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Amino acid release from biopsy samples of temporal neocortex from patients with Alzheimer's disease
138
Brain Research, 264 (1983) 138-141 Elsevier Biomedical Press
Amino acid release from biopsy samples of temporal neocortex from patients with Alzheimer's disease C. C. T. SMITH, D. M. BOWEN, N. R. SIMS, D. NEARY and A. N. DAVISON
Miriam Marks Department of Neurochemistry, Institute of Neurology, Queen Square, London WCI N 3BG and (D.N.) Department of Neurology, Manchester Royal Infirmary, Manchester 13 (U. K.) (Accepted November 16th, 1982)
Key word~: neurotransmitter release - amino acids - human brain - temporal neocortex - Alzheimer's disease
Tissue prisms prepared from neurosurgical samples of temporal neocortex of Alzheimer and control patients, upon depolarization preferentially released aspartate, glutamate and y-aminobutyrate (GABA). The Alzheimer and control samples did not significantly differ in the pattern of amino acid release, although acetylcholine synthesis by the Alzheimer tissue prisms was greatly reduced. There was no correlation between the efflux of any amino acid and acetylcholine synthesis. These observations suggest that in Alzheimer's disease there are no major changes in the extracellular concentrations of these putative amino acid transmitters.
Few of the techniques used to investigate neurotransmitter metabolism in human brain are suitable for studying amino acid transmitter systems 19. Moreover, methods routinely used to study these transmitters in animal brain 5,9 are difficult to apply to h u m a n brain. Thus, apart from limited data on markers of 3,-aminobutyrate (GABA) synapses in autopsy samples and amino acids in body fluids, there are no data on the functional status of amino acid transmitter systems in diseased h u m a n brain. A previous study suggested that a useful marker of such transmitters is the K÷-evoked release of amino acids from tissue prisms prepared from control human samples ofneocortex obtained at neurosurg e r y 19. These results are compared in the present paper with amino acid release data obtained with tissue prisms of neurosurgical samples of neocortex from patients with Alzheimer's disease (AD). Acetylcholine synthesis 16 and a marker of overall glucose oxidation ~7 in tissue prisms are both altered in AD and were examined for possible correlations with amino acid release. Samples of temporal neocortex were obtained at diagnostic craniotomy ~7from 7 patients with a clinical diagnosis of AD. Histology revealed the
pathological hallmarks of AD (senile plaques and neurofibrillary tangles) in all samples. Control specimens of temporal neocortex 17,19 were from patients of similar age to those with AD, undergoing surgery for removal of deep-seated tumors (2 gliomas, 1 craniopharyngioma, 1 pituitary tumor and 1 suprasella tumor, probably secondary to breast carcinoma) or aneurysm (1 patient). Immediately on removal the tissue was placed in ice-cold, oxygenated modified KrebsRinger phosphate medium 17 (pH 7.4, hereafter Krebs-medium) and processed to yield suspensions of tissue prisms tT. Amino acid release experiments were conducted according to Smith et al. 19 (see Table I). The medium fractions containing the amino acids were desalted by methanolic extraction 7 and amino acids determined fluorometrically using an autoanalyzer 12. The recovery of each amino acid was similar for AD and control samples and all results have been adjusted for recoveryt9 and are expressed relative to the protein content ~°of the prisms. []4C]Acetylcholine synthesis from [uJnC]glucose and a marker of overall glucose oxidation (the production of 14CO2 from [Ul4C]glucose) in medium containing high (31 mM) K ÷ were determined according to Sims et al. 17. All values are express-
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
139 TABLEI
Amino acid release (nmol/100 mg protein) and acetylcholine synthesis (d.p.m./mg protein/min) by tissue prisms of temporal neocortex from control neurosurgical samples and patients with A lzheimer's disease Vials containing tissue prism suspensions (0.5 1.0 mg of prism protein in Krebs-medium) were oxygenated for 30 s, sealed and incubated at 37 °C for 30 min. Stimulation began at 20 min by elevation of the K + concentration from 5 mM (unstimulated incubations) to 55 mM (K ÷-stimulated incubations). The prisms were then sedimented by centrifugation (12,000 g 2, min) and the resulting medium fractions frozen at - 7 0 ° C for later analysis (see text). Incorporation of [u-lac]glucose into [laC]actylcholine by depolarized tissue prisms was also determined (see text). Values are for 7 AD samples from patients of age 60 ± 5 years and 6 control samples from patients of similar age (57 ± 8 years), except for acetylcholine synthesis by 5 controls.
Indices t~f"
Control samples
A lzheimer's disease samples
6.33±1.23
3.21 ± 0.66~
95 ± 43 185±55"
126 ___35 216±55"*
129 ± 36 702±233**
152 ± 32 987±255**
60 ± 24 181 ±57**
65 ± 21 169±24"*
131 ___68 150 ___68
173 ± 26 187 ___41
262 + 121 382 ___239
368 ___1! 8 443 ___109
599 ___117 533 ± 124
456 ___168 475 ± 145
303___ 131 431 __.236
322___ 109 434___ 101
293 ± 132 386± 105
331 __+73 462± 132
Cortical transmitters Acetylcholine synthesis (31 m M K + ) Aspartate release Unstimulated K+ -stimulated Glutamate release Unstimulated K+-stimulated GABA release Unstimulated K+ -stimulated
Other amino acids Threonine release Unstimulated K + -stimulated Serine release Unstimulated K ÷ -stimulated Glutamine release Unstimulated K + -stimulated Glycine release Unstimulated K + -stimulated Alanine release Unstimulated K+-stimulated
Asterisks identify values for K + -stimulated release that are significantly different from the corresponding value for unstimulated release with *P < 0.0l and **P < 0.001. § Identifies the only significant difference (P ~ 0.001) between values for AD and control samples. The release of valine, isoleucine, leucine, tyrosine and phenylalanine were also measured. No significant differences were observed for these amino acids between AD and control samples.
ed as mean __.S.D. Data were analyzed by the Student's t-test for unpaired observations unless otherwise stated. On K ÷ -stimulation, tissue prisms of temporal neocortex from both AD and control samples, responded with an enhanced and preferential efflux of only 3 of the 13 amino acids measured. These were the putative transmitters aspartate, glutamate and GABA (Table I). The numerical difference between the values for each putative amino acid transmitter released in unstimulated and K ÷-stimulated incubations were examined for correlations (linear regression analysis) with acetylcholine synthesis and glucose oxidation by depolarized prisms. For both AD and control samples there were no correlations, except curiously for an unexplained correlation (r = -0.98 by linear regression analysis, n = 7) between glutamate release and glucose oxidation in incubations containing the AD samples. We have previously concluded that the release of aspartate from control samples may not be a good indicator of the value for normal tissue 19, so quantitative comparisons cannot be made for this amino acid. However, the release of GABA and glutamate by control samples appear to be good indicators of the values for normal tissue 19. The results in Table I show that for control preparations the K ÷-stimulated release of GABA was 202% of the unstimulated value, while the increase for the AD samples (160% of the unstimulated value) was slightly lower. The absolute amount of GABA released by the AD samples was not significantly altered from control (Table I), although acetylcholine synthesis was greatly reduced (to 50% of control; Table I). Under the conditions used to assay acetylcholine synthesis, animal experiments 18 indicate that most of the acetylcholine that is determined has been released into the medium. Therefore, the data suggests that in AD the extracellular concentration of GABA (from probable intrinsic neurons8), is selectively preserved. The glutamate decarboxylase activities of biopsy samples also suggest that intrinsic GABA-containing neurons are not affected3, whereas in such samples all markers of cholinergic terminals (acetylcholine synthesis, choline uptake and choline
140 acetyltransferase) are reduced4,t7. Data on autopsy samples indicate that GABAergic cells in comparison with cholinergic nerve endings are either less uniformly affected or are relatively intact6.~5, although Perry et al? 3 suggest that there may be some reduction of glutamate decarboxylase activity other than that caused by the terminal state of the patient 2. The results in Table I show that for control preparations the K ÷ -stimulated release of glutamate was 444% of the unstimulated value, whereas for the AD samples the increase (549% of the unstimulated value) was slightly greater. The absolute amount of glutamate released was not significantly altered from control (Table I). This provides the first data on the extracellular concentration of glutamic acid in AD. It is possible that an alteration in release may have been masked by tissue shrinkage such that the concentration of the transmitter remains unchanged. However, glutamate release may be a marker of neurons intrinsic to the neocortex ~9. There is already evidence in AD of sparing of in-
trinsic GABAergic neurons and two other types of intrinsic cortical neuron (vasoactive intestinal polypeptide and cholecystokinin but possibly not somatostatin-containing cellslt,15). Moreover, the finding of markedly reduced presynaptic cholinergic markers4 (Table I) indicates a predominant loss of an ascending pathway to the neocortex 14. Hence in view of this and reduced uptakes of noradrenaline and serotonin by tissue prisms of neocortex of AD patients it is suggested that ascending pathways are altered 1 before the disease process affects intrinsic cortical neurons.
1 Benton, J. S., Bowen, D. M., Allen, S. J., Haan, E. A., Murphy, R. P. and Snowden, J. S., Alzheimer's disease as a disorder ofisodendritic core, Lancet, i (1982) 456. 2 Bowen, D. M., Smith, C. B., White, P. and Davison, A. N., Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99 (1976) 459-496. 3 Bowen, D. M., Biochemical evidence for selective vulnerability in Alzheimer's disease. In P. J. Roberts (Ed.), Biochemistry of Dementia, John Wiley, Chichester, 1980, pp. 7 ~ 90. 4 Bowen, D. M., Benton, J. S., Spillane, J. A., Smith, C. C. T. and Allen, S. J., Choline acetyltransferase activity and histopathology of frontal neocortex from biopsies of demented patients, J. Neurol. Sci., in press. 5 Cotman, C. W., Foster, A. and Lanthorn, T., An overview of glutamate as a neurotransmitter. In G. DiChiara and G. L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press, New York, 1981, pp. 413-422. 6 Davies, P., Neurotransmitter related enzymes in senile dementia of Alzheimer's type, Brain Research, 177 (1979) 319-327. 7 Dodd, P. R.,Hardy, J. A., Oakley, A. E., Edwardson, J. A., Perry, E. K. and Delaunoy, J.-P., A rapid method for preparing synaptosomes: comparison with alternative procedures, Brain Research, 226 (1981) 107-118. 8 Emson, P. C. and Lindvall, O., Distribution of putative neurotransmitters in the neocortex, Neuroscience, 4 (1979) 1-30. 9 Fonnum, F. and Malthe-S¢renssen, D., Localization of glutamate neurons. In P. J. Roberts, J. Storm-Mathisen
and G. A. R. Johnson (Eds.), Glutamate: Transmitter in the Central Nervous System, John Wiley, Chichester, 1981, pp. 205-222. Lowry, O. H., Rosenbrough, N. S., Farr, A. C. and Randall, R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. McKinney, M., Davies, P. and Coyle, J. T., Somatostatin is not co-localized in cholinergic neurons innervating the rat cerebral cortex-hippocampal formation, Brain Research, 243(1982) 169-172. Norris, P. J., Smith, C. C. T., DeBelleroche, J., Bradford, H. F., Mantle, P. G., Thomas, A. J. and Penny, R. H. C., Actions of tremorgenic fungal toxins on neurotransmitter release, J. Neurochem., 34 (1980) 33- 42. Perry, E. K., Gibson, P. H., Blessed, G., Perry, R. H. and Tomlinson, B. E., Neurotransmitter enzyme abnormalities in senile dementia, J. neurol. Sci., 34 (1977) 247- 265. Rossor, M. N., Parkinson's disease and Alzheimer's disease as disorders of the isodendritic core, Brit. Med. J., 283 (198 !) 1588-1590. Rossor, M. N., Emson, P. C., Mountjoy, C. Q., Roth, M. and Iversen, L. L., Neurotransmitters of the cerebral cortex in senile dementia of Alzheimer type. In S. Hoyer (Ed.) The Aging Brain. Physiological and PathophysiologicalA spects, Springer-Verlag, Berlin, 1982, pp. 153-157. Sims, N. R., Bowen, D. M., Smith, C. C. T., Flack, R. H. A., Davison, A. N., Snowden, J. S. and Neary, D., Glucose metabolism and acetylcholine synthesis in relation to neuronal activity in Alzheimer's disease, Lancet, i (1980) 333-336. Sims, N. R., Bowen, D. M. and Davison, A. N., [14C]Ace-
We thank Dr. R. Balfizs for providing the amino acid analyzer and Mr. D. J. Atkinson and Mr. A. Hunt for assistance in its use. The help and co-operation is gratefully acknowledged of the large number of people involved in the collection and classification of human samples. The work was supported by the Medical Research Council, Brain Research Trust and the Miriam Marks Charitable Trust.
10 11
12
13 14 15
16
17
141 tylcholine synthesis and [14C]carbon dioxide production from [U-laC]glucose by tissue prisms from human neocortex, Biochem. J., 196 (198l) 867-876. 8 Sims, N. R., Marek, K. L., Bowen, D. M. and Davison, A. N., Production of [14C]acetylcholine and [14C]carbon dioxide from [U-14C]glucose in tissue prisms from aging rat
brain, J. Neurochem., 38 (1982) 488-492. 19 Smith, C. C. T., Bowen, D. M. and Davison, A. N., The evoked release of endogenous neurotransmitter amino acids from tissue prisms of human neocortex, Brain Research, in press.
Brain Research, 264 (1983) 138-141 Elsevier Biomedical Press
Amino acid release from biopsy samples of temporal neocortex from patients with Alzheimer's disease C. C. T. SMITH, D. M. BOWEN, N. R. SIMS, D. NEARY and A. N. DAVISON
Miriam Marks Department of Neurochemistry, Institute of Neurology, Queen Square, London WCI N 3BG and (D.N.) Department of Neurology, Manchester Royal Infirmary, Manchester 13 (U. K.) (Accepted November 16th, 1982)
Key word~: neurotransmitter release - amino acids - human brain - temporal neocortex - Alzheimer's disease
Tissue prisms prepared from neurosurgical samples of temporal neocortex of Alzheimer and control patients, upon depolarization preferentially released aspartate, glutamate and y-aminobutyrate (GABA). The Alzheimer and control samples did not significantly differ in the pattern of amino acid release, although acetylcholine synthesis by the Alzheimer tissue prisms was greatly reduced. There was no correlation between the efflux of any amino acid and acetylcholine synthesis. These observations suggest that in Alzheimer's disease there are no major changes in the extracellular concentrations of these putative amino acid transmitters.
Few of the techniques used to investigate neurotransmitter metabolism in human brain are suitable for studying amino acid transmitter systems 19. Moreover, methods routinely used to study these transmitters in animal brain 5,9 are difficult to apply to h u m a n brain. Thus, apart from limited data on markers of 3,-aminobutyrate (GABA) synapses in autopsy samples and amino acids in body fluids, there are no data on the functional status of amino acid transmitter systems in diseased h u m a n brain. A previous study suggested that a useful marker of such transmitters is the K÷-evoked release of amino acids from tissue prisms prepared from control human samples ofneocortex obtained at neurosurg e r y 19. These results are compared in the present paper with amino acid release data obtained with tissue prisms of neurosurgical samples of neocortex from patients with Alzheimer's disease (AD). Acetylcholine synthesis 16 and a marker of overall glucose oxidation ~7 in tissue prisms are both altered in AD and were examined for possible correlations with amino acid release. Samples of temporal neocortex were obtained at diagnostic craniotomy ~7from 7 patients with a clinical diagnosis of AD. Histology revealed the
pathological hallmarks of AD (senile plaques and neurofibrillary tangles) in all samples. Control specimens of temporal neocortex 17,19 were from patients of similar age to those with AD, undergoing surgery for removal of deep-seated tumors (2 gliomas, 1 craniopharyngioma, 1 pituitary tumor and 1 suprasella tumor, probably secondary to breast carcinoma) or aneurysm (1 patient). Immediately on removal the tissue was placed in ice-cold, oxygenated modified KrebsRinger phosphate medium 17 (pH 7.4, hereafter Krebs-medium) and processed to yield suspensions of tissue prisms tT. Amino acid release experiments were conducted according to Smith et al. 19 (see Table I). The medium fractions containing the amino acids were desalted by methanolic extraction 7 and amino acids determined fluorometrically using an autoanalyzer 12. The recovery of each amino acid was similar for AD and control samples and all results have been adjusted for recoveryt9 and are expressed relative to the protein content ~°of the prisms. []4C]Acetylcholine synthesis from [uJnC]glucose and a marker of overall glucose oxidation (the production of 14CO2 from [Ul4C]glucose) in medium containing high (31 mM) K ÷ were determined according to Sims et al. 17. All values are express-
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
139 TABLEI
Amino acid release (nmol/100 mg protein) and acetylcholine synthesis (d.p.m./mg protein/min) by tissue prisms of temporal neocortex from control neurosurgical samples and patients with A lzheimer's disease Vials containing tissue prism suspensions (0.5 1.0 mg of prism protein in Krebs-medium) were oxygenated for 30 s, sealed and incubated at 37 °C for 30 min. Stimulation began at 20 min by elevation of the K + concentration from 5 mM (unstimulated incubations) to 55 mM (K ÷-stimulated incubations). The prisms were then sedimented by centrifugation (12,000 g 2, min) and the resulting medium fractions frozen at - 7 0 ° C for later analysis (see text). Incorporation of [u-lac]glucose into [laC]actylcholine by depolarized tissue prisms was also determined (see text). Values are for 7 AD samples from patients of age 60 ± 5 years and 6 control samples from patients of similar age (57 ± 8 years), except for acetylcholine synthesis by 5 controls.
Indices t~f"
Control samples
A lzheimer's disease samples
6.33±1.23
3.21 ± 0.66~
95 ± 43 185±55"
126 ___35 216±55"*
129 ± 36 702±233**
152 ± 32 987±255**
60 ± 24 181 ±57**
65 ± 21 169±24"*
131 ___68 150 ___68
173 ± 26 187 ___41
262 + 121 382 ___239
368 ___1! 8 443 ___109
599 ___117 533 ± 124
456 ___168 475 ± 145
303___ 131 431 __.236
322___ 109 434___ 101
293 ± 132 386± 105
331 __+73 462± 132
Cortical transmitters Acetylcholine synthesis (31 m M K + ) Aspartate release Unstimulated K+ -stimulated Glutamate release Unstimulated K+-stimulated GABA release Unstimulated K+ -stimulated
Other amino acids Threonine release Unstimulated K + -stimulated Serine release Unstimulated K ÷ -stimulated Glutamine release Unstimulated K + -stimulated Glycine release Unstimulated K + -stimulated Alanine release Unstimulated K+-stimulated
Asterisks identify values for K + -stimulated release that are significantly different from the corresponding value for unstimulated release with *P < 0.0l and **P < 0.001. § Identifies the only significant difference (P ~ 0.001) between values for AD and control samples. The release of valine, isoleucine, leucine, tyrosine and phenylalanine were also measured. No significant differences were observed for these amino acids between AD and control samples.
ed as mean __.S.D. Data were analyzed by the Student's t-test for unpaired observations unless otherwise stated. On K ÷ -stimulation, tissue prisms of temporal neocortex from both AD and control samples, responded with an enhanced and preferential efflux of only 3 of the 13 amino acids measured. These were the putative transmitters aspartate, glutamate and GABA (Table I). The numerical difference between the values for each putative amino acid transmitter released in unstimulated and K ÷-stimulated incubations were examined for correlations (linear regression analysis) with acetylcholine synthesis and glucose oxidation by depolarized prisms. For both AD and control samples there were no correlations, except curiously for an unexplained correlation (r = -0.98 by linear regression analysis, n = 7) between glutamate release and glucose oxidation in incubations containing the AD samples. We have previously concluded that the release of aspartate from control samples may not be a good indicator of the value for normal tissue 19, so quantitative comparisons cannot be made for this amino acid. However, the release of GABA and glutamate by control samples appear to be good indicators of the values for normal tissue 19. The results in Table I show that for control preparations the K ÷-stimulated release of GABA was 202% of the unstimulated value, while the increase for the AD samples (160% of the unstimulated value) was slightly lower. The absolute amount of GABA released by the AD samples was not significantly altered from control (Table I), although acetylcholine synthesis was greatly reduced (to 50% of control; Table I). Under the conditions used to assay acetylcholine synthesis, animal experiments 18 indicate that most of the acetylcholine that is determined has been released into the medium. Therefore, the data suggests that in AD the extracellular concentration of GABA (from probable intrinsic neurons8), is selectively preserved. The glutamate decarboxylase activities of biopsy samples also suggest that intrinsic GABA-containing neurons are not affected3, whereas in such samples all markers of cholinergic terminals (acetylcholine synthesis, choline uptake and choline
140 acetyltransferase) are reduced4,t7. Data on autopsy samples indicate that GABAergic cells in comparison with cholinergic nerve endings are either less uniformly affected or are relatively intact6.~5, although Perry et al? 3 suggest that there may be some reduction of glutamate decarboxylase activity other than that caused by the terminal state of the patient 2. The results in Table I show that for control preparations the K ÷ -stimulated release of glutamate was 444% of the unstimulated value, whereas for the AD samples the increase (549% of the unstimulated value) was slightly greater. The absolute amount of glutamate released was not significantly altered from control (Table I). This provides the first data on the extracellular concentration of glutamic acid in AD. It is possible that an alteration in release may have been masked by tissue shrinkage such that the concentration of the transmitter remains unchanged. However, glutamate release may be a marker of neurons intrinsic to the neocortex ~9. There is already evidence in AD of sparing of in-
trinsic GABAergic neurons and two other types of intrinsic cortical neuron (vasoactive intestinal polypeptide and cholecystokinin but possibly not somatostatin-containing cellslt,15). Moreover, the finding of markedly reduced presynaptic cholinergic markers4 (Table I) indicates a predominant loss of an ascending pathway to the neocortex 14. Hence in view of this and reduced uptakes of noradrenaline and serotonin by tissue prisms of neocortex of AD patients it is suggested that ascending pathways are altered 1 before the disease process affects intrinsic cortical neurons.
1 Benton, J. S., Bowen, D. M., Allen, S. J., Haan, E. A., Murphy, R. P. and Snowden, J. S., Alzheimer's disease as a disorder ofisodendritic core, Lancet, i (1982) 456. 2 Bowen, D. M., Smith, C. B., White, P. and Davison, A. N., Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99 (1976) 459-496. 3 Bowen, D. M., Biochemical evidence for selective vulnerability in Alzheimer's disease. In P. J. Roberts (Ed.), Biochemistry of Dementia, John Wiley, Chichester, 1980, pp. 7 ~ 90. 4 Bowen, D. M., Benton, J. S., Spillane, J. A., Smith, C. C. T. and Allen, S. J., Choline acetyltransferase activity and histopathology of frontal neocortex from biopsies of demented patients, J. Neurol. Sci., in press. 5 Cotman, C. W., Foster, A. and Lanthorn, T., An overview of glutamate as a neurotransmitter. In G. DiChiara and G. L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press, New York, 1981, pp. 413-422. 6 Davies, P., Neurotransmitter related enzymes in senile dementia of Alzheimer's type, Brain Research, 177 (1979) 319-327. 7 Dodd, P. R.,Hardy, J. A., Oakley, A. E., Edwardson, J. A., Perry, E. K. and Delaunoy, J.-P., A rapid method for preparing synaptosomes: comparison with alternative procedures, Brain Research, 226 (1981) 107-118. 8 Emson, P. C. and Lindvall, O., Distribution of putative neurotransmitters in the neocortex, Neuroscience, 4 (1979) 1-30. 9 Fonnum, F. and Malthe-S¢renssen, D., Localization of glutamate neurons. In P. J. Roberts, J. Storm-Mathisen
and G. A. R. Johnson (Eds.), Glutamate: Transmitter in the Central Nervous System, John Wiley, Chichester, 1981, pp. 205-222. Lowry, O. H., Rosenbrough, N. S., Farr, A. C. and Randall, R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. McKinney, M., Davies, P. and Coyle, J. T., Somatostatin is not co-localized in cholinergic neurons innervating the rat cerebral cortex-hippocampal formation, Brain Research, 243(1982) 169-172. Norris, P. J., Smith, C. C. T., DeBelleroche, J., Bradford, H. F., Mantle, P. G., Thomas, A. J. and Penny, R. H. C., Actions of tremorgenic fungal toxins on neurotransmitter release, J. Neurochem., 34 (1980) 33- 42. Perry, E. K., Gibson, P. H., Blessed, G., Perry, R. H. and Tomlinson, B. E., Neurotransmitter enzyme abnormalities in senile dementia, J. neurol. Sci., 34 (1977) 247- 265. Rossor, M. N., Parkinson's disease and Alzheimer's disease as disorders of the isodendritic core, Brit. Med. J., 283 (198 !) 1588-1590. Rossor, M. N., Emson, P. C., Mountjoy, C. Q., Roth, M. and Iversen, L. L., Neurotransmitters of the cerebral cortex in senile dementia of Alzheimer type. In S. Hoyer (Ed.) The Aging Brain. Physiological and PathophysiologicalA spects, Springer-Verlag, Berlin, 1982, pp. 153-157. Sims, N. R., Bowen, D. M., Smith, C. C. T., Flack, R. H. A., Davison, A. N., Snowden, J. S. and Neary, D., Glucose metabolism and acetylcholine synthesis in relation to neuronal activity in Alzheimer's disease, Lancet, i (1980) 333-336. Sims, N. R., Bowen, D. M. and Davison, A. N., [14C]Ace-
We thank Dr. R. Balfizs for providing the amino acid analyzer and Mr. D. J. Atkinson and Mr. A. Hunt for assistance in its use. The help and co-operation is gratefully acknowledged of the large number of people involved in the collection and classification of human samples. The work was supported by the Medical Research Council, Brain Research Trust and the Miriam Marks Charitable Trust.
10 11
12
13 14 15
16
17
141 tylcholine synthesis and [14C]carbon dioxide production from [U-laC]glucose by tissue prisms from human neocortex, Biochem. J., 196 (198l) 867-876. 8 Sims, N. R., Marek, K. L., Bowen, D. M. and Davison, A. N., Production of [14C]acetylcholine and [14C]carbon dioxide from [U-14C]glucose in tissue prisms from aging rat
brain, J. Neurochem., 38 (1982) 488-492. 19 Smith, C. C. T., Bowen, D. M. and Davison, A. N., The evoked release of endogenous neurotransmitter amino acids from tissue prisms of human neocortex, Brain Research, in press.