周边γ-氨基丁酸通过GABA_B受体调控骶髓后联合核神经元谷氨酸能突触

目的研究突触周边γ-氨基丁酸(ambient GABA)通过GABAB受体调控骶髓后联合核(SDCN)神经元谷氨酸能突触的机制。方法在急性切取的骶段脊髓薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs),将GABAB受体用其特异性受体拮抗剂CGP52432阻断,观察谷氨酸突触终末上的GABAB受体被周边GABA作用的影响。结果在突触后GABAB受体被从胞内阻断的条件下,再灌流CGP52432阻断谷氨酸能突触前GABAB受体,可增加刺激引发的EPSCs(eEPSCs)幅度;改变配对刺激的两个EPSC比率(paired-pulse ratio,PPR),并激发沉默突触(silent synapse)。但CGP52432对微小兴奋性突触后电流(mEPSCs)无影响。结论位于SDCN神经元谷氨酸能突触前的GABAB受体受周边GABA调控。这种影响参与调节谷氨酸释放并可能参与痛觉信息在脊髓水平的传递。     

SKF97541抑制大鼠骶髓后联合核神经元

目的研究GABAB受体特异性激动剂SKF97541对骶髓后联合核(SDCN)神经元的作用。方法在大鼠骶段脊髓横切薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元。电流钳记录模式下,观察SKF97541对神经元膜电位和动作电位发放的影响。电压钳模式下,观察谷氨酸能兴奋性突触后电流(EPSCs)对SKF97541处理的变化。结果SKF97541(0.5μmol.L-1)通过作用于GABAB受体,减少SDCN神经元动作电位发放,同时促进细胞膜超极化。SKF97541在电压钳模式下,减少谷氨酸介导的微小EPSCs的频率,但对振幅无影响,提示SKF97541通过作用于突触前GABAB受体抑制谷氨酸释放。突触前刺激引起的突触后电位,也被SKF97541抑制。结论在骶髓后联合核,SKF97541通过作用于突触后GABAB受体,直接抑制神经元的兴奋性和动作电位发放;并通过突触前GABAB受体,抑制谷氨酸的释放。以上结果提示SKF97541的抑制作用可能抑制骶髓后联合核神经元对伤害性信息的传递。     

大鼠骶髓后连合核神经元的分离

To isolate rat sacral dorsal commissural neurons (SDCN). METHODS: Using enzymatic and mechanical dissociation techniques to isolate the neurons and using nystatin perforated patch technique to evaluate their functional state. RESULTS: The isolated neurons exhibited good responses to excitatory and inhibitory amino acids. The responses of SDCN to N-methyl-D-aspartate were markedly potentiated by substance P and trans-1-aminocyclopentane-1,3-dicarboxylate, whereas GABA responses were significantly potentiated by diazepam, pregnenolone, and pentobarbital. CONCLUSION: This preparation provides a satisfactory model for exploring the mechanisms of the SDCN in nociception and antinociception.     

锌离子对大鼠骶髓后连合核神经元甘氨酸激活的全细胞电流的调控

目的研究锌离子对急性分离的大鼠骶髓后连合核神经元甘氨酸激活全细胞电流的调控。方法采用制霉菌素穿孔膜片钳方法。结果锌离子（10～1000μmol·L-1）呈浓度依赖性地抑制甘氨酸激活的全细胞电流。结论在大鼠骶髓后连合核，锌离子对甘氨酸激活的电流呈单相调控作用，即高浓度（≥10μmol·L-1）的锌离子抑制甘氨酸的反应，而低浓度（＜10μmol·L-1）的锌离子则无此作用。     

髓系白血病患者骨髓单个核细胞β-连环蛋白mRNA的表达

目的观察急慢性髓系白血病患者骨髓单个核细胞β-连环蛋白的表达及其意义。方法 41例髓系白血病患者,其中25例急性髓系白血病（AML）,16例慢性髓系白血病（CML）。同时选择18例非恶性血液病患者作为对照。肝素抗凝骨髓2 ml,Ficoll液分离骨髓单个核细胞,荧光定量逆转录聚合酶链反应法检测β-连环蛋白的表达。结果β-连环蛋白在AML组表达量明显高于CML组及对照组（P〈0.01）;CML组表达量高于对照组表达量,差异有统计学意义（P〈0.05）;CML组4例急变患者的β-连环蛋白表达量也较高。β-连环蛋白表达量与患者年龄、性别无关;与骨髓原始细胞含量有关,含量≥30%的患者β-连环蛋白表达量较高。结论β-连环蛋白在AML和CML急变的患者骨髓单个核细胞中异常高表达,Wnt/β-连环蛋白通路在AML和CML急变病例中异常激活可能与白血病细胞的异常增殖有关。     

三环哌酯对海马脑片神经元烟碱受体及谷氨酸能兴奋性突触传递的阻断作用

目的研究中枢抗胆碱药三环哌酯(TCPN)对海马脑片神经元烟碱受体(nAChR)及谷氨酸能兴奋性突触传递的阻断作用。方法采用海马脑片盲法全细胞记录技术,以自发兴奋性与抑制性突触后电流(sEPSC和sIPSC)为观测指标。结果TCPN(10～500μmol.L-1)浓度依赖地对抗nAChR激动剂碘化二甲基苯基哌嗪(DMPP)增强海马脑片CA1锥体神经元sEPSC的作用,500μmol.L-1完全阻断DMPP的增强作用。同时,TCPN浓度依赖地直接抑制神经元的sEPSC。但TCPN不抑制神经元的sIPSC,也不阻断DMPP对sIPSC的增强作用。结论TCPN对海马脑片神经元突触传递的影响具有双重作用,既能通过阻断谷氨酸能突触前末梢nAChR而抑制sEPSC,同时还能直接抑制sEPSC。     

丙泊酚对丘脑室旁核谷氨酸能神经元活性的影响

本研究利用全细胞膜片钳技术探索丙泊酚对丘脑室旁核(paraventricular thalamus, PVT)谷氨酸能神经元活性的影响及作用机制。在8周龄C57BL/6J小鼠急性脑片上,用单细胞逆转录PCR技术鉴定PVT神经元类型。记录丙泊酚给药前、后和洗脱后PVT神经元的放电频率(firing frequencies before, during, and after, FB, FD and FW)及给药前、后的膜电位(membrane potential before and during, MPB and MPD)。探索木防己苦毒素(picrotoxin, PTX)阻断γ-氨基丁酸A型(gamma-aminobutyric acid type A, GABAA)受体后对丙泊酚作用的影响,以及丙泊酚对PVT神经元上自发和微小抑制性突触后电流(spontaneous and miniature inhibitory postsynaptic currents, sIPSCs and mIPSCs)的影响。动物实验已获得复旦大学上海医学院动物实验伦理委员会批准。结果显示,在脂肪乳组和2...     

脊髓薄片制备方法的改进及其脊髓Ⅱ板层神经元突触后电流的记录

目的探讨记录脊髓Ⅱ板层神经元兴奋性和抑制性突触后电流的方法。方法SD雄性大鼠,160～180 g,2%氟烷和100%氧气吸入麻醉后快速取腰段脊髓,迅速放入冰冷充氧的人工脑脊液,将约1 cm长的脊髓粘立于振动切片机载物台上,同时覆以冰冷的人工脑脊液进行横切片,片厚350～450μm,Kreb’S液提前30 min用95%CO2和5%O2预充氧,将切好的脊髓薄片转移其中,34～35℃孵育至少1 h后,以备实验记录用。结果在倒置显微镜下可清楚分辨出脊髓Ⅱ板层神经元。采用全细胞电压钳记录技术,给予选择性受体阻断剂以分离不同的突触后受体电流。在钳制电压为-70 mV时,可以记录到谷氨酸能的兴奋性突触后电流（EPSCs）;而在钳制电压为0 mV时,可以记录到抑制性突触后电流（IPSCs）。结论该方法为研究脊髓Ⅱ板层神经元递质释放提供了有效的途径。     

Neurokinin 1受体对脊髓后角疼痛传递和调控作用的研究进展

脊髓后角在伤害性刺激传递中起着重要的作用,将初级传入含有的疼痛信息传递到大脑。伤害性疼痛的初级传入主要终止于Ⅰ层和Ⅱ层,其中一些初级传入包含P物质。许多Ⅰ层的投射神经元发出纤维到大脑的不同区域,包括延髓腹外侧区,导水管周围灰质,丘脑。Neurokinin 1受体(NK1R)在脊髓后角的许多神经元表达,这些神经元接受含有P物质的初级传入。80%的Ⅰ层投射神经元表达NK1R阳性。在Ⅲ层和Ⅳ层,同样存在着NK1R阳性神经元,它们不但发出纤维投射到延髓腹外侧区,而且发出纤维调控Ⅰ层的投射神经元。脊髓后角的NK1R阳性神经元既受到局部抑制性神经元的调控,又受到下行调控系统的控制。     

Dual effects of pentobarbital on rat sacral dorsal commissural neurons in vitro

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Potentiation of inhibitory amino acid receptors-mediated responses by lanthanum in rat sacral dorsal commissural neurons

Lanthanum is one of rare earth cations with extremely active chemical property and has been reported to influence neuronal transmitter systems. To date, little attention has been directed towards the sacral dorsal commissural nucleus (SDCN), which serves as a relay of sensory information from the pelvic viscera in the spinal cord. Therefore, the effect of lanthanum on the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and glycine (Gly) responses in neurons acutely dissociated from the rat SDCN was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. At a holding potential of − 40 mV, La³⁺ reversibly potentiated GABA (3 μM)-activated currents (I_GABA) in a concentration-dependent manner over the concentration range of 10 μM to 30 mM, with the EC₅₀ value of 67.3 ± 16.4 μM. Similarly, La³⁺ reversibly potentiated glycine (10 μM)-activated currents (I_Gly) in a concentration-dependent manner over the concentration range of 1 μM to 1 mM, with the EC₅₀ value of 52.3 ± 10.9 μM. The effects of La³⁺ on I_GABA and I_Gly were voltage-independent. Moreover, both of the potentiations were not use-dependent and were overcome by increasing the concentration of agonist. Our results indicate that La³⁺ potentiates the inhibitory amino acid receptors-mediated responses in SDCN, which may reduce the transmission of the pelvic visceral information. The information provided by this work may help to elucidate the mechanisms and effects of lanthanum on brain functions.     

氯苯氨丁酸抑制脊髓背角神经元谷氨酸量子释放的机制

目的　研究GABAB 受体特异性激动剂氯苯氨丁酸(baclofen)在脊髓背角神经元抑制谷氨酸量子释放的机制。方法　在脊髓薄片标本上 ,采用全细胞电压钳法记录脊髓背角神经元谷氨酸能的微兴奋性突触后电流 (miniatureexcita torypostsynapticcurrents;mEPSCs) ,通过分析这些电流的变化来研究baclofen影响谷氨酸量子释放的机制。结果　ba clofen抑制mEPSCs的发放频率 ,但对平均幅度无明显影响 ,表明baclofen抑制谷氨酸释放的作用部位在突触前。在无钙溶液或者K+ 通道阻滞剂 4 AP存在的条件下 ,baclofen对mEPSCs发放频率的抑制作用不受影响 ,但腺苷酸环化酶激动剂foskolin (可使cAMP保持在较高水平 )能降低其抑制作用。而蛋白激酶C (PKC)激动剂PDBu对baclofen的抑制作用无影响。用NEM破坏G蛋白 ,则可取消baclofen的抑制效果。结论　baclofen不是通过影响突触前Ca2 + 通道或K+通道 ,或PKC途径 ,而是通过作用于G蛋白和 (或 )cAMP途径抑制谷氨酸的释放 ;这种抑制作用可能参与baclofen在脊髓水平的镇痛     

5-HT potentiation of the GABAA response in the rat sacral dorsal commissural neurones

1. The modulatory effect of 5-hydroxytryptamine (5-HT) on the γ-aminobutyric acid_A (GABA_A) response was investigated in the neurones freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions.
2. 5-HT potentiated GABA-induced Cl⁻ current (I_GABA) without affecting the reversal potential of I_GABA and the apparent affinity of GABA to its receptor.
3. α-Methyl-5-HT mimicked the potentiation effect of 5-HT on I_GABA while ketanserine blocked it. 1-Oleoyl-2-acetyl-glycerol (OAG) potentiated I_GABA, and the effect of 5-HT on I_GABA was occluded by OAG pretreatment. In the presence of chelerythrine, 5-HT failed to potentiate I_GABA, suggesting that protein kinase C (PKC) is involved in the pathway through which the activation of the 5-HT₂ receptor potentiates the I_GABA.
4. The facilitatory effect of 5-HT on I_GABA remained in the presence of BAPTA-AM. LiCl also had no effect on 5-HT-induced potentiation of I_GABA.
5. H-89, genistein, okadaic acid and pervanadate all had no effects on 5-HT potentiation of I_GABA. Pertussis toxin treatment for 6–8 h did not block the facilitatory effect of 5-HT on I_GABA.
6. The present results show that GABA_A receptor in the rat SDCN could be modulated in situ by 5-HT, one of the major transmitters involved in the supraspinal control of nociception, and that the phosphorylation of GABA_A receptor by PKC may be sufficient to support such modulation. The results also strongly support the hypothesis that the cotransmission by 5-HT and GABA has an important role in the spinal cord.

     

Involvement of GABA and opioid peptide receptors in sevoflurane-induced antinociception in rat spinal cord

AIM: The spinal cord is pivotal in immobility induced by volatile anesthetics because the anesthetics depress the activity of motor neurons in the spinal cord. The aim of this study was to observe the effects of sevoflurane on pain processing at the spinal level. METHODS: The firing of the gastrocnemius muscle was evoked by electrical stimulation to the ipsilateral hindpaw in rats. The nociceptive C response of electromyography (EMG) was selected to study. The GABAA receptor antagonist bicuculline (0.1 mg/kg) and opioid receptor antagonist naloxone (0.4 mg/kg) were administered intravenously, either in the presence or in the absence of 1.0% sevoflurane. RESULTS: In rats with transected spinal cord, sevoflurane produced a profound reduction in the C response in a dose- and time-dependent manner. In the presence of 1.0% sevoflurane, the C responses were increased after injections of bicuculline and naloxone. CONCLUSION: Sevoflurane is a volatile anesthetic that acts directly on the spinal cord to suppress the nociceptive reflex. The sevoflurane-induced suppression of the C response is antagonized by either bicuculline or naloxone. The results suggest that spinal GABAA receptors and opioid peptide receptors are involved in the sevoflurane-induced suppression of spinal nociception.     

龙血素B抑制大鼠背根神经节细胞辣椒素诱发的电流反应

目的探讨龙血素B对大鼠背根神经节细胞辣椒素诱发的辣椒素受体电流的影响。方法采用全细胞膜片钳技术在急性分离的大鼠背根神经节细胞上观察龙血素B对辣椒素诱发的辣椒素受体电流的影响。结果①在-60mV的钳制电位下,辣椒素受体拮抗剂辣椒卓平可以完全抑制辣椒素受体电流;②不同浓度的龙血素B溶液对辣椒素诱发的受体电流具有浓度依赖的抑制作用;2.0、4.0、8.0和16.0μmol.L-1的龙血素B溶液对辣椒素诱发的受体电流峰值的抑制率分别为15.36%±2.12%、36.41%±2.43%、76.26%±2.16%和96.69%±3.21%(n=10,P<0.05),半数抑制浓度(IC50)为4.9μmol.L-1,Hill系数为2.29。结论龙血素B可以明显抑制辣椒素诱发的辣椒素受体电流,影响痛觉信息的传入,这可能是以龙血素B为重要成分的中药血竭产生镇痛作用的机制之一。     

川芎嗪对大鼠背根神经节细胞P2X嘌呤受体介导反应的作用

目的探讨川芎嗪(tetram ethy1pyrazine,TMP)对嘌呤2X(P2X)受体介导反应的作用。方法在大鼠新鲜分离的背根神经节(dorsal root ganglion,DRG)神经元标本上应用全细胞膜片钳技术记录川芎嗪对P2X受体激动剂激活电流的影响。结果外加ATP(1~1 000μmol.L-1)可引起DRG神经元产生激活电流(n=102),ATP-激活电流(IATP)显示快失敏和慢失敏两种形式的内向电流。预加川芎嗪(0.1~10mmol.L-1)后,大部分(89.2%,91/102)受检细胞可观察到ATP(100μmol.L-1)-激活电流出现明显的抑制作用。川芎嗪(1 mmol.L-1)使α,-βm eATP(10μmol.L-1)-激活电流减小。预加川芎嗪(1 mmol.L-1)后ATP(1~1 000μmol.L-1)激活电流的剂量-效应曲线明显下移。预加川芎嗪(1 mmol.L-1)前后ATP(100μmol.L-1)的I-V曲线反转电位值不变,均接近0 mV。川芎嗪(1 mmol.L-1)可明显抑制被前列腺素E2(100μmol.L-1)或P物质(0.1μmol.L-1)增大的ATP激活电流。通过微电极胞内透析注入PKA抑制剂H89(10μmol.L-1)至胞内,使川芎嗪(1 mmol.L-1)抑制ATP(100μmol.L-1)激活电流的作用减小。结论川芎嗪可能是通过PKA系统以及P2X受体离子通道复合体细胞外环的变构调制点影响P2X受体激动剂在大鼠DRG神经元的激活电流。