氯胺酮在大鼠脊髓背角胶状质内对突触前神经递质释放的影响

为探讨临床有效浓度的氯胺酮(ketamine,KTM)在脊髓背角胶状质(substansia gelatinosa,SG)内对突触前神经递质释放的影响及其作用机制,本研究应用红外可视神经组织薄片全细胞膜片钳记录方法,在电压钳模式下,观察了KTM对自发性抑制性和兴奋性突触后电流(spontaneous inhibitory and excitatory postsynaptic currents,sIPSCs and sEPSCs)的频率和幅值的影响。结果显示:(1)钳制电压在0mV时,在人工脑脊液(artificial cerebrospinal fluid,ACSF)中加入10-5mol/LAP-V和10-6mol/LCNQX,可记录到sIPSCs。将此时记录到的频率和幅值都作为前对照组的基础值(100%)。给予10-4mol/LKTM后,与前对照组相比,sIPSCs频率为127.93%±25.17%(P<0.05),幅值为104.78%±11.35%(P>0.05,n=7);(2)钳制电压为-70mV时,在ACSF中加入3×10-7mol/L士的宁和10-6mol/L荷包牡丹碱后,可观察到sEPSCs。加入10-4mol/LKTM后,与前对照组相比,sEPSCs的频率和幅值分别为97.89%±4.06%和101.63%±7.66%(P>0.05,n=8)。以上结果提示:(1)KTM增加了sIPSCs的频率,而对幅值没有明显影响,即KTM引起突触前抑制性神经递质的释放增加,而对突触后神经元的作用不明显;(2)KTM对sEPSCs的频率和幅值均未见明显影响,说明KTM在SG内对兴奋性神经递质的释放无显著影响。由此我们推测KTM在脊髓SG内主要通过增强抑制性信息传递发挥作用,KTM增强SG内突触前抑制性神经递质释放可能与其在脊髓背角发挥麻醉和镇痛作用有关。     

内源性大麻素类似物NAGly对大鼠脊髓胶状质神经元的突触传递的抑制作用

目的:探讨内源性大麻素类似物N-花生四烯酰甘氨酸(N-arachidonylglycine,NAGly)对脊髓后角II层胶状质(SG)神经元突触兴奋性的影响。方法:选用生后4~6周雄性SD大鼠,深麻醉后蔗糖人工脑脊液快速心脏灌注处死,取脊髓腰膨大段,制备保留脊髓腰膨大处一侧后根的脊髓矢状切片。利用膜片钳技术,对脊髓后角II层SG神经元进行全细胞记录,通过分析后根刺激诱发的兴奋性突触后电流(eEPSC)的变化情况,观察NAGly(20μmol/L)对SG神经元突触传递兴奋性的影响,以及对自发性兴奋性突触后电流(sEPSC)的发放频率及幅度的影响。结果:通过诱发刺激的强度、潜伏期以及纤维传导速度我们将记录到的SG神经元分为Aδ纤维/C纤维投射神经元,NAGly对Aδ纤维和C纤维介导的eEPSC的幅度都有明显的抑制作用(P0.001),并且这种作用可以被洗脱。NAGly对SG神经元的sEPSC的频率有明显的抑制,但不明显改变其幅度,提示其作用部位在突触前。结论:内源性大麻素类似物NAGly可以抑制脊髓后角浅层Aδ纤维及C纤维介导的突触传递,并通过突触前机制抑制SG神经元的兴奋性。提示内源性大麻素类似物NAGly可通过抑制伤害性C和A纤维介导的突触传递发挥镇痛作用。     

大鼠桡神经感觉纤维在脊髓胶状质的纵横定位研究

目的　研究大鼠桡神经感觉纤维在脊髓胶状质的定位投射。方法　按照跨神经节溃变原理 ,采用酸性磷酸酶(ACP)法在 10只切断桡神经的昆明大鼠观察桡神经感觉纤维在颈髓 5～ 8和胸髓 1上、中、下段胶状质内ACP的变化。结果　大鼠桡神经感觉纤维纵向投射 ,主要至脊髓C6上段至T1中段胶状质。其横向投射 ,桡神经主要位于整个胶状质的中 1/ 3区域。结论　桡神经投射区居外 ,尺神经投射区居内     

缺血缺氧对神经细胞突触后电流的影响及其作用机制

神经细胞缺血缺氧后突触后电流的频率或幅度出现变化 ,提示氨基酸类神经递质释放的变化。急性缺血缺氧时 ,自发性兴奋性突触后电流和抑制性突触后电流均受到抑制 ,诱发性兴奋性突触后电流幅度增高。长期慢性缺氧情况下 ,NMDA受体介导的兴奋性突触后电流受到抑制。缺氧预适应后 ,γ 氨基丁酸释放增加 ,谷氨酸释放减少。多次重复急性缺氧抑制诱发性兴奋性突触后电流的幅度 ,说明长期慢性缺氧及缺氧预适应启动神经细胞的保护机制。腺苷通过调节氨基酸递质的释放 ,对神经细胞具有明显的保护作用 ,但对于缺血缺氧所造成的损伤却未见修复作用。     

琥珀酸在海马CA1区对突触前GABA释放的影响

为了观察琥珀酸在大鼠海马CA1区对突触前GABA释放的影响,我们采用红外可视全细胞膜片钳技术记录了琥珀酸对γ-氨基丁酸(GABA)能自发性微小抑制性突触后电流(miniature inhibitory postsynaptic currents,mIPSCs)的作用。结果显示不同浓度的琥珀酸(10-6mol/L、10-5mol/L、10-4mol/L和10-3mol/L)在海马CA1区均能以浓度依赖的方式增强GABA能mIPSCs的频率,而对其电流幅度没有影响。10-4mol/L琥珀酸组GABA能mIPSCs的频率为2.25±0.99Hz,与正常对照组相比有显著性差异(n=8,P<0.01),而其电流幅度为31.63±6.16pA,与正常对照组相比没有差异(n=8,P>0.05)。以上实验结果表明琥珀酸能通过增强突触前GABA的自发性释放,对海马CA1区神经元产生超极化作用,此作用可能是琥珀酸抑制癫痫形成的主要方式之一。     

大鼠脊髓背角向丘脑和外侧臂旁核的神经降压素能投射

本研究应用荧光金(FG)逆行追踪结合神经降压素(NT)免疫荧光组织化学染色的双标记技术,观察了大鼠脊髓背角向丘脑(TH)和外侧臂旁核(LPb)的NT能投射。将FG注入一侧TH或LPb后,FG逆标神经元主要见于脊髓背角的I层;NT阳性神经元主要分布于脊髓背角的I层、II层外侧部及II层内侧部与III层交界处;脊髓背角I层内可观察到FG逆标记并呈NT阳性的双标记神经元。上述结果表明脊髓背角I层的NT阳性神经元向TH和LPb投射,提示脊髓背角I层内的NT阳性神经元可能向TH和LPb传递伤害性信息。     

大鼠后根第一级传入纤维在脊髓胶状质内的溃变和突触联系

用切断后根方法在透射电镜下观察了大鼠后根第一级传入纤维在脊髓胶状质内的溃变和突触联系。切断脊神经后根24小时后,胶状质内出现明显的溃变终末。量最多又持续时间最长的溃变是电子致密型溃变,此外,也观察到了少数的电子透明型溃变和神经微丝型溃变;溃变终末大部分位于胶状质突触小球的中央。据统计,电子致密型溃变作为突触前成分与胶状质内的树突或树突棘形成轴—树突触者占98.8％,作为突触后成分与周围轴突形成轴—轴突触者占1.2％。     

大鼠腓浅神经传入纤维在脊髓胶状质的定位投射--FRAP法

为了解大鼠腓浅神经传入纤维在脊髓胶状质的投射定位投射按照跨神经节溃变原理，用抗氟化物酶性酸酶法和显微测量进行定量研究。结果显示：大鼠腓浅神经向ＳＧ的纵向投射主要为为Ｌ２－６，Ｌ２－６各节段ＳＧ水平切面“眉毛状反应带”弧形长均值分别为０．７９５、０．８４９、０．９１３、０．９２１、０．８５２ｍｍ；而腓浅神经向Ｌ２－６各节段ＳＧ水平切面投射均值分别为０．４４７－０．５３３、０．３８４－０．５６１、０．     

大鼠中缝大核向脊髓投射神经元与神经降压素能终末的突触联系

神经降压素(NT)是中枢下行抑制系统的重要神经活性物质。本研究运用免疫组织化学与逆行追踪法相结合的双标技术,在电镜下观察NT能终末与中缝大核(NRM)向脊髓投射神经元的突触联系。在光镜下可见NT阳性纤维和终末散在分布于NRM,但未见NT阳性神经元;将HRP注入腰髓背角后,在NRM内可见比较密集的HRP逆标神经元。在电镜下可见NT阳性终末与HRP逆标神经元的胞体和树突形成以非对称性为主的轴-体突触和轴-树突触。上述结果说明NT可能调控NRM向脊髓背角投射神经元的活动,借此对伤害性信息向中枢的传递发挥抑制效应。     

Influences of contralateral nerve and skin stimulation on neurones in the substantia gelatinosa of the rat spinal cord

Stimulation of high threshold A delta and C fibre peripheral afferents inhibits dorsal horn cells on the other side of the spinal cord. The substantia gelatinosa (SG) is an area full of interneurones known to have commissural connections across the spinal cord. The role of SG in this contralateral inhibitory pathway is investigated here. Forty-three SG cells were recorded in the lumbar dorsal horn of decerebrate spinal rats. Their ipsilateral excitatory receptive fields and responses to sciatic nerve stimulation were recorded. Repetitive electrical stimulation was then applied to the contralateral sciatic nerve. Eight (19%) units were excited by such stimulation. A brief tetanus was followed by an increase of ongoing activity lasting 30 s to 10 min. These cells did not, however, have excitatory contralateral fields. A small separate group of 4 cells (9%) were mildly inhibited by heating or pinching the contralateral limb. The significance of contralateral excitation of some SG cells is discussed in the light of the predominantly inhibitory contralateral effect on dorsal horn cells in laminae 4 and 5. It is suggested that some SG cells may be inhibitory interneurones in their effect on deeper cells.     

Responses to 5-HT in morphologically identified neurons in the rat substantia gelatinosa in vitro

Bath application of 5-HT (1-1000 μM) induced a tetrodotoxin (TTX)-resistant outward current at the holding membrane potential (V_H) of −50 mV in 104/162 (64.2%) of substantia gelatinosa (SG) neurons from the rat spinal cord in vitro. The 5-HT-induced outward current was suppressed by an external solution containing Ba²⁺, or a pipette solution containing Cs₂SO₄ and tetraethylammonium. It was reversed near the equilibrium potential of the K⁺ channel. The response to 5-HT was abolished 30 min after patch formation with a pipette solution containing guanosine-5-O-(2-thiodiphosphate)-S. The 5-HT-induced outward current was mimicked by a 5-HT_1A agonist, (±)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide, and suppressed by a 5-HT_1A antagonist, WAY100635, suggesting the 5HT_1A receptor-mediated activation of K⁺ channels in the outward current. In 11/162 (6.8%) SG neurons, 5-HT produced an inward current, which was mimicked by a 5-HT₃ agonist, 1-(m-chlorophenyl)-biguanide (mCPBG). The 5-HT-induced outward currents were observed in vertical cells (21/34) and small islet cells (11/34), while inward currents were induced in islet cells (1/5) and small islet (4/5) cells, but not in vertical cells. It is known that most vertical cells and islet cells in the SG are excitatory (glutamatergic) and inhibitory interneurons, respectively, while small islet cells consist of both excitatory and inhibitory neurons. Bath application of 5-HT or mCPBG increased the amplitude and the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs), but no neurons showed a decrease in sIPSC. Furthermore, frequency, but not amplitude, of miniature IPSCs increased with perfusion with 5-HT in the presence of TTX. These findings, taken together, suggest that 5-HT induces outward currents through 5-HT_1A receptors in excitatory SG neurons. These findings also suggest that the inward currents are post- and presynaptically evoked through 5-HT₃ receptors, probably in inhibitory neurons.     

Neurons with asymmetrical dendritic arbors in the substantia gelatinosa of the rat spinal cord

Summary Two types of neurons were observed in the substantia gelatinosa (SG) of the rat spinal cord which exhibit wide variations in dendritic symmetry. As demonstrated with the Golgi technique, islet cells with short dendritic arbors and type III stalk cells display dendritic patterns which vary from a bipolar type arrangement with two dendritic arbors of nearly equal dimensions to a unipolar arrangement with a dendritic arbor which extends in only one direction. Examination of the morphology and dendritic development of these neurons shows that they are unique compared with other SG neurons in that they have short, longitudinal dendritic arbors which undergo maturation relatively late in the postnatal period. As is discussed, variations in dendritic symmetry are probably dependent on the location of the terminal fields of primary and/or other types of afferents which are formed earlier in development.     

Activation of presynaptic group I metabotropic glutamate receptors enhances glutamate release in the rat spinal cord substantia gelatinosa

The activation of group I metabotropic glutamate receptors (mGluRs) produces a long-term potentiation of sensory transmission in the substantia gelatinosa (SG) region of the spinal cord (Prog. Brain Res. 129 (2000) 115). The mechanism(s) responsible for the induction of this potentiation is not known. Using rat spinal cord slice preparation and patch-clamp recordings, here we show, that the activation of the group I mGluRs by (S)-3,5-dihydroxyphenylglycine (DHPG, 1 microM), the mGluR1/5 agonist, increased the frequency of both activity-dependent spontaneous EPSCs, and activity-independent miniature EPSCs (mEPSCs). However, DHPG did not affect amplitude of mEPSCs. The effects of DHPG were not seen in the presence of the preferential mGluR1 antagonist CPCCOEt (10 microM). On the other hand, 2-methyl-6-(phenylethynyl)-pyridine (10 microM), a selective mGluR5 antagonist, blocked the DHPG facilitation present during the wash-out of the drug. This novel facilitating effect of the group I mGluR activation on glutamate release is the first report of a direct facilitatory action of both mGluR1 and mGluR5 subtypes on sensory transmission in the spinal cord SG region. These results indicate the potential contribution of synaptic activation of these facilitatory autoreceptors in plasticity of primary afferent neurotransmission.     

Effect of resiniferatoxin on glutamatergic spontaneous excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord

The transient receptor potential (TRP) vanilloid type 1 (TRPV1) agonist, capsaicin, enhances glutamatergic spontaneous excitatory synaptic transmission in CNS neurons. Resiniferatoxin (RTX) has a much higher affinity for TRPV1 than capsaicin, but its ability to modulate excitatory transmission is unclear. We examined the effect of RTX on excitatory transmission using the whole-cell patch-clamp technique in substantia gelatinosa (SG) neurons of adult rat spinal cord slices. Bath-applied RTX dose-dependently increased the frequency, but not the amplitude, of spontaneous excitatory postsynaptic current (sEPSC), independent of its application time. In about a half of the neurons tested, this effect was accompanied by an inward current at −70 mV that was sensitive to glutamate-receptor antagonists. Repeated application of RTX did not affect excitatory transmission. RTX was more potent than capsaicin but showed similar efficacy. RTX activity could be blocked by capsazepine or SB-366791, a TRPV1 antagonist, but not tetrodotoxin, a Na⁺-channel blocker, and could be inhibited by pretreatment with capsaicin but not the TRPA1 agonist, allyl isothiocyanate. RTX enhances the spontaneous release of l-glutamate from nerve terminals with similar efficacy as capsaicin and produces a membrane depolarization by activating TRPV1 in the SG, with fast desensitization and slow recovery from desensitization. These results indicate a mechanism by which RTX can modulate excitatory transmission in SG neurons to regulate nociceptive transmission.     

Cannabinoid-induced presynaptic inhibition of glutamatergic EPSCs in substantia gelatinosa neurons of the rat spinal cord.

The effect of cannabinoids on excitatory transmission in the substantia gelatinosa was investigated using intracellular recording from visually identified neurons in a transverse slice preparation of the juvenile rat spinal cord. In the presence of strychnine and bicuculline, perfusion of the cannabinoid receptor agonist WIN55,212-2 reduced the frequency and the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Furthermore, the frequency of miniature EPSCs (mEPSCs) was also decreased by WIN55,212-2, whereas their amplitude was not affected. Similar effects were reproduced using the endogenous cannabinoid ligand anandamide. The effects of both agonists were blocked by the selective CB(1) receptor antagonist SR141716A. Electrical stimulation of high-threshold fibers in the dorsal root evoked a monosynaptic EPSC in lamina II neurons. In the presence of WIN55,212-2, the amplitude of the evoked EPSC (eEPSCs) was reduced, and the paired-pulse ratio was increased. The reduction of the eEPSC following CB(1) receptor activation was unlikely to have a postsynaptic origin because the response to AMPA, in the presence of 1 microM TTX, was unchanged. To investigate the specificity of this synaptic inhibition, we selectively activated the nociceptive C fibers with capsaicin, which induced a strong increase in the frequency of EPSCs. In the presence of WIN55,212-2, the response to capsaicin was diminished. In conclusion, these results strongly suggest a presynaptic location for CB(1) receptors whose activation results in inhibition of glutamate release in the spinal dorsal horn. The strong inhibitory effect of cannabinoids on C fibers may thereby contribute to the modulation of the spinal excitatory transmission, thus producing analgesia at the spinal level.     

Transient receptor potential vanilloid type 1 receptor regulates glutamatergic synaptic inputs to the spinothalamic tract neurons of the spinal cord deep dorsal horn

The spinothalamic tract (STT) neurons in the spinal dorsal horn play an important role in transmission and processing of nociceptive sensory information. Although transient receptor potential vanilloid type 1 (TRPV1) receptors are present in the spinal cord dorsal horn, their physiological function is not fully elucidated. In this study, we examined the role of TRPV1 in modulating neuronal activity of the STT neurons through excitatory and inhibitory synaptic inputs. Whole-cell patch-clamp recordings were performed on STT neurons labeled by a retrograde fluorescent tracer injected into the ventral posterior lateral (VPL) nucleus of the thalamus. Capsaicin (1 μM) increased the frequency of miniature excitatory postsynaptic currents (mEPSC) without changing the amplitude or decay time constant of mEPSC. In contrast, capsaicin had no distinct effect on GABAergic miniature inhibitory postsynaptic currents (mIPSC). Capsazepine (10 μM), a TRPV1 receptor antagonist, abolished the effect of capsaicin on mEPSCs. Capsazepine itself did not affect the baseline amplitude and frequency of mEPSC. The effect of capsaicin on mEPSC was also abolished by removal of external Ca²⁺, but not by treatment with Cd²⁺. Furthermore, capsaicin increased the firing activity of the STT neurons and this increase in neuronal activity by capsaicin was abolished in the presence of non–N-methyl-d-aspartic acid (NMDA) and NMDA receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and (R)-amino-5-phosphonovaleric acid (APV). These data suggest that activation of TRPV1 potentiates the glutamate release from excitatory terminals of primary afferent fibers and subsequently increases the neural activity of STT neurons of the rat spinal cord deep dorsal horn.     

Activity-dependent release of adenosine inhibits the glutamatergic synaptic transmission and plasticity in the hypothalamic hypocretin/orexin neurons

The hypocretin (orexin) neurons in the lateral hypothalamus play a crucial role in the promotion of arousal. Adenosine, an endogenous sleep-promoting factor, modulates both neuronal excitatory and synaptic transmission in the CNS. In this study, the involvement of endogenous adenosine in the regulation of excitatory glutamatergic synaptic transmission to hypocretin neurons was investigated in the hypothalamic slices from transgenic mice by using different frequencies of stimulation. A train of low-frequency stimulation (0.033, 1 Hz) had no effect on the amplitude of evoked excitatory postsynaptic currents (evEPSCs) in hypocretin neurons. Blockade of adenosine A1 receptors with selective A1 receptor antagonist 8-cyclopentyltheophylline (CPT), the amplitude of evEPSCs did not change during 0.033 and 1 Hz stimuli. When the frequency of stimulation was increased upto 2 Hz, a time-dependent depression of amplitude was recorded in hypocretin neurons. Administration of CPT caused no significant change in depressed synaptic response induced by 2 Hz stimulus. While depression induced by 10 and 100 Hz stimuli was partially inhibited by the CPT but not by the selective A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine. Further findings have demonstrated that high-frequency stimulation could induce long-term potentiation (LTP) of glutamatergic synaptic transmission to hypocretin neurons in acute hypothalamic slices. The experiments with CPT suggested that A1 receptor antagonist could facilitate the induction of LTP, indicating that endogenous adenosine, acting through A1 receptors, may suppress the induction of LTP of excitatory synaptic transmission to hypocretin neurons. These results suggest that in the hypothalamus, endogenous adenosine will be released into extracellular space in an activity-dependent manner inhibiting both basal excitatory synaptic transmission and LTP in hypocretin neurons via A1 receptors. Our data provide further support for the notion that hypocretin neurons in the lateral hypothalamus may be another important target involved in the endogenous adenosine modulating the sleep and wakefulness cycle in the mammalian CNS.