鞘内注射促皮质素抑制甲醛痛敏大鼠脊髓NOS阳性神经元增多

目的:研究鞘内注射促皮质素(Cor)对甲醛痛敏大鼠脊髓背角一氧化氮合酶(NOS)阳性神经元增多的影响。方法:采用痛级均数(PIR)测定、NADPH-d组织化学法、Fos免疫组织化学法染色,观察鞘内注射(ith)Cor对甲醛痛敏大鼠脊髓背角NOS阳性神经元、Fos免疫反应神经元、NOS/Fos双标记神经元及痛敏的影响。结果:ith Cor(0.5-1.5U)均能显著抑制甲醛引起的大鼠脊髓背角NOS、Fos、NOS/Fos阳性神经元的增多和痛敏反应,其作用为ith NOS底物左旋精氨酸(Arg,5-15nmol)部分翻转。结论:Cor通过抑制大鼠脊髓背角NOS阳性神经元的增多抑制痛敏。     

鞘内注射P物质拮抗氯胺酮的抗伤害作用

目的观察脊髓P物质对氯胺酮抗伤害作用的影响。方法在小鼠福尔马林实验中,结合行为学和Fos蛋白表达,观察鞘内注射(it)不同剂量的P物质对氯胺酮抗伤害作用的影响。结果氯胺酮20、30mg·kg-1ip可剂量依赖性地减少小鼠舔足时间(P<0.05)。P物质0.25、0.5ngit可增加注射氯胺酮小鼠舔足时间(P<0.05)。小鼠注射福尔马林后,注射侧脊髓背角Fos免疫样(Foslikeimmunoreactive,FLI)阳性神经元数量明显增加(P<0.01),预先给于氯胺酮30mg.kg-1ip可以明显减少脊髓背角FLI阳性神经元数量(P<0.01),而P物质0.5ngit能明显削弱氯胺酮对脊髓背角Fos表达的抑制(P<0.01)。结论鞘内注射P物质能拮抗脊髓水平氯胺酮抗伤害作用。     

脊髓背角M胆碱能和GABAB受体在糖尿病神经痛形成中的作用机制研究进展

脊髓背角作为中枢神经系统痛觉信息整合加工的重要部位,是接受和调控伤害性信息由外周向中枢传递的关键部位。背角浅层含有大量的谷氨酸能、胆碱能、GABA能及甘氨酸能神经元,这些神经元的轴突末梢及胞体上同时表达丰富的M胆碱能受体和GABAB受体,激活这些受体可调控兴奋性/抑制性神经递质的释放过程。而在糖尿病神经痛的形成过程中,     

鞘内注射左旋布比卡因下调甲醛炎性痛大鼠远位触液神经元P物质的表达

目的观察鞘内注射左旋布比卡因对甲醛炎性痛大鼠P物质(substanceP,SP)在脊髓背角及远位触液神经元(thedistal cerebrospinal fluid contacting neuron,dCSF-CN)表达的影响。方法采用CB-HRP大鼠侧脑室注射示踪标记dCSF-CN;48h后先鞘内注射0.5%左旋布比卡因10μl(并以鞘内注射10μl人工脑脊液作对照),大鼠左后掌足跖部皮下注射2.5%福尔马林建立炎性痛模型,记录大鼠舔足时间,1h后测定机械缩足阈值(mechanical withdrawal threshold,MWT)评估机械痛敏;免疫组织化学光镜镜检及免疫电镜镜检P物质在脊髓背角(L4~5)及dCSF-CN的表达。结果鞘内注射左旋布比卡因减少大鼠福尔马林试验的舔足时间(P<0.01),MWT值无变化(与基础值对比,P>0.05),SP在脊髓背角及dCSF-CN的表达弱于对照组(P<0.01)。结论鞘内注射左旋布比卡因减低脊髓伤害性疼痛反应及下调SP在dCSF-CN的表达。     

降钙素基因相关肽与疼痛和免疫炎症反应

降钙素基因相关肽(calcitoningene relatedpeptide,CGRP)是一种含有37个氨基酸的神经肽,广泛分布于哺乳动物和人的中枢和外周神经系统感觉神经C-纤维末梢及组织器官中,生理作用广泛而多样。本文仅就CGRP与疼痛、炎症反应及免疫调节的研究进展作一综述。1在脊髓水平参与伤害性信息的传递和痛觉过敏形成在甲醛致痛实验中,发现投射到脊髓背角浅层的初级传入神经末梢中CGRP样免疫活性物质明显增多;佐剂性关节炎痛时,脊髓背根神经节(DRG)神经元内CGRP mRNA表达明显增强;镇痛药可使DRG内CGRP免疫活性物质含量和脊髓内释放CGRP明显减…     

大鼠脑室管膜下区包埋前免疫电镜双标技术的探索

目的 观察脑室管膜下区(SVZ)神经干细胞上N-甲基-D-天冬氨酸受体亚单位1(NR1)的超微分布.方法 成年大鼠灌注固定,冠状面振动切片,片厚150 μm.取含SVZ区域的脑片,行巢蛋白(nestin)和NR1双标免疫组织化学染色.其中,巢蛋白为辣根过氧化物酶标记,NR1为免疫金银法标记.取1.5mm×1.0mm大小的SVZ双标阳性组织,常规电镜制样、观察、拍照.结果 电镜下观察到标记NR1的纳米金-银标颗粒电子密度高,呈颗粒状,分布于细胞膜上,偶见于胞质中;巢蛋白免疫阳性产物呈絮状,分布于胞质中.结论 包埋前免疫电镜双重标记技术对组织的抗原性保存较好,免疫金颗粒对抗原定位较精确,有利于研究两种递质在同一个细胞或终末内的共存或分析递质与其相应受体之间的联系,特别是对于中枢神经系统的研究有独到之处.     

bFGF，GDNF，PDGF，IGF-I在大鼠脊髓中的定位表达

目的　碱性成纤维细胞生长因子 (bFGF)、胶质细胞源性神经营养因子 (GDNF)、血小板源性生长因子 (PDGF)和胰岛素样生长因子 -I (IGF -I)对神经细胞起特殊的营养作用 ,但它们在脊髓中的定位分布和生物学作用还不十分清楚。本研究旨在探索这一问题 ,方法　取正常成年SD大鼠脊髓分别用抗bFGF ,GDNF ,PDGF ,IGF -I抗体行免疫组化染色 ,观察上述 4种神经营养因子免疫阳性反应物在大鼠脊髓中的分布。结果　bFGF ,GDNF ,PDGF ,IGF -I的免疫阳性反应物主要见于脊髓灰质内各板层神经元 ,腹角和背角深部明显 ,背角浅层较少见。但II板层还可见bFGF阳性细胞和GDNF阳性膨体 ,中央管可见PDGF、bFGF阳性的室管膜细胞。结论　bFGF ,GDNF ,PDGF ,IGF -I在维持脊髓神经元的生理功能中可能起重要作用     

利多卡因对致痛大鼠L4～5脊髓P物质分布影响的实验研究

目的研究鞘内(IT)利多卡因对福尔马林致痛大鼠脊髓P物质(SP)含量的影响,探讨其镇痛的可能机制.方法取雄性SD大鼠15只,分为三组假手术组、对照组和治疗组.后足皮下注射3%福尔马林50ml致痛造膜.对照组造膜前IT0.9%氯化钠20μl;治疗组造模前IT2%利多卡因20μl.采用免疫组织化学SABC法结合图像分析技术.结果大鼠脊髓背角Ⅰ层和Ⅱ层有密集SP分布,治疗组与对照组相比含量明显减少,差异非常显著(P＜0.01).结论在大鼠外周炎性疼痛模型中,超前鞘内给利多卡因的镇痛作用与其抑制SP在脊髓背角的释放有关.     

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。     

生长抑素和电针抑制关节炎大鼠脊髓c—fos的表达

目的：观察鞘内注射生长抑素及电针“夹脊”穴对病理性疼痛情况下大鼠脊髓ｃ－ｆｏｓ原癌基因蛋白表达的影响。方法：用急性佐剂性关节炎大鼠作为病理性疼痛的实验模型，免疫组织化学法检测ｃ－ｆｏｓ样免疫反应细胞。结果：佐剂性关节炎可诱发大鼠脊髓内多层神经元ｃ－ｆｏｓ蛋白表达，接种后２４ｈ，以背角Ｉ－Ⅱ，Ⅴ－Ⅵ层内最多，而生长抑素和电针均可抑制其表达，表现为ｃ－ｆｏｓ蛋白形成减少。结论：生长抑素和电针缓解病理性     

Immunocytochemical technique--Application for identifying GABA neurons (author's transl)]

Immunocytochemical techniques locating neurotransmitter-synthsizing enzymes are currently being employed to determine the nature of transmitters associated with individual neurons. The use of peroxidase-anti-peroxidase Fab (PAP Fab) complex modified from Sternberger's PAP method, among several other immunocytochemical methods is recommended for the visualization of antigens in cerebral tissues. The enzyme fixed in nervous tissues is reacted with anti-enzyme produced in rabbits followed by incubation with goat-anti-rabbit serum. Subsequent application of PAP Fab complex prepared separately results in a formation of a complex composed of enzyme: anti-enzyme: goat-anti-rabbits: PAP-Fab. The enzymes can be visualized under light and electron microscope by the deposition produced by the action of peroxidase on 3,3'-diaminobenzidine. Thus, the antibody to glutamate decarboxylase (GAD), the enzyme that synthesizes gamma-aminobutyric acid (GABA) was employed to identify GABAergic neurons in central nervous system of rodents. Specific staining for GAD was highly localized in close association with synaptic vesicles in certain axon terminals including basket, Golgi and the Purkinje cell terminals in the cerebellum. The distribution of GAD observed in immunocytochemical preparations was consistent with indirect biochemical, physiological and morphological data dealing with the synaptic role of GABA neurons in the cerebellum. The correlation of the immunocytochemical distribution of GABA neurons in the spinal cord, substantia nigra, olfactory bulb, retina and Ammon's horn with physiological and biochemical results can also been obtained. The method has been successfully employed to visualize dopamine-beta-hydroxylase (DBH) and substance P. DBH, as an indicative enzyme for noradrenergic (NA) neurons, was highly localized in the neuronal soma of the locus coeruleus and in synaptic varicosities in the stria terminalis associated with synaptic vesicles. Association of substance P in probable primary afferent terminals with large vesicles also supports the synaptic function of the compound in the spinal cord.     

GABA in the control of sympathetic preganglionic neurons

1. Amino acid neurotransmitters are critical for controlling the activity of most central neurons, including sympathetic preganglionic neurons (SPN), the spinal cord neurons involved in controlling blood pressure and other autonomic functions. 2. In studies reviewed here, SPN were identified either by retrograde tracing from a peripheral target (superior cervical ganglion or adrenal medulla) or by detection of immunoreactivity for choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme that is a marker for all SPN, in intact or completely transected rat spinal cord. 3. Postembedding immunogold labelling on ultrathin sections was then used to detect GABA and sometimes glutamate in nerve terminals on SPN or near them in the neuropil of the lateral horn. 4. In some cases, the terminals were prelabelled to show an anterograde tracer or immunoreactivity for ChAT or neuropeptide Y. 5. This anatomical work has provided information that is helpful in understanding how SPN are influenced by their GABAergic innervation. 6. Immunogold studies showed that the proportion of input provided by GABAergic terminals varies between different groups of SPN. For some groups, this input may be preferentially targeted to cell bodies. 7. Anterograde tracing demonstrated that supraspinal as well as intraspinal GABAergic neurons innervate SPN and investigations on completely transected cord suggested that supraspinal neurons may provide a surprisingly large proportion of the GABAergic terminals that contact SPN. 8. The double-labelling studies in which other amino acids, ChAT or neuropeptide Y were localized along with GABA indicate that GABAergic terminals contain other neurochemicals that could modulate the actions of GABA, depending on the complement of receptors that are present pre- and post-synaptically. 9. Taken together, these data indicate that GABAergic transmission to SPN may be much more complicated than suggested by the currently available electrophysiological studies.     

Lack of effect of microinjection of noradrenaline or medetomidine on stimulus-evoked release of substance P in the spinal cord of the cat: a study with antibody microprobes.

1. Experiments were performed on barbiturate anaesthetized, spinalized cats to investigate the effect of microinjected noradrenaline or medetomidine on the release of immunoreactive substance P in the dorsal spinal cord following peripheral nerve stimulation. The presence of immunoreactive substance P was assessed with microprobes bearing C-terminus-directed antibodies to substance P. 2. Noradrenaline or medetomidine were microinjected into the grey matter of the spinal cord, near microprobe insertion sites, at depths of 2.5, 2.0, 1.5 and 1.0 mm below the spinal cord surface with volumes of approximately 0.125 microliters and a concentration of 10(-3) M. 3. In the untreated spinal cord, electrical stimulation of the ipsilateral tibial nerve (suprathreshold for C-fibres) elicited release of immunoreactive substance P which was centred in and around lamina II. Neither noradrenaline nor medetomidine administration in the manner described produced significant alterations in this pattern of nerve stimulus-evoked release. 4. In agreement with recent ultrastructural studies these results do not support a control of substance P release by catecholamines released from sites near to the central terminals of small diameter primary afferent fibres.     

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。     

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.     

Differential effects of wortmannin on the release of substance P and amino acids from the isolated spinal cord of the neonatal rat

1. Effects of wortmannin, an inhibitor of myosin light chain kinase, on the release of substance P and amino acids, GABA and glutamate, were investigated in the isolated spinal cord preparation of the neonatal rat.
2. Wortmannin at 0.5–10 μM depressed the release of substance P evoked by high-K⁺ (90 mM) medium from the spinal cord (IC₅₀=1.1 μM). Wortmannin also depressed the high-K⁺ (70 mM)-evoked release of substance P from cultured dorsal root ganglion neurons of neonatal rats. In contrast, the high-K⁺ (90 mM)-evoked release of GABA and glutamate from the spinal cord was not affected by wortmannin (0.1–10 μM).
3. Upon stimulation of a dorsal root, a monosynaptic reflex and a subsequent slow ventral root depolarization were evoked in the ipsilateral ventral root of the same segment in the isolated spinal cord preparation. The magnitude of the slow ventral root depolarization was depressed gradually to about 70% of the control during the course of 30 min under wortmannin (1 μM). In contrast, the monosynaptic reflex was unaffected by wortmannin.
4. Immunofluorescent staining revealed that immunoreactivities of substance P and myosin II were colocalized at presynaptic terminals in the dorsal horn of the neonatal rat spinal cord.
5. The present results suggest that myosin phosphorylation by myosin light chain kinase may play a crucial role in the release of substance P, but not in the release of GABA and glutamate in the neonatal rat spinal cord. This may reflect a difference in the exocytic mechanisms of substance P-containing large dense core vesicles and amino acid-containing small clear vesicles.

     

GABA and central neuropathic pain following spinal cord injury

Spinal cord injury induces maladaptive synaptic transmission in the somatosensory system that results in chronic central neuropathic pain. Recent literature suggests that glial-neuronal interactions are important modulators in synaptic transmission following spinal cord injury. Neuronal hyperexcitability is one of the predominant phenomenon caused by maladaptive synaptic transmission via altered glial-neuronal interactions after spinal cord injury. In the somatosensory system, spinal inhibitory neurons counter balance the enhanced synaptic transmission from peripheral input. For a decade, the literature suggests that hypofunction of GABAergic inhibitory tone is an important factor in the enhanced synaptic transmission that often results in neuronal hyperexcitability in dorsal horn neurons following spinal cord injury. Neurons and glial cells synergistically control intracellular chloride ion gradients via modulation of chloride transporters, extracellular glutamate and GABA concentrations via uptake mechanisms. Thus, the intracellular “GABA-glutamate-glutamine cycle” is maintained for normal physiological homeostasis. However, hyperexcitable neurons and glial activation after spinal cord injury disrupts the balance of chloride ions, glutamate and GABA distribution in the spinal dorsal horn and results in chronic neuropathic pain. In this review, we address spinal cord injury induced mechanisms in hypofunction of GABAergic tone that results in chronic central neuropathic pain.This article is part of a Special Issue entitled ‘Synaptic Plasticity & Interneurons’.     

Functional significance of co-localization of GABA and Glu in nerve terminals: a hypothesis

Salient features of the co-transmission by GABA and Glu in neural signaling are summarized. Experimental data have been accumulating which demonstrate; i) GABA-immunoreactivity in and GABA-release from constitutively Gluergic hippocampal mossy fibre terminals, ii) plasticity of the GABAergic phenotype of constitutively Gluergic granule cells of the Dentate Gyrus, iii) expression of GABA(A) receptor gamma(3) subunit in the mossy fibre termination zone in the CA3 subfield, iv) co-labeling of terminals for GABA and Glu in the retina, brain stem and spinal cord, and v) functional compatibility of vesicular Glu (VGLUT3) and GABA (VIAAT) transporters. It is not clear, however, whether or not Glu and GABA are released from the same terminals, and packaged in the same vesicles. Using multiple transmitters neurons may serve to reduce the metabolic cost and errors of signaling.     

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。     

Opposing functions of spinal M2, M3, and M4 receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.