Journal of Neuroscience Research 1 : 19-30 (1975)
CELLULAR COMPARTMENTATION OF BRAIN METABOLISM AND ITS FUNCTIONAL SIGNIFICANCE Steven P. R. Rose Brain Research Group, Open University, Walton Hall, Milton Keynes MK7 6AA, England
This paper reviews work from our laboratory on the metabolism and interrelations of isolated neuronal and neuropil fractions. The cell preparations are evaluated according t o criteria of yield, purity, and integrity. Differences in the levels of activity of six groups of enzymes, of glucose metabolism, amino-acid metabolism, transmitter metabolism, acid hydrolysis, alkaline hydrolysis, and carbonic anhydrase have been followed, and neuronal and glial marker enzymes are proposed. Lysosomes and their enzymes are concentrated in neuronal perikarya. Metabolically, although n o major differences in glucose oxidation have been found, there is considerable evidence of compart mentation of amino acids and their metabolism. At short times after injection of 3H-lysine as precursor in vivo, neuronal incorporation is high as compared with neuropil; at longer times the ratio is reversed, and we interpret this as evidence for the presence of a rapidly labeling protein fraction present in the neuronal perikarya but subsequently transported out. Neuronal protein incorporation is suppressed in the visual but not the motor cortex of dark-reared rats and is switched on following exposure to light; there is evidence that the suppressed fraction of neuronal protein synthesis includes t h e rapidly labeling component. A model for neuronal-glial metabolic interaction and its state-dependence in response t o changes in the organism’s environment and behavior is sketched out. INTRODUCTION
It is today a commonplace that neurochemistry is not divorced from the classical biological dialectic of structure and function. Whatever biochemical measure one makes in the nervous system, one must expect t o find it differentially distributed amongst t h e different structures. With t h e broad pathways, at least of primary metabolism, well mapped out, attention has turned increasingly t o investigating the role of structural compartmentation both in regulating ongoing basic metabolic processes and in providing a mechanism for coding changes in the external environment and in the behavior of the organism at the cellular and biochemical levels. We must recognize that t o report a biochemical change as occurring in “whole cortex” is t o describe the algebraic sum of events that in fact take place in many partially intercommunicating compartments. In this context we may define a “compartment” as “any small region of tissue of uniform phase of homogeneous biochemical properties bounded by a region of phase change” (for example, b y a membrane) (Rose, 1972).
19
0 1975 Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. 10011
20
Rose
At the time of the first ISN meeting in 1967, the approach to the problem of the functional biochemistry of the two principal cell types in cortex, neurons and glia, was still virtually confined to the pioneering microdissection studies of HydCn and his collaborators. At this meeting there are several sessions devoted to neuronal and glial biochemistry, with reports from laboratories throughout the world. In this paper, we will concentrate on describing our own work, in which we used the method for separation of neurons and neuropil in bulk that was first reported in 1965 (Rose, 1965).
PREPARATION, CHARACTERISTICS, AND EVALUATION O F ISOLATED CELL FRACTIONS
Any cell (or subcellular) fractionation technique must be judged against three criteria: the yield, purity, and integrity of the isolated fractions. According to our technique, the cell fractions are isolated by first disaggregating cleaned cerebral cortical tissue in a medium containing 110 rnM KC1, 10 mM potassium phosphate buffer, pH 7.4, and Ficoll, then by teasing through meshes of defined aperture size, and finally by centrifugation on a discontinuous Ficoll/sucrose gradient. The procedure we currently use is exactly that of Rose (1967) with the following modifications: (a) 11% Ficoll replaces 10%Ficoll in the initial disaggregation medium and (b) the nylon mesh is mounted on a sawn-off syringe and the tissue and medium passed backward and forward through it several times. For certain purposes, the following further modifications may also be introduced: (a) a preliminary low-speed spin to remove subcellular contaminants (Sinhaand Rose, 1972a) and (b) passage through a siliconized glass bead column to remove lipid material and capillary endothelial cells (Rose and Sinha, 1970). These modifications affect both yield and purity of the fractions. The basic procedure yields four fractions: (a) myelin, undisrupted tissue, and debris, including 60-70% of total protein; (b) glia and neuropil, 10-20% of total protein; (c) neurons, 5-l% of total protein; and (d) red cells and nuclei, 5-1 0% of total protein. The centrifugation procedure is not isopycnic; distribution depends o n ionic as well as density factors, and recovery and protein yield depend in part upon the loading of the gradient. The intention behind the development of the technique was to obtain a method for providing as rapidly as possible, and in milligram quantities, neuron- and glia-enriched cell fractions in which the cells, or rather their perikarya, are present in a relatively undamaged state. This result would make possible two broad lines of investigation: metabolic studies in vivo followed by fractionation and examination of distribution of metabolites, and studies of metabolism or enzyme activities in vitro following separation. This specification demands a playoff of factors - speed of separation against relative damage to the cells, and yield against purity. Subsequent methods have been developed that depend on enzyme tissue dissociation (for example, Norton and Poduslo, 1970) or hypotonic media and the use of PVP/BSA in gradients (for example, Sellinger et al., 197 1). These methods, whilst producing somewhat purer fractions (notably glial), also result either in metabolically more damaged preparations or
21
Metabolism of Neurons and Glia
a lower yield, making difficult the controlled comparison of cell properties in fractions recovered from the same gradient and derived from the same animal (Sinha and Rose, 1971). One disadvantage of the relative proliferation of separation techniques, whether the differences between them are major or minor, is that it makes it harder to interpret the sometimes conflicting results that the techniques generate. On the one hand, in the context of bulk separation (as opposed to microdissection techniques) the question of yield is of relatively minor importance, although where subcellular fractions are being made from the cells or where, as in behavioral experiments, regional fractionations on single brains are required, yield may once again become limiting. On the other hand, the question of purity is of extreme significance. We have attempted to specify the purity of the fractions using three criteria: (1) morphological appearance of the cells under phase-contrast light microscopy; (2) counting of neuronal and glial nuclei in fixed preparations stained with cresyl violet and by the use of enzymic markers (see below); and (3) integrity. On this basis, the cell population in the neuronal fraction is 60-70% neurons and in the glial-enriched fraction 6 0 4 6 % glia. However, electron-microscopic examination makes it clear that there are other cellular contaminants present, especially in the glial-enriched fraction, including axonal and dendritic processes and synaptosomes. It is for this reason that we have preferred to describe the glial-enriched fraction as neuropil; other authors have retained the “glial” nomenclature. The third criterion, that of integrity, is the most vexed. During disaggregation and centrifugation the cells lose a certain amount (about 10%)of soluble protein; cytoplasmic enzymes such as lactic dehydrogenase and glutamine synthetase tend t o leak from the cell. There is also a loss of low-molecular-weight constituents, such as amino acids, of up to 6076, although these are subsequently resynthesized (Rose, 1968a). Thus, interpretation of experiments describing the distribution of a property or substance between cell types always requires that recovery data be presented, though regrettably this is not always done. Such losses, and the fact that cells that appear well preserved to light microscopy seem more damaged under the electron microscope, has led to some criticism of the possibility of making any meaningful interpretation of data obtained from the fractions (e.g., Cremer et al., 1968). Whilst scepticism is desirable, the facts that the cell fractions show integrated metabolic behavior in vitro and that they preserve such properties as the ability to accumulate potassium ions (Rose and Sinha, 1969) and amino acids (Hamberger, 1971) against a concentration gradient, to respire with glucose as substrate at rates not much different from the tissue slice (Rose, 1967), and to synthesize and maintain high ATP levels (Rose and Sinha, 1969), may all be taken as evidence that biochemically the cells appear t o reseal after disaggregation and that extrapolation from in vitro properties to the in vivo situation may justifiably be made. These issues have been discussed elsewhere and will not be further referred to here (Rose, 1968b, 1969, 1972). ENZYME ACTIVITIES OF ISOLATED CELLS
Table I groups the various enzymes whose activities we have measured in the
Glutamate dehydrogenase Aspartate aminotransferase Glutamate decarboxylase Glutamine synthetase
Monoamine oxidase Acetylcholinesterase Butyrylcholinesterase
(b) Amino acid metabolism
(c) Transmitter
Alkaline DNase Alkaline phosphatase
Carbonic anhydrase
(e) Alkaline hydrolases
(0
25.9
398.8
13.0 3.2
3.4 5.8 6.8 6 .O 9.9 2.7 6.8 3.4 1.4 0.6
9 .0 6.9 39.7 62.9 20.1 15 1.3 4.3 6.6 188.9 64.9
31.1 40.2 270.1 383.1 199.3 414.8 29.2 22.3 267.0 40.0
55.4 19.3
1.8 2.1 3.6
21.9 39.2 8.0
13.5
4.2 6.0
1.0 1.2 1.8
40.2 82.8 29.1
4 .O
0.6 1.1 1.8
Neuronal/neuropil ratio
5 .O 57.5 20.0 7.9
11.1 14.9 29.6
neuropil
19.9 57.3 24.0 14.0
7.1 16.7 54.3
neurons
Enzyme activity (units/mg prot)
*All enzyme activities are in arbitrary units per mg protein except for the hydrolases. which are nM substrate released per hr per mg protein. Data abstracted (and errors omitted for clarity) from the following. where assay methods are also described: ( 1 ) Rose (1967): (2) Rose (1968a); (3) Sinha and Rose (1972a); (4) Sinha and Rose (1972b); ( 5 ) Sinha and Rose (1973).
Arab inosidase ArylSulphdtaSe Cathepsin Acid DNase p-galactosidase Glucosaminidase Glucosidase Glu cur0 niddse Acid phosphatase 0-N-acetyl-galacto saminidase
(d) Acid hydrolases
metabolism
Lactic dehydrogenase Succinic dehydrogenase Cytochronie oxidase
Enzyme
(a) Glucose metabolism
Group
TABLE I. Enzyme Activites in Neuronal and Neuropil Fractions*
1
4 4
4 4 4 4 4 4 4 4 4 5
3 3 3
2 2 2 2
1
1 1
Ref.
N N
23
Metabolism of Neurons and Glia
isolated cells. These include (a) enzymes of glucose metabolism, (b) enzymes of amino acid metabolism, (c) enzymes of putative transmitter systems, (d) acid hydrolases (lysosomal enzymes), (e) alkaline hydrolases, and (f) carbonic anhydrase. The table reveals wide differences in the distribution of the enzymes between the cells. In general, we have found that there are no great differences between fractions as regards the activity of SDH and LDH, the enzymes of glucose metabolism, and that cytochrome oxidase is somewhat higher in neurons than neuropil. We have not measured Na', K'-ATPase, but there have been reports that it is more active in glia than neurons (Cummins and HydCn, 1962; Medzihradsky et al., 1972). In the absence of detergent activation, glutamate dehydrogenase is four times as high in neurons as in neuropil, whilst asparate aminotransferase, glutamate decarboxylase, and glutamine synthetase do not preatly differ between the fractions (though the recovery of glutamine synthetase is low, suggesting a loss through leakage). Of the transmitter enzymes studied, M A 0 is 84%higher in the neuronal fraction and AChE 110%higher, which is perhaps not surprising; what is more surprising is that BChE is also higher - by 260% in the neurons. The AChE/BChE difference cannot then serve as a neuronal marker, as is sometimes assumed, and, in fact, our observations are supported by histological evidence (Pavlin, 1965). Amongst the ten acid hydrolases studied, nine are more active in neurons than neuropil and one, 0-N-acetyl galactosaminidase, is more active in neuropil. These enzymes are generally lysosomal in localization, and there is also supportive histochemical evidence for the proposition that lysosomes and their enzymes are more concentrated in neurons than in glia or neuropil (Holtzman, 1969; Hirsch, 1969). However, the very different neuronal/neuropil activity ratios of the enzymes is evidence of the microheterogeneity of lysosomal particles in the cortex, and we have proposed (Sinha and Rose 1972a, 1973) that at least four types of particle could be identified. One particle contains acid phosphatase, glucosaminidase, arabinosidase, and glucuronidase (activity ratio 1.4-3.4); a second contains aryl sulphatase, acid DNase, cathepsin, and glucosidase (activity ratio 5 2 - 6 3 ) ; a third contains one enzyme, 0-galactosidase (activity ratio 9-10), which we have therefore suggested may serve as a neuronal marker; and a fourth contains the single enzyme P-N-acetyl galactosaminidase. The fifth general group of enzymes studied, the alkaline hydrolases, is a disparate collection, and perhaps does not warrant special comment. The sixth contains one enzyme, carbonic anhydrase, included because of evidence that it may serve as a glial marker; the activity ratio does indeed make such a use of the enzyme possible. GLUCOSE METABOLISM In vitro, in a high K', Ca2'-free medium, both cell fractions metabolize glucose t o a comparable extent, about 80%of the rate of slice (Table 11) (Rose, 1967). This observation must be contrasted with the finding of Hertz, who, using microdiver techniques, noted that on a wet-weight basis, the glial respiration rate was only some 23% of neuronal (e.g., Dittmann et al., 1973; Hertz et al., 1973). Hertz has argued that the glia are high potassium cells, accumulating ions released from the neurons during, physiological activity. The discrepancy between bulk-separated and microdiver preparations may be due to differences in incubation media, but we have no adequate explana-
24
Rose
TABLE 11. Oxidative and Ion Metabolism in Neuronal and Neuropil Fractions
Property
Whole cell suspension
Neuron
Neuropil
Neuron/neuropil ~
0, uptake (glucose) 0, uptake (pyruvate) ATP K+accumulation
Ref. ~~
4 30
394
378
1.04
1
330 5.1 3.2
345 12.3 3.3
368 7.4 2.7
0.94 1.66 1.22
1 2 2
Experimental details are in references: (1) Rose (1967); (2) Rose and Sinha (1969). Units are: 0, uptake in the presence of glucose or pyruvate, nm/mg dry wt/hr; ATP, nM/mg protein; K+ accumulation, concentration ratio tissue/medium, poisoned vs unpoisoned tissue.
tion at present. After in vitro incubation, ATP levels in neurons are 66%higher than in neuropil. AM1NO-ACID METABOLISM
We have examined the conversion of 14C-labeledsubstrates into the amino acids ' glutamate, glutamine, GABA, aspartate, and alanine in vitro. On the one hand, the pool sizes of the free amino acids are all about twice as high in neurons as in neuropil, either directly after preparation of the fractions or after up to 2 hr incubation.with glucose. On the other hand, the specific activity of the amino acids is 3- t o %fold higher in neuropil than in neurons, whether glucose or pyruvate is substrate. When TABLE 111. Amino Acid Metabolism in Isolated Neuronal and Neuropil Fractions ~
~
Glutamate (a) Pool size
2 34
(b) Specific rddioactivitysubstrate, glucose
12
(c) Specific radioactivitysubstrate, pyruvate
57
(d) Specific radioactivitysubstrate, glutamate + glucose
Neuronal/neuropil ratio for Glutamine GABA Aspartate 176
Alanine
202
167
2 30
18
34
33
23
28
24
106
102
CELLULAR COMPARTMENTATION OF BRAIN METABOLISM AND ITS FUNCTIONAL SIGNIFICANCE Steven P. R. Rose Brain Research Group, Open University, Walton Hall, Milton Keynes MK7 6AA, England
This paper reviews work from our laboratory on the metabolism and interrelations of isolated neuronal and neuropil fractions. The cell preparations are evaluated according t o criteria of yield, purity, and integrity. Differences in the levels of activity of six groups of enzymes, of glucose metabolism, amino-acid metabolism, transmitter metabolism, acid hydrolysis, alkaline hydrolysis, and carbonic anhydrase have been followed, and neuronal and glial marker enzymes are proposed. Lysosomes and their enzymes are concentrated in neuronal perikarya. Metabolically, although n o major differences in glucose oxidation have been found, there is considerable evidence of compart mentation of amino acids and their metabolism. At short times after injection of 3H-lysine as precursor in vivo, neuronal incorporation is high as compared with neuropil; at longer times the ratio is reversed, and we interpret this as evidence for the presence of a rapidly labeling protein fraction present in the neuronal perikarya but subsequently transported out. Neuronal protein incorporation is suppressed in the visual but not the motor cortex of dark-reared rats and is switched on following exposure to light; there is evidence that the suppressed fraction of neuronal protein synthesis includes t h e rapidly labeling component. A model for neuronal-glial metabolic interaction and its state-dependence in response t o changes in the organism’s environment and behavior is sketched out. INTRODUCTION
It is today a commonplace that neurochemistry is not divorced from the classical biological dialectic of structure and function. Whatever biochemical measure one makes in the nervous system, one must expect t o find it differentially distributed amongst t h e different structures. With t h e broad pathways, at least of primary metabolism, well mapped out, attention has turned increasingly t o investigating the role of structural compartmentation both in regulating ongoing basic metabolic processes and in providing a mechanism for coding changes in the external environment and in the behavior of the organism at the cellular and biochemical levels. We must recognize that t o report a biochemical change as occurring in “whole cortex” is t o describe the algebraic sum of events that in fact take place in many partially intercommunicating compartments. In this context we may define a “compartment” as “any small region of tissue of uniform phase of homogeneous biochemical properties bounded by a region of phase change” (for example, b y a membrane) (Rose, 1972).
19
0 1975 Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. 10011
20
Rose
At the time of the first ISN meeting in 1967, the approach to the problem of the functional biochemistry of the two principal cell types in cortex, neurons and glia, was still virtually confined to the pioneering microdissection studies of HydCn and his collaborators. At this meeting there are several sessions devoted to neuronal and glial biochemistry, with reports from laboratories throughout the world. In this paper, we will concentrate on describing our own work, in which we used the method for separation of neurons and neuropil in bulk that was first reported in 1965 (Rose, 1965).
PREPARATION, CHARACTERISTICS, AND EVALUATION O F ISOLATED CELL FRACTIONS
Any cell (or subcellular) fractionation technique must be judged against three criteria: the yield, purity, and integrity of the isolated fractions. According to our technique, the cell fractions are isolated by first disaggregating cleaned cerebral cortical tissue in a medium containing 110 rnM KC1, 10 mM potassium phosphate buffer, pH 7.4, and Ficoll, then by teasing through meshes of defined aperture size, and finally by centrifugation on a discontinuous Ficoll/sucrose gradient. The procedure we currently use is exactly that of Rose (1967) with the following modifications: (a) 11% Ficoll replaces 10%Ficoll in the initial disaggregation medium and (b) the nylon mesh is mounted on a sawn-off syringe and the tissue and medium passed backward and forward through it several times. For certain purposes, the following further modifications may also be introduced: (a) a preliminary low-speed spin to remove subcellular contaminants (Sinhaand Rose, 1972a) and (b) passage through a siliconized glass bead column to remove lipid material and capillary endothelial cells (Rose and Sinha, 1970). These modifications affect both yield and purity of the fractions. The basic procedure yields four fractions: (a) myelin, undisrupted tissue, and debris, including 60-70% of total protein; (b) glia and neuropil, 10-20% of total protein; (c) neurons, 5-l% of total protein; and (d) red cells and nuclei, 5-1 0% of total protein. The centrifugation procedure is not isopycnic; distribution depends o n ionic as well as density factors, and recovery and protein yield depend in part upon the loading of the gradient. The intention behind the development of the technique was to obtain a method for providing as rapidly as possible, and in milligram quantities, neuron- and glia-enriched cell fractions in which the cells, or rather their perikarya, are present in a relatively undamaged state. This result would make possible two broad lines of investigation: metabolic studies in vivo followed by fractionation and examination of distribution of metabolites, and studies of metabolism or enzyme activities in vitro following separation. This specification demands a playoff of factors - speed of separation against relative damage to the cells, and yield against purity. Subsequent methods have been developed that depend on enzyme tissue dissociation (for example, Norton and Poduslo, 1970) or hypotonic media and the use of PVP/BSA in gradients (for example, Sellinger et al., 197 1). These methods, whilst producing somewhat purer fractions (notably glial), also result either in metabolically more damaged preparations or
21
Metabolism of Neurons and Glia
a lower yield, making difficult the controlled comparison of cell properties in fractions recovered from the same gradient and derived from the same animal (Sinha and Rose, 1971). One disadvantage of the relative proliferation of separation techniques, whether the differences between them are major or minor, is that it makes it harder to interpret the sometimes conflicting results that the techniques generate. On the one hand, in the context of bulk separation (as opposed to microdissection techniques) the question of yield is of relatively minor importance, although where subcellular fractions are being made from the cells or where, as in behavioral experiments, regional fractionations on single brains are required, yield may once again become limiting. On the other hand, the question of purity is of extreme significance. We have attempted to specify the purity of the fractions using three criteria: (1) morphological appearance of the cells under phase-contrast light microscopy; (2) counting of neuronal and glial nuclei in fixed preparations stained with cresyl violet and by the use of enzymic markers (see below); and (3) integrity. On this basis, the cell population in the neuronal fraction is 60-70% neurons and in the glial-enriched fraction 6 0 4 6 % glia. However, electron-microscopic examination makes it clear that there are other cellular contaminants present, especially in the glial-enriched fraction, including axonal and dendritic processes and synaptosomes. It is for this reason that we have preferred to describe the glial-enriched fraction as neuropil; other authors have retained the “glial” nomenclature. The third criterion, that of integrity, is the most vexed. During disaggregation and centrifugation the cells lose a certain amount (about 10%)of soluble protein; cytoplasmic enzymes such as lactic dehydrogenase and glutamine synthetase tend t o leak from the cell. There is also a loss of low-molecular-weight constituents, such as amino acids, of up to 6076, although these are subsequently resynthesized (Rose, 1968a). Thus, interpretation of experiments describing the distribution of a property or substance between cell types always requires that recovery data be presented, though regrettably this is not always done. Such losses, and the fact that cells that appear well preserved to light microscopy seem more damaged under the electron microscope, has led to some criticism of the possibility of making any meaningful interpretation of data obtained from the fractions (e.g., Cremer et al., 1968). Whilst scepticism is desirable, the facts that the cell fractions show integrated metabolic behavior in vitro and that they preserve such properties as the ability to accumulate potassium ions (Rose and Sinha, 1969) and amino acids (Hamberger, 1971) against a concentration gradient, to respire with glucose as substrate at rates not much different from the tissue slice (Rose, 1967), and to synthesize and maintain high ATP levels (Rose and Sinha, 1969), may all be taken as evidence that biochemically the cells appear t o reseal after disaggregation and that extrapolation from in vitro properties to the in vivo situation may justifiably be made. These issues have been discussed elsewhere and will not be further referred to here (Rose, 1968b, 1969, 1972). ENZYME ACTIVITIES OF ISOLATED CELLS
Table I groups the various enzymes whose activities we have measured in the
Glutamate dehydrogenase Aspartate aminotransferase Glutamate decarboxylase Glutamine synthetase
Monoamine oxidase Acetylcholinesterase Butyrylcholinesterase
(b) Amino acid metabolism
(c) Transmitter
Alkaline DNase Alkaline phosphatase
Carbonic anhydrase
(e) Alkaline hydrolases
(0
25.9
398.8
13.0 3.2
3.4 5.8 6.8 6 .O 9.9 2.7 6.8 3.4 1.4 0.6
9 .0 6.9 39.7 62.9 20.1 15 1.3 4.3 6.6 188.9 64.9
31.1 40.2 270.1 383.1 199.3 414.8 29.2 22.3 267.0 40.0
55.4 19.3
1.8 2.1 3.6
21.9 39.2 8.0
13.5
4.2 6.0
1.0 1.2 1.8
40.2 82.8 29.1
4 .O
0.6 1.1 1.8
Neuronal/neuropil ratio
5 .O 57.5 20.0 7.9
11.1 14.9 29.6
neuropil
19.9 57.3 24.0 14.0
7.1 16.7 54.3
neurons
Enzyme activity (units/mg prot)
*All enzyme activities are in arbitrary units per mg protein except for the hydrolases. which are nM substrate released per hr per mg protein. Data abstracted (and errors omitted for clarity) from the following. where assay methods are also described: ( 1 ) Rose (1967): (2) Rose (1968a); (3) Sinha and Rose (1972a); (4) Sinha and Rose (1972b); ( 5 ) Sinha and Rose (1973).
Arab inosidase ArylSulphdtaSe Cathepsin Acid DNase p-galactosidase Glucosaminidase Glucosidase Glu cur0 niddse Acid phosphatase 0-N-acetyl-galacto saminidase
(d) Acid hydrolases
metabolism
Lactic dehydrogenase Succinic dehydrogenase Cytochronie oxidase
Enzyme
(a) Glucose metabolism
Group
TABLE I. Enzyme Activites in Neuronal and Neuropil Fractions*
1
4 4
4 4 4 4 4 4 4 4 4 5
3 3 3
2 2 2 2
1
1 1
Ref.
N N
23
Metabolism of Neurons and Glia
isolated cells. These include (a) enzymes of glucose metabolism, (b) enzymes of amino acid metabolism, (c) enzymes of putative transmitter systems, (d) acid hydrolases (lysosomal enzymes), (e) alkaline hydrolases, and (f) carbonic anhydrase. The table reveals wide differences in the distribution of the enzymes between the cells. In general, we have found that there are no great differences between fractions as regards the activity of SDH and LDH, the enzymes of glucose metabolism, and that cytochrome oxidase is somewhat higher in neurons than neuropil. We have not measured Na', K'-ATPase, but there have been reports that it is more active in glia than neurons (Cummins and HydCn, 1962; Medzihradsky et al., 1972). In the absence of detergent activation, glutamate dehydrogenase is four times as high in neurons as in neuropil, whilst asparate aminotransferase, glutamate decarboxylase, and glutamine synthetase do not preatly differ between the fractions (though the recovery of glutamine synthetase is low, suggesting a loss through leakage). Of the transmitter enzymes studied, M A 0 is 84%higher in the neuronal fraction and AChE 110%higher, which is perhaps not surprising; what is more surprising is that BChE is also higher - by 260% in the neurons. The AChE/BChE difference cannot then serve as a neuronal marker, as is sometimes assumed, and, in fact, our observations are supported by histological evidence (Pavlin, 1965). Amongst the ten acid hydrolases studied, nine are more active in neurons than neuropil and one, 0-N-acetyl galactosaminidase, is more active in neuropil. These enzymes are generally lysosomal in localization, and there is also supportive histochemical evidence for the proposition that lysosomes and their enzymes are more concentrated in neurons than in glia or neuropil (Holtzman, 1969; Hirsch, 1969). However, the very different neuronal/neuropil activity ratios of the enzymes is evidence of the microheterogeneity of lysosomal particles in the cortex, and we have proposed (Sinha and Rose 1972a, 1973) that at least four types of particle could be identified. One particle contains acid phosphatase, glucosaminidase, arabinosidase, and glucuronidase (activity ratio 1.4-3.4); a second contains aryl sulphatase, acid DNase, cathepsin, and glucosidase (activity ratio 5 2 - 6 3 ) ; a third contains one enzyme, 0-galactosidase (activity ratio 9-10), which we have therefore suggested may serve as a neuronal marker; and a fourth contains the single enzyme P-N-acetyl galactosaminidase. The fifth general group of enzymes studied, the alkaline hydrolases, is a disparate collection, and perhaps does not warrant special comment. The sixth contains one enzyme, carbonic anhydrase, included because of evidence that it may serve as a glial marker; the activity ratio does indeed make such a use of the enzyme possible. GLUCOSE METABOLISM In vitro, in a high K', Ca2'-free medium, both cell fractions metabolize glucose t o a comparable extent, about 80%of the rate of slice (Table 11) (Rose, 1967). This observation must be contrasted with the finding of Hertz, who, using microdiver techniques, noted that on a wet-weight basis, the glial respiration rate was only some 23% of neuronal (e.g., Dittmann et al., 1973; Hertz et al., 1973). Hertz has argued that the glia are high potassium cells, accumulating ions released from the neurons during, physiological activity. The discrepancy between bulk-separated and microdiver preparations may be due to differences in incubation media, but we have no adequate explana-
24
Rose
TABLE 11. Oxidative and Ion Metabolism in Neuronal and Neuropil Fractions
Property
Whole cell suspension
Neuron
Neuropil
Neuron/neuropil ~
0, uptake (glucose) 0, uptake (pyruvate) ATP K+accumulation
Ref. ~~
4 30
394
378
1.04
1
330 5.1 3.2
345 12.3 3.3
368 7.4 2.7
0.94 1.66 1.22
1 2 2
Experimental details are in references: (1) Rose (1967); (2) Rose and Sinha (1969). Units are: 0, uptake in the presence of glucose or pyruvate, nm/mg dry wt/hr; ATP, nM/mg protein; K+ accumulation, concentration ratio tissue/medium, poisoned vs unpoisoned tissue.
tion at present. After in vitro incubation, ATP levels in neurons are 66%higher than in neuropil. AM1NO-ACID METABOLISM
We have examined the conversion of 14C-labeledsubstrates into the amino acids ' glutamate, glutamine, GABA, aspartate, and alanine in vitro. On the one hand, the pool sizes of the free amino acids are all about twice as high in neurons as in neuropil, either directly after preparation of the fractions or after up to 2 hr incubation.with glucose. On the other hand, the specific activity of the amino acids is 3- t o %fold higher in neuropil than in neurons, whether glucose or pyruvate is substrate. When TABLE 111. Amino Acid Metabolism in Isolated Neuronal and Neuropil Fractions ~
~
Glutamate (a) Pool size
2 34
(b) Specific rddioactivitysubstrate, glucose
12
(c) Specific radioactivitysubstrate, pyruvate
57
(d) Specific radioactivitysubstrate, glutamate + glucose
Neuronal/neuropil ratio for Glutamine GABA Aspartate 176
Alanine
202
167
2 30
18
34
33
23
28
24
106
102
