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Distribution of freed -serine in vertebrate brains
BRAIN RESEARCH ELSEVIER
Brain Research 634 (1994) 291-295
Research Report
Distribution of free o-serine in vertebrate brains Yoko Nagata a,., Kihachiro Horiike b, Toshihiro Maeda c a Department of Life Science, Himeji Institute of Technology, 2167 Shosha, Himeji 671-22, Japan Departments of b Biochemistry and CAnatomy, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-21, Japan
(Accepted 7 September 1993)
Abstract Free D-serine distribution in vertebrate brains was investigated. In various brain regions of the lower vertebrate species, carp, frog and chick, free D-serine levels were low. On the contrary, in the mammals, mouse, rat and bull, the contents of free D-serine were high in the forebrain (around 400 nmol/g wet weight, and the ratio of D-serine to L-serine, was O/L = 0.4), and low in the hindbrain. In developing mice, D-serine levels in the cerebrum increased with age and attained the adult level (D/L = 0.40) 8 weeks after birth. In the cerebellum and brain stem, the free D-serine levels increased with age until 2 weeks, followed by a decrease to the adult levels: the D/L ratios remained constant until 2 weeks of age, then decreased to 0.03 in the cerebellum and 0.12 in the brain stem. Free D-serine was shown not to be of microbial origin using germ-free mice. In the rat forebrain, D-serine was evenly distributed in two cerebral regions, namely frontal and occipital lobes. The D/L ratios in other regions of forebrain, hippocampus and hypothalamus, were comparable to the cerebrum (D/L = 0.4), while that in the olfactory bulb was lower (D/L = 0.12). In the rat cerebrum, the D-serine content in the grey matter was significantly higher than that in the white matter. The contents of free o-serine in bovine cerebrum and cerebellum were similar to those in other mammalian brains, but the D/L ratio for bovine cerebral grey matter was lower than that for the cerebral white matter. The D-serine level was discussed in terms of D-amino-acid oxidase activity. Key words: D-Serine; Distribution; Vertebrate; Brain; Cerebrum; Cerebellum; NMDA receptor; D-Amino-acid oxidase
1. Introduction Neutral D-amino acids are present in microorganisms, some insects, marine invertebrates and higher plants, but it is generally believed that neutral D-amino acid is neither synthesized nor occurs in vertebrates [12]. Recently, we devised a sensitive method for the enantiomeric analysis of amino acids [17]. This method is based on the resolution of D- and L-enantiomers of amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide ( F D A A ) using a combination of two-dimensional thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). We have detected substantial amounts of free Dalanine, o-proline and D-serine in the kidney and serum of a mutant mouse lacking D-amino-acid oxidase (EC 1.4.3.3) [18], and in human plasma from patients with renal diseases [16]. Moreover, we have recently found
free D-serine at surprisingly high concentrations, D/L ratio of 0.44, in the mouse cerebrum [13], in concert with the results on rat brain [5]. Since o-serine is a potent activator of the N-methylD-aspartate (NMDA) receptor complex by binding to the glycine modulatory site in vitro [2,9,21] and in vivo [1,20], and since Hashimoto et al. [6] recently reported N M D A receptor-related distribution of D-serine in rat brain, we investigated the distribution of free o-serine in several vertebrate brains, in the brain of the developing mouse, and especially in detail in rat forebrain. As the results, it was, however, indicated that the localization was mainly ascribable to the function of D-amino-acid oxidase.
2. Materials and methods 2.1. Brain separation from animals
* Corresponding author. Fax: (81) (792) 66-8868. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E 1260-A
Carp (Cyprinus carpio, female, 35-45 cm; infants, 8-12 cm) and bullfrogs (Rana catesbeiana, 400-500 g) were collected from local
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Y. Nagata et al. / Brain Research 634 (1994) 291-295
ponds, and brought to the laboratory alive. They were decapitated under hypothermic anesthesia by dipping them in crushed ice. The cerebrum, cerebellum, optic lobe, hypophysis and medulla oblongata were obtained from the carp. The cerebrum and cerebellum were separated from the frogs. Chick (White Leghorn, male, adult, 2.5-3.0 kg) and bull (male, adult, 550-600 kg) brains were obtained from local slaughter houses. Three hours after the chick was decapitated, the cerebrum, cerebellum, medulla oblongata and the remainder of the brain (designated as midbrain) were separated and kept at -79°C until use. The bovine brain was separated immediately after death and kept in an ice bucket for 1 h before excision of the cerebrum and cerebellum, which were subsequently stored at -79°C. BALB/cA (1 day to 23 weeks old) mice, germ-free (IQI/Jcl, 8 weeks old) and specific pathogen-free (IQI/Jc|, 8 weeks old) mice, all females, were obtained from Japan Clea (Tokyo, Japan). BALB/ cA mice were maintained in an animal room of the Himeji Institute of Technology on a standard diet (Type MF and NMF, Oriental Yeast, Tokyo). The mice were starved, but allowed water, for 16-19 h before being killed by collecting blood from the axillary vessels under anesthesia with diethyl ether. Germ-free and specific pathogen-free mice were killed in the same manner as above immediately after axenic transportation from the supplier. The brains were separated rapidly and frozen at -79°C until use. Each brain was separated on an ice-cooled glass plate into the cerebellum, cerebrum including olfactory bulbs (designated as cerebrum in the present paper) and the remaining region (designated as brain stem). The spinal cord was not examined. Male Wistar rats (9 weeks old, 240-260 g), obtained from Japan Clea and bred with CE-2 diet (Japan Clea), were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg), and perfused through the left ventricle after ligation of the descending aorta and incision of the right heart auricle, initially with 100 ml of 150 mM NaCI in 10 mM sodium phosphate, pH 7.4 (phosphatebuffered saline), and subsequently with 150 ml of the same ice-cold solution at a flow rate of 70 ml/min. The whole brain was frozen with solid CO 2 immediately after removal, and kept at -20°C on a stainless steel table in a Cryocut 1800 (Reichert-Jung, NuBloch, Germany). To obtain the results described in Table 4, the brain was dissected into olfactory bulbs, hippocampus, hypothalamus, frontal lobe and the occipital lobe, according to Glowinski and Iversen [3]. The frontal and occipital lobes were subsequently separated into right and left hemispheres. To obtain the results shown in Table 5, the cerebrum was cut into I-ram-thick coronal sections, then the right and left halves of the sections were separated into grey and white matter by hand with a razor blade. The grey matter prepared in this manner excluded white matter, whereas the white matter section was contaminated with some grey matter.
Table 1 Contents of free D- and L-serine, and D/L ratios in various brain regions of several vertebrate species Vertebrate species
Brain region Free serine (nmol/g wet weight) 1.
D
(D/L)
× 1000 Carp
(n = 2) Cerebrum Optic lobe Hypophysis Medulla oblongata Cerebellum Infantcarp ( n = 3 ) Cerebrum Frog ( n = 3 ) Cerebrum Cerebellum Chick (n = 3) Cerebrum Midbrain Medulla oblongata Cerebellum Mouse, 8w ( n = 3 ) Cerebrum Brain stem Cerebellum Rat, 9w ( n = 3 ) Cerebrum Bovine (n = 2) Cerebrum Cerebellum
1170,1160 0, 6.3 1290,895 6.5, 0 796, 917 3.0, 7.5
3* 3* 6*
1660, 1870 1500, 1110 646_+194 377_+27 606 733_+216 1190_+95
3.5, 5.3 4.2, 0 1.9_+1.9 1.1+_1.9 3.6 7.0+_0.7 16_+4
2 * 2 * 2_+2 2_+3 6 10+_2 14+_5
1510+_82 1040+_10 12905:16 1110_+55 989+_123 928+_102 917, 1020 835,801
185:2 11+_0.0 520_+98 127_+32 28+-16 395+_74 355,430 20, 24
12-+2 11-+0 425_+69 115+_34 29 +- 20 425+_52 405 * 27 *
Values are the means +-S.D. for the number of animals shown in parentheses, except for the cerebellum of frog where tissues from three animals were combined. * Values are calculated by using the mean values for two animals.
are described elsewhere [17]. Briefly, the FDAA-amino acids were separated on a silica gel-plate by two-dimensional TLC. FDAA-serine recovered from the plate was analyzed by HPLC with a reversedphase column, Nova-Pak C18 (150×3.9 mm i.d., Waters, Milford, MA, USA). The sample was eluted with a linear gradient of acetonitrile in 50 mM triethylamine-phosphate buffer (pH 3.5) from 10 to 25% over 20 min at a flow rate of 1.0 ml/min at 22°C. The eluate was monitored at 340 nm with a D-2500 Chromato-lntegrator (Hitachi, Tokyo, Japan), and peak areas of FDAA-D- and -L-serine were obtained automatically. Amounts of n- and L-serine were calculated on the basis of the peak areas and standard curves [17].
3. Results 2.2. Analysis of o- and L-serine Each brain section was homogenized with 4 vols. of phosphatebuffered saline in a glass homogenizer in an ice bucket at 1100 rpm for 1 min, and centrifuged at 16000× g for 1 h at 4°C. To the supernatant extract, a 6% (w/v) cold trichloroacetic acid solution was added to a final concentration of 5%. Alternatively, the brain sections were homogenized with 10 vols. of the 6% trichloroacetic acid solution, and centrifuged. Both procedures yielded the same results. The supernatant fraction containing free amino acids was passed through a Dowex 1 x 8 column (acetate form; Muromachi Chemicals, Tokyo) to remove trichloroacetic acid and acidic amino acids. The effluent fraction was brought to dryness with a centrifugal evaporator (VC-360, Taitec, Saitama, Japan) in vacuo. The residue was dissolved in distilled water, and derivatized with FDAA (Pierce, Rockford, IL, USA) to resolve and determine D- and L-serine. Details of the analytical method that gives a 20-pmol detection limit
T h e f r e e D - s e r i n e l e v e l s in v a r i o u s r e g i o n s o f b r a i n s o f s e v e r a l v e r t e b r a t e s p e c i e s a r e s h o w n in T a b l e 1. A l t h o u g h h i g h l e v e l s o f L - s e r i n e w e r e p r e s e n t in t h e c e r e b r u m , optic lobe, hypophysis, m e d u l l a o b l o n g a t a a n d c e r e b e l l u m o f t h e c a r p as w e l l as in t h e c e r e b r u m and c e r e b e l l u m of the frog, almost no D-serine was d e t e c t e d . T h e D - s e r i n e c o n t e n t w a s a l s o low in t h e infant c a r p c e r e b r u m . In s o m e s p e c i m e n s of c a r p a n d frog, no D-serine was d e t e c t a b l e . In the c e r e b r u m , m i d b r a i n , m e d u l l a o b l o n g a t a a n d c e r e b e l l u m of chick, D - s e r i n e w a s p r e s e n t a t low levels. T h e D / L r a t i o in t h e c h i c k b r a i n w a s a r o u n d 0.01. O n t h e o t h e r h a n d , in c e r e b r a o f m a m m a l s , m o u s e , r a t a n d bull, h i g h c o n tents of o-serine were found; 400-500 nmol/g wet
Y. Nagata et a l . / Brain Research 634 (1994) 291-295 Table 2 Contents of free D- and L-serine in the cerebrum, brain stem and cerebellum of mice of different ages Brain region
Age
Cerebrum
1 day (n = 3) 8 day (n = 3) 2 w e e k s (n = 3) 3 weeks (n = 1) 4 w e e k s (n = 3) 8 w e e k s (n = 3) 23weeks ( n = 2 ) 1 day (n = 3) 8day (n=3) 2weeks (n=3) 3 w e e k s ( n = 1) 4 weeks (n = 3) 8weeks (n=3) 23weeks (n = 2) 1 day (n = 3) 8 day (n = 3) 2 w e e k s (n = 3) 3weeks (n=l) 4 weeks (n = 3) 8 weeks (n = 3) 23weeks (n = 2)
Brain stem
Cerebellum
293
Free serine ( n m o l / g wet weight)
Table 3 Contents of free o- and L-serine, and D/L ratios in the cerebrum, brain stem and cerebellum of germ-free and specific pathogen-free mice (8 weeks old, n = 2)
L
D
(D/L)
Brain region
442±267 489±120 891±57 999 817±230 1290± 16 1090, 1090 ND 641±172 910± 19 737 923±429 1110±55 653, 989 314 568±60 1190±80 765 741±54 989± 123 991,763
61±31 9 3 ± 15 230±6 226 263± 19 520±98 449,388 ND 108±32 160±43 103 53±38 127±32 68, 106 30 4 8 ± 19 94±72 16 12±7 2 8 ± 16 26, 11
0.142±0.016 0.192±0.018 0.258±0.024 0.226 0.331±0.070 0.403±0.069 0.384 * 0.167±0.005 0.176±0.051 0.140 0.054±0.016 0.115 ±0.034 0.106 * 0.096 0.084±0.024 0.078±0.055 0.021 0.016±0.004 0.029±0.020 0.021 *
Values are the m e a n s + S . D , for the n u m b e r of animals shown in parentheses, except for the cerebellum of day 1, in which tissues from three mice were combined. ND, not determined. * Values are calculated by using the m e a n values for two animals.
weight (D/L = 0.4). The free L-serine levels in the cerebella and brain stems of mouse and bull were comparable to those in the cerebra, whereas the Oserine levels in the cerebella and brain stems were considerably lower than those in the cerebra, resulting in low D/L ratios for the mammalian cerebella (D/L 0.03). Free D- and L-serine levels in the cerebrum, brain stem and cerebellum from mice of various ages, ranging from 1 day to 23 weeks, are presented in Table 2. The contents of L-serine increased with age in the brain regions examined, and reached the level of the adult mouse (8 weeks old), namely about 1 /zmol/g wet weight. At each developing stage, no prominent difference in the L-serine level was observed among the three brain regions. The D-serine level in the cerebrum also increased with age. In the cerebellum and brain stem, by contrast, the peak D-serine levels were attained at 2 weeks of age, followed by a decrease to the respective adult levels. The value of D/L ratio for the cerebrum increased with age and approached the adult level of 0.4 by age 8 weeks, whereas those for the brain stem and cerebellum decreased 2 weeks after birth. The D/L ratios in adult mice were 0.12 for the brain stem and 0.03 for the cerebellum. To determine if the free D-serine detected in the present study originated from microorganisms, e.g. enterobacteria, the free D-serine content in the brain was
Cerebrum Brain stem Cerebellum
Free serine ( n m o l / g wet weight) Germ-free mouse
Specific pathogenfree mouse
L
D
D/L
L
D
D/L
743 1070 798 899 777 885
418 365 86 133 4.4 10.6
0.432 *
1190 792 1030 806 940 701
488 443 185 48 8.6 2.9
0.470 *
0.129 * 0.009 *
0.127 * 0.007 *
* Values are calculated by using the mean values for two animals.
compared between germ-free and specific pathogenfree mice (Table 3). In the brain regions examined, no significant difference was found in either the o- and L-serine levels and in the O/L ratio between the two groups. Since the mammalian forebrain contained free oserine at a high level (400-500 nmol/g, O/L -----0.4), as revealed in the above investigations, the distribution of free o-serine was investigated in detail in the rat forebrain, i.e. olfactory bulb, right and left frontal lobes, right and left occipital lobes, hippocampus and hypothalamus (Table 4) as well as in the grey and white matter of the cerebrum (Table 5). No significant difference was found in the o- and L-serine levels or in the O/L ratio between the two regions of the rat cerebrum, namely the frontal and occipital lobes. Although the Dand L-serine contents in both hippocampus and hypothalamus were respectively lower than those in the above two cerebral regions, the D/L ratios were similar to those in the same cerebral regions. The D/L ratio for the olfactory bulb was lower than those for the other regions of the forebrain. Next, the level of free D-serine was compared between grey and white matter in the cerebrum. In the rat, the D/L ratio for the grey matter was higher than that for the white matter (Table 5). No significant differences in the o- and L-serine
Table 4 Contents of free D- and L-serine, and D / L ratios in rat forebrain Forebrain region
Olfactory bulb * Right frontal lobe Left frontal lobe Right occipital lobe Left occipital lobe Hippocampus * Hypothalamus t
Free serine ( n m o l / g wet weight) L
D
874 1030 + 148 827 + 55 1000 d: 96 853 ± 124 464 614
108 480 + 377 + 419 + 305 + 208 250
D//L
135 42 101 19
0.123 0.465 + 0.065 0.456 -I-0.025 0.419 + 0.081 0.358 + 0.036 0.448 0.406
Values are the m e a n s + S.D. for three animals except where tissues from two ( * ) or three (t) animals were combined.
Y. Nagata et al. / Brain Research 634 (1994) 291-295
294
Table 5 Contents of free D- and L-serine, and D/L ratios in the grey and white matter of the rat cerebrum Free serine ( n m o l / g w e t w e i g h t ) Right cerebrum
Grey matter White matter
Leficerebrum
c
D
D/c
c
D
D/L
947 + 50 851 + 86
407 _+ 20 249 _+ 47
0.430 +_ 0.004 0.293 + 0.014 P < 0.02 *
1000 _+ 40 982 + 2
416 +_ 61 241 _+ 66
0.416 _+ 0.046 0.245 + 0.054 P < 0.05 *
Values are the means + S.D. for three animals. * t-test for differences between the two groups, after examination by F-test.
contents were observed between the right and left cerebral halves, respectively. In the bull, the D/C ratio for cerebral grey matter (D/L = 0.27) was lower than that for the cerebral white matter (D/L = 0.62), whereas the ratios for the cerebellar grey and white matter were similar (D/L = 0.01-0.03).
4. Discussion
The analytical method used in the present study is capable of determining 20-50 pmol of D- and L-amino acids [17]. The sample was identified as D-serine by using a combination of two-dimensional TLC and HPLC, and furthermore the identification was confirmed by the treatment of the sample with D-aminoacid oxidase: the FDAA-amino acid migrated to the same location as FDAA-serine on the TLC-plate, on which amino acids were separated well from one another [17], was recovered from the plate, and co-chromatographed with authentic FDAA-D- and FDAA-Lserine in HPLC. The peaks of the sample from the TLC-plate overlapped completely with D- and L-serine peaks, respectively. On the other hand, the brain extract was treated with D-amino-acid oxidase from porcine kidney (Sigma, St. Louis, MO, USA) at 37°C for 30 min, and then subjected as described under Materials and Methods. On HPLC analysis, one of two peaks of the sample, which corresponded to that of D-serine, completely disappeared (data not shown). The oxidase has high specificity to D-amino acids [14]. Furthermore, both the amount of free D-serine and D/L ratio of free serine in rat cerebrum (Table 1) were, respectively, found to be similar to those obtained by using gas chromatographic-mass spectrometric analysis [5]. The presence of considerable amounts of free Dserine has been demonstrated in cerebra of mouse (Tables 1-3), rat (Tables 1, 4 and 5) and bull (Table 1). A high content of D-serine was also observed in human cerebrum (unpublished work). D-Serine level was low in cerebellum (Tables 1-3). Although species variation and regional differences in the distribution of D-serine in the brain were observed, it appears that the distribution of free D-serine is general in mammalian cerebra.
D-Serine level in the mouse cerebellum is low (Tables 1-3), whereas it is high in the cerebellum [13] of a mutant mouse [10,11] lacking D-amino-acid oxidase. Furthermore, we recently obtained some evidence that D-amino-acid oxidase catabolizes neutral free D-amino acids in vivo in the mouse [15,18] and in humans [16]: substantial amounts of free D-serine, D-alanine and D-proline were observed in plasma and kidney specimens obtained from where the oxidase activity was very low or absent. Together with these facts, the present results were interpreted in terms of D-amino-acid oxidase activity as follows. (1) The oxidase is distributed evenly over the whole brain in the lower vertebrate species, and the activity in the chick forebrain is lower than those in the fish and frog [4]. In mammals, the oxidase is highly concentrated in the hindbrain, particularly in the cerebellum [4,7,8,19]. The oxidase activity is absent or scarce in the forebrain, and low in the midbrain of mammals [4]. Therefore, the distribution of free D-serine in the various brain regions of vertebrates described in Table 1, indicates that the D-serine content depends on D-amino-acid oxidase activity. (2) The oxidase activity in the rat cerebellum and brain stem was first detected on day 14 postnatally, and then increased rapidly to reach the adult level 4 to 5 weeks later [22]. The oxidase activity in the brain stem is 39% of that in the cerebellum. In the cerebrum, the activity was undetectable until day 29, and the maximal activity reached less than 2% of the cerebellar activity in adult rat [22]. In adult mice, the oxidase activity in the cerebrum is also remarkably lower than that in the cerebellum [4,13]. Therefore, the changes in the D / e ratio among different brain regions of developing mice (Table 2) suggest that D-serine is oxidatively deaminated in situ by D-amino-acid oxidase. Recently, Hashimoto et al. [6] also revealed a substantial amount of free D-serine in various regions of rat brain with a similar distribution pattern to that shown in the present study. They communicated that the regional variations in the amount of free D-serine were closely correlated with that of N M D A receptor, and proposed that D-serine was a novel candidate as an intrinsic ligand for the glycine site of the N M D A receptor [6]. However, in the mutant mouse cerebellum, free D-serine level is remarkably high (D/L =
Y. Nagata et aL / Brain Research 634 (1994) 291-295
0.256) [13]. It is of interest if the amount of N M D A receptor is increased in that cerebellum. The mutant mice show no abnormal behavior by our observation, and the life span and body weight are not different from the normal mouse, in spite that free D-serine may not be metabolized in the mutant animals. Furthermore, in the bull, the D/L ratio of free D-serine for cerebral grey matter was much lower than that for the cerebral white matter. Hence, it is possible that the distribution pattern of free D-serine in the brain is mainly a consequence of D-amino-acid oxidase activity. Since D-serine was detected in the brains of germfree mice (Table 3), the free D-serine did not originate from bacteria, which possess some D-amino acids as cell wall components and products [12]. However, since 1% of the total serine in the diets used in the present study for mouse and rat was the o-enantiomer (D/L----0.01, our unpublished data), we cannot rule out that the free D-serine in the brain is of dietary origin. Further investigations are required to elucidate the source of the D-serine as well as its physiological, pharmacological and pathological significance, since, in in vitro experiments, D-serine potentiates the action of N M D A receptor by binding to a strychnine-insensitive glycine binding site on the receptor complex [1,2,9,20, 21].
Acknowledgements. We thank Dr. Mineko Fujimiya of the Department of Anatomy, Shiga University of Medical Science, for her cooperation in the preparation of brain sections. Thanks are also due to Prof. Mitsuhiro Nozaki of the Department of Biochemistry, Shiga University of Medical Science, for his continuous encouragement during the course of this study.
References [1] Contreras, P.C., D-Serine antagonized phencyclidine- and MK801-induced stereotyped behavior and ataxia, Neuropharmacology, 29 (1990) 291-293. [2] Fadda, E., Danysz, W., Wroblewski, J.T. and Costa, E., Glycine and D-serine increase the affinity of N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes, Neuropharmacology, 27 (1988) 1183-1185. [3] Glowinski, J. and Iversen, L.L., Regional studies of catecholamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]dopa in various regions of the brain, J. Neurochern., 13 (1966) 655-669.
295
[4] Goldstein, D.B., D-Amino acid oxidase in brain: distribution in several species and inhibition by pentobarbitone, J. Neurochem., 13 (1966) 1011-1016. [5] Hashimoto, A., Nishikawa, T., Hayashi, T., Fujii, N., Harada, K., Oka, T. and Takahashi, K., The presence of free D-serine in rat brain, FEBS Lett., 296 (1992) 33-36. [6] Hashimoto, A., Nishikawa, T., Oka, T. and Takahashi, K., Endogenous D-serine in rat brain: N-methyl-D-aspartate receptor-related distribution and aging, J. Neurochem., 60 (1993) 783-786. [7] Horiike, K., Arai, R., Tojo, H., Yamano, T., Nozaki, M. and Maeda, T., Histochemical staining of cells containing flavoenzyme D-amino acid oxidase based on its enzymatic activity: application of a coupled peroxidation method, Acta Histochem. Cytochem., 18 (1985) 539-550. [8] Horiike, K., Tojo, H., Arai, R., Yamano, T., Nozaki, M. and Maeda, T., Localization of D-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum, Brain Res. Bull., 19 (1987) 587-596. [9] Kleckner, N.W. and Dingledine, R., Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes, Science, 241 (1988) 835-837. [10] Konno, R. and Yasumura, Y., Mouse mutant deficient in Damino acid oxidase activity, Genetics, 103 (1983) 277-285. [11] Konno, R. and Yasumura, Y., Brain and kidney D-amino acid oxidases are coded by a single gene in the mouse, J. Neurochem., 42 (1984) 584-586. [12] Man, E.H. and Bada, J.L., Dietary D-amino acids, Annu. Rev. Nutr., 7 (1987) 209-225. [13] Nagata, Y., Involvement of D-amino acid oxidase in elimination of D-serine in mouse brain, Experientia, 48 (1992) 753-755. [14] Nagata, Y., Akino, T. and Ohno, K., Microdetermination of serum D-amino acids, Anal. Biochem., 150 (1985) 238-242. [15] Nagata, Y., Konno, R., Yasumura, Y. and Akino, T., Involvement of D-amino acid oxidase in elimination of free D-amino acids in mice, Biochem. J., 257 (1989) 291-292. [16] Nagata, Y., Masui, R. and Akino, T., The presence of free D-serine, D-alanine and D-proline in human plasma, Experientia, 48 (1992) 986-988. [17] Nagata, Y., Yamamoto, K. and Shimojo, T., Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography, J. Chromatogr., 575 (1992) 147-152. [18] Nagata, Y., Yamamoto, K., Shimojo, T., Konno, R., Yasumura, Y. and Akino, T., The presence of free D-alanine, D-proline and D-serine in mice, Biochim. Biophys. Acta, 1115 (1992) 208-211. [19] Neims, A.H., Zieverink, W.D. and Smilack, J.D., Distribution of D-amino acid oxidase in bovine and human nervous tissues, J. Neurochem., 13 (1966) 163-168. [20] Salt, T.E., Modulation of NMDA receptor-mediated responses by glycine and D-serine in the rat thalamus in vivo, Brain Res., 481 (1989) 403-406. [21] Snell, L.D., Morter, R.S. and Johnson, K.M., Structural requirements for activation of the glycine receptor that modulates the N-methyl-o-aspartate operated ion channel, Eur. J. Pharmacol., 156 (1988) 105-110. [22] Weimar, W.R. and Neims, A.H., The development of D-amino acid oxidase in rat cerebellum, J. Neurochem., 29 (1977) 649-656.
Brain Research 634 (1994) 291-295
Research Report
Distribution of free o-serine in vertebrate brains Yoko Nagata a,., Kihachiro Horiike b, Toshihiro Maeda c a Department of Life Science, Himeji Institute of Technology, 2167 Shosha, Himeji 671-22, Japan Departments of b Biochemistry and CAnatomy, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-21, Japan
(Accepted 7 September 1993)
Abstract Free D-serine distribution in vertebrate brains was investigated. In various brain regions of the lower vertebrate species, carp, frog and chick, free D-serine levels were low. On the contrary, in the mammals, mouse, rat and bull, the contents of free D-serine were high in the forebrain (around 400 nmol/g wet weight, and the ratio of D-serine to L-serine, was O/L = 0.4), and low in the hindbrain. In developing mice, D-serine levels in the cerebrum increased with age and attained the adult level (D/L = 0.40) 8 weeks after birth. In the cerebellum and brain stem, the free D-serine levels increased with age until 2 weeks, followed by a decrease to the adult levels: the D/L ratios remained constant until 2 weeks of age, then decreased to 0.03 in the cerebellum and 0.12 in the brain stem. Free D-serine was shown not to be of microbial origin using germ-free mice. In the rat forebrain, D-serine was evenly distributed in two cerebral regions, namely frontal and occipital lobes. The D/L ratios in other regions of forebrain, hippocampus and hypothalamus, were comparable to the cerebrum (D/L = 0.4), while that in the olfactory bulb was lower (D/L = 0.12). In the rat cerebrum, the D-serine content in the grey matter was significantly higher than that in the white matter. The contents of free o-serine in bovine cerebrum and cerebellum were similar to those in other mammalian brains, but the D/L ratio for bovine cerebral grey matter was lower than that for the cerebral white matter. The D-serine level was discussed in terms of D-amino-acid oxidase activity. Key words: D-Serine; Distribution; Vertebrate; Brain; Cerebrum; Cerebellum; NMDA receptor; D-Amino-acid oxidase
1. Introduction Neutral D-amino acids are present in microorganisms, some insects, marine invertebrates and higher plants, but it is generally believed that neutral D-amino acid is neither synthesized nor occurs in vertebrates [12]. Recently, we devised a sensitive method for the enantiomeric analysis of amino acids [17]. This method is based on the resolution of D- and L-enantiomers of amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide ( F D A A ) using a combination of two-dimensional thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). We have detected substantial amounts of free Dalanine, o-proline and D-serine in the kidney and serum of a mutant mouse lacking D-amino-acid oxidase (EC 1.4.3.3) [18], and in human plasma from patients with renal diseases [16]. Moreover, we have recently found
free D-serine at surprisingly high concentrations, D/L ratio of 0.44, in the mouse cerebrum [13], in concert with the results on rat brain [5]. Since o-serine is a potent activator of the N-methylD-aspartate (NMDA) receptor complex by binding to the glycine modulatory site in vitro [2,9,21] and in vivo [1,20], and since Hashimoto et al. [6] recently reported N M D A receptor-related distribution of D-serine in rat brain, we investigated the distribution of free o-serine in several vertebrate brains, in the brain of the developing mouse, and especially in detail in rat forebrain. As the results, it was, however, indicated that the localization was mainly ascribable to the function of D-amino-acid oxidase.
2. Materials and methods 2.1. Brain separation from animals
* Corresponding author. Fax: (81) (792) 66-8868. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E 1260-A
Carp (Cyprinus carpio, female, 35-45 cm; infants, 8-12 cm) and bullfrogs (Rana catesbeiana, 400-500 g) were collected from local
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ponds, and brought to the laboratory alive. They were decapitated under hypothermic anesthesia by dipping them in crushed ice. The cerebrum, cerebellum, optic lobe, hypophysis and medulla oblongata were obtained from the carp. The cerebrum and cerebellum were separated from the frogs. Chick (White Leghorn, male, adult, 2.5-3.0 kg) and bull (male, adult, 550-600 kg) brains were obtained from local slaughter houses. Three hours after the chick was decapitated, the cerebrum, cerebellum, medulla oblongata and the remainder of the brain (designated as midbrain) were separated and kept at -79°C until use. The bovine brain was separated immediately after death and kept in an ice bucket for 1 h before excision of the cerebrum and cerebellum, which were subsequently stored at -79°C. BALB/cA (1 day to 23 weeks old) mice, germ-free (IQI/Jcl, 8 weeks old) and specific pathogen-free (IQI/Jc|, 8 weeks old) mice, all females, were obtained from Japan Clea (Tokyo, Japan). BALB/ cA mice were maintained in an animal room of the Himeji Institute of Technology on a standard diet (Type MF and NMF, Oriental Yeast, Tokyo). The mice were starved, but allowed water, for 16-19 h before being killed by collecting blood from the axillary vessels under anesthesia with diethyl ether. Germ-free and specific pathogen-free mice were killed in the same manner as above immediately after axenic transportation from the supplier. The brains were separated rapidly and frozen at -79°C until use. Each brain was separated on an ice-cooled glass plate into the cerebellum, cerebrum including olfactory bulbs (designated as cerebrum in the present paper) and the remaining region (designated as brain stem). The spinal cord was not examined. Male Wistar rats (9 weeks old, 240-260 g), obtained from Japan Clea and bred with CE-2 diet (Japan Clea), were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg), and perfused through the left ventricle after ligation of the descending aorta and incision of the right heart auricle, initially with 100 ml of 150 mM NaCI in 10 mM sodium phosphate, pH 7.4 (phosphatebuffered saline), and subsequently with 150 ml of the same ice-cold solution at a flow rate of 70 ml/min. The whole brain was frozen with solid CO 2 immediately after removal, and kept at -20°C on a stainless steel table in a Cryocut 1800 (Reichert-Jung, NuBloch, Germany). To obtain the results described in Table 4, the brain was dissected into olfactory bulbs, hippocampus, hypothalamus, frontal lobe and the occipital lobe, according to Glowinski and Iversen [3]. The frontal and occipital lobes were subsequently separated into right and left hemispheres. To obtain the results shown in Table 5, the cerebrum was cut into I-ram-thick coronal sections, then the right and left halves of the sections were separated into grey and white matter by hand with a razor blade. The grey matter prepared in this manner excluded white matter, whereas the white matter section was contaminated with some grey matter.
Table 1 Contents of free D- and L-serine, and D/L ratios in various brain regions of several vertebrate species Vertebrate species
Brain region Free serine (nmol/g wet weight) 1.
D
(D/L)
× 1000 Carp
(n = 2) Cerebrum Optic lobe Hypophysis Medulla oblongata Cerebellum Infantcarp ( n = 3 ) Cerebrum Frog ( n = 3 ) Cerebrum Cerebellum Chick (n = 3) Cerebrum Midbrain Medulla oblongata Cerebellum Mouse, 8w ( n = 3 ) Cerebrum Brain stem Cerebellum Rat, 9w ( n = 3 ) Cerebrum Bovine (n = 2) Cerebrum Cerebellum
1170,1160 0, 6.3 1290,895 6.5, 0 796, 917 3.0, 7.5
3* 3* 6*
1660, 1870 1500, 1110 646_+194 377_+27 606 733_+216 1190_+95
3.5, 5.3 4.2, 0 1.9_+1.9 1.1+_1.9 3.6 7.0+_0.7 16_+4
2 * 2 * 2_+2 2_+3 6 10+_2 14+_5
1510+_82 1040+_10 12905:16 1110_+55 989+_123 928+_102 917, 1020 835,801
185:2 11+_0.0 520_+98 127_+32 28+-16 395+_74 355,430 20, 24
12-+2 11-+0 425_+69 115+_34 29 +- 20 425+_52 405 * 27 *
Values are the means +-S.D. for the number of animals shown in parentheses, except for the cerebellum of frog where tissues from three animals were combined. * Values are calculated by using the mean values for two animals.
are described elsewhere [17]. Briefly, the FDAA-amino acids were separated on a silica gel-plate by two-dimensional TLC. FDAA-serine recovered from the plate was analyzed by HPLC with a reversedphase column, Nova-Pak C18 (150×3.9 mm i.d., Waters, Milford, MA, USA). The sample was eluted with a linear gradient of acetonitrile in 50 mM triethylamine-phosphate buffer (pH 3.5) from 10 to 25% over 20 min at a flow rate of 1.0 ml/min at 22°C. The eluate was monitored at 340 nm with a D-2500 Chromato-lntegrator (Hitachi, Tokyo, Japan), and peak areas of FDAA-D- and -L-serine were obtained automatically. Amounts of n- and L-serine were calculated on the basis of the peak areas and standard curves [17].
3. Results 2.2. Analysis of o- and L-serine Each brain section was homogenized with 4 vols. of phosphatebuffered saline in a glass homogenizer in an ice bucket at 1100 rpm for 1 min, and centrifuged at 16000× g for 1 h at 4°C. To the supernatant extract, a 6% (w/v) cold trichloroacetic acid solution was added to a final concentration of 5%. Alternatively, the brain sections were homogenized with 10 vols. of the 6% trichloroacetic acid solution, and centrifuged. Both procedures yielded the same results. The supernatant fraction containing free amino acids was passed through a Dowex 1 x 8 column (acetate form; Muromachi Chemicals, Tokyo) to remove trichloroacetic acid and acidic amino acids. The effluent fraction was brought to dryness with a centrifugal evaporator (VC-360, Taitec, Saitama, Japan) in vacuo. The residue was dissolved in distilled water, and derivatized with FDAA (Pierce, Rockford, IL, USA) to resolve and determine D- and L-serine. Details of the analytical method that gives a 20-pmol detection limit
T h e f r e e D - s e r i n e l e v e l s in v a r i o u s r e g i o n s o f b r a i n s o f s e v e r a l v e r t e b r a t e s p e c i e s a r e s h o w n in T a b l e 1. A l t h o u g h h i g h l e v e l s o f L - s e r i n e w e r e p r e s e n t in t h e c e r e b r u m , optic lobe, hypophysis, m e d u l l a o b l o n g a t a a n d c e r e b e l l u m o f t h e c a r p as w e l l as in t h e c e r e b r u m and c e r e b e l l u m of the frog, almost no D-serine was d e t e c t e d . T h e D - s e r i n e c o n t e n t w a s a l s o low in t h e infant c a r p c e r e b r u m . In s o m e s p e c i m e n s of c a r p a n d frog, no D-serine was d e t e c t a b l e . In the c e r e b r u m , m i d b r a i n , m e d u l l a o b l o n g a t a a n d c e r e b e l l u m of chick, D - s e r i n e w a s p r e s e n t a t low levels. T h e D / L r a t i o in t h e c h i c k b r a i n w a s a r o u n d 0.01. O n t h e o t h e r h a n d , in c e r e b r a o f m a m m a l s , m o u s e , r a t a n d bull, h i g h c o n tents of o-serine were found; 400-500 nmol/g wet
Y. Nagata et a l . / Brain Research 634 (1994) 291-295 Table 2 Contents of free D- and L-serine in the cerebrum, brain stem and cerebellum of mice of different ages Brain region
Age
Cerebrum
1 day (n = 3) 8 day (n = 3) 2 w e e k s (n = 3) 3 weeks (n = 1) 4 w e e k s (n = 3) 8 w e e k s (n = 3) 23weeks ( n = 2 ) 1 day (n = 3) 8day (n=3) 2weeks (n=3) 3 w e e k s ( n = 1) 4 weeks (n = 3) 8weeks (n=3) 23weeks (n = 2) 1 day (n = 3) 8 day (n = 3) 2 w e e k s (n = 3) 3weeks (n=l) 4 weeks (n = 3) 8 weeks (n = 3) 23weeks (n = 2)
Brain stem
Cerebellum
293
Free serine ( n m o l / g wet weight)
Table 3 Contents of free o- and L-serine, and D/L ratios in the cerebrum, brain stem and cerebellum of germ-free and specific pathogen-free mice (8 weeks old, n = 2)
L
D
(D/L)
Brain region
442±267 489±120 891±57 999 817±230 1290± 16 1090, 1090 ND 641±172 910± 19 737 923±429 1110±55 653, 989 314 568±60 1190±80 765 741±54 989± 123 991,763
61±31 9 3 ± 15 230±6 226 263± 19 520±98 449,388 ND 108±32 160±43 103 53±38 127±32 68, 106 30 4 8 ± 19 94±72 16 12±7 2 8 ± 16 26, 11
0.142±0.016 0.192±0.018 0.258±0.024 0.226 0.331±0.070 0.403±0.069 0.384 * 0.167±0.005 0.176±0.051 0.140 0.054±0.016 0.115 ±0.034 0.106 * 0.096 0.084±0.024 0.078±0.055 0.021 0.016±0.004 0.029±0.020 0.021 *
Values are the m e a n s + S . D , for the n u m b e r of animals shown in parentheses, except for the cerebellum of day 1, in which tissues from three mice were combined. ND, not determined. * Values are calculated by using the m e a n values for two animals.
weight (D/L = 0.4). The free L-serine levels in the cerebella and brain stems of mouse and bull were comparable to those in the cerebra, whereas the Oserine levels in the cerebella and brain stems were considerably lower than those in the cerebra, resulting in low D/L ratios for the mammalian cerebella (D/L 0.03). Free D- and L-serine levels in the cerebrum, brain stem and cerebellum from mice of various ages, ranging from 1 day to 23 weeks, are presented in Table 2. The contents of L-serine increased with age in the brain regions examined, and reached the level of the adult mouse (8 weeks old), namely about 1 /zmol/g wet weight. At each developing stage, no prominent difference in the L-serine level was observed among the three brain regions. The D-serine level in the cerebrum also increased with age. In the cerebellum and brain stem, by contrast, the peak D-serine levels were attained at 2 weeks of age, followed by a decrease to the respective adult levels. The value of D/L ratio for the cerebrum increased with age and approached the adult level of 0.4 by age 8 weeks, whereas those for the brain stem and cerebellum decreased 2 weeks after birth. The D/L ratios in adult mice were 0.12 for the brain stem and 0.03 for the cerebellum. To determine if the free D-serine detected in the present study originated from microorganisms, e.g. enterobacteria, the free D-serine content in the brain was
Cerebrum Brain stem Cerebellum
Free serine ( n m o l / g wet weight) Germ-free mouse
Specific pathogenfree mouse
L
D
D/L
L
D
D/L
743 1070 798 899 777 885
418 365 86 133 4.4 10.6
0.432 *
1190 792 1030 806 940 701
488 443 185 48 8.6 2.9
0.470 *
0.129 * 0.009 *
0.127 * 0.007 *
* Values are calculated by using the mean values for two animals.
compared between germ-free and specific pathogenfree mice (Table 3). In the brain regions examined, no significant difference was found in either the o- and L-serine levels and in the O/L ratio between the two groups. Since the mammalian forebrain contained free oserine at a high level (400-500 nmol/g, O/L -----0.4), as revealed in the above investigations, the distribution of free o-serine was investigated in detail in the rat forebrain, i.e. olfactory bulb, right and left frontal lobes, right and left occipital lobes, hippocampus and hypothalamus (Table 4) as well as in the grey and white matter of the cerebrum (Table 5). No significant difference was found in the o- and L-serine levels or in the O/L ratio between the two regions of the rat cerebrum, namely the frontal and occipital lobes. Although the Dand L-serine contents in both hippocampus and hypothalamus were respectively lower than those in the above two cerebral regions, the D/L ratios were similar to those in the same cerebral regions. The D/L ratio for the olfactory bulb was lower than those for the other regions of the forebrain. Next, the level of free D-serine was compared between grey and white matter in the cerebrum. In the rat, the D/L ratio for the grey matter was higher than that for the white matter (Table 5). No significant differences in the o- and L-serine
Table 4 Contents of free D- and L-serine, and D / L ratios in rat forebrain Forebrain region
Olfactory bulb * Right frontal lobe Left frontal lobe Right occipital lobe Left occipital lobe Hippocampus * Hypothalamus t
Free serine ( n m o l / g wet weight) L
D
874 1030 + 148 827 + 55 1000 d: 96 853 ± 124 464 614
108 480 + 377 + 419 + 305 + 208 250
D//L
135 42 101 19
0.123 0.465 + 0.065 0.456 -I-0.025 0.419 + 0.081 0.358 + 0.036 0.448 0.406
Values are the m e a n s + S.D. for three animals except where tissues from two ( * ) or three (t) animals were combined.
Y. Nagata et al. / Brain Research 634 (1994) 291-295
294
Table 5 Contents of free D- and L-serine, and D/L ratios in the grey and white matter of the rat cerebrum Free serine ( n m o l / g w e t w e i g h t ) Right cerebrum
Grey matter White matter
Leficerebrum
c
D
D/c
c
D
D/L
947 + 50 851 + 86
407 _+ 20 249 _+ 47
0.430 +_ 0.004 0.293 + 0.014 P < 0.02 *
1000 _+ 40 982 + 2
416 +_ 61 241 _+ 66
0.416 _+ 0.046 0.245 + 0.054 P < 0.05 *
Values are the means + S.D. for three animals. * t-test for differences between the two groups, after examination by F-test.
contents were observed between the right and left cerebral halves, respectively. In the bull, the D/C ratio for cerebral grey matter (D/L = 0.27) was lower than that for the cerebral white matter (D/L = 0.62), whereas the ratios for the cerebellar grey and white matter were similar (D/L = 0.01-0.03).
4. Discussion
The analytical method used in the present study is capable of determining 20-50 pmol of D- and L-amino acids [17]. The sample was identified as D-serine by using a combination of two-dimensional TLC and HPLC, and furthermore the identification was confirmed by the treatment of the sample with D-aminoacid oxidase: the FDAA-amino acid migrated to the same location as FDAA-serine on the TLC-plate, on which amino acids were separated well from one another [17], was recovered from the plate, and co-chromatographed with authentic FDAA-D- and FDAA-Lserine in HPLC. The peaks of the sample from the TLC-plate overlapped completely with D- and L-serine peaks, respectively. On the other hand, the brain extract was treated with D-amino-acid oxidase from porcine kidney (Sigma, St. Louis, MO, USA) at 37°C for 30 min, and then subjected as described under Materials and Methods. On HPLC analysis, one of two peaks of the sample, which corresponded to that of D-serine, completely disappeared (data not shown). The oxidase has high specificity to D-amino acids [14]. Furthermore, both the amount of free D-serine and D/L ratio of free serine in rat cerebrum (Table 1) were, respectively, found to be similar to those obtained by using gas chromatographic-mass spectrometric analysis [5]. The presence of considerable amounts of free Dserine has been demonstrated in cerebra of mouse (Tables 1-3), rat (Tables 1, 4 and 5) and bull (Table 1). A high content of D-serine was also observed in human cerebrum (unpublished work). D-Serine level was low in cerebellum (Tables 1-3). Although species variation and regional differences in the distribution of D-serine in the brain were observed, it appears that the distribution of free D-serine is general in mammalian cerebra.
D-Serine level in the mouse cerebellum is low (Tables 1-3), whereas it is high in the cerebellum [13] of a mutant mouse [10,11] lacking D-amino-acid oxidase. Furthermore, we recently obtained some evidence that D-amino-acid oxidase catabolizes neutral free D-amino acids in vivo in the mouse [15,18] and in humans [16]: substantial amounts of free D-serine, D-alanine and D-proline were observed in plasma and kidney specimens obtained from where the oxidase activity was very low or absent. Together with these facts, the present results were interpreted in terms of D-amino-acid oxidase activity as follows. (1) The oxidase is distributed evenly over the whole brain in the lower vertebrate species, and the activity in the chick forebrain is lower than those in the fish and frog [4]. In mammals, the oxidase is highly concentrated in the hindbrain, particularly in the cerebellum [4,7,8,19]. The oxidase activity is absent or scarce in the forebrain, and low in the midbrain of mammals [4]. Therefore, the distribution of free D-serine in the various brain regions of vertebrates described in Table 1, indicates that the D-serine content depends on D-amino-acid oxidase activity. (2) The oxidase activity in the rat cerebellum and brain stem was first detected on day 14 postnatally, and then increased rapidly to reach the adult level 4 to 5 weeks later [22]. The oxidase activity in the brain stem is 39% of that in the cerebellum. In the cerebrum, the activity was undetectable until day 29, and the maximal activity reached less than 2% of the cerebellar activity in adult rat [22]. In adult mice, the oxidase activity in the cerebrum is also remarkably lower than that in the cerebellum [4,13]. Therefore, the changes in the D / e ratio among different brain regions of developing mice (Table 2) suggest that D-serine is oxidatively deaminated in situ by D-amino-acid oxidase. Recently, Hashimoto et al. [6] also revealed a substantial amount of free D-serine in various regions of rat brain with a similar distribution pattern to that shown in the present study. They communicated that the regional variations in the amount of free D-serine were closely correlated with that of N M D A receptor, and proposed that D-serine was a novel candidate as an intrinsic ligand for the glycine site of the N M D A receptor [6]. However, in the mutant mouse cerebellum, free D-serine level is remarkably high (D/L =
Y. Nagata et aL / Brain Research 634 (1994) 291-295
0.256) [13]. It is of interest if the amount of N M D A receptor is increased in that cerebellum. The mutant mice show no abnormal behavior by our observation, and the life span and body weight are not different from the normal mouse, in spite that free D-serine may not be metabolized in the mutant animals. Furthermore, in the bull, the D/L ratio of free D-serine for cerebral grey matter was much lower than that for the cerebral white matter. Hence, it is possible that the distribution pattern of free D-serine in the brain is mainly a consequence of D-amino-acid oxidase activity. Since D-serine was detected in the brains of germfree mice (Table 3), the free D-serine did not originate from bacteria, which possess some D-amino acids as cell wall components and products [12]. However, since 1% of the total serine in the diets used in the present study for mouse and rat was the o-enantiomer (D/L----0.01, our unpublished data), we cannot rule out that the free D-serine in the brain is of dietary origin. Further investigations are required to elucidate the source of the D-serine as well as its physiological, pharmacological and pathological significance, since, in in vitro experiments, D-serine potentiates the action of N M D A receptor by binding to a strychnine-insensitive glycine binding site on the receptor complex [1,2,9,20, 21].
Acknowledgements. We thank Dr. Mineko Fujimiya of the Department of Anatomy, Shiga University of Medical Science, for her cooperation in the preparation of brain sections. Thanks are also due to Prof. Mitsuhiro Nozaki of the Department of Biochemistry, Shiga University of Medical Science, for his continuous encouragement during the course of this study.
References [1] Contreras, P.C., D-Serine antagonized phencyclidine- and MK801-induced stereotyped behavior and ataxia, Neuropharmacology, 29 (1990) 291-293. [2] Fadda, E., Danysz, W., Wroblewski, J.T. and Costa, E., Glycine and D-serine increase the affinity of N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes, Neuropharmacology, 27 (1988) 1183-1185. [3] Glowinski, J. and Iversen, L.L., Regional studies of catecholamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]dopa in various regions of the brain, J. Neurochern., 13 (1966) 655-669.
295
[4] Goldstein, D.B., D-Amino acid oxidase in brain: distribution in several species and inhibition by pentobarbitone, J. Neurochem., 13 (1966) 1011-1016. [5] Hashimoto, A., Nishikawa, T., Hayashi, T., Fujii, N., Harada, K., Oka, T. and Takahashi, K., The presence of free D-serine in rat brain, FEBS Lett., 296 (1992) 33-36. [6] Hashimoto, A., Nishikawa, T., Oka, T. and Takahashi, K., Endogenous D-serine in rat brain: N-methyl-D-aspartate receptor-related distribution and aging, J. Neurochem., 60 (1993) 783-786. [7] Horiike, K., Arai, R., Tojo, H., Yamano, T., Nozaki, M. and Maeda, T., Histochemical staining of cells containing flavoenzyme D-amino acid oxidase based on its enzymatic activity: application of a coupled peroxidation method, Acta Histochem. Cytochem., 18 (1985) 539-550. [8] Horiike, K., Tojo, H., Arai, R., Yamano, T., Nozaki, M. and Maeda, T., Localization of D-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum, Brain Res. Bull., 19 (1987) 587-596. [9] Kleckner, N.W. and Dingledine, R., Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes, Science, 241 (1988) 835-837. [10] Konno, R. and Yasumura, Y., Mouse mutant deficient in Damino acid oxidase activity, Genetics, 103 (1983) 277-285. [11] Konno, R. and Yasumura, Y., Brain and kidney D-amino acid oxidases are coded by a single gene in the mouse, J. Neurochem., 42 (1984) 584-586. [12] Man, E.H. and Bada, J.L., Dietary D-amino acids, Annu. Rev. Nutr., 7 (1987) 209-225. [13] Nagata, Y., Involvement of D-amino acid oxidase in elimination of D-serine in mouse brain, Experientia, 48 (1992) 753-755. [14] Nagata, Y., Akino, T. and Ohno, K., Microdetermination of serum D-amino acids, Anal. Biochem., 150 (1985) 238-242. [15] Nagata, Y., Konno, R., Yasumura, Y. and Akino, T., Involvement of D-amino acid oxidase in elimination of free D-amino acids in mice, Biochem. J., 257 (1989) 291-292. [16] Nagata, Y., Masui, R. and Akino, T., The presence of free D-serine, D-alanine and D-proline in human plasma, Experientia, 48 (1992) 986-988. [17] Nagata, Y., Yamamoto, K. and Shimojo, T., Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography, J. Chromatogr., 575 (1992) 147-152. [18] Nagata, Y., Yamamoto, K., Shimojo, T., Konno, R., Yasumura, Y. and Akino, T., The presence of free D-alanine, D-proline and D-serine in mice, Biochim. Biophys. Acta, 1115 (1992) 208-211. [19] Neims, A.H., Zieverink, W.D. and Smilack, J.D., Distribution of D-amino acid oxidase in bovine and human nervous tissues, J. Neurochem., 13 (1966) 163-168. [20] Salt, T.E., Modulation of NMDA receptor-mediated responses by glycine and D-serine in the rat thalamus in vivo, Brain Res., 481 (1989) 403-406. [21] Snell, L.D., Morter, R.S. and Johnson, K.M., Structural requirements for activation of the glycine receptor that modulates the N-methyl-o-aspartate operated ion channel, Eur. J. Pharmacol., 156 (1988) 105-110. [22] Weimar, W.R. and Neims, A.H., The development of D-amino acid oxidase in rat cerebellum, J. Neurochem., 29 (1977) 649-656.