Newoscieme Vol 4, pp. 995 to 1005 Perptxxt Press Ltd 1979. Printed in Great Bntarn



J. L. MADERDRUT’ Neuroembryology Laboratory, Division of Research, North Carolina Department of Mental HeaIth, Raleigh, NC 27611, U.S.A. Neurobiolo~ Curriculum, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. Abstract-A simple method for purifying r_-[3H]glutamic acid and incubation conditions suitable for estimating L-glutamic acid decarboxylase activity are described. Routine and recycled cation-exchange procedures for separating y-aminobutyric acid from L-glutamate are outlined and compared. Blanks using the routine cation-exchange method average approximately 0.05% of the radioactivity in the substrate; product recovery is approximately 890/,. Recycling increases the sensitivity of the cationexchange method by 6-7-fold. L-Glutamate decarboxylase activity can be measured reliably in samples of embryonic neural tissue having wet-weights of approximately 1 pg using the routine cation-exchange method. The relationship between substrate purity and the sensitivity of the cation-exchange procedures is assessed. Radiochemicaf purity is the critical determinant of sensitivity for the recycled method. The ~tion~xchange method is compared with the anion-exchange and CO,-trapping methods. The cation-exchange method is approximately 2-3 orders of magnitude more sensitive than the CO&rapping method and l-2 orders of magnitude more sensitive than the anion-exchange method. The cationexchange method is also more specific than either of the other 2 methods. L-Glutamate decarboxylase activity has been detected in the lumbar spinal cord of the chick embryo at Day 24 (Stage 14) using the cation-exchange method. This is 5-6 days earlier than L-glutamate decarboxylase activity has been detected in embryonic neural tissue by previous investigators. L-Glutamate decarboxylase is present in the lumbar spinal cord at least as early as the birth of the first lumbar spinal cord neurons and at least 1-2 days before the initiation of synaptogenesis.

v-AMMINOBUTIXIC acid (GABA) is an inhibitory transmitter in both the vertebrate and invertebrate nervous system (ROBERTS,CHASE & TOWER, 1976). Since GABA-secreting neurons cannot be visualized using classical neurohistologic~ techniques, a marker(s) for the uniquely expressed gene(s) that distinguish GABAsecreting neurons from all other neuronal types is needed to study the regional distribution (ALBBRSBEBRADY,1959), hodology (FONNIJM,GROFOVA,Rmvm, STORM-MATHISEN t WALBERG,1974) and embryological development (COYLE & ENNA, 1976) of this specific class of neurons. L-Glutamic acid decarboxylase (L-glutamic acid l-carboxy-lyase, EC (GAD), the enzyme responsible for the synthesis of GABA from r_-glutamic acid, is a more reliable marker for GABA-s~reting neurons than GABA. GAD is a more direct marker of gene expression than GABA. GAD measurement obviates problems associated with the rapid postmortem rise in GABA levels in most neural tissues (ALDERMAN & SHELLEN~ERGER, 1974; BAXTER,1970) and the diffu’ Present address: Section of Neurosurgery


Mayo Clinic, Rochester, MN 55901, U.S.A. Abbreviations: AET, 2-aminoethylisothiouronium


mide; ChAc, choline acetyltransferase; DTT, dithiothreitol; GABA, y-aminobutyric acid; GAD, L-glutamic acid dmar-

boxylase: PCMS, p-chloromercuriphenyl sulfonic acid; PLP, Pyridoxai 5‘-phosphate; TCA, trichloroacetic acid.

sion of GABA during tissue preparation (FONNUM, 1972). A pathway for the biosynthesis of GABA that does not involve r.-glutamic acid as an intermediate has been described in both neural and non-Nepal tissues (SELLER & AL-T-~, 1974); this pathway appears to be a major source of GABA in embryonic neural tissues (DE MELLQ BACHRACH& NIRENBERG, 1976; SOBUE& NAKAJIMA,1978). The 2 most commonly used radiometric assays for estimating GAD activity do not measure GABA formation directly. The method of ALBERS& BRADY (1959) is based on the measurement of r4C02 evolution from [r4C]carboxyl-labelled L-glutamic acid. Although this method is sensitive and relatively convenient, it does not discriminate between CO2 formed by the dec~boxylation of L-glutamate from that of one of its metabolites. The COG-trapping method is blind to the route of decarboxylation and has repeatedly been shown to yield spuriously high GAD estimates in several tissues (DRUMMOND& PHILLIPS, 1974; MACDONNELL& GRBENGARD,1975; WILSON, SCHRIER,FARBER,THOMPSON,R~SENBWG,BLUME& NIRENBERG,1972). The method of MOLINOFP& KRAvtrz (1968) is based on the binding of r_-[r4C]glutamic acid to an anion-exchange resin at neutral pH. This method is convenient and sensitive; however, it measures the conversion of L-glutamate to any metabolite that does not bind to an anion-exchange resin at neutral pH. Data presented below will show that


1 L.


will also yield spuriously high GAD estimates in some tissues. Neither method is suitable for use in tissues having very low GAD levefs and/or very high levels of contaminating enzymes (cf. MACthis method


The following cation-exchange



GABA formation rather than L-glutamate catabolism;

it is relatively convenient and substantially more sensitive and specific than either of the other 2 methods. EXPER~ME~AL


Radioisotopes were purchased from New England Nuclear (Boston, MA); ion-exchange rams were purchased from Bio-Rad (Richmond, CA). Triton X-100 and X-114 were obtained from Robm & Haas (Philadeiphia PA); 2.5diphenyloxazole (PPO) and 1,4-bis (2,5diphenyloxazoiyl) benzene (PQPOP) were purchased from Fisher (Raleigh, NC). The following chemicals were purchased from Sigma (St. Louis, MO): pyridoxal 5’-phosphate; semicarbazide HCI; p-chloromercuriphenyl sutfonic acid, monosodium salt; EDTA-tetra sodium salt; Drdithiothreitol; DL-~-a~~y~-giyCiIIe. The fotfowing chemicals were purchased from Calbiochem (La Jolla, CA): 2-ammoethyl~sothiouron~um bromide, hydrobr~mide~ I.-aspartic acid; y-aminobutyric acid; L-gtutamtc acid. All other chemicals and solvents were reagent grade. Doub~d~t~~ water was used for all procedures. Scintillation


Aqueous samples were counted using a xylene;Trlton X-114 (2: 1, v/v) liquid sciatillation cucktai1 (GREENE. 1970) containing OSP, PPO and 0.02”/, POPOP. Quenched standards were used for all comparisons between heterogeneous samples.

AG SC-X4 ~rnon~um form (lO@-200 mesh) resin was prepared from the corresponding hydrogen form resin by washing with 2~ ammonium hydroxide. AG 50-X4 sodium form (lO@-200 mesh) and AG l-X2 acetate form (10&2OOmesh) resins were_ prepared as previously described (MADERDRUT,I978). Resins were exhaustively washed with distilled water and stored in 4-5 volumes of distilled water at 5°C. Columns were fashioned from disposable Pasteur pipettes plugged with glass wool. So& tions used in chromatographic procedures were kept in crushed ice. Gktwnic

acid purijkztion


and L-[3-%]Gfutamic acid were purified by cation~xch~e chromat~~apb~ using a 3cm column of AG .50-X4 (lOt%200 mesh, NH: form) resin. The radioisotope (25-250&i) was added to 5O@d oft 576 TCA and the sample was pipetted onto a column that had previously been equilibrated with 4 ml 0.8 M formic acid. The column was washed with 3m1 of distilled water. Glut;rmic acid

1A sodium bromide concentration that gives less than loOo/, inhibition of GAD a&iv&y was chosen to enhance the abiiity of the control sample to detect any contamination of the assay by a ‘GABA’-producing enzyme(s) that has a lower Kg for sodium bromide than GAD.

was eluted wrth 8ml of 0.8 M amrn~~murn acr‘tate huger ipH 3.5) and 500 pi fractions were c&lested. Ttie fracrion~ comprising t-he peak of radioactivt\ were Identified and the corresponding samples were evidc .wpral. Experiments using localized excision and transplantation or explantation of neural tissue during early embryogenesis have suggested that regional differences in the nervous system become determined duiing the neural plate stage (CORNEE. ~~~~:~NARAYANAN & HAMBURGER, 1971; WENGER, I951). These expertments do not discriminate between the determination of a ‘field’ (HARRISON, 1918) and the determination of the individual ceils within that held. The synthesis of a gene product that IS characteristic of a specialized cell type has often been consider& to mdicati: that the fate of that cell has been determtned (WIISONc? cd.1972) Biochemicaf and tmmunohtstnchemiccaJ ewdence suggests that the somata of GABA-secreting neurons are concentrated in the dorsolater% p&t of the dorsal horn (BERGEK, CARTER & LOWRY. 1977; MC-LAUGHLIN,BARBER.SAITO, ROBERTS& WV, 1975a; M~YATA & OTSUKA, 1975). A few cells m the dorsal horn of the chick lumbar spinal cord become post-mitotic as early as Stage 14 (Day 24) (HOLLYDAY & HAMBURGER, 1977). Thus, the preliminary experiments illustrated in Fig. 3 do not discrimmate between 3 alternative (albeit not necessarily mutually exclusive) interpretations: (1) GABA-secreting neurons in the lumbar dorsal horn become pnst-mitotic eat Stage I4 and determination occurs coincident with or +.hortIy after their final mitosis: (2) GABA-secreting neurons

L-Glutamate decarboxylase radiometric microassay method


become determined before their final mitosis; (3) all synapses have been formed. Thus, physical contact between the nascent GABA-secreting neuron and its neurons express small amounts of the GAD gene until target cells is not required for either the initial inducthey become determined, and complete suppression tibn of GAD synthesis or for the earliest increases of the synthesis of inappropriate gene products occurs with determination (cf. CAPLAN & ORDAHL, 1978). in GAD activity (cf. MCLAUGHLIN,WOOD, SAITO & ROBERTS,1975b; OPPENHEIM,&U-WANG & MADERCholine acetyltransferase (acetyl_CoA: choline O-acetyltransferase, EC (ChAc) activity has also DRUT, 1978). Behavioral experiments indicate that GABA recepbeen detected in the lumbar spinal cord at Stage 14 tors are present at least as early as the onset of spon(J. L. MADERDRUT& R. W. OPPENHEIM,unpublished observations); this is apprbximately 6 h (3 stages) taneous motility (BEITZELet al., 1979); electrophysiological experiments indicate that post-synaptic recepbefore any neurons in the ventral horn have undertors may be present before the initiation of synaptogone their final mitosis (HOLLYDAY& HAMBURGER, genesis (D~OND & MILDI, 1962; WWDWARD, 1977). Concurrent measurement of both GAD and HOFFER,SIGGINS & BLOOM,1971). Whether GABA ChAc activity using homogenates from isolated dorsal receptors are present as early as GAD activity is unand ventral halves of the lumbar spinal cord beginknown. ning at Stages 12-13 should resolve these ambiguities. The first synapses in the embryonic chick lumbar spinal cord are detected in the ventrolateral marginal CONCLUSION zone at Day 4; the initial synapses are all axodendritic and contain spherical synaptic vesicles (OPPENHEIM There are other areas of neurobiology besides et al., 1975; SINGER,SKOFF & PRICE, 1978). Some of neuroembryology where the cation-exchange method these early synapses appear to be propriospinal in should be uniquely useful. The cation-exchange origin (SINGER et al., 1978). Axosomatic synapses have method should be useful for estimating the GAD activity of identified somata in invertebrate ganglia and not been detected in the embryonic lumbar spinal of subcellular fractions from discrete regions of the cord before Day 6 (OPPENHEIMet al., 1975); synapses mammalian brain. The method should also be conhave not been detected in the lumbar dorsal horn venient for examining the stoichiometry between earlier than Day 5-6 (SINGERef al., 1978). The initial and CO,-production when GABA formation synapses in the ventrolateral white matter are unlikely is used to estimate GAD activity. to be GABA-secreting synapses (cf. MCLAUGHLINet L-~-‘4Cjglutamate al., 1975a). GAD activity is present in the lumbar spinal cord Acknowledgements-This research was supported by a at least l-2 days before the initiation of synaptogrant from the Alfred P. Sloan Foundation to the Neurogenesis and the total GAD activity of the lumbar biology Program, a grant from Sigma Xi and NIMH spinal cord appears to increase %fold (presumably 16598. I would like to thank R. W. OPPENHEIM for assistdue to both cell proliferation and growth) before any ance and for critically reading a draft of this manuscript.

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L-Glutamate decarboxylase radiometric microassay method


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A radiometric microassay for glutamic acid decarboxylase.

Newoscieme Vol 4, pp. 995 to 1005 Perptxxt Press Ltd 1979. Printed in Great Bntarn A RADIOMETRIC MICROASSAY FOR ACID DECARBOXYLASE GLUTAMIC J. L. M...
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