Abstract
Conventional subcellular fractionation techniques have been applied to human fetal brain (13–15 weeks gestation) and the fractions have been characterized by assaying for marker enzymes, cholinergic binding sites and electron microscopy. Fractionation of the homogenate resulted in a nuclear pellet (P1), a crude mitochrondrial pellet (P2) and a supernatant (S2). Further resolution of the P2 fraction by density gradient centrifugation resulted in two bands at the gradient interfaces and a pellet. The P2 and subsequently the P2B fraction contained intact plasma membrane profiles as judged by the predominance of adenylate cyclase activity and the presence of occluded lactate dehydrogenase which constituted over 70% of the total activity in these fractions. Morphological examination of the gradient fractions revealed that the P2B fraction contains membrane bound structures which resembie synaptosomes prepared from neonatal rat brain. These structures have a granular matrix in which mitochondria and frequently, neurofilaments were observed. Very few synaptic vesicles were present and there was no evidence for post synaptic attachments. The cholinergic markers choline acetyltransferase, acetylcholinesterase and receptor sites defined by quinuclidinyl benzilate and α-bungarotoxin binding were enriched in fractions P2 and P2B which contained the bulk of nerve ending particles. This enriched preparation of fetal synaptosomes may be valuable for functional studies on pre-synaptic terminals in developing brain.
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References
Bull, G., Hebb, C., andRakovic, D. 1970. Choline acetyltransferase activity of human brain tissue during development and at maturity. J. Neurochem. 17:1505–1516.
Brooksbank, B. W., Martinez, M., Atkinson, D. J. andBalazs, R. 1978. Biochemical development of human brain I. some parameters of the cholinergic system. Dev. Neurosci. 1:267–284.
Aguilar, J. S., andLunt, G. G. 1985. Muscarinic receptor sites in human fetal brain. Neurochem. Int. 7:509–514.
Ravikumar, B. V., andSastry, P. S. 1985. Muscarinic cholinergic receptors in human fetal brain: Characterisation and ontegeny of [3H]quinuclidinyl benzilate binding sites in frontal cortex. J. Neurochem. 44:240–246.
Whyte, J., Harrison, R., Lunt, G. G., andWonnacott, S. 1985. Properties of α-bungarotoxin binding sites in fetal human brain. Neurochem. Int. 7:515–523.
Barnard, E. A., andDolly, J. O. 1982. Peripheral and central acetylcholine receptors—how similar are they? Trends in Neurosci. 5:325–327.
Wonnacott, S., Harrison, R., andLunt, G. G. 1982. Immunological cross-reactivity between the α-bungarotoxin binding component from rat brain and nicotinic acetylcholine receptor. J. Neuroimmun. 3:1–13.
Oswald, R. E., andFreeman, J. A. 1981. Alpha-bungarotoxin binding and central nervous system acetylcholine receptors. Neuroscience 6:1–14.
Heilbronn, E., andBartfai, T. 1978. Muscarinic acetylcholine receptor. Progress in Neurobiology. 11:171–188.
De Robertis, E., Pellegrino de Iraldi, A., Rodriguez de Lores Arnaiz, G., andGomez, C. J. 1961. On the isolation of nerve endings and synaptic vesicles. J. Biophys. Biochem. Cytol. 9:229–235.
De Robertis, E., Pellegrino de Iraldi, A., Rodriguez de Lores Arnaiz, R. G., andSalganicoff, L. 1962. Cholinergic and non-cholinergic nerve endings in rat brain I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J. Neurochem. 9:23–35.
Gray, E. G., andWhittaker, V. P. 1962. The isolation of nerve endings from brain; an electron-microscopic study of cell fragments derived by homogenisation and centrifugation. J. Anat. 96:79–88.
Gibson, G. andBlass, J. 1982. Synaptosomes, Pages 289–321,in Hannin, I., andGoldberg, A. M. (eds.), Progress in cholinergic biology; model cholinergic synapses. Raven Press, New York.
Dodd, P., Hardy, J. A., Oakley, A. E., andStrong, A. J. 1981. Synaptosomes prepared from fresh human cerebral cortex; morphology, respiration and release of transmitter amino acids. Brain Res. 224:419–425.
Garey, R. E., Harper, J. W., andHeath, R. G. 1974. Postmortem isolation of synaptosomes from human brain. Brain Res. 82:151–162.
Van Kempen, G. M. J. andVrensen, G. F. M. 1974. Biochemical and morphological integrity of subcellular organelles from human postmortem brain. Neurobiol. 4:12–20.
Hickey, S. M., Ansell, G. B., Mitchel, K., andPearce, G. A. 1976. Subcellular fractions of normal brain substantia nigra and caudate nucleus; a study of their morphology and some enzymes including glutamate decarboxylase and choline acetyltransferase. J. Neurochem. 27:957–962.
Hardy, J. A., Dodd, P. R., Oakley, A. E., Kidd, A. M., Perry, R. H., andEdwardson, J. A. 1982. Use of post-mortem human synaptosomes for studies of metabolism and transmitter amino acid release. Neurosci. Lett. 33:317–322.
Hardy, J. A., Dodd, P. R., Oakley, A. E., Perry, R. H., Edwardson, J. A. andKidd, A. M. 1983. Metabolically active synaptosomes can be prepared from frozen rat and human brain. J. Neurochem. 40:608–614.
Yamamura, H. I., andSnyder, S. H. 1974. Postsynaptic localisation of muscarinic cholinergic receptor binding in rat hippocampus. Brain. Res. 78:320–326.
Fonnum, F. 1969. Radiochemical microassays for the determination of ChAC and AChE activities. Biochem. J. 115:465–472.
Ellman, G. L., Courtney, D., Andres, S. V., andFeatherstone, R. M. 1961. A new and rapid colorimetric determination of acetylcholonesterase activity. Biochem. Pharmac. 7:88–95.
Johnson, M. K. 1960. The intracellular distribution of glycolytic and other enzymes in rat brain homogenates and mitochondrial preparations. Biochem. J. 77:610–618.
Marchbanks, R. M. 1967. The osmotically sensitive potassium and sodium compartments of synaptosomes. Biochem. J. 104:148–157.
Furman, M. A., andShulman, K. 1977. Cyclic AMP and adenyl cyclase in brain tumours. J. Neurosurg. 46:477–483.
Brown, B., Albano, J., Ekins, R. P., andSgherzi, A. M. 1971. A simple and sensitive saturation assay method for the measurement of adenosine 3′:5′-cyclic monophosphate. Biochem. J. 121:561–562.
Porteus, J. W., andClark, B. 1965. The isolation and characterisation of subcellular components of the epithelial cells of rabbit small intestine. Biochem. J. 96:159–171.
Forsdyke, J. B. 1979. A simple apparatus for staining and washing a batch of grids. J. Microscopy 117:437–440.
Wonnacott, S., Harrison, R., andLunt, G. G. 1980. Interrelationship of carbohydrate and the α-toxin binding site on the acetylcholine receptor from Torpedo marmorata. Life. Sci. 27:1769–1775.
De Robertis, E., Rodriguez De Lores Arnaiz, R. G., Alberici, M., Butcher, R. W., andSutherland, E. W. 1967. Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. J. Biol. Chem. 242:3487–3493.
Sinha, A. K., Mahajan, R. G., Mandel, S., Mukopadhyaya, S., Naryanswam, A. andMukherjee, K. L. 1980. Ontogeny of soluble proteins in fetal human brain. Indian J. of Biochem. Biophys. 17:37–41.
Dobbing, J., andSands, J. 1973. The quantitative growth and development of the human brain. Arch. Diseases Childhood 48:757–767.
Sarma, M. K. J., andSubba Rao, K. 1976. Growth and development in different regions of human fetal brain: Changes in wet weight, moisture content and nucleic acids. Indian J. Med. Res. 64:154–161.
Savolainen, H., Palo, J., Riekkinen, P., Moronen, P., andBrody, L. E. 1971. Maturation of myelin protein in human brain. Brain Res. 37:253–263.
Jones, D. G. 1975. Synapses and synaptosomes. Chapman & Hall, London.
Jones, D. G., andRevell, E. 1970. The postnatal development of the synapse: a morphological approach utilising synaptosomes. I. general features. Z. Zellforsch 111:179–194.
Gonatas, N. K., Autillo-Gambetti, L., Gambetti, P., andShafer, B. 1971. Morphological and biochemical changes in rat synaptosome fractions during neonatal development. J. Cell Biol. 51:484–498.
Marks, N., Stern, F., andLajtha, A. 1975. Changes in proteolytic enzymes and proteins during maturation of the brain. Brain Res. 86:307–322.
Barrantes, F. J., andLunt, G. G. 1970. Enzymatic digestion of synapses in rat cerebral cortex. Brain Res. 23:305–313.
Rees, S. 1977. The incidence of ultrastructural abnormalities in the cortex of two retarded human brains (Down's syndrome). Acta Neuropath. 37:65–68.
Swanson, P. D., Harvey, F. H., andStahl, W. L. 1973. Subcellular fractionation of postmortem brain. J. Neurochem. 20:465–475.
Fonnum, F. 1967. The “compartmentation” of choline acetyltransferase within the synaptosome. Biochem. J. 103:262–270.
Wong, P., andPrince, A. K. 1979. The membrane binding of choline acetyltransferase: uptake and acetylation of3H-choline in rabbit cortical slices. Neuropharmacol. 18:511–513.
Spence, M. W., andWolfe, L. S. 1967. Gangliosides in developing rat brain. Isolation and composition of subcellular membranes enriched in gangliosides. Canadian J. of Biochem. 43:671–688.
Ivanshina, Z. 1978. Development of synaptic contacts in the human brain at early stages of embryogenesis. Zhurnol Neuropatologiii Psikhiatrii 76:292–298.
Huttenlocher, P. R., de Courten, C., Garey, L. J. andVan der Loos, H. 1982. Synaptogenesis in human visual cortex-evidence for synapse elimination during normal development. Neurosci. Lett. 33:247–252.
Petit, T. L., Le Boutillier, J. C., Alfano, D. P., andBecher, L. E. 1984. Synaptic development in the human fetus: A morphometric analysis of Normal and Down's syndrome neocortex. Experimental Neurology 83:13–23.
Mollivar, M. E., Kostovic, I. andVan der Loos, H. 1973. The development of synapses in cerebral cortex of the human fetus. Brain. Res. 50:403–407.
De Belleroche, J. S., andBradford, H. F. 1978. Biochemical evidence for the presence of presynaptic receptors on dopaminergic nerve terminals. Brain Res. 142:53–68.
Mills, A., andWonnacott, S. 1984. Antibodies to nicotinic acetylcholine receptors used to probe the structural and functional relationships between brain α-bungarotoxin binding sites and nicotinic receptors. Neurochem. Int. 6:249–257.
Rapier, C., Harrison, R., Lunt, G. G., andWonnacott, S. 1985. Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain. Neurochem. Int. 7:389–396.
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Whyte, J., Harrison, R., Lunt, G.G. et al. Subcellular fractionation and distribution of cholinergic binding sites in fetal human brain. Neurochem Res 11, 1011–1023 (1986). https://doi.org/10.1007/BF00965590
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DOI: https://doi.org/10.1007/BF00965590