Neuronal and Glial Perikarya Preparations: An Appraisal of Present Methods

Neuronal and Glial Perikarya Preparations : An Appraisal of Present Methods PATRICIAV. JOHNSTON AND BETTYI. ROOTS Children’s Research Center, and the Burn~ide~ Research Laboratory, Univarsi& of Illinois at Urbana-Champaign, Urbana, Illinois and Department of Zoologv, University of Toronto, Toronto, Canada

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assessment of Purity of Fractions . . . . . . . . . . . . .

I. Introduction.

11. The Preparation of Neuronal and/or Glial Suspensions 111. Isolation of Cell Types from Suspensions . . . . . .

265 261 268 269

IV. V. The Question of Cellular Integrity . . . . . . . . . . . . . 27‘ VI. The Future of Research on Isolated Neuronal and Glial Perikarya 276 References. . . . . . . . . . . . . . . . . . . . . . . . 279

I. Introduction The particularly close association of most neurons with their supporting cells, the extent of their processes, and their entanglement within the neuropile preclude the possibility that samples of complete neurons and glia can be obtained. Nevertheless, studies on the nervous system would be enhanced if the chemical and physical properties of neurons or glia could be studied in isolation. Methods have been devised for sampling neurons and/or glia by Lowry (1953), Chu (1954), Hyden (1959), and Roots and Johnston (1964). These procedures provide neurons from frozen (Lowry, 1953) or fresh (Chu, 1954; Hydkn, 1959; Roots and Johnston, 1964) tissue which retain processes of considerable length, up to 300-400 p (Roots and Johnston, 1964). None of the methods is rapid and the number of cells that can be obtained is limited, however. There are an increasing number of reports that claim the macroscale preparation of mammalian neuronal and/or glial fractions that are of sufficient purity and morphological integrity to consider their biochemical properties meaningful. The procedures described closely resemble each other, yet the authors invariably claim inadequacy and even failure of previous techniques. Clearly this is a controversial field. There seem to be several problems, not least among them being definition of the term “isolated neuronal and glial perikarya.” Other problems involve the definition of “pure” or “enriched” when referring to fractions and in the assessment of the morphological and biochemical integrity of cells. In this chapter, we will review the literature and suggest the adoption of criteria which may assist in communication and understanding between interested investigators and hopefully lead to the development of standard preparations that are more widely acceptable. 26 5

HAND DISSECTION Deiters,

1865

/ Lowry, '913

\ Giacobini, 1956; Hpdtn, 1959;and others

MICROMANIPULATION Chu,

-

I954

MACROSCALE PREPARATIONS McIlwain, 195 4 (cell suspensions enzyme digestion)

Korey,

I 95

7

/

Roots and Johnston, I 964 (sieving, electron microscopy)

&a

P

(white matter glial prep. centrifugation on gradients)

Rose, 1965 ;Bocci, 1966

-

/

Satakeand Abe, 1966 Freysz e t al., 1967 (organic solvents)

\

Satake e t al., 1968; Norton and Poduslo, 1969 Flangas and Bowman, 1968 (enzyme digestion) (zonal centrifugation)

Fewster, Scheibel and Mead, 1967

FIG.I. An illustration of the development of procedures for the isolation of neuronal and glial perikarya.

?-

3 3

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Neuronal perikarya were first isolated by Deiters (1865). Since Deiters’ time, investigators seeking to isolate neurons and glia in a histologically and/or biochemically useful state have used microdissection in one form or another. Thus, between 1950 and 1960 several investigators began sampling neurons and/or glia using microdissection techniques (Lowry, I 9 5 3 ; Chu, I 9 5 4; Giacobini, 1956; HydCn, 1959). Other approaches were undoubtedly tried, but during this time the only account of attempts to prepare nerve cell fractions on a bulk scale appears to be that of McIlwain ( I Y S ~ )who , used enzyme digestion of the tissue. McIlwain did not attempt to prepare pure cell populations, however. Korey (1957) described the preparation of a glial fraction obtained by centrifugation on a sucrose gradient, but no claim of a pure fraction was made. This history of efforts to isolate neuronal and glial perikarya is illustrated in Fig. I . The preparation of neuronal and glial fractions from mammalian nervous systems1 can be divided into three stages: ( I ) preparation of a suspension in which cells are floating free; (2) isolation of a single cell type free of extraneous debris; and (3) assessment of the morphological purity of the fractions and of the integrity of the cells obtained.

11. The Preparation of Neuronal and/or Glial Suspensions Classically, cell suspensions have been prepared either by the use of media free of divalent cations and/or containing cation-sequestering agents, or by the use of enzymes or other methods that reduce intercellular adhesion. Success in the use of these methods, however, has mainly been confined to special cases, namely, to the disaggregation of embryonic tissues, the dispersion of cells growing in culture and therefore in contact with a “foreign” surface such as glass, and the prevention of flocculation of cells in tissues in which they do not natively adhere. When these approaches have been applied to other tissues, problems have arisen (Berry and Simpson, 1962; Carr e t al., 1967). This is particularly true in the case of nervous tissue, in which there is an unusually close association of cells and intertwining of processes. We found that central nervous system tissue and peripheral ganglia did not readily dissociate when treated with trypsin, pronase, and papain. In our experience, once some disaggregation of tissue was obtained, disruption of the cells themselves and/or decomposition of cellular components such as phosphoglycerides had already advanced (Johnston and Roots, unpublished observations). Since we were interested in obtaining cells that could be used for chemical as well as morThe qualification “mammalian” is inserted here because mammalian brains have been the sole objects of this kind of study. The discussion that follows could apply equally well to vertebrate brains in general, however.

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phological studies, these findings presented a major disadvantage. We therefore attempted mechanical means of disrupting the tissue while it was maintained in an isotonic medium. Passing the tissue through a series of monofilament nylon cloths with apertures of decreasing size proved to be a successful approach. The aperture size of the finest cloth is chosen so that the average cell in the population will pass through. In this way, large capillary fragments, connective tissue, and myelin fragments are retained on the sieves, and a suspension containing neurons, glia, erythrocytes, and small debris is obtained. As a dispersion procedure, sieving appears to be standing the test of time since it has generally been adopted, in one form or another, by subsequent investigators (Rose, 1961, 1967; Satake and Abe, 1966; Bocci, 1966; Flangas and Bowman, 1968). In a procedure recently reported by Norton and Poduslo (1969)~ the two approaches are combined, and disruption of the tissue by sieving is preceded by a period of incubation in a medium containing 1 % trypsin. In this method trypsin does not appear to have caused decomposition of cellular components. It is possible that the particular composition of the incubation medium, especially the use of bovine serum albumin, had a protective effect.

111. Isolation of Cell Types from Suspensions As in the isolation of subcellular particles, the manipulation of tissues in artificial media is unavoidable when preparation of cell fractions is attempted. As a general rule, when dealing with cells one tries to maintain an isotonic environment. As soon as isolation by centrifugation on gradients is contemplated, this criterion must be abandoned. For this reason, we tried various aqueous two-phase polymer systems as described by Albertsson (I 960) for the separation of chloroplasts, erythrocytes, and various subcellular particles in which relatively physiologically compatible environments can be maintained. These efforts were abortive, however. Some degree of enrichment of neurons over glia and debris could be obtained in some systems, especially those employing methylcellulose and polyethylene glycol as the aqueous polymers. The phases took several hours to separate, however, and as a consequence the condition of the cells deteriorated. Although we tried a large number of phase systems, it cannot be stated that this approach was tried exhaustively since the variations in phase composition are infinite and, furthermore, several new highpurity polymers have since become available. We also tried a simple mechanical approach to effect separation, namely, by sieving alone. It was considered that by using a series of sieves of suitable aperture size and by starting with a neuron population of a limited size range it might be possible to sieve out all contaminating material. Here we met numerous problems involving coacervation of the suspended cells and clogging

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of the sieve apertures. We also found it impossible to retrieve an adequate number of neurons from sieves even when they were treated with silicones. Satake e t af. (1968) d o not appear to have encountered this problem. Possibly the medium these investigators employed in their recent studies conferred different surface properties on the cells, and sticking to sieves was avoided. Electrophoresis as a means of separation was also considered and tried. Neurons, glia, and extraneous material in the suspension all moved toward the anode in an applied field of Z I O V dc, although at slightly different speeds. We did not find conditions under which the collection of any practical quantity of one cell type could be obtained and in a reasonable time to avoid serious cell damage. Again, however, we would not regard the repetitive sieving o r the electrophoretic approach as having been examined exhaustively. Further studies in the light of more recent knowledge may prove rewarding. Recently, in an appraisal (Cremer e t al., 1968) of a centrifugation procedure (Rose, 1 9 6 5 , 1967) for the preparation of neuronal and glial enrichments, we have discussed some of the difficulties encountered. In our earlier attempts to isolate neurons by centrifugation, we employed a variety of gradients containing high-molecular weight dextrans and sucrose concentrations up to 1.75 M. In general, we obtained more satisfactory fractions employing low centrifugation speeds (10-300 x g) similar to the speeds used by Bocci (1966) to obtain neuron fractions. Careful assessment of our fractions by light and electron microscopy, however, led us to conclude that the amount and type of contamination was such that use of these fractions for biochemical studies could lead to erroneous conclusions regarding differences between neurons and glia. Furthermore, the cells suffered considerable cytoplasmic damage, which made leakage of cellular components inevitable. These findings are consistent with those of Bocci (1966), who concluded that the cells obtained by his technique were essentially “dead” as a result of morphological impairment and the loss of enzymic activity incurred. The term dead is used here in a relative sense, that is, metabolically dead relative to the activity in vivo or in slices. Most preparations show some metabolic activity, indeed some recent preparations compare favorably with brain slices (see next section). Two facts clearly emerge, namely, that presently the methods of choice for tissue disruption and cell separation involve some form of sieving and centrifugation and that neither procedure is free of hazards. Just what these facts involve is discussed further when we consider cellular integrity.

IV. Assessment of Purity of Fractions Undoubtedly, initial assessment of the relative purity of a cell fraction with respect to cell type and freedom from extraneous material is most easily obtained by examination of the fractions by light microscopy. Even when

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attempts are made to assess contamination by use of differential staining and dark ground illumination, it is often impossible to determine its real nature. As a general rule, we employed light microscopy to assess fractions initially and electron microscopy to place determination of the nature of contamination on a more definite level and, more significantly, to assess cellular integrity. Light microscope examination (with and without staining) is used generally to assess purity of cell fractions. I n the case of neurons, there has been little difficulty regarding identification and, with one exception (Rose, 1961, 1967), erroneous identities do not appear to have been assigned. Glia, however, are more difficult to identify, especially when they have been removed from their locations in the brain, and selective staining combined with electron microscopy is necessary. Fewster, Scheibel, and Mead (1967) designated their preparation glial largely on the basis that the particles were surrounded by a birefringent layer. This does not appear to be an adequate basis for identification since many fragments of tissue, including myelin, exhibit this phenomenon. A major difficulty lies in the adoption of uniform criteria for the description of fractions, and dispute arises regarding use of the terms pure and enriched which can mean very different things to different investigators. This problem is compounded when biochemical studies are employed with a view toward facilitating the understanding of neuronal and glial functions. It is obvious that if the interpretations of these studies are to have any meaning contamination should be very low. We agree with the conclusions of Bocci (1966) that even slight glial contamination of a neuronal enrichment may lead to erroneous evaluation of enzyme content. It is difficult to see how impure fractions of neurons and glia can either compete with, or usefully supplement, carefully monitored studies on microdissected cells and regional neurochemical studies such as those of Hess and Pope (1960, 1961; Hess and Lewin, 1965; Hess and Thalheimer, 1 9 6 5 ) ~This is particularly true when one considers that the source of the cells is in itself usually histologically and physiologically heterogeneous (e.g., whole cortex), whereas when microdissection is employed it is usual to use discrete nuclei as a source. As we have discussed elsewhere (Cremer e t a/., 1968), fractions prepared by the original (1965, 1967) Rose procedure are markedly heterogeneous. The neuronal fraction contains a large proportion of fragmented capillaries and other non-neuronal material, while the glial enrichment is heavily contaminated with nerve endings. Commenting on our findings, Rose (1968) suggested that our level of contamination was unusually high because of the constitution of the Ficoll used. In a reappraisal of his own preparations, however, he places contamination of the neuronal fraction as high as 30% and of the glial fraction These and numerous other papers by these investigators, many of which have appeared in the Journal of Neurochemistry over the last ten years.

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at 17% (Rose, 1968). More recently Rose and Sinha (1969) have discussed in more detail the problems which may be encountered due to differences between batches of Ficoll. They point out that the isolation procedure is not based solely on density differences and that changes in the ionic content of the medium and/or variations in the molecular weight of the Ficoll may effect the distribution of the material on the gradients. While such factors might account for variation in the degree of contamination found by different workers they do not account for its presence. For this reason Rose has recently introduced a step in his procedure to decrease the contamination by capillary material. This involves pouring the neuronal fraction through a bed of small glass beads to which capillaries preferentially stick (Rose, I 970). The use of acetone-glycerol-water as an isolation medium severely limited the use of the neuronal perikarya preparations of Satake and Abe (1966). The more recent preparations of Satake e t al. (1968) do not suffer from this disadvantage, and information regarding these preparations is promising. The very recent report on neuronal and glial preparations by Norton and Poduslo (1969, 1970) is equally promising. As yet, there has been insufficient time for an appraisal of the degree of reproducibility of these fractions by other workers, a criterion of considerable import in preparations of this nature. Comments on relative purity would, therefore, be premature. Information regarding cellular integrity is also lacking. Hopefully, however, these preparations at least come near to solving many earlier difficulties. A further note of caution should be interjected here.It has recentlybeen shown (Dounce and Ickowicz, 1969) that the composition of cell nuclei depends on the medium in which they are prepared. Differences in the isolation medium are re-, flected by dramatic differences in the chemical composition of the nuclei. Clearly, cell preparations should be checked for redistribution of cellular components.

V.

The Question of Cellular Integrity

While the level and type of contamination alone may be considered to negate neuronal and glial fractions as useful biochemical preparations, even more serious is the question of the degree of damage suffered by the cells during the isolation process. Light microscopy can be misleading in the assessment of cellular integrity. Cells isolated by the method we developed for collecting several hundred neurons in a relatively short time (Roots and Johnston, 1964) had an acceptable appearance under the light microscope (Fig. 2). Before using these preparations for chemical studies, however, we examined them by electron microscopy and found them to be extensively damaged. Neurons isolated in media (pH 7.4) such as Ringer-Locke and 0.25 M sucrose were found to lack the usual image of a surface membrane over the greater part of the soma and its processes.

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Neurons isolated in the same media, but hand dissected, also lacked the surface membrane image (Roots and Johnston, 1964, 1965; Johnston and Roots, I 96 5 and unpublished observations). When one considers the shear stress developed when neurons are freed of adhering glia (even by relatively gentle hand manipulation) this observation is not too surprising. Indeed, this type of damage is known to occur when other cells are isolated, for example, liver cells (Berry and Simpson, 1962; Carr e t al., 1967). Nevertheless, some investigators (Rose, 1965, 1967; Bradford and Rose, 1967; Bondareff and Hydtn, 1969) have questioned whether this damage is “real,” and they have suggested

FIG.2 . A phase-contrast photograph of a neuronal perikaryon isolated from the lateral vestibular nucleus of ox brain (Roots and Johnston, 1964).

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that it is an artifact of the fixation and/or the dehydration procedure used for electron microscopy. Recently, Bondareff and HydCn (1969) showed that the plasma membranes of cells isolated in 0 . 2 5 M sucrose are not necessarily fragmented during the isolation procedure. This is in keeping with our previous observations that in 20% of the cells isolated in media lacking divalent ions there was considerable retention of the membrane (Johnston and Roots, 1965 and unpublished observations). Bondareff and HydCn suggest that damage to a cell may occur after isolation if it is mechanically manipulated or is subjected to interfacial tensions during dehydration. The latter may be minimized by the use of a water-miscible epoxy resin (Durcupan A) as a dehydrating agent. Clearly, in future studies care should be taken to eliminate the possibility of disruption of the plasma membranes by interfacial forces during dehydration. However, the experiments of Bondareff and Hydin, d o not warrant their conclusion that the previously observed damage to isolated nerve cells resulted primarily from preparative procedures for electron microscopy, and indeed the evidence against this conclusion is strong. Hillman and HydCn (1965) have recorded a potential from isolated rabbit neuronal perikarya and this observation has been cited in support of the view that damage to cell surfaces occurs during preparation for electron microscope examination rather than during the isolation procedure. This potential is, however, as yet undefined, and from the data available it cannot be described with certainty as a membrane potential. Potentials attributable to the adsorption of ions into intracellular phases (Simon et a]., 1957) may exist or be established in the isolated perikarya. The fact that in Hillman and HydCn’s experiments an increased potential was recorded when gangliosides were added to the medium is consistent with the potential-creating effects of adsorbed ions. The acidic groups of adsorbed gangliosides may be expected to produce this effect. The chemical environments that favor surface retention are now known to be quite numerous and diverse. They include the use of media lacking divalent ions, containing gangliosides, albumin, or dextrans, and having a low pH (3.0-5.5). Figure 3 shows the surface of a nerve cell prepared in a medium containing gangliosides. None of the particular features of isolation media in which a membrane image is retained has, however, been shown to be specific and, furthermore, few of these media favor the retention of other desirable features within the cell. The use of dextrans, for example, is accompanied by anomalous osmotic effects leading to cell shrinkage. In general, environments favoring surface retention are consistent with those known to reduce cell adhesion (Curtis, I 967). That certain media protect against surface membrane damage is, in itself, insufficient evidence for claiming an absence of fixative or dehydration effects, but taken together with the other findings discussed below, this fact lends strong support to the theory that the observed damage to cells is not primarily a fixation/dehydration artifact.

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FIG. 3. An electron micrograph of part of the surface of a neuronal perikaryon isolated in 0.9% sodium chloride solution containing 0.55 mg per milliliter of gangliosides (pH 1.0). Inset: Part of the surface at higher magnification showing the trilaminate membrane structure.

When we found that neurons obtained by hand dissection may suffer surface membrane damage, we suggested that metabolic examination of isolated neurons and glia be monitored in order to establish the extent to which these systems reflect other features of the in vivo situation. Such studies were undertaken by Brzin, Tennyson, and Duffy (1966). These investigators showed that there was an unequivocal co.rrelation between biochemical data and damage to the plasmalemma suffered by neurons during hand dissection. Thus, they found that in cells having high acetylcholinesterase activity the neural plasmalemma and sheath was badly ruptured or absent, whereas in neurons having low activity these structures were intact. O n the basis of experiments on intact cells and cells disrupted by various means, Giacobini (1969) has recently suggested that the results obtained by Brzin, Tennyson, and Duffy may not be entirely attributable to the degree of integrity of the plasmalemma. Since Giacobini did not apply any criteria of integrity to his intact cells, however, his experiments cannot be regarded as definitive. Some appraisal of cellular integrity can be made by determining particular enzymic activities. If the leakage of lactic dehydrogenase (LDH) is taken as an indication of abnormal permeability (Zimmerman et a/., 1960; Berry, 1962; Exton, 1964), then it follows that neurons isolated by both the Bocci (1966) and Rose (I 96 j , I 767) procedures suffer surface membrane damage. Bocci found considerable leakage of L D H from his neuronal fraction and, in agreement with Rose (1965, 1967), we estimated in collaboration with others

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(Cremer e t al., 1968) (on the basis of LDH data) that in using the Rose technique yoyo of the cells are damaged in the original dispersion. Flangas and Bowman (1968) claim to have obtained intact neurons by a modification of Rose’s procedure using zonal centrifugation. In view of the findings of Bocci (1966), Rose (1961, 1967), and Cremer e t al. (1968), and the more drastic isolation conditions imposed by Flangas and Bowman, it would be surprising if these preparations contained a large number of intact cells. A more detailed assessment of the biochemical integrity of some macropreparations has been made by Rose and Sinha (1969). Neuronal and glial fractions were prepared by the Rose (1967) procedure but the glial fraction was redesignated neuropil. Both fractions were found to synthesize ATP in vitro and were shown to have considerable resistance to loss of free amino acids on repeated washing. After incubation with glucose, potassium accummulation was shown in both fractions ( ~ O O / ~of that in brain slices in the neuronal fraction and 6yy0 in the neuropil). Oxygen uptake, carbon dioxide, and lactate production of whole washed cell suspensions prepared by three different procedures were compared. All these metabolic properties were substantially lowered when acetone-glycerol-water (Satake and Abe, I 966) or tetraphenylboron (Rappaport and Howze, 1966) were used for tissue disaggregation in place of the Rose medium. These findings illustrate that some degree of cellular integrity remains when a Ficoll-sucrose medium is used but that the use of organic solvents or an ion complexing agent such as tetraphenylboron is deleterious to metabolic reaction systems and/or cellular integrity. Precisely what cellular integrity means when based on the activity of biochemical reaction sequences is not clear. It is interesting that we have observed that nerve cells gel on isolation, that is they can be severed and show no flow of cytoplasm. The gelling of the cytoplasm-itself would tend to retain small molecules and ions, allow for ion accumulation and metabolic activity. The loss of surface molecules due to shearing stress during tissue disaggregation and the redistribution of components during centrifugation is not precluded however. As noted earlier, information regarding the integrity of the neuronal perikarya isolated by the method of Norton and Poduslo (1969, 1970) is not yet available. One disturbing feature of this isolation procedure is the use of incubation with trypsin as part of the tissue disruption step. Trypsin has been shown to have an adverse effect upon the properties of neural membranes (Somogyi, 1968; Sellinger et al., 1969). Fragmentation of the membranes, a drop in their electrophoretic mobility, and an appreciable loss of acetylcholinesterase activity have been observed. Very recently neurons prepared by this procedure have been shown, by electron microscopy, to have a surface membrane (Norton e t al., I 970). The hexose-albumin-serum medium used in this method possibly serves as a protective agent. It is difficult to comment on the integrity of the cells in glial enrichments, at least

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since few attempts to isolate glia have been reported and there is considerable controversy regarding the identity of the preparations. Fewster e t al. (1967) report that electron microscope examination of the glial cells they prepared revealed that the surface membrane was for the most part lacking, but they comment that this loss of membrane may have occurred subsequent to the initial separation by centrifugation. They do not provide any evidence in support of this opinion, however. Considerable membrane damage and loss of cytoplasm was found (M. C. MacBrinn, unpublished obsertions) in this preparation, the yield consisting largely of glial nuclei. These results are in keeping with our own observations on similar systems. This series of investigations provides an excellent example of the importance that must be attached not only to fraction purity but also to cellular integrity. Fewster and Mead (1968) have provided lipid analyses for their glial preparations. If, as the authors state, the galactolipid content of the cells arises from both cytoplasmic lipids and (surface) membrane lipids, this observation provides new clues regarding the proliferation of myelin by glial cells. Thus, their finding of an increased proportion of cerebroside sulfate relative to cerebroside in the glia, compared to that in myelin, may mean that sulfation is a prerequisite step which facilitates solubilization and incorporation of cerebrosides into myelin. Desulfation would presumably occur later within the sheath. If on the other hand, the loss of glial surface membrane seen in their electron micrographs is “real,” other interpretations of their lipid analyses could be offered. In this regard, it is interesting that Norton and Poduslo (1969, 1970) also report a low cerebroside level in their glial fraction. It is imperative that questions regarding the cellular integrity of these fractions be answered before they are extensively used for postulations regarding the chemical events during the proliferation of myelin. Davison et al. (1966) have reported that early myelin in the rat is rich in phospholipids and poor in cerebrosides as compared to adult myelin. They also reported that glial fractions prepared by the Rose procedure contained more phospholipid and much less cerebroside than is present in myelin. The validity of the latter observation is of course in some doubt since, according to Cremer et al. (1768),the Rose glial fraction is contaminated by nerve endings, neurons, and other debris, and Rose himself places this contamination at 17%. Furthermore, the damage to the glial surface membrane has not been fully assessed and it is conceded (Rose and Sinha, 1969) that in reality this fraction is neuropil rather than glial and, therefore, heterogeneous.

VI. The Future of Research on Isolated Neuronal and Glial Perikarya Indisputably, we have much information to gain from successful neuronal and glial preparations regarding the individual roles of the cells and their

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interrelationship in the operation and control of nervous systems. Belief in this approach has been demonstrated by the increasing interest in this area. Uncertainties lie in questions such as: What is the most profitable line of attack-conventional analysis of macroscale preparations or microanalysis of microscale preparations ? Hopefully, the answer is that in the long run both approaches will prove profitable. The utilization of micro preparations will necessitate the development of new analytical methods and biochemical assays. This is another problem and is beyond the subject of the present discussion. There is, however, infinite scope in the field of new analytical approaches and there is every reason to believe that analysis for many substances in the nanogram and picogram range may well become routine. Clearly, as far as macroscale preparations are concerned, there is still considerable disagreement among investigators regarding what constitutes a pure or enriched cell fraction, and/or intact cells. Furthermore, controversy exists regarding the usefulness of fractions that are not pure and contain rather severely damaged cells3 These problems would, to some extent, be alleviated if all investigators described what, in their view, constituted pure or enriched in terms of percent cell density and offered some biochemical and microscopical evidence regarding their view of the intactness of cells. Some standardization of macroscale fractions might then be achieved. For example, L D H assays may be informative regarding degree of leakage from cells, as may assays of the apparent glial marker (Giacobini, I 964) carbonic anhydrase. Staining and examination by light microscopy could provide a determination of the density of the nuclei present in a population. Random selection of cells from suspensions and their examination by electron microscopy could provide an overall picture of surface and cytoplasmic integrity. Another potential criterion would be the demonstration of action potentials in neuronal cells. It might be thought that invertebrate ganglia in which neuronal somata are generally arranged in a peripheral layer are suitable material for the separation of cell types. Not only do our remarks on the heterogeneity of neuronal and glial cell populations also apply to invertebrate nervous systems, but the additional difficulty of invagination, often extensive, of neurons by glial cell processes exists. It is difficult to see how this problem can be overcome. The search for new methods will undoubtedly continue. Reexamination of earlier approaches in the light of new knowledge may prove profitable. Use may now be made of conditions known to reduce damage by shearing forces to cell surfaces (Johnston and Roots, 1965 and unpublished observations; Wallach, 1967). As a consequence, manipulation of membrane charge may be It is emphasized that isolated neuronal and glial perikarya represent quite severely damaged cells in any event. Most investigators report the cells in their preparations as shorn of processes to varying degrees.

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utilized to improve separation by relatively less traumatic procedures such as by two-phase aqueous polymer systems (Albertsson, 1960) and by electrophoresis. The preparation of fractions of a generally acceptable degree of purity would mark a great advance over the homogenates of whole brain or whole cortex currently in use and would contribute greatly to our knowledge of the chemical and biochemical constitution of nervous systems. Preparations in which the surface integrity of the cells has been lost may still be used to make preparations of subcellular particles, e.g., mitochondria and nuclei. Such preparations would be a useful supplement to subcellular fractions prepared from mixed cell populations (Siakotos e t al., 1969). While the neuronal and glial cells in these preparations may themselves have been derived from heterogeneous populations such as whole cortex, refinements such as the selection of small groups of cells of known physiological function should follow. An approach involving the selection of anatomical regions relatively free from other cell types, as in glial preparations from white matter, might also be further exploited. Bulk scale preparations of neurons and glia that receive universal acceptance may not be found. Certainly for some refined studies microdissection of cells will continue to be the method of choice since cells may be individually checked for damage. I t seems likely that macroscale preparations will become tailored to individual research needs. If it is desired to obtain detailed knowledge of the amounts of specific components in each cell type then clearly the preparations must be checked carefully for surface damage and redistribution of components. If in vitro biochemical studies are the aim, preparations that involve the use of organic solvents are not suitable. Such preparations may be useful when cells are separated for study subsequent to an in vivo treatment, however. For example, the procedure of Freysz et a/. (1967, 1968) involves the use of an acetone-glycerol-water medium and this preparation has been used to study the kinetics of the biosynthesis of phospholipids in neurons and glia (Freysz e t a/., 1969). In this study the radioactive precursor was given in vivo and the turnover of phospholipids was assessed on subsequent isolation of cell fractions. Blomstrand and Hamberger (1969) have carried out similar in vdvo studies of protein turnover using their modified Rose (1967) preparations. As illustrated in Fig. I progress in the area of nervous system cell preparations has been very rapid in the last decade. Although there are many problems associated with the various preparations, it is felt that consideration of the points discussed here will assist in the development of standardized preparations tailored to meet various research needs. ACKNOWLEDGMENTS This review was prepared while Dr. Roots was associated with the Department of Neurosciences, University of California, San Diego, La Jolla, California, and supported by Grant

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