Localization of elevated glutaminase immunoreactivity in small DRG neurons

Brain Research, 336 (1985) 158-1 (al

158

Elsevier BRE 20842

Localization of elevated glutaminase immunoreactivity in small DRG neurons CHARLES B. CANGRO 1, PAUL M. SWEETNAM l, JEAN R. WRATHALL2, WAYNE B. HASER 3, NORMAN P. CURTHOYS 3 and JOSEPH H. NEALE 1

Departments of lBiology and 2Anatorny, Georgetown University, Washington, DC 20057 and 3Department of Biochemistry, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (U.S.A.) (Accepted January 3rd, 1985)

Key words: spinal sensory neurons - - glutamate - - glutaminase - - immunohistochemistry

Glutamate has long been considered to be a neurotransmitter candidate in vertebrate spinal sensory nerve cells. We report here the first immunohistochemical evidence in support of this hypothesis. We find that up to 30% of the moderately small dorsal root ganglion neurons in the rat contain elevated levels of glutaminase immunoreactivity. This enzyme, which mediates the synthesis of glutamate from glutamine, is not found at these high levels in large diameter neurons of the same ganglia. In contrast, another enzyme associated with glutamate metabolism, aspartate aminotransferase, is rather uniformly distributed within neurons of the sensory ganglia. These data define a subpopulation of sensory neurons which appear to contain an elevated capacity to synthesize glutamate through the glutamine cycle and suggest that glutaminase immunoreactivity may be an indicator of glutamatergic function in some nerve cells.

Neurons of the dorsal root ganglia ( D R G ) subserve a variety of sensory modalities and subpopulations of these cells a p p e a r to contain different neurotransmitters, including peptides15,22,30, purines9, 22 and catecholamines 23. Evidence suggesting that glutamate or a closely related molecule is an excitatory neurotransmitter in D R G cells is derived from its relative distribution in dorsal roots 13,26, D R G 13,16 and spinal cord 17 as well as its physiological action on spinal neuronsS, 21. The ubiquitous cellular distribution of glutamate, the m y r i a d pathways available for its biosynthesis and the c o m p a r t m e n t a t i o n 3,11 of its metabolism have militated against attempts to localize enzymes which might specifically identify glutamatergic neurons. The g l u t a m i n e - g l u t a m a t e cycle, mediated by glial cell glutamine synthetase and neuronal glutaminase, has been p r o p o s e d as a potentially significant pathway for the synthesis of glutamate for neurotransmission4,5, 27. In D R G , glutamate is efficiently taken up by satellite cells11,12,28, while the small, type B, sensory neurons within the ganglia efficiently accumulate glutaminel2. This, together with the effective conversion of glutamine to glutamate by D R G 12, suggests the possibility that glutaminase ~s

may have a significant role in the synthesis of glutamate for neurotransmission in some sensory neurons. To test this hypothesis, we have utilized antibody p r e p a r e d against glutaminase6, 7 to examine tissue sections of rat D R G . P h o s p h a t e - d e p e n d e n t glutaminase was purified from rat kidney 7. New Z e a l a n d white rabbits were immunized with the enzyme and i m m u n e sera obtained with characteristics similar to those previously described 6. Dorsal root ganglia were o b t a i n e d from the cervical, thoracic and l u m b a r spinal regions of adult male Wistar rats following transcardial perfusion with 4% freshly d e p o l y m e r i z e d p a r a f o r m a l d e hyde in p h o s p h a t e buffer (100 mM, p H 7.4). Ganglia were postfixed for 1 h, rinsed for 3 days in phosphate-buffered saline with 30% sucrose and frozen at - 9 0 °C in isopentane. T w e n t y - f i v e / ~ m frozen sections of D R G were o b t a i n e d and incubated for 1 h at 37 °C in antiserum diluted 1:800 with phosphate-buffered saline containing 2.5% bovine serum albumin and 0.3% Triton X-100 followed by incubation with a 1:40 dilution of fluoresceinated swine anti-rabbit serum (Accurate Chemical) for 1 h at 22 °C. Primary and secondary antisera were a d s o r b e d with 5 mg/ml

Correspondence: J. H. Neale, Georgetown University, Washington. DC 20057, U.S.A. 0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)

159

Fig. 1. A subpopulation of DRG neurons in these sections through sensory ganglia exhibit intense immunofluorescence following reaction with antiserum prepared against glutaminase. More than 80% of the cells in the ganglia present relatively weak fluorescence under these conditions. The bar is 30/~m.

of rat liver powder after dilution. Reactions were observed and photographed under epi-illumination with immersion objective lenses using a Zeiss Photoscope. Three ganglia were sectioned from each of 3 spinal levels. A subpopulation of cell bodies exhibited intense glutaminase immunoreactivity (IR) in each of these ganglia (Fig. 1). The reaction product was punctate in nature and rather evenly distributed throughout the cytoplasm. This intracellular distribution is consistent with a mitochondrial localization of the enzyme 7, but the level of resolution presented here is not sufficient to confirm this. A limited number of apparently reactive neurites were also observed within the bundles of neurites near the ganglia. We found that 16% (n = 1405) of the cervical neurons, 18% (n = 1892) of the thoracic neurons and 18% (n = 1984) of the lumbar neurons, which we observed, exhibited elevated glutaminase-IR. That the IR represents the enzyme glutaminase is supported by the observation that when this same serum was adsorbed over a solid phase preparation of highly purified enzyme, the serum components which remained unbound to the enzyme failed to produce an immunofluorescent reaction on D R G sections. In order to test the possibility that these cells might

simply be metabolically more active and thus possess generally elevated enzyme levels, we incubated D R G sections from the same ganglia with murine antiserum which had been prepared against the enzyme, aspartate aminotransferase. Very little D R G IR was observed with this serum under conditions which we have used to detect differentially elevated aspartate aminotransferase levels in sections of rat retina similar to that reported by others1, 20. At higher antibody concentrations, aspartate aminotransferase-IR could be detected in D R G neurons but there was no differential localization of the type observed with the serum against glutaminase. D R G neurons have been morphologically characterized through analysis of their histochemistry9,22, 29, size 19 and ultrastructure10,25. In order to correlate our data with these observations, we have analyzed the size and staining characteristics of the neurons containing elevated glutaminase-IR. We observed neuronal perikarya with diameters ranging from about 15 ~m to 80/~m in D R G sections (Fig. 2). The glutaminase-IR cells were generally between 20 ~tm and 40/~m in diameter with very few of the IR cells being smaller than 20/~m or larger than 45/~m. In some cases, sections were stained with silver according to the method of Palmgren24 following immunohisto-

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while a group of nonfluorescent cells, presumably those containing more concentrated neurofilaments, were highly stained. In contrast, toluidine blue, a Nissl stain which has been used to identify the small, type B, neurons 12 reacted well with most of the gluta-

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minase-IR cells. However, many non-fluorescent cells were also stained with toluidine blue. Using a

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Fig. 2. Several sections from each of 3 DRG from cervical, thoracic and lumbar regions (9 ganglia) were evaluated for glutaminase-IR. The diameters of those cells containing a distinct nucleus in the tissue section were measured in the longest apparent axis and these cells were scored for the presence (solid columns) or absence (stippled columns) of intense glutaminaseIR. Each column represents, at its maximum, the total number of cells within an estimated diameter range of 5/~m. The values at the top of each column are the percent of the neurons within that size range which contained elevated glutaminase-IR. Note that of the more than 300 neurons with diameters greater than 45/~m, only 3 possessed elevated glutaminase-IR. chemical evaluation. Cells with elevated glutaminase-IR did not stain intensely with this method

1 Altschuler, R. A., Mosinger, J. L., Harmison, G. C., Parakkal, M. H. and Wenthold, R. J., Aspartate aminotransferase-like immunoreactivity as a marker for aspartate/glutamate in guinea pig photoreceptors, Nature (Lond.), 298 (1982) 657-659. 2 Altschuler, R. A., Wenthold, R. J., Schwartz, A. M., Haser, W. G., Curthoys, N. P., Parakkal, M. H. and Fex, J., Immunocytochemical localization of glutaminase-like immunoreactivity in the auditory nerve, Brain Research, 291 (1984) 173-178. 3 Berl, S. and Clark, D. D., The metabolic compartmentation concept. In Glutamine, Glutamate and G A B A in the Central Nervous System, Alan R. Liss, New York, 1983, pp.:205-217. 4 Bradford, H. F., Ward, H. K. and Thanki, C. M., Glutamine as a neurotransmitter precursor: complementary studies in vivo and in vitro on the synthesis and release of transmitter glutamate and G A B A . In Glutamine, Glutamate and GABA in the Central Nervous System, Alan R. Liss, New York, 1983, pp. 249-260.

small D R G neurons with elevated glutaminase-IR. These cells may well be those which have been identified radioautographically as efficiently taking up [3H]glutamine from extracellular medium 12, They appear to have the size and Nissl staining characteristics of the type B cells but do not include the smallest diameter neurons. In contrast to the putative glutamatergic neurons of the auditory system in which both aspartate aminotransferase and glutaminase-IR are elevated 2, we have detected only the differential elevation of the latter enzyme in these spinal sensory cells. The role of elevated glutaminase in these cells remains to be clearly established. It may signify an elevated metabolic d e m a n d which relates to the sensory modality served by these neurons or possibly a differential participation in the g l u t a m i n e - g l u t a m a t e cycle for the synthesis of glutamate or a derivative14 to be utilized in neurotransmission.

5 Cotman, C. W., Foster, A. and Lanthorn, T., An overview of glutamate as a neurotransmitter. In Glutamate as a Neurotransmitter, Raven Press, New York, 1981, pp. 1-27. 6 Curthoys, N. P., Kuhlenschmidt, T. and Godfrey, S. S., Phosphate-dependent glutaminase from rat kidney: cause of increased activity in response to acidosis and identity with glutaminase from other tissues, Arch. Biochem. Biophys., 172 (1976) 162-167. 7 Curthoys, N. P , Kuhlenschmidt, T. and Godfrey, S. S., Regulation of renal ammoniagenesis: purification and characterization of phosphate-dependent glutaminase from rat kidney, Arch. Biochem. Biophys., 174 (1976) 82-89. 8 Davies, J. and Watkins, J. C., Actions of o and Lforms of 2amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate in the cat spinal cord, Brain Research, 235 (1982) 378. 9 Dodd, J., Jahr, C. E., Hamilton, P. N., Heath, M. J. S., Matthew, W. D. and JesseU, T. M., Cytochemical and physiologicalproperties of sensory and dorsal horn neurons that transmit cutaneous sensation. In Cold Spring Harbor

161 Symposium on Quatitative Biology, Cold Spring Harbor Laboratory, New York, 1983, pp. 685-695. 10 Duce, I. R. and Keen, P., An ultrastructural classification of the neuronal cell bodies of the rat dorsal root ganglion using zink iodide-osmium impregnation, Cell Tissue Res., 185 (1977) 263-277. 11 Duce, I. R. and Keen, P., Morphological evidence for compartmentation of glutamine and glutamate metabolism in sensory ganglia, Neurosci. Lett., Suppl. 1 (1978) 257. 12 Duce, I. R. and Keen, P., Selective uptake of [3H]glutamine and [3H]glutamate into neurons and satellite cells of dorsal root ganglia in vitro, Neuroscience, 8 (1983) 861-866. 13 Duggan, A. W. and Johnston, G. A. R., Glutamate and related amino acids in cat spinal roots, dorsal root ganglia and peripheral nerves, J. Neurochem., 17 (1970) 1205-1208. 14 Ffrench-Mullen (sic), J. M. H., Zaczek, R., Koller, K. J., Coyle, J. T. and Carpenter, D. O., Actions of N-acetylaspartylglutamate on mammalina neurons, Soc. Neurosci. Abstr., 9 (1983) 444. 15 Hfkfelt, T., Elde, R., Johansson, O., Luft, R., Nilson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 16 Johnson, J. L. and Aprison, M. H., "l'he distribution of glutamic acid, a transmitter candidate, and other amino acids in the dorsal sensory neuron of the cat, Brain Research, 24 (1970) 285-292. 17 Jones, I. M., Jordan, C. C., Morton, I. K. M., Stagg, C. J. and Webster, R. A., The effect of chronic dorsal root section on the concentration of free amino acid in the rabbit spinal cord, J. Neurochem., 23 (1974) 1239-1244. 18 Kvamme, E., Glutaminase (PAG). In Glutamine, Glutamate and G A B A in the Central Nervous System, Alan R. Liss, New York, 1983, pp. 51-67. 19 Lawson, S. N., The postnatal development of large light and small dark neurons in mouse dorsal root ganglia: a statistical analysis of cell numbers and size, J. Neurocytol., 8 (1979) 275-294.

20 Lin, C.-T., Li, H.-Z. and Wu, J.-Y., Immunocytochemical localization of L-glutamate decarboxylase, gamma-aminobutyric acid transaminase, cysteine-suifinic acid decarboxylase, aspartate aminotransferase and somatostatin in rat retina, Brain Research, 270 (1983) 273-284. 21 Mayer, M. L., Westbrook, G. L. and Guthrie, P. B., Voltage-dependent block by Mg ÷2 of NMDA responses in spinal cord neurones, Nature (Lond.), 309 (1984) 261-263. 22 Nagy, J. I. and Hunt, S. P., Fluoride-resistant acid phosphatase containing neurons in dorsal root ganglia are separate from those containing substance P or somatostatin, Neuroscience, 7 (1982) 89-97. 23 Price, J. and Mudge, A.W., A subpopulation of rat dorsal root ganglion neurones is catecholaminergic, Nature (Lond.), 301 (1983) 241-243. 24 Palmgren, A., A rapid method for selective silver staining of nerve fibers and nerve endings in mounted paraffin sections, Acta zool., 29 (1984) 378-392. 25 Rambourg, A. M., Clermont, Y. and Beaudet, A. J., UItrastructural features of six types of neurons in rat dorsal root ganglia, J. Neurocytol., 12 (1983) 47-66. 26 Roberts, P. J. and Keen, P., High-affinity uptake system for glutamine in rat dorsal roots but not in nerve-endings, Brain Research, 67 (1974) 352-357. 27 Schank, R. P. and Aprison, M. H., Present status and significance of the glutamine cycle in neural tissues, Life Sci., 28 (1981) 837-842. 28 Schon, F. and Kelly, J. S., Autoradiographic localization of [3H]GABA and [3H]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. 29 Spater, H. W., Schnitzer, J. A., Quintana, N., Spater, S. H. and Novikoff, A. B., Neuronal phosphatase activities with ARA-AMP and ARA-ATP as substrates, J. Histochem. Cytochem., 29 (1981) 639-702. 30 Sweetnam, P. M., Neale, J. H., Barker, J. L. and Goldstein, A., Localization of immunoreactive dynorphin in neurons cultured from spinal cord and dorsal root ganglia, Proc, nat. Acad. Sci. U.S.A., 79 (1982) 6742-6746.