大鼠脊髓背角浅层GABA能轴突与SP受体阳性神经元的突触联系(英文)

目的:研究大鼠脊髓背角浅层内GABA能神经元与P物质受体(SPR)阳性神经元之间的相互关系.方法:应用SPR包埋前免疫电镜结合GABA胶体金技术.结果:SPR样免疫阳性神经元主要分布于脊髓背角Ⅰ层和Ⅱ层背侧部的内侧半.电镜下,浅层内SPR样免疫反应阳性产物主要定位于细胞体和树突.免疫反应产物在树突呈片或颗粒状,常常与线粒体外膜表面、粗面内质网、高尔基氏体、树突膜、胞浆膜内面和核膜外表面等相连.双标记显示SPR样免疫反应树突接受GABA样免疫金标记的轴突传入,形成对称性轴-树突触.此外,GABA还可与SPR共存于同一树突中.结论:GABA和P物质受体在脊髓背角神经元的突触联系,为以往GABA调制脊髓伤害感受功能的药理学研究提供了形态学证据.     

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。     

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.     

Glutamate Decarboxylase 67 Deficiency in a Subset of GABAergic Neurons Induces Schizophrenia-Related Phenotypes

Decreased expression of the GABA synthetic enzyme glutamate decarboxylase 67 (GAD67) in a subset of GABAergic neurons, including parvalbumin (PV)-expressing neurons, has been observed in postmortem brain studies of schizophrenics and in animal models of schizophrenia. However, it is unclear whether and how the perturbations of GAD67-mediated GABA synthesis and signaling contribute to the pathogenesis of schizophrenia. To address this issue, we generated the mice lacking GAD67 primarily in PV neurons and characterized them with focus on schizophrenia-related parameters. We found that heterozygous mutant mice exhibited schizophrenia-related behavioral abnormalities such as deficits in prepulse inhibition, MK-801 sensitivity, and social memory. Furthermore, we observed reduced inhibitory synaptic transmission, altered properties of NMDA receptor-mediated synaptic responses in pyramidal neurons, and increased spine density in hippocampal CA1 apical dendrites, suggesting a possible link between GAD67 deficiency and disturbed glutamatergic excitatory synaptic functions in schizophrenia. Thus, our results indicate that the mice heterozygous for GAD67 deficiency primarily in PV neurons share several neurochemical and behavioral abnormalities with schizophrenia, offering a novel tool for addressing the underlying pathophysiology of schizophrenia.     

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’.     

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.     

Glutamic Acid Decarboxylase 65: A Link Between GABAergic Synaptic Plasticity in the Lateral Amygdala and Conditioned Fear Generalization

An imbalance of the gamma-aminobutyric acid (GABA) system is considered a major neurobiological pathomechanism of anxiety, and the amygdala is a key brain region involved. Reduced GABA levels have been found in anxiety patients, and genetic variations of glutamic acid decarboxylase (GAD), the rate-limiting enzyme of GABA synthesis, have been associated with anxiety phenotypes in both humans and mice. These findings prompted us to hypothesize that a deficiency of GAD65, the GAD isoform controlling the availability of GABA as a transmitter, affects synaptic transmission and plasticity in the lateral amygdala (LA), and thereby interferes with fear responsiveness. Results indicate that genetically determined GAD65 deficiency in mice is associated with (1) increased synaptic length and release at GABAergic connections, (2) impaired efficacy of GABAergic synaptic transmission and plasticity, and (3) reduced spillover of GABA to presynaptic GABA_B receptors, resulting in a loss of the associative nature of long-term synaptic plasticity at cortical inputs to LA principal neurons. (4) In addition, training with high shock intensities in wild-type mice mimicked the phenotype of GAD65 deficiency at both the behavioral and synaptic level, indicated by generalization of conditioned fear and a loss of the associative nature of synaptic plasticity in the LA. In conclusion, GAD65 is required for efficient GABAergic synaptic transmission and plasticity, and for maintaining extracellular GABA at a level needed for associative plasticity at cortical inputs in the LA, which, if disturbed, results in an impairment of the cue specificity of conditioned fear responses typifying anxiety disorders.     

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.

     

Calcium channel subtypes mediating central synaptic transmission

It is well established that neurotransmitter release is triggered by Ca2+ entry into the presynaptic terminals through voltage-dependent Ca2+ channels. In the mammalian central nervous system, multiple types of Ca2+ channels including N-type, P/Q-type and other types mediate fast synaptic transmission. Electrophysiological studies using type-specific antagonists for Ca2+ channels have estimated the relative contribution of N-, P/Q- and other types of Ca2+ channels in excitatory and inhibitory synaptic transmission in the hippocampus, cerebellum, spinal cord, brain stem, and striatum. A recent study has demonstrated that activation of presynaptic dopamine D2-like receptors selectively block N-type Ca2+ channels to reduce GABA release onto cholinergic interneurons in the rat striatum. In addition, it has been recently clarified that the contribution of N-type Ca2+ channels to synaptic transmission is restricted to the early postnatal period at synapses in auditory brain stem, cerebellum, or thalamus. Advanced morphological studies are necessary for the further understanding of the subcellular localization of each subtype of Ca2+ channels and receptors modulating the transmitter release through Ca2+ channel activity in relation to the release sites in the presynaptic terminals.     

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。     

The effects of 5-hydroxytryptamine-depleting drugs on peptides in the ventral spinal cord

The ventral spinal cord content of several neuronally localised peptides was measured after treatment with a number of drugs which deplete spinal cord monoamines. Reserpine and tetrabenazine, but not p-chlorophenylalanine caused a partial depletion of ventral spinal cord substance P (SP) and thyrotropin-releasing hormone (TRH). Two other peptides, methionine-enkephalin and somatostatin were not depleted by any of the drugs. The rates of loss and recovery of SP and TRH after reserpine and tetrabenazine were different from that of 5-hydroxytryptamine (5-HT), though in the ventral spinal cord these two peptides probably coexist with 5-HT in the terminals of bulbospinal neurones. The results are discussed in relation to the possible costorage of SP and TRH with 5-HT in the same vesicles in nerve terminals in the ventral spinal cord.     

Quisqualamine,a novel γ-aminobutyric acid (GABA)-related depressant amino acid

A new substance, quisqualamine, the decarboxylated analogue of quisqualic acid, predictably depressed electrical activity of neurons of the frog and rat spinal cord in vitro and of the mouse spinal cord in vivo. In the in vitro preparations, the action of quisqualamine was associated with a prolonged depolarization of primary afferent terminals which was sensitive to blockade by picrotoxin and bicuculline and which was also depressed by strychnine. This suggests an interaction of quisqualamine with presynaptic receptors for both GABA and β-alanine. Post-synaptic actions of quisqualamine, which were less marked than those at pre-synaptic sites, also appeared to be predominantly GABA-mimetic in vitro, though a sensitivity to the GABA-antagonist bicuculline could not be demonstrated in vivo.     

Postnatal development of the GABA system in the rat spinal cord

To assess the postnatal development of the GABA system in the rat spinal cord, GABA levels and GAD activity, as well as specific [3H]-muscimol binding, were determined in the dorsal and ventral areas. GABA levels and GAD activities did not vary in parallel from 1 to 8 postnatal days since the former decreased and the latter increased. After 8 days, however, increases in GABA levels and GAD activities were observed in tissues from the dorsal area and decreases were observed in tissues from the ventral area. Specific [3H]muscimol binding was unexpectedly high in both areas at 1 and 8 days but decreased linearly up to 22 days. Our findings show that there are distinct differences in the development of the GABA system in the rat spinal cord and brain.     

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.     

Primary afferent terminal excitability in the normal and spastic mutant mouse spinal cord

A microcomputer-based system has been used to apply the technique of excitability testing to the study of the actions of a range of pharmacological agents on the excitability of single primary afferent terminals in the mouse spinal cord in vitro. GABAA analogues all evoked increases in excitability that were bicuculline sensitive. GABA itself also evoked biphasic changes in excitability, or occasionally only suppressed terminal excitability. This latter effect was often enhanced in the presence of bicuculline, and resembled the action of the GABAB agonist, baclofen. The GABAA action could be enhanced by concurrent application of either benzodiazepine, midazolam or flurazepam. Bicuculline alone frequently decreased excitability. This action could be abolished by blocking synaptic activity with a low Ca2+ high Mg2+ superfusate, and was therefore considered to be due to reduction of the tonic action of GABA released at synaptic connections. Comparison of the action of these agents on terminals in the spastic mutant mouse showed an increased sensitivity of the GABA response to the benzodiazepines in mutant animals.     

Development of tolerance to the effects of vigabatrin (gamma-vinyl-GABA) on GABA release from rat cerebral cortex, spinal cord and retina.

1. The effects of acute and chronic vigabatrin (gamma-vinyl-GABA) (GVG) administration on gamma-aminobutyric acid (GABA) levels and release in rat cortical slices, spinal cord slices and retinas were studied. 2. GVG (250 mgkg-1 i.p.) administered to rats 18 h before death (acute administration) produced an almost 3 fold increase in GABA levels of the cortex and spinal cord and a 6 fold increase in retinal GABA. The levels of glutamate, aspartate, glycine and taurine were unaffected. 3. When GVG (250 mgkg-1 i.p.) was administered daily for 17 days (chronic administration) a similar (almost 3 fold) increase in cortical GABA occurred but the increases in spinal and retinal GABA were reduced by approximately 40%. 4. Acute administration of GVG strikingly increased the potassium-evoked release (KCl 50 mM) of GABA from all three tissues. This enhanced evoked release was reduced by about 50% in tissues taken from rats that had been chronically treated with GVG. 5. Acute administration of GVG reduced GABA-transaminase (GABA-T) activity by approximately 80% in cortex and cord and by 98% in the retina. Following the chronic administration of GVG, there was a trend for GABA-T activities to recover (significant only in cortex). Acute administration of GVG had no effect on glutamic acid decarboxylase (GAD) activity in cortex or spinal cord. However, chronic treatment resulted in significant decreases in GAD activity in both the cortex and cord (35% and 50% reduction respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic AMPA and kainate receptors increase the size of GABAergic terminals and enhance GABA release

In the developing cerebellum, NMDA receptors promote the maturation of axonal terminals of inhibitory interneurons. We compared the effects of AMPA/kainate receptor agonists in cultured cerebellar cells from GAD65-eGFP mice. Both AMPA and kainate augmented granule cell survival without affecting interneurons. The action of kainate was blocked by an AMPA but not by a NMDA receptor antagonist, suggesting AMPA receptor involvement. AMPA and kainate increased the size of the GABAergic terminals and the action of kainate was insensitive to NMDA blockers. Whole cell recordings in granule neurons revealed that chronic treatment for 5 days with kainate as well as NMDA decreased AMPA receptor expression while interneuronal kainate receptors were depressed by kainate treatment. Acute kainate application increased mIPSCs frequency in both granule neurons and interneurons and this effect was only partially blocked by an AMPA receptor antagonist. In contrast to what was reported for NMDA, chronic treatment with kainate induced a significant decrease of the basal mIPSCs frequency but increased the acute action of kainate on mIPSCs. Direct recordings from presynaptic GABAergic terminals suggest that AMPA and kainate receptors are present in developing GABAergic terminals and their activation affects the size of GABAergic terminals and spontaneous GABA release.     

THERE ARE FEW CATECHOLAMINE- OR NEUROPEPTIDE Y-CONTAINING SYNAPSES IN THE INTERMEDIOLATERAL CELL COLUMN OF RAT THORACIC SPINAL CORD

1. A quantitative electron microscopic immunocytochemical technique was used to assess the number of synapses immunoreactive for tyrosine hydroxylase (TH), phenylethanolamine N-methyl-transferase (PNMT) and neuropeptide Y (NPY) in the intermediolateral cell column in segments T2 and T3 of rat thoracic spinal cord. 2. TH synapses comprised about 5%; PNMT synapses 1-2%; and NPY synapses 1-2% of the total number of synapses in the intermediolateral cell column. All three types of synapses were predominantly or exclusively on dendrites. 3. Our results suggest that catecholamine/NPY neurons may not provide a major synaptic input to sympathetic preganglionic neurons in rat upper thoracic spinal cord.     

The influence of manipulations to alter ambient GABA concentrations on the hypnotic and immobilizing actions produced by sevoflurane, propofol, and midazolam

Recent studies have suggested that extrasynaptic GABA(A) receptors, which contribute tonic conductance, are important targets for general anesthetics. We tested the hypothesis that manipulations designed to alter ambient GABA concentrations (tonic conductance) would affect hypnotic (as indicated by loss of righting reflex, LORR) and immobilizing (as indicated by loss of tail-pinch withdrawal reflex, LTWR) actions of sevoflurane, propofol, and midazolam. Two manipulations studied were 1) the genetic absence of glutamate decarboxylase (GAD) 65 gene (GAD65-/-), which purportedly reduced ambient GABA concentrations, and 2) the pharmacological manipulation of GABA uptake using GABA transporter inhibitor (NO-711). The influence of these manipulations on cellular and behavioral responses to the anesthetics was studied using behavioral and electrophysiological assays. HPLC revealed that GABA levels in GAD65-/- mice were reduced in the brain (76.7% of WT) and spinal cord (68.5% of WT). GAD65-/- mice showed a significant reduction in the duration of LORR and LTWR produced by propofol and midazolam, but not sevoflurane. NO-711 (3?mg/kg, ip) enhanced the duration of LORR and LTWR by propofol and midazolam, but not sevoflurane. Patch-clamp recordings revealed that sevoflurane (0.23?mM) slightly enhanced the amplitude of tonic GABA current in the frontal cortical neurons; however, these effects were not strong enough to alter discharge properties of cortical neurons. These results demonstrate that ambient GABA concentration is an important determinant of the hypnotic and immobilizing actions of propofol and midazolam in mice, whereas manipulations of ambient GABA concentrations minimally alter cellular and behavioral responses to sevoflurane.     

A novel translocator protein 18 kDa ligand,ZBD‐2, exerts neuroprotective effects against acute spinal cord injury

Traumatic spinal cord injury (SCI) happens accidently and often leads to motor dysfunction due to a series of biochemical and pathological events and damage, either temporarily or permanently. Translocator protein 18 (TSPO) has been found to be involved in the synthesis of endogenous neurosteroids which have multiple effects on neurons, but the internal mechanisms are not clear. N‐benzyl‐N‐ethyl‐2‐(7,8‐oxo‐2‐phenyl‐9H‐purin‐9‐yl) acetamide (ZBD‐2), a newly reported ligand of TSPO, shows some neuroprotective effect against focal cerebral ischemia in vivo and NMDA‐induced neurotoxicity in vitro. The present study aims to examine the role of ZBD‐2 in SCI mice and elucidate the underlying molecular mechanisms. The SCI model was established by crushing spinal cord. ZBD‐2 (10 mg/kg) significantly enhanced the hindlimb locomotor functions after SCI and decreased the tissue damage and conserved the white matter of the spinal cord. High‐dose ZBD‐2 alleviated the oxidative stress induced by SCI and regulated the imbalance between NR2B‐containing NMDA and GABA receptors by increasing the levels of GAD67 in the spinal cord of SCI mice. Additionally, ZBD‐2 (10 mg/kg) increased phosphorylated Akt (p‐Akt) and decreased the ratio of Bax/Bcl‐2. These results demonstrate that ZBD‐2 performs neuroprotection against SCI through regulating the synaptic transmission and the PI3K/AKT signaling pathway.