Brain Research, 158 (1978) 295-302 © Elsevier/North-HollandBiomedicalPress

295

GLUTAMIC ACID AND ASPARTIC ACID IN THE COCHLEAR NUCLEUS OF THE WALTZING GUINEA PIG

ROBERT J. WENTHOLDand ROBERT L. GULLEY Laboratory of Neuro-otolaryngology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)

(Accepted March 2nd, 1978)

SUMMARY Glutamic acid and aspartic acid were measured in the cochlear nucleus of the genetically deaf waltzing guinea pig. Morphologically, degeneration of spiral ganglion cells and of primary auditory terminals in the cochlear nucleus occurs between 30 and 90 days of ageL Glutamic acid and aspartic acid decrease when primary auditory terminals degenerate. At 3, 10 and 30 days of age, glutamic acid and aspartic acid levels (nmoles per mg protein) in the cochlear nucleus of the waltzer do not differ significantly from those of the normal. Both amino acids are lower in the waltzer cochlear nucleus than in the normal at 60 and 90 days. At 90 days, glutamic acid and aspartic acid are, respectively, 11.2 and 17.0~ lower in the cochlear nucleus of the waltzer than in the cochlear nucleus of the normal. The magnitude of decrease in glutamic acid and aspartic acid after auditory nerve lesion is greatly reduced in the 60and 90-day waltzing guinea pig. In the 90-day waltzer, aspartic acid decreases only 5.3 ~ compared to 30.4 Y/ooin the normal and glutamic acid decreases 4.8 ~o compared to 16.2~ in the normal. Alanine, glutamate decarboxylase and choline acetyltransferase levels in the cochlear nucleus of the waltzer do not differ significantlyfrom those of the normal except at 3 days, when glutamate decarboxylase is slightly elevated in the waltzer. Total protein content of the cochlear nucleus of the waltzer is consistently lower than that of the normal. These results suggest that glutamic acid and aspartic acid are closely associated with primary auditory terminals in the cochlear nucleus.

INTRODUCTION Glutamic acid and aspartic acid decrease in the cochlear nucleus after lesion of the auditory nerve by cochlear ablation 18. The mode of decrease of these amino acids suggests that aspartic acid, and possibly glutamic acid, may be concentrated in the primary auditory terminals in the cochlear nucleus. Several other amino acids and the

296 enzymes choline acetyltransferase (ChAc), glutamate decarboxylase (GAD) and tyrosine hydroxylase, do not decrease in the cochlear nucleus after cochlear ablation 17. An additional method of demonstrating an association between a population of synapses and a particular putative neurotransmitter is to show a selective loss of the substance in mutant animals lacking the specific population of synapses. Such studies have been carried out on the cerebella of micO0,14. The N I H strain of waltzing guinea pig displays a progressive degeneration of auditory function beginning shortly after birth and extending to one to two months of age when the animal is totally deaf 7. Morphological examination shows degeneration beginning with hair cells in the organ of Corti and later progressing to the spiral ganglion cells6,9 and their terminals in the cochlear nucleus 9. Most spiral ganglion cells and primary terminals degenerate between the ages of 30 and 90 days 9. This degeneration allows one to measure decreases in suspected neurotransmitters in the cochlear nucleus as primary auditory terminals degenerate. In this study we have further characterized the terminals of the auditory nerve by measuring glutamic acid and aspartic acid in the cochlear nucleus of the waltzing guinea pig over the time period when the primary auditory terminals degenerate. Furthermore, this represents the initial step in our study of the biochemistry of genetic hearing disorders in experimental animals. METHODS Waltzing guinea pigs are from the stock maintained at the National Institutes of Health. The mutation arose spontaneously from the N I H guinea pig in 1953 and has been maintained by breeding the waltzer with the normal NIH tricolor guinea pig. The genetic defect of the strain is dominant. The waltzing guinea pig described by Ernston 5-7 was initially of this strain. Normal N I H guinea pigs were used as controls. The auditory nerve was lesioned by cochlear ablation as previously described 16. One cochlea was ablated; the contralateral cochlear nucleus served as a control. Cochlear nuclei were dissected and amino acids extracted and assayed as previously described 16. ChAc and G A D were assayed as described s except in the measurement of GAD, ~,-aminobutyric acid (GABA) formed in the reaction was separated on a Dowex column 1. Protein was measured by the method of Lowry et al. 13. Statistical analyses were done using the two-tailed Student's t-test. RESULTS Glutamic acid and aspartic acid levels in the cochlear nucleus of the normal and waltzing guinea pig are shown in Fig. 1. In the normal animal, the levels of both amino acids show little age dependence over the time period studied. In the waltzing guinea pig, glutamic acid and aspartic acid levels did not differ significantly from those of the normal until after 30 days of age. Both amino acids decrease in the waltzer between 30 and 90 days with the greatest decreases occurring between 30 and 60 days. At 60 days of age, glutamic acid levels in the waltzer were

297

H

Normal

o----(3 Waltzer Glutamic Acid 64

z [] I--(D tr tt O

60

rr

52

m

z

56

0 28

Aspartic Acid 26 24 22 20.

o't 3

1

I

I

I

10

30

60

90

AGE, DAYS

Fig. 1. Glutamic acid and aspartic acid levels in the cochlear nucleus of the normal and waltzing guinea pig. Values represent mean ± S.E.M. of 8-12 experiments. For 60-day and 90-day glutamic acid and aspartic acid, P < O.001.

10.4~ lower than in the normal and aspartic acid was 13.3 ~o lower, while at 90 days, glutamic acid and aspartic acid were 11.2~o and 17.0~ lower, respectively, in the waltzer. No further decreases occurred in glutamic acid or aspartic acid in the cochlear nucleus of the waltzing guinea pig up to 120 days (data not shown). Alanine levels in the cochlear nucleus of the waltzing guinea pig did not differ significantly from those of the normal over this time period (Table I). Glutamic acid and aspartic acid were measured in the cerebellum and olfactory bulb to determine if the decrease in these amino acids in the cochlear nucleus was due to general metabolic differences in the animals. Both amino acids (expressed as nmoles/mg protein) were the same in the waltzer (n ~ 4) as in the normal (n ~ 4) (data not shown). ChAc and GAD, synthetic enzymes for acetylcholine and GABA, respectively, showed similar specific activities for the normal and waltzer except at 3 days, GAD in the waltzer was slightly higher than in the normal (Fig. 2). Total protein content of the cochlear nucleus of the waltzer was consistently lower than that of the normal (Fig. 3). Ablation of the normal guinea pig cochlea leads to a rapid decrease in aspartic acid in the cochlear nucleus and to a somewhat slower decrease in the level of glutamic

298 TABLE I

Alanine in the cochlear nucleus of the normal and waltzing guinea pig Data is presented as nmoles alanine per mg protein. Value is parentheses is the number of experiments.

Age (days)

Normal (mean ± S.E.M.)

Waltzer (mean ± S.E.M.)

10 30 90

8.36 :~ 0.42 (8) 8.74 ~ 0.18 (11) 8.51 ~ 0.34 (9)

8.47 ± 0.34 (8) 8.94 ± 0.42 (8) 8.54 ± 0.48 (6)

2.40

[ ] Normal [ ] Waltzer

ZOO 1.60 (.9

1.20 0.80

0.40 0

iiiii

~iili

120 L o

100

L)

80

40

ii

20

ii

60

0

Iili~i 60

90

AGE, DAYS Fig. 2. ChAc and G A D levels in the cochlear nucleus of the normal and waltzing guinea pig. G A D is expressed as nmoles GABA formed per minute per mg protein; ChAc is expressed as pmoles Ach formed per minute per mg protein. Values represent mean ± S.E.M. of 5-8 experiments. For 3-day G A D , P < 0.02.

299

H

NORMAL WALTZER

0.8 "-~ 0.7 0'~

E 0.6 z o

rr O,.

0.5

0.4

0.3 [

3 10

I

30

I

]

60 AGE, DAYS

90

Fig. 3. Total protein content of the cochlear nucleus of the normal and waltzing guinea pig. Values represent mean i S.E.M. of 11-26 experiments.

TABLE II Effect o f cochlear ablation on the levels o f aspartic acid and glutamic acid in the cochlear nucleus o f the normal and waltzing guinea pig Animals were sacrificed 3 days after unilateral cochlear ablation; controls represent levels in the cochlear nucleus on the unoperated side. Data is presented as nmoles amino acid per mg protein. All values are the average of 4 experiments except for the 90°day waltzer which is the average of 6 experiments. Age (days)

Aspartic acid Normal Mean qS.E.M.

10 Lesion Control 30 Lesion Control 60 Lesion Control 90 Lesion Control

18.44-1.8 26.84-4-2.0 16.94-0.4 24.24-0.5 19.2±0.6 26.94-1.2 17.64-0.6 25.3i0.9

Glutamic acid Waltzer

Normal

Per cent Mean 4change S.E.M.

Per cent Mean -4change S.E.M.

--31.3

--20.2

--30.2 --28.6 --30.4

17.84-0.3 22.34-0.3 20.64-0.3 25.44-0.6 19.74-1.0 22.34-0.3 21.64-1.1 22.8-4-1.0

--18.9 --11.7 - - 5.3

Waltzer Per cent Mean -4change S.E.M.

51.7±2.0 --18.2 63.24-0.8 50.14-1.4 --13.0 57.64-0.7 52.44-1.2 --17.9 63.84-0.5 48.84-1.0 --16.2 58.2±0.6

Per cent change

54.64-1.6 --13.2 62.94-2.3 54.64-1.0 - - 9.6 60.44-1.0 53.24-1.3 - - 5.0 56.04-1.5 54.04-1.7 - - 4.8 56.74-2.0

300 acid 16. Although the decrease in both is substantial, the amount represents only a fraction of the total glutamic acid or aspartic acid in the cochlear nucleus. To establish whether the age-dependent decrease in the levels of these amino acids in the cochlear nucleus of the waltzing guinea pig involves the same pool of amino acid as does the decrease after cochlear ablation in the normal animal, we studied the effect of cochlear ablation in the waltzing guinea pig. A three-day survival time was chosen because most primary endings have degenerated in both the normal and waltzer by this timeg, 16. Furthermore, aspartic acid has decreased and reached a plateau and glutamic acid has decreased 16. Table II shows the effect of cochlear ablation on glutamic acid and aspartic acid in the cochlear nucleus of the normal and waltzing guinea pig as a function of age. The per cent decrease of both amino acids in the normal animal changes little over the age studied with aspartic acid decreasing about 30 ~ and glutamic acid about 15 ~. As early as 10 days of age, the per cent decrease of both glutamic acid and aspartic acid in response to cochlear ablation is less in the cochlear nucleus of the waltzer than in that of the corresponding normal. The per cent decrease in aspartic acid remains constant to 30 days and then decreases until at 90 days of age, there is only a 5.3 ~ decrease after cochlear ablation compared to a 30.4 ~ decrease in the normal. The per cent decrease in glutamic acid drops slightly at 30 days in the waltzer, paralleling a decrease which also occurs in the normal animal at this time. The per cent decrease in glutamic acid continues to drop at 60 days in the waltzer and remains at this level at 90 days. At this time glutamic acid decreases only 4.8 ~ in response to cochlear ablation compared to 16.2 ~ for the normal. There were no further changes in the per cent decrease of glutamic acid or aspartic acid in the waltzing guinea pig up to 120 days (n = 4) (data not shown). DISCUSSION Morphologically, degeneration of the cochlea of the waltzing guinea pig begins with the hair cell6,a. Although subtle morphological abnormalities are observed in hair cells of newborn waltzers, hair cell loss is not observed until after 10 days of age with most hair cell loss occurring between 10 and 60 days of age. Degeneration of spiral ganglion cells and corresponding degeneration of primary terminals in the cochlear nucleus occurs mostly between 30 and 90 days of age with the most intense degeneration occurring between 30 and 60 days. About 50 ~ of the spiral ganglion cells remain at 90 days and this number does not decrease up to 170 days9. Remaining spiral ganglion cells appear normal, morphologically, at all ages investigatedL However, remaining primary endings in the anteroventral cochlear nucleus undergo morphological changes; there is a decrease in the number of synaptic vesicles and the synaptic junction becomes flattened9. Glutamic acid and aspartic acid levels in the cochlear nucleus of the waltzing guinea pig decrease between 30 and 90 days of age, the time period when spiral ganglion cells are degenerating. At 90 days, when only about 50 ~ of the spiral ganglion cells remain, glutamic acid and aspartic acid are, respectively, 11.2 ~ and 17.0~ lower in the cochlear nucleus of the waltzer than in the normal. Cochlear

301 ablation, which leads to a decrease in glutamic acid and aspartic acid in the cochlear nucleus of the normal guinea pig 16, causes only a slight decrease in these amino acids in the cochlear nucleus of the 90-day waltzing guinea pig; aspartic acid decreases 5.3 compared to 30.3 ~ in the normal and glutamic acid decreases 4.8 ~ compared to 16.2 ~ in the normal. In the young waltzing guinea pig, before degeneration of spiral ganglion cells and primary terminals, glutamic acid and aspartic acid levels in the cochlear nucleus do not differ significantly from those of the normal animals of the same age. Furthermore, the decrease in these amino acids after cochlear ablation is much closer in magnitude to that of the normal than in the older animals. However, even in the young animals, the decrease in the waltzer is less than in the normal. While the young waltzers (10 and 30 days) have the same levels of glutamic acid and aspartic acid as corresponding normals (Fig. 1), the decrease in these amino acids after cochlear ablation is about 60-70 ~ that of the normal (Table I|). This suggests the waltzing guinea pig may have less aspartic acid and glutamic acid associated with primary terminals in the cochlear nucleus than corresponding normal animals even before morphological degeneration of the primary terminals. ChAc and GAD do not decrease in the cochlear nucleus of the waltzing guinea pig, suggesting a lack of degeneration of neurons containing these enzymes. Furthermore, it supports earlier work suggesting acetylcholine and GABA are not transmitters for the auditory nerveS,15,17. After cochlear ablation the decrease in aspartic acid in the cochlear nucleus parallels the morphological degeneration of primary auditory terminals, suggesting that aspartic acid may be concentrated in the terminals16. Glutamic acid decreases slightly later and its decrease may be secondary to the aspartic acid decrease or glutamic acid may be associated with a specific group of terminals which degenerate more slowly. In the waltzing guinea pig, both glutamic acid and aspartic acid decrease between 30 and 90 days of age with the largest decreases in each occurring between 30 and 60 days. However, aspartic acid continues to decrease between 60 and 90 days in the waltzer, relative to the normal, while glutamic acid remains constant during this time. This point is also illustrated in the response of these amino acids to cochlear ablation. The magnitude of aspartic acid decrease is reduced both between 30 and 60 days as well as 60 and 90 days, while the decrease in glutamic acid, relative to normal, is reduced only between 30 and 60 days. This differential decrease in glutamic acid and aspartic acid may indicate these amino acids are associated with separate populations of primary endings which degenerate at different times in the waltzer. However, our morpholigical data do not show two types of primary terminals in the cochlear nucleus of the waltzing guinea pig9. The present results, showing a decrease in glutamic acid and aspartic acid concomitant with the morphological degeneration of spiral ganglion cells and primary terminals in the waltzing guinea pig, support our earlier study showing a decrease in these amino acids after cochlear ablation 16. By using the waltzing guinea pig we have shown that glutamic acid and aspartic acid decrease in the cochlear nucleus with degeneration of primary auditory terminals without surgically lesioning the auditory nerve. This indicates that the decrease in these amino acids after surgical lesion is not

302 due to u n i n t e n t i o n a l damage to the cochlear nucleus d u r i n g surgery. I n the waltzing guinea pig degeneration of p r i m a r y terminals in the cochlear nucleus occurs over a twom o n t h period, while in the n o r m a l animal, degeneration after cochlear a b l a t i o n is essentially complete after three days. I n b o t h cases the decreases in glutamic acid a n d aspartic acid coincide with the degeneration of the p r i m a r y auditory terminals. Together, the results suggest these a m i n o acids are closely associated with the p r i m a r y a u d i t o r y terminals a n d are possibly c o n c e n t r a t e d within the terminal. Since glutamic acid a n d aspartic acid are considered as possible excitatory neurotransmitters2-4,11,12, our results are consistent with a possible n e u r o t r a n s m i t t e r f u n c t i o n for one or b o t h of these a m i n o acids in the cochlear nucleus. ACKNOWLEDGMENTS We t h a n k Dr. J. Fex for his support a n d S. W. Jones for excellent technical assistance. REFERENCES 1 Chude, O. and Wu, J. Y., A rapid method for assaying enzymes whose substrates and products differ by charge. Application to brain L-glutamate decarboxylase, J. Neurochem., 27 (1976) 83-86. 2 Curtis, D. R., Gamma-aminobutyric and glutamic acids as mammalian central transmitters. In S. Bed, D. D. Clarke and D. Schneider (Eds.), Metabolic Compartmentation and Neurotransmission: Relation to Brain Structure and Functions, Plenum Press, New York, 1975, 11-36. 3 Curtis, D. R. and Johnston, G. A. R., Amino acid transmitters in the mammalian central nervous system. In R. H. Adrian, E. Helmreich, H. Holzer, R. Jung, K. Kramer, O. Krayer, F. Lynen, P. A. Miescher, H. Rasmussen, A. E. Renold, U. Trendelenburg,K. Ullrich, W. Vogt and A. Weber (Eds.), Reviews of Physiology, 69 (1974) 97-188. 4 DeFeudis, F. V.,Amino acids as central neurotransmitters,Ann. Rev. Pharmacol., 15 (1975) 105-130. 5 Ernstson, S., Heredity in a strain of the waltzing guinea pig, Acta. otolaryng., 69 (1970) 358-362. 6 Ernstson, S., Cochlear morphology in a strain of the waltzing guinea pig, Acta. otolaryng., 71 (1971) 469~82. 7 Ernstson, S., Cochlear physiology and hair cell population in a strain of the waltzing guinea pig, Acta. otolaryng., Supplement 297 (1972) 1-24. 8 Fex, J. and Wenthold, R. J., Choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the cochlea and cochlear nucleus of the guinea pig, Brain Research, 109 (1976) 575-585. 9 Gully, R. L., Wenthold, R. J. and Neises, G. R., Changes in the synapses of spiral ganglion cells in the rostral anteroventral cochlear nucleus of the waltzing guinea pig following hair cell loss, Brain Research, 158 (1978) 279-294. 10 Hudson, D. B., Valcana, T., Bean, G. and Timeras, P. S., Glutamic acid: A strong candidate as the neurotransmitter of the cerebellar granule cell, Neurochem. Res., 1 (1976) 73-81. 11 Johnson, J. L., Glutamic Acid as a synaptic transmitter in the nervous system. A review, Brain Research, 37 (1972) 1-19. 12 Krnjevic, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418540. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 14 McBride, W. J., Aprison, M. H. and Kasano, K., Contents of several amino acids in the cerebellum, brain stem and cerebrum of the 'staggerer', 'weaver' and 'nervous' neurologically mutant mice, J. Neurochem., 26 (1976) 867-870. 15 Wenthold, R. J. and Fex, J., Transmitter related enzymes in cochlea and cochlear nucleus of guinea pig, Trans. Amer. Soc. Neurochem., 7 (1976) 193. 16 Wenthold, R. J., Gulley, R. L., Aspartic acid and glutamic acid levels in the cochlear nucleus after auditory nerve lesion, Brain Research, 138 (1977) 111-123. 17 Wenthold, R. J. and Morest, D. K., Transmitter related enzymes in the guinea pig cochlear nucleus, Neurosci., Abstr., 2 (1976) 28.

Glutamic acid and aspartic acid in the cochlear nucleus of the waltzing guinea pig.

Brain Research, 158 (1978) 295-302 © Elsevier/North-HollandBiomedicalPress 295 GLUTAMIC ACID AND ASPARTIC ACID IN THE COCHLEAR NUCLEUS OF THE WALTZI...
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