Journal of Neuroscience Research 1:37-56 (1975)
EVIDENCE FOR DIFFERENTIAL FUNCTION OF NEURONAL AND GLIAL CELLS IN PROTEIN METABOLISM AND AMINO ACID TRANSPORT Anders Hamberger, Joseph A. Babitch, Christian Blomstrand, Hans-Arne Hansson, and ake Sellstrom Institute of Neurobiology, University o f Goteborg, S-40033 Goteborg, Sweden
Amino acid incorporation in neuronal and glial cells has been investigated in several laboratories employing bulk-separation techniques to obtain cell-enriched fractions. The relative rates of incorporation into the proteins of both cell types vary substantially with the method of isotope administration. Through the use of single-pulse perfusions with a duration of 30-40 sec the early time course of labeling has been studied. The difference between neuronal and glial cells with respect to indicating cell interactions will be discussed. The in vitro amino acid incorporation has been measured in neurons and glia after slice incubation. In material from animals developing experimental allergic encephalitis the rate of 3H-leucine incorporation more than doubles in the unfractionated brain. Glial cells increased their rate of incorporation by approximately 400% under the same conditions. The involvement of specific proteins in the cells and organelles has been studied by gel electrophoresis. The high uptake capacity of glial cells for certain amino acids with possible transmitter function has been further characterized. The release of these substances is measured in a superfusion system where beds of cells, preloaded with the labeled substance, are used. High potassium pulses stimulate release of, for example, GABA in both neuronal and glial cells. INTRODUCTION
The field of bulk separation and isolation of cellular fractions from the brain has developed in a number of laboratories more or less along the path outlined by Rose (1965, 1967). Preparations enriched in neurons and glia with somewhat differing composi. tion and purity have been produced (Satake and Abe, 1966; Azcurra et al., 1969; Blomstrand and Hamberger, 1969; Norton and Poduslo, 1970). The possibility of doing separate analyses of neurons and glia has generated much enthusiasm, but also much discussion about the nature of the fractions. The two most important factors are the purity of the fractions and the state of the cells which are obtained in the fractions. Other aspects involve the question of how representative the fractions are. For example, all
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0 1975 Alan R. Liss, Inc.,
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neuron-enriched fractions consist of perikarya which retain only the most proximal parts of their processes. In extreme cases these parts represent only a small percentage of the functioning neuron. This makes extrapolations concerning the whole neuronal compartment in the brain somewhat hazardous. Another limitation of the methods for bulk separation is that in their present state of development they require large amounts of starting material - one brain or cerebral cortex or often the pooled brains of several laboratory animals. Analyses at the cellular level of discrete functional units of the nervous system are thus difficult or impossible. The characteristic features of neuronal and glial metabolism that can be demonstrated are those common to large and in many ways heterogeneous populations. In all work with nervous tissue it is difficult to obtain absolutely pure fractions, particularly so with whole cell fractions, as the dissociation of the organ must be carried only about half-way. Such cell fractions are referred t o by most authors as enriched that is, they normally contain the desired cell types t o 80-90%, as judged by light microscopy. In addition to whole-cell contamination, subcellular particles are present. These can be determined only by electron microscopy. In many cases the amount and origin of contaminating material can be quantified better by biochemical methods. While the degree of purity of cellular fractions is critically important in all studies, the importance of cell integrity may vary according to the biological problems to be studied. ~
CELL FRACTIONS
The bulk-separation technique employed in our laboratory (Blomstrand and Hamberger, 1969, 1970) produces a neuronal and a glial fraction from a single discontinuous ficoll-sucrose gradient (Fig. 1) which is run at high speed for 2 hr. The gradient
15.7
FlCOLL GLIAL
19.1
%
FRACTION
-11-
NEURONAL F R A C T ION 20
+25
-11-
%
SUCROSE
Fig. 1. Ficoll-sucrose density gradient used for the separation of neuronal and glial fractions. The photo shows the gradient after centrifugation for 120 mm at 25,000 rpm in an SW 27 rotor (Reckman Spinco). The mixed suspension was loaded in the 24.6%ficoll.
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Neuronal and Glial Function
was loaded in the middle part with a mixed cell suspension obtained by incubation of chopped tissue, which was then further dissociated and filtered. Brains from rabbits, guinea pigs, rats, monkeys, and humans have been used. Figure 2 shows the neuronal fraction photographed with low power in the light microscope. Few processes are seen on the perikarya, but little nonneuronal material is present. As most of the perikarya are
Fig. 2. Neuronal fraction, light microscopy.
spheres, the fraction can be concentrated by low-speed centrifugation to allow electron microscopic (EM) examination of many cells in one field (Fig. 3). A high degree of purity is also seen at this magnification, but it is obvious that the plasma membrane is not continuous around the perikarya. It is hard to tell at which step this damage occurred - that is, during cell separation or EM preparation. The apparently almost intact surface structure seen in scanning EM (Fig. 4) indicates that most of the damage was caused by the dehydration and embedding processes used for transmission EM. The glial fraction often contains clumped cell aggregates, but where single cells are seen (Fig. 5 ) it is possible to recognize astrocytes (the main component) and oligodendrocytes. This fraction is more heterogeneous than the neuronal fraction; capillary endothelial cells are seen, and torn processes devoid of nuclei are also found. Surprisingly, very long processes are frequently seen in the astrocytic population. When viewed in transmission EM most plasma membranes appear to be damaged, but scanning EM indicates more well preserved surface.structures (Figs. 6 and 7). Free mitochondria or nerve endings are rarely observed. Absolute EM quantification of purity, which would be feasible for the neuronal fraction, is considerably more difficult in the case of the glial fraction. Approximate values of purity are 90% for the neuronal fraction and 80%for the glial fraction. Biochemical data to be presented support these morphological estimations.
Fig. 3. Neuronal fraction. Transmission electron microscopy. ( X 3200)
Fig. 4. Neuronal fraction. Scanning electron microscopy. ( X 2200)
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Neuronal and Glial Function
Fig. 5. Glial fraction. Light microscopy.
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Fig. 7. Glial fraction. Scanning electron microscopy. ( X 6400)
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Neuronal and Glial Function
TABLE 1. Summary of Some Characteristics of Fractions from Rabbit Brain Fractions
Neurons DNA/ protein Na+/K*/ATPase/protein Amino acid incorporation Amino acid transport Amino acid content Ion transport SlOO (pg/mg soluble protein) Ethanolamine exchange (nM/rng/30 min): Ho mogenat e Mcrosomes Glutamic acid decarboxylase ( p moles CO,/g/h)
Glia
S ynaptosomes
high low high low low low 1.2
low high low high high high 7.4
low high high -
6.1 10.7 1.5
2.3 1.4 1.7
1.8 13.5
Whole brain
-
5.4
GENERAL BIOCHEMICAL CHARACTERISTICS OF THE CELL FRACTIONS
In Table I some basic characteristics, a summary of the main topics studied, and two possible “markers” are presented. The high DNA/protein ratio of the neuronal fraction and the Na”-K+-ATPaseactivity of the glial fraction most probably reflect the fact that the relative contribution of the cell nucleus is large in the former and membrane elements more abundant in the latter. Nevertheless, it may be worth pointing out that the specific activity of ATPase is higher in glial membranes (Henn et al., 1972). Of the markers, SlOO is thought to be a glial protein (Moore et al., 1968). The glial fraction contains six times as much SlOO as the neuronal fraction (Haglid et al., manuscript in preparation), but a disadvantage of SlOO as a marker substance is its high diffusability. Thus not even the glial fraction contains as high a level of SlOO as does unfractioned brain. The small amount of SlOO present in the neuronal fraction seems at least partly to be due to neuronal S100, as judged by immunofluorescence (Haglid e t al., manuscript in preparation). The second parameter, the calcium-dependent base-exchange system for phospholipid biosynthesis, is localized within the microsomal fraction from whole brain (Porcellati et al., 197 1). The enzyme activity of the neuronal microsomal fraction is eight times higher than that of glial microsomes (Goracci et a]., 1973). As this enzyme is low in nerve endings, it seems promising as a marker for neuronal perikaryal membranes. Glutamic acid decarboxylase (GAD) is localized almost exclusively in nerve endings (Salganicoff and De Robertis, 1965) and could serve as a marker for nerve-ending contamination in the cell-enriched fractions. The enzyme activity in the neuronal and glial fractions is approximately 10% of that in nerve endings (Sellstram et al., manuscript in preparation); so a maximal contamination of 10%may be assumed. The finding that “glial” GAD behaves differently from nerve-ending GAD with respect to aminooxyacetic acid inhibition suggests that the contaminating factor could be reduced to a few percent. Although this assortment of morphological and biochemical
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criteria does not take account of all possible contaminating factors, we feel that the fractions provide a valuable tool for investigating the compartmentalization of some aspects of brain metabolism. INCORPORATION OF AMINO ACIDS
We have used this cellular system for evaluating the compartmentalization of protein synthesis by following the incorporation of labeled amino acids. The autoradiographic findings of a high labeling of neuronal perikaryal proteins by amino acids (Altman, 1963; Droz and Leblond, 1963) naturally made us eager t o confirm these data with the use of bulk preparations. When labeled amino acids are given in vivo - for example, by an intraperitoneal or intravenous injection - the neuronal fraction incorporates radioactivity into protein at about twice the rate of the glial fraction (Blomstrand and Hamberger, 196Y). However, when amino acids are given during incubation of slices prior t o cell separation - for example, in an in vitro system - the neuronal incorporation is x
3
10
10
30
75
180
min
Fig. 8. Protein bound (top) and trichloroacetic-acid-soluble (bottom) radioactivity on perfused 0 ) and contralateral ( ) sides of whole rabbit brain. Time after a 30 sec perfusion.
=
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Neuronal and Glial Function
approximately five times that of glia (Blomstrand and Hamberger, 1970). The findings are supported by the work of Tiplady and Rose (1971). Corresponding findings on developing animals suggest high rates of amino acid incorporation also in glial cells (Johnsson and Sellinger, 1971). In a recent study we reinvestigated the cellular incorporation of amino acids and applied a technique which by a physiological route of entry provides the brain with high levels of radioactivity in a short pulse without the problems of diffusion gradients resulting from local application of the precursor. After administration of the precursor unilaterally in the carotid artery as a 30 sec perfusion, brain proteins are labeled approximately 50 times higher than when a corresponding dose is given intravenously (Blomstrand et al., submitted for publication). Protein-bound and acid-soluble radioactivity in whole brain is shown in Fig. 8. The labeling of neuronal and glial proteins is shown in Table 11. The incorporated activity in the neuronal fraction is higher than in the glial fraction at all times studied. However, the ratio is not even as high TABLE 11. Protein-Bound Radioactivity in Neuronal and Glial Fractions and Whole Brain in Rabbits During the First Two Hours After Intracarotid Perfusion with H-Leucine Minutes after perfusion
5 15 30 45 60 120
Neuronal fraction
34,839 i 1,065 * (3)t 37,906 i 7,444 (4) (5) 41,834 i 767 41,691 (46,486; 36,896) 31,472 5 9,450 (3) 17,487 (13,014; 2 1,960)
Glial fraction 20,719 * 3,025 (3) 27,918 i 2,815 (4) 28,357 i 5,308 (5) 25,786 (29,970; 2 1,602) 21,064 i 4,175 (3) 14,867 (10,130; 19,604)
Whole brain
13,312 5 1,772 (3) 1 5 , 2 1 4 2,059 ~ (4) 19,630 f 3,066 (5) 17,076 (19,580; 14,572) 12,798 2 2,643 (3) 10,191 (71,081; 13,274)
*Mean ? S.E.M. ?Number o f experiments.
as 2: 1 and is very small two hours after perfusion. A similar change with time in the ratio of neuronal to glial labeling was shown by Rose (1973). Table I1 shows that the peak of protein-bound radioactivity was reached between 15 and 30 min in the glial fraction and between 30 and 45 min in the neuronal fraction. This could be due to a number of factors - for example, glial cells may be reached by the precursor earlier than neurons - or it may be due to the high proportion of nuclei in the neuronal fraction. Whole brain nuclei are labeled with a time lag in this system (unpublished results). AMINO ACIDS
The question of pool sizes in the cells of the living animal is difficult to answer, as the cells have to go through the long separation procedure which gives leakage of both small molecules and larger molecules such as protein and RNA. It is thus a bit imprecise
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slice
-
synaptosome
.- . ....
- glia
neuron
I
Fig. 9. Amino acid content of rabbit brain slices, incubated in buffered saline media for 30 min, and subfractions obtained from the slice.
to extrapolate back from data obtained on isolated fractions, since neuronal and glial cell bodies may leak differently as a result of damage. Intracellular levels of amino acids were several times lower in the neuronal fraction than in the glial fraction, which in turn had a similar concentration t o nerve endings (Fig. 9) (Sellstriim et al., unpublished data). These results refer to the amount of amino acid per unit protein, which may be misleading, as the neuronal perkarya contain relatively less cytoplasm. The first attempts to estimate transport rates of amino acid were done on the isolated neuronal and glial fractions, which were incubated with L4C-labeledamino acids (Hamberger, 1971). The distribution ratios indicated that all amino acids were accumulated by the glial fraction, while the neuronal perikarya concentrated amino acids only t o quite a low tissue-to-medium ratio. Particularly those amino acids grouped as putative transmitters were concentrated by the glial fraction.
UPTAKE AND RELEASE OF y-AMINOBUTYRATE The amino acid uptake work has thus been concentrated on y-aminobutyrate, glutamate, aspartate, and glycine. In contrast to serotonin, norepinephrine, and dopamine, which are accumulated almost exclusively by the nerve ending fraction, y-aminobutyrate (GABA) uptake by glial cells was 30-50% of that of synaptosomal preparations (Table Ill) (Henn and Hamberger, 1971). This uptake was temperature-sensitive and sodium-dependent. The results were interpreted as evidence that the glial cell is involved in limiting the
Neuronal and Glial Function
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TABLE 111. Uptake of GABA by Neuronal, Glial, and Synaptosomal Fractions as a Function of Time of Incubation*
(
Fractions cpm/mg tissue cpm/mgmediumt
Incubation time
Neuronal
Glial
0 5 10 20 40
1 5 8 12 14
3 25 44 91 106
Synaptosomal
12 88 150 224 246
*Means of three experiments. Incubation at 37" C. ?Medium: Tris-HC1, 35 mM, pH 7.4; NaCl 120 mM; KC1 5 mM; CaC1, 2 mM; MgC1, 2.5 mM; glucose 20 mM; and (2.3-3H)-GABA 0.6 fiM.
extracellular buildup of substances that might trigger synaptic transmission and that it acts by removing certain transmitters that may diffuse out of the synaptic cleft during the transmission of impulses. This role of glial cells had been suggested long ago and received its initial support from the studies of Koelle (1955). Most work on GABA uptake has centered on the nerve ending as the only compartment that gives findings similar to those with most other transmitters (Neal and Iversen, 1969; Bloom and Iversen, 1971; Iversen and Johnston, 197 1). The role of glia in glutamate and GABA accumulation in nerve-muscle preparations has been demonstrated by autoradiographic techniques (Faeder and Salpeter, 1970; Orkand and Kravitz, 1971). The particular importance of glial cells for GABA uptake has now been confirmed in tissue culture (Haber et al., 1973) and by autoradiography on spinal ganglia (Kelly et al., 1973). As extracellular GABA is reaccumulated mainly in two compartments, glia and nerve endings, we are presently attempting to characterize the transport mechanism of these Compartments. Most evidence indicates the presence of two systems. One uptake system is temperature- and sodium-dependent and represents the larger part. This system is probably based on the accumulation of GABA in the cytoplasm, possibly after an initial binding process. The other uptake system is neither energydependent nor ion-dependent but is based on binding of GABA to receptor proteins in the membrane. The present results mainly reflect the first system. As shown in Fig. 10 the uptake in all fractions is magnesium-dependent. Addition of calcium to the medium had no appreciable effect on synaptosomal or neuronal perikaryal uptake, but it definitely inhibited glial uptake. Figure 11 illustrates the activities of both glutamic acid decarboxylase (GAD) - by which GABA is formed by 1-decarboxylation of glutamate - and GABA-T, which is in the major pathway of metabolism of GABA (Balazs et al., 1970). That GAD is concen-
Hamberger et al.
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neurons
glia
synaptosornes
Fig. 10. Accumulation of 3H-GABA into neuronal, glial, and synaptosomal fractions. The fractions were incubated at 37" C for 15 min, after which the distribution of radioactivity between tissue ; standard medium minus Mg++v&# ; pellet and medium was determined. Standard medium standard medium plus 1.5 mM Cat+=.
0
-
GAD
15i
GAEA
50
whole brain
neurons
plia
synaptosomes
1
-
T
n
neurons
glia
synapto-
sornes
Fig. 11. Glutamic acid decarboxylase (GAD) and y-aminobutyric-acid-glutarnic-acid transminase ; standard (GABA-T) activity in neuronal, glial, and synaptosomal fractions. Standard medium medium plus 10- M aminooxyacetic acid (AOAA) .
0
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Neuronal and Glial Function
1;1 3
Incubation w i t h 2 . 3 - H gaba
15 min
Incubation w l t h 1-
I
3 0 niiii
neurons
14 C gaba
R
synaplosomes
Fig. 12. Distribution of radioactivity after incubation of neuronal, glial, and synaptosomal fractions with 3H or I 4 C y-aminobutyrate. Percent of total radioactivity.
trated in nerve terminals has been established in several studies (Salganicoff and De Robertis, 1965; Fonnum, 1968); neither glial cells nor neuronal perikarya have much activity. However, GABA-T is highest in the glial fraction, suggesting that the glial cell is designed more to metabolize GABA than to produce it. An expected strong inhibition of both enzymes by AOAA was seen in both neurons and nerve endings, while the “glial” enzyme activity was much less sensitive to the inhibitor. As mentioned before, glial and neuronal GAD activity may reflect contamination of these fractions by nerve endings, since there are contaminating elements of a subcellular nature in the fractions. It is thus only with extreme caution that we may talk about “glial” GAD or “neuronal” GAD, since their apparent existence may be solely a result of the deficiency of the cellseparation method. This possibility is, however, contradicted by the difference in AOAA sensitivity between “glial” and synaptosomal GAD (Haber et al., 1970a,b). The uptake data for GABA are measured as 3H or I4C distribhtion, and in view of the metabolic data we felt that it was important t o characterize the radioactivity in the cell compartments. Figure 12 shows the distribution of radioactivity after 15 and 30 min incubation. The different cells and nerve endings have similar rates of GABA breakdown, and the main labeled metabolites are glutamate and aspartate as expected (Balazs et al., 1970). One of the unanswered questions in this context is what happens to the accumu-
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Hamberger et al.
lated GABA. It is easy to understand that material reaccumulated into nerve endings serves as a transmitter supply for future impulse transmission. The glial role is not easily understood. The transmitter could be completely broken down inside the cell, or some of it could be transported back to neurons. Release of amino acids from brain slices can be produced by electrical stimulation or by a high concentration of K' in
b
Fig. 13. Superfusion chamber. The cell fractions which were preloaded with the labeled substance are centrifuged to form a layer on a filter paper which is placed in the center of the chamber (dotted area). The perfusion fluid is continuously gassed with oxygen and pumped with a peristaltic pump through the cell layer. The perfusate is finally collected in fractions. The system is submerged in 37" C water bath.
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Neuronal and Glial Function
120-
f-'e
V
":0 w
.
I I I I
.-
,. .. I \
40.
Fig. 14. Typical washout curve after 7-aminobutyrate preloading. Each point represents t h e amount of radioactivity in 1 rnin fraction (2 rnl). The thick black line represents a 3 rnin period in which the K' concentration in the perfusion solution was raised from 5 to 15 rnM.
1
slice
neurons
-
glia
synaptosomeo
Fig. 1 5 . Stimulated release of radioactivity by a 3 rnin period of 15 mM K+ in the perfusion solution. The results are presented as percent of the radioactivity in t h e cells at the start of the experiment. Standard medium with 1.5 rnM Ca++ D ;without Ca++
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Hamberger et al.
the medium. This release is selectively greater for GABA, glutamate, and aspartate in synaptosornes (Bradford, 1970; Belleroche and Bradford, 1972). As the ionic dependence for release is similar to that for acetylcholine or catecholamine, the GABA released from brain slices has been thought of only as synaptosomal. In order to investigate the compartmentation of the release process, we preloaded neuronal, glial, and synaptosoma1 fractions with radioactive GABA and centrifuged the material down as a thin layer on a 25 mm disc of filter paper. The discs were placed in superfusion chambers (Fig, 13) and the fluid superfusing the cells passed t o a fraction collector (Sellstriim and Hamberger, manuscript in preparation). A washout curve is seen in Fig. 14. The peak on the curve occurs when the K' concentration of the superfusion medium was raised from 5 to 15 mM. The amount of radioactivity released in a 3 min K' pulse is seen in Fig. 15. The most prominent release was noted for the neuronal perikaryal fraction, less for the synaptosomes, and least for t h e glial fraction. Neuronal and synaptosomal release was calcium-dependent, while the addition of calcium apparently inhibited glial release. Calcium ion is the factor which has a qualitatively different effect on neuronal perikarya and nerve endings on the one hand and on glial cells on the other. These effects concern both uptake and release of GABA, as shown schematically in Fig. 16. Nerve-ending-neuronal-perikaryal uptake and release was high at high calcium levels, while glial activity was high at low calcium levels. Such findings could have functional 0 mM Ca"
11
1,s
mM
Ca"
Fig. 16. Schematic presentation of the calcium effect on uptake and release of y-aminobutyrate. The upper central part is a nerve ending, the lower central part is a neuronal perikaryon (or nerve ending), and t h e lateral parts are glial cells.
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Neuronal and Glial Function
3
10
17
Days after E A E induction Fig. 17. 14C-leucineincorporation into neuronal and glial proteins at different stages of EAE. Guinea pig brain slices were incubated (Blomstrand and Hamberger, 1970) for 60 min. Cell separation ;glial fraction T AM. was carried out after incubation. Neuronal fraction
D
significance. If the extracellular calcium level which is necessary for optimal transmitter release is reduced as a function of the nerve impulse, glial-cell uptake of transmitter could be activated (Fig. 17). The possibility that glial cells participate in the modulation of impulse transmission in the central nervous system may have some pharmacological importance. Drugs which affect glial metabolism may prove to be active in psychiatric and neurological disorders. EXPERIMENTAL ALLERGIC ENCEPHALITIS
Few attempts have been made to apply bulk separation techniques to pathological conditions, mainly because of the limited use of human nervous tissue. We have studied an experimental 'animal model of multiple sclerosis, a demyelinating disease where I glia seem to be particularly involved. The technique for investigating the system for in vitro incorporation of amino acids into proteins - the addition of isotope during incubation of slices prior to cell separation (Blomstrand and Hamberger, 1970) - has been applied to a study of experimental allergic encephalitis (EAE) in guinea pigs. Earlier work in this field (Smith, 1969) was done with labeled glucose as protein precursor and indicated a slight stimulation of myelin protein synthesis. In the present series of experiments, brain slices from animals inoculated 3, 10, and 18 days previously with the basic myelin protein with Freund's adjuvant were incubated with 14C-leucinein a phosphatebuffered saline medium (Babitch et al., 1974). Figure 18 shows that no appreciable effect was present in the early or middle stage of the disease, while slices from animals in the late (paralytic) stage incorporated considerably more leucine into proteins than did the controls. The uptake of the free amino acid did not differ markedly between the
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Hamberger et al.
/
/
/
/
/
500
0
15
30
60
min
Fig. 18. 14C-leucineincorporation with time into whole brain slices at different stages of EAE. Each point represents the average of three animals. (0-9)3 days postinduction 10 days postinduction (e +) 18 days postinduction )-( control.
(v-.-v)
different groups. An analysis of the subcellular distribution of this increase revealed that the largest increase was in the nuclear fraction and the least change in the nerve-ending fraction. Figure 17 shows the reactions of the neuronal and glial cell fractions in the system. The glial fraction was affected even in the intermediate stage of the disease. In late EAE, the incorporated radioactivity in the glial fraction was approximately four times that in the control (Babitch et al., in press). It is also noteworthy that in these sick animals the incorporated activity in the glial fraction was higher than in the neuronal fraction; this has not previously been observed in our system. Nevertheless, it is apparent that both neuronal perikarya and glia are involved in late EAE. Other experimental data using electrophoretic analysis of the incorporated radioactivity seem to rule out invading lymphatic cells as the site of the high amino acid incorporation. We have very tentatively concluded that in addition t o reflecting phagocytic activity on behalf of glial cells, the results represent part of the repair process.
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Neuronal and Glial Function
CONCLUSIONS
Fractions enriched in neuronal perikarya and in glial cells produced by gradient centrifugation contain the desired cell types to 80-90%. Cross-contamination, admixture of capillary endothelial cells, broken cell processes, and some free subcellular organelles demand a careful analysis of the influence of these factors OD the results in any type of experiment. Plasma-membrane discontinuity, as revealed by the electron microscope and supported by biochemical findings, indicates that both small and large molecules may leak out of the cells during preparation. The transport capacities of the isolated cell fractions suggest that appropriate resealing of the cells can occur. The technique applied to the study of compartmentation of amino acid incorporation shows that neuronal perikarya have the highest rate of incorporation. We have not yet been able to calculate the intercellular pools in the living animal. Uptake studies on amino acids have shown that particularly those considered to be transmitters are very actively accumulated by glial cells. Inactivation of amino acid transmitters may be largely carried out by glial cells.
AC KNO WL E DGM ENTS
This work was supported by a grant from the Swedish Medical Research Council (grant B74-12X-164-10A)and by a grant from Ollie and Elof Ericssons Stiftelse.
R E F ER ENC ES Altman, J. (1963). Differences in the utilization of tritiated leucine by single neurons in normal and exercised rats: an autoradiographic investigation with microdensitometry. Nature (Lond.) 1991777-80. Azcurra, J. M., Lodin, Z . , and Sellinger, 0. Z. (1969). Enzymes in glial cells and in neurons: a comparison of intracellular distribution patterns. Abstr. Second Int. Meeting Int. SOC.Neurochem., Milan, Italy, p. 76. Babitch, J.A., Blomstrand, C., and Hamberger, A. (1974). Protein synthesis in experimental allergic encephalomyelitis. Brain Research 79 :477-87. Babitch, J.A., Blomstrand, C., and Hamberger, A. Amino acid incorporation into neurons and glia of guinea pigs afflicted with experimental allergic encephalitis. Brain Research, in press. Balazs, R., Machiyama, Y., Hammond, B. J., Julian, T., and Richter, D. (1970). The operation of the yaminobutyrate by-path of the tricarboxylic acid cycle in brain tissue in vitro. Biochem. J. 116 1445-67. Belleroche, J. S. De, and Bradford, H. S. (1972). Metabolism of beds of mammalian cortical synaptosomes: response to depolarizing influences. J. Neurochem. 19:585 -602. Blomstrand, C., and Hamberger, A. (1969). Protein turnover in cell-enriched fractions from rabbit brain. J . Neurochem. 16:1401-07. Blomstrand, C., and Hamberger, A. (1970). Amino acid incorporation in vitro in proteins of neuronal and glial cell enriched fraction? J. Neurochem. 17:1187-95. Blomstrand, C., Hamberger, A,, Sellstram, A,, and Steinwall, 0. (submitted). Protein bound radioactivity in neuronal and glial fractions following intracarotid 3H-leucine perfusion. Bloom, F. E., and Iversen, L. L. (1971). Localizing 3HGABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography. Nature 229:628-30.
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Bradford, H. S. (1970). Metabolic response of synaptosomcs to electrical stimulation: release of amino acids. Brain Res. 19 :2 39 -47. Droz, B., and Leblond, C. P. (1963). Axonal migration of proteins in the central nervous system and peripheral nerves shown by radioautogrdphy. J. Comp. Neurol. 121 :325-45. Faeder, I. R., and Salpeter, M. M. (1970). Glutamate uptake by a stimulated insect nerve muscle preparation. J. Cell Biol. 46:300-7. Fonnum, F. (1968). The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea pig brain. Biochem. J. 106:401-12. Goracci, G., Blomstrand, C., Arienti, G., Hamberger, A,, and Porcellati, G. (1973). Base exchange enzymic system for the synthesis of phospholipids in neuronal and neuroglial cells and their subfractions: a possible marker for neuronal membranes. J. Neurochem. 20: 1167-80. Haber, G., Kuriyama, K., and Roberts, E. (1970a). An anion stimulated L-glutamic acid decarboxylase in non-neural tissues. Biochem. Pharmacol. 19:1119-36. Haber, B., Kuriyama, K., and Roberts, E. (1970b). L-glutamic acid decarboxylasc: a new type in glial cells and human brain gliomas. Science 168598-9. Haber, B., Werrbach, K., Vance, C., and Hutchison, H. T. (1973). Uptake of y-aminobutyric acid and norepinephrine by clonal astrocytoma and neurobldstoma cell lines in culture. Abstr. Fourth Int. Meeting Int. SOC.Neurochem., Tokyo, Japan, p. 324. Hamberger, A. (1971). Amino acid uptake in neuronal and glial cell fractions from rabbit cerebral cortex. Brain Res. 31:169-78. Henn, F. A., and Hamberger, A. (1971). Glial cell function: uptake of transmitter substances. Proc. Nat. Acad. Sci. U.S. 68:2686-90. Henn, F. A., Haljamk, H., and Hamberger, A. (1972). Glial cell function: active control of extracellular K+ concentration. Brain Res. 43:437-43. Iversen, L. L., and Johnson, G. A. R. (197 1). GABA uptake in rat central nervous system: comparison of uptake in slices and homogenates and the effects of some inhibitors. J. Neurochem. 18 :1939-50. Johnsson, D. E., and Sellinger, 0. 2. (1971). Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study. J. Neurochem. 18:1445-60. Kelly, J. S., Iversen, L. L., Minchin, M., and Schon, F. (1973). Inactivation of amino acid transmitters, Abstr. Fourth Int. Meeting Int. SOC.Neurochem., Tokyo, Japan, p. 27. Koelle, G. B. (1955). The histochemical identification of acetylcholinesterase in cholinergic, adrenergic, and sensory neurons. J. Pharmacol. Exp. Ther. 114:167-84. Moore, B. W., Perez, V. J., and Gehring, M. (1968). Assay and regional distribution of a soluble protein characteristic of the nervous system. J. Neurochem. 15 :265-72. Neal, M. J., and Iversen, L. L. (1969). Subcellular distribution of endogenous and (3H)y-aminobutyric ,acid;in rat cerebral cortex. J. Neurochem. 16:1245-52. Norton, W. T., and Poduslo, S. E. (1970). Neuronal soma and whole neuroglia of rat brain: a new isolation technique. Science 167 :1i44-6. Orkand, P., and Kravitz, E. A. (1971). Localization of the sites of y-aminobutyric acid (GABA) uptake in lobster nerve muscle preparations. J. Cell Biol. 49:75-89. Porcellati, G., Arienti, G., Pirotta, M., and Giorgini, D. (1971). Base-exchange reactions for the synthesis of phospholipids in nervous tissue: the incorporation of serine and ethanolamine into the phospholipids of isolated brain microsomes. J. Neurochem. 18:1395-1417. Rose, S. P. R. (1965). Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal cells. Nature (Lond.) 206:621. Rose, S. P. R. (1967). Preparations of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells. Biochem. J. 102:33-43. Rose, S. P. R. (1973). Cellular compartmentation of brain metabolism and its functional significance. Abstr. Fourth Int. Meeting Int. SOC.Neurochem., Tokyo, Japan, p. 43. Salganicoff, L., and De Robertis, E. (1965). Subcellular distribution of the enzymes of the glutamic acid glutamine and yaminobutyric acid cycles in rat brain. J. Neurochem. 12:287-309. Satake, M., and Abe, S. (1966). Preparation and characterization of nerve cell perikaryon from rat cerebral cortex. J . Biochem. (Tokyo) 59:72-5. Smith, M. E. (1969). Myelin metabolism in vitro in experimental allergic encephalitis. J. Neurochem. 16 ~1099-1104. Tiplady, B., and Rose, S. P. R., (1971). Amino acid incorporation into protein in neuronal cell body and neuropil fractions in vitro. J. Neurochem. 18549-58.
EVIDENCE FOR DIFFERENTIAL FUNCTION OF NEURONAL AND GLIAL CELLS IN PROTEIN METABOLISM AND AMINO ACID TRANSPORT Anders Hamberger, Joseph A. Babitch, Christian Blomstrand, Hans-Arne Hansson, and ake Sellstrom Institute of Neurobiology, University o f Goteborg, S-40033 Goteborg, Sweden
Amino acid incorporation in neuronal and glial cells has been investigated in several laboratories employing bulk-separation techniques to obtain cell-enriched fractions. The relative rates of incorporation into the proteins of both cell types vary substantially with the method of isotope administration. Through the use of single-pulse perfusions with a duration of 30-40 sec the early time course of labeling has been studied. The difference between neuronal and glial cells with respect to indicating cell interactions will be discussed. The in vitro amino acid incorporation has been measured in neurons and glia after slice incubation. In material from animals developing experimental allergic encephalitis the rate of 3H-leucine incorporation more than doubles in the unfractionated brain. Glial cells increased their rate of incorporation by approximately 400% under the same conditions. The involvement of specific proteins in the cells and organelles has been studied by gel electrophoresis. The high uptake capacity of glial cells for certain amino acids with possible transmitter function has been further characterized. The release of these substances is measured in a superfusion system where beds of cells, preloaded with the labeled substance, are used. High potassium pulses stimulate release of, for example, GABA in both neuronal and glial cells. INTRODUCTION
The field of bulk separation and isolation of cellular fractions from the brain has developed in a number of laboratories more or less along the path outlined by Rose (1965, 1967). Preparations enriched in neurons and glia with somewhat differing composi. tion and purity have been produced (Satake and Abe, 1966; Azcurra et al., 1969; Blomstrand and Hamberger, 1969; Norton and Poduslo, 1970). The possibility of doing separate analyses of neurons and glia has generated much enthusiasm, but also much discussion about the nature of the fractions. The two most important factors are the purity of the fractions and the state of the cells which are obtained in the fractions. Other aspects involve the question of how representative the fractions are. For example, all
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0 1975 Alan R. Liss, Inc.,
150 Fifth Avenue, New York, N.Y. 10011
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Hamberger et al.
neuron-enriched fractions consist of perikarya which retain only the most proximal parts of their processes. In extreme cases these parts represent only a small percentage of the functioning neuron. This makes extrapolations concerning the whole neuronal compartment in the brain somewhat hazardous. Another limitation of the methods for bulk separation is that in their present state of development they require large amounts of starting material - one brain or cerebral cortex or often the pooled brains of several laboratory animals. Analyses at the cellular level of discrete functional units of the nervous system are thus difficult or impossible. The characteristic features of neuronal and glial metabolism that can be demonstrated are those common to large and in many ways heterogeneous populations. In all work with nervous tissue it is difficult to obtain absolutely pure fractions, particularly so with whole cell fractions, as the dissociation of the organ must be carried only about half-way. Such cell fractions are referred t o by most authors as enriched that is, they normally contain the desired cell types t o 80-90%, as judged by light microscopy. In addition to whole-cell contamination, subcellular particles are present. These can be determined only by electron microscopy. In many cases the amount and origin of contaminating material can be quantified better by biochemical methods. While the degree of purity of cellular fractions is critically important in all studies, the importance of cell integrity may vary according to the biological problems to be studied. ~
CELL FRACTIONS
The bulk-separation technique employed in our laboratory (Blomstrand and Hamberger, 1969, 1970) produces a neuronal and a glial fraction from a single discontinuous ficoll-sucrose gradient (Fig. 1) which is run at high speed for 2 hr. The gradient
15.7
FlCOLL GLIAL
19.1
%
FRACTION
-11-
NEURONAL F R A C T ION 20
+25
-11-
%
SUCROSE
Fig. 1. Ficoll-sucrose density gradient used for the separation of neuronal and glial fractions. The photo shows the gradient after centrifugation for 120 mm at 25,000 rpm in an SW 27 rotor (Reckman Spinco). The mixed suspension was loaded in the 24.6%ficoll.
39
Neuronal and Glial Function
was loaded in the middle part with a mixed cell suspension obtained by incubation of chopped tissue, which was then further dissociated and filtered. Brains from rabbits, guinea pigs, rats, monkeys, and humans have been used. Figure 2 shows the neuronal fraction photographed with low power in the light microscope. Few processes are seen on the perikarya, but little nonneuronal material is present. As most of the perikarya are
Fig. 2. Neuronal fraction, light microscopy.
spheres, the fraction can be concentrated by low-speed centrifugation to allow electron microscopic (EM) examination of many cells in one field (Fig. 3). A high degree of purity is also seen at this magnification, but it is obvious that the plasma membrane is not continuous around the perikarya. It is hard to tell at which step this damage occurred - that is, during cell separation or EM preparation. The apparently almost intact surface structure seen in scanning EM (Fig. 4) indicates that most of the damage was caused by the dehydration and embedding processes used for transmission EM. The glial fraction often contains clumped cell aggregates, but where single cells are seen (Fig. 5 ) it is possible to recognize astrocytes (the main component) and oligodendrocytes. This fraction is more heterogeneous than the neuronal fraction; capillary endothelial cells are seen, and torn processes devoid of nuclei are also found. Surprisingly, very long processes are frequently seen in the astrocytic population. When viewed in transmission EM most plasma membranes appear to be damaged, but scanning EM indicates more well preserved surface.structures (Figs. 6 and 7). Free mitochondria or nerve endings are rarely observed. Absolute EM quantification of purity, which would be feasible for the neuronal fraction, is considerably more difficult in the case of the glial fraction. Approximate values of purity are 90% for the neuronal fraction and 80%for the glial fraction. Biochemical data to be presented support these morphological estimations.
Fig. 3. Neuronal fraction. Transmission electron microscopy. ( X 3200)
Fig. 4. Neuronal fraction. Scanning electron microscopy. ( X 2200)
41
Neuronal and Glial Function
Fig. 5. Glial fraction. Light microscopy.
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Hamberger et al.
Fig. 7. Glial fraction. Scanning electron microscopy. ( X 6400)
43
Neuronal and Glial Function
TABLE 1. Summary of Some Characteristics of Fractions from Rabbit Brain Fractions
Neurons DNA/ protein Na+/K*/ATPase/protein Amino acid incorporation Amino acid transport Amino acid content Ion transport SlOO (pg/mg soluble protein) Ethanolamine exchange (nM/rng/30 min): Ho mogenat e Mcrosomes Glutamic acid decarboxylase ( p moles CO,/g/h)
Glia
S ynaptosomes
high low high low low low 1.2
low high low high high high 7.4
low high high -
6.1 10.7 1.5
2.3 1.4 1.7
1.8 13.5
Whole brain
-
5.4
GENERAL BIOCHEMICAL CHARACTERISTICS OF THE CELL FRACTIONS
In Table I some basic characteristics, a summary of the main topics studied, and two possible “markers” are presented. The high DNA/protein ratio of the neuronal fraction and the Na”-K+-ATPaseactivity of the glial fraction most probably reflect the fact that the relative contribution of the cell nucleus is large in the former and membrane elements more abundant in the latter. Nevertheless, it may be worth pointing out that the specific activity of ATPase is higher in glial membranes (Henn et al., 1972). Of the markers, SlOO is thought to be a glial protein (Moore et al., 1968). The glial fraction contains six times as much SlOO as the neuronal fraction (Haglid et al., manuscript in preparation), but a disadvantage of SlOO as a marker substance is its high diffusability. Thus not even the glial fraction contains as high a level of SlOO as does unfractioned brain. The small amount of SlOO present in the neuronal fraction seems at least partly to be due to neuronal S100, as judged by immunofluorescence (Haglid e t al., manuscript in preparation). The second parameter, the calcium-dependent base-exchange system for phospholipid biosynthesis, is localized within the microsomal fraction from whole brain (Porcellati et al., 197 1). The enzyme activity of the neuronal microsomal fraction is eight times higher than that of glial microsomes (Goracci et a]., 1973). As this enzyme is low in nerve endings, it seems promising as a marker for neuronal perikaryal membranes. Glutamic acid decarboxylase (GAD) is localized almost exclusively in nerve endings (Salganicoff and De Robertis, 1965) and could serve as a marker for nerve-ending contamination in the cell-enriched fractions. The enzyme activity in the neuronal and glial fractions is approximately 10% of that in nerve endings (Sellstram et al., manuscript in preparation); so a maximal contamination of 10%may be assumed. The finding that “glial” GAD behaves differently from nerve-ending GAD with respect to aminooxyacetic acid inhibition suggests that the contaminating factor could be reduced to a few percent. Although this assortment of morphological and biochemical
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Hamberger et al.
criteria does not take account of all possible contaminating factors, we feel that the fractions provide a valuable tool for investigating the compartmentalization of some aspects of brain metabolism. INCORPORATION OF AMINO ACIDS
We have used this cellular system for evaluating the compartmentalization of protein synthesis by following the incorporation of labeled amino acids. The autoradiographic findings of a high labeling of neuronal perikaryal proteins by amino acids (Altman, 1963; Droz and Leblond, 1963) naturally made us eager t o confirm these data with the use of bulk preparations. When labeled amino acids are given in vivo - for example, by an intraperitoneal or intravenous injection - the neuronal fraction incorporates radioactivity into protein at about twice the rate of the glial fraction (Blomstrand and Hamberger, 196Y). However, when amino acids are given during incubation of slices prior t o cell separation - for example, in an in vitro system - the neuronal incorporation is x
3
10
10
30
75
180
min
Fig. 8. Protein bound (top) and trichloroacetic-acid-soluble (bottom) radioactivity on perfused 0 ) and contralateral ( ) sides of whole rabbit brain. Time after a 30 sec perfusion.
=
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Neuronal and Glial Function
approximately five times that of glia (Blomstrand and Hamberger, 1970). The findings are supported by the work of Tiplady and Rose (1971). Corresponding findings on developing animals suggest high rates of amino acid incorporation also in glial cells (Johnsson and Sellinger, 1971). In a recent study we reinvestigated the cellular incorporation of amino acids and applied a technique which by a physiological route of entry provides the brain with high levels of radioactivity in a short pulse without the problems of diffusion gradients resulting from local application of the precursor. After administration of the precursor unilaterally in the carotid artery as a 30 sec perfusion, brain proteins are labeled approximately 50 times higher than when a corresponding dose is given intravenously (Blomstrand et al., submitted for publication). Protein-bound and acid-soluble radioactivity in whole brain is shown in Fig. 8. The labeling of neuronal and glial proteins is shown in Table 11. The incorporated activity in the neuronal fraction is higher than in the glial fraction at all times studied. However, the ratio is not even as high TABLE 11. Protein-Bound Radioactivity in Neuronal and Glial Fractions and Whole Brain in Rabbits During the First Two Hours After Intracarotid Perfusion with H-Leucine Minutes after perfusion
5 15 30 45 60 120
Neuronal fraction
34,839 i 1,065 * (3)t 37,906 i 7,444 (4) (5) 41,834 i 767 41,691 (46,486; 36,896) 31,472 5 9,450 (3) 17,487 (13,014; 2 1,960)
Glial fraction 20,719 * 3,025 (3) 27,918 i 2,815 (4) 28,357 i 5,308 (5) 25,786 (29,970; 2 1,602) 21,064 i 4,175 (3) 14,867 (10,130; 19,604)
Whole brain
13,312 5 1,772 (3) 1 5 , 2 1 4 2,059 ~ (4) 19,630 f 3,066 (5) 17,076 (19,580; 14,572) 12,798 2 2,643 (3) 10,191 (71,081; 13,274)
*Mean ? S.E.M. ?Number o f experiments.
as 2: 1 and is very small two hours after perfusion. A similar change with time in the ratio of neuronal to glial labeling was shown by Rose (1973). Table I1 shows that the peak of protein-bound radioactivity was reached between 15 and 30 min in the glial fraction and between 30 and 45 min in the neuronal fraction. This could be due to a number of factors - for example, glial cells may be reached by the precursor earlier than neurons - or it may be due to the high proportion of nuclei in the neuronal fraction. Whole brain nuclei are labeled with a time lag in this system (unpublished results). AMINO ACIDS
The question of pool sizes in the cells of the living animal is difficult to answer, as the cells have to go through the long separation procedure which gives leakage of both small molecules and larger molecules such as protein and RNA. It is thus a bit imprecise
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Hamberger et al.
slice
-
synaptosome
.- . ....
- glia
neuron
I
Fig. 9. Amino acid content of rabbit brain slices, incubated in buffered saline media for 30 min, and subfractions obtained from the slice.
to extrapolate back from data obtained on isolated fractions, since neuronal and glial cell bodies may leak differently as a result of damage. Intracellular levels of amino acids were several times lower in the neuronal fraction than in the glial fraction, which in turn had a similar concentration t o nerve endings (Fig. 9) (Sellstriim et al., unpublished data). These results refer to the amount of amino acid per unit protein, which may be misleading, as the neuronal perkarya contain relatively less cytoplasm. The first attempts to estimate transport rates of amino acid were done on the isolated neuronal and glial fractions, which were incubated with L4C-labeledamino acids (Hamberger, 1971). The distribution ratios indicated that all amino acids were accumulated by the glial fraction, while the neuronal perikarya concentrated amino acids only t o quite a low tissue-to-medium ratio. Particularly those amino acids grouped as putative transmitters were concentrated by the glial fraction.
UPTAKE AND RELEASE OF y-AMINOBUTYRATE The amino acid uptake work has thus been concentrated on y-aminobutyrate, glutamate, aspartate, and glycine. In contrast to serotonin, norepinephrine, and dopamine, which are accumulated almost exclusively by the nerve ending fraction, y-aminobutyrate (GABA) uptake by glial cells was 30-50% of that of synaptosomal preparations (Table Ill) (Henn and Hamberger, 1971). This uptake was temperature-sensitive and sodium-dependent. The results were interpreted as evidence that the glial cell is involved in limiting the
Neuronal and Glial Function
41
TABLE 111. Uptake of GABA by Neuronal, Glial, and Synaptosomal Fractions as a Function of Time of Incubation*
(
Fractions cpm/mg tissue cpm/mgmediumt
Incubation time
Neuronal
Glial
0 5 10 20 40
1 5 8 12 14
3 25 44 91 106
Synaptosomal
12 88 150 224 246
*Means of three experiments. Incubation at 37" C. ?Medium: Tris-HC1, 35 mM, pH 7.4; NaCl 120 mM; KC1 5 mM; CaC1, 2 mM; MgC1, 2.5 mM; glucose 20 mM; and (2.3-3H)-GABA 0.6 fiM.
extracellular buildup of substances that might trigger synaptic transmission and that it acts by removing certain transmitters that may diffuse out of the synaptic cleft during the transmission of impulses. This role of glial cells had been suggested long ago and received its initial support from the studies of Koelle (1955). Most work on GABA uptake has centered on the nerve ending as the only compartment that gives findings similar to those with most other transmitters (Neal and Iversen, 1969; Bloom and Iversen, 1971; Iversen and Johnston, 197 1). The role of glia in glutamate and GABA accumulation in nerve-muscle preparations has been demonstrated by autoradiographic techniques (Faeder and Salpeter, 1970; Orkand and Kravitz, 1971). The particular importance of glial cells for GABA uptake has now been confirmed in tissue culture (Haber et al., 1973) and by autoradiography on spinal ganglia (Kelly et al., 1973). As extracellular GABA is reaccumulated mainly in two compartments, glia and nerve endings, we are presently attempting to characterize the transport mechanism of these Compartments. Most evidence indicates the presence of two systems. One uptake system is temperature- and sodium-dependent and represents the larger part. This system is probably based on the accumulation of GABA in the cytoplasm, possibly after an initial binding process. The other uptake system is neither energydependent nor ion-dependent but is based on binding of GABA to receptor proteins in the membrane. The present results mainly reflect the first system. As shown in Fig. 10 the uptake in all fractions is magnesium-dependent. Addition of calcium to the medium had no appreciable effect on synaptosomal or neuronal perikaryal uptake, but it definitely inhibited glial uptake. Figure 11 illustrates the activities of both glutamic acid decarboxylase (GAD) - by which GABA is formed by 1-decarboxylation of glutamate - and GABA-T, which is in the major pathway of metabolism of GABA (Balazs et al., 1970). That GAD is concen-
Hamberger et al.
48
neurons
glia
synaptosornes
Fig. 10. Accumulation of 3H-GABA into neuronal, glial, and synaptosomal fractions. The fractions were incubated at 37" C for 15 min, after which the distribution of radioactivity between tissue ; standard medium minus Mg++v&# ; pellet and medium was determined. Standard medium standard medium plus 1.5 mM Cat+=.
0
-
GAD
15i
GAEA
50
whole brain
neurons
plia
synaptosomes
1
-
T
n
neurons
glia
synapto-
sornes
Fig. 11. Glutamic acid decarboxylase (GAD) and y-aminobutyric-acid-glutarnic-acid transminase ; standard (GABA-T) activity in neuronal, glial, and synaptosomal fractions. Standard medium medium plus 10- M aminooxyacetic acid (AOAA) .
0
49
Neuronal and Glial Function
1;1 3
Incubation w i t h 2 . 3 - H gaba
15 min
Incubation w l t h 1-
I
3 0 niiii
neurons
14 C gaba
R
synaplosomes
Fig. 12. Distribution of radioactivity after incubation of neuronal, glial, and synaptosomal fractions with 3H or I 4 C y-aminobutyrate. Percent of total radioactivity.
trated in nerve terminals has been established in several studies (Salganicoff and De Robertis, 1965; Fonnum, 1968); neither glial cells nor neuronal perikarya have much activity. However, GABA-T is highest in the glial fraction, suggesting that the glial cell is designed more to metabolize GABA than to produce it. An expected strong inhibition of both enzymes by AOAA was seen in both neurons and nerve endings, while the “glial” enzyme activity was much less sensitive to the inhibitor. As mentioned before, glial and neuronal GAD activity may reflect contamination of these fractions by nerve endings, since there are contaminating elements of a subcellular nature in the fractions. It is thus only with extreme caution that we may talk about “glial” GAD or “neuronal” GAD, since their apparent existence may be solely a result of the deficiency of the cellseparation method. This possibility is, however, contradicted by the difference in AOAA sensitivity between “glial” and synaptosomal GAD (Haber et al., 1970a,b). The uptake data for GABA are measured as 3H or I4C distribhtion, and in view of the metabolic data we felt that it was important t o characterize the radioactivity in the cell compartments. Figure 12 shows the distribution of radioactivity after 15 and 30 min incubation. The different cells and nerve endings have similar rates of GABA breakdown, and the main labeled metabolites are glutamate and aspartate as expected (Balazs et al., 1970). One of the unanswered questions in this context is what happens to the accumu-
50
Hamberger et al.
lated GABA. It is easy to understand that material reaccumulated into nerve endings serves as a transmitter supply for future impulse transmission. The glial role is not easily understood. The transmitter could be completely broken down inside the cell, or some of it could be transported back to neurons. Release of amino acids from brain slices can be produced by electrical stimulation or by a high concentration of K' in
b
Fig. 13. Superfusion chamber. The cell fractions which were preloaded with the labeled substance are centrifuged to form a layer on a filter paper which is placed in the center of the chamber (dotted area). The perfusion fluid is continuously gassed with oxygen and pumped with a peristaltic pump through the cell layer. The perfusate is finally collected in fractions. The system is submerged in 37" C water bath.
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Neuronal and Glial Function
120-
f-'e
V
":0 w
.
I I I I
.-
,. .. I \
40.
Fig. 14. Typical washout curve after 7-aminobutyrate preloading. Each point represents t h e amount of radioactivity in 1 rnin fraction (2 rnl). The thick black line represents a 3 rnin period in which the K' concentration in the perfusion solution was raised from 5 to 15 rnM.
1
slice
neurons
-
glia
synaptosomeo
Fig. 1 5 . Stimulated release of radioactivity by a 3 rnin period of 15 mM K+ in the perfusion solution. The results are presented as percent of the radioactivity in t h e cells at the start of the experiment. Standard medium with 1.5 rnM Ca++ D ;without Ca++
52
Hamberger et al.
the medium. This release is selectively greater for GABA, glutamate, and aspartate in synaptosornes (Bradford, 1970; Belleroche and Bradford, 1972). As the ionic dependence for release is similar to that for acetylcholine or catecholamine, the GABA released from brain slices has been thought of only as synaptosomal. In order to investigate the compartmentation of the release process, we preloaded neuronal, glial, and synaptosoma1 fractions with radioactive GABA and centrifuged the material down as a thin layer on a 25 mm disc of filter paper. The discs were placed in superfusion chambers (Fig, 13) and the fluid superfusing the cells passed t o a fraction collector (Sellstriim and Hamberger, manuscript in preparation). A washout curve is seen in Fig. 14. The peak on the curve occurs when the K' concentration of the superfusion medium was raised from 5 to 15 mM. The amount of radioactivity released in a 3 min K' pulse is seen in Fig. 15. The most prominent release was noted for the neuronal perikaryal fraction, less for the synaptosomes, and least for t h e glial fraction. Neuronal and synaptosomal release was calcium-dependent, while the addition of calcium apparently inhibited glial release. Calcium ion is the factor which has a qualitatively different effect on neuronal perikarya and nerve endings on the one hand and on glial cells on the other. These effects concern both uptake and release of GABA, as shown schematically in Fig. 16. Nerve-ending-neuronal-perikaryal uptake and release was high at high calcium levels, while glial activity was high at low calcium levels. Such findings could have functional 0 mM Ca"
11
1,s
mM
Ca"
Fig. 16. Schematic presentation of the calcium effect on uptake and release of y-aminobutyrate. The upper central part is a nerve ending, the lower central part is a neuronal perikaryon (or nerve ending), and t h e lateral parts are glial cells.
53
Neuronal and Glial Function
3
10
17
Days after E A E induction Fig. 17. 14C-leucineincorporation into neuronal and glial proteins at different stages of EAE. Guinea pig brain slices were incubated (Blomstrand and Hamberger, 1970) for 60 min. Cell separation ;glial fraction T AM. was carried out after incubation. Neuronal fraction
D
significance. If the extracellular calcium level which is necessary for optimal transmitter release is reduced as a function of the nerve impulse, glial-cell uptake of transmitter could be activated (Fig. 17). The possibility that glial cells participate in the modulation of impulse transmission in the central nervous system may have some pharmacological importance. Drugs which affect glial metabolism may prove to be active in psychiatric and neurological disorders. EXPERIMENTAL ALLERGIC ENCEPHALITIS
Few attempts have been made to apply bulk separation techniques to pathological conditions, mainly because of the limited use of human nervous tissue. We have studied an experimental 'animal model of multiple sclerosis, a demyelinating disease where I glia seem to be particularly involved. The technique for investigating the system for in vitro incorporation of amino acids into proteins - the addition of isotope during incubation of slices prior to cell separation (Blomstrand and Hamberger, 1970) - has been applied to a study of experimental allergic encephalitis (EAE) in guinea pigs. Earlier work in this field (Smith, 1969) was done with labeled glucose as protein precursor and indicated a slight stimulation of myelin protein synthesis. In the present series of experiments, brain slices from animals inoculated 3, 10, and 18 days previously with the basic myelin protein with Freund's adjuvant were incubated with 14C-leucinein a phosphatebuffered saline medium (Babitch et al., 1974). Figure 18 shows that no appreciable effect was present in the early or middle stage of the disease, while slices from animals in the late (paralytic) stage incorporated considerably more leucine into proteins than did the controls. The uptake of the free amino acid did not differ markedly between the
54
Hamberger et al.
/
/
/
/
/
500
0
15
30
60
min
Fig. 18. 14C-leucineincorporation with time into whole brain slices at different stages of EAE. Each point represents the average of three animals. (0-9)3 days postinduction 10 days postinduction (e +) 18 days postinduction )-( control.
(v-.-v)
different groups. An analysis of the subcellular distribution of this increase revealed that the largest increase was in the nuclear fraction and the least change in the nerve-ending fraction. Figure 17 shows the reactions of the neuronal and glial cell fractions in the system. The glial fraction was affected even in the intermediate stage of the disease. In late EAE, the incorporated radioactivity in the glial fraction was approximately four times that in the control (Babitch et al., in press). It is also noteworthy that in these sick animals the incorporated activity in the glial fraction was higher than in the neuronal fraction; this has not previously been observed in our system. Nevertheless, it is apparent that both neuronal perikarya and glia are involved in late EAE. Other experimental data using electrophoretic analysis of the incorporated radioactivity seem to rule out invading lymphatic cells as the site of the high amino acid incorporation. We have very tentatively concluded that in addition t o reflecting phagocytic activity on behalf of glial cells, the results represent part of the repair process.
55
Neuronal and Glial Function
CONCLUSIONS
Fractions enriched in neuronal perikarya and in glial cells produced by gradient centrifugation contain the desired cell types to 80-90%. Cross-contamination, admixture of capillary endothelial cells, broken cell processes, and some free subcellular organelles demand a careful analysis of the influence of these factors OD the results in any type of experiment. Plasma-membrane discontinuity, as revealed by the electron microscope and supported by biochemical findings, indicates that both small and large molecules may leak out of the cells during preparation. The transport capacities of the isolated cell fractions suggest that appropriate resealing of the cells can occur. The technique applied to the study of compartmentation of amino acid incorporation shows that neuronal perikarya have the highest rate of incorporation. We have not yet been able to calculate the intercellular pools in the living animal. Uptake studies on amino acids have shown that particularly those considered to be transmitters are very actively accumulated by glial cells. Inactivation of amino acid transmitters may be largely carried out by glial cells.
AC KNO WL E DGM ENTS
This work was supported by a grant from the Swedish Medical Research Council (grant B74-12X-164-10A)and by a grant from Ollie and Elof Ericssons Stiftelse.
R E F ER ENC ES Altman, J. (1963). Differences in the utilization of tritiated leucine by single neurons in normal and exercised rats: an autoradiographic investigation with microdensitometry. Nature (Lond.) 1991777-80. Azcurra, J. M., Lodin, Z . , and Sellinger, 0. Z. (1969). Enzymes in glial cells and in neurons: a comparison of intracellular distribution patterns. Abstr. Second Int. Meeting Int. SOC.Neurochem., Milan, Italy, p. 76. Babitch, J.A., Blomstrand, C., and Hamberger, A. (1974). Protein synthesis in experimental allergic encephalomyelitis. Brain Research 79 :477-87. Babitch, J.A., Blomstrand, C., and Hamberger, A. Amino acid incorporation into neurons and glia of guinea pigs afflicted with experimental allergic encephalitis. Brain Research, in press. Balazs, R., Machiyama, Y., Hammond, B. J., Julian, T., and Richter, D. (1970). The operation of the yaminobutyrate by-path of the tricarboxylic acid cycle in brain tissue in vitro. Biochem. J. 116 1445-67. Belleroche, J. S. De, and Bradford, H. S. (1972). Metabolism of beds of mammalian cortical synaptosomes: response to depolarizing influences. J. Neurochem. 19:585 -602. Blomstrand, C., and Hamberger, A. (1969). Protein turnover in cell-enriched fractions from rabbit brain. J . Neurochem. 16:1401-07. Blomstrand, C., and Hamberger, A. (1970). Amino acid incorporation in vitro in proteins of neuronal and glial cell enriched fraction? J. Neurochem. 17:1187-95. Blomstrand, C., Hamberger, A,, Sellstram, A,, and Steinwall, 0. (submitted). Protein bound radioactivity in neuronal and glial fractions following intracarotid 3H-leucine perfusion. Bloom, F. E., and Iversen, L. L. (1971). Localizing 3HGABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography. Nature 229:628-30.
56
Hamberger et al.
Bradford, H. S. (1970). Metabolic response of synaptosomcs to electrical stimulation: release of amino acids. Brain Res. 19 :2 39 -47. Droz, B., and Leblond, C. P. (1963). Axonal migration of proteins in the central nervous system and peripheral nerves shown by radioautogrdphy. J. Comp. Neurol. 121 :325-45. Faeder, I. R., and Salpeter, M. M. (1970). Glutamate uptake by a stimulated insect nerve muscle preparation. J. Cell Biol. 46:300-7. Fonnum, F. (1968). The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea pig brain. Biochem. J. 106:401-12. Goracci, G., Blomstrand, C., Arienti, G., Hamberger, A,, and Porcellati, G. (1973). Base exchange enzymic system for the synthesis of phospholipids in neuronal and neuroglial cells and their subfractions: a possible marker for neuronal membranes. J. Neurochem. 20: 1167-80. Haber, G., Kuriyama, K., and Roberts, E. (1970a). An anion stimulated L-glutamic acid decarboxylase in non-neural tissues. Biochem. Pharmacol. 19:1119-36. Haber, B., Kuriyama, K., and Roberts, E. (1970b). L-glutamic acid decarboxylasc: a new type in glial cells and human brain gliomas. Science 168598-9. Haber, B., Werrbach, K., Vance, C., and Hutchison, H. T. (1973). Uptake of y-aminobutyric acid and norepinephrine by clonal astrocytoma and neurobldstoma cell lines in culture. Abstr. Fourth Int. Meeting Int. SOC.Neurochem., Tokyo, Japan, p. 324. Hamberger, A. (1971). Amino acid uptake in neuronal and glial cell fractions from rabbit cerebral cortex. Brain Res. 31:169-78. Henn, F. A., and Hamberger, A. (1971). Glial cell function: uptake of transmitter substances. Proc. Nat. Acad. Sci. U.S. 68:2686-90. Henn, F. A., Haljamk, H., and Hamberger, A. (1972). Glial cell function: active control of extracellular K+ concentration. Brain Res. 43:437-43. Iversen, L. L., and Johnson, G. A. R. (197 1). GABA uptake in rat central nervous system: comparison of uptake in slices and homogenates and the effects of some inhibitors. J. Neurochem. 18 :1939-50. Johnsson, D. E., and Sellinger, 0. 2. (1971). Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study. J. Neurochem. 18:1445-60. Kelly, J. S., Iversen, L. L., Minchin, M., and Schon, F. (1973). Inactivation of amino acid transmitters, Abstr. Fourth Int. Meeting Int. SOC.Neurochem., Tokyo, Japan, p. 27. Koelle, G. B. (1955). The histochemical identification of acetylcholinesterase in cholinergic, adrenergic, and sensory neurons. J. Pharmacol. Exp. Ther. 114:167-84. Moore, B. W., Perez, V. J., and Gehring, M. (1968). Assay and regional distribution of a soluble protein characteristic of the nervous system. J. Neurochem. 15 :265-72. Neal, M. J., and Iversen, L. L. (1969). Subcellular distribution of endogenous and (3H)y-aminobutyric ,acid;in rat cerebral cortex. J. Neurochem. 16:1245-52. Norton, W. T., and Poduslo, S. E. (1970). Neuronal soma and whole neuroglia of rat brain: a new isolation technique. Science 167 :1i44-6. Orkand, P., and Kravitz, E. A. (1971). Localization of the sites of y-aminobutyric acid (GABA) uptake in lobster nerve muscle preparations. J. Cell Biol. 49:75-89. Porcellati, G., Arienti, G., Pirotta, M., and Giorgini, D. (1971). Base-exchange reactions for the synthesis of phospholipids in nervous tissue: the incorporation of serine and ethanolamine into the phospholipids of isolated brain microsomes. J. Neurochem. 18:1395-1417. Rose, S. P. R. (1965). Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal cells. Nature (Lond.) 206:621. Rose, S. P. R. (1967). Preparations of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells. Biochem. J. 102:33-43. Rose, S. P. R. (1973). Cellular compartmentation of brain metabolism and its functional significance. Abstr. Fourth Int. Meeting Int. SOC.Neurochem., Tokyo, Japan, p. 43. Salganicoff, L., and De Robertis, E. (1965). Subcellular distribution of the enzymes of the glutamic acid glutamine and yaminobutyric acid cycles in rat brain. J. Neurochem. 12:287-309. Satake, M., and Abe, S. (1966). Preparation and characterization of nerve cell perikaryon from rat cerebral cortex. J . Biochem. (Tokyo) 59:72-5. Smith, M. E. (1969). Myelin metabolism in vitro in experimental allergic encephalitis. J. Neurochem. 16 ~1099-1104. Tiplady, B., and Rose, S. P. R., (1971). Amino acid incorporation into protein in neuronal cell body and neuropil fractions in vitro. J. Neurochem. 18549-58.
