Recovery potential in glucose deprived astrocytes

llEUROSCIENCE RESEARCH ELSEVIER

Neuroscience Research 26 (1996) 133-139

Recovery potential in glucose deprived astrocytes Jeannine Mertens-Strijthagen*, Josianne Lacremans-Pirsoul, Georges Baudoux Laboratory of Pharmacology and Physiology, Faculty of Medicine, Faeultks Universitaires, rue de Bruxelles, 61, B-5000 Namur, Belgium

Received 13 May 1996; accepted 4 July 1996

Abstract

D-glucose deprivation for a 45 min period reduces the ATP and creatine phosphate concentrations of astrocytes. Recovery experiments were initiated by reincubating the cells with D-glucose and glucose replacement metabolites. No recovery of ATP concentration could be obtained even after 1 h of reincubation with the replacement metabolites. After a 45 min incubation period without D-glucose, 14CO2 production fell to 36% and 21% of controls when the cells were reincubated respectively with D-[U-14C]-glucose and L-[2-14C]-pyruvate as substrate marker. When reincubated for 1 h in the presence of L-malate (1 mM) + L-pyruvate (10 raM) with L-[2-14C]-pyruvate as marker, a total recovery of 14CO2 production was ascertained. Reincubation of the glucose deprived cells in the presence of D-glucose (10 mM) did not increase the 14CO2 production indicating that the cells were unable to use D-glucose for oxidative purposes. As pyruvate concentration was dramatically decreased in glucose deprived cells, astrocytes were treated with c~-ketovalerate (25 raM) which led to an 8-fold increase in pyruvate concentration. In these conditions ~4CO2 production did not increase when the cells were incubated in the presence of L-malate (1 mM). 02 consumption of State 4 in astrocytes, submitted to glucose deprivation, decreased. These cells treated with FCCP could not be uncoupled and when reincubated in the presence of replacement metabolites only a 20% increase of oxygen consumption took place. Keywords: Astrocytes; Glucose deprivation; Recovery from glucose deprivation; 02 consumption; Postdeprivation glucose

metabolism; Malate-pyruvate

1. Introduction

Preservation of the central nervous system in hypoglycemia requires a p r o m p t increase of glucose production by the liver (Cryer and Gerich, 1985). A profound and prolonged hypoglycemic coma is known to produce cellular injuries in the cerebral cortex (Auer, 1986; Auer and Siesj6, 1988) which persist during recovery thus suggesting an irreversible lesion (Agardh et al., 1980). Despite a final c o m m o n exhaustion of energy, the type of nerve cell injury reported in hypoglycemia

Abbrev&tions: DMEM, Dubelco modified Eagle medium; FCCP, Carbonyl-cyanide-p-trifloromethoxyphenylhydrazone; GFAP, glial fibrillary acidic protein; Hepes, N-[2-hydroxyethyl]piperazine-N'-[2ethanesulfonic acid]; ATP, adenosine triphosphate; Tris, 2-amino-2(hydroxymethyl)-l,3propanediol. * Corresponding author. Fax: + 32 81 230391.

seemed to be different from ischemia (Agardh et al., 1980; Brooks et al., 1989). In hypoglycemia a selective neuronal necrosis was claimed to be present due to a release of an excitotonic endogenous c o m p o u n d (Weil et al., 1938; Auer, 1986; Sandberg et al., 1986; Butcher et al., 1987; Auer and Siesj6, 1988; Siesj6, 1988). Morphological studies have shown that glia and other non neuronal cells are spared in severe hypoglycemic conditions (Lawrence et al., 1942; Agardh et al., 1980). M a n y authors have reported that A T P and creatine phosphate decreased dramatically in vivo during hypoglycemia experiments (Harris et al., 1984; Hertz and Schousboe, 1986; Kauppinen et al., 1988; Baudoux et al., 1989). Although in isolated cell preparations the A T P concentration in synaptosomes were not affected in the initial stage of glucose deprivation, the astrocytes elicited, in the same conditions, an alteration of their

0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO168-0102(96)01081-4

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respiratory capabilities leading to an important reduction of ATP (Kauppinen and Nicholls, 1986; Kauppinen et al., 1988; Baudoux et al., 1989). A considerable decrease of high energy compounds should alter the glutamate and K + uptake (Harris et al., 1984; Kauppinen et al., 1988). Since glia cells have a supportive role in neuronal activities, the impaired function of astrocytes would expected to impact on the functional capabilities of neurons. In the present work, the problem of recovery from hypoglycemia was examined by incubating isolated astrocytes from the cerebral cortex of adult rats in the presence of D-glucose or L-glucose, the glucose deprivation model, for a period of 45 min. Subsequently, the cells were reincubated with D-glucose and several parameters were monitored in order to ascertain whether the cells were able to recover from the effects of glucose deprivation.

2. Material and methods

All the chemicals used were purchased from Sigma (St. Louis, USA). Experiments were performed on male Sprague-Dawley rats, 3 - 4 months of age. The animals were fasted for 18 h before the experiments. Rats were killed by decapitation, the brains were excised, the cerebral cortex was dissected and meninges and superficial blood vessels removed. Then, the cortex were cut in small pieces and mechanically dissociated by up and down pumping in a Pasteur pipette. Subsequently, the cortex suspension was filtered through a series of nylon filters of different mesh ranging from 1000 mm to 82 mm. All procedures were performed at 4°C. Furthermore, astrocytes were isolated on a Ficoll gradient according to the method of Nagata et al., 1974 and of Hamberger et al., 1975. The preparation was analysed for contaminations by electron microscopy and no neurofilaments were found by immunofluorescence with reagents of Dakopatts, Denmark, according to the manufacturer's instructions. Subsequently, the preparation was observed by fluorescence microscopy for G F A P according to the method of Bignami (Bignami et al., 1972). The integrity of cellular membranes was ascertained by measuring the free activity of lactate dehydrogenase of the incubation media at various times and in the experimental conditions as described below (Johnson, 1960). The astrocytes were incubated for 45 min at 37°C in a Krebs-type solution buffered with HEPES-TRIS (10 mM) at pH 7.4 in the presence of D-glucose (10 mM) as controls or with L-glucose (10 mM) in the glucose deprived model. The incubation was carried out with 3 mg of proteins and the medium bubbled with a gas mixture containing 95% oxygen and 5% carbon dioxide.

In order to evaluate whether L-glucose, an isomer not metabolized in cells, had a specific effect in the experiments, some incubations were run in the absence of D- and L-glucose. Identical results were obtained as with L-glucose. Recovery from glucose deprivation experiments were designed as follows: astrocytes were submitted to two incubation periods. The cells were first incubated for 45 min at 37°C in the incubation medium with L-glucose. Subsequently, the astrocytes were centrifuged, washed and again sedimented. Then, the cells were reincubated in the same medium with either D-glucose or any other replacement metabolites. The results were compared to control experiments in which both incubations were carried out in the presence of D-glucose. Glucose transport into the cells was assayed with [1-3H]-2-deoxy-D-glucose (25 mCi/mmol) according to the method of Foley et al., 1980. Very small quantities of [1-3H]-2-deoxy-D-glucose were used in the experiments in order to avoid an inhibition of hexokinase by the accumulation of 2-deoxy-D-glucose-6-phosphate. Since the activity of glucose-6-phosphatase is relatively low in the brain, the accumulation of 2-deoxy-D-glucose-6-phosphate reflects relatively well the transport of D-glucose into the cell (Sokoloff et al., 1977; Mori et al., 1990). Glycolysis was estimated by the rate of formation of 3H20 from [6-3H glucose (Katz and Rognstad, 1975) and separation of 3H20 water from glucose was carried out as described by Bontemps et al., 1978. ATP was assayed according to Lamprecht and Trautschold, 1974, creatine phosphate according to Bergmeyer et al., 1983 and lactate and pyruvate as described by Passonneau and Lowry, 1974 and Passonneau, 1974. Glucose-6-phosphate was estimated with the method of Lang and Michal, 1974. Pyruvate dehydrogenase activity was measured by the method of Dennis et al., 1978 and Ksiezak-Reding et al., 1982. Proteins were estimated by the method of Miller, 1959. CO2 production was measured according to Yu et al., 1982. Those measurements were carried out on cells first incubated with L-glucose for 45 min. Then, the cells were reincubated either with D-[U-~4C]-glucose (100 mCi/mmol) or with L-[2-~4C]-pyruvate (25 mCi/ mmol) in a sealed environment. At the end of the incubation period HC104 (1 M) was added to the incubation medium and the released CO2 was absorbed on K O H for 90 min. KH14CO3 was then counted in a scintillation counter. 88% of ~4CO2 was recovered by KOH. Astrocytes were incubated with D-glucose and L-glucose and analysed for their respiration characteristics in a Gilson oxypolarograph supplied with a Clark electrode at 30°C. Measurements were carried out on State 4 with succinate as substrate. Cells were incubated in a slightly hypoosmotic incubation medium (240 mosmole) containing sucrose (200 mM), MgC12 (5

J. Mertens-Str~#hagen et al. / Neuroscience Research 26 (1996) 133 139

mM), Na2HPO4 (5 mM) and buffered with HEPES HCI (10 mM) at pH 7.5. Statistical analysis was performed according to Snedecor, 1964. Unless otherwise stated, P values are the results of the analysis between controls and experiments.

Fig. 1 shows the effect of glucose deprivation on the D-glucose transport capabilities of astrocytes. The transport of 3H-2-deoxy-D-glucose into the cells after an incubation with L-glucose was significantly increased after 20 min. Intracellular glycolysis was estimated and was identical in both incubation conditions. In glucose deprived conditions, the concentration of glucose-6-phosphate was very low and hardly measurable (not shown). Fig. 2 shows that glucose-6-phosphate increases during the recovery period. As seen in Fig. 3, astrocytes preincubated for 45 min in the presence o f L-glucose and subsequently incubated for another 45 min with D-glucose, showed a reduction of the ATP content when compared with cells preincubated with D-glucose. Similar data were registered for creatine phosphate. Similar results were also obtained when the second incubation period with D-glucose was extended to 1 h. This suggests that the astrocytes once

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3. Results

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Fig. 1. Transport o f [1-3H]-2-deoxy-D-glucose 25 (mCi/mmol) as a function of time in astrocytes after a deprivation period of 45 min. Cells were first incubated for 45 min at 37°C in a Krebs-type medium buffered with HEPES (10 m M ) in the presence o f L-glucose (10 m M ) (full circular symbols). Control experiments were carried out with D-glucose (square symbols). Then, the astrocytes were washed and reincubated with 2-deoxy-D-glucose (1 raM). Results are shown by m e a n s of experiments + the standard error of the mean. Each point represents 5 to 6 experiments. Statistical significance is indicated by P < 0.05, P < 0.01, and N.S. for non significant.

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Fig. 2. Glucose-6 phosphate concentration in isolated astrocytes during recovery experiments following a 45 min glucose depletion period with L-glucose. Astrocytes were first incubated for 45 min at 37°C in a Krebs-type medium buffered with HEPES (10 m M ) either in the presence of D-glucose (10 raM) (controls) or with L-glucose (10 m M ) (glucose deprivation model). Then, the cells were washed and resuspended in the same medium in the presence of D-glucose 10 m M and incubated for another 45 min (recovery period). The substances indicated in the histogram are related to the first incubation period preceding the recovery experiment, n stands for the n u m b e r of experiments. Results are shown by histograms _+ the standard error of the mean. Statistical significance is indicated by P < 0.01.

subjected to a 45 min glucose deprivation experiment do not recover their ability to synthesize ATP. When astrocytes were incubated twice with L-glucose, pyruvate concentrations fell to 29% of control values; however, pyruvate concentrations of cells submitted to a second incubation with D-glucose resulted in 48% of control values. Lactate concentrations were identical to control values during the recovery period. Previously, we were able to show that among several putative replacement metabolites for glucose, L-malate (1 mM) + L-pyruvate (10 raM) or L-glutamate (10 mM) were particularly efficient to maintain the high energy nucleotides concentration in astrocytes (Baudoux et al., 1993). The same metabolites were tested in our setup for recovery experiments as shown in Fig. 3. As seen in Fig. 4, when cells were submitted to glucose deprivation, the replacement metabolites in the recovery incubation hardly increased the ATP concentration when compared to controls. Similar results were observed after a recovery period of 1 h. In the presence of L-pyruvate (10 mM), the recovery during a 45 min period for ~4CO2 production was 21% of the controls; when L-malate (1 raM) was added the

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14CO2production rose to 70% of controls. As seen in Fig. 5, after 1 h of recovery the 14CO2 production of the cells incubated with L-malate (1 m M ) + L-pyruvate (10 mM) was not different from controls. However, when glucose deprived astrocytes were reincubated with D-glucose (10 mM) no such recovery took place. The addition of L-malate (1 mM) to D-glucose (10 mM) did not change the ~4CO2 production (Fig. 6). Total and active pyruvate dehydrogenase activity were assayed in the presence of D-glucose and L-glucose. For total and active enzyme activity the results were not different in the presence of D-glucose and L-glucose (5.27 + 0.44 and 4.75 + 0.11 mmol CO2/h/mg of proteins for total enzyme and 1.37+0.06 and 1.37_ 0.07mmol CO2/h/mg of proteins for the active enzyme). Since a L-glucose incubation lead to an important decrease of intracellular pyruvate concentration, cells were treated with e-ketovalerate (25 mM) which inhibits the transformation of pyruvate to lactate (Nakamura et al., 1986). In these conditions, the pyruvate concentration in the glucose deprived cells increased 8 fold. During the recovery experiments however, Lmalate (1 mM) did not increase their 14CO2production. 1,0 (n=4)

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Fig. 3. ATP concentration in astrocytes during a recovery experiment following a 45 min glucose depletion period with L-glucose. Astrocytes were first incubated for 45 min at 37°C in a Krebs-type medium buffered with HEPES 10 m M either in the presence of D-glucose (10 mM) (controls) or with L-glucose (10 mM) (glucose deprivation model). Then, the cells were washed and resuspended in the same medium in the presence of D-glucose (10 mM) and incubated for another 45 min (recovery period). The substances indicated in the histogram are related to the first incubation period, n stands for the number of experiments. Results are shown by a histogram + the standard error of the mean. Statistical significance is indicated by P < 0.001.

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Fig. 4. ATP concentration in astrocytes during a recovery experiment following a 45 min glucose depletion period with L-glucose. Astrocytes were first incubated for 45 min at 37°C in a Krebs-type medium buffered with HEPES (10 mM) either in the presence of D-glucose (10 mM) (controls) or with L-glucose (10 mM) (glucose deprivation model). Then, the cells were washed and resuspended in the same medium in the presence of substrates and incubated for another 45 min (recovery period). The substances indicated in the histogram are related to the second incubation period, n stands for the number of experiments. Results are shown by histograms __+the standard error of the mean. Statistical significance is indicated by P < 0.001. A N O V A 1 showed an F value was 127. Besides confirmation of the previous analysis by a t-test of Scheff6 showed that L-pyruvate + malate was different from the L-glucose group with a P value < 0.01.

The oxygen consumption in controls was 36.29 + 3.02 nanoatoms/mg of proteins. When cells were incubated with L-glucose (10 mM) the oxygen consumption was 30.16+0.91 nanoatoms/mg of proteins. In the recovery experiments, respiration measurements showed a slight increase to 33.49 nanoatoms/mg of proteins (mean of two experiments). As expected, when FCCP (0.2 mM) was added to control experiments, a 43% increase of oxygen consumption was recorded. No such increase of oxygen consumption could be elicited when the cells were incubated twice with L-glucose (10 mM). In a similar way, astrocytes incubated with L-glucose (10 mM) and reincubated in a recovery experiment with D-glucose (10 mM) did not react to the FCCP treatment. Only when the recovery reincubation was carried out in the presence of L-malate (1 m M ) + Lpyruvate (10 mM) did the oxygen consumption increase 20% only with FCCP when compared to the D-glucose recovery incubation (Fig. 7). A statistical analysis was carried out comparing the means of State 4 of respiration between controls or FCCP treated cells and different incubation conditions.

4. Discussion As described in Section 2, a bulk isolated astrocyte model from adult rats' cerebral cortex was chosen for

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J. Mertens-Strij'thagen et al. / Neuroscience Research 26 (1996) 133 139 0,06.

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Time (minutes) Fig. 5. ~4COz production at different times by astrocytes in the recovery medium. Astrocytes were first incubated for 45 rain in a Krebs medium at 37°C buffered with HEPES (10 mM) at pH 7.4 in the presence of L-glucose. Subsequently, the cells were washed, centrifuged and reincubated at different times either with L-malate (1 mM) + L-pyruvate (10 mM) with L-[2J4C]pyruvate (25 mCi/mmole) (square symbols) or with D-glucose (10 raM) with D-[UJ4C]-glucose (100 mCi/mmol) (filled circular symbols). Controls were incubated first for 45 min with D-glucose (10 raM) and at different times in the presence of D-glucose (10 raM) with D-[UJ4C]-glucose (I00 mCi/ mmol) (empty circular symbols). Eacla point is the mean of two experiments. o u r experiments instead o f c u l t u r e d astrocytes isolated from n e w b o r n rats. Previous investigations have s h o w n that c u l t u r e d astrocytes i n c u b a t e d in glucose-free

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Fig. 6. CO2 production (mmoles/mg of proteins) in astrocytes during a recovery experiment following a 45 min glucose depletion period with L-glucose. Astrocytes were first incubated for 45 min at 37°C in a Krebs-type medium buffered with HEPES (10 mM) either in the presence of D-glucose (10 mM) (controls) or with L-glucose (10 mM) (glucose deprivation model). Then, the cells were washed and resuspended in the same medium in the presence of substrates and incubated for another 45 rain (recovery period). The substances indicated in the histogram are related to the second incubation period. D-glucose (10 mM)+ L-malate (1 mM) did not increase the CO2 production. Differences between D-glucose (10 mM) and D-glucose (10 mM) + L-malate (l mM) was statistically non significant, n stands for the number of experiments. Results are shown by histograms + the standard error of the mean. Statistical significance between controls and experiments is indicated by P<0.001 and P < 0.05.

L-gl ue0ae 10mH

D-glmo~ 10ra.M L-pyr 10mbl + L-II~I lrt#l

Fig. 7. Oxygen consumption in isolated astrocytes. Oxygen consumption in astrocytes were first incubated in a medium as described in materials and methods during 45 min with L-glucose (10 raM) and then washed and reincubated for 45 min in the presence of either L-glucose (10 mM) or D-glucose (10 raM) or L-malate (1 mM) + Lpyruvate (10 mM). Controls are cells submitted to both incubation periods of 45 min in the presence of D-glucose. n stands for the number of experiments. Results are shown by histograms + the standard error of the mean. Means were compared by a student t-test in State 4 of respiration between controls or FCCP treated cells and different incubation conditions. The incubation substances indicated in the histogram are recovery conditions. Statistical significance is indicated by P < 0.001 and N.S. for non significant. The number of experiments being different among the different groups, a one way ANOVA on the difference between State 4 and FCCP groups was performed with a F value of 9.80. The test of Scheff6 confirmed the results of the t-test analysis. D M E M b u t c o n t a i n i n g other metabolites did n o t react to glucose d e p r i v a t i o n to the same extend as b u l k isolated astrocytes (Rosier et al., 1996). T h e r e d u c t i o n o f A T P in p r i m a r y cultures was 30% after 48 h o f i n c u b a t i o n as c o m p a r e d to 60% in b u l k isolated astrocytes i n c u b a t e d in a salt s o l u t i o n for 45 m i n (Fig. 3). W h e n c u l t u r e d cells were i n c u b a t e d in a salt s o l u t i o n ( H a n k s b a l a n c e d salt solution) the same type of A T P depletion was recorded. However, at that time, cells were m o r p h o l o g i c a l l y altered a n d some l o o s e n i n g from the dish was apparent. A similar p h e n o m e n o n was also observed in c u l t u r e d so-called 'reactive' astrocytes pretreated with d i b u t y r y l cyclic A M P ( F e d o r o f f et al., 1984). W h e t h e r the freshly isolated astrocytes are m e t a b o l i c a l l y m o r e active t h a n the c u l t u r e d cells is an o p e n question. A s s u m i n g that a m a j o r a m o u n t o f glycogen is p r i m a r i l y f o u n d in the astrocytes ( C a t a l d o a n d Broadwell (1985); C a m b r a y - D e a k i n et al., 1988), a r o u g h c a l c u l a t i o n allows us to estimate that the b r a i n glycogen c o n t e n t in p r o f o u n d hypoglycemic c o m a (unp u b l i s h e d results) is c o m p a r a b l e to o u r intracellular glycogen c o n c e n t r a t i o n . Since, in several experiments, D-glucose was u n a b l e to restore the astrocytes initially s u b m i t t e d to a period o f glucose d e p r i v a t i o n , it seemed m a n d a t o r y to investigate whether d u r i n g the i n c u b a t i o n with L-glucose, the glucose t r a n s p o r t into the cells was affected. As one can observe from Fig. 1, after a 45 miD period o f glucose

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deprivation, the transport of 3H-2-deoxy-D-glucose was increased in the astrocytes. This is in accordance with hypoglycemia experiments on total rat brain in which glucose extraction was increased in a chronic hypoglycemic environment (McCall et al., 1986). According to our results, when D-glucose did not allow a recovery from a 45 min glucose deprivation, it was unrelated to a decrease of glucose transport into the cells neither to a modification of intracellular glycolysis. As presented in Fig. 3, once astrocytes were subjected to glucose deprivation conditions for 45 min, they do not recover at all their ability to synthesize high energy nucleotides like ATP and creatine phosphate when placed in the presence of D-glucose. Efficient replacement metabolites for D-glucose only slightly increased the ATP content of the cells (Fig. 4). This is consistent with the morphological data suggesting an irreversible brain damage but at variance with the selective neuronal necrosis effect associated with a glia sparing concept in hypoglycemia (Agardh et al., 1980; Auer, 1986). When incubated with L-glucose (10 mM) the production of 14CO2 fell sharply. An attempt to recover the production of 14C02 in the presence of L-pyruvate (10 mM) only led to a production which was 21% of that of the control cells. When L-malate (1 mM) was added to L-pyruvate (10 mM) an increase of 14CO2 production of 70% was registered and after 1 h total recovery was obtained. This experiments would be consistent with an exhaustion of Krebs cycle endogenous substrates during the glucose deprivation period. However, unexpectedly, when L-malate (1 mM) was added to the incubation medium with D-glucose (10 raM) no recovery of ~4CO2 production was shown (Fig. 6). This situation is similar to a phenomenon described by Hertz and Schousboe, 1986 in which 50% of glucose was transformed in lactate, 15% in glycogen and only 5% in CO2. Furthermore, Passonneau and Crites showed that the glycogen content of astroglial cells was dependent upon the concentration of glucose in the incubation medium (Passonneau and Crites, 1976). In a glycogen depleted situation like glucose deprivation the metabolism towards CO2 is likely to be further reduced. According to Nakamura et al., spermatids treated with a-ketovalerate increased their CO2 production (Nakamura et al., 1986). Since in glucose deprivation an important decrease of pyruvate concentration was recorded, c~-ketovalerate (25 mM) was added to the incubation medium. This increased dramatically the intracellular pyruvate concentration at the expense of lactate. However after a glucose deprivation, no increase of CO2 production could be elicited even in the presence of L-malate (1 mM). After 1 h, the Krebs cycle in the glucose deprived cells seems to function like in control astrocytes but no recovery could be ascertained for the ATP production.

This would suggest that some permanent alteration at the respiratory level may be responsible for the irreversible ATP depletion. As Fig. 7 shows, respiration decreases when astrocytes are incubated with L-glucose and no uncoupling effect was demonstrated with FCCP. As opposed to what was expected from the efficiency of glucose replacement metabolites (Baudoux et al., 1993), only a 20% increase of respiration was noticeable during the recovery experiment with a Lmalate + L-pyruvate incubation. In conclusion, the problem of hypoglycemia was investigated by using a D-glucose deprivation model in bulk isolated rat astrocytes. After being submitted to a 45 min incubation period with L-glucose, astrocytes could not recover from their ATP depletion with D-glucose and with glucose replacement metabolites. When compared to control cells D-glucose, and to some extend L-pyruvate, could not allow a recovery in the 'posthypoglycemic' cells of Krebs cycle activity and some functional lesions were detected at the respiratory level. Although structural lesions of glia have not been reported in hypoglycemic encephalopathy, our experiments strongly suggest that some functional impairment affects the glucose deprived astrocytes (Agardh et al., 1980). However, caution should be exercised in drawing conclusions from studies on isolated astrocytes separated from their cerebral environment with all their trophic and the reciprocal metabolic influences on neurones. The isolated astrocytes elicited an alteration of their respiratory capabilities leading probably to a reduction of ATP production. Whether these effects may be extrapolated to hypoglycemia in the brain is a matter of conjecture.

Acknowledgements The authors wish to thank Dr C. D e Schryver for valuable discussions and Dr E. Depiereux of the division of Biostatistics for resolving the statistical problems. The excellent technical assistance of C. Fautr6 is gratefully aknowledged.

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