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Brain Research, 133 (1977) 172-176 © Elsevier/North-Holland Biomedical Press

Glutamic acid decarboxylase and choline acetyltransferase in human foetal brain

JEAN S. GALE MRC Neurochemical Pharmacology Unit, Department of Pharmacology, University of Cambridge, Hills Road, Cambridge, CB2 2QQ (Great Britain)

Accepted May 18th, 1977)

The development of neurotransmitter systems in the human foetal brain is of interest to those investigating hereditary neurological diseases. The appearance o f cell-specific proteins, such as the neurotransmitter biosynthetic enzymes, may indicate cell differentiation and neuronal pathway formation in the growing brain. The measurement of enzyme activity, as opposed to the level of the transmitter itself, can give a more accurate representation of the changes occurring, as these proteins are more stable post-mortem 13. The neurological disorder Huntington's chorea, is known to cause decreases in the levels of glutamic acid decarboxylase (GAD) and, to a lesser extent, choline acetyltransferase (CAT) in post-mortem brains analyzed 2. This hereditary disease shows dominant gene inheritance and it would be of interest to determine if the brains of foetuses from parents either suffering from Huntington's chorea, or 'at risk' of developing it, also show the decrease in these enzyme activities. This study represents an initial investigation into the activities of G A D and CAT in neurologically normal foetal brains. Foetal heads from therapeutic abortions were used in this study. Brains from foetuses delivered by hysterotomy were obtained from 11 to 20 weeks after conception; older foetuses were delivered after prostaglandin infusion. Prostaglandin terminations often produced mis-shaped heads, making dissection more difficult. Foetal age was determined from the menstrual history of the mother and from foetal size. Foetuses were placed at 4 °C immediately after delivery. Heads were removed within 24 h of delivery and frozen quickly in the vapour phase of liquid nitrogen, before being stored at - - 2 0 °C. Previous studies with mouse and adult post-mortem brains show that the activity of G A D and CAT is relatively stable under these storage conditions, the critical stage being the time between death and placement of the tissues at 4 °C 2. Frozen foetal heads were first sliced sagittally, using a domestic meat slicer. A slice, about 5 mm in width, from the right hand side of the medial sagittal cut was dissected on a freezing surface at approximately - - 5 °C. Frontal cortical samples were dissected from cortical regions corresponding to Brodmann's areas 9, 10, and 11.

173 TABLE I GAD activity of the human foetal brain Age (weeks)

N*

11-14

4

15-17

4

17-20

4

21-23

4

24-26

4

28 Adult***

1

GAD (ktmoles/h/g protein) ** Frontal cortex

Basal ganglia

Cerebellum

2.4 ± 2.1 (0-8.5) 1.8 ± 0.5 (0.8-2.9) 4.1 ± 1.8 (2.0-9.5) 3.7 ± 1.4 (1.045.1) 6.4 -4- 2.3 (1.1-10.8) 3.0 37.8 4- 4.3 (34)

10.0 -4- 2.5 (5.0-17.0) 39.7 ± 10.7 (26.4-71.5) 30.6 ± 10.5 (7.0-52.0) 18.7 ± 8.1 (9.0-43.0) 13.6 -4- 4.3 (6.5-25.8) 5.0 43.9 4- 3.0 (99)

10.8 ± 4.0 (3.5-18.0) 45.6 ± 18.1 (14.0-93.6) 16.2 ± 6.3 (8.5-35.0) 8.4 ± 1.3 (6.5-10.8) 17.7 4- 8.9 (3.5-34.0) 2.5 16.04- 4.3 (5)

* N is the number of foetal brains in each age group. ** Mean ± S.E. (range). *** The numbers of adult brains assayed are given in parenthesis for each area.

The whole cerebellum was removed from the slice at its junction with the pons. The basal ganglia samples were taken from the mid-basal region of the brain, just ventral to the developing ventricles and dorsal to the brain stem. Discrete dissection of the nuclei was difficult and, therefore, for the larger brains this area was sectioned into small squares which were removed for assay. The squares with the highest G A D activity corresponded anatomically to the region of the basal ganglia. Frozen samples were weighed and homogenized in ice-cold deionized distilled water. A 20 ~ (w/v) homogenate was used for all assays. Both enzymes were assayed in duplicate on the same homogenate. Values of G A D and CAT activity in normal adult human brains are from results collected in this laboratory since 1973, on a total of 99 post-mortem control brains, and expressed in #moles/h/g protein. Collection, storage and dissection of these brains has been described previously% For comparison with whole foetal cerebellum activity, 5 control adult cerebella were homogenized whole at 20 ~ (w/v) in deionized distilled water, and an aliquot assayed for G A D and CAT. G A D was assayed by the 14CO2 trapping method of Roberts and Simonsen 14. With smaller samples a microadaption of this method, using only 5/zl of homogenate, was used 11. CAT activity was measured by the method of Fonnum 8. Protein was measured by the method of Lowry et al. 12, using bovine serum albumin as the standard. Glutamic acid decarboxylase. The activity of G A D in the frontal cortex, basal ganglia and cerebellum of 21 foetal brains is shown in Table I. G A D activity in the foetal frontal cortex was low when compared to adult cortical values (37.8 -k 4.3 (34)). There appeared to be no significant change in activity with foetal age.

174 TABLE II CAT activity of the human foetal brain Age (weeks)

N*

11-14

4

15-17

4

18-20

4

21-23

4

24 26

4

28 Adult***

1

CAT (ttmoles/h/g protein) * * Frontal cortex

Basal ganglia

Cerebellum

3.2 ± 0.6 (1.8~,.8) 5.9 ± 1.09 (4.0-8.5) 4.7 ~ 1.2 (1.2-6.4) 1.9 ± 0.5 (1.1-3.4) 2.3 ± 0.9 (0.14.5) 1.8 5.2 ± 0.6 (20)

4.1 i 1.2 11.7 ± 3.8 (0.9-6.4) (1.9-18.2) 17.8 ± 3.199 73.4 ± 23.6 ( 1 1 . 7 - 2 3 . 4 ) (23.4-127.0) 10.6 ± 4.2 44.3 :~ 21.5 (2.7-18.4) (6.7-96.8) 6.0 ± 1.9 17.6 ± 7.6 (2.9-10.8) (7.1-39.5) 1.6 ± 0.4 12.5 i 8.1 (0.4-2.1) (0.5-36.0) 7.6 1.7 183.4± 10.7 (75) 3.0 ~ 0.3 (5)

* N is the number of foetal brains in each age group. ** Mean 4- S.E. (range). *** The numbers of adult brains assayed are given in parentheses for each area. 9p < 0.05 and 99 p < 0.0l when compared to the 24-26 week age group. In the basal ganglia the maximal G A D activity was found in the brains of foetuses aged from 15 to 17 weeks (39.7 ± 10.7 (4)/zmoles/h/g protein). Activity remained at this level until 20 weeks, after which it dropped to below 20 #moles/h/g protein. Control adult caudate has a comparable G A D activity of 43.9 ± 3.0 (99). Cerebellar samples also showed high G A D activity between 15 and 17 weeks, although the difference between activity at this age and at 24 to 26 weeks was not significant. The activity at 15 to 17 weeks (45.6 :~ 18.1 (4) ftmoles/h/g protein) was higher than the activity found in whole adult cerebella, (16.0 ~- 4.3 (5)), but again this difference was not significant. Choline acetyltransferase. The activity of CAT in the frontal cortex, basal ganglia and cerebellum of the same foetal brains is shown in Table II. Frontal cortical CAT shows maximal activity (5.9 ± 1.0 (4) #moles/h/g protein) at 15-17 weeks and this level is comparable to the activity found in the adult frontal cortex (5.2 ! 0.6 (20)). Again levels of activity appear to drop significantly (P < 0.05) in foetal brains of 24-26 weeks. CAT shows the same significant peak of activity in the basal ganglia between 15 and 17 weeks and maximal levels at this age (17.8 ~ 3.1 (4) #moles/h/g protein) are about 10 ~ of the levels in adult caudate, (183.4 ~ 10.7 (75) #moles/h/g protein). The cerebellum was the region with the highest CAT activity in 20 out of the 21 brains assayed. Maximal activity, again between 15 and 17 weeks, was 73.4 ± 23.6 (4) #moles/h/g protein. After 20 weeks levels appeared to drop, but activity at 24-26 weeks was not significantly lower. When whole adult cerebellum was assayed a level of 3.0 ~ 0.3 (5) #moles/h/g protein was found. Foetal CAT activity at 15-17 weeks was significantly higher than adult cerebellar CAT (P < 0.025).

175 Proteins. The average protein concentration of the foetal tissues increased slowly over the interval studied, from 35 #g/mg tissue at 13.5 weeks up to about 50/zg/mg tissue at 28 weeks. Dobbing and Sands 5, measuring DNA concentrations, have shown two phases of cellular multiplication in the human foetal brain, the first between 15 and 20 weeks, and the second after 25 weeks until birth. It was postulated that the early growth spurt represented neuronal division, and the latter phase glial cell proliferation in the human foetal brain. A peak in activity of neuronal cell marker enzymes such as CAT and G A D would be expected at the point of maximal growth of these ceils, the specific activity dropping as the rate of glial cell division increased. These enzymes are thought to be located specifically in the cell bodies, axons and synapses of GABA-containing and cholinergic neurones 9, although small amounts of G A D are also detectable in glial cells 11,15. The results presented here show a peak in CAT activity between 15 and 17 weeks in the three brain regions studied, and of G A D activity in the basal ganglia and cerebellum (Tables I and II). In an earlier investigation Bull et al. a measured CAT activity in several areas of 7 human foetal brains aged from 8 to 32 weeks and found increasing activity with age. It is possible that the lower activities in foetal brains older than 20 weeks may reflect ,the poor state of preservation of tissue enzymes in the greater proportion of prostaglandin terminations in this age group. However, the two older hysterotomy terminations obtained, aged 24 and 28 weeks, also had lower activity than the 15-20 week group, and prostaglandin-terminated cases between 21 and 23 weeks showed higher activities than those above 23 weeks. High activities of CAT and G A D were measured in the foetal cerebellum from 15 to 17 weeks. Activities fell after 20 weeks but remained higher than in normal adult whole cerebella. Different growth rates have been described for cerebellum and cerebrum in manT, TM,pig 4 and guinea pig6; the cerebellum appears to have a very fast rate of growth relative to the rest of the brain. Dendritic aborization appears to cease in this region after 20 weeks TM. The early peak of G A D and CAT activity may reflect this rapid development of neurones. High CAT activity in foetal cerebellum was also measured by Bull et al. 3. Their data show a 20-fold difference between foetal and adult cerebellar CAT. This investigation also noted a low CAT activity in foetal caudate when compared to adult values, as shown in this paper. The appearance of a peak of measurable G A D activity between 15 and 20 weeks in the basal ganglia and cerebellum may be useful in future work with foetal material carrying the gene for Huntington's chorea.

The author is grateful for the supply of brain tissue from the Royal Marsden Hospital Foetal Tissue Bank, London, which is supported by MRC funds, and the Public Health Laboratory of Addenbrookes Hospital, Cambridge. I also thank Dr. E. D. Bird and Dr. L. L. Iversen for helpful discussions and assistance in the preparation of the manuscript.

176 1 Bird, E. D., Gale, J. S. and Spokes, E. G., Huntington's chorea: postmortem activity of enzymes involved in cerebral glucose metabolism, J. Neurochem., in press. 2 Bird, E. D. and Iversen, L. L., Huntington's chorea: post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia, Brain, 97 (1974) 457-472. 3 Bull, G., Hebb, C. and Ratkovic, D., Choline acetyltransferase activity of human brain tissue during development and at maturity, J. Neurochem., 17 (1970) 1505-1516. 4 Davison, A. N. and Dobbing, J., The developing blain. In A. N. Davison and J. Dobbing (Eds.), Applied Neurochemistry, Blackwell Scientific Publications, Oxford, 1968 pp. 253-286. 5 Dobbing, J. and Sands, J., Timing of neuroblast multiplication in developing human brain, Nature (Lond.), 226 (1970) 639-640. 6 Dobbing, J. and Sands, J., Growth and development of the brain and spinal cord of the guinea pig, Brain Research, 17 (1970) 115-123. 7 Dunn, H. L., The growth of the central nervous system in the human foetus as expressed by graphic analysis and empirical formulae, J. comp. Neurol., 18 (1921) 345-393. 8 Fonnum, F., Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465-472. 9 Fonnum, F., The localisation of glutamate decarboxylase, choline acetyltransferase, and aromatic amino acid decarboxylase in mammalian and invertebrate nervous tissue. In S. Bed, D. D. Clarke and D. Schneider (Eds.), Metabolic Compartmentation and Neurotransmission, Plenum Press, New York, 1975, pp. 99-122. 10 Howard, E., Granoff, D. M. and Buynovszky, P., DNA, RNA and cholesterol increases in cerebrum and cerebellum during development of human fetus, Brain Research, 14 (1969) 697-706. 11 Kanazawa, I., Iversen, L. L. and Kelly, J. S., Glutamate decarboxylase activity in the rat posterior pituitary, pineal gland, dorsal root ganglion and superior cervical ganglion, J. Neurochem., 27 (1976) 1267-1269. 12 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with th~ Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 13 McGeer, P. L. and McGeer, E. G., Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea, J. Neurochem., 26 (1976) 65 76. 14 Roberts, E. and Simonsen, D. G., Some properties of L-glutarnic decarboxylase in mouse brain, Biochem. Pharmacol., 12 (1963) 113-134. 15 Schon, F., Beart, P. H., Chapman, D. and Kelly, J. S., On GABA metabolism in the gliocyte cells of the rat pineal gland, Brain Research, 85 (1975) 479-490.

Glutamic acid decarboxylase and choline acetyltransferase in human foetal brain.

172 Brain Research, 133 (1977) 172-176 © Elsevier/North-Holland Biomedical Press Glutamic acid decarboxylase and choline acetyltransferase in human...
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