Functional Biochemistry of Neurons and Glial Cells

Functional Biochemistry of Neurons and Glial Cells S.P.R. ROSE Brain Research Group, Open University, Walton Hall, Milton Keynes (Great Britain)

INTRODUCTION The title of the present paper is so broad that it could contain virtually every aspect of our biochemical knowledge of brain activity. What I will discuss, however, will be much more restricted, as biochemistry’s ignorance concerning the metabolic and structural - - let alone functional -- interrelationships between neurons and their surrounding cellular milieu remains profound. I do not even propose to attempt to review systematically what knowledge we have. I have tried (as have others) t o do this elsewhere (e.g., Johnston and Roots, 1970; Rose, 1972, 1975; Hamberger and Sellstrom, 1975). Instead it seems t o me more fitting t o discuss here some particular problems and methods which our laboratory is developing. Our aim is t o describe the biochemical dynamics of neuronal/glial interactions in the adult brain, and to relate these both to those genetic and developmental pathways through which they arise and by which they are determined, and also to the capacity of the brain as a system t o make transient and longer-term plastic responses to changed environmental circumstances. Thus throughout our work we are concerned not so much with static structures as with flux and process. Our approach over the last decade - now adopted in a considerable number of laboratories -- has been that of the separation and preparation, on a relatively large scale, of neuronal and glial cell fractions, from developing and adult brain regions, in conditions which enable their metabolism, in vivo or in vitro, t o be followed. The basic method has been derived, with only minor modifications, from that first published 1 0 years ago (Rose, 1965, 1967) and will not be described in detail. Essentially, brain tissue is mechanically disaggregated into an isotonic, buffered Ficoll-containing medium and subjected to density gradient centrifugation. Four fractions are recovered, of which two principally concern us here; a glial cell-enriched fraction, containing some 10-2076 of the starting tissue protein, which we call B, or neuropil, and a neuronal cell body-enriched fraction, C, containing some 5-10% of the starting protein. These and the other fractions can be collected and further subfractionated - for example into subcellular fractions, or into individual protein components by gel or chromatographic methods.

68

Enzymes or structural substances present in the cell fractions can be compared, and cellular metabolism studied in vitro, or separation may follow in vivo administration of isotopes. Such techniques have enabled us t o report on, for instance the neuronal concentration of lysosomal enzymes (Sinha and Rose, 1972), and the compartmentation of glucose and amino acid metabolism (Rose, 1970). I do not propose t o review this work in detail, but merely t o make here the point that interpretation of data derived from cell fractions in terms of properties of the in vivo system demands circumspection. First, it must be remembered that in the intact tissue the cell body fractions whose metabolism is being studied represent only a few per cent of the total tissue volume, the rest being composed of processes, synaptic junctions, capillaries, endothelial cells - and extracellular space (Blinkov and Glezer, 1968; Rose, 1972). Second, cell fractions may give misleading results because of the presence of contaminants and of cell damage, sometimes adversely affecting one cell type differentially. The precautions which enable these problems t o be, if not circumvented, then at least minimized, have been reviewed elsewhere (Rose, 1972, 1975); the crucial points are that comparisons should always be between cell fractions derived from the same gradient; and that recovery studies and light microscopic examination of the fractions and/or marker enzyme assay, for instance of /I-galactosidase for neurons and carbonic anhydrase for glia, should be routinely carried out. It is regrettable how often one still reads papers reporting results for which these precautions have been omitted. With this background, I would like t o describe briefly 4 recent developments from our laboratory, the first two still primarily a t the methodological stage and the others revealing an important aspect of metabolic interaction between neurons and glia and its sensitivity to changes in the environment of the organism. ISOLATION O F CELL FRACTIONS FROM THE DEVELOPING BRAIN We have known for several years that our cell isolation method works well only with relatively adult brain tissue: in rats, above about 3 weeks of age. Below this age, little or n o glial material can be obtained. This is partly because in the young rat there is, of course, very much less neuropil present, and partly because the cells appear to change their properties on the gradient with age, perhaps as a function of their changing lipid content. We have, therefore (A Sinha, L. Sinha, Spears and Rose, unpublished; Sinha e t aL, 1975), developed a modified gradient system from which neuronal and neuropil fractions can be obtained from forebrain (cortex) of rats from just neonatal t o 21 days and older. In this procedure, the tissue is disaggregated by chopping in a buffered medium containing 8% Ficoll and polyvinylpyrrolidone, passing through successively finer nylon meshes, and finally filtering through a 40 (um stainless steel mesh. The gradient system is still a two-stage one, as for the adult system, but its composition is changed, the bottom layer consisting of 1.7 M sucrose, above which is 30% Ficoll. Centrifugation is for 30 min a t 13,000 x g. This procedure yields a neuropil fraction at the 8%-30% Ficoll interface in which

69

Fig. 1. Unfixed neuronal perikarya and neuroblasts from 1-day-old rat forebrain. Phase contrast, x 1500.

TABLE I PROTEIN, DNA AND RNA RATIOS IN NEURON AND GLIAL ENRICHED FRACTIONS FROM CEREBRAL CORTEX IN DIFFERENT AGED RATS Each gradient was derived from 3-6 rat brains of the stated ages (day of birth is day 0)as starting material, and each ratio is the mean of the 1 4 determinations not differing by more than f 10% S.E.M. (F ro m Sinha et al., 1975.) The elevation in RNA/DNA ratio in both cell types at day 1 0 is significant (P < 0.01).

Neurons ' RNA/DNA Protein/DNA

2.4 2.6

2.8 2.6

6.9 2.7

7.1 3.2

9.4 3.3

Neuropil RNA/DNA Protein/DNA

11.3 2.1

8.2 3.5

20.6 4.4

21.7 3.2

13.1 4.4

70

Fig. 2. Neuronal cell body observed in initial tissue suspension after disruption of 1-day-old rat forebrain. The suspension was fixed with 1.5% aqueous gluturaldehyde for 30 min, centrifuged at 10,000 revs/min for 5 min and postfixed in osmium tetroxide for 1 hr. Pellets were embedded in durcurpan, sectioned on a Reichert OM U 3 ultramicrotome and strained with uranyl acetate and lead citrate. x 15.000.

78% of the identifiable cell bodies are glial, and a neuronal fraction at the 30% Ficoll-sucrose interface in which 80% of the identifiable cells are neuronal. The criteria for this identification have been discussed elsewhere (Sinha and Rose, 1971). Fig. 1 shows a field of unfixed, isolated neurons from 1-day-old rat forebrain viewed under phase contrast; Fig. 2 is an electron micrograph of a single immature neuron showing well preserved morphology, including an external cell membrane and organized cytoplasmic structure. For comparison purposes a similar cell, as it appears in a sectioned tissue block, is presented in Fig. 3. The electron microscopic appearance of these cells is better than we have yet been able to achieve with material from adult cortex, and we have been able t o use the neuronal and neuropil fractions to follow the ontogeny of RNA and protein metabolism, both in vivo and in vitro. Some data on protein, DNA and RNA levels during development are presented in Table I.

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Fig. 3. A tissue block from l-day-old rat forebrain was sliced into 1 mm pieces, fixed, embedded and stained as in the caption t o Fig. 2. x 10,000.

ISOLATION OF CEREBELLAR CELL TYPES The unique organization of the cerebellum, with its distinctive neuronal types and well-defined wiring diagram, has over the past few years begun t o make it a favored preparation for both physiological and biochemical study (e.g., Eccles et al., 1967). What is more, the existence of a series of neurologically mutant mouse strains in which there seems to be a very specific defect in cellular ontogeny in the cerebellum, associated with characteristic behavioral abnormalities (the weaver, Staggerer, Reeler etc. strains), also suggests that the availability of homogeneous preparations of particular cell types, derived from normal and mutant cerebellar tissue at different stages during development, could represent a powerful contribution to the elucidation of key features of cerebellar specificity. Making use of a novel technique in which cell separation is based largely on size, rather than on mass or density:

72 the unit gravity sedimentation method, Cohen, DuLton and Currie, working in collaboration with Balazs at the M.R.C. laboratories at Carshalton, Great Britain, have developed a method for the preparation, in adequate amounts for biochemical analysis, of isolated cell fractions. This includes a cerebellar h r k i n j e cell fraction either from normal rat or mouse, or from mutant mouse. The procedure involves chopping the cerebellum into 400 pm slices and dispersing the tissue blocks in a Krebs-Ringer-bicarbonate buffer containing bovine serum albumin and glucose in the presence of low (0.025% w/v) trypsin concentrations at 37°C for 1 5 min. At the end of this period a trypsin inhibitor is added and the tissue further dissociated by incubation in a Ca2+- and Mg2+-free medium in the presence of EDTA and of DNAse t o prevent clumping. The dissociated perikarya are pelleted at about 50 x g for 5 min through a bovine serum albumin cushion, after which they can be further sedimented through the unit gravity chamber for size separation (Cohen et al., 1973, 1974; Dutton e t al., 1975a, b). The cells have been shown t o be viable in vitro, and aspects of their protein and glycoprotein metabolism, in vivo and in vitro, have begun t o be studied. PROTEIN SYNTHESIS IN NEURONS AND NEUROPIL

I turn now t o the main biochemical theme I wish to discuss, that of protein synthesis in neurons and glia. It has been long suspected, on the basis both of histochemical observation of the rich ribosomal content of cortical neurons (e.g., Palay and Chan-Palay, 1972) and of autoradiographic results (e.g., Droz and Koenig, 1970), that the predominant portion of the rapid protein synthesis known t o occur even in the adult brain is neuronally located, and for the last few years work in both our laboratory and that of Hamberger in Goteborg has been directed towards a study of protein metabolism in isolated neurons and neuropil. Polyacrylamide gel fractionations of even crude neuronal and neuropil soluble and insoluble protein fractions reveal, scarcely surprisingly, the presence of characteristically different protein bands in the two cell types (e.g., Packman e t al., 1971; Sinha,, Rose and Sinha, unpublished), In vitro labeled amino acids are incorporated into neuronal protein a t rates of between 2 and 6 times higher than into neuropil, depending on the precursor and incubation conditions (Blomstrand and Hamberger, 1969; Tiplady and Rose, 1971). Similarly, if a 30 min or 1hr pulse of intraperitoneally injected [ HI lysine is given in vivo and the cell fractions subsequently separated, the specific radioactivity of neuronal proteins is up to 1.8 times that of the neuropil proteins (Rose and Sinha, 1974a). However, when we followed up these observations by extending the time course of radioactive labeling in vivo from 1hr t o 4 hr or more, the picture changed. Whereas at 1hr neuronal protein specific radioactivity was higher than neuropil, by 4 hr it had fallen substantially, whilst neuropil incorporation continued t o rise. The net result was that at 4 hr neuronal specific radioactivity was some 40% of that of neuropil, and, furthermore, extending the in vivo incorporation period as far as 1 9 2 hr was without further effect on the ratio of neuronal t o neuropil incorporation, although in absolute terms both fell

73 slightly over the period. This phenomenon was not specific t o the precursor used, as it could also be found with [ 14C]phenylalanine. These results suggested t o us that there is present in the neuronal fraction a protein component which, incorporating label rapidly at early times after injection of precursor, subsequently disappears from the perikaryon. A simple calculation shows that this cannot merely represent turnover since, if we assume that incorporation of labeled lysine proceeds 1.5 times faster in neurons than in glia, and that the two cell types comprise similar proportions of total tissue protein, then a 25% decline in neuronal specific radioactivity between 1hr and 4 hr after administration of label should produce a 17% decline in whole cortex specific radioactivity. However, this does not occur; instead there is a 24% increase. It seems likely that the decline therefore represents the transport of labeled material from the cell body to a compartment which is separated from the perikaryon during the cell fractionation procedure. It is of interest to go back at this point to the autoradiographic evidence of Droz and Koenig (1970). They observed a similar phenomenon with [3H]leucine as precursor: intense labeling of the perikaryon at short times after injection of precursor, followed by a relative decline, compatible with, in their words: “a transfer of protein from the nerve cell bodies into regions made up with nerve cell processes”. Further, the data of Blomstrand and Hamberger (1969) and of Babitch et al. (1975) support this interpretation. Two general questions follow: what more can one say about the nature of the protein fraction which interests us, identified, as it is, purely on the basis of kinetic analysis; and t o where, in the neuropil, does the label migrate? Concerning the first question, we have two relevant pieces of information. By homogenizing the neuronal and neuropil fractions after isolation and separating into soluble and particulate components, we have concluded that the rapidly labeling and transported component is particulate rather than soluble. We then substituted [ HI fucose, a glycoprotein precursor, for [ HI lysine in the intraperitoneal injection. Table I1 shows that the incorporation of labeled fucose into neuronal and neuropil fractions follows a similar pattern to that for labeled lysine; a neuronal/neuropil incorporation ratio of 1.37 after 1hr of incorporation had fallen by 4 hr to 0.77. Thus the time course of fucose labeling suggests that the rapidly labeling and transported neuronal protein fraction includes glycoprotein. Concerning the fate of the migrating label, the fact that neuropil specific radioactivity continues to rise over the 4 hr period suggests that we may be observing a migration of labeled protein from the neuronal perikarya to the neuropil. The neuropil fraction contains glial cells and also dendritic and axonal processes and synaptosomes. We have made a variety of experiments which together enable us to suggest which of these compartments might be the recipient of the neuronal protein. To rule out the synaptosomes we performed a standard subcellular fractionation using 1hr and 4 hr in vivo lysine-labeled cerebral cortex homogenates. As expected from the earlier work of Barondes (1968) there was no net accumulation of radioactivity in the synaptosomes over the 4 hr period. Thus this compartment can be eliminated as recipient. We then attempted t o perform a “chase” experiment, pulse labeling the tissue in vivo for 1hr with [ HI lysine, then taking cortex slices and incubating them in

74 vitro with [ 14C]lysine for a further 1 hr. In this type of experiment, over the hour of in vitro incubation new 14C-labeled protein is being synthesized whilst previously labeled [ HI protein is turning over, undergoing metabolism or transport. At the end of the in vitro incubation, slices are fractionated into neuronal and neuropil fractions in the usual way and the 3H/'4C ratio in the fractions determined. The results showed that during the in vitro incubation period considerable incorporation of 14C occurred in all fractions, but that there was a disproportionate loss of already synthesized highly H-labeled protein from the slice to the medium. High specific radioactivity protein was thus leaking out of the neurons during incubation, although it was not being reaccumulated in the neuropil fraction under these in vitro conditions. TABLE I1 [ 3H]FUCOSE INCORPORATION INTO NEURONAL AND

NEUROPIL FRACTIONS

[ H I Fucose was injected intraperitoneally into rats, the animals killed 1 or 4 h r later, cortex fractionated into neurons and neuropil, and t h e cell fractions precipitated with TCA, washed and counted. Results are means f S.E.M. of 1 2 determinations. The decline in neuronal specific radioactivity, and the elevation in neuropil, is significant ( P 0.001). (Fucose data are from Rose and Sinha, unpublished. Data in last row, from a separate set of experiments with [ 3H]lysine precursor, are from Rose and Sinha, 1974a.)

<

_

~

_

Fraction

~-

~

__

~.

~

Specific radioactivity ( d i s i n l . / m i n / mg-~~ protein) 1 hr 4 hr ~

Suspension Neuron Neuropil

602 L 4 1 535 f 37 440 2 31

583 f 43 386 2 3 6 587 f 108

Neuronlneuropil

1.37 ? 0.12

0.77 f 0 . 1 1

Neuron/neuropil ( [ H ] Lysine precursor)

1.38 f 0.20

0.42

* 0.04

We next examined the effect of two substances which interfere with protein synthesis and distribution: cycloheximide, which is an inhibitor of protein synthesis, and colchicine, which is without overall effect on protein synthesis but is known t o inhibit axonal transport. We predicted that in the presence of cycloheximide transport of prelabeled protein would still occur and thus over a 4 hr period the neuronal/neuropil incorporation ratio should be lower than that of saline controls. On the other hand, with colchicine to block axonal transport, if this was the mechanism of migration, then the neuronal/neuropil incorporation ratio at 4 hr should be higher than in controls. Animals were injected with [ HI lysilie intraperitoneally ; 15 min later either cycloheximide (approximately 6 mg/kg) or saline was also injected, and animals killed after a further 45 or 225 min and neuronal and neuropil fractions prepared. The

75 cycloheximide dose was enough to inhibit more than 90% of protein synthesis in whole cortex for up to 4 hr. The results of this series of experiments (Table 111) show that neuronal protein incorporation seems more sensitive to cycloheximide than that of neuropil, so that the residual neuronal/neuropil incorporation ratio, even at 1hr, was only 0.64 and did not change much in the period to 4 hr. This pattern of results was more complex than we had predicted and does not permit a simple interpretation - the differential sensitivity of a fraction of neuronal protein synthesis to cycloheximide, however, is certainly an interesting observation. TABLE 111 EFFECT OF CYCLOHEXIMIDE ON [ 3 H ] LYSINE INCORPORATION IN NEURONS AND NEUROPIL Rats were injected intraperitoneally with [ H ] lysine, followed after 15 min with cycloheximide ( 6 mg/kg) or saline, and killed after a further 45 or 225 min. Neuronal and neuropil fractions were prepared, precipitated with TCA and counted. Results are expressed here as incorporation in cycloheximide-injected animals as a percentage of saline controls at 1 or 4 hr of incorporation, and are means of 1 2 determinations not differing by more than If: 15%. Note the differential sensitivity of neuronal protein synthesis. (Rose and Sinha, unpublished.) im_ide _C_y clo _ _hex ___ _ _incorporation ~____ x 100

Fraction

Saline incorporation 1 hr

4 hr

Suspension A (debris etc.) B (neuropil) C (neurons) D (nuclei etc.)

42.0 36.7 40.5 15.1 32.4

57.5 63.0 49.5 34.0 46.3

Neuron/neuropil (cycloheximide)

0.64

0.53

Neuron/neuropil (saline)

1.96

0.73

__

~~

-

The effect of injecting colchicine at 35 pg/kg, a dose sufficient to block axonal transport for many hours, but without effect on the overall [3H]ly~ine incorporation rate, is shown in Table IV. In this experiment the inhibitor was injected 1hr prior to the isotope, and we expected to find neuronal protein synthesis unimpaired whilst, if axonal transport was blocked, there would be a steady rise of specific radioactivity of neuronal protein over the 1 to 4 hr period, instead of the fall found in the absence of the inhibitor. The results of Table IV show a slightly more complex picture; the specific radioactivity of neuronal protein at 1hr was lower in the presence of colchicine than in the saline controls, even though in the total brain suspension no inhibitory effect could be detected. Once again, therefore, it appears as if a fraction of neuronal protein synthesis is differentially sensitive to an added drug. However, if

76 incorporation into the cell fractions in the presence of colchicine is compared, at 1hr and 4 hr, with that into controls, it can be seen that our more simple-minded prediction is fulfilled. Between 1hr and 4 hr the overall specific radioactivity of the total cell suspension, in the presence or absence of colchicine, rises some 20%. In the absence of colchicine, neuronal specific radioactivity falls slightly, whilst that of neuropil rises markedly. In the presence of colchicine, neuronal specific radioactivity rises by 40%, whilst the rise in neuropil specific radioactivity is markedly reduced. TABLE IV EFFECT O F COLCHICINE ON [ HI LYSINE INCORPORATION IN NEURONS AND NEUROPIL Rats were injected ( 3 5 pg/kg) with colchicine or saline intraperitoneally. After 1 hr [3H]lysine was injected and animals killed after a further 1 or 4 hr. Neuronal and neuropil fractions were prepared, precipitated with TCA and counted. Results are expressed here as (incorporation a t 4 hr/incorporation at 1 h r ) x 100, and are the means of 12 determinations not differing by more than + 15%. Note the accumulation of radioactivity in neurons for colchicine-injected animals a t 4 hr compared t o the saline controls. In these experiments colchicine was without effect on overall protein incorporation in the whole cell suspension (incorporation + colchicine/incorporation + saline at 1 hr, 103%, at 4 hr, 101%). (Rose and Sinha, unpublished.)

(Incorporation at 4 hrlincorporation at 1 hr) x 100

Suspension Neurons Neuropil

Saline

Colchicine

123 94 260

120 140 135

We conclude, therefore, from these studies that synthesis of the rapidly labeling neuronal protein fraction whose fate we have been following is especially sensitive to the effects of cycloheximide and of colchicine, and that its transport from the neuronal perikaryon is inhibited by colchicine. If blocking of axonal flow is the only effect of colchicine, the rapidly labeling component must indeed be part of the axonally transported protein under normal circumstances and, presumably, ci:rer the 4 hr period of our analysis will still be present in axons, not yet having measurably accumulated at the synapse. We have no evidence that neuronally synthesized protein accumulates in the glial cells.

77 EFFECTS OF SENSORY DEPRIVATION AND STIMULATION ON NEURONAL PROTEIN SYNTHESIS The final example I want to give of our current work on neuronal/glial biochemical interrelations moves us firmly into the field of function. Up till now I have been discussing what we may describe as “normal” neuronal biochemistry, without reference to what must be one of our major long-term goals, the cellular description of the mechanisms of neuronal plasticity. For several years now we have, alongside our cellular studies, been examining the effects of modifying the environment of the organism on such neurochemical parameters as RNA and protein synthesis and levels of activity of transmitter enzymes (for review, see Rose et al., 1976). In these experiments we have been studying the biochemical sequelae of learning (using the chick imprinting system: Horn e t al., 1973) and of visual deprivation and stimulation in the young rat. In the latter system we have shown that 50 days of dark-rearing followed by visual stimulation results in a transient elevation of incorporation of amino acids into a number of protein fractions in the visual cortex, lateral geniculate and retina (Richardson and Rose, 1973); the elevation is maximal between 1 and 3 hr after onset of light stimulation and, in the whole visual cortex, is of the order of 20-30% above the level in control animals maintained in the dark. It was an obvious question to ask whether the elevation in incorporation was confined to one cell type, or was a more general effect. Fractionation of visual cortex in the dark-reared and subsequently light-exposed animals, after 1hr of incorporation showed that the elevation in incorporation is an entirely neuronal phenomenon (Rose et al., 1973). The neuropil fraction is thus being unaffected by light-exposure following dark-rearing. This cellular specificity was very exciting to us. The observation became still more intriguing when we looked at the results in terms of the neuronal/neuropil incorporation ratio. For a 60 min pulse of [ HI lysine, whereas in the normally reared animal an incorporation ratio of up t o 2.0 would be observed, the ratio in the dark-reared rat visual cortex was only 0.6, while in the 1hr-light exposed rat it rose to around 1.0. The ratio after 1hr in the dark-reared animals was thus close to that observed after a 4 hr pulse in the normal animals. Was this because dark-rearing suppressed the rapidly labeling fraction? If so, then prolonging the pulse length in the dark-reared animals should be without effect on the ratio. If, on the other hand, there is a general suppression of incorporation into visual cortex neuronal protein in the dark-reared animals, then increasing the pulse length to 4 hr should result in a reduction of the neuronal/neuropil ratio by some 70%, to about 0.2. Table V shows the results of an experiment to test this possibility. By contrast with normally reared littermate controls, in the visual cortex of the dark-reared animals there was no change in the neuronal/neuropil incorporation ratio at either 4 hr or 24 hr as compared with 1hr. At 4 hr the ratio was the same as that at 1hr, whereas in the normals the ratio at 4 hr was 26% of that at 1hr. As a consequence of this, whereas at 1hr the incorporation rate in dark-reared animals was only 54% of that in normally reared animals, by 4 hr

TABLE V NEURONAL/NEUROPIL INCORPORATION RATIO WITH [ HI LYSINE PRECURSOR IN NORMAL AND DARK-REARED ANIMALS FOLLOWING PULSE-LABELING [ 3 H ] Lysine was injected intraperitoneally into 50-day-old, dark-reared rats and littermate controls reared in a 12 hr light/dark animal house cycle. Dark-reared animals were replaced in the dark for the period of incorporation. One hour later animals were killed and neuronal and neuropil fractions from visual and motor cortex regions prepared, precipitated with TCA and counted. The number of animals used for each measure with the dark-reared animals were: visual cortex: 1 hr, 8; 4 hr, 7; 24 hr, 5 and motor cortex 6. Data represent means f S.E.M. in each case. Differences in ratios between 1 and 4 or 24 hr are significant (P 0.001) for normal animals but not for visual cortex in dark-reared animals. Differences between neuronal and neuropil specific activities (depression) in visual cortex in dark-reared animals is significant (P 0.01) at all times. Difference (elevation) in dark-reared motor cortex is significant at the 0.05 level. (From Rose and Sinha, 1974b.)

<

<

Specific radioactivity (disint./min/mg protein) Normal Ratio 1 hr 4 hr 24 hr

1.28 f 0.18 0.33 f 0.04 0.44 f 0.06

Dark-reared (visual cortex) Neuronal Neuropil Ratio 3074 f 186 4405 f 427 4365 f 677

5090 f 420 5859 f 453 6186 f 625

0.69 f 0.03 0.75 f 0.03 0.70 f 0.05

Dark-reared ( m o t o r cortex) Neuronal Neuropil R a ti0 4954 f 356 -

4266 f 239 -

-

1-27 f 0.07 -

79 the figure has risen to 227%. On the other hand, in the frontal (motor) cortex region, where n o elevation in protein incorporation occurs when dark-reared animals are exposed t o the light, the neuronal/neuropil incorporation ratio at 1 hr was almost exactly the normal level. Further fractionation showed that, as with the rapidly labeled fraction in neurons from normal animals, the fraction whose labeling is suppressed in dark-reared animals and enhanced when the dark-reared animals are exposed to light belongs to the particulate (water-insoluble) proteins of the neurons. These results are consistent with the hypothesis that incorporation into the rapidly labeling insoluble protein fraction present in cortical neurons in normally reared animals is specifically suppressed in the visual but not the motor cortex of dark-reared rats. It is relevant t o the interpretation of these data to note that elevations in incorporation of orotic acid into RNA have also been found in the visual cortex of dark-reared and subsequently light-exposed rats in an analogous experimental situation to ours (Dewar e t al., 1973). This would suggest that for the rapidly labeling neuronal protein fraction t o be synthesized, new messenger RNA may be involved.

CONCLUSION AND SUMMARY This paper is a report of work in progress, part of an ongoing attempt t o describe the dynamic state of cerebral cellular metabolism. It is based on a particular methodology, the bulk separation of fractions containing purified, viable populations of a specific cellular type. We have satisfied ourselves of the relative high degree of biochemical integrity and purity of the fractions which we can obtain, and have begun to describe some of the ways in which metabolism is compartmented between the cell types. In relation t o protein metabolism in general, we believe that we have been able t o identify a neuronally synthesized particulate glycoprotein fraction which is probably axonally transported. We have shown that the synthesis of this fraction is (rather) specifically sensitive not merely to inhibitors of protein synthesis and axonal flow, but also t o the environmental circumstances of the organism. We have proposed elsewhere (Rose, 1974; Rose et al., 1976) that neuronal and glial metabolism is state-dependent, that is, it fluctuates with the changing environmental circumstances of the organism. The dynamic biochemical interactions between the neuronal perikaryon, its processes and synapses, and the surrounding glial cells are presumably, at the cellular level, correlates of neuronal functional plasticity at the physiological level, and thus of altered behavior at the organismic level.

ACKNOWLEDGEMENTS This paper draws on the work of and discussions with all the members of the Brain Research Group at the Open University, but in particular A r m Sinha,

80 Layla Sinha, Dave Spears, Gary Dutton, Jim Cohen and Neil Currie. I thank all of them, especially for their agreement to my drawing on some still unpublished work. Grants from the Medical Research Council and Science Research Council are also gratefully acknowledged. REFERENCES Babitch, J.A. Blomstrand, C. and Hamberger A. (1975) Amino acid incorporation into neurons and glia of guinea pigs with experimental allergic encephalomyelitis. Brain Res., 86: 459-467. Barondes, S. (1968) Further studies of the transport of protein to nerve endings. J. Neurochem., 15: 343-350. Blinkov, S.M. and Glezer, 1.1. (1968) The Human Brain in Figures and Tables. Plenum Press, New York. Blomstrand, C. and Hamberger, A. (1969) Protein turnover in cell-enriched fractions from rabbit brain. J. Neurochem., 16: 1401-1407. Cohen, J. Marex, V. and Lodin, Z. (1973) DNA content of purified preparations of mouse Purkinje neurons isolated by a velocity sedimentation technique. J. Neurochem., 20: 651-657. Cohen, J., Dutton, G.R., Wilkin, G.P., Wilson, J.E. and B a l k , R.A. (1974) Preparation of viable cell perikarya from developing rat cerebellum with preservation of a high degree of morphological integrity. J. Neurochem., 23: 899-902. Dewar, A.J., Reading, H.W. and Winterburn, A.K. (1973) RNA metabolism in the cortex of newly weaned rats following first exposure to light. Life Sci., 13: 565-573. Droz, B. and Koenig, H.L. (1970) Localization of protein metabolism in neurons. In Profein Metabolism of the Nervous System, A. Lajtha (Ed.), Plenum Press, New York, pp. 93-108. Dutton, G.R., Cohen, J. and Currie, D.N. (1975a) Some properties of viable perikarya from weaver and litter-mate cerebella. Trans. Amer. SOC. Neurochem., 6: 97. Dutton, G.R., Cohen, J. and Wilkin, G.P. (1975b) In vivo incorporation of labeled fucose and glucosamine into glycoproteins from cerebellar neuronal perikarya and nerve endings. Trans. Amer. SOC.Neurochem., 6: 98. Eccles, J.C., Ito, M. and Szentagothai, J. (1967) The Cerebellum as a Neuronal Machine. Springer, New York. Hamberger, A. and Sellstrom, A. (1975) Techniques for separation of neurons and glia and their application to metabolic studies. In Metabolic Compartmentation and Neurotransmitters, S. Berl, D.D. Clark and D. Schneider (Eds.), Plenum, New York, pp. 145-166. Horn, G., Rose, S.P.R. and Bateson, P.P.G. (1973) Experience and plasticity in the nervous system. Science, 181: 506-514. Johnston, P.V. and Roots, B.I. (1970) Neuronal and glial perikarya preparations: an appraisal of present methods. I n t . Rev. Cytol., 29: 265-295. Packman, P.M., Blomstrand, C. and Hamberger, A. (1971) Disc electrophoresis separation of proteins in neuronal, glial and subcellular fractions from cerebral cortex. J. Neurochem., 18: 1-9. Palay, S.L. and Chan-Palay, V. (1972) In Metabolic Compartmentation in the Brain, R. Balazs and J.E. Cremer (Eds.), Macmillan, London, pp. 187-208. Richardson, K. and Rose, S.P.R. (1973) Differential incorporation of H-lysine into visual cortex protein fractions during first exposure to light. J. Neurochem., 21: 531-537. Rose, S.P.R. (1965) Preparation of enriched fractions of isolated metabolically active neuronal cells. Nature (Lond.), 208: 621-622. Rose, S.P.R. (1967) Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells. Biochem. J., 102: 33-43. Rose, S.P.R. (1970) The compartmentation of glutamate and its metabolites in fractions of

81 neuronal cell bodies and neuropil studied by intraventricular injection of U - l 4 c gluiamate. J. Neurochem., 1 7 : 809-816. Rose, S.P.R. (1972) Cellular compartmentation of metabolism in the brain. I n Metabolic Compartmentation in the Brain, R. B a l k s and J.E. Cremer (Eds.), Macmillan, London, pp. 287-304. Rose, S.P.R. ( 1 9 7 4 ) Neuronal protein synthesis and environmental stimulation: state dependent and longer term effects. Trans. biochem. Soc., 2 : 30-33. Rose, S.P.R. ( 1 9 7 5 ) Cellular compartmentation of brain metabolism and its functional significance. Int. J. neurosci. Res., 1: 19-30. Rose, S.P.R. and Sinha A.K. (1974a) Incorporation of amino acids into proteins in neuronal and neuropil fractions of rat cerebral cortex: presence of a rapidly labeling neuronal fraction. J. Neurochem., 23: 1055-1076. Rose, S.P.R. and Sinha, A.K. (1974b) Incorporation of 3H-lysine into a rapidly labelling neuronal protein fraction in visual cortex is suppressed in dark-reared rats. L i f e Sci., 1 5 : 223-230. Rose, S.P.R. and Sinha, A.K. (1976) Rapidly labeling and exported neuronal protein; 3 H-fucose as a precursor and effects of cycloheximide and colchicine. J. Neurochem., in press. Rose, S.P.R., Sinha, A.K. and Broomhead, S. (1973) Precursor incorporation into cortical protein during first exposure of rats t o light: cellular localisation of effects. J. Neurochem., 21: 539-546. Rose, S.P.R., Hambley, J. and Haywood, J. ( 1976) Neurochemical approaches to learning and memory. In Neural Approaches t o Learning and M e m o r y , E. Bennett and M.R. Rosenzweig (Eds.), M.I.T. Press, Cambridge, Mass., in press. Sinha, A.K. and Rose, S.P.R. ( 1 9 7 1 ) Bulk separation of neurons and glia: a comparison of techniques. Brain Res., 3 3 : 205-217. Sinha, A.K. and Rose, S.P.R. ( 1 9 7 2 ) Compartmentation of lysosomes in neurons and neuropil; and a new neuronal marker. Brain R e s . , 39: 181-196. Sinha, A.K., Rose, S.P.R. and Sinha, L. (1975) Separation of neuronal and neuropil fractions from developing rat cerebral cortex. Trans. biochem. Soc., 3 : 97-98. Tiplady, B. and Rose, S.P.R. (1971) Amino acid incorporation into protein in neuronal cell body and neuropil fractions in vitro. J. Neurochem., 18: 549-558.

DISCUSSION R. BALAZS: It would be worthwhile to spend a little time a bout one question which is very relevant, and basic to other conclusions that you can draw from isolated cell populations: that is, the integrity of t h e preparations. Unfortunately, in most of the preparations tha t have been used u p till now, t h e cells were gravely damaged; cell membranes are torn apart, most of t h e mitochondria are small or completely lost. What is remaining from the cell structure is only t h e nucleus and t h e cytoplasm. Even in our most recent preparations, which are very much better than most of t h e preparations which are presently available, we are not able to get from adult animals a sufficient yield of good cells in order to d o reasonable biochemical work. And I should like to p u t up a plea here, and that is tha t one should always get electron microscopic low magnification pictures in order t o appreciate what properties the preparations have, because what you really can see is also a question of how long you look. Until we have got reasonable preparations where the integrity is preserved, it is very difficult t o p u t any interpretation at all o n the biochemical data.

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S.P.R. ROSE: course, all t h e co.ncern a bout the morphological integrity of cell preparations is quite right -this is a point we have repeatedly emphasized. The problem has always been that t h e biochemical data was compatible with intact cells, the morphological data less so. We have always had good evidence for biochemical integrity of the cells. We feel now that in fact a lot of the bad morphology was due t o the inadequacy of the electron microscopic techniques! A great deal depends o n the exact conditions under which you maintain the preparations for electron microscopy, under which you fix them, un&r which

a2 you observe them, and so on. George Gray’s experience with albumin would bear out the problem of finding structures by means of t h e electron microscope, even though they may be there at the biochemical level.

V.P. WHITTAKER: I agree with you, because our own first electron micrographs of cells from the electric layer of t h e Torpedo were just terrible, b u t they got better and better as E.M. techniques have improved. There is, I think, a great deal that we still d o not know about the best way of fixing isolated structures for E.M., without the support that you get in a whole tissue block from the surrounding structures. For t h e isolation of neuronal and glial cell bodies we are using Hamberger’s techniques. Whereas the synaptosome preparations are very reproducible, the cell body preparations are sometimes good but sometimes quite bad. So just because you get a bad result it does not mean that the whole preparation is bad: it may just be that the particular technique is bad. Dr. Hamberger admits that in his own laboratory, where they are very experienced in making these preparations, about 50% of the preparations are not what they would call really good (while the other 50% are really excellent preparations). So there is an intrinsic variability, and we can only hope t o find out in the future what t h e tricks are. S.P.R. ROSE: I think you are right: there is a fair bit of “noise” in the system, though I believe we d o a good bit better than 5 0 / 5 0 . Hamberger’s technique is very similar t o ours and we get variation in our system as well. That was why I emphasized the need for routine screening and routine use of either light microscopy or enzyme markers.