A comparison of microdistributions of taurine and cysteine sulphinate decarboxylase activity with those of GABA and L-glutamate decarboxylase activity in rat spinal cord and thalamus

Abstract— Distribution profiles of taurine and activity of cysteine sulphinate decarboxylase (CSD), the enzyme catalysing the formations of hypotaurine from cysteine sulphinate and of taurine from cysteate respectively, in the rat spinal cord and thalamus were studied in comparison with those of GABA and activity of l -glutamate decarboxylase (GAD), the rate limiting enzyme for GABA formation. In the spinal cord (L₂-L₃), it was found that taurine is fairly evenly distributed, whereas the activity of CSD is higher in the dorsal half of the spinal cord than in the ventral half. The highest CSD activity was found in the dorsal part of the dorsal horn. In the anterior part (A 5.4) of the thalamus, taurine and CSD activity were also distributed evenly and no areas having high taurine content and CSD activity were detected. In contrast with the even distributions of taurine and CSD activity, both GABA and GAD activity were distributed unevenly in the same CNS areas examined: The areas having high GABA content and GAD activity in the thalamus (A 5.4) coincided with the ventrolateral part of the ventral nucleus of thalamus (VM), entopeduncular nucleus (EP) and nucleus reuniens thalami (RE), whereas those in the spinal cord were found to be in the dorsal part of the dorsal horn and surrounding parts of the central canal, respectively. Considering a probable role of GABA in mammalian central nervous system (CNS) as an inhibitory neurotransmitter, it seems unlikely that taurine acts as an inhibitory neurotransmitter at least in the rat spinal cord and thalamus.     

Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2 + GAD67 mRNAs were not significantly different, we concluded that glycine and GABA coexisted in the various projection neurons. After injections in the cervical cord, we found that 29% ± 1 (SEM) of the retrogradely labeled neurons in the VM were Gly/GABA (RVM: 43%; CVM: 21%). After lumbar dorsal horn injections 31% ± 3 of the VM neurons were Gly/GABA (RVM: 45%; CVM: 12%), and after lumbar ventral horn injections 25% ± 2 were Gly/GABA (RVM: 35%; CVM: 17%). In addition, we have identified a novel ascending Gly/GABA pathway originating from neurons in the area around the central canal (CC) throughout the spinal cord and projecting to the RVM, emphasizing the interaction between the ventromedial medulla and the spinal cord. The present study has now firmly established that GABA and glycine are present in many VM neurons that project to the spinal cord. These neurons strongly influence spinal processing, most notably the inhibition of nociceptive transmission.     

Expression of GABA transporters,GAT-1 and GAT-3, in the cerebral cortex and thalamus of the rat during postnatal development

The cellular and subcellular localization of two GABA transporters, GAT-1 and GAT-3, was investigated using immunocytochemical methods in the rat cerebral cortex and thalamus during postnatal development. The distribution of the transporters is compared with that of the neuronal marker GABA, and with that of vimentin and of glial fibrillary acidic protein, which identify immature and mature astrocytes, respectively. Our observations show that the two transporters are already expressed at birth in both brain areas with the same cellular localization as in adult rats, as GAT-1 is present in growth cones and terminals only in the cortex, whereas both transporters are expressed in astrocytes in the cortex and thalamus. The distribution of GAT-1 and GAT-3 undergoes postnatal changes reflecting in general the neurogenetic events of the neocortex and thalamus and, more specifically, the maturation of GABAergic innervation. The adult-like pattern of expression is achieved in the third postnatal week in the cortex and in the second postnatal week in the thalamus. The early expression of GAT-1 in GABAergic terminals confirms previous studies showing the existence of neuronal mechanisms of GABA uptake from the embryonic stages. As for the glial localization, the precocious existence of two astrocytic GABA transporters suggests that they operate through different functional mechanisms from birth, whereas their exclusively glial expression in the thalamus indicates that the astroglia plays a major role in the transport, recycling and metabolism of thalamic GABA.     

乙酰胆碱,谷氨酸与GABA对丘脑腹内侧核神经元活动的影响

本文采用微电泳方法观察到在大鼠丘脑腹内侧核（ＶＭ）,微电泳给予乙酰胆碱（ＡＣＨ）使所有受试神经元自发放电频率加快,谷氨酸（ＧＬＵ）使大多数神经元放电加快,它们的作用依赖于电流强度;而γ氨基丁酸（ＧＡＢＡ）和氯苯氨丁酸则抑制大多数神经元的放电活动,但前者的作用快速而暂短,而后者的作用相对缓慢而持久。在微电泳ＡＣＨ或ＧＬＵ的过程中,给予ＧＡＢＡ可拮抗它们的兴奋作用。双钴碱使大多数神经元的自发放电频率加快,而阿托品和ＭＫ８０１对自发放电的影响较小。这些结果表明ＧＡＢＡ,ＡＣＨ和ＧＬＵ等递质活动在同一ＶＭ神经元有重要的会聚作用;ＧＡＢＡ对ＶＭ神经元有紧张性抑制作用。     

Physiological presynaptic facilitation of dopamine release in the cat caudate nucleus

Using halothane-anaesthetized cats implanted with push-pull cannulae, we investigated the effects of GABA application into the VM/VL on the release of [3H] DA continuously synthetized from [3H] tyrosine in the ipsilateral CN and SN and on single unit activity of nigral DA cells. GABA was applied (30 min) at a concentration of 10(-3) or 10(-5) M since the higher concentration reduces the local multi-unit activity in the VM/VL while the opposite response is observed with the lower one. The application of GABA into the VM/VL at a concentration of 10(-3) M resulted in an increase in nigral [3H] DA release, an inhibition of DA cell firing and a decrease in [3H] DA release in the CN. The latter effect is likely due to the inhibition of DA neuron activity which is triggered through DA autoreceptors by DA released from dendrites. In contrast, when applied at a concentration of 10(-5) M into the VM/VL, GABA stimulated [3H] DA release in the CN despite its inhibitory effect on single unit activity of DA cells. Furthermore, the nigral release of [3H] DA was no longer affected. These results indicated that DA release from nerve terminals was not dependent on nerve activity and they favour the existence of a potent facilitatory presynaptic regulation of DA release. The intervention of a presynaptic mechanism was further established by examining the effect of GABA (10(-5) M) application into the VM/VL on [3H] DA release in the CN shortly after a complete ipsilateral hemisection of the brain made at the meso-diencephalic level.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thalamus of the monotremes: cyto- and myeloarchitecture and chemical neuroanatomy

Echidna and platypus brains were sectioned and stained by Nissl or myelin stains or immunocytochemically for calcium-binding proteins, gamma aminobutyric acid (GABA) or other antigens. Cyto- and myeloarchitecture revealed thalami that are fundamentally mammalian in organization, with the three principal divisions of the thalamus (epithalamus, dorsal thalamus and ventral thalamus) identifiable as in marsupials and eutherian mammals. The dorsal thalamus exhibits more nuclear parcellation than hitherto described, but lack of an internal medullary lamina, caused by splaying out of afferent fibre tracts that contribute to it in other mammals, makes identification of anterior, medial and intralaminar nuclear groups difficult. Differentiation of the ventral nuclei is evident with the ventral posterior nucleus of the platypus enormously expanded into the interior of the cerebral hemisphere, where it adopts a relationship to the striatum not seen in other mammals. Other nuclei such as the lateral dorsal become identifiable by expression of patterns of calcium-binding proteins identical to those found in other mammals. GABA cells are present in the ventral and dorsal thalamic nuclei, and in the ventral thalamus form a remarkable continuum with GABA cells of the two segments of the globus pallidus and pars reticulata of the substantia nigra.     

Preincubation of homogenates from rat brain thalamus and striatum affects the rate of [3H]GABA uptake

The effect of preincubation of brain homogenates on the subsequent uptake of [³H]GABA was studied in rat thalamus and corpus striatum. The results show that in both brain regions preincubation causes a decrease in the initial rate of [³H]GABA influx, the phenomenon being most evident in the thalamus.     

Comparative effects of the GABA uptake inhibitors,tiagabine and NNC-711, on extracellular GABA levels in the rat ventrolateral thalamus

The primary mechanism by which the action of synaptically released GABA is thought to be terminated is by re-uptake into neurones and glial cells, and the pharmacological inhibition of this uptake may be beneficial in conditions where decreased GABAergic transmission has been implicated, such as epilepsy. We have compared the effects of two of these uptake inhibitors, tiagabine and NNC-711, on extracellular GABA levels in the thalamus of the rat, after both systemic and local administration. Both compounds produced dose-dependent increases in GABA concentration irrespective of the route of administration, but the concentrations required to produce increased extracellular GABA levels were considerably higher than those known to be effective for anticonvulsant purposes. These data suggest that, initially at least, alternative GABA transporters, not susceptible to inhibition by the compounds used, may still be able to remove synaptically released GABA from the extracellular space. Special issue dedicated to Dr. Kinya Kuriyama.     

Local gating of information processing through the thalamus

Inhibitory sculpting of afferent signals in the thalamus is exerted by two types of neurons using gamma-amino butyric acid (GABA) as neurotransmitter. Of them, local-circuit neurons exert their functions via two outputs: axons and presynaptic dendrites. In this issue of Neuron, Govindaiah and Cox reveal that synaptic activation of metabotropic glutamate receptors selectively increases the output of presynaptic dendrites of local interneurons in rat visual thalamus, without affecting the axonal output.     

Noisy galvanic vestibular stimulation promotes GABA release in the substantia nigra and improves locomotion in hemiparkinsonian rats

Background

The vestibular system is connected to spinal, cerebellar and cerebral motor control structures and can be selectively activated with external electrodes. The resulting sensation of disturbed balance can be avoided by using stochastic stimulation patterns. Adding noise to the nervous system sometimes improves function. Small clinical trials suggest that stochastic vestibular stimulation (SVS) may improve symptoms in Parkinson''s disease. We have investigated this claim and possible mechanisms using the 6-hydroxydopamine (6-OHDA) hemilesion model of Parkinson''s disease.

Methodology/Principal Findings

Animals were tested in the accelerating rod test and the Montoya staircase test of skilled forelimb use. In 6-OHDA hemilesioned animals, SVS improved rod performance by 56±11 s. At group level L-DOPA treatment had no effect, but positive responders improved time on rod by 60±19 s. Skilled forelimb use was not altered by SVS. To investigate how SVS may influence basal ganglia network activity, intracerebral microdialysis was employed in four regions of interest during and after SVS. In presence of the γ-amino buturic acid (GABA) transporter inhibitor NNC 711, SVS induced an increase in GABA to 150±15% of baseline in the substantia nigra (SN) of unlesioned animals, but had no effect in the pedunculopontine nucleus (PPN), the striatum or the ventromedial thalamus (VM). Dopamine release remained stable in all areas, as did GABA and amine concentrations in the SN of unstimulated controls. Following SVS, a sustained increase in GABA concentrations was observed in the ipsilesional, but not in the contralesional SN of 6-OHDA hemilesioned rats. In contrast, L-DOPA treatment produced a similar increase of GABA in the ipsi- and contra-lesional SN.

Conclusions/Significance

SVS improves rod performance in a rat model of Parkinson''s disease, possibly by increasing nigral GABA release in a dopamine independent way. We propose that SVS could be useful for treating symptoms of Parkinson''s disease.     

Regional distribution of gamma-aminobutyric acid (GABA) in brain of the rhesus monkey

—The concentrations of γ-aminobutyric acid (GABA) in twenty different regions of Rhesus monkey brain were studied. The highest levels were found in the substantia nigra, globus pallidus, and hypothalamus. Regions of the cerebral cortex and thalamus contained low amounts and white matter the lowest. Indirect evidence supporting an inhibitory transmitter role for GABA is discussed.     

电刺激大鼠束旁核对底丘脑核和丘脑腹内侧核神经元的影响

本工作旨在探讨电刺激束旁核（parafascicular nucleus,PF）对帕金森病模型（Parkinson’s disease,PD）大鼠神经行为的改善作用及其机制。成年雄性Sprague—Dawley大鼠黑质致密部注射6一羟基多巴胺建立PD大鼠模型。采用行为学方法观察电刺激PF对阿朴吗啡诱发的大鼠旋转行为的作用,并应用在体细胞外记录法观察电刺激PF对大鼠底丘脑核（subthalamic nucleus,STN）及丘脑腹内侧核（ventromedial nucleus,VM）神经元放电的影响。结果发现,高频电刺激（130Hz,0．4mA,5s）PF一周,明显改善PD大鼠旋转行为。细胞外放电记录显示,高频电刺激PF使PD大鼠STN神经元自发放电减少,且该作用具有频率依赖性。另外,高频电刺激PF可使VM神经元兴奋,该作用也是频率依赖性的。我们在实验中同时观察到微电泳谷氨酸（glutamicacid,Glu）受体拮抗剂MK-801使STN神经元放电频率减少或完全抑制,微电泳t氨基丁酸（T-amino butyricacid,GABA）受体拮抗剂印防己毒素（picrotoxin,Pic）则使神经元放电频率增加。以上结果表明,GABA能和GIu能传入纤维可会聚于同-STN神经元,并对后者有紧张性作用。高频刺激PF,使该核团到STN神经元的Glu能兴奋性输出减少,导致STN的失活。这一作用通过基底神经节的间接通路,最终释放了丘脑运动核团VM的活性。高频刺激PF经PF,STN和VM的神经通路而改善PD大鼠神经行为。     

Distribution of GABA-immunoreactive neurons in the forebrain of the goldfish,Carassius auratus

Summary The distribution of gamma-aminobutyric acid (GABA) immunoreactivity was studied in the forebrain (tel-and diencephalon) of the goldfish by means of immunocytochemistry on Vibratome sections using antibodies against GABA. Positive perikarya were detected in the olfactory bulbs and in all divisions of the telencephalon, the highest density being found along the midline. In the diencephalon, GABA-containing cell bodies were found in the hypothalamus, in particular in the preoptic and tuberal regions. The inferior lobes, the nucleus recessus lateralis, and more laterodorsal regions, such as the nucleus glomerulosus and surrounding structures, also exhibited numerous GABA-positive perikarya. Cell bodies were also noted in the thalamus, in particular in the dorsomedial, dorsolateral and ventromedial nuclei. The relative density of immunoreactive fibers was evaluated for each brain nucleus and classified into five categories. This ubiquitous distribution indicates that, as in higher vertebrates, GABA most probably represents one of the major neurotransmitters in the brain of teleosts.     

Reversible Alterations of Cerebral γ-Aminobutyric Acid in Pyrithiamine-Treated Rats: Implications for the Pathogenesis of Wernicke''s Encephalopathy

Treatment of rats with the central thiamine antagonist, pyrithiamine, results in severe neurological symptoms such as loss of righting reflex. Measurement of gamma-aminobutyric acid (GABA) content of brain tissue from symptomatic pyrithiamine-treated (PT) rats revealed significant reductions in thalamus, cerebellum, and pons. GABA content of cerebral cortex, however, was unaltered. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were reduced in parallel with the GABA changes. On the other hand, activities of the GABA-synthetic enzyme glutamic acid decarboxylase (GAD) remained within normal limits, with the exception of a small but significant decrease in thalamus of symptomatic PT rats. Affinities and densities of high-affinity [3H]muscimol binding sites on crude cerebral membrane preparations from symptomatic PT rats were unchanged. Thiamine administration to symptomatic animals resulted in correction of abnormal righting reflexes and in normalization of decreased GABA levels and reduced alpha KGDH activities in cerebellum and pons. Thalamic GABA levels and alpha KGDH activities, on the other hand, remained significantly lower than normal. These results suggest that the reversible symptoms of pyrithiamine treatment may result from imparied GABA synthesis in cerebellum and pons of these animals. Similar mechanisms may play a role in the pathogenesis of the reversible symptoms of Wernicke's encephalopathy in man.     

Regional alterations in brain amino acids during the estrous cycle of the rat

Concentrations of 11 amino acids, including the neurotransmitters GABA, glutamate, aspartate, glycine and taurine, were determined in 12 brain regions of female rats during different stages of the estrous cycle. In addition, amino acids and sex hormone levels were determined in plasma. All sample collections were done in the forenoon between 9 and 11 a.m. Most regional amino acid levels measured did not change signficantly during estrous cycle, but significant alterations were found for GABA and glutamate in hypothalamus. Both amino acids were slightly decreased in hypothalamus during proestrus, which might reflect an alteration of GABA turnover in response to the high estrogen levels during this stage. A decreased glutamate level during proestrus was also found in thalamus, while both glutamate and GABA did not vary throughout estrous cycle in any of the other examined regions, including substantia nigra, amygdala, striatum, cortex and hippocampus. When diestrus was subdivided according to progesterone levels, high levels of this hormone seemed to be associated with effects on metabolism of certain amino acids, including glycine in substantia nigra, alanine in thalamus and threonine in pons/medulla. However, the few changes in regional amino acid concentrations found during the estrous cycle were so small that the functional significance of these changes cannot be ascertained without further determination of the cellular or subcellular compartments of brain tissue involved.     

Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ-Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat

We evaluated whether regional differences in the magnitude of glutamate, gamma-aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia-induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10- and 20-min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathological neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathological outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10-min insult but severely damaged after 20 min of ischemia. Ischemia-induced increase in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different. Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10- and 20-min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20-min insult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral differences in GABA binding sites in rat brain

An asymmetric distribution of GABA binding sites was found in the cerebral cortex, hippocampus, cerebellar hemispheres, striatum, and thalamus. Higher levels of [³H]GABA binding were observed in the left-side of most brain areas and in a greater percentage of adult rats, but the opposite asymmetry was found in the thalamus. A similar left-right difference in cerebral hemispheres was also found in five day-old rats, suggesting the genetic predetermination of asymmetry.     

Neurotransmitter systems of some brain regions of rats subjected to chronic morphine intoxication

The content of neurotransmitters and their metabolites was investigated in brain cortex hemispheres, thalamus and brainstem of rats subjected to chronic morphine intoxication (7–21 days). Morphine administration for 7–14 days was accompanied by changes of the catecholamine system functioning, which was the most pronounced in the thalamus and the brainstem. These changes included increased secretion of dopamine and noradrenaline, their decrease in the brain tissue, and an increased content of their metabolites. The changes in serotonin and GABA content were less pronounced and included a decrease of serotonin level and the increase of the GABA content in different periods of opiate administration.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Effect of vitamin B6 on gamma-aminobutyric acid level in rat brain.

Vitamin B6 injected intraperitoneally into rats 400 mg/kg body weight, has produced a statistically significant decrease in GABA level concentrations in hippocampus and cerebellum. Cerebral cortex, caudate nucleus and thalamus have shown the decrease in GABA concentrations, but these changes were not statistically significant. No remarkable behavioural changes were noted under such circumstances. The possible functional meaning of these results is discussed in relation to the role of GABA distribution in different brain regions and development of convulsions.