Characteristics of Flunitrazepam Binding to Intact Primary Cultured Spinal Cord Neurons and Its Modulation by GABAergic Drugs

The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]-flunitrazepam binding was rapid and saturable. The benzodiazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 +/- 1.6 nM and Bmax of 822 +/- 194 fmol/mg protein. The association and dissociation of [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15-1788, and beta-carbolines like methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration-dependent manner by gamma-aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)-etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor-ionophore complex. Since these cells can also be studied in parallel for characterizing GABA-induced 36Cl-influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.     

Synaptogenesis in Co-Culture of Neurons of Dorsal Root Ganglia and Spinal Cord

In co-culture of spinal cord and dorsal root ganglion (DRG) neurons, we studied at different terms of culturing postsynaptic currents in DRG neurons evoked by direct electrical stimulation of single spinal neurons using a voltage-clamp technique in the whole-cell configuration. According to the reversal potential and sensitivity to bicuculline, these currents were classified as inhibitory postsynaptic currents (IPSC) carried by Cl^- ions through GABA_A receptors. During neuronal development in dissociated co-culture, the amplitude of evoked IPSC and their time to peak significantly increased. The time to peak of spontaneous IPSC (sIPSC) in DRG neurons remained unchanged, while the frequency of these currents increased with increasing culturing time. It is concluded that under culturing conditions spinal neurons establish inhibitory synaptic contacts with the somata of DRG neurons, and the number of such functional contacts increases in the course of culturing. Our findings show that in dissociated co-culture the process of formation of inhibitory synapses on the axon terminals of primary afferent neurons is akin to that realized in vivo, but with dissimilar topography of distribution of such synapses.     

Postsynaptic Currents in Dorsal Root Ganglion Neurons Co-cultured with Spinal Cord Neurons

In co-cultured dorsal root ganglion (DRG) neurons and spinal cord neurons from newborn rats, using a voltage-clamp technique in the whole-cell configuration enabled us to observe in DRG neurons the effects evoked by extracellular local electrical stimulation of cells corresponding to spinal cord neurons in their morphological characteristics. Such stimulation caused the appearance of postsynaptic currents (PSC) in DRG neurons in 9% of the cases. The mean delay of these currents (measured from the stimulus leading edge) was 4.7 ± 0.29 msec, the mean time to peak was 2.6 ± 0.77 msec, and the decay time constant = 14.5 ± 1.04 msec. The reversal potential of evoked PSC (ePSC) was close to the equilibrium potential for chloride ions estimated by the Nernst equation. Application of 20 M bicuculline induced practically complete and reversible ePSC block. The conclusion was drawn that these currents arise due to activation of the chloride channels operated by GABA receptors and, hence, represent an inhibitory PSC. Thus, one may deem it proved that spinal cord neurons can establish functional inhibitory synapses with DRG neurons.     

大鼠脊髓细胞膜胰岛素受体的结合特性

大鼠脊髓细胞膜胰岛素受体的结合特性朱尚权,徐明华,张新堂,叶莺（中国科学院上海生物化学研究所,２０００３１）姜新建,林淑琼（上海市第一人民医院康复科,２０００８５）关键词胰岛素受体,脊髓细胞膜免疫组织化学、放射免疫自显影和放射受体测定技术已证明脑各区...     

Enzyme Immunoassay for Tachykinin-Like Immunoreactivity in the Guinea Pig Spinal Cord

A solid-phase enzyme immunoassay for quantitation of tachykinin-like immunoreactivity (TK-LI) is presented. Because the antiserum K-12 recognizes various tachykinins, such as neurokinin A (100%), kassinin (103%), eledoisin (51%), neurokinin B (18%), physalaemin (0.7%), and substance P (0.7%), the immunoreactivity detected in this enzyme immunoassay has been termed TK-LI. The assay was performed on 96-well microtiter plates coated with a mouse monoclonal second antibody. After preincubation of soluble neurokinin A or samples and K-12 antiserum for 3 h at room temperature, acetylcholinesterase-labelled neurokinin A was allowed to react overnight at 4 degrees C. Samples were finally incubated with Ellman's reagent for 2 h and the absorbance was measured at 414 nm. The threshold for detection of TK-LI was 2 fmol/well. TK-LI release from guinea pig dorsal spinal cord slices was evoked by capsaicin or high K+ medium. The capsaicin-evoked TK-LI release was increased in the presence of thiorphan, but not in that of captopril.     

器官型培养的脊髓与体内脊髓的对比

旨在观察体外器官型培养的脊髓薄片是否与同龄大鼠体内生长的脊髓具有相似的形态和恒定的前角a运动神经元数目,建立能模拟体内生长环境的稳定的脊髓器官培养模型。利用出生后8天乳鼠的腰段脊髓组织切片建立脊髓器官型培养模型,用神经元的特异性免疫组化染色SMI-32对脊髓前角a运动神经元加以鉴定并与同龄大鼠体内生长的脊髓做比较。结果发现脊髓体外生长良好,形态完整,a运动神经元数目恒定,与同龄大鼠比较无显著差异,并可长期存活达2个月。脊髓的器官培养技术为研究脊髓生理、病理改变及神经保护提供了有效的方法。     

大鼠放射性脊髓损伤脊髓血流量变化规律

目的:放射性脊髓损伤(Radiation spinal cord injury,RSCI)是头颈部、胸部及上腹部肿瘤放射治疗和射线意外照射时的常见并发症,一般认为,白质坏死、脱髓鞘为其主要的病理学变化.然而,越来越多的证据表明血-脊髓屏障破裂和血管通透性增加等血管损伤远早于白质坏死和脱髓鞘改变.所以本文阐明大鼠放射性脊髓损伤病理生理过程中脊髓血流量变化规律.方法:将60只Sprague-Dawley (SD)大鼠随机分为12组,1组为对照,其余11组采用60Co放射治疗机行30 Gy大鼠颈髓C2-T2单次照射,剂量率为153 cGy/min,源皮距为80 cm,照射时长为1153 s,照射范围为2.0× 1.0 cm,对照组大鼠于麻醉后置于60Co放射治疗机下,佯照,照射前及照射后分别采用激光多普勒法测量脊髓血流量,11组大鼠于照射前以及照射后1、3、7、14、21、30、60、90、120、150、180天进行测量,以照射前测量值为基数,各时间点以基数的百分比表示该时间点脊髓血流量.结果:大鼠放射性脊髓损伤后,脊髓血流量在照射早期即有降低,照射后90天达到最低,随后脊髓血流量进入平台期.结论:阐明了大鼠放射性脊髓损伤后脊髓血流量的变化规律.大鼠放射性脊髓损伤可影响脊髓血流量,导致脊髓长期处于持续低灌流、缺血缺氧状态,最终导致脊髓不可逆性损伤.临床上放射性脊髓损伤的病人感到疲乏无力,出现神经系统的症状体征,通常死于脑疝.本文为临床上疲乏无力,出现神经系统的症状体征,死于脑疝放射性脊髓损伤的病人的早期防治提供病理生理基础.     

Decreased Uptake and Release of D-Aspartate in the Guinea Pig Spinal Cord After Dorsal Root Section

This study attempts to determine if L-glutamate and/or L-aspartate may be transmitters of dorsal sensory neurons. The uptake and the electrically evoked release of D-[3H]aspartate, a putative marker for L-glutamate and L-aspartate, were measured in the cervical enlargement (segments C4-T1) of the guinea pig spinal cord before and after cutting dorsal roots C5-T1 on the right side. The uptake and the release of gamma-aminobutyric acid (GABA) also were measured as indices of the integrity of GABAergic neurons in the spinal cord. The cervical enlargement was excised and divided into left and right halves, then into dorsal and ventral quadrants. Quadrants from unlesioned animals took up D-aspartate and GABA, achieving concentrations in the tissues which were 14-25 times that in the medium. Subsequently, electrical stimulation evoked a Ca2+-dependent release of D-aspartate and of GABA. The uptake and release of D-aspartate and GABA were similar in tissues taken from intact and sham-operated animals. However, dorsal rhizotomy, without damage to dorsal radicular or spinal blood vessels, depressed the uptake (by 22-29%) and the release (by 50%) of D-aspartate only in quadrants ipsilateral to the lesion. The uptake and the release of GABA were unchanged. In transverse sections of the cervical enlargement, stained to reveal degenerating fibers, by far the heaviest loss of axons occurred in the cuneate fasciculus and in the gray matter ipsilateral to the cut dorsal roots. These findings suggest that the synaptic endings of dorsal sensory neurons probably mediate the uptake and the release of D-aspartate and, therefore, may use L-glutamate or L-aspartate as a transmitter. When spinal blood vessels were damaged during dorsal rhizotomy, the deficits in D-aspartate uptake and release were larger than those in the absence of vascular damage and were accompanied by deficits in GABA uptake and release. These findings imply that vascular damage results in the loss of intraspinal neurons, some of which probably mediate the uptake and release of D-aspartate and, therefore, may use L-glutamate and/or L-aspartate as a transmitter.     

Identification and Distribution of Neuroleptic Binding Sites in the Rat Spinal Cord

Identification of neuroleptic receptor sites in the rat spinal cord could be achieved by the binding of [3H]haloperidol to membranes taken from the different horns. The use of pooled frozen microdiscs punched from these different spinal cord areas allowed the detection of saturable stereospecific binding, as defined in the presence of (+)- and (-)-butaclamol. Comparison of the binding constants with those obtained in the corpus striatum resulted in similar dissociation constants and Hill's slopes. Maximal binding capacity was quite different, being the greatest in the whole striatum (157 +/- 8 fmol/mg protein) followed by the dorsal horn (56 +/- 3 fmol/mg protein) and the lateral (34 +/- 5 fmol/mg protein) and ventral ones (31 +/- 2 fmol/mg protein). The displacement of the labelled ligand by different dopaminergic and nondopaminergic drugs at various concentrations gave similar results in the whole striatum and the spinal cord, giving further support for the existence of a dopaminergic innervation of the spinal cord and showing that dopaminergic receptor sites are distributed through the different spinal horns, with a maximal density in the dorsal horn--as for dopamine levels. No detectable stereospecific binding could be obtained from the surrounding spinal white matter, even at high tissue concentrations. Owing to poor sensitivity of the binding technique, no stereospecific neuroleptic binding could be demonstrated in the whole spinal cord, even at very high tissue concentration, whereas it could be detected in spinal cord tissue sampled from restricted areas of dense dopaminergic innervation.     

Spinal Cord Regeneration in Amphibians: A Historical Perspective

In some vertebrates, a grave injury to the central nervous system (CNS) results in functional restoration, rather than in permanent incapacitation. Understanding how these animals mount a regenerative response by activating resident CNS stem cell populations is of critical importance in regenerative biology. Amphibians are of a particular interest in the field because the regenerative ability is present throughout life in urodele species, but in anuran species it is lost during development. Studying amphibians, who transition from a regenerative to a nonregenerative state, could give insight into the loss of ability to recover from CNS damage in mammals. Here, we highlight the current knowledge of spinal cord regeneration across vertebrates and identify commonalities and differences in spinal cord regeneration between amphibians.     

Localization of Endomorphin-2-Like Immunoreactivity in the Rat Medulla and Spinal Cord

Martin-Schild, S., J. E. Zadina, A. A. Gerall, S. Vigh and A. J. Kastin. Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord. Peptides 18(10) 1641–1649, 1997.—Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) are endogenous ligands that have greater affinity and selectivity for the μ-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin–biotin–peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing μ receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.     

Novel Therapeutic Effects in Rat Spinal Cord Injuries: Recovery of the Definitive and Early Spinal Cord Injury by the Administration of Pentadecapeptide BPC 157 Therapy

Recently, marked therapeutic effects pertaining to the recovery of injured rat spinal cords (1 min compression injury of the sacrocaudal spinal cord (S2-Co1) resulting in tail paralysis) appeared after a single intraperitoneal administration of the stable gastric pentadecapeptide BPC 157 at 10 min post-injury. Besides the demonstrated rapid and sustained recovery (1 year), we showed the particular points of the immediate effect of the BPC 157 therapy that began rapidly after its administration, (i) soon after injury (10 min), or (ii) later (4 days), in the rats with a definitive spinal cord injury. Specifically, in counteracting spinal cord hematoma and swelling, (i) in rats that had undergone acute spinal cord injury, followed by intraperitoneal BPC 157 application at 10 min, we focused on the first 10–30 min post-injury period (assessment of gross, microscopic, and gene expression changes). Taking day 4 post-injury as the definitive injury, (ii) we focused on the immediate effects after the BPC 157 intragastric application over 20 min of the post-therapy period. Comparable long-time recovery was noted in treated rats which had definitive tail paralysis: (iii) the therapy was continuously given per orally in drinking water, beginning at day 4 after injury and lasting one month after injury. BPC 157 rats presented only discrete edema and minimal hemorrhage and increased Nos1, Nos2, and Nos3 values (30 min post-injury, (i)) or only mild hemorrhage, and only discrete vacuolation of tissue (day 4, (ii)). In the day 4–30 post-injury study (iii), BPC 157 rats rapidly presented tail function recovery, and no demyelination process (Luxol fast blue staining).     

The Adhesive Role of Acetylcholinesterase (AChE): Detection of AChE Binding Proteins in Developing Rat Spinal Cord

Acetylcholinesterase (AChE) is expressed by dorsal root ganglion (DRG) neurons during developmental periods when their central axons are growing into and through the spinal cord. Importantly, our previous studies have shown that AChE induces DRG axonal outgrowth by an adhesive mechanism and thus, have now employed a blot overlay technique to screen for potential AChE binding proteins in the developing spinal cord. Our results show that: (1) AChE binds to proteins with apparent molecular weights of 200, 110, 35, and 33k Da; (2) these proteins are developmentally expressed during periods of axonal outgrowth from DRG neurons; (3) all four proteins are synthesized by astrocytes; and (4) AChE binding to these proteins is highly dependent on ionic strength supporting an electrostatic mechanism of adhesion. Taken together, these data provide further documentation for the participation of AChE in adhesive interactions during morphogenesis of the central nervous system and suggest a role for astrocytes in regulating AChE-mediated axonal growth.Special issue dedicated to Lawrence. F. Eng.     

NMDA受体通道参与大鼠脊髓背角C纤维诱发电位LTP的表达

以往研究表明,激动NMDA受体是引起海马长时程增强(LTP)的必备条件,而LTP的表达主要与AMPA受体的磷酸化及其受体组装到突触后膜有关.但是,近年来有研究表明NMDA受体通道也参与了LTP的表达.为探讨NMDA受体通道是否参与了脊髓背角C纤维诱发电位LTP的表达,诱导LTP后,分别静脉或脊髓局部给予NMDA受体拮抗剂MK801或APV,观察其作用.发现静脉注射非竞争性NMDA受体MK801(0.1mg/kg)对脊髓LTP无影响,注射0.5mg/kg显著抑制LTP,但是当剂量增高到1.0mg/kg时,抑制作用并未进一步增大.脊髓局部给予MK801也能抑制脊髓背角LTP.为验证上述结果,使用了竞争性NMDA受体拮抗剂APⅤ.结果显示,脊髓局部给予50μmol/LAPⅤ对LTP无影响,100μmol/L对LTP有显著的抑制作用,当浓度升至200μmol/L时,抑制作用并未见进一步增强.因此认为,NMDA受体通道部分地参与了脊髓背角C纤维诱发电位LTP的表达.     

Effects of Peripheral Axotomy on Cholecystokinin Neurotransmission in the Rat Spinal Cord

Abstract : Because cholecystokinin (CCK) acts as a "functional" endogenous opioid antagonist, it has been proposed that changes in central CCKergic neurotransmission might account for the relative resistance of neuropathic pain to the analgesic action of morphine. This hypothesis was addressed by measuring CCK-related parameters 2 weeks after unilateral sciatic nerve section in rats. As expected, significant decreases (-25-38%) in the tissue concentrations and in vitro release of both substance P and calcitonin gene-related peptide were noted in the dorsal quadrant of the lumbar spinal cord on the lesioned side. In contrast, the tissue levels and in vitro release of CCK were unchanged in the same area in lesioned rats. Measurements in dorsal root ganglia at L4-L6 levels revealed no significant changes in proCCK mRNA after the lesion. However, sciatic nerve section was associated with a marked ipsilateral increase in both CCK-B receptor mRNA levels in these ganglia (+70%) and the autoradiographic labeling of CCK-B receptors by [³H]pBC 264 (+160%) in the superficial layers of the lumbar dorsal horn. Up-regulation of CCK-B receptors rather than CCK synthesis and release probably contributes to increased spinal CCKergic neurotransmission in neuropathic pain.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.     

Regional Distribution of Calcitonin Gene-Related Peptide-, Substance P-, Cholecystokinin-, Met5-Enkephalin-, and Dynorphin A (1–8)-Like Materials in the Spinal Cord and Dorsal Root Ganglia of Adult Rats: Effects of Dorsal Rhizotomy and Neonatal Capsaicin

Biochemical mapping of five different peptide-like materials--calcitonin gene-related peptide (CGRP), substance P (SP), Met5-enkephalin (ME), cholecystokinin (CCK), and dynorphin A (1-8) (DYN)--was conducted in the dorsal and ventral zones of the spinal cord at the cervical, thoracic, and lumbar levels in 3-month-old rats 10 days after unilateral dorsal rhizotomy at the cervical level (C4-T2) or after neonatal administration of capsaicin (50 mg/kg s.c.). In control rats, all peptide-like materials were more abundant in the dorsal than in the ventral zone all along the spinal cord. However, in both zones, absolute concentrations of CGRP, SP, ME, and CCK were significantly higher at the lumbar than at the cervical level. Rhizotomy-induced CGRP depletion (-85%) within the ipsilateral dorsal zone of the cervical cord was more pronounced than that due to neonatal capsaicin (-60%), a finding suggesting that this peptide is contained in both capsaicin-sensitive (mostly unmyelinated) and -insensitive (myelinated) primary afferent fibers. In contrast, similar depletions of SP (-50%) were observed after dorsal rhizotomy and neonatal capsaicin treatment, as expected from the presence of SP only in the capsaicin-sensitive small-diameter primary afferent fibers. Although the other three peptides remained unaffected all along the cord by either intervention, evidence for the existence of capsaicin-insensitive CCKergic primary afferent fibers could be inferred from the increased accumulation of CCK (together with SP and CGRP) in dorsal root ganglia ipsilateral to dorsal root sections.     

NADPH-Diaphorase-Positive Nerve Cells in Heterotopic Spinal Cord Transplants

The method of ectopic transplantation of embryonic CNS rudiments makes it possible to study the mechanisms underlying adaptation of the transplanted embryonic rudiments. The production of nitric oxide by cells is considered as one of such mechanisms. NADPH-diaphorase is an index of the presence of nitric oxide synthase in cells. It was shown that the nerve cells of rat embryonic spinal cord transplants preserved their capacity to express NADPH-diaphorase after transplantation in the sciatic nerve of an adult animal for six months. The dynamics of NADPH-diaphorase-positive neurons of rat embryonic spinal cord developing after transplantation and in situwere studied. In spinal cord neck region, small bipolar NADPH-diaphorase-positive neurons were visualized on day 17 of prenatal development. After transplantation of the embryonic (day 15) spinal cord in the nerve, NADPH-diaphorase-positive neurons were formed later than in situ: within seven days. The results of histochemical studies carried out within six months after the operation suggest a protective role of NADPH-diaphorase in the neurons of allotransplants developing under the conditions of altered microenvironment and insufficient innervation and also suggest that nitric oxide can cause the death of neurons in long surviving transplants.     

脊髓损伤的基因治疗

基因治疗脊髓损伤（SCI）既不存在胎儿神经组织移植的组织来源问题,且比外周神经组织移植引起的排异性低,是目前脊髓损伤治疗中最有前途的方法.基因治疗的转基因方式有两种：一是将目的基因直接导入体内靶细胞令其表达;二是将基因在体外导入适当的细胞内,并筛选出高效表达的移植细胞作为转基因中介移植到体内靶组织.不论采用何种方式,将基因导入细胞又可用多种手段实现：如微注射、脂质体等物理或化学手段;利用缺陷病毒作为载体感染细胞的生物学手段.因为用生物学手段转基因的细胞移植方法空间定位明确,所以目前最常采用它作为基因治疗效果的研究.虽然SCI基因治疗目前仍停留在实验探索阶段,一些问题尚待解决,但随着基因治疗技术方法的不断提高,它的临床应用前景可以预见.