神经肽优洛可定对丘脑底核神经元自发放电影响与CRF-2R关系研究

目的研究神经内分泌生物活性肽优洛可定(urocortin,UCN)对丘脑底核(subthalamic nucleus,STN)神经元放电及STN中多巴胺(dopamine,DA)能、谷氨酸(glutamic acid,GLU)能神经传递的影响,研究UCN在STN中是否与GLU及DA存在汇聚作用,探讨UCN在帕金森疾病(Parkinson’s disease,PD)发生中可能发挥的作用。方法取40只SD大鼠,采用神经电生理实验方法中的微电泳技术,在微电泳UCN过程中,观察STN神经元放电的波形及频率变化,同时,微电泳Astressin(AST)及蛋白激酶A(protein kinase A,PKA)抑制剂,观察促皮质素释放因子2型受体(corticotropin releasing factor 2 receptor,CRF-2R)是否参与UCN对STN神经元放电的影响。另外,在微电泳DA和GLU过程中,给予UCN及AST,观察STN神经元放电的变化,明确是否存在DA能及GLU能与UCN的交汇作用。结果微电泳UCN过程中,82%(27/33)STN神经元频率减慢。STN平均放电频率由(3.65±0.27)Hz减少到(2.05±0.33)Hz,微电泳UCN前、后STN放电频率明显降低(P<0.01)。另外,UCN抑制作用可被AST明显拮抗(P<0.01),UCN抑制作用也可被PKA阻断剂部分阻断。在微电泳抑制性神经递质DA和兴奋性GLU过程中,UCN能进一步协同DA的抑制效应,并明显拮抗GLU的兴奋作用(P<0.01)。结论 UCN与DA能及GLU能在STN神经元中存在汇聚作用,UCN可能是通过与CRF-2R结合,进而通过PKA信号通路,影响DA能、GLU能神经递质活动,抑制STN神经元放电。     

微量注射6-OHDA损毁大鼠黑质对丘脑底核神经元电活动影响的研究

为了观察黑质内注射 6-OHDA损毁黑质多巴胺 (DA)能神经元后大鼠丘脑底核 (STN)神经元自发放电活动的变化 ,以探讨 STN神经元功能紊乱与帕金森病 (PD)运动障碍的关系 ,选择单侧黑质致密部 (SNC)微量注射 6-OHDA方法建立 PD大鼠模型 ,采用微电极细胞外记录神经元放电 ,观察对照组大鼠和 PD模型大鼠不同时期的 STN神经元放电频率及放电形式的变化。结果显示 ,对照组大鼠 STN神经元放电频率平均为 11.16± 1.5 2 Hz,其放电形式主要表现为规则型 (3 9/4 4) ,其次为混合型 (3 /4 4)和爆发型 (2 /4 4) ;而 PD模型大鼠 (SNC损毁 )不同时期的 STN神经元的放电形式发生明显变化 ,呈爆发型放电的神经元数较对照组大鼠明显增多 (P<0 .0 5和 P<0 .0 1) ;平均放电频率较对照组大鼠明显升高 (P<0 .0 5和 P<0 .0 1)。     

吗啡对吗啡依赖大鼠腹侧苍白球神经元放电的影响

目的 :研究吗啡对吗啡依赖大鼠腹侧苍白球的电生理影响 ,探讨其在成瘾机制中的作用。方法 :通过单细胞细胞外记录吗啡依赖大鼠腹侧苍白球的电信号 ,观察吗啡对放电的影响。结果 :吗啡依赖大鼠腹侧苍白球神经元放电频率给药前为 3 76Hz±s 1 18Hz,注射吗啡后为 13 2 2Hz±s 2 37Hz;并且纳洛酮可以逆转这种兴奋效应。结论 :吗啡显著兴奋吗啡依赖大鼠腹侧苍白球神经元 ,腹侧苍白球在药物强化过程中起着重要作用。     

不同频率电刺激VTA对吗啡成瘾大鼠ACbSh的放电活动的影响

目的:观察电刺激大鼠腹侧被盖区(VTA)对吗啡成瘾大鼠伏隔核壳部(ACbSh)神经元的影响,为应用脑深部电刺激技术治疗阿片类药物成瘾提供实验依据。方法:采用单管玻璃微电极细胞外记录法,观察不同频率电刺激VTA对吗啡成瘾大鼠ACbSh神经元放电的影响。结果:低频(50Hz)电刺激VTA,吗啡组ACbSh神经元主要表现为无反应(47.83%);高频(135Hz)电刺激VTA,吗啡组多数ACbSh神经元表现为抑制性作用(50.00%)。结论:高频电刺激VTA有可能作为新方法用于治疗阿片类药物成瘾。     

三七总皂苷对大鼠星状神经节后超极化电位的影响

目的研究三七总皂苷(PNS)对神经元放电频率的影响是否与改变膜电位的超极化程度有关。方法向大鼠星状神经节(SG)细胞内输入去极化电流,诱发动作电位,区分相位式发放(phasic)细胞和紧张式发放(ton ic)细胞,观察PNS灌流SG后,细胞放电类型和放电频率、后超极化电位(AHP)、高钙易化的快兴奋性突触后电位(f-EPSP)的变化。结果PNS使ton ic细胞重复发放的频率下降。在0.12~0.16 g.L-1浓度范围内,PNS对SG细胞的AHP有浓度依赖性抑制作用。PNS还能可逆性拮抗高钙对f-EPSP的易化作用。结论PNS降低大鼠SG细胞的放电频率并非通过强化AHP电位引起,PNS对神经细胞兴奋性的抑制是因拮抗胞膜钙内流所致。     

谷氨酸对吗啡依赖大鼠戒断症状的影响及其作用机制

目的:探讨谷氨酸(Glu)对吗啡依赖大鼠戒断症状的影响及其作用机制。方法:建立大鼠吗啡依赖及戒断模型并给予评分,研究Glu对吗啡依赖大鼠戒断症状的影响;通过电生理,原位杂交,免疫组化方法研究丘脑束旁核神经元动作电位变化和c-fos基因表达的变化。结果:Glu剂量依赖性增加大鼠戒断症状的总评分(P<0.05);Glu使吗啡依赖大鼠丘脑束旁核的神经元自发放电和诱发放电频率增加(P<0.05),而诱发放电抑制时程缩短(P<0.05);在Glu诱发吗啡戒断过程中,c-fos基因表达水平显著增高(P<0.01,P<0.01)。结论:Glu可通过增加细胞电脉冲的形式兴奋神经元,提高依赖状态下神经元的兴奋性,加重吗啡依赖大鼠的戒断症状;Glu也可通过c-fos间接影响Glu受体和其他的神经递质的表达起作用。     

神经肽Urocortin2对吗啡成瘾大鼠VTA神经元放电及DA能神经传递的影响

目的明确神经肽urocortin2(UCN2)在吗啡成瘾中抑制丘脑腹侧背盖区(ventral tegmental area,VTA)神经活动的机制。方法建立吗啡成瘾大鼠模型,采用七管微电泳方法,电泳UCN2对吗啡成瘾大鼠VTA神经元自发放电的改变,及UCN2对多巴胺(dopamine,DA)能神经元簇发放电模式的影响,明确UCN2在VTA神经元中与DA存在汇聚作用。另外,给予促皮质激素释放因子(coticortropin-releasing factor,CRF)受体阻断剂及蛋白激酶A(protein kinase A,PKA)抑制剂,明确UCN抑制吗啡成瘾中发挥关键作用。结果UCN2使82%(31/38)吗啡成瘾大鼠VTA神经元放电频率减慢,平均放电频率由微电泳前的(20.89±2.86)Hz减少到(13.66±3.93)Hz,给药前后放电频率降低具有显著性(P<0.01),UCN2抑制作用可被PKA抑制剂H89及CRF-2R阻断剂AST-2B取消。另外,微电泳UCN2可抑制吗啡成瘾大鼠VTA中DA神经元的爆发放电,AST-2B可增强DA的兴奋效应。结论 UCN2与CRF-2R结合后,通过PKA信号途径,抑制VTA内DA神经元异常放电,在吗啡类药物成瘾中发挥抑制作用,为其用于临床治疗阿片类药物成瘾提供理论依据。     

PTD-BDNF对海马神经元兴奋性突触活动的作用*

［摘要］　目的：研究具有跨血脑屏障功能的PTD-BDNF对培养海马神经元细胞自发电活动的作用。方法：应用全细胞膜片钳技术,观察脑源性神经营养因子(BDNF)和蛋白转导结构域-脑源性神经营养因子(PTD-BDNF)对原代培养大鼠海马神经元自发突触活动的影响。结果：与对照组比较，BDNF与PTD-BDNF作用后均可明显增加海马神经元自发兴奋性突触后电流(sEPSC)和微小兴奋性突触后电流(mEPSC)的产生频率［sEPSC的频率：(1.4±0.5)vs(4.0±0.6)，(3.9±0.5)Hz，均为P<0.01；mEPSC的频率：(4.2±1.0)vs(7.9±1.9)，(7.6±1.8) Hz,均为P<0.01］，并增加sEPSC的幅度但并不影响mEPSC的幅度［sEPSC的幅度：(415.7±48.0)vs(743.7±95.7)，(693.7±99.9)pA，均为P<0.01；mEPSC的幅度：(34.7±4.7)vs(36.3±6.2)，(36.2±4.4)pA，均为P>0.05］。BDNF与PTD-BDNF两者之间比较差异均无统计学意义(P>0.05)。结论：PTD-BDNF可以增强大鼠海马神经元兴奋性突触活动，说明重组PTD-BDNF与BDNF有相似的电生理活性。     

针刺对2型糖尿病大鼠弓状核的调整作用

目的探讨针刺对2型糖尿病(T2DM)大鼠弓状核功能的作用,进一步深化治疗T2DM的中枢神经作用机制。方法以普通全阶鼠饲料喂养的大鼠作为正常组,将造模成功的实验性T2DM大鼠随机分为对照组和针刺组。针刺组大鼠给以针刺治疗28天,正常组和对照组大鼠分别每天放入大鼠固定器中适应10分钟,持续28天。采用神经电生理及神经生化技术观察针刺治疗前后T2DM大鼠空腹血糖(FPG)、空腹胰岛素(FINS)、C-肽(C-P)、胰岛素敏感性指数(ISI)、胰岛素抵抗指数(Homa-IR)、胰岛β细胞功能指数(Homa-β)和下丘脑弓状核神经细胞自发放电频率及弓状核组织神经递质的变化。结果对照组下丘脑弓状核神经细胞自发放电频率显著低于正常组水平([3.59±0.92)Hz,(10.32±2.95)Hz,P<0.01],而针刺组较对照组明显增高[(12.17±3.54)Hz,P<0.01]。大鼠下丘脑弓状核神经细胞自发放电频率与FPG、FINS、C-P、Homa-IR均呈负相关,而与ISI和Homa-β呈正相关。结论针刺对T2DM大鼠下丘脑弓状核功能的良性作用可能是纠正胰岛素抵抗和调整异常内分泌代谢的中枢神经作用机制之一。     

杨梅黄酮对炎性痛大鼠的外周镇痛作用

目的研究杨梅黄酮对炎性痛大鼠的外周镇痛作用及机制。方法采用大鼠左侧后肢足底中心皮下注射完全弗氏佐剂(complete Freund's adjuvant,CFA)建立慢性炎性痛模型。检测给予杨梅黄酮前后大鼠热缩足反射潜伏期(thermal withdrawal latency,TWL)的改变。电生理学方法分析杨梅黄酮对背根节神经元放电频率及电压依赖性钾电流的影响。结果大鼠左侧足底皮下注射CFA致炎后,TWL明显降低(P<0.05),腹腔注射50、500 mg·kg-1杨梅黄酮使CFA组大鼠TWL明显升高(P<0.05),电流钳记录显示杨梅黄酮抑制背根节小神经元的放电频率(P<0.01),且该抑制作用是通过增加钙依赖性钾通道电流(P<0.05)产生的。结论杨梅黄酮通过增加背根节神经元钙依赖性钾电流,抑制神经元兴奋性而发挥外周镇痛作用。     

Bidirectional activity-dependent plasticity of membrane potential and the influence on spiking in rat hippocampal dentate granule cells

Granule cells of the dentate gyrus in the hippocampus generally fire at low frequencies but are known to respond to sensory cues by increasing their rate of firing. We have previously shown that a burst of action potentials in synaptically isolated granule cells can induce a long-term depolarisation (LTDepol) of the neuronal membrane potential. This form of excitability plasticity could be an important mechanism for learning and memory. Here we demonstrate that this depolarisation can be reversed by physiologically relevant firing patterns. At a basal action potential frequency of 0.1Hz the membrane potential depolarises in response to brief high frequency stimulation (HFS) but this depolarisation is blocked or reversed by 1Hz action potential firing. The depolarisation of the neurones did not, however, affect the input-output function of the dentate gyrus measured by field or single cell recordings. HFS or brief forskolin application that mimics LTDepol did not alter the excitatory input-output spike coupling measured using field potential recordings. Similarly, LTDepol did not change the probability of spike firing in response to a given input. The mechanism underlying the lack of change in input-output relationship was found to be a concurrent depolarisation of the threshold potential for the initiation of action potentials. We demonstrate that physiologically relevant patterns of activity regulate the membrane potential of dentate gyrus granule cells in a bidirectional manner but the changes in membrane potential do not alter the excitability of these neurones measured by their response to excitatory inputs.     

Effects of some antiepileptic drugs on the repetitive activity of the node of Ranvier

1 Effects of some antiepileptic drugs on the repetitive activity of the node of Ranvier have been tested on frog myelinated nerve fibres. 2 Nerve fibres were stimulated by supraliminal direct current pulses of long duration. Motor fibres responded with a single action potential; sensory fibres responded with repetitive firing at a frequency of about 300/s. Chlordiazepoxide hydrochloride (0.1 mM), phenobarbitone sodium (0.25 mM) or diphenylhydantoin sodium (0.5 mM) suppressed the repetitive activity. 3 Sensory and motor nerve fibres stimulated by a 10 kHz alternating current of strength twice the threshold responded with repetitive firing at a frequency of 400-500/s. Superfusion of the node with chlordiazepoxide hydrochloride (0.2 mM), phenobarbitone sodium (0.5 mM) or diphenylhydantoin sodium (1.7 mM) reduced the frequency of firing by 50% either in sensory or in motor fibres activated by a.c. stimulation; at the same concentrations, the drugs altered amplitude of the action potential and threshold for electric excitability by less than 10%. 4 Unlike local anaesthetics, chlordiazepoxide, phenobarbitone and diphenylhydantoin are more selective in inhibiting repetitive firing than in reducing the amplitude of the action potential or increasing the threshold for electric excitability. 5 Trimethadione (up to 5 mM) was ineffective on repetitive firing elicited either with direct or with alternating current.     

Excitability of nodose ganglion cells and their role in vago-vagal reflex control of gastrointestinal function

A large body of evidence has demonstrated that vagal afferent neurones show non-uniform properties and that distinct neuronal populations can be identified within the nodose ganglia. Of particular interest is recent work illustrating the significant degree of plasticity displayed by vagal sensory neurones; alterations in vagal afferent neuronal excitability might be important in the development and maintenance of gastrointestinal pathological states. Although it is unclear whether such adaptations are mimicked centrally, recent studies suggest that tonic afferent vagal inputs act as a 'brake' on inputs to the dorsal motor nucleus of the vagus. It would be reasonable to assume, therefore, that plasticity in the excitability of vagal afferent neurones would have dramatic consequences for the regulation and modulation of gastrointestinal vago-vagal reflexes.     

Multi-neuronal recordings reveal a differential effect of thapsigargin on bicuculline- or gabazine-induced epileptiform excitability in rat hippocampal neuronal networks

The present study was performed to investigate the effects of depleting intracellular Ca(2+) stores on bicuculline- or gabazine-induced epileptiform excitability. Studies were performed on monolayer rat hippocampal neuronal networks utilising a system that allowed simultaneous multiple extracellular single-unit recordings of neuronal activity. Hippocampal neuronal networks were prepared from enzymatically dissociated hippocampi from 18-day-old fetal Wistar rats. The cells were cultured in Neurobasal medium with B27 serum-free supplements directly onto the surface of planar multiple microelectrode arrays with a central recording array of 64 (4 x 16) indium-tin thin-film recording electrodes. All cells recorded at 21 days-in-vitro exhibited spontaneous discharge activity with firing rates between 0.3-30.7 Hz. gamma-aminobutyric acid (GABA) produced a concentration-dependent decrease in firing (EC(50)=9.1 microM) which could be blocked by pre-application of bicuculline methobromide (10 microM). Addition of the GABA(A)-receptor antagonists gabazine (10 microM) or bicuculline (10 microM) resulted in the rapid generation of synchronised bursting within all the cells recorded. Bicuculline exhibited heterogeneity of action on firing rate, whereas gabazine always increased firing. Pre-incubation with thapsigargin, which depletes intracellular calcium stores, resulted in a decrease in the amount of neuronal excitation produced by bicuculline, but not by gabazine, suggesting that bicuculline-induced neuronal excitation requires release of Ca(2+) from intracellular stores.     

Clonidine fails to modify dopaminergic neuronal activity during morphine withdrawal

RATIONALE: Cellular substrates of opiate withdrawal syndrome involve several brain areas, in particular the mesolimbic dopaminergic and noradrenergic systems, but the interactions between the two pathways remain unclear. OBJECTIVES: The aim of the present work was to investigate the effects of the alpha2-agonist clonidine on ventral tegmental area dopamine neurons during morphine withdrawal syndrome by recording their neuronal activity before and after the administration of low and relatively high doses of clonidine (from 5 to 100 microg/kg). METHODS: The spontaneous neuronal activity of meso-accumbens dopaminergic neurons, identified by antidromical stimulation from the nucleus accumbens, was recorded by use of in vivo extracellular single-unit recordings in control and morphine-withdrawn rats after chronic administration (15 days). RESULTS: Control rats showed a mean spontaneous firing frequency of 2.47+/-0.48 Hz, percentage of burst firing of 22+/-12 and an increase in firing after the administration of cumulative doses of clonidine (5, 10, 20, 40, 100 microg/kg). Conversely, both spontaneous firing rate (1.55+/-0.25 Hz) and the percentage of burst firing (5+/-2) were found to be significantly reduced in rats abstinent for 24 h, and increasing doses of clonidine did not re-establish electrophysiological activity observed in the controls. CONCLUSION: The results indicate that: 1) clonidine did not restore the decreased firing activity of DA neurons in morphine-withdrawn rats, and 2) high doses of clonidine increased firing in control rats but not in morphine-withdrawn rats.     

延髓甩尾相关神经元参与化学性刺激大鼠外侧缰核衣发的下行易化作用

AIM: To study effects of sodium L-glutamate microinjection into lateral habenular nucleus (LHN) of rats on the firing of medullary tail-flick related neurons and tail-flick reflex (TF). METHODS: Using synchronous recording of unitary neuronal discharges and TF induced by noxious heat. RESULTS: Chemical stimulation of LHN induced an excitement of the on-cell spontaneous activity, an inhibition of the off-cell spontaneous activity with an enhancement of their TF related responses. The spontaneous firing rate of on-cells increased from 5.8 +/- 2.2 Hz to 10.9 +/- 3.4 Hz while the spontaneous firing rate of off-cells decreased from 11.8 +/- 2.2 Hz to 6.1 +/- 2.2 Hz. Meanwhile the TFL was shortened from 4.04 +/- 0.17 s to 2.97 +/- 0.13 s. CONCLUSION: The chemical stimulation of LHN produced a facilitating action on nociceptive spinal defensive reflex. This effect is brought out by the cooperation of on- and off-cells.     

Inhibition of mechanical activation of guinea-pig airway afferent neurons by amiloride analogues

1. The aim of this study was to investigate a role for Epithelial Sodium Channels (ENaCs) in the mechanical activation of low-threshold vagal afferent nerve terminals in the guinea-pig trachea/bronchus. 2. Using extracellular single-unit recording techniques, we found that the ENaC blocker amiloride, and its analogues dimethylamiloride and benzamil caused a reduction in the mechanical activation of guinea-pig airway afferent fibres. 3. Amiloride and it analogues also reduced the sensitivity of afferent fibres to electrical stimulation such that greater stimulation voltages were required to induce action potentials from their peripheral terminals within the trachea/bronchus. 4. The relative potencies of these compounds for inhibiting electrical excitability of afferent nerves were similar to that observed for inhibition of mechanical stimulation (dimethylamiloride approximately benzamil > amiloride). This rank order of potency is incompatible with the known rank order of potency for blockade of ENaCs (benzamil > amiloride > dimethylamiloride). 5. As voltage-gated sodium channels play an important role in determining the electrical excitability of neurons, we used whole-cell patch recordings of nodose neuron cell bodies to investigate the possibility that amiloride analogues caused blockade of these channels. At the concentration required to inhibit mechanical activation of vagal nodose afferent fibres (100 microM), benzamil caused significant inhibition of voltage-gated sodium currents in neuronal cell bodies acutely isolated from guinea-pig nodose ganglia. 6. Combined, our findings suggest that amiloride and its analogues did not selectively block mechanotransduction in airway afferent neurons, but rather they reduced neuronal excitability, possibly by inhibiting voltage-gated sodium currents.     

Nitric oxide modulation of the spontaneous firing of rat medial vestibular nuclear neurons

Modulation of the spontaneous activity of rat medial vestibular nuclear neurons by nitric oxide was investigated using the whole-cell patch-clamp technique. The spike frequency was increased by sodium nitroprusside (SNP), a nitric oxide liberating agent, and it was also increased by another nitric oxide liberating agent, sodium-nitroso-N-acetylpenicillamine. L-Arginine, the substrate of nitric oxide synthase, increased the firing of the neurons. The increased SNP-induced firing was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one (ODQ), a specific inhibitor of guanylate cyclase. These results suggest that nitric oxide increases the neuronal excitability of the neurons by a cGMP-dependent mechanism.     

Effects of intravenous kainic acid,N-methyl-d-aspartate,and (-)-nuciferine on the cat spinal cord

Summary Kainic acid (a rigid conformational analogue of glutamate), N-methyl-d-aspartate (the methylated derivative of aspartate), and (-)-nuciferine (an aporphine alkaloid with a depressant effect on glutamate-induced neuronal firing), which, so far, have been examined in microiontophoretic studies, were investigated in spinal cats for their effects on some spinal cord activities after intravenous injections.At low doses, kainic acid (0.3 mg kg^–1) enhanced segmental monosynaptic but not polysynaptic ventral root reflexes and increased the excitability of motoneurones, whereas N-methyl-d-aspartate (3 mg kg^–1) facilitated polysynaptic but not monosynaptic reflexes. Higher doses of the two amino acids depolarized motoneurones and primary afferent endings, enhanced monosynaptic reflexes and depressed polysynaptic reflexes.(-)-Nuciferine (1–10 mg kg^–1) depressed monosynaptic but not polysynaptic ventral root reflexes in a dose-dependent manner and antagonized the effects of kainic acid but not of N-methyl-d-aspartate on the spinal cord.The results are consistent with the hypothetical excitatory transmitter role of glutamate in primary afferents and of aspartate in excitatory spinal cord interneurones; the findings also suggest that (-)-nuciferine may be used as a systemically effective, rather selective blocker of central glutamate receptors.