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。     

盐酸埃他卡林对大鼠海马神经元谷氨酸受体功能及突触活动的影响

目的 :研究盐酸埃他卡林 (Ipt)对脑神经元谷氨酸受体功能及突触活动的影响。方法 :采用原代培养的大鼠海马神经元 ,应用膜片钳全细胞记录技术 ,记录Ipt对培养的海马神经元谷氨酸或天冬氨酸(NMDA)诱发电流及神经元突触后电流的影响。结果 :Ipt(1～ 1 0 0 μmol·L- 1)可浓度依赖性地对抗培养的海马神经元谷氨酸或NMDA诱发电流 ,并为ATP敏感性钾通道拮抗剂格列本脲 30 μmol·L- 1所对抗。Ipt抑制培养的海马神经元之间突触联系形成的自发兴奋性突触后电流 ,降低其发放频率 ,抑制其电流幅度 ;但对微小兴奋性突触后电流无显著性影响。结论 :Ipt可阻断脑神经元谷氨酸受体功能 ,抑制脑神经元谷氨酸的兴奋性突触传递 ,其作用与ATP敏感性钾通道相关     

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。     

脊髓背角PKC在慢性炎性疼痛中的作用及其机制

目的探讨蛋白激酶C(protein kinase C,PKC)的抑制剂和激动剂对痛觉超敏的影响及其分子机制。方法小鼠后趾皮下注射完全弗氏佐剂(complete Freund's adjuvant,CFA)建立炎性疼痛模型;鞘内给予PKC抑制剂白屈菜赤碱(chelerythrine,CHE)或激动剂Phorbol 12-myristate 13-acetate(PMA)前后,测定小鼠缩足阈值;随后立即分离脊髓背角,免疫印迹法检测NMDA(N-methyl-D-aspartate)型谷氨酸受体的突触表达。结果 PKC抑制剂CHE在缓解炎性痛觉超敏的同时,明显翻转脊髓NMDA受体NR2B亚基的突触表达亢进;而正常小鼠鞘内给予PKC激动剂PMA,可模拟CFA的效应,即:诱发痛觉超敏,并特异性增加NR2B亚基的突触含量。结论 PKC通过调节脊髓NMDA受体NR2B亚基的突触表达,参与炎性疼痛的形成。     

辣椒素受体参与骶髓后联合核神经元突触传递

目的研究辣椒素受体对大鼠骶髓后联合核(SDCN)神经元突触传递的影响。方法在脊髓骶段横切薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs)和γ-氨基丁酸(GABA)能抑制性突触后电流(IPSCs),比较激动辣椒素受体后上述突触电流的变化;观察激动辣椒素受体对SDCN神经元动作电位发放的影响。结果辣椒素受体被其特异性激动剂辣椒素(1μmol.L-1)激动后,自发EPSCs(sEPSCs)的频率和振幅均有明显增加(P<0.05,n=17)。在河豚毒素(0.5μmol.L-1)存在的条件下,辣椒素明显增加微小EPSCs(mEPSCs)的频率(P<0.01,n=13),但对mEPSCs的振幅无影响(P>0.05,n=13),提示辣椒素的作用在突触前。辣椒素也明显增加动作电位发放(P<0.05,n=19)。上述作用均可被辣椒素受体特异性拮抗剂capsazepine(10μmol.L-1)阻断。辣椒素也增加GABA能的自发IPSCs(sIPSCs)的频率(P<0.05,n=20),但对其不依赖动作电位的微小IPSCs(mIPSCs)的频率或振幅均无作用(P>0.05,n=9)。结论在SDCN,辣椒素受体主要表达于兴奋性突触终末;激动辣椒素受体影响兴奋性和抑制性突触活动,并可能参与痛觉信息在脊髓水平的传递和调制。     

褪黑激素对大脑皮层谷氨酸释放及其神经毒性的拮抗作用(英文)

目的:观察褪黑激素(Mel)对老年小鼠大脑皮层突触体谷氨酸(Glu)释放以及KCl,Glu在原代培养胎鼠脑细胞诱发的神经毒性的影响,以探讨Mel抗衰老的作用机制。方法:制备老年小鼠大脑皮层突触体,用RF-5000型双波长荧光分光光度计检测谷氨酸释放量。应用原代培养的大鼠皮层细胞MTT染色和乳酸脱氢酶(LDH)测定法评估神经元活性。结果:Mel能够抑制高浓度氯化钾(30mmol·L~(-1))诱发的老年小鼠大脑皮层突触体钙依赖性及非依赖性谷氨酸释放,抵抗KCl和Glu诱发的皮层细胞损伤,对神经元有保护作用。结论:Mel对大脑皮层突触体谷氨酸释放的抑制作用以及对大脑皮层神经元的保护作用可能是其抗衰老作用机制之一。     

巴氯芬致驰张热合并白细胞减少1例

巴氯芬 (baclofen) ,其化学名 :4 氨基 3 (4 氯苯基 ) 丁酸 ,商品名 :力奥来素 (Lioresal) ,是一类肌松药物 ,γ -氨基丁酸 (GABA)受体激动剂[1,2 ] 。巴氯芬作用于脊髓后 ,由于其对GABA受体有较高的亲和力 ,能与之结合 ,从而产生抑制兴奋性氨基酸释放的作用 ,如天门冬氨酸、谷氨酸 ;抑制脊髓的单突触反射和多突触反射 ,从而缓解骨骼肌痉挛状态 ,使肌张力降低 ,改善肌肉的功能状态等作用[3 ,4] 。治疗开始时常出现日间镇静、嗜睡和恶心等中枢神经系统的不良反应 ,偶尔出现欣快、抑郁、感觉异常、肌痛、肌无力、共济失调、震颤、眼球…     

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。     

P2X7受体介导异丙酚对缺氧海马突触前膜谷氨酸Ca~(2+)依赖性释放的抑制作用

目的观察嘌呤P2X7受体激动剂和拮抗剂以及异丙酚对大鼠缺氧海马突触前膜谷氨酸释放的影响及P2X7受体是否介导异丙酚的作用。方法选用SD♂大鼠,制备突触体,置入无糖并充以95%N2/5%CO2的人工脑脊液(aCSF)中进行缺氧实验。向无糖aCSF中加入P2X7受体特异性拮抗剂BBG(终浓度1μmol·L-1)或P2X7受体特异性激动剂BZATP(终浓度100μmol·L-1)。于缺氧前、缺氧20min及40min时测定孵育液中谷氨酸浓度,谷氨酸Ca2+非依赖性释放试验中将Ca2+、DHK(谷氨酸转运体抑制剂)从aCSF中去除。观察异丙酚对谷氨酸Ca2+依赖性释放的影响时,分别向无糖aCSF中加入1～100μmol·L-1的异丙酚,然后进行缺氧40min,测定孵育液中谷氨酸浓度并计算异丙酚的半效抑制浓度(IC50)。观察P2X7受体在异丙酚抑制效应中的作用时,施加异丙酚(2倍IC50水平)、异丙酚和BBG(终浓度1μmol·L-1)或异丙酚和BZATP(终浓度100μmol·L-1)。缺氧40min后测定孵育液中谷氨酸浓度。结果缺氧不同时点海马突触体谷氨酸Ca2+依赖性释放增加(P<0.01),BBG可完全抑制谷氨酸释放(P<0.01),BZATP则进一步使谷氨酸释放增加(P<0.01)。谷氨酸Ca2+非依赖性释放在缺氧各时点无变化。异丙酚可浓度依赖性的抑制谷氨酸Ca2+依赖性释放,IC50为(27.4±5.2)μmol·L-1。55μmol·L-1(2倍IC50)异丙酚与BBG或与BZATP共同作用下的谷氨酸Ca2+依赖性释放水平与单独应用异丙酚比较差异无显著性(P>0.05)。结论缺氧状态下,P2X7受体介导海马突触前膜谷氨酸Ca2+依赖性释放,异丙酚通过抑制P2X7受体而呈浓度依赖性的抑制谷氨酸Ca2+依赖性释放。     

甘丙肽对幼年大鼠蓝斑神经元兴奋性的影响及作用机制

目的探讨甘丙肽(galanin,GAL)对幼年大鼠蓝斑神经元兴奋性的影响,研究甘丙肽与其受体(GalR)和钾离子通道的作用机制。方法制备幼年大鼠脑片,采用全细胞记录法记录灌流给予甘丙肽后,蓝斑神经元静息膜电位水平和自发动作电位的发放频率;灌流给予不同浓度甘丙肽2型受体(GalR2)激动剂AR-M1896和不同类型钾离子通道阻断剂后,研究甘丙肽对幼年大鼠蓝斑神经元兴奋性的影响。结果甘丙肽能够诱导蓝斑神经元膜电位发生超级化并抑制其动作电位的发放,但GalR2激动剂AR-M1896只有在高浓度(1μM)时才能够诱导蓝斑神经元膜电位发生轻微的超级化并减少动作电位的发放;电压依赖性钾通道阻断剂四乙胺(Tetraethylammonium,TEA)能够部分阻断甘丙肽的抑制作用,内向整流钾通道阻断剂Ba Cl2能够显著阻断甘丙肽的抑制作用,而其他钾通道阻断剂,如ATP敏感性钾通道阻断剂格列本脲(Glybenclamide)、大电导钙依赖的钾通道(BK通道)阻断剂北非蝎毒素(Charybdotoxin)、小电导钙依赖的钾通道(SK通道)阻断剂蜂毒明肽(Apamin)均未能阻断甘丙肽的抑制作用。结论甘丙肽能够抑制幼年大鼠蓝斑神经元的兴奋性,其抑制作用可能主要是通过甘丙肽1型受体(GalR1)产生的,并且这种抑制作用可能与TEA敏感的钾通道和内向整流钾通道相关,而ATP敏感的钾通道和钙依赖的钾通道不参与其中。     

Role of Presynaptic Glutamate Receptors in Pain Transmission at the Spinal Cord Level

Nociceptive primary afferents release glutamate, activating postsynaptic glutamate receptors on spinal cord dorsal horn neurons. Glutamate receptors, both ionotropic and metabotropic, are also expressed on presynaptic terminals, where they regulate neurotransmitter release. During the last two decades, a wide number of studies have characterized the properties of presynaptic glutamatergic receptors, particularly those expressed on primary afferent fibers. This review describes the subunit composition, distribution and function of presynaptic glutamate ionotropic (AMPA, NMDA, kainate) and metabotropic receptors expressed in rodent spinal cord dorsal horn. The role of presynaptic receptors in modulating nociceptive information in experimental models of acute and chronic pain will be also discussed.     

Effects of beta-(p-chlorophenyl)-GABA (baclofen) on spinal synaptic activity.

In the isolated perfused spinal cord of the bullfrog, baclofen caused a reduction in the rate of spontaneous discharges in the ventral root at lower concentrations than GABA. The inhibitory effect of baclofen was fully present in a chloride-free medium, whereas that of GABA was markedly reduced. In contrast to GABA, which depolarized the dorsal root, baclofen produced a hyperpolarization in the dorsal root. Moreover, baclofen produced a hyperpolarization in the ventral roots. In the rat, the development of spinal reflex inhibition by baclofen was slow and the effect was long-lasting. Baclofen decreased dorsal root reflexes, suggesting a decreased excitability of presynaptic fibres. Baclofen produced a more pronounced and faster inhibitory effect on monosynaptic reflexes than on polysynaptic reflexes. In the monoamine depleted rat, the effect of baclofen on both types of reflexes was markedly reduced. It appears that catecholamines are involved in the effects produced by baclofen.     

Pharmacology and physiological function of gamma-aminobutyric acid B type receptor]

Baclofen, a beta-chlorophenyl derivative of gamma-aminobutyric acid (GABA), depresses neuronal excitability in various parts of the central nervous system. The site of action for this drug had once been considered to be distinct from GABA recognition sites. In addition to the classical GABA recognition site (GABAA site), a new class of GABA receptor (GABAB site) has been characterized. GABAB sites are mainly present on nerve terminals and, when activated, result in diminished transmitter release, probably through a reduction in Ca2+ influx. Baclofen was shown to be a selective agonist for this novel GABAB recognition. Baclofen also directly hyperpolarizes the membrane of mammalian brain neurons, in addition to its presynaptic action. This postsynaptic action of baclofen was shown to result from an increase in K+ conductance when studied in hippocampal pyramidal neurons through postsynaptic GABAB receptors. Thus, the inhibitory neurotransmitter GABA activates two receptor subtypes that can be distinguished by their physiological and pharmacological properties. GABAA receptors mediate rapid alterations in the distribution of Cl- across the membrane. GABAA receptors are linked directly to an ion channel, thus contributing to the prompt inhibition of cellular excitability. On the contrary, the GABAB receptor does not contain an integral ion channel and is thus responsible for slower responses through receptor-G-protein-effector complexes. G-protein may be directly coupled to K+ or Ca2+ channels. In addition, G-protein may modulate a variety of regulatory proteins or second messengers, thus contributing to the slower alteration of cellular excitability or to the modulation of neurotransmitter release.     

GABA, glutamate and substance P-like immunoreactivity release: effects of novel GABAB antagonists.

1. The effects of various GABA receptor ligands on the electrically-evoked release of endogenous GABA, glutamate and substance P-like immunoreactivity from the dorsal horn of rat isolated spinal cord were examined. 2. Exogenous GABA (10-300 microM) significantly decreased the evoked, but not basal, release of endogenous glutamate in a concentration-dependent manner. The GABAA agonist, isoguvacine (1-100 microM), failed to decrease the release of glutamate although it did reduce the release of GABA. Baclofen (0.1-1000 microM), the GABAB agonist, reduced the release of GABA and glutamate in a stereospecific and concentration-dependent manner. 3. The actions of five GABAB antagonists on these release systems were compared. CGP36742, CGP52432, CGP55845A and CGP57250A significantly increased the evoked release of GABA and glutamate. They also reversed the effects of (-)-baclofen in a concentration-dependent manner. On the other hand, while CGP56999A had no effect on glutamate release, it was an effective antagonist of the baclofen-induced inhibition of GABA and substance P release. 4. These results suggest that GABAB receptors on nerve terminals within the dorsal horn spinal cord may be heterogeneous. However, this is based solely on the data obtained with CGP56999A which affected only GABA and substance P, but not glutamate, release.     

5-HT(1B) receptors inhibit glutamate release from primary afferent terminals in rat medullary dorsal horn neurons

BACKGROUND AND PURPOSE Although 5-HT(1B) receptors are expressed in trigeminal sensory neurons, it is still not known whether these receptors can modulate nociceptive transmission from primary afferents onto medullary dorsal horn neurons. EXPERIMENTAL APPROACH Primary afferent-evoked EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices using a conventional whole-cell patch clamp technique under a voltage-clamp condition. KEY RESULTS CP93129, a selective 5-HT(1B) receptor agonist, reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, CP93129 reduced the frequency of spontaneous miniature EPSCs without affecting the current amplitude. The CP93129-induced inhibition of EPSCs was significantly occluded by GR55562, a 5-HT(1B/1D) receptor antagonist, but not LY310762, a 5-HT(1D) receptor antagonist. Sumatriptan, an anti-migraine drug, also decreased EPSC amplitude, and this effect was partially blocked by either GR55562 or LY310762. On the other hand, primary afferent-evoked EPSCs were mediated by the Ca(2+) influx passing through both presynaptic N-type and P/Q-type Ca(2+) channels. The CP93129-induced inhibition of EPSCs was significantly occluded by ω-conotoxin GVIA, an N-type Ca(2+) channel blocker. CONCLUSIONS AND IMPLICATIONS The present results suggest that the activation of presynaptic 5-HT(1B) receptors reduces glutamate release from primary afferent terminals onto medullary dorsal horn neurons, and that 5-HT(1B) receptors could be, at the very least, a potential target for the treatment of pain from orofacial tissues. LINKED ARTICLE This article is commented on by Connor, pp. 353-355 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01963.x.     

Molecular neuropharmacology of nociceptive transmission and opioid receptors

This review summarizes our studies using pharmacological, neurochemical and molecular biological methods on the nociception in the CNS and opioid receptors (OPRs). We designed an in vitro fluorometric on-line monitoring system including an immobilized glutamate dehydrogenase column, and for the first time actually demonstrated that capsaicin induced the release of glutamate from rat dorsal horn slices containing the terminal area of primary afferents, in concentration-dependent, extracellular Ca(2+)-dependent and tetrodotoxin-resistant manners. Further, such a release was shown to be inhibited through mu- and delta-opioid receptors and alpha 2-adrenoceptors. On the other hand, we found that intracerebroventricular injections of interleukin (IL)-1 beta in rats produced biphasic effects on the mechanical nociception in rats (hyperalgesia in lower concentrations but analgesia in higher ones) and that similar injections of cytokine-induced neutrophil chemoattractant-1 (CINC-1) facilitated mechanical nociception in rats. The above described facts suggest that glutamate and some sorts of cytokines (IL-1 beta and CINC-1) contribute to nociception at least from the primary afferents to the spinal dorsal horn neurons and in higher brain, respectively. We have cloned rat kappa- and mu-opioid receptors. Using cloned cDNA for OPRs, we demonstrated (1) the distribution of mRNAs for OPRs in the rat central nervous system, (2) coexistence of each type of mRNA for mu-, delta- and kappa-OPRs and pre-protachykinin A mRNA in the dorsal root ganglion neurons, (3) an increased expression of mu- and kappa-OPR mRNAs in the I-II layers of rat lumbar dorsal horn with an adjuvant arthritis in the hind limb, (4) the inhibitions of N- and Q-types of Ca2+ channels by mu- and kappa-OPR agonists and (5) cross-desensitization of the inhibition through a common intracellular phosphorylation-independent mechanism, (6) pharmacological characterization of "antagonist analgesics" as partial agonists at every type of OPRs, and (7) the key-structure(s) of OPRs for discriminative binding of DAMGO to mu-OPR.     

Activation of presynaptic 5-HT3 receptors facilitates glutamatergic synaptic inputs to area postrema neurons in rat brain slices

Whole-cell voltage-clamp recordings were performed to investigate the serotonergic modulation of neurotransmitter release onto rat area postrema neurons in vitro. The bath application of serotonin (5-HT; 50 microM) or phenylbiguanide (PBA; 50 microM), a potent 5-HT3 receptor agonist, increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) or miniature EPSCs (mEPSCs) in 35 of 83 neurons (42%). These increases occurred in all electrophysiological cell classes. No cells exhibited a decrease in EPSC frequency. The majority of responding cells showed no inward currents during the application of serotonergic agonists (n = 34/35). However, the amplitude of mEPSCs was increased in 11/11 cells with 5-HT or 3/11 cells with PBA. ICS-205,930, a potent 5-HT3 receptor antagonist, markedly suppressed the 5-HT-induced facilitation of sEPSCs (n = 5) or mEPSCs (n = 5). An increase in the frequency of mEPSCs after PBA exposure was found, even with media containing Cd2+ (50 microM) or zero Ca2+. mEPSCs and evoked EPSCs were completely blocked in media containing the non-NMDA ionotropic receptor antagonist, CNQX (10 microM), indicating that EPSCs were glutamate events. These results suggest that glutamate release is increased in the area postrema by presynaptic 5-HT3 receptor activation. Furthermore, we present evidence that 5-HT3 receptor activation may be able to directly release glutamate from terminals, bypassing a requirement for voltage-dependent calcium entry into terminals. Such a mechanism may contribute to the chemosensitive function of area postrema neurons.     

Metabotropic glutamate receptors potentiate ionotropic glutamate responses in the rat dorsal horn.

The effects of the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] were examined on responses mediated by the ionotropic glutamate receptor agonists N-methyl D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and kainic acid (KA), in neurons acutely isolated from the dorsal horn of the rat spinal cord. (1S,3R)-ACPD produced an increase in the intracellular Ca2+ concentration in 50% of acutely isolated dorsal horn neurons, which could be prevented by blockers of voltage-sensitive Ca2+ channels. (1S,3R)-ACPD markedly potentiated increases in the intracellular Ca2+ concentration induced by NMDA, AMPA, and KA but not by 10-50 mM KCl. This potentiation occurred in all cells, required the simultaneous presence of both agonists, and was rapidly reversible. In the spinal cord slice preparation, (1S,3R)-ACPD potentiated the inward currents evoked by pressure application of AMPA, NMDA, and KA, an effect that was also rapidly reversible. These short term effects of (1S,3R)-ACPD may play an important role in the regulation of ionotropic responses mediated by glutamate in the spinal cord.