Effect of antemortem and postmortem factors on [3H]glutamate binding in the human brain

312

Neuroscience Letters, 93 (1988) 312--317 Elsevier ScientificPublishers Ireland Ltd.

NSL 05635

Effect of antemortem and postmortem factors on [3H]glutamate binding in the human brain Johannes Kornhuber, Wolfgang Retz, Peter Riederer, Helmut Heinsen and Jiirgen Fritze Department of Psychiatry, Clinical Neurochemistry. University of Wiirzburg ( F.R.G. ) (Received 22 April 1988; Revised version received 28 June 1988; Accepted 2 July 1988) Key words: [3H]Glutamatebinding; Receptor; Postmortem human brain; Age; Development;Glutamate; N-Methyl-D-aspartate (NMDA) The effect of a number of antemortem and postmortem factors on both N-methyl-o-aspartate (NMDA) sensitive and NMDA insensitive[3H]glutamatebinding was investigated in the frontal cortex and putamen of human brains. There was a high correlation between both binding sites (r=0.86, P<0.001) and both binding sites increased during the early postnatal period reaching a maximum between age 1 and 2 years. After age 10 years NMDA sensitive sites disappeared at 9.2% per decade while the NMDA insensitive sites disappeared at 7.4% per decade only. Therefore, the ratio between NMDA sensitive and NMDA insensitive sites changed in favor of the NMDA insensitive site with increasing age. The storage time of brain tissue had a strong reducing effect on both binding sites, again affecting the NMDA sensitive sites more severely. There was no obvious effect of gender, brain weight or postmortem time interval on either binding site. Furthermore, there was no differencebetween frontal cortex and putamen. Both binding sites were bilateral symmetricallydistributed in either frontal cortex and putamen.

A substantial p o r t i o n of central m a m m a l i a n n e u r o n s use glutamate as an excitatory n e u r o t r a n s m i t t e r a n d a variety o f disease states has been associated with a n imp a i r m e n t of the glutamatergic n e u r o t r a n s m i s s i o n . Therefore, m e a s u r e m e n t of glutamate receptors has been performed by several groups in p o s t m o r t e m h u m a n b r a i n s using [3H]glutamate as the ligand [ 2 4 ] . However, p r o b l e m s m a y arise from the u n a v o i d a b l e delays in processing the tissue for dissection a n d the necessity for prolonged storage prior to analysis. The aim o f the present investigation was, therefore, to evaluate the effects of age, gender, b r a i n weight, laterality, p o s t m o r t e m interval (time between death a n d freezing of tissue) a n d storage time of b r a i n tissue on [3H]glutamate b i n d i n g in the frontal cortex a n d p u t a m e n of h u m a n brains. Tissue was t a k e n at a u t o p s y from subjects with n o history of neurological or psychiatric disorder (causes of death included sudden i n f a n t death syndrome, t r a u m a

Correspondence." J. Kornhuber, Klinische Neurochemie, Psychiatrische Klinik der Universit/it Wfirzburg, Ffichsleinstrasse 15, 8700 Wiirzburg, F.R.G. 0304-3940/88/$ 03.50 © 1988 Elsevier ScientificPublishers Ireland Ltd.

313

exclusive of craniocerebral trauma, carcinoma, pneumonia, cardiac arrest, homicide). The samples were placed in a freezer at - 80°C until analysis. The fight frontal cortex (Brodmann areas 8 and 9) was taken from all brains (n = 30). Furthermore, in order to obtain information concerning region- and side-specificity of [3H]glutamate binding, tissue samples from the left frontal cortex and from both sides of the putamen were taken from a subgroup of 12 brains. Tissue pieces were homogenized in about 50 vols of buffer using a glass-Teflon homogenizer. Tris-acetate buffer (pH 6.9, 50 mM) was used throughout. The homogenate was pelleted (15,000 g, 20 min) and resuspended by vortexing and pelleted again and stored thereafter at -80°C until assay. On the day of assay the pellet was thawed and resuspended in 0.1% Triton X-100 and incubated at 37°C for 15 min. Thereafter, the homogenate was pelleted and resuspended 4 times in buffer as described above. Binding was done in microfuge tubes in a total volume of 0.5 ml with protein concentrations around 0.34).5 mg/ml. After incubation (30 min, 21°C) bound ligand was separated by centrifugation at 13,000 g for 3 rain. The pellet was quickly and superficially rinsed once with 0.75 ml ice-cold buffer and treated with 0.5 ml Protosol overnight. 0.1 ml of glacial acetic acid and 5 ml of a toluene-based scintillation cocktail were added and monitored for tritium in a Beckman LS 1801 counter at about 44% efficacy. Non-specific binding was defined as that not displaced by 1 mM of unlabelled glutamate. All samples were assayed at least in quadruplicate within a single experiment. Protein was determined using the BioRad assay [1]. Using this procedure specific [3H]glutamate binding was linear with tissue concentration, stereoselective, heatlabile, region-specific and fully reversible. A detailed saturation analysis (5-1500 nM [3H]glutamate) was performed in 7 experiments using 5 different brains (age range 3 months to 82 years). In Scatchard analysis of saturation studies a high-affinity (Kd 19.3 + 5.0 nM, Bmax0.77_+0.10 pmol/mg protein) and a low-affinity (Kd 797 _+237 nM, Bmax3.41 +_ 1.48 pmol/mg protein) binding site appeared and Kd values were similar for all age groups. N-Methyl-D-aspartate (NMDA, 100/tM) nearly completely and selectively blocked the high-affinity site. In the present study we used two concentrations of [3H]glutamate, 10 nM in order to preferentially label the high-affinity NMDA sensitive site, and 500 nM in order to preferentially label the low-affinity NMDA insensitive site. The influence of brain region (frontal cortex vs putamen) and laterality (left vs fight) was tested (paired Wilcoxon rank test) in the subgroup of 12 brains in which the 4 brain regions had been dissected. The influence of all other measures was estimated in the right frontal cortex samples (regression analysis, Mann-Whitney U-test, Spearman's rank correlation). Mean values are given _+S.D. Case data and mean values for both binding sites in the right frontal cortex are given in Table I. There was a high correlation between NMDA sensitive and NMDA insensitive binding sites (r=0.86, P<0.001) and the effect of the antemortem and postmortem factors on [3H]glutamate binding was qualitatively similar for either binding site. The figures refer, therefore, only to the NMDA sensitive binding site. There was a sharp, short-lasting increase in [3H]glutamate binding in the postnatal period (Fig. IA). This is in good agreement with investigations on other receptor sys-

314 TABLE I CASE DATA AND VALUES FOR BOTH N M D A SENSITIVE A N D N M D A INSENSITIVE [3H]GLUTAMATE BINDING SITES IN THE RIGHT F R O N T A L CORTEX Mean values are given + S.D. (range), n = 30. Case data

M/F Age (yrs)

16/14 44.1 + 27.6

(0.125-8b Brain weight (g)

1211 + 270

(570-1600) Postmortem time (h) Freezing time (yrs) N M D A sensitive binding (pmol/mg protein) N M D A insensitive binding (pmol/mg protein)

39.4 + 20.9 (3-72) 0.939 + 1.620 (0.011-7.227) 0.214+0.161 (0.0304).674) 1.536 _+0.826 (0.469~,. 121)

terns [6, 9] and synapsogenesis [5]. In adult patients (age > 10 yrs, n = 24) the correlations between age (x) and [3H]glutamate binding sites (y) were best represented by linear equations ( y = - 0 . 0 0 2 8 2 x + 0 . 3 0 6 , r = - 0 . 5 8 , P<0.01 for N M D A sensitive sites and y = - 0 . 0 1 6 x + 2 . 1 2 6 , r = - 0 . 5 7 , P<0.01 for N M D A insensitive sites). N M D A sensitive binding sites gradually diminished at 9.2% per decade whereas the N M D A insensitive binding sites diminished at 7.4% per decade only. These different age-dependent processes resulted in a significant linear reduction of the N M D A sensitive/NMDA insensitive binding ratio (y) with age (x) (n=30, y = 0.001079x+0.179, r = - 0 . 6 3 , P<0.01, Fig. IC). Increased storage time of the brain tissue had a reducing effect on [3H]glutamate binding (Fig. 1B). The best fit was a logarithmic relationship between [3H]glutamate binding (_v) and freezing time (x) (n=30; y = - 0 . 1 6 9 1 o g x + 0 . 1 2 8 , r = - 0 . 7 3 , P < 0.001 for N M D A sensitive sites and y = - 0.7481ogx + 1.157, r = - 0.64, P < 0.001 for N M D A insensitive sites). Again this effect was more pronounced for the N M D A sensitive binding site compared to the N M D A insensitive site (Fig. 1D), but this relationship could not easily be linearized. Our data indicate a higher vulnerability of the N M D A sensitive sites. We found a small positive correlation between age and storage time (r = 0.45, P < 0.05, n : 30). Higher binding values in infants thus might in part be due to the shorter storage time of these brain tissues. However, even after correction of binding values for storage time (using linear regression) glutamate binding remained higher in infants and still exhibited a transient elevation during early childhood. There was a positive correlation between postmortem interval and N M D A sensitive glutamate binding (r = 0.44, P < 0.05, n = 30). However, this was mainly due to

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316 TABLE II NMDA SENSITIVE AND NMDA INSENSITIVE [3H]GLUTAMATE BINDING IN THE FRONTAL CORTEX AND PUTAMEN OF EITHER SIDE These 4 brain regions were investigated in 12 brains. Values are given as mean + S.D. [3H]glutamate binding (pmol/mg protein) NMDA sensitive

Fr. cortex Putamen

NMDA insensitive

Left

Right

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Right

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the fact that the 6 infants (with high glutamate binding due to age and shorter storage time) had longer postmortem time intervals than the adults (60.0+20.1 h vs 34.1 + 17.9 h; P<0.05). Looking at both subgroups, infants and adult patients, the correlation between postmortem time interval and N M D A sensitive binding did not reach significance any more ( r = - 0 . 3 7 , P > 0 . 0 5 , n = 6 for the infants and r---0.26, P>0.05, n = 24 for the adult patients). Therefore, we feel that there is no influence of postmortem time interval on [3H]glutamate binding. This would be in concordance with previous observations in a rat model [3]. There was no effect of gender on either binding site (0.195+0.138 in males vs 0.235+0.187 in females for N M D A sensitive binding; 1.362+0.758 in males vs 1.749+0.885 in females for N M D A insensitive binding; pmol/mg protein). Furthermore, there was no relationship between brain weight and [3H]glutamate binding. In agreement with previous investigations both on rodents (7) and humans (4) we found similar amounts of [3H]glutamate binding in the putamen and the frontal cortex (Table II). Although suggested from behavioral experiments in animals [8], we found no laterality of either [3H]glutamate binding site in the frontal cortex or putamen of postmortem human brains (Table II). J.K. received a D F G scholarship. 1 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein~lye binding, Anal. Biochem., 72 (1976) 248 254. 2 Cross, A., Skan, W. and Slater, P., Binding sites for [3H]glutamate and [3H]aspartate in human cerebellum, J. Neurochem., 47 (1986) 1463 1468. 3 Geddes, J.W., Chang-Chui, H., Cooper, S.M., Lott, I.T. and Cotman, C.W., Density and distribution of N M DA receptors in the human hippocampus in Alzheimer's disease, Brain Res., 399 (1986) 156-161. 4 Greenamyre, J.T., Penney, J.B., Young, A.B., D'Amato, C.J., Hicks, S.P. and Shoulson, I., Alterations in L-glutamate binding in Alzheimer's and Huntington's diseases, Science, 227 (1985) 1496-1499. 5 Huttenlocher, P.R., Synaptic density in human frontal cortex developmental changes and effects of aging, Brain Res., 163 (1979) 195 205. 6 Johnston, M.V., Silverstein, F.S., Reindel, F.O., Penney, J.B. and Young, A.B., Muscarinic cholinergic receptors in human infant forebrain: [3H]quinuclidinyl benzilate binding in homogenates and quantita-

317 tive autoradiography in sections, Dev. Brain Res., 19 (1985) 195 203. 7 Monaghan, D.T. and Cotman, C.W., Distribution of N-methyl-D-aspartate-sensitive L-[3H]glutamate binding sites in rat brain, J. Neurosci., 5 (1985) 2909-2919. 8 Roos, D.A. and Glick, S.D., Lateralized effects of bilateral frontal cortex lesions in rats, Brain Res., 210 (1981) 379-382. 9 Seeman, P., Bzowey, N.H., Guan, H.C., Bergeron, C., Becker, L.E., Reynolds, G.P., Bird, E.D., Riederer, P., Jellinger, K., Watanabe, S. and Tourtellotte, W.W., Human brain dopamine receptors in children and aging adults, Synapse, 1 (1987) 399-404.