Journal of Neuroscience Research 26: 217-223 (1990)
Changes With Aging in the Levels of Amino Acids in Rat CNS Structural Elements: IV. Methionine and Basic Amino Acids M. Banay-Schwartz, A. Lajtha, and M. Palkovits The Nathan S. Kline Institute for Psychiatric Research, Ward's Island (M.B.-S., A.L.); New York University Medical Center, New York (A.L.); First Department of Anatomy, Semmelweis University Medical School, Budapest, Hungary (M.P.), Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland (M.P.)
This paper reports the distribution of methionine, histidine, lysine, arginine, and ornithine in 53 discrete brain areas of 3- and 29-month-old male Fischer 344 rats microdissected by the punch technique. Like that of the other amino acids we reported in previous papers of this series, the distribution of methionine and the basic amino acids was regionally highly heterogeneous. The ratios of levels in the areas of highest concentrations to levels in the areas of lowest concentration varied from 10 to 15 for these amino acids, except that it was 23 for arginine. This heterogeneity is also illustrated by the finding that in some areas arginine was more than 5 % of the total amino acid content and in others was less than 0.5%. The distribution of methionine differed from that of the basic amino acids, which were high in hypothalamic areas and low in the limbic system. With aging, methionine and basic amino acids, like the other amino acids studied in this series, mainly decreased in level, although in a few cases increases could be seen. Significant decreases were noted more than five times as often as increases.
Details of the method used are described in previous papers (Banay-Schwartz et al., 1989a,b, 1990). Briefly, 3-month-old and 27-month-old male Fischer 344 rats were decapitated, and in the cytostat corona1 sections were prepared for microdissection (Palkovits, 1973) of 53 areas by the punch technique. The dissected pieces were homogenized by sonication in perchloric acid, and aliquots were taken for protein determination; the rest was centrifuged, and amino acids were determined in the supernatant with precolumn derivatization and reverse-phase chromatography on a C,, silica column using a fluorescence detector and a UV monitor (Neidle et al., 1989).
Key words: brain, regional amino acid levels, basic amino acid levels, aging
RESULTS
INTRODUCTION This paper is part of a series in which we analyze changes with age in levels of cerebral amino acids in discrete areas of rat brain. Previous papers (BanaySchwartz et al., 1989a,b, 1990) dealt with acidic and large and small neutral amino acids. The present paper describes the findings with methionine and the basic amino acids in the same tissue samples in which the other amino acids were assayed. The tissue samples from 53 specific brain areas were small enough to give an estimate of the heterogeneity of distribution of these compounds and the changes in such distribution with age. 0 1990 Wiley-Liss, Inc.
The purpose of these studies, rather than to assay whole brain, was to measure functional elements for comparison. The previous papers show that when such areas are compared, the heterogeneity of distribution is much greater than when larger brain sections are compared, although the unavoidable margin of error also increases.
MATERIALS AND METHOD
The levels of amino acids in the various areas of young and old rat brain are given in Tables I-V, according to sensory areas (Table I), motor areas (Table 11), hypothalamic areas (Table III), limbic areas (Table IV), and lower brainstem areas (Table V). Concentrations are given in nmol of amino acidimg of protein. Each value given is the average of determinations in six separate tissue samples. In several cases amino acids at low levels
Received July 18, 1989; revised December I , 19x9; accepted Deccmber 8, 1Y89. Address reprint requests to M . Banay-Schwartz, Center for Neurochemisty, Ward's Island, New York, N Y 10035.
218
Banay-Schwartz et al.
TABLE I. Sensory Brain Areas ~
~~
~~~~
~~~~
nmol amino acid oer me orotein Methionine Areas General Sensory Sensory cortex Ventral thalamic nucleus Sensory trigeminal nucleus Nucleus gracilia Nucleus cuneatus Dorsal horn (spinal cord) Special Sensory Visual cortex Lateral geniculate body Superior colliculus Cochlear nuclei Medial geniculate body Inferior colliculus Vestibular nuclei
Histidine
Lysine
Or ni t hine
Arginine
3 mo
29mo
3mo
29mo
3 mo
29mo
3 mo
29mo
3 mo
29 mo
2.0
2.7
0.37
0.26
I .2 2.7 3.4
4.0 3.7 3.1 3. I 3.7 4.0
2.5 4.8 2.5 3.3 5.2 5.2
2.2 II 15 6.3 6.2 5.8
0.94 II 10 5.3 7.1 4.7
I .6 0.64 0.67 I .2 0.78
I .7 0.62 0.56 0.69
I .3 1.6
I .3 3.4 2.8 I .o 1.3 2.2 0.79 0.68 I .7 1.6 2.9 2.2 1.5
2.6 2.9 2.4 2.6 6.1 5.0
I.8 2.8 I .9 3.4 6.7 3.4 2.7
3.6 6.9 9.6 13 14 9.6 4.5
3.3 5.6 6.6 16 II 8.8 4.4
0.36
0.83
0.92 2.6 I .6 4.6 I .5 0.96
0.33 0.47
0.24 0.53 0.82 0.60 0.48
0.30 I .3
1.1
1.9 0.66 2.0
I .2 0.65 I .8
I .6
3.0 0.40
1 .o
1 .o
0.90 0.36
1.1
TABLE 11. Motor Brain Areas nmol amino acid oer me orotein Methionine Areas Pyramidal and Pxapyramidal Motor cortex Pontine nuclei Ventral horn (spinal cord) Extrapyramidal Caudate nucleus Putamen Globus pallidus Substantia nigra Cerebellar cortex Cerebellar nuclei
Hihtidine
Lysine
Arginine
Ornithine
3mo
29 mo
3mo
29mo
3 mo
29mo
3 mo
29mo
3 mo
29mo
1.2
1.5
2.0 11 2.4
2.5 8.9 2.5
2.0 21 4.2
1.9 14 3.6
0.79
0.37
I .o 2.7 0.37
0.17 I .5
0.52
I.3 5.2 0.56
2.5 0.77 0.76 2.2
I .8 0.60 0.55 2. I 0.54 I .o
I .4
I .6 0.89 0.42 1.3 I .3 0.64
3.7 3.4 I .8 2.9 2.3 3.7
4.9 3.2 1.4 3.8 2.3 4.2
3.6 6.4 4.5 7.5 2.0 9.9
I .7 4.4 2.9 7.3 1.3 7.4
1.1
1.2
I .4 0.52 I .4
0.91
1 .s
0.59
1 .5 0.42
0.33
0.44
0.48
0.26
1.1
TABLE 111. Hypothalamic Brain Areas nmol amino acid per mg protein Methionine Areas Medial preoptic nucleus Anterior hypothalamic nucleus Supraoptic nucleus Paraventricular nucleus Arcuate nucleus Median eminence Ventromedial nucleus Dorsomedial nucleus Lateral hypothalamus (MFB) Posterior hypothalamic nucleus
3 mo
29 mo
0.98
0.97 0.41 0.53
0.59
0.5 2.9
0.6 0.33 2.1 1.1
Histidine
3
in0
3.0 1.7 2.0 2.6 2.6 I .5 3.8 I .7 1.8 I .o
did not give well-defined peaks on the chromatograms or were not well separated. These uncertainties are not due to the low sensitivity of the assay method but to the presence of some unidentified peaks not well separated
Lysine
Arginine
29 in0
3 mo
29 mo
3 mo
29 mo
3.3 I .3 2.8 2.2 2.9 I .8 2.4 2.0
5.7 4.7 4.3 2.9 3.8 2.1 5. I 3.0 3.4 2.4
7. I 3.7 6.2 3.6 2.3 3.9 3.8 2.8 2.2 2.6
8.7 5.7 5.5 7.3 5.5 5.3 27 6.7 7. I 4.9
8.5 4.5 8.3 9.3 6. I 4.8 12 6.0 4.5 3.5
0.92
Ornithine 3 mo
29 mo
1 .s
I .o 0.91 I .4
0.89 I .5 1.1
1.1
I .6 0.76 I .8 0.93 0.99 0.64
0.7 1 1.1
I .7 0.85 0.31 0.78
each time on the chromatogram. If fewer than three reliable determinations were available, no value for that area is given (blank spaces in the Tables). The standard deviations of the values were greater than that for the
219
Aging and Amino Acid Levels in Brain TABLE IV. Limbic Brain Areas nmol amino acid Der mg protein Methionine Areas Olfactory bulb Olfactory tubercle Cingulate cortex Pyriform cortex Hippocanipus Dentate gyrus Medial septal nucleus Lateral septal nucleus Medial amygdaloid nucleus Lateral amygdaloid nucleus Central amygdaloid nucleus Bed nucleus, stria terminals Habenula lnterpeduncular nucleus
Histidine
Lysine
Arginine
Ornithine
3 mo
29 mo
3 mo
29 mo
3 nio
29 mo
3 mo
29 mo
3 mo
29 nio
I.2 0.7 I 0.55 0.93 2.4 2.0 0.48 0.85 0.52 0.88 0.67 1 .5
1.1 0.81 0.33 0.49 2.4 I.7 0.50
4.7 I.2 0.50
2.9 I .o 0.63 0.91 0.68 2.5 0.50
4.6 2.2 6.2 3.5 2.6 I .8
5.8 2.6 6.2 3.4 2.0 1.2 I.2 2.0 2.0 1 .0 2. I I.0 2.6 2.7
3.8 3.4 I .2 2.5 1.9 4.3 3. I 3.5 2.5 2.6 4.2 3.5 8.5 7.0
3.0 2.9 0.93
1.2 I.2
0.77 0.35 0.x0
0.83 0.70 0.33 0.51 0.34
0.72 0.46 0.37 0.49 0.44
0.26
0.30 0.23 0.48 I.7 I .4 0.22
0.83 0.42 I .2 0.68 I.3
0.93 0.65 0.59
1.1
2.7 2.2 I.6 3.7 1.1
I.9
I.4
1.9
1.2
3.4 I.9
I.s 1.1
3.4 2.2 5.6 I.x I.6 I .7
3.1 6.9 4.5
0.63 0.64 0.62
0.56 0.28 0.96
TABLE V. Lower Brainstem Areas nmol amino acid per mg protein Methionine Areas Periaqueductal central gray Dorsal raphe nucleus Parabrachial nuclei Locus coeruleus Reticular formation Nucleus of the solitary tract Central -gray (sninal ~ cord) .
Histidine
Lysine
Arginine
Ornithine
3 mo
29mo
3 mo
29 mo
3 mo
29mo
3 mo
29nio
3 mo
29rno
2.4 I.5
1.8 1.2
I.6 1 .5 I.3
I.2
0.74 0.96 I .2 1 .0
7.9 6.3 8.6 I0 9.5 6.8 4.3
6.0 6.4 6.5 6.0 5.6 3. I 3.4
0.48
0.33 0.64
2.3 2.6 4.8 0.83 3.4 3.8 3.9
0.55 0.45 0.61
0.33
3.4 4. I 4.0 0.72 3.8 5.0 4.0
3.4 0.6
0.5
1.1 0.99 0.70 0.65 0.76 0.7 -
other amino acids; they were below 10% for histidine, lysine, and arginine and between I0 and 20% for methionine and ornithine. The heterogeneity of the distribution of the amino acids studied in this paper was similar to that of the large neutral amino acids presented in the previous paper (Banay-Schwartz et al., 1990). The ratio of the concentration of the area with the highest level to that at the lowest level varied between I 1 and 23, being 13 for methionine, 12 for histidine, 15 for lysine, 23 for arginine, and 11 for ornithine. This was not due to unusually high or low content of a single area; even if the average of the 5 highest areas was compared to the average of the 5 lowest areas, the difference was over six-fold, the ratios being 6.9, 8.0, 6.2, 9.5, and 5.5 for methionine, histidine, lysine. arginine, and ornithine, respectively. Among basic amino acids, concentrations of arginine are the highest, followed by lysine, histidine, methionine, and ornithine. Basic amino acid levels are generally high in the hypothalamic nuclei and low in the limbic system (Table VI). In contrast to the other amino acids, high methionine levels were measured in the brainstem, and relatively low values were measured in the hypothalamic nuclei (Table VI).
0.3 0.65
0.56 0.3 I 0.37 0.59
With a few exceptions, there was good correlation in the distribution of the basic amino acids, in that in areas of high or low content, each of them was similarly at high or low levels, respectively; methionine distribution varied more frequently from the basic amino acids. Areas in which amino acids were at high levels included the sensory trigeminal nucleus, medial geniculate body, pontine nuclei, and medial preoptic and ventromedial nucleus. These are areas in which the large neutral amino acids (Banay-Schwartz et al., 1990) were also at high levels. Areas where most of these amino acids were at low levels include the limbic areas (Table IV), the medial septal nucleus, the medial and lateral amygdaloid nucleus, the cingulate cortex and hippocampus, and, in the sensory areas (Table I ) , the vestibular nuclei. Again, these are the areas in which the large neutral amino acids were also present at low levels (Banay-Schwartz et al., 1990). It has to be emphasized that the levels were not parallel in all areas, and in a number of areas there were ornithine at low levels in the cochlear nuclei, cerebellar nuclei, and locus coerulus, while arginine was low and ornithine was high in the sensory cortex; in a few areas similar differences can be seen with the other amino acids. It is noteworthy that subcortical centers of the
220
Banay-Schwartz et al.
TABLE VI. Ranking by Concentration Methionine and Basic Amino Acids in Major Brain Systems I (highest) Methionine Hiatidine Lysine Arginine Ornithine
Brainstem Hypothalarnic Hypothalamic Hypothalamic Hypothalamic
2
3
4
S (lowest)
Sensory Sensory Motor Sensory Sensory
Motor Motor Brainstern Brainstern Motor
Hypothalamic Limbic Sensory Motor Lirnbic
Lirnbic Brainstem Lirnhic Lirnbic Brainstem
auditory system (cochlear nuclei, inferior colliculus, medial geniculate body) are rich in arginine but not in other basic amino acids (Table I). The changes with age in various areas expressed as percent alteration in the old brain are shown in Tables V11 and VIII. The magnitude of the change itself was somewhat variable, although usually in the same direction. For example, in a particular area there was a decrease in each sample with age, but the size of the decrease was variable, therefore the numerial value for the decrease could be of low statistical significance. Although both increases and decreases with age have been found in the various areas, decreases were found more often. The number of areas in which the levels changed with age and the ratio of the number of areas with decrease to those with increase varied with each amino acid, but significant decrease was noted more than five times as frequently as significant increase. This was similar to the changes with age with the large neutral amino acids described in the previous paper where decrease with age in general also occurred almost three times as frequently as increase. Highly significant changes ( P < .OOl), calculated using Student’s f-test analysis, were always decreases in aged rats, never increases. Highly significant decrease occurred 8 times with arginine, in one area each with methionine and histidine, and nowhere with lysine or ornithine (Tables VlI and VIII). Almost one-half of brain areas investigated (26 of 53) showed significantly (from P < .OS up to P < .001) decreased arginine levels in aged rats. This number was much lower with histidine (12), lysine ( 7 ) , methionine (6), ornithine ( 5 ) . Slightly increased ( P < .05) levels were measured in 8 areas (three each with lysine and arginine, one each with methionine and ornithine) (Tables VII and VIII).
DISCUSSION The distribution of the amino acids assayed in this paper in the selected specific brain areas is, like that of the amino acids assayed in the previous papers of this series (Banay-Schwartz et al., 1989a,b, 1990), surprisingly heterogeneous, with a more than ten-fold difference between the highest and lowest values and still more than five-fold difference between the average val-
ues of the five highest and five lowest areas. When the distribution of all the amino acids that were measured in this and the previous papers of this series is compared, several areas are seen to have many (or most) at high levels, and some to have most at low levels. Areas with generally high amino acid levels include the sensory trigeminal nucleus, medial geniculate body, inferior colliculus, medial preoptic nucleus, arcuate nucleus, and lateral hypothalamus. There are two brain regions where basic amino acid levels are relatively high: 9.8% of the total amino acids in the pontine nuclei and 8.2% in the ventromedial nucleus are basic amino acids. This percentage does not exceed 5 % in any other brain regions investigated. Areas in which most amino acids are low levels include the spinal cord, ventral horn and central gray, globus pallidus, cerebellar cortex, cingulate cortex, medial septal nucleus, lateral amygdaloid nucleus, and reticular formation (Banay-Schwartz et al., 1989a,b, 1990). There are also some areas in which some amino acids are near their highest and some near their lowest level; an example of this is the olfactory bulb, in which most of the neutral amino acids are at low levels and the acidic and basic amino acids are at high levels, or the medial amygdaloid nucleus, in which glutamate, glutamine, and GABA are high, and the large neutral and basic amino acids are low. Concentrations of basic amino acids in the brain nuclei are generally low; they constitute together no more than 1 4 % of the total amino acid levels. The relative concentration (in percentage of the total amino acid level) of certain basic amino acids also shows high heterogeneity: the arginine content is over 5% in the ventromedial (5.7%) and pontine nuclei (5.6%) but only 0.49% in the cingulate cortex or 0.56% in the medial amygdala. Similarly, the lysine content in the pontine nuclei is 2.8% of the total amino acid content, 2.5% in the cingulate cortex, but only 0.23% in the locus coeruleus and 0.31% in the vestibular nuclei. Our studies reveal only one aspect of amino acid heterogeneity, that of levels in discrete structures. It is likely that the heterogeneity is considerably higher than shown here in that, even within the structures examined, at least some of the amino acids may not be distributed evenly, in the synaptic region levels of GABA, taurine,
Aging and Amino Acid Levels in Brain
221
TABLE V11. Percent Change in Amino Acid Levels in Old Rats (Motor and Sensory Areas)* Met Sensory Areas Sensory cortex Ventral thalamic nucleus Sensory trigeminal nucleus Nucleus gracilis Nucleus cuneatus Dorsal horn (spinal cord) Visual cortex Lateral geniculate body Superior colliculus Cochlear nuclei Medial geniculate body Inferior colliculus Vestibular nuclei Motor areas Motor cortex Pontinc nuclei Ventral horn (spinal cord) Caudate nucleus Putamen Globus pallidus Substantia nipra Cerebellar cortex Cerebellar nuclei *O
=
not significant. I
=
P
His
+ 30"
0
Lys
Arg
-38'
-573 0 -33" - 16"
+ 26"
+ 30'
-30"
- 19"
0 + 38"
+ 4 I" + 30"
0
- 15" -37' 0 0
-31'
-29' - 28" - 22" - 28" 0 -51" - 17" P
< .02, 3
-23" -483 34" + 14" -36'
+ 25"
+
+ 25"
=
0 -21" + 3 I" 0 -32" + 170'
-
+ 881
< .OS, 2
-26" 35' 0 -37' 47" + 56"
-
Changes With Aging in the Levels of Amino Acids in Rat CNS Structural Elements: IV. Methionine and Basic Amino Acids M. Banay-Schwartz, A. Lajtha, and M. Palkovits The Nathan S. Kline Institute for Psychiatric Research, Ward's Island (M.B.-S., A.L.); New York University Medical Center, New York (A.L.); First Department of Anatomy, Semmelweis University Medical School, Budapest, Hungary (M.P.), Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland (M.P.)
This paper reports the distribution of methionine, histidine, lysine, arginine, and ornithine in 53 discrete brain areas of 3- and 29-month-old male Fischer 344 rats microdissected by the punch technique. Like that of the other amino acids we reported in previous papers of this series, the distribution of methionine and the basic amino acids was regionally highly heterogeneous. The ratios of levels in the areas of highest concentrations to levels in the areas of lowest concentration varied from 10 to 15 for these amino acids, except that it was 23 for arginine. This heterogeneity is also illustrated by the finding that in some areas arginine was more than 5 % of the total amino acid content and in others was less than 0.5%. The distribution of methionine differed from that of the basic amino acids, which were high in hypothalamic areas and low in the limbic system. With aging, methionine and basic amino acids, like the other amino acids studied in this series, mainly decreased in level, although in a few cases increases could be seen. Significant decreases were noted more than five times as often as increases.
Details of the method used are described in previous papers (Banay-Schwartz et al., 1989a,b, 1990). Briefly, 3-month-old and 27-month-old male Fischer 344 rats were decapitated, and in the cytostat corona1 sections were prepared for microdissection (Palkovits, 1973) of 53 areas by the punch technique. The dissected pieces were homogenized by sonication in perchloric acid, and aliquots were taken for protein determination; the rest was centrifuged, and amino acids were determined in the supernatant with precolumn derivatization and reverse-phase chromatography on a C,, silica column using a fluorescence detector and a UV monitor (Neidle et al., 1989).
Key words: brain, regional amino acid levels, basic amino acid levels, aging
RESULTS
INTRODUCTION This paper is part of a series in which we analyze changes with age in levels of cerebral amino acids in discrete areas of rat brain. Previous papers (BanaySchwartz et al., 1989a,b, 1990) dealt with acidic and large and small neutral amino acids. The present paper describes the findings with methionine and the basic amino acids in the same tissue samples in which the other amino acids were assayed. The tissue samples from 53 specific brain areas were small enough to give an estimate of the heterogeneity of distribution of these compounds and the changes in such distribution with age. 0 1990 Wiley-Liss, Inc.
The purpose of these studies, rather than to assay whole brain, was to measure functional elements for comparison. The previous papers show that when such areas are compared, the heterogeneity of distribution is much greater than when larger brain sections are compared, although the unavoidable margin of error also increases.
MATERIALS AND METHOD
The levels of amino acids in the various areas of young and old rat brain are given in Tables I-V, according to sensory areas (Table I), motor areas (Table 11), hypothalamic areas (Table III), limbic areas (Table IV), and lower brainstem areas (Table V). Concentrations are given in nmol of amino acidimg of protein. Each value given is the average of determinations in six separate tissue samples. In several cases amino acids at low levels
Received July 18, 1989; revised December I , 19x9; accepted Deccmber 8, 1Y89. Address reprint requests to M . Banay-Schwartz, Center for Neurochemisty, Ward's Island, New York, N Y 10035.
218
Banay-Schwartz et al.
TABLE I. Sensory Brain Areas ~
~~
~~~~
~~~~
nmol amino acid oer me orotein Methionine Areas General Sensory Sensory cortex Ventral thalamic nucleus Sensory trigeminal nucleus Nucleus gracilia Nucleus cuneatus Dorsal horn (spinal cord) Special Sensory Visual cortex Lateral geniculate body Superior colliculus Cochlear nuclei Medial geniculate body Inferior colliculus Vestibular nuclei
Histidine
Lysine
Or ni t hine
Arginine
3 mo
29mo
3mo
29mo
3 mo
29mo
3 mo
29mo
3 mo
29 mo
2.0
2.7
0.37
0.26
I .2 2.7 3.4
4.0 3.7 3.1 3. I 3.7 4.0
2.5 4.8 2.5 3.3 5.2 5.2
2.2 II 15 6.3 6.2 5.8
0.94 II 10 5.3 7.1 4.7
I .6 0.64 0.67 I .2 0.78
I .7 0.62 0.56 0.69
I .3 1.6
I .3 3.4 2.8 I .o 1.3 2.2 0.79 0.68 I .7 1.6 2.9 2.2 1.5
2.6 2.9 2.4 2.6 6.1 5.0
I.8 2.8 I .9 3.4 6.7 3.4 2.7
3.6 6.9 9.6 13 14 9.6 4.5
3.3 5.6 6.6 16 II 8.8 4.4
0.36
0.83
0.92 2.6 I .6 4.6 I .5 0.96
0.33 0.47
0.24 0.53 0.82 0.60 0.48
0.30 I .3
1.1
1.9 0.66 2.0
I .2 0.65 I .8
I .6
3.0 0.40
1 .o
1 .o
0.90 0.36
1.1
TABLE 11. Motor Brain Areas nmol amino acid oer me orotein Methionine Areas Pyramidal and Pxapyramidal Motor cortex Pontine nuclei Ventral horn (spinal cord) Extrapyramidal Caudate nucleus Putamen Globus pallidus Substantia nigra Cerebellar cortex Cerebellar nuclei
Hihtidine
Lysine
Arginine
Ornithine
3mo
29 mo
3mo
29mo
3 mo
29mo
3 mo
29mo
3 mo
29mo
1.2
1.5
2.0 11 2.4
2.5 8.9 2.5
2.0 21 4.2
1.9 14 3.6
0.79
0.37
I .o 2.7 0.37
0.17 I .5
0.52
I.3 5.2 0.56
2.5 0.77 0.76 2.2
I .8 0.60 0.55 2. I 0.54 I .o
I .4
I .6 0.89 0.42 1.3 I .3 0.64
3.7 3.4 I .8 2.9 2.3 3.7
4.9 3.2 1.4 3.8 2.3 4.2
3.6 6.4 4.5 7.5 2.0 9.9
I .7 4.4 2.9 7.3 1.3 7.4
1.1
1.2
I .4 0.52 I .4
0.91
1 .s
0.59
1 .5 0.42
0.33
0.44
0.48
0.26
1.1
TABLE 111. Hypothalamic Brain Areas nmol amino acid per mg protein Methionine Areas Medial preoptic nucleus Anterior hypothalamic nucleus Supraoptic nucleus Paraventricular nucleus Arcuate nucleus Median eminence Ventromedial nucleus Dorsomedial nucleus Lateral hypothalamus (MFB) Posterior hypothalamic nucleus
3 mo
29 mo
0.98
0.97 0.41 0.53
0.59
0.5 2.9
0.6 0.33 2.1 1.1
Histidine
3
in0
3.0 1.7 2.0 2.6 2.6 I .5 3.8 I .7 1.8 I .o
did not give well-defined peaks on the chromatograms or were not well separated. These uncertainties are not due to the low sensitivity of the assay method but to the presence of some unidentified peaks not well separated
Lysine
Arginine
29 in0
3 mo
29 mo
3 mo
29 mo
3.3 I .3 2.8 2.2 2.9 I .8 2.4 2.0
5.7 4.7 4.3 2.9 3.8 2.1 5. I 3.0 3.4 2.4
7. I 3.7 6.2 3.6 2.3 3.9 3.8 2.8 2.2 2.6
8.7 5.7 5.5 7.3 5.5 5.3 27 6.7 7. I 4.9
8.5 4.5 8.3 9.3 6. I 4.8 12 6.0 4.5 3.5
0.92
Ornithine 3 mo
29 mo
1 .s
I .o 0.91 I .4
0.89 I .5 1.1
1.1
I .6 0.76 I .8 0.93 0.99 0.64
0.7 1 1.1
I .7 0.85 0.31 0.78
each time on the chromatogram. If fewer than three reliable determinations were available, no value for that area is given (blank spaces in the Tables). The standard deviations of the values were greater than that for the
219
Aging and Amino Acid Levels in Brain TABLE IV. Limbic Brain Areas nmol amino acid Der mg protein Methionine Areas Olfactory bulb Olfactory tubercle Cingulate cortex Pyriform cortex Hippocanipus Dentate gyrus Medial septal nucleus Lateral septal nucleus Medial amygdaloid nucleus Lateral amygdaloid nucleus Central amygdaloid nucleus Bed nucleus, stria terminals Habenula lnterpeduncular nucleus
Histidine
Lysine
Arginine
Ornithine
3 mo
29 mo
3 mo
29 mo
3 nio
29 mo
3 mo
29 mo
3 mo
29 nio
I.2 0.7 I 0.55 0.93 2.4 2.0 0.48 0.85 0.52 0.88 0.67 1 .5
1.1 0.81 0.33 0.49 2.4 I.7 0.50
4.7 I.2 0.50
2.9 I .o 0.63 0.91 0.68 2.5 0.50
4.6 2.2 6.2 3.5 2.6 I .8
5.8 2.6 6.2 3.4 2.0 1.2 I.2 2.0 2.0 1 .0 2. I I.0 2.6 2.7
3.8 3.4 I .2 2.5 1.9 4.3 3. I 3.5 2.5 2.6 4.2 3.5 8.5 7.0
3.0 2.9 0.93
1.2 I.2
0.77 0.35 0.x0
0.83 0.70 0.33 0.51 0.34
0.72 0.46 0.37 0.49 0.44
0.26
0.30 0.23 0.48 I.7 I .4 0.22
0.83 0.42 I .2 0.68 I.3
0.93 0.65 0.59
1.1
2.7 2.2 I.6 3.7 1.1
I.9
I.4
1.9
1.2
3.4 I.9
I.s 1.1
3.4 2.2 5.6 I.x I.6 I .7
3.1 6.9 4.5
0.63 0.64 0.62
0.56 0.28 0.96
TABLE V. Lower Brainstem Areas nmol amino acid per mg protein Methionine Areas Periaqueductal central gray Dorsal raphe nucleus Parabrachial nuclei Locus coeruleus Reticular formation Nucleus of the solitary tract Central -gray (sninal ~ cord) .
Histidine
Lysine
Arginine
Ornithine
3 mo
29mo
3 mo
29 mo
3 mo
29mo
3 mo
29nio
3 mo
29rno
2.4 I.5
1.8 1.2
I.6 1 .5 I.3
I.2
0.74 0.96 I .2 1 .0
7.9 6.3 8.6 I0 9.5 6.8 4.3
6.0 6.4 6.5 6.0 5.6 3. I 3.4
0.48
0.33 0.64
2.3 2.6 4.8 0.83 3.4 3.8 3.9
0.55 0.45 0.61
0.33
3.4 4. I 4.0 0.72 3.8 5.0 4.0
3.4 0.6
0.5
1.1 0.99 0.70 0.65 0.76 0.7 -
other amino acids; they were below 10% for histidine, lysine, and arginine and between I0 and 20% for methionine and ornithine. The heterogeneity of the distribution of the amino acids studied in this paper was similar to that of the large neutral amino acids presented in the previous paper (Banay-Schwartz et al., 1990). The ratio of the concentration of the area with the highest level to that at the lowest level varied between I 1 and 23, being 13 for methionine, 12 for histidine, 15 for lysine, 23 for arginine, and 11 for ornithine. This was not due to unusually high or low content of a single area; even if the average of the 5 highest areas was compared to the average of the 5 lowest areas, the difference was over six-fold, the ratios being 6.9, 8.0, 6.2, 9.5, and 5.5 for methionine, histidine, lysine. arginine, and ornithine, respectively. Among basic amino acids, concentrations of arginine are the highest, followed by lysine, histidine, methionine, and ornithine. Basic amino acid levels are generally high in the hypothalamic nuclei and low in the limbic system (Table VI). In contrast to the other amino acids, high methionine levels were measured in the brainstem, and relatively low values were measured in the hypothalamic nuclei (Table VI).
0.3 0.65
0.56 0.3 I 0.37 0.59
With a few exceptions, there was good correlation in the distribution of the basic amino acids, in that in areas of high or low content, each of them was similarly at high or low levels, respectively; methionine distribution varied more frequently from the basic amino acids. Areas in which amino acids were at high levels included the sensory trigeminal nucleus, medial geniculate body, pontine nuclei, and medial preoptic and ventromedial nucleus. These are areas in which the large neutral amino acids (Banay-Schwartz et al., 1990) were also at high levels. Areas where most of these amino acids were at low levels include the limbic areas (Table IV), the medial septal nucleus, the medial and lateral amygdaloid nucleus, the cingulate cortex and hippocampus, and, in the sensory areas (Table I ) , the vestibular nuclei. Again, these are the areas in which the large neutral amino acids were also present at low levels (Banay-Schwartz et al., 1990). It has to be emphasized that the levels were not parallel in all areas, and in a number of areas there were ornithine at low levels in the cochlear nuclei, cerebellar nuclei, and locus coerulus, while arginine was low and ornithine was high in the sensory cortex; in a few areas similar differences can be seen with the other amino acids. It is noteworthy that subcortical centers of the
220
Banay-Schwartz et al.
TABLE VI. Ranking by Concentration Methionine and Basic Amino Acids in Major Brain Systems I (highest) Methionine Hiatidine Lysine Arginine Ornithine
Brainstem Hypothalarnic Hypothalamic Hypothalamic Hypothalamic
2
3
4
S (lowest)
Sensory Sensory Motor Sensory Sensory
Motor Motor Brainstern Brainstern Motor
Hypothalamic Limbic Sensory Motor Lirnbic
Lirnbic Brainstem Lirnhic Lirnbic Brainstem
auditory system (cochlear nuclei, inferior colliculus, medial geniculate body) are rich in arginine but not in other basic amino acids (Table I). The changes with age in various areas expressed as percent alteration in the old brain are shown in Tables V11 and VIII. The magnitude of the change itself was somewhat variable, although usually in the same direction. For example, in a particular area there was a decrease in each sample with age, but the size of the decrease was variable, therefore the numerial value for the decrease could be of low statistical significance. Although both increases and decreases with age have been found in the various areas, decreases were found more often. The number of areas in which the levels changed with age and the ratio of the number of areas with decrease to those with increase varied with each amino acid, but significant decrease was noted more than five times as frequently as significant increase. This was similar to the changes with age with the large neutral amino acids described in the previous paper where decrease with age in general also occurred almost three times as frequently as increase. Highly significant changes ( P < .OOl), calculated using Student’s f-test analysis, were always decreases in aged rats, never increases. Highly significant decrease occurred 8 times with arginine, in one area each with methionine and histidine, and nowhere with lysine or ornithine (Tables VlI and VIII). Almost one-half of brain areas investigated (26 of 53) showed significantly (from P < .OS up to P < .001) decreased arginine levels in aged rats. This number was much lower with histidine (12), lysine ( 7 ) , methionine (6), ornithine ( 5 ) . Slightly increased ( P < .05) levels were measured in 8 areas (three each with lysine and arginine, one each with methionine and ornithine) (Tables VII and VIII).
DISCUSSION The distribution of the amino acids assayed in this paper in the selected specific brain areas is, like that of the amino acids assayed in the previous papers of this series (Banay-Schwartz et al., 1989a,b, 1990), surprisingly heterogeneous, with a more than ten-fold difference between the highest and lowest values and still more than five-fold difference between the average val-
ues of the five highest and five lowest areas. When the distribution of all the amino acids that were measured in this and the previous papers of this series is compared, several areas are seen to have many (or most) at high levels, and some to have most at low levels. Areas with generally high amino acid levels include the sensory trigeminal nucleus, medial geniculate body, inferior colliculus, medial preoptic nucleus, arcuate nucleus, and lateral hypothalamus. There are two brain regions where basic amino acid levels are relatively high: 9.8% of the total amino acids in the pontine nuclei and 8.2% in the ventromedial nucleus are basic amino acids. This percentage does not exceed 5 % in any other brain regions investigated. Areas in which most amino acids are low levels include the spinal cord, ventral horn and central gray, globus pallidus, cerebellar cortex, cingulate cortex, medial septal nucleus, lateral amygdaloid nucleus, and reticular formation (Banay-Schwartz et al., 1989a,b, 1990). There are also some areas in which some amino acids are near their highest and some near their lowest level; an example of this is the olfactory bulb, in which most of the neutral amino acids are at low levels and the acidic and basic amino acids are at high levels, or the medial amygdaloid nucleus, in which glutamate, glutamine, and GABA are high, and the large neutral and basic amino acids are low. Concentrations of basic amino acids in the brain nuclei are generally low; they constitute together no more than 1 4 % of the total amino acid levels. The relative concentration (in percentage of the total amino acid level) of certain basic amino acids also shows high heterogeneity: the arginine content is over 5% in the ventromedial (5.7%) and pontine nuclei (5.6%) but only 0.49% in the cingulate cortex or 0.56% in the medial amygdala. Similarly, the lysine content in the pontine nuclei is 2.8% of the total amino acid content, 2.5% in the cingulate cortex, but only 0.23% in the locus coeruleus and 0.31% in the vestibular nuclei. Our studies reveal only one aspect of amino acid heterogeneity, that of levels in discrete structures. It is likely that the heterogeneity is considerably higher than shown here in that, even within the structures examined, at least some of the amino acids may not be distributed evenly, in the synaptic region levels of GABA, taurine,
Aging and Amino Acid Levels in Brain
221
TABLE V11. Percent Change in Amino Acid Levels in Old Rats (Motor and Sensory Areas)* Met Sensory Areas Sensory cortex Ventral thalamic nucleus Sensory trigeminal nucleus Nucleus gracilis Nucleus cuneatus Dorsal horn (spinal cord) Visual cortex Lateral geniculate body Superior colliculus Cochlear nuclei Medial geniculate body Inferior colliculus Vestibular nuclei Motor areas Motor cortex Pontinc nuclei Ventral horn (spinal cord) Caudate nucleus Putamen Globus pallidus Substantia nipra Cerebellar cortex Cerebellar nuclei *O
=
not significant. I
=
P
His
+ 30"
0
Lys
Arg
-38'
-573 0 -33" - 16"
+ 26"
+ 30'
-30"
- 19"
0 + 38"
+ 4 I" + 30"
0
- 15" -37' 0 0
-31'
-29' - 28" - 22" - 28" 0 -51" - 17" P
< .02, 3
-23" -483 34" + 14" -36'
+ 25"
+
+ 25"
=
0 -21" + 3 I" 0 -32" + 170'
-
+ 881
< .OS, 2
-26" 35' 0 -37' 47" + 56"
-
