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Regional distribution of postsynaptic receptor binding for gamma-aminobutyric acid (GABA) in monkey brain
168
Bruin Re~warch, 93 (1975) 168-174 ~f) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Regional distribution of poatsynaptic receptor binding for gamma-aminobutyric acid (GABA) in monkey brain
S. J. ENNA, MICHAEL J. K U H A R AND SOLOMON H. SNYDER
Departments of Pharmacology and Experimental Therapeutics, and Psychiatry and the Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore Md. 21205 ( U.SM,) (Accepted April 14th, 1975)
Recently we have identified sodium independent binding of y-aminobutyric acid (GABA) to synaptic membranes of mammalian brain which appears to represent an interaction with the postsynaptic receptor for GABAI°,2L This interpretation is supported by the fact that the relative potencies of drugs and amino acids for receptor binding sites parallels closely the neurophysiologic activity of these compounds as mimickers of GABA. Besides GABA receptor binding, in the presence of sodium GABA binds to membrane particlesa-10,~2,16,~9-~1,23-~5, and this binding may involve sites related to GABA uptake by brain tissue, since the relative potency of drugs as inhibitors of sodium dependent GABA binding parallels their potency as inhibitors of uptakO 0. In brain, GABA can be accumulated by distinct systems involving nerve terminals or glia and these systems can be distinguished by autoradiographic studies or by the effects of fl-alanine, which is 200 times more potent as an inhibitor of glial than of nerve terminal uptake and diaminobutyric acid which is 10 times more potent an inhibitor of the nerve terminal than of the glial uptake process5,15,~. We observed that fl-alanine is 1000 times more potent an inhibitor of sodium dependent GABA binding than of synaptosomal GABA uptake and regional variations in synaptosomal GABA uptake in rat brain do not correlate well with variations in sodium dependent GABA binding x°. These discrepancies in drug effects and regional distribution suggest that the sodium dependent GABA binding process may involve glial rather than neuronal uptake sites 1°. In the present study we have evaluated the regional distribution in monkey brain of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), the synaptosomal uptake of GABA, sodium independent binding of GABA to its postsynaptic receptor sites, and the sodium dependent GABA binding process, The findings reveal that sodium independent GABA receptor binding correlates with synaptosomal uptake of GABA and with GAD activity but not with sodium dependent GABA binding. Seven rhesus monkeys (Macaca mulatta) were injected intraperitoneally with 40 mg/kg sodium pentobarbital, a drug which has no influence on GABA receptor binding 2v, and were decapitated 1 h later. The skull and spinal vertebrae were opened
169 TABLE I REGIONAL DISTRIBUTION OF SODIUM DEPENDENT AND SODIUM INDEPENDENTGABA BINDING, GABA UPTAKE AND GLUTAMICACID DECARBOXYLASE(GAD) IN THE MONKEYBRAIN Data are the means/rag wet weight 4- S.E. of 7 determinations.
Region
Binding (fmoles/mg) Na dependent
Olfactory bulb
Na independent
Uptake (fmoles/mg)
GAD (nmoles/mg/h)
5.1 4- 0.2
0.04 -4- 0.03
232 4- 38
11.06 4- 1.20
17.1 i 1.9 15.8 4- 1.9 12.8 4- 1.8 9.4 4- 0.4 8.6 4- 1.0 10.5 4- 0.6 18.8 4- 0.7 13.1 4- 1.0 6.0 4- 0.5
1.53 ± 0.19 1.82 4- 0.20 2.03 4- 0.33 0.67 4- 0.12 0.40 ± 0.03 1.33 4- 0.13 2.18 4- 0.26 1.58 :~ 0.24 1.52 4- 0.27
607 641 533 739 535 563 815 746 764
9.07 8.50 14.60 7.87 11.23 11.40 8.70 9.53 9.93
White matter areas Corpus callosum Corona radiata Optic chiasm
3.4 4- 0.4 2.6 4- 0.2 3.2 4- 0.2
0.23 4- 0.13 0.11 4- 0.04 0.13 4- 0.08
97 4- 3 68 d: 11 55 4- 11
0.43 4- 0.09 0.30 4- 0.12 0.47 4- 0.34
Limbic cortex Amygdala Hippocampus Septum
4.7 4- 0.5 7.3 4- 0.6 5.4 4- 0.8
1.40 4- 0.11 0.74 4- 0.11 1.21 4- 0.11
847 4- 49 390 4- 38 491 4- 44
9.10 4- 0.86 4.87 4- 0.77 8.60 4- 0.37
Hypothalamus
12.8 4- 1.2
0.41 4- 0.07
787 4- 75
20.00 4- 1.43
Thalamus
43.8 4- 2.7
1.02 :~ 0.08
224 4- 30
7.50 ~ 0.37
Extrapyramidal areas Caudate Putamen Globus pallidus Substantia nigra
11.7 15.3 4.8 7.9
2.52 2.37 2.00 1.14
339 299 852 587
Midbrain
31.6 4- 3.5
0.70 4- 0.11
405 4- 25
14.40 4- 1.99
Cerebellum - - lower brain stem Cortex Nodule and uvula Deep cerebellar nuclei
23.9 4- 1.9 4.7 4- 1.0 12.4 4- 0.99
1.72 4- 0.28 0.55 4- 0.03 0.30 4- 0.13
229 4- 26 286 4- 46 148 4- 14
8.07 4- 1.20 8.67 4- 0.96 8.67 4- 0.63
Ports
15.8 ± 1.9
0.20 4- 0.07
158 4- 12
1.17 4- 0.17
Medulla oblongata
21.4 4- 0.8
0.06 4- 0.02
270 4- 35
4.67 4- 0.30
9.8 4- 0.5 4.4 4- 1.1 4.1 4- 0.2
0.07 4- 0.02 0.09 4- 0.05 0.08 4- 0.04
154 4- 21 102 4- 9 196 4- 13
1.17 4- 0.17 0.83 4- 0.02 2.60 4- 0.80
Cerebrum Frontal cortex Frontal pole Occipital pole Temporal pole Precentral gyrus Postcentral gyrus Superior temporal gyrus Medial temporal gyrus Cingulate cortex
Spinal cord Cervical Thoracic Lumbar
± 0.8 4- 1.0 4- 0.3 4- 0.9
4- 0.49 4- 0.40 4- 0.11 4- 0.16
4- 21 4- 34 4- 25 4- 51 4- 39 4- 26 ! 69 4- 34 ± 61
4- 16 4- 8 4- 68 4- 75
13.23 13.00 47.87 27.20
4- 0.72 ± 0.99 ± 1.60 4- 0.77 4- 0.66 4- 0.57 4- 0.87 4- 0.89 4- 1.47
4± d: !
1.33 1.23 2.16 3.97
170 with an autopsy saw and the entire central nervous system was transferred to ice-cold 0.9~ NaCI within 5-10 min. After dissection, brain and spinal cord regions were homogenized in at least 20 vol. of 0.32 M sucrose and aliquots of the homogenate were analyzed for glutamic acid decarboxylase (GAD) 1. After centrifuging the homogenate for 10 min at 1000 × g, [aH]GABA (New England Nuclear, 10 Ci/mmole) uptake was studied in samples of the supernatant3,17, and the remainder of the supernatant was used for the preparation of crude synaptic membranes 1°,27. One-half of the freshly prepared synaptic membranes from each region was assayed immediately for Na dependent GABA binding, and the other half was frozen for at least 24 h at --20 °C before measurement of Na independent GABA receptor binding 1°. In confirmation of previous findings1, is, GAD activity is highest in extrapyramidal areas of the brain (Table I). The greatest activity is observed in the globus pallidus which is 1.8 times that of the substantia nigra, the second highest region. The hypothalamus, an area rich in endogenous GABA 1~, is the third highest region with values which are about 40 ~ those of the globus pallidus. The caudate and putamen are also among the most active areas with values 30 ~o of the globus pallidus. Within the cerebral cortex, GAD activity varies almost 2-fold between the occipital pole, which displays the greatest activity, and the temporal pole, with lowest values. White matter areas such as corpus callosum, corona radiata and optic chiasm, possess the lowest GAD levels in the brain, only about 1 ~o of those in the globus pallidus. GAD activity is also quite low in the spinal cord, where it is about 2-4 times greater than in the white matter regions. Biosynthetic enzymes for neurotransmitters as well as nerve terminal uptake systems are usually most enriched in nerve endings. Thus one might anticipate a close correlation between GAD activity and synaptosomal GABA uptake. While there is a correlation, there are considerable differences in the regional distribution of GABA uptake and GAD activity. As with GAD, GABA uptake is highly enriched in extrapyramidal areas, the globus pallidus displaying the highest levels of both GABA uptake and GAD and the substantia nigra possessing considerable GABA uptake activity. Similarly, the hypothalamus is highly enriched in both GAD and GABA uptake. However, the amygdala, which possesses the second greatest GABA uptake levels, is only moderately active in GAD. Similarly, among the cerebral cortical regions, the occipital pole, which has the highest GAD activity, is the lowest in GABA uptake, while the superior temporal gyrus, which displays the highest cortical uptake value, has one of the lower GAD levels. As with GAD, GABA uptake is lowest in white matter areas and the spina cord. Sodium independent GABA binding, presumably associated with the postsynaptic receptor, correlates significantly with both GABA uptake and with GAD. Thus, sodium independent GABA binding is most enriched in extrapyramidal areas. However, the highest levels of sodium independent GABA binding occur in the caudate with values in the globus pallidus being about 20 ~ lower, while GAD activity and GABA uptake are about 2.5-3 times higher in the globus pallidus than in the caudate. One marked discrepancy between sodium independent GABA binding and GAD and GABA uptake occurs in the hypothalamus which is one of the lower regions for
171 sodium independent GABA binding, although it is highly enriched in GAD and GABA uptake. Within the cerebral cortex, sodium independent GABA binding is highest in the occipital pole and the superior temporal gyrus, which are the highest cerebral cortical regions respectively for GAD and GABA uptake. The cerebellar cortex, which possesses the highest sodium independent GABA binding in rat brain, also has substantial GABA binding in the absence of sodium in the monkey brain, though values are lower than those in extrapyramidal areas and some parts of the cerebral cortex of the monkey. While spinal cord areas contain 2-4 times more GAD and GABA uptake than the white matter regions, the reverse situation occurs with sodium independent GABA binding, which is 2-3 times greater in white matter areas than in the spinal cord. Highest values for sodium dependent GABA binding occur in the thalamus, which is one of the lower regions for sodium independent GABA binding, uptake, and GAD. Thus thalamic sodium dependent GABA binding is 10 times the value in the globus pallidus, while sodium independent binding, GABA uptake and GAD are 2-6 times greater in the globus pallidus than in the thalamus. The midbrain possesses the second highest sodium dependent GABA binding, three-fourths of the value in the thalamus. The cerebellar cortex and medulla oblongata have similar levels of sodium dependent GABA binding, about half those of the thalamus, while sodium independent GABA binding, uptake and GAD are among the lowest in these regions. Among cerebral cortical areas there is about a three-fold variation in sodium dependent GABA binding with highest values in the superior temporal gyrus and lowest in the cingulate cortex. While sodium independent GABA binding is 2-3 times higher in white matter areas than in the spinal cord, the situation is reversed for sodium dependent GABA binding which is 2-3 times higher in various areas of the spinal cord than in the white matter regions. Moreover, within the spinal cord, sodium dependent GABA binding is more than twice as high in the cervical than in the thoracic and lumbar regions of the spinal cord. In contrast, there are no marked variations among spinal cord regions for sodium independent GABA binding, and GAD and GABA uptake are substantially higher in the lumbar spinal cord than in the cervical and thoracic cords. Using the Spearman Rank Correlation analysis it was found that sodium independent GABA binding correlates to a high statistical degree with GABA uptake and with GAD (P < 0.01) and GABA uptake and GAD are well correlated with each other (P < 0.01). By contrast the sodium dependent GABA binding process shows no statistically significant correlation with sodium independent GABA receptor binding, with GABA uptake into synaptosomal fractions or with GAD activity. In studies of both sodium dependent and independent GABA binding, the concentrations of GABA used are substantially lower than the dissociation constant. Thus it is conceivable that regional variations might derive from variations in affinity for GABA and not from differences in the total number of sites. Accordingly, we estimated the number of sites and affinity for sodium dependent and independent GABA binding in the cerebellar cortex, frontal cerebral cortex, thalamus and putamen. These experiments involved measurement of binding with 4-5 concentrations
172 of GABA in these regions. No difference in the dissociation constant calculated for sodium dependent and independent GABA binding was observed in any of these areas. The KD values for sodium dependent GABA binding are 147 nM, 156 nM and 132 nM, for cerebellar cortex, cerebral frontal cortex and thalamus, respectively and for sodium independent GABA binding the K9 values for the cerebellar cortex, cerebral frontal cortex and putamen are 63 nM, 69 nM and 46 nM. By contrast, the total number of binding sites for both sodium dependent and independent GABA binding differs among these three regions in close proportion to variations in binding obtained using single concentrations of GABA (Table I). Thus, although detailed dose-response curves were not obtained for all 31 regions of the monkey brain, it is likely that the variations in binding reflect differences in the number of sites and not in affinity. Interestingly, the average dissociation constants for both sodium dependent and sodium independent GABA binding in the monkey are about one-eighth of those seen in the rat brain 1°. Most of these experiments were performed with a crude synaptic membrane preparation, which is most enriched in GABA receptor binding27. However, it is possible that different membrane populations may vary in their centrifugal characteristics in various areas of the brain. For instance, in the cerebellum, the giant mossy fiber nerve terminals sediment in the crude nuclear fraction 14. Accordingly, in some experiments, 4 areas, cerebral cortex, putamen, thalamus and cerebellar cortex, were assayed for sodium dependent and independent GABA binding both in the crude synaptic membrane preparation and in whole homogenates. The relative values among these brain regions are the same using either preparation. In summary, sodium independent GABA receptor binding correlates with synaptosomal GABA uptake and GAD activity in the monkey brain. These observations are consistent with the conclusion based on pharmacologic correlations 1°,'~7 that this binding does involve postsynaptic receptor sites for GABA. In the rat brain, only 8 areas were examined and sodium independent binding did not correlate as well with GABA uptake 2v. Despite the generally positive relationship between GAD activity, GABA uptake and sodium independent GABA binding, there are some discrepancies among these three parameters. This is not altogether surprising, since the concentration of the biosynthetic enzyme for a neurotransmitter within a nerve terminal may vary among the nerve terminals which store the transmitter independently of variations in presynaptic nerve terminal membrane surface area, which may be a major determinant of transmitter uptake, and postsynaptic membrane surface, which presumably determines receptor binding. Thus, there are some differences in the regional distribution of muscarinic acetylcholine receptor binding and regional variations in endogenous levels of acetylcholine and choline acetyltransferase1~,26. Similarly, binding of LSD to presumed postsynaptic serotonin receptors in various regions of rat and monkey brain does not correlate well with endogenous levels of serotonin2,4. On the basis of the relative affinities of several amino acids, we postulated that the sodium dependent GABA binding may involve sites associated with the glial accumulation of GABA 1°. If this hypothesis is correct, then the marked regional
173
variations in sodium dependent GABA binding may provide valuable information about the disposition of proposed GABA accumulating glia. We wish to thank Drs. J. P. Bennett and J. T. Coyle for technical advice and assistance. This research was supported by USPHS Grants MH-18501, MH-25951, a grant of the John A. Hartford Foundation, Research Scientist Development Award MH33128 to S. H. S. and USPHS Fellowship MH-01598 to S.J.E.
1 ALBERS, R. W., AND BRADY, R. O., The distribution of glutamic decarboxylase in the nervous system of the rhesus monkey, J. bioL Chem., 234 (1959) 926-928. 2 BENNETT,J. L., AND AGHAJANIAN,G. K., D-LSD binding to brain homogenates : possible relationship to serotonin receptors, Life Sci., 15 (1974) 1935-1944. 3 BENNETT,J. P., LOGAN, W. J., AND SNYDER, S. H., Amino acids as central nervous neurotransmitters: the influences of ions, amino acid analogues, and ontogeny on transport systems for Lglutamic and L-aspartic acids, and glycine into the central nervous synaptosomes of the rat, J. Neurochem., 21 (1973) 1533-1550. 4 BENNETT,J. P., AND SNYDER,S. H., Stereospecific binding of o-lysergic acid diethylamide (LSD) to brain membranes: relationship to serotonin receptors, Brain Research, (1975) in press. 5 BLOOM, F. E., AND IVERSEN,L. L., Localizing 8H-GABA in nerve terminals of rat central cortex by electron microscopic autoradiography, Nature (Lond.), 229 (1971) 629-630. 6 DEFEUDIS, F. V., Sodium dependency of 7-aminobutyric acid binding to particulate fractions of mouse brain, Exp. NeuroL, 41 (1973) 54-62. 7 DEFEuoIs, F. V., Binding of aH-7-aminobutyric acid and 14C-glycine to synaptosomal-mitochondrial fractions of rat cerebral cortex and spinal cord, Canad. J. Physiol. PharmacoL, 51 (1973) 873-878. 8 DEFEumS, F. V., Preferential 'binding' of 7.aminobutyric acid and glycine to synaptosome-enriched fractions of rat cerebral cortex and spinal cord, Canad. J. PhysioL Pharmacol., 52 (1974) 138-147. 9 DEFEUDIS,F. V., ANDBLACK,W. C., A comparison of the binding of [14C]v-aminobutyric acid and [aH]acetylcholine to particulate fractions of 4 regions of rat brain in the presence of 40 m M NaCl, Brain Research, 49 (1973) 218-222. l0 ENNA, S. J., AND SNYDER,S. H., Properties of 7-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions, Brain Research, (1975) in press. 11 FAHN, S., AND COTI~,L. J., Regional distribution of 7-aminobutyric acid (GABA) in brain of the rhesus monkey, J. Neurochem., 15 (1968) 209-213. 12 GOTTESFELD,Z., AND ELLIOTT, K. k. C., Factors that affect the binding and uptake of GABA by brain tissue, J. Neurochem., 18 (1971) 683-690. 13 HILEY, C. R., AND BURGEN,A. S. V., The distribution of muscarinic receptor sites in the nervous system of the dog, J. Neurochem., 22 (1974) 159-162. 14 ISRAEL,i . , AND WHITTAKER,V. P., The isolation of mossy fiber endings from the granular layer of the cerebellar cortex, Experientia (Basel), 15 (1965) 325-326. 15 IVERSEN,L. L., AND BLOOM,F. E., Studies of the uptake of ZH-GABA and ZH-glycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 16 KURIYAMA,K., ROBERTS,E., AND VOS, J., Some characteristics of binding of 7-aminobutyric acid and acetylcholine for synaptic vesicle fraction from mouse brain, Brain Research, 9 (1968) 231-252. 17 LOGAN,W. J., AND SNYDER,S. H., High affinity uptake systems for glycine, glutamic and aspartic acids in synaptosomes of rat central nervous tissue, Brain Research, 42 (1972) 413431. 18 LOWE, I. P., ROBINS, E., AND EYERMAN,G. S., The fluorimetric measurement of glutamic decarboxylase and its distribution in brain, d. Neurochem., 3 (1958) 8-18. 19 ROBERTS, E., AND KURIYAMA, K., Biochemical-physiological correlations in studies of the ~,aminobutyric acid system, Brain Research, 8 (1968) 1-35.
174 20 SANO, K., AND ROBERTS, E., Binding of 7-aminobutyric acid by brain preparations, Biachcm.
biophys. Res. Commun., 4 (1961) 358 361. 21 Sat
Bruin Re~warch, 93 (1975) 168-174 ~f) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Regional distribution of poatsynaptic receptor binding for gamma-aminobutyric acid (GABA) in monkey brain
S. J. ENNA, MICHAEL J. K U H A R AND SOLOMON H. SNYDER
Departments of Pharmacology and Experimental Therapeutics, and Psychiatry and the Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore Md. 21205 ( U.SM,) (Accepted April 14th, 1975)
Recently we have identified sodium independent binding of y-aminobutyric acid (GABA) to synaptic membranes of mammalian brain which appears to represent an interaction with the postsynaptic receptor for GABAI°,2L This interpretation is supported by the fact that the relative potencies of drugs and amino acids for receptor binding sites parallels closely the neurophysiologic activity of these compounds as mimickers of GABA. Besides GABA receptor binding, in the presence of sodium GABA binds to membrane particlesa-10,~2,16,~9-~1,23-~5, and this binding may involve sites related to GABA uptake by brain tissue, since the relative potency of drugs as inhibitors of sodium dependent GABA binding parallels their potency as inhibitors of uptakO 0. In brain, GABA can be accumulated by distinct systems involving nerve terminals or glia and these systems can be distinguished by autoradiographic studies or by the effects of fl-alanine, which is 200 times more potent as an inhibitor of glial than of nerve terminal uptake and diaminobutyric acid which is 10 times more potent an inhibitor of the nerve terminal than of the glial uptake process5,15,~. We observed that fl-alanine is 1000 times more potent an inhibitor of sodium dependent GABA binding than of synaptosomal GABA uptake and regional variations in synaptosomal GABA uptake in rat brain do not correlate well with variations in sodium dependent GABA binding x°. These discrepancies in drug effects and regional distribution suggest that the sodium dependent GABA binding process may involve glial rather than neuronal uptake sites 1°. In the present study we have evaluated the regional distribution in monkey brain of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), the synaptosomal uptake of GABA, sodium independent binding of GABA to its postsynaptic receptor sites, and the sodium dependent GABA binding process, The findings reveal that sodium independent GABA receptor binding correlates with synaptosomal uptake of GABA and with GAD activity but not with sodium dependent GABA binding. Seven rhesus monkeys (Macaca mulatta) were injected intraperitoneally with 40 mg/kg sodium pentobarbital, a drug which has no influence on GABA receptor binding 2v, and were decapitated 1 h later. The skull and spinal vertebrae were opened
169 TABLE I REGIONAL DISTRIBUTION OF SODIUM DEPENDENT AND SODIUM INDEPENDENTGABA BINDING, GABA UPTAKE AND GLUTAMICACID DECARBOXYLASE(GAD) IN THE MONKEYBRAIN Data are the means/rag wet weight 4- S.E. of 7 determinations.
Region
Binding (fmoles/mg) Na dependent
Olfactory bulb
Na independent
Uptake (fmoles/mg)
GAD (nmoles/mg/h)
5.1 4- 0.2
0.04 -4- 0.03
232 4- 38
11.06 4- 1.20
17.1 i 1.9 15.8 4- 1.9 12.8 4- 1.8 9.4 4- 0.4 8.6 4- 1.0 10.5 4- 0.6 18.8 4- 0.7 13.1 4- 1.0 6.0 4- 0.5
1.53 ± 0.19 1.82 4- 0.20 2.03 4- 0.33 0.67 4- 0.12 0.40 ± 0.03 1.33 4- 0.13 2.18 4- 0.26 1.58 :~ 0.24 1.52 4- 0.27
607 641 533 739 535 563 815 746 764
9.07 8.50 14.60 7.87 11.23 11.40 8.70 9.53 9.93
White matter areas Corpus callosum Corona radiata Optic chiasm
3.4 4- 0.4 2.6 4- 0.2 3.2 4- 0.2
0.23 4- 0.13 0.11 4- 0.04 0.13 4- 0.08
97 4- 3 68 d: 11 55 4- 11
0.43 4- 0.09 0.30 4- 0.12 0.47 4- 0.34
Limbic cortex Amygdala Hippocampus Septum
4.7 4- 0.5 7.3 4- 0.6 5.4 4- 0.8
1.40 4- 0.11 0.74 4- 0.11 1.21 4- 0.11
847 4- 49 390 4- 38 491 4- 44
9.10 4- 0.86 4.87 4- 0.77 8.60 4- 0.37
Hypothalamus
12.8 4- 1.2
0.41 4- 0.07
787 4- 75
20.00 4- 1.43
Thalamus
43.8 4- 2.7
1.02 :~ 0.08
224 4- 30
7.50 ~ 0.37
Extrapyramidal areas Caudate Putamen Globus pallidus Substantia nigra
11.7 15.3 4.8 7.9
2.52 2.37 2.00 1.14
339 299 852 587
Midbrain
31.6 4- 3.5
0.70 4- 0.11
405 4- 25
14.40 4- 1.99
Cerebellum - - lower brain stem Cortex Nodule and uvula Deep cerebellar nuclei
23.9 4- 1.9 4.7 4- 1.0 12.4 4- 0.99
1.72 4- 0.28 0.55 4- 0.03 0.30 4- 0.13
229 4- 26 286 4- 46 148 4- 14
8.07 4- 1.20 8.67 4- 0.96 8.67 4- 0.63
Ports
15.8 ± 1.9
0.20 4- 0.07
158 4- 12
1.17 4- 0.17
Medulla oblongata
21.4 4- 0.8
0.06 4- 0.02
270 4- 35
4.67 4- 0.30
9.8 4- 0.5 4.4 4- 1.1 4.1 4- 0.2
0.07 4- 0.02 0.09 4- 0.05 0.08 4- 0.04
154 4- 21 102 4- 9 196 4- 13
1.17 4- 0.17 0.83 4- 0.02 2.60 4- 0.80
Cerebrum Frontal cortex Frontal pole Occipital pole Temporal pole Precentral gyrus Postcentral gyrus Superior temporal gyrus Medial temporal gyrus Cingulate cortex
Spinal cord Cervical Thoracic Lumbar
± 0.8 4- 1.0 4- 0.3 4- 0.9
4- 0.49 4- 0.40 4- 0.11 4- 0.16
4- 21 4- 34 4- 25 4- 51 4- 39 4- 26 ! 69 4- 34 ± 61
4- 16 4- 8 4- 68 4- 75
13.23 13.00 47.87 27.20
4- 0.72 ± 0.99 ± 1.60 4- 0.77 4- 0.66 4- 0.57 4- 0.87 4- 0.89 4- 1.47
4± d: !
1.33 1.23 2.16 3.97
170 with an autopsy saw and the entire central nervous system was transferred to ice-cold 0.9~ NaCI within 5-10 min. After dissection, brain and spinal cord regions were homogenized in at least 20 vol. of 0.32 M sucrose and aliquots of the homogenate were analyzed for glutamic acid decarboxylase (GAD) 1. After centrifuging the homogenate for 10 min at 1000 × g, [aH]GABA (New England Nuclear, 10 Ci/mmole) uptake was studied in samples of the supernatant3,17, and the remainder of the supernatant was used for the preparation of crude synaptic membranes 1°,27. One-half of the freshly prepared synaptic membranes from each region was assayed immediately for Na dependent GABA binding, and the other half was frozen for at least 24 h at --20 °C before measurement of Na independent GABA receptor binding 1°. In confirmation of previous findings1, is, GAD activity is highest in extrapyramidal areas of the brain (Table I). The greatest activity is observed in the globus pallidus which is 1.8 times that of the substantia nigra, the second highest region. The hypothalamus, an area rich in endogenous GABA 1~, is the third highest region with values which are about 40 ~ those of the globus pallidus. The caudate and putamen are also among the most active areas with values 30 ~o of the globus pallidus. Within the cerebral cortex, GAD activity varies almost 2-fold between the occipital pole, which displays the greatest activity, and the temporal pole, with lowest values. White matter areas such as corpus callosum, corona radiata and optic chiasm, possess the lowest GAD levels in the brain, only about 1 ~o of those in the globus pallidus. GAD activity is also quite low in the spinal cord, where it is about 2-4 times greater than in the white matter regions. Biosynthetic enzymes for neurotransmitters as well as nerve terminal uptake systems are usually most enriched in nerve endings. Thus one might anticipate a close correlation between GAD activity and synaptosomal GABA uptake. While there is a correlation, there are considerable differences in the regional distribution of GABA uptake and GAD activity. As with GAD, GABA uptake is highly enriched in extrapyramidal areas, the globus pallidus displaying the highest levels of both GABA uptake and GAD and the substantia nigra possessing considerable GABA uptake activity. Similarly, the hypothalamus is highly enriched in both GAD and GABA uptake. However, the amygdala, which possesses the second greatest GABA uptake levels, is only moderately active in GAD. Similarly, among the cerebral cortical regions, the occipital pole, which has the highest GAD activity, is the lowest in GABA uptake, while the superior temporal gyrus, which displays the highest cortical uptake value, has one of the lower GAD levels. As with GAD, GABA uptake is lowest in white matter areas and the spina cord. Sodium independent GABA binding, presumably associated with the postsynaptic receptor, correlates significantly with both GABA uptake and with GAD. Thus, sodium independent GABA binding is most enriched in extrapyramidal areas. However, the highest levels of sodium independent GABA binding occur in the caudate with values in the globus pallidus being about 20 ~ lower, while GAD activity and GABA uptake are about 2.5-3 times higher in the globus pallidus than in the caudate. One marked discrepancy between sodium independent GABA binding and GAD and GABA uptake occurs in the hypothalamus which is one of the lower regions for
171 sodium independent GABA binding, although it is highly enriched in GAD and GABA uptake. Within the cerebral cortex, sodium independent GABA binding is highest in the occipital pole and the superior temporal gyrus, which are the highest cerebral cortical regions respectively for GAD and GABA uptake. The cerebellar cortex, which possesses the highest sodium independent GABA binding in rat brain, also has substantial GABA binding in the absence of sodium in the monkey brain, though values are lower than those in extrapyramidal areas and some parts of the cerebral cortex of the monkey. While spinal cord areas contain 2-4 times more GAD and GABA uptake than the white matter regions, the reverse situation occurs with sodium independent GABA binding, which is 2-3 times greater in white matter areas than in the spinal cord. Highest values for sodium dependent GABA binding occur in the thalamus, which is one of the lower regions for sodium independent GABA binding, uptake, and GAD. Thus thalamic sodium dependent GABA binding is 10 times the value in the globus pallidus, while sodium independent binding, GABA uptake and GAD are 2-6 times greater in the globus pallidus than in the thalamus. The midbrain possesses the second highest sodium dependent GABA binding, three-fourths of the value in the thalamus. The cerebellar cortex and medulla oblongata have similar levels of sodium dependent GABA binding, about half those of the thalamus, while sodium independent GABA binding, uptake and GAD are among the lowest in these regions. Among cerebral cortical areas there is about a three-fold variation in sodium dependent GABA binding with highest values in the superior temporal gyrus and lowest in the cingulate cortex. While sodium independent GABA binding is 2-3 times higher in white matter areas than in the spinal cord, the situation is reversed for sodium dependent GABA binding which is 2-3 times higher in various areas of the spinal cord than in the white matter regions. Moreover, within the spinal cord, sodium dependent GABA binding is more than twice as high in the cervical than in the thoracic and lumbar regions of the spinal cord. In contrast, there are no marked variations among spinal cord regions for sodium independent GABA binding, and GAD and GABA uptake are substantially higher in the lumbar spinal cord than in the cervical and thoracic cords. Using the Spearman Rank Correlation analysis it was found that sodium independent GABA binding correlates to a high statistical degree with GABA uptake and with GAD (P < 0.01) and GABA uptake and GAD are well correlated with each other (P < 0.01). By contrast the sodium dependent GABA binding process shows no statistically significant correlation with sodium independent GABA receptor binding, with GABA uptake into synaptosomal fractions or with GAD activity. In studies of both sodium dependent and independent GABA binding, the concentrations of GABA used are substantially lower than the dissociation constant. Thus it is conceivable that regional variations might derive from variations in affinity for GABA and not from differences in the total number of sites. Accordingly, we estimated the number of sites and affinity for sodium dependent and independent GABA binding in the cerebellar cortex, frontal cerebral cortex, thalamus and putamen. These experiments involved measurement of binding with 4-5 concentrations
172 of GABA in these regions. No difference in the dissociation constant calculated for sodium dependent and independent GABA binding was observed in any of these areas. The KD values for sodium dependent GABA binding are 147 nM, 156 nM and 132 nM, for cerebellar cortex, cerebral frontal cortex and thalamus, respectively and for sodium independent GABA binding the K9 values for the cerebellar cortex, cerebral frontal cortex and putamen are 63 nM, 69 nM and 46 nM. By contrast, the total number of binding sites for both sodium dependent and independent GABA binding differs among these three regions in close proportion to variations in binding obtained using single concentrations of GABA (Table I). Thus, although detailed dose-response curves were not obtained for all 31 regions of the monkey brain, it is likely that the variations in binding reflect differences in the number of sites and not in affinity. Interestingly, the average dissociation constants for both sodium dependent and sodium independent GABA binding in the monkey are about one-eighth of those seen in the rat brain 1°. Most of these experiments were performed with a crude synaptic membrane preparation, which is most enriched in GABA receptor binding27. However, it is possible that different membrane populations may vary in their centrifugal characteristics in various areas of the brain. For instance, in the cerebellum, the giant mossy fiber nerve terminals sediment in the crude nuclear fraction 14. Accordingly, in some experiments, 4 areas, cerebral cortex, putamen, thalamus and cerebellar cortex, were assayed for sodium dependent and independent GABA binding both in the crude synaptic membrane preparation and in whole homogenates. The relative values among these brain regions are the same using either preparation. In summary, sodium independent GABA receptor binding correlates with synaptosomal GABA uptake and GAD activity in the monkey brain. These observations are consistent with the conclusion based on pharmacologic correlations 1°,'~7 that this binding does involve postsynaptic receptor sites for GABA. In the rat brain, only 8 areas were examined and sodium independent binding did not correlate as well with GABA uptake 2v. Despite the generally positive relationship between GAD activity, GABA uptake and sodium independent GABA binding, there are some discrepancies among these three parameters. This is not altogether surprising, since the concentration of the biosynthetic enzyme for a neurotransmitter within a nerve terminal may vary among the nerve terminals which store the transmitter independently of variations in presynaptic nerve terminal membrane surface area, which may be a major determinant of transmitter uptake, and postsynaptic membrane surface, which presumably determines receptor binding. Thus, there are some differences in the regional distribution of muscarinic acetylcholine receptor binding and regional variations in endogenous levels of acetylcholine and choline acetyltransferase1~,26. Similarly, binding of LSD to presumed postsynaptic serotonin receptors in various regions of rat and monkey brain does not correlate well with endogenous levels of serotonin2,4. On the basis of the relative affinities of several amino acids, we postulated that the sodium dependent GABA binding may involve sites associated with the glial accumulation of GABA 1°. If this hypothesis is correct, then the marked regional
173
variations in sodium dependent GABA binding may provide valuable information about the disposition of proposed GABA accumulating glia. We wish to thank Drs. J. P. Bennett and J. T. Coyle for technical advice and assistance. This research was supported by USPHS Grants MH-18501, MH-25951, a grant of the John A. Hartford Foundation, Research Scientist Development Award MH33128 to S. H. S. and USPHS Fellowship MH-01598 to S.J.E.
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biophys. Res. Commun., 4 (1961) 358 361. 21 Sat