达的影响,阐述塞络通在脑缺血后恢复期对脑组织保护的作用机制。通过大鼠颈内动脉注射微球血管栓塞剂的方法建立多发性脑梗死大鼠模型,在脑梗死后10天采用不同剂量的塞络通(16.5及33.0 mg.kg 1)连续干预60天。采用透射电子显微镜观察脑组织超微病理结构改变,高效液相色谱法检测脑组织Glu和GABA含量,免疫组织化学方法分析脑组织NMDA受体NR1、NR2A和NR2B亚型表达的变化。结果表明,与假手术组相比,模型组恢复期脑组织不仅存在神经元、神经胶质细胞和突触超微结构异常改变,而且神经递质Glu和GABA合成显著减少,同时NMDA受体NR1、NR2A和NR2B亚型表达明显升高;塞络通能够改善脑组织超微结构,明显促进神经元Glu和GABA合成,同时下调NMDA受体NR1、NR2A和NR2B亚型表达。以上结果提示,塞络通在大鼠多发性脑梗死恢复期通过促进Glu和GABA神经递质合成以及抑制NMDA受体表达而产生一定程度的神经保护作用。     

阿魏酸钠对抗Aβ25-35诱导的鼠神经元凋亡作用研究

目的 探讨阿魏酸钠对β淀粉样蛋白(Aβ25-35)通过谷氨酸诱导的鼠皮层神经元凋亡的影响.方法 培养的皮层神经元分别与谷氨酸(20μmol/L)、Aβ25-35(5μmol/L)、Aβ25-35(5μmol/L)+谷氨酸(20μmol/L)孵育,采用Hoechst 33258荧光染色法分析神经细胞凋亡;然后用谷氨酸( 50μmol/L)诱导神经元凋亡,采用荧光染色法和Westem blot观察阿魏酸钠的保护作用.结果 单独应用Aβ25-35(5μmol/L)和单独加入谷氮酸(20μmol/L)引起的皮层神经元凋亡率与对照组无明显差异;Aβ25-35与皮层神经元共同孵育5天,接着用谷氨酸处理24h,细胞凋亡率从正常的9.2%+1.5%增加到43%±8%:阿魏酸钠能够显著降低谷氨酸诱导的神经细胞凋亡百分比至21%±5%,并对抗谷氨酸引起的Bcl-2蛋白表达的降低.结论 阿魏酸钠能够减弱Aβ25-35提高谷氨酸毒性诱导的鼠皮层神经元凋亡.     

早期听觉剥夺对幼鼠听皮层NMDA受体亚单位的影响

目的 观察听觉剥夺对幼鼠听皮层N-甲基-D-天冬氨酸(NMDA)受体亚单位NR1、NR2A及NR2B蛋白表达的影响.方法 将36只新生幼鼠随机分为听觉剥夺组和对照组各18只,每组分别按出生周龄不同分3、5、7周龄组,每周龄组6只.听觉剥夺组在出生第7天开始用阿米卡星(Amikacin)500 mg/(kg·d)皮下注射,直到生后第16天.对照组皮下注射相同次数、同等容量的生理盐水.造模成功后,以10%水合氯醛腹腔注射麻醉后,分离出双侧听皮层组织,提取总蛋白.以Western blot法检测听皮层NR1、NR2A、NR2B蛋白表达水平.结果 与同龄对照组比较,听觉剥夺组各周龄组听皮层NR1蛋白表达均显著降低(P＜0.05);3周龄及5周龄组听皮层NR2A蛋白表达水平显著降低(P＜0.05);3周龄组听皮层NR2B蛋白表达水平显著降低(P＜0.05).结论 生后早期听觉剥夺降低了听皮层NMDA受体蛋白的表达,提示NMDA受体可能参与了听觉剥夺后认知功能障碍的发病机制.     

边缘下皮层在海洛因觅药行为消退和恢复中的作用

目的研究边缘下皮层在海洛因觅药行为消退和恢复中的作用及可能的作用途径。方法成年SD雄性大鼠采用固定比率程序1(fixed ratio,FR1)进行海洛因自身给药训练,连续14 d,建立海洛因自身给药模型。环境消退12 d,第1～5天消退训练15 min,实验处理组动物在消退训练后立即向双侧边缘下皮层区注射GABA受体激动剂Baclofen+Mus-cimol(BM,0.3+0.03 nmol/侧),可逆性失活该核团。第7～12天消退训练2 h,测定觅药行为消退记忆的保留程度;而另一部分大鼠用同样程序建立稳定的海洛因自身给药模型后,然后,每天进行2 h的环境消退,共14 d,以达到消退标准。实验处理组动物在条件性线索诱导海洛因觅药行为恢复测试前,向双侧边缘下皮层区分别注射PEPA(AMPA受体激动剂,30 ng/侧)和Clenbuterol(β2肾上腺素受体激动剂,8 ng/侧)。测试结束,动物立即断头取脑,分离核团,包括边缘下皮层、伏隔核(NAc)壳部及NAc核部。采用Western blotting检测GluR1,p-GluR1,β2受体和p-CREB的表达水平变化。结果经双因素重复测量方差分析发现:第6～12天的消退训练中,对照组、15 min实验组有效鼻触数均随着时间的推移而下降(P=0.000),组间无显著差异(P=0.071),但时间与分组的交互作用有统计学意义(P=0.02)。与对照组相比,消退训练第6天,15 min实验组的有效鼻触数(76±16)显著高于对照组(33±6)的有效鼻触数(P=0.037);而条件性线索诱导的海洛因觅药行为恢复测定中,PEPA处理组(13±4)和Clenbuterol组(10±3)的有效鼻触数分别显著低于相应复吸对照组(65±18)的有效鼻触数(P=0.042,P=0.046);Western blotting检测发现,与复吸对照组相比,PEPA处理组边缘下皮层区GluR1的表达显著上调,p-GluR1的表达无明显变化,但NAc中GluR1和p-GluR1的表达均显著上调;Clenbuterol组边缘下皮层及NAc壳部中β2受体的表达均上调,p-CREB的表达均显著下调,而NAc核部中的β2受体表达下调,p-CREB的表达上调。结论边缘下皮层区神经元失活对消退过程不影响,但减弱海洛因觅药行为消退学习记忆的保留;边缘下皮层区分别注射AMPA和β2肾上腺素受体激动剂可抑制海洛因觅药行为的恢复;而边缘下皮层和NAc区的GluR1,β2受体和CREB磷酸化不同变化可能分别介导了上述作用过程。     

治疗2型糖尿病的新靶点药物研究进展

糖尿病是一种代谢障碍性疾病,患病率逐年上升,已成为全球慢性非传染性疾病中最具流行性的疾病。糖尿病患者中以2型糖尿病患者为主,占比超过90%。现有糖尿病治疗药物主要包括磺脲类、双胍类、格列奈类、噻唑烷二酮类、α-葡萄糖苷酶抑制剂、胰高血糖素样肽-1(GLP-1)受体激动剂、二肽基肽酶IV(DPP-4)抑制剂和钠–葡萄糖协同转运蛋白2(SGLT2)抑制剂。它们在降糖控糖作用机制上各具特点和优势,但仍不能满足临床治疗的需求。随着研究人员的不断探索,一些治疗2型糖尿病的新靶点化合物已进入临床I、II期研究,并有多个化合物处于临床前研发中,有望成为2型糖尿病的治疗药物。介绍了葡萄糖激酶激动剂、胰高血糖素受体拮抗剂、G蛋白偶联受体119(GPR119)激动剂、腺苷酸活化蛋白激酶(AMPK)激动剂、游离脂肪酸受体1(FFAR1)激动剂、蛋白酪氨酸磷酸酶-1B(PTP-1B)抑制剂和11β-羟类固醇脱氢酶1(11β-HSD1)抑制剂的作用机制、研发进程和注意事项,希望为2型糖尿病治疗药物的研发提供参考。     

谷氨酸损伤大鼠皮层神经元及抑制钙/钙调蛋白依赖性蛋白激酶Ⅱ活性

利用原代培养的大鼠胎鼠皮层神经元 ,采用乳酸脱氢酶 (LDH)测定和32 P参入方法 ,研究谷氨酸 (Glu)对皮层神经元损伤 ,钙 /钙调蛋白依赖性蛋白激酶 (Ca MK )活性的影响及其机理 .Glu(50- 1 0 0 0μmol· L-1)作用 1 0 min,导致皮层神经元Ca MK 活性立即明显下降 ,神经元形态及 LDH释放早期无明显改变 ,2 4 h时 LDH释放显著增加 ,神经元损伤 .N-甲基 - D-天冬氨酸 (NMDA)受体拮抗剂地佐环平 (MK- 80 1 )明显降低 LDH释放 ,保护Ca MK 活性 ,而非 NMDA受体拮抗剂 6,7-二硝基喹口恶啉二酮 (DNQX)无保护作用 .去除胞外 Ca2 +可明显保护 Ca MK 活性 .Ca MK 抑制剂 KN- 62可明显拮抗 Ca MK 活性下降 ,部分保护神经元损伤 .结果提示 ,Glu兴奋毒引起皮层神经元迟发性损伤和 Ca MK 活性早期明显下降 ,主要由 NMDA受体介导和胞外 Ca2 +内流增加有关 .     

Additive neuroprotection of GABA A and GABA B receptor agonists in cerebral ischemic injury via PI-3K/Akt pathway inhibiting the ASK1-JNK cascade

Co-activation of GABA A and GABA B receptors results in neuroprotection during in vitro ischemia. However, it is unclear whether this mode of action is responsible for its neuroprotective effects in animal models of ischemia in vivo, and the precise mechanisms are also unknown. This study compared the neuroprotective efficacies of muscimol, a GABA A receptor agonist, and a GABA B receptor agonist baclofen in rat brain ischemia. The additive neuroprotection could be obtained in the hippocampal CA1 pyramidal cells prominently when muscimol and baclofen were co-applied. In particular, our study showed that co-activation of GABA A and GABA B receptors could strongly increase Akt activation and inhibit ASK1 activation by phosphorylation of serine 83 of ASK1. PI-3K inhibitor LY294002 reversed the increasing Akt activation and ASK1 (S83) phosphorylation. Moreover, MKK4/MKK7-JNK signaling activation was inhibited during ischemia/reperfusion (I/R) by co-treatment of muscimol with baclofen. JNK substrate, Bcl-2 and c-jun phosphorylation were also attenuated. Our results indicated that co-activation of GABA A receptor and GABA B receptor exerted neuroprotective effect via PI-3K/Akt pathway, which could inhibit the ASK1-c-Jun N-terminal protein kinase (JNK) cascade.     

Effect of experimental diabetes on GABA-mediated inhibition of neurally induced contractions in rat isolated trachea

1. In the present study, we investigated the effect of GABA and selective GABA agonists and antagonists on neurally induced tracheal contractions in streptozotocin (STZ) diabetic rats. 2. Contractile responses to electrical field stimulation (EFS) in rat tracheal rings were completely abolished by atropine and tetrodotoxin, but were unaffected by the ganglion blocker hexamethonium, indicating that they were mediated via neuronal release of acetylcholine (ACh). 3. Contractions induced by EFS, but not by exogenous ACh, were inhibited by GABA and the selective GABA(B) receptor agonist baclofen, but not by the selective GABA(A) receptor agonist 3-aminopropane sulphonic acid. The inhibitory effects of GABA or baclofen were not affected by the GABA(A) antagonist bicuculline, but were significantly reversed by the GABA(B) antagonist phaclofen. 4. The inhibitory effects of both GABA and baclofen were found to be significantly greater in trachea from control rats compared with tissues from diabetic rats. 5. Non-adrenergic, non-cholinergic relaxation responses elicited by EFS in precontracted tracheal rings from diabetic and control rats were similar in magnitude and were unaffected by GABA or GABA analogues. 6. These results suggest that GABA decreases the response to EFS by directly inhibiting the evoked release of ACh through GABA(B) receptors in rat trachea and that STZ-induced diabetes causes an impairment in the inhibitory effect of GABA on neurally induced contractions in this tissue.     

mGluR7-like receptor and GABA(B) receptor activation enhance neurotoxic effects of N-methyl-D-aspartate in cultured mouse striatal GABAergic neurones.

Presynaptic metabotropic glutamate receptors (mGluRs) of group III constitute possible targets for putative neuroprotective drugs acting against glutamate excitotoxic insults. Indeed, in glutamatergic cerebellar granule neurones in culture, high concentrations of L-2-amino-4-phosphonobutyrate (L-AP4, above 0.3 mM, thus activating mGluR7) inhibit NMDA-induced cell death. In contrast, in striatal cultures which are enriched in GABAergic neurones, we show that high concentrations of L-AP4 increased neuronal death in control as well as in NMDA-stimulated cultures. Moreover, similar results were obtained with the GABA(B)R agonist. baclofen. Both the neuroprotective effects in cerebellar granule cells and the neurotoxic effects in striatal neurones were mediated via Gi-Go-coupled mGluRs, suggesting that these effects were probably mediated by mGluR7a or b and GABA(B)R expressed in these neurones. In striatal neurones, we found that L-AP4 and baclofen inhibited both basal and NMDA-stimulated GABA release. These inhibitions of GABA release may be responsible for the increase in basal and NMDA-stimulated neuronal death. Indeed, blockade of GABA(A) receptors with bicuculline increased neuronal death of control and NMDA-treated striatal cultures. Taken together, these results suggest that L-AP4 and baclofen, via mGluR7 and GABA(B)R, reduced the neuroprotective effect of GABA present in striatal cultures acting via GABA(A) receptors. Although caution must be taken when extrapolating from in vitro to in vivo situations, the present experiments and the recent observations that mGluR7 and GABA(B)R are expressed in heterologous synapses, should be taken into consideration when evaluating the neuroprotective action of future mGluR7 specific agonists or GABA(B)R specific antagonists.     

Trimethylolpropane phosphate induces epileptiform discharges in the CA1 region of the rat hippocampus

The actions of trimethylolpropane phosphate (TMPP), an ethyl bicyclophosphate convulsant produced during the partial pyrolysis of some phosphate ester-based lubricants, were tested on CA1 neurons of rat hippocampal slices using intracellular recording techniques. Bath application of TMPP (0.1-100 microM) induced spontaneous paroxysmal depolarizing shifts and the associated spontaneous epileptiform bursts followed by after-hyperpolarizations in 63% of neurons tested. The TMPP-induced epileptiform bursts were blocked by muscimol, a gamma-aminobutyric acid A (GABA(A)) receptor agonist, diazepam (DZP), a GABA(A)-benzodiazepine ionophore complex agonist, or baclofen, a GABA(B) receptor agonist. While bath application of muscimol, DZP, or baclofen suppressed spontaneous activity in CA1 neurons not previously exposed to TMPP, subsequent application of TMPP (10 microM) reversed the actions of muscimol and diazepam, but not baclofen. TMPP (0.1-100 microM) also induced membrane hyperpolarization associated with an increase in peak input resistance and inward rectification in 33% of neurons tested or membrane depolarization associated with an increase in input resistance in 17% of neurons tested. In summary, TMPP induced epileptiform activities in hippocampal CA1 neurons. The epileptogenic effects of TMPP are consistent with its interaction with GABA(A)-benzodiazepine receptors.     

GABAergic modulation of hippocampal glutamatergic neurons: an in vivo microdialysis study

We have demonstrated the effects of activation of presynaptic gamma-aminobutyric acid (GABA) receptors on glutamate release using in vivo brain microdialysis. A dialysis probe inserted into the hippocampus CA2 area of freely moving rats was perfused with Ringers solution containing 100 mM potassium chloride (KCl) or 0.05 mM veratridine for 20 min. Extracellular concentrations of amino acids were monitored by measuring their levels in dialysates by high performance liquid chromatography (HPLC) fluorometry. Perfusion with depolarizing agents, such as KCl or veratridine, increased extracellular glutamate levels in the hippocampus. Pretreatment with 1 mM GABA, before perfusion with depolarizing agents, significantly suppressed the depolarizing agent-induced increase in glutamate levels. The GABA(B) receptor agonist baclofen (1 mM) also significantly inhibited the depolarizing agent-induced increase in glutamate levels, whereas the GABA(A) receptor agonist, muscimol, had no affect. Similarly, baclofen (0.5 mM) decreased the KCl (13.5 mM)-induced 45Ca(2+) influx into cortical synaptosomes to 57% of the level induced in the absence of baclofen. On the other hands, GABA did not affect the increases in glycine and taurine level by depolarizing agents. These results suggest that GABA modulates depolarization-evoked glutamate release in the hippocampus by inhibiting Ca(2+) entry into neurons, an effect mediated by presynaptic GABA(B) receptors.     

Metabotropic glutamate 1 (mGlu1) receptor antagonists enhance GABAergic neurotransmission: a mechanism for the attenuation of post-ischemic injury and epileptiform activity?

Selective antagonists of mGlu1 metabotropic glutamate receptors attenuate neuronal death in models of cerebral ischemia. Because GABAergic mechanisms have recently been proposed to contribute to these neuroprotective effects, we examined the effects of selective mGlu1 antagonists characterized in our laboratory on GABAergic transmission in three different models of neuropathology. In rat organotypic hippocampal slices exposed to oxygen-glucose deprivation, the mGlu1 antagonists AIDA, CBPG and 3-MATIDA reduced CA1 pyramidal cell loss when added to the medium during the insult and the subsequent recovery period. This effect was mimicked by the GABA(A) and GABA(B) agonists muscimol and baclofen and partially prevented by the antagonists bicuculline and CGP 55845. In gerbils subjected to global ischemia, protection of CA1 pyramidal cells by transdialytic perfusion of AIDA and CBPG was associated with a significant increase in the basal and ischemic output of GABA and minor changes in the output of glutamate. In a mouse cortical wedge model, both muscimol and 3-MATIDA reduced the frequency of spontaneous bursts induced by 4-aminopyridine and this reduction was prevented by co-perfusion with bicuculline. Taken together, our results suggest that the release of GABA, and the subsequent activation of GABA receptors, may contribute to the attenuation of post-ischemic neuronal damage and epileptiform activity induced by mGlu1 receptor antagonists.     

Effects of an endothelin B receptor agonist on secretory phospholipase A2-IIA-induced apoptosis in cortical neurons

Endothelin (ET), a vasoconstrictive peptide, acts as an anti-apoptotic factor, and endothelin receptor B (ETB receptor) is associated with neuronal survival in the brain. Human group IIA secretory phospholipase A2 (sPLA2-IIA) is expressed in the cerebral cortex after brain ischemia and causes neuronal cell death via apoptosis. In primary cultures of rat cortical neurons, we investigated the effects of an ETB receptor agonist, ET-3, on sPLA2-IIA-induced cell death. sPLA2-IIA caused neuronal cell death in a concentration- and time-dependent manner. ET-3 significantly prevented neurons from undergoing sPLA2-IIA-induced cell death. These agonists reversed sPLA2-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Before cell death, sPLA2-IIA potentiated the influx of Ca2+ into neurons. Blockers of the L-type voltage-dependent calcium channel (L-VSCC) not only suppressed the Ca2+ influx, but also exhibited neuroprotective effects. As well as L-VSCC blockers, ET-3 significantly prevented neurons from sPLA2-IIA-induced Ca2+ influx. An ETB receptor antagonist, BQ788, inhibited the effects of ET-3. The present cortical cultures contained few non-neuronal cells, indicating that the ETB receptor agonist affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that the ETB receptor agonist rescues cortical neurons from sPLA2-IIA-induced apoptosis. Furthermore, the present study suggests that the inhibition of L-VSCC contributes to the neuroprotective effects of the ETB receptor agonist.     

Mu-opioid receptor modulation of calcium channel current in periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR-1.

1. The actions of opioid receptor agonists on the calcium channel currents (IBa) of acutely dissociated periaqueductal grey (PAG) neurons from C57B16/J mice and mutant mice lacking the first exon of the mu-opioid receptor (MOR-1) were examined using whole cell patch clamp techniques. These effects were compared with the GABA(B)-receptor agonist baclofen. 2. The endogenous opioid agonist methionine-enkephalin (met-enkephalin, pEC50 6.8, maximum inhibition 40%), the putative endogenous mu-opioid agonist endomorphin-1 (pEC50 6.2, maximum inhibition 35%) and the mu-opioid selective agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin, pEC50 6.9, maximum inhibition 40%) inhibited IBa in 70% of mouse PAG neurons. The inhibition of IBa by each agonist was completely prevented by the mu-receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2). The delta-opioid receptor agonists DPDPE ([D-Pen2,5]enkephalin, 1 microM) and deltorphin II (1 microM), and the kappa-opioid receptor agonist U-69593 (1-10 microM), did not affect IBa in any cell tested. 3. The GABA(B) agonist baclofen inhibited IBa in all neurons (pEC50 5.9, maximum inhibition 42%). 4. In neurons from the MOR-1 deficient mice, the mu-opioid agonists met-enkephalin, DAMGO and endomorphin-1 did not inhibit IBa, whilst baclofen inhibited IBa in a manner indistinguishable from wild type mice. 5. A maximally effective concentration of endomorphin-1 (30 microM) partially (19%), but significantly (P<0.005), occluded the inhibition of IBa normally elicited by a maximally effective concentration of met-enkephalin (10 microM). 6. This study indicates that mu-opioid receptors, but not delta- or kappa-opioid receptors, modulate somatic calcium channel currents in mouse PAG neurons. The putative endogenous mu-agonist, endomorphin-1, was a partial agonist in mouse PAG neurons.     

Lamina-specific differences in GABA(B) autoreceptor-mediated regulation of spontaneous GABA release in rat entorhinal cortex

Spontaneous synaptic inhibition plays an important role in regulating the excitability of cortical networks. Here we have investigated the role of GABA(B) autoreceptors in regulating spontaneous GABA release in the entorhinal cortex (EC), a region associated with temporal lobe epilepsies. We have previously shown that the level of spontaneous inhibition in superficial layers of the EC is much greater than that seen in deeper layers. In the present study, using intracellular and whole cell patch clamp recordings in rat EC slices, we have demonstrated that evoked GABA responses are controlled by feedback inhibition via GABA(B) autoreceptors. Furthermore, recordings of spontaneous, activity-independent inhibitory postsynaptic currents in layer II and layer V neurones showed that the GABA(B) receptor agonist, baclofen, reduced the frequency of GABA-mediated currents indicating the presence of presynaptic GABA(B) receptors in both layers. Application of the antagonist, CGP55845, blocked the effects of baclofen and also increased the frequency of GABA-mediated events above baseline, but the latter effect was restricted to layer V. This demonstrates that GABA(B) autoreceptors are tonically activated by synaptically released GABA in layer V, and this may partly explain the lower level of spontaneous GABA release in the deep layer.     

Effects of GABA agonists and GABA-A receptor modulators on cocaine discrimination in rhesus monkeys

RATIONALE: Dopaminergic systems thought to mediate the abuse-related effects of cocaine are under inhibitory control by GABAergic systems. These findings suggest that GABA agonists may attenuate some abuse-related effects of cocaine. OBJECTIVE: To assess the effects of GABA receptor agonists and GABA-A receptor modulators on cocaine discrimination in rhesus monkeys. METHODS: Rhesus monkeys were trained to discriminate 0.4 mg/kg cocaine from saline in a two-key, food-reinforced drug discrimination task. The effects of the GABA-A agonist muscimol, the GABA-B agonist baclofen, the barbiturate GABA-A receptor modulator pentobarbital, and the benzodiazepine GABA-A modulators triazolam and imidazenil were examined alone and as pretreatments to cocaine. For comparison, the effects of pentobarbital pretreatment on the cocaine-like discriminative stimulus effects of amphetamine were also examined. RESULTS: When administered alone, the GABA agonists and GABA-A receptor modulators produced primarily saline-appropriate responding. When administered as pretreatments to cocaine, pentobarbital attenuated the discriminative stimulus effects of cocaine in all monkeys tested, and the high efficacy benzodiazepine agonist triazolam attenuated cocaine's effects in three of five monkeys. Muscimol, baclofen and the low efficacy benzodiazepine agonist imidazenil did not alter cocaine's discriminative stimulus effects. Although pentobarbital blocked the effects of the monoamine reuptake blocker cocaine, it did not alter the cocaine-like effects of the monoamine releaser amphetamine. CONCLUSIONS: These results are consistent with the hypothesis that GABA-A receptor modulators attenuate the discriminative stimulus effects of cocaine in rhesus monkeys by decreasing the activity of dopaminergic systems. Direct GABA receptor agonists may be less effective in blocking the abuse-related effects of cocaine in rhesus monkeys.