Immunocytochemical localization of glutamate decarboxylase in rat spinal cord

McLaughlin, Barbara J.; Barber, Robert P.; Saito, Kihachi; Roberts, Eugene; Wu, Jang‐Yen

doi:10.1002/cne.901640304

Cited by 281 publications

(92 citation statements)

References 33 publications

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“…In addition, all laminae contain GABAergic terminals (McLaughlin et al, 1975;Mackie et al, 2003). Thus, it would be expected that there are many different populations of GABAergic interneurons with distinct functions.…”

Section: Distribution Of Gabaergic Interneurons In Gad65::gfp Micementioning

confidence: 99%

Genetically Defined Inhibitory Neurons in the Mouse Spinal Cord Dorsal Horn: A Possible Source of Rhythmic Inhibition of Motoneurons during Fictive Locomotion

Wilson¹,

Blagovechtchenski²,

Brownstone³

2010

J. Neurosci.

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To ensure alternation of flexor and extensor muscles during locomotion, the spinal locomotor network provides rhythmic inhibition to motoneurons. The source of this inhibition in mammals is incompletely defined. We have identified a population of GABAergic interneurons located in medial laminae V/VI that express green fluorescent protein (GFP) in glutamic acid decarboxylase-65::GFP transgenic mice. Immunohistochemical studies revealed GFPϩ terminals in apposition to motoneuronal somata, neurons in Clarke's column, and in laminae V/VI where they apposed GFPϩ interneurons, thus forming putative reciprocal connections. Whole-cell patch-clamp recordings from GFPϩ interneurons in spinal cord slices revealed a range of electrophysiological profiles, including sag and postinhibitory rebound potentials. Most neurons fired tonically in response to depolarizing current injection. Calcium transients demonstrated by two-photon excitation microscopy in the hemisected spinal cord were recorded in response to low-threshold dorsal root stimulation, indicating these neurons receive primary afferent input. Following a locomotor task, the number of GFPϩ neurons expressing Fos increased, indicating that these neurons are active during locomotion. During fictive locomotion induced in the hemisected spinal cord, two-photon excitation imaging demonstrated rhythmic calcium activity in these interneurons, which correlated with the termination of ventral root bursts. We suggest that these dorsomedial GABAergic interneurons are involved in spinal locomotor networks, and may provide direct rhythmic inhibitory input to motoneurons during locomotion.

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Section: Distribution Of Gabaergic Interneurons In Gad65::gfp Micementioning

confidence: 99%

Genetically Defined Inhibitory Neurons in the Mouse Spinal Cord Dorsal Horn: A Possible Source of Rhythmic Inhibition of Motoneurons during Fictive Locomotion

Wilson¹,

Blagovechtchenski²,

Brownstone³

2010

J. Neurosci.

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“…The reagent ( 1 ) and presence ( 2 ) of L-glutamate (10 m M ) at p H 7.2. The sample of enzyme was allowed to incubate for 1 h at 25°C in the presence of L-glutamate prior to recording the spectra was a solution containing 0.25 g cyclohexane 1,3-dione, 10 g ammonium acetate and 5 ml glacial acetic acid in 100 ml of water.…”

Section: Determination Of Succinic Semialdehydementioning

confidence: 99%

Glutamate decarboxylase side reactions catalyzed by the enzyme

Choi

Churchich

1986

European Journal of Biochemistry

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A homogeneous glutamate decarboxylase isolated from pig brain contains 0.8 mol of tightly bound pyridoxal 5-phosphate/enzyme dimer. Upon addition of exogenous pyridoxal 5-phosphate (pyridoxal-5-P), the enzyme acquires maximum catalytic activity.Preincubation of the enzyme with L-glutamate (10 mM) brings about changes in the absorption spectrum of bound pyridoxal-5-P with the concomitant formation of succinic semialdehyde. However, the rate of this slow secondary reaction, i. e. decarboxylative transamination, is times the rate of normal decarboxylation. It is postulated that under physiological conditions enzymatically inactive species of glutamate decarboxylase, generated by the process of decarboxylative transamination, are reconstituted by pyridoxal-5-P produced by the cytosolic enzymes pyridoxal kinase and pyridoxine-5-P oxidase.The catalytic activity of resolved glutamate decarboxylase is recovered by preincubation with phosphopyridoxyl-ethanolamine phosphate. The experimental evidence is consistent with the interpretation that the resolved enzyme binds the P-pyridoxyl analog, reduces the stability of the covalent bond of the phospho-pyridoxyl moiety, and catalyzes the formation of pyridoxal-5-P.Glutamate decarboxylase is mainly located in the soluble component of the nerve endings, but it has been shown that in the presence of CaZf the enzyme binds to synaptic vesicle membranes [l]. Localization studies of the enzyme by means of electron microscopy, using antibodies against the decarboxylase, have shown that the enzyme can be associated to synaptic vesicle membranes 121.Glutamate decarboxylase has been purified from several brain tissues [3 -5] and it is generally accepted that the dimeric protein possesses a relative molecular mass of around 110000. The stoichiometry of binding of the cofactor to the protein has not been elucidated, though the presence of non-equivalent binding sites has been postulated to explain the catalytic behavior of the decarboxylase [6] (general review).The substrate L-glutamate, at concentrations slightly higher than the K,,, value, promotes slow inactivation of the decarboxylase [7]. This inactivation process has been attributed to a secondary reaction, i. e. decarboxylation-dependent transamination.In order to assess the metabolic significance of a transamination reaction catalyzed by glutamate decarboxylase, it is essential (a) to demonstrate the formation of pyridoxamine-5-P and succinic semialdehyde during the substrate-induced inactivation of the enzyme and (b) to measure the rates of normal decarboxylation and transamination.In this paper, we examine multiple reactions catalyzed by purified glutamate decarboxylase, and provide proof that succinic semialdehyde is generated during the substrate-promoted inactivation of the decarboxylase. EXPERIMENTAL PROCEDURES Purification of glutamate decarboxylaseThe purification of glutamate decarboxylase from pig brain was performed following procedures developed for the purification of the enzyme from brain tissues [3 -51 except that a...

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“…GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD), and Abs against GAD have been used to identify GABAergic axonal boutons in the spinal cord (2,3). More recently, two forms of the enzyme have been identified, and based on their molecular weights these forms have been named GAD65 and GAD67 (4).…”

mentioning

confidence: 99%

P boutons in lamina IX of the rodent spinal cord express high levels of glutamic acid decarboxylase-65 and originate from cells in deep medial dorsal horn

Hughes

Mackie

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

112

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Presynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population of GABAergic axon terminals known as P boutons. Despite extensive investigation, the cells that mediate this control have not yet been identified. In this work, we use immunocytochemistry with confocal microscopy and EM to demonstrate that P boutons can be distinguished from other GABAergic terminals in the ventral horn of rat and mouse spinal cord by their high level of the glutamic acid decarboxylase (GAD) 65 isoform of GAD. By carrying out retrograde labeling from lamina IX in mice that express green fluorescent protein under the control of the GAD65 promoter, we provide evidence that the cells of origin of the P boutons are located in the medial part of laminae V and VI. Our results suggest that P boutons represent the major output of these cells within the ventral horn and are consistent with the view that presynaptic inhibition of proprioceptive afferents is mediated by specific populations of interneurons. They also provide a means of identifying P boutons that will be important in studies of the organization of presynaptic control of Ia afferents.GABA ͉ Ia afferent ͉ presynaptic inhibition T he inhibitory transmitter GABA is used by many neurons in the spinal cord and generates both postsynaptic inhibition at axo-dendritic and axo-somatic synapses and presynaptic inhibition at axo-axonic synapses (1). GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD), and Abs against GAD have been used to identify GABAergic axonal boutons in the spinal cord (2, 3). More recently, two forms of the enzyme have been identified, and based on their molecular weights these forms have been named GAD65 and GAD67 (4). Both forms are present in the ventral horn of the rat spinal cord but have a very different distribution (5, 6). The majority of GABAergic boutons throughout the ventral horn show strong GAD67 immunoreactivity and a low level of GAD65. However, there are clusters of boutons in lamina IX that contain very high levels of GAD65. We have suggested (6) that these clusters may correspond to the P boutons that form axo-axonic synapses with terminals of group Ia afferents (7-10). In this work, we have used a variety of approaches to confirm this hypothesis and to identify the cells of origin of these boutons. MethodsAll experiments were approved by the Ethical Review Process Applications Panel of the University of Glasgow or the Animal Care and Protection Committee at the University of Debrecen and were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and the European Communities Council Directives. Immunocytochemical reactions were performed on free-floating 60-m transverse Vibratome sections that had been treated with 50% ethanol for 30 min to enhance Ab penetration. Abs were diluted in PBS with 0.3% Triton X-100 (except on sections used for EM).Primary Afferent Labeling. Transgang...

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Immunocytochemical localization of glutamate decarboxylase in rat spinal cord

Cited by 281 publications

References 33 publications

Genetically Defined Inhibitory Neurons in the Mouse Spinal Cord Dorsal Horn: A Possible Source of Rhythmic Inhibition of Motoneurons during Fictive Locomotion

Genetically Defined Inhibitory Neurons in the Mouse Spinal Cord Dorsal Horn: A Possible Source of Rhythmic Inhibition of Motoneurons during Fictive Locomotion

Glutamate decarboxylase side reactions catalyzed by the enzyme

P boutons in lamina IX of the rodent spinal cord express high levels of glutamic acid decarboxylase-65 and originate from cells in deep medial dorsal horn

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