Journal of Neurochemistry, 1977. Vol. 29, pp. 1039-1051. Pergamon Press. Printed in Great Britain.

LOCALIZATION OF HEXOKINASE IN NEURAL TISSUE: LIGHT MICROSCOPIC STUDIES WITH IMMUNOFLUORESCENCE AND HISTOCHEMICAL PROCEDURES G . P. WILKIN’ and 3. E. WILSON Biochemistry Department, Michigan State University, East Lansing, MI 48824, U S A . (Received 25 April 1977. Accepted 18 May 1977)

Abstract-The distribution of hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) in rat cerebellum, retina, hippocampus, choroid plexus and ependymal cells of the cerebral ventricles, and dorsal root ganglion has been determined, at the light microscopic level, by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. With the exception of an artifactual staining of the outer photoreceptor segments of retina when the histochemical procedure was used, both methods gave comparable results, from which the following conclusions are drawn: (a) The cytoplasm of neuronal cell bodies clearly contained hexokinase, although the relative levels varied markedly among different types of neurons; such variations have previously been detected by direct assay of hexokinase in dissected neuronal cell bodies (KATO& LOWRY,1973a). (b) Glial cells contained readily detectable levels of hexokinase: the immunofluorescence technique revealed spidery glial processes within the myelinated tracts; in other areas, glial cell cytoplasms were indistinguishable from surrounding neuropil, indicating comparable levels of hexokinase; the satellite glia of dorsal root ganglia actually contained higher levels than did adjacent large neurons. The present results, therefore, do not support previous suggestions that glia are characteristically low and neurons characteristically high in hexokinase content. (c) Hexokinase was distributed throughout neuropil areas, with a somewhat speckled appearance suggesting the existence of small localizations of relatively higher activity, the nature of which could not be determined at this level of resolution; the hexokinase level in neuropil was clearly higher than that of white fiber tracts, in agreement with previous direct biochemical measurements (BUELL et al., 1958). (d) No detectable levels of hexokinase were found in cell nuclei. (e) Regions expected to be rich in nerve terminals (e.g. the cerebellar glomeruli, the plexiform layers of retina) showed relatively high hexokinase levels compared to the cytoplasm of adjacent neuronal perikarya, in agreement with previous subcellular fractionation experiments which indicated relatively high levels of hexokinase in nerve endings (WILSON,1972). Considered along with the ‘high affinity’ glucose transport system in nerve endings (DIAMOND & FISHMAN,1973), these results suggest nerve terminals are well adapted for the.efficient acquisition and introduction of glucose into metabolism. (f) In addition, high levels of hexokinase were observed in the inner photoreceptor segments of retina, and in the ependymal and choroid plexus cells of the ventricles.

IT IS now well known that enzymes and their related metabolic activities are not uniformly distributed throughout the brain, but are ‘compartmented.’ Such metabolic compartmentation is a reflection at the biochemical level of the structural and functional heterogeneity so evident in neural tissue [for many pertinent references, see BALAZS & CREMER(1972) and BERL et al. (197511. The utilization of glucose appears to be no exception since it is readily demonstrated that there are multiple metabolic pools into which glucose may enter, with the rate of utilization a n d metabolic fate of the glucose being different in these distinct metabolic pools (BALAZSet al., 1972). BALAZS et al. (1972) have proposed a correlation of these different pools with discrete morphological structures.

* Present address: Medical Research Council, Developmental Neurobiology Unit, 33 St. John’s Mews, London, wc1.

Since glucose is normally the principal source of energy in mature neural tissues (BACHELARD,1970; BALAZS, 1970), variations in glucose metabolism among various elements of neural tissues may reasonably be expected to reflect variations in energy requirments of the different structures. Although most of the glucose is oxidized via the glycolytic pathway, appreciable metabolism via the hexose monophosphate pathway may also occur (BALAZS, 1970). The first step in metabolism of glucose by either glycolysis or the hexose monophosphate pathway is the phosphorylation catalyzed by hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The importance of hexokinase as a regulator of the cerebral glycolytic rate was first pointed out by LOWRY et al. (1964) a n d now seems t o be generally accepted (BALAZS, 1970; BACHELARD, 1970; GARFINKEL et Q i . 9 1972). Although regulation of glucose metabolism via the hexose monophosphate pathway has, to our knowledge,

1039

I040

G . P. WILIUNand J. E. WILSON

received less attention, it seems reasonable that hexokinase, as the first step of that pathway, would also play a prominent role. It is frequently assumed (BUELLet al., 1958; LOWRY et al., 1956, 1961; MCDOUGALet al., 1961; MATSCHINSKY, 1970; SUGDEN & NEWSHOLME, 1973) that the level of an enzyme in a particular structure may be taken as an indication of the importance of the pathway utilizing that enzyme in the overall metabolism of the structure. (This is not to imply that the metabolic potential, as represented by cellular enzyme levels, is fully employed at all times; certainly it is well-accepted that cellular enzyme activity is controlled, with metabolic activity varying in response to cellular function.) Based on the above assumption, it may be inferred that the level of hexokinase should reflect the relative importance of glucose metabolism and hence the relative energy requirements of various, neural structures. Thus, although determining the distribution of hexokinase among various defined neural structures has its own intrinsic interest, it is also reasonable to expect that such a study may provide information relevant to an understanding of differences in energy metabolism between the various structures which comprise neural tissue. Previous studies of hexokinase distribution in neural tissue have demonstrated marked variations in the hexokinase activity of various brain regions (BENNETT et a/., 1962; BIGL et al., 1971) and of the different layers of retina (LOWRYeta/., 1961) and cerebellum (BUELLet al., 1958). Subcellular fractionation of brain homogenates has suggested that a major portion of the total hexokinase activity of brain is located in the nerve endings (WILSON,1972). Although these previous investigations have provided us with useful information about the distribution of hexokinase in various neural structures, this information is clearly at a rather gross level, e.g. such studies would not permit any conclusion as to possible variations in hexokinase content of different structural elements within a given layer of the cerebellum. Although studies of specific structural elements, obtained by microdissection, would permit such comparisons and have, in fact, provided direct evidence for variations in hexokinase levels in various types of neuronal cell 1973a), such studies are diffibodies (KATO& LOWRY, cult, tedious, and generally of rather restricted applicability due to practical considerations. The studies described here give a light microscopic picture of the distribution of hexokinase in the retina, cerebellum, hippocampus, choroid plexus and ependymal cells, and dorsal root ganglia of the rat. The techniques used, immunofluorescence and conventional histochemical procedures, permit semiquantitative‘ comparison of the hexokinase levels in various

neural elements at a level of resolution not possible with previously used methods. It should be noted that these represent two quite distinct methods, with immunofluorescence depending on the antigenic properties of the enzyme while the histochemical procedure depends on the catalytic activity. Their application in parallel offers a useful crosscheck on the results obtained.

MATERIALS AND METHODS Materials

Phenazine methosulfate, nitro blue tetrazolium, and all biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Fluorescein isothiocyanate-conjugated IgC, prepared from serum of goats immunized with rabbit IgG, was obtained from Miles Laboratories (Elkhart, IN). Freud’s Adjuvant was a product of Difco Laboratories (Detroit, MI). Rats (150-25Og) of either sex were used. The animals were obtained from Spartan Research Animals (Haslett, MI) or bred in the departmental animal colony. Animals were maintained on standard commercially available diet and water ad lib. Methods Anti-rat brain hexokinase serum. Pure rat brain hexokinase was prepared according to CHOU& WILSON(1972).

Hexokinase (1.7mg), emulsified in Freud’s complete adjuvant, was injected subcutaneously in the back region and into the toe pads of a New Zealand white rabbit. A second injection (1.4mg enzyme) was done 10 days later. Collection of antiserum was begun t week after the second injection. Preimmune serum had been collected from the same rabbit prior to immunization. Both preimmune and immune serum were heated at 56°C for 20min, then centrifuged at 100,000g for 20min before use. Adsorption by acetone powders. In order to reduce possible interference by non-specific adsorption artifacts (JOHNSON & HOLBOROW, 1973; MCLAUCHLIN et al., 1974), prior to use each 0.5 ml portion of both immune and preimmune serum was adsorbed with 100mg of PBS-washed (PBS--O.9% NaCl plus 10 mwpotassium phosphate, pH 7.5) rat liver acetone powder at room temperature for 30 min. Fluorescein conjugated goat anti-rabbit IgG (purchased from Miles Laboratories, Elkhart, IN) was adsorbed with rat brain acetone powder at room temperature for 30min. Tissue sectioning. At least three animals were used to study each area by both immunofluorescence and histochemistry. Rats were stunned and decapitated, and the required tissues quickly removed and frozen for 1 min in isopentane cooled by cither liquid nitrogen or an acetonedry ice bath. The frozen tissue was sectioned at 4 or 6 pm in an Ames cryostat at -20°C. Sections were picked up on glass cover slips and air dried. If sections were not to be used on the same day, they were stored in a dessicator at room temperature in uacuo, a procedure which has been shown previously by LOWRYet al. (1961) to result in no loss of hexokinase activity for up to 3 days. Nitro blue tetrazolium histochemical procedure. Two proIf the microscope were equipped with photometric capability, these techniques could be made quantitative; cedures were used and, since they gave identical staining the immunofluorescence procedure would seem particu- patterns, were judged to be equally useful in determining the distribution of HK. lady amenable to such applications.

Localization of hexokinase

1041

(a) Sections on cover slips were immersed in acetone Histochemistry at -20°C for 30min, then air-dried for IS min at room It is known that hexokinase exists in two forms, et a/., 1960). In prelimitemperature (AoE, 1974; NOVIKOFF the cytoplasmic enzyme and the particulate or mitonary experiments, hexokinase activity was assayed (CHOU chondria] form (KELLOGGet al., 1974). While it might & WILSON,1972) in homogenates (prepared in 0.25 M-SUCbe expected that the particulate enzyme would remain r O S d . 5 % Triton X-100) of unfixed and acetone-fixed sections; acetone fixation resulted in less than 10% decrease in the section during the incubation procedures, the in hexokinase activity of the sections. The sections were cytoplasmic (soluble) enzyme could easily be lost into stained for hexokinase by incubating for 2 M 5 min at 37°C the medium. This is a problem common to histoin a medium containing 3.3 mM-gluCOSe, 6.7 rnM-ATP, chemistry when such enzymes as lactate dehydrogen6.7 mM-MgCI,, 40 mM-N-2-hydroxyethylpiperazine-N’-2- ase and aldolase are investigated (WACHSMUTH, 1976). ethanesulfonic acid (HEPES), pH 6.5, 0.64 mM-NADP, 2 Indeed, in preliminary experiments it was found that units/ml of glucose-6-phosphate dehydrogenase, there was a substantial amount of reaction product 10 mM-KCN, 0.4 mg/ml nitro blue tetrazolium (NBT) and 0.08 mg/ml phenazine methosulphate. This medium rep- in the staining medium following incubation of unresents a modification of that used routinely for spectro- fixed sections. However, when cryostat sections were photometric assay of hexokinase (CHOU& WILSON,1972). prefixed in cold acetone or treated by the procedure (1976) (See Methods), no Control sections were incubated in media lacking ATP or advocated by WACHSMUTH glucose. Following incubation the sections were rinsed in reaction product was seen in the medium. In addition, distilled water and mounted in glycerol/PBS, 9 : l . direct assay for hexokinase in the histochemical stain(b) This method is essentially as recommended by ing medium overlying acetone-fixed sections disclosed WACHSMUTH (1976). Cryostat sections were covered with no detectable release of the enzyme during the incubarabbit anti-rat brain hexokinase antiserum, diluted 1 :10 tion. Hence, the distribution of both soluble and parwith PBS, for 30min at room temperature. They were ticulate forms of the enzyme should be represented washed twice (2 x 15 min) by immersion in PBS and then incubated in a solution of purified HK ( - 3 u/ml). The in the sections. hexokinase was removed and the sections given two washes as before. They were then incubated in the staining Cerebellum medium as in (a) above. The methylene blue stained cryostat section in Fig. Immunojuorescence procedure. Acetone-fixed sections’ were incubated in a 1:10 dilution of antiserum or preim- 2A clearly shows the typical layered structure of the mune serum (diluted with PBS) for 30min at room tem- cerebellar cortex; apparent are the molecular, Purperature (2045°C) in a humid chamber. The cover slips kinje cell and granule cell layers as well as white fiber were then washed twice by immersion in PBS for 15 min tracts. The myelinated fiber tracts were only faintly each time. The sections were covered with fluorescein con- fluorescent (Fig. 2B, C). The spidery fluorescent projugated goat anti-rabbit IgG, at a dilution of 1 : l O with cesses of glial cells were seen interspersed among the PBS, for 30 min. Following this incubation the cover slips fibers; while these cells cannot be identified with cerwere washed as described previously and mounted in gly- tainty by the present procedures, it seems most likely cerol/PBS, 9 : l . Sections were viewed with a Zeiss Photothat these are fibrous astrocytes. The distribution of microscope 111 and photographs taken using Kodak Ektachrome film. Black and white negatives and prints fluorescence within the granule cell layer was distinctly punctate (Fig. 2B). This pattern of fluorescence were made from the original color exposures. is in keeping with the distribution of the cerebellar glomeruli, terminal structures containing high conRESULTS centrations of mitochondria (PALAY& CHAN-PALAY, Rabbit anti-rat hexokinase antiserum 1974). At higher magnifications (Fig. 2C) the nonTypical Ouchterlony double immunodiffusion fluorescent nuclei of the granule cells were seen to results are shown in Fig. I. Both crude and purified be clearly outlined by fluorescent cytoplasm ; the flumitochondria1 hexokinase (CHOU& WILSON,1972) as orescence of the glomeruli was much more intense well as the cytoplasmic enzyme (see below) gave a than that observed in granule cell cytoplasm. Except single, continuous immunoprecipitin band against for the non-fluorescent cell nuclei, the molecular layer rabbit anti-rat brain hexokinase serum, indicating showed a rather even distribution although a slightly that the antiserum was able to selectively react with speckled appearance suggested some small local conboth the mitochondria1 and cytoplasmic forms of the centrations of hexokinase. A similar speckled appearance was also noted in other brain regions (e.g. the enzyme. periventricular region shown in Fig. 4A); at the level of resolution afforded by light microscopy it is not possible to make any substantive comment about the ’ Although acetone-fixed sections were used in the ex- possible morphological origin of these apparent locaperiments presented here, the distribution of hexokinase was indistinguishable in acetone-fixed and unfixed sec- lizations. The cytoplasm of the Purkinje cell bodies tions; thus the initial incubations with antiserum appeared (Fig. 2B, C) displayed a fluorescence somewhat less to have successfully prevented release of the enzyme from bright than the adjacent molecular layer but considerthe sections which is consistent with the findings of ably brighter than that of the white fiber tracts. SecWACHSMUTH (1976). tions treated with preimmune serum, o r with immune

1042

G . P. WILKINand J. E. WILSON

serum from which the antibodies had been removed with an excess of pure hexokinase, displayed no fluorescence (Fig. 2D). Consistent with the immunofluorescence pattern, the most concentrated deposits of purple formazan in the histochemical study were over the cerebellar glomeruli (Fig. 2E). The general distribution and intensity of staining over the molecular layer, Purkinje cell bodies and white fiber tracts paralleled the relative intensities of fluorescence, with the first two staining shades of light purple while the latter appeared more pink. Purple formazan has been determined as being the fully reduced diformazan and the pink or red color as the half reduced form (ALTMAN& BUTCHER,1973). ALTMAN& BUTCHER(1973) have demonstrated that the red, half-reduced form is produced initially and the purple diformazan second. Hence, it might be expected that in areas of low enzyme activity the pink form would be seen while in areas of high activity the purple form would predominate. This appeared to be true in the cerebellum where the cerebellar glomeruli were purple while the less reactive myelinated fiber tracts were pink. Sections incubated without ATP or without glucose showed no staining other than a uery faint pink stain in the white fiber tracts that would appear to be artifactual (Fig. 2F). Hippocampus Cryostat sections of the rat brain cut to reveal the hippocampus also included the lateral ventricles. Although the hippocampus possesses a layered structure not unlike the cerebellum (Fig. 3A), the fluorescence, in contrast to that in the cerebellum, was rather uniformly distributed, broken only by the nonfluorescent nuclei of the various cell bodies (Fig. 3B). Myelinated fiber tracts coursing around the hippocampal region were only faintly fluorescent (Fig. 3B), as were the fiber tracts of the cerebellum (Figs. 2B, C). The ependymal cells lining the lateral ventricles and the choroid plexus exhibited an exceptionally bright fluorescence, an observation described more fully below. When examined by the histochemical procedure, the hippocampus was stained throughout its entire area except for the readily observable cell nuclei. Closer examination of the pyramidal cell layer revealed that the lightly stained cytoplasm of the neuronal cell bodies was surrounded by more darkly

' An examination of the ventricle regions shown in Figs. 3 8 and 4C and D reveals that portions of the ventricular surface are devoid of the normal lining by ependymal cells. Such regions were seen in several sections prepared in the course of the present studies, but we have not made a specific attempt to search for them nor to delineate their exact distribution in the ventricular system. An absence of ependymal cells in some regions of the lateral ventricle of the dog has previously been reported (ALLEN & LOWE, 1973) but we are not aware of previous descriptions of similarly deficient regions in the rat.

stained deposits (Fig. 3C) which could possibly be due to the various nerve terminals (including the large mossy terminals originating in the granule cell layer) which are known to synapse on cells in the pyramidal layer (for review, see ISAACSON & PRIBRAM,1975). Ventricles Both the ependymal cells lining the ventricles and the cell bodies of the choroid plexus epithelium displayed brightly fluorescent cytoplasm surrounding the darker nuclei (Fig. 3B, 4A-D).' The cuboidal ependyma1 and choroid plexus cells also displayed highly localized accumulations of fluorescence following treatment with preimmune serum (Figs. 4B, D). The staining was not found throughout the cytoplasm as was the case with the immune serum (Fig. 4A, C), but appeared to be localized at a point near the ventricular surface of the cells. The fluorescent spot was always smaller in size in the choroid plexus cells compared with the cuboidal ependyma. At the present time we have no explanation for this phenomenon. Histochemical staining of the ventricular region (Fig. 4E and F) confirmed the conclusion drawn from immunofluorescence. There was a pink staining of the fiber tracts surrounding the ventricle, while the ependymal lining was more purple. Dense formazan deposits were clearly apparent in the cytoplasm of the choroid plexus cells. In all regions, the unstained nuclei were highly visible against their darker surroundings. Retina The various layers of the retina may be seen in the methylene blue stained cryostat section (Fig. 5A). Immunofluorescence was obvious in every layer with the exception of the outer segments of the photoreceptor cells (Fig. 5B), the brightest accumulations being in the inner segments of the receptor layer and the outer and inner plexiform layers. The dark nuclei of the cell bodies of the outer and inner nuclear layers and ganglion layer were surrounded by fluorescent cytoplasmic areas. Section exposed to preimmune instead of immune serum were almost devoid of fluorescence apart from a relatively slight green fluorescence in the outer plexiform layer and very faint green fluorescence most noticeable near the outer portions of the inner segments of the photoreceptors (Fig. 5C). The deposition of formazan following the histochemica1 procedure was again in good agreement with the fluorescence data except that dark pink deposits of the formazan were also seen in the outer segments of the receptor cell layer in contrast to the total lack of fluorescence seen in this region. Though not obvious in the black and white photograph (Fig. 5D), this pink color was different from the purple formazan of the other deposits throughout the retina section. Sections incubated without ATP or glucose revealed only a uery light pink stain throughout the cell layers (Fig. 5E); hence the staining in the outer

1043

FIG.1. Ouchterlony double immunodiffusion analysis of rabbit anti-rat brain hexokinase serum. The center well contained antiserum prepared against pure mitochondrial hexokinase. Wells 1 and 3 contained crude (80 u/ml) and pure (65 u/ml) mitochondrial hexokinase, respectively. Well 2 contained cytoplasmic hexokinase (8.9 u/ml). The mitochondrial hexokinase samples were purposely run at high concentrations ( - 1 mg/ml) in order to better detect possible additional precipitin bands (none were seen); this resulted in a slight diffusiveness on l h e antigen side of the precipitin line (wells 1 and 3) not seen if lower concentrations were used. It was not practical to run the cytoplasmic enzyme at higher concentration due to the already high protein concentrations in the sample. The plate was photographed after 18 h at 23°C.

.?

1

1

1

--I.--

-A

^"

FIG. 2. Cerebellum. A, Methylene blue stained cryostat section: f, white fiber tracts; g, granule cell layer; p, Purkinje cell layer; m, molecular layer. Bar = 160pm. B, Localization of hexokinase by immunofluorescence. Bar = 160pm. C, Finer details of fluorescence distribution in cerebellum. Arrows indicate Purkinje cells. Bar = 60 pm. D, Immunofluorescence control. Immune serum from which the antibodies had been precipitated with pure hexokinase was used; comparable results were obtained with preimmune serum. Bar = 60 pm. E, Histochemical staining for hexokinase. Reactive Purkinje cell bodies (arrows) with unstained nuclei are seen at the border of the granule cell and molecular layers. Bar = 160prn. F, Histochemical stain for hexokinase with ATP deleted from reaction mixture. Identical results were obtained if glucose . . * . * A"--.A..L.-A L. A nnr- im..,

FIG.3. Hippocampus and lateral ventricle. A, Methylene blue stained section of part of hippocampus showing pyramidal cell (p) and granule cell (g) layers. Small dark nuclei of glial cells are interspersed among the neuronal cell bodies. Bar = 160pm. B, Fluorescence micrograph of part of the hippocampus (p, pyramidal cell layer; s, stratum oriens) and the adjacent lateral ventricle lined in part by ependymal cells (e) and containing the choroid plexus (arrow). The neuropil of the hippocampus, ependymal cell bodies and choroid plexus are brightly fluorescent in contrast to the dull fiber tracts (f). Bar = 160 pm. C, Histochemical staining of the hippocampal pyramidal cell layer. Arrows indicate dense formazan deposits at periphery of neuronal cell bodies. Bar = 60 pm.

1046

FIG.4. Choroid plexus and ependymal cells. A, lmmunofluorescent staining of ependymal cells lining the Duct olsylvius. Portions of the surface become quite tortuous in this region. B, Immunofluorescence control, using preimmune serum. C, Immunofluoresceiit staining of choroid plexus epithelial cells. Note the markedly diminished fluorescence of the internal, endothelial cells (arrows). D, Immunofluorescence control, using preimmune serum. E, Histochemical staining of choroid plexus (in center of picture) and adjacent ependymal lining (arrows). Surrounding fiber tracts appear more pink (see text). F, Histochemical control, in which ATP was deleted from the reaction mixture. In A, B, E, and F. bar = 160 pm: in C and D, bar = 6 0 p n .

1047

FIG.5. Retina. A, Methylene blue stained section: 0,outer segments of receptor cells; i, inner segments of receptor cells; on, outer nuclear layer; op, outer plexiform layer; in, inner nuclear layer; ip, inner plexiform layer; gl, ganglion layer. B, Fluorescence micrograph showing fluorescence in every layer except the outer segments of the receptor cells. Brightest accumulations are in the inner segments of the receptor cells and in the outer and inner plexiform layers. C , Fluorescence micrograph of section treated with preimmune serum. D, Histochemical staining for hexokinase. All layers are stained including probably artifactual staining (see text) of the outer segments of the receptor cells. E, Histochemical staining with ATP deleted from the reaction mixture. In A-E, bar = 60pm.

FIG.6 . Dorsal Root Ganglion. A, Methylene blue stained section. B, lmmunofluorescent staining_pat. tern. C. Control (preimmune serum). D, Histochemical staining of dorsal root ganglion section. E, Control, ATP deleted from histochemical staining medium. In A, bar = 160 pm; B E at same magnification as A.

Localization of hexokinase receptor segments was dependent on these substrates being present in the medium. It seems clear that the histochemical staining of the outer segments, though dependent on hexokinase activity, is nevertheless artifactual in origin since both immunofluorescence (this paper) and microdissection techniques (LOWRYet al., 1961) indicate the absence of appreciable amounts of this enzyme in the outer segments. We suggest that the source of this artifact may lie in the existence of a region having high hexokinase activity (the inner segments) adjacent to the extremely lipid-rich outer segments (RAVIOLA & RAVIOLA, 1962; FLEISCHER & MCCONNELL, 1966). Abundant formazan would be generated in the inner segments, while the lipid-rich outer segments may act as a ‘sink’due to the appreciable lipid solubility of the formazan, particularly the monoformazan (PEARSE, 1972) which would account for the pink color (characteristic of the monoformazan) of the outer segments. Dorsal root ganglion As is readily seen in the methylene blue stained section (Fig. 6A), the dorsal root ganglion contains a markedly heterogeneous population of neurons, with the largest being several times the size of the large cerebellar Purkinje cells (cf. Figs. 2A or B and 6A); in addition to the larger, more readily distinguishable neuronal population the much smaller satellite (or glial) cells m a y be seen interspersed among the neurons, and in the fiber tracts. Both immunofluorescence (Fig. 6B, C) and histochemical (Fig. 6D, E) methods demonstrated a marked variation in hexokinase levels with the smaller neurons being particularly rich in this enzyme. In addition, brightly fluorescent processes (Fig. 6B) were seen to surround the periphery of many of the neurons (this contrast was most evident with the large neurons whose own cytoplasm was relatively dimly fluorescent) and are attributed to the processes of the satellite cells which ensheath the neurons (PETERS et al., 1976). In accord with this, the histochemical procedure also demonstrated densely stained satellite cells surrounding and interspersed among the neurons (Fig. 6D). DISCUSSION

The various layers within the cerebellum were strikingly differentiated by both histochemical and immunofluorescence procedures. The relatively low hexokinase content of fiber tracts, seen in both the immunofluorescence and histochemical experiments, is in accord with previous direct measurements of hexokinase activity in white fiber tracts (BIGLet a/., 1971 ; BUELLet al., 1958; MCDOUGALet al., 1961). Perhaps the most notable aspect of the distribution of hexokinase in the cerebellum was the punctate appearance within the granule cell layer. This distribution conforms to that of the large cerebellar glomeruli. The concentration of hexokinase in such discrete locations assumes greater import when con-

104Y

sidered against the backdrop of the microdissection studies of BUELLet al. (1958) in which the hexokinase activity of the ‘cell body layer’ (i.e. granule cell layer) was found to be intermediate between that of the molecular layer and the white fiber tracts. The present results now show that this intermediate activity in the entire layer must in fact be a composite of the lower levels found in the bulk of the layer and the concentrations found in the cerebellar glomeruli; the hexokinase activity in the latter must be very high indeed. Cerebellar glomeruli are complex structures composed of mossy fiber terminals, dendritic digits of the granule cells, and axon terminals of Golgi neurons (HAJOS& WILKIN,1975). At the level of resolution provided by the light microscope, it could not be determined whether or not hexokinase was pkesent in all of these component structures. Previous work (WILSON,1972) has strongly suggested that a high proportion (4&50%) of the total hexokinase of brain is localized in the nerve endings, being entrapped within synaptosomes (i.e. pinched off nerve endings) when brain is homogenized; hence it would appear likely that at least the mossy fiber terminals and Golgi endings contain substantial levels of hexokinase. Additional information concerning distribution of hexokinase among the discrete structural components of the cerebellar glomeruli will have to await the results of planned electron microscopic studies. The latter will also provide better information concerning the distribution of hexokinase among the various structural elements of the molecular layer. Aside from the artifactual staining of the outer segments by the histochemical procedure, as noted above, the distribution of hexokinase in the various layers of the retina was found to be in excellent agreement with the results obtained by LOWRYet al. (1961), with the inner segments of the photoreceptor cells being particularly rich in hexokinase, while the outer segments and outer nuclear layer were notably deficient in this enzyme; the remaining layers exhibited intermediate levels of activity. Although the hippocampus, like the cerebellum and retina, possesses a distinctly layered structure, both the immunofluorescence and histochemical procedures (this paper) as well as direct assay of hexokinase in the dissected layers (BUELLet al., 1958) indicated a rather uniform distribution of hexokinase throughout the hippocampus which was certainly a dramatic difference from the marked variations among the cerebellar and retinal layers. We are not aware of previous reports of a relatively high level of hexokinase in ependymal cells or the choroid plexus. This was, however, dramatically evident in the present studies. The ciliated ependymal cells of the ventricular lining (PETERS, 1974) may reasonably be expected to require an abundant supply of energy for maintenance of ciliary motion. Similarly, the secretory role of the choroid plexus must also represent a substantial energy demand and, in fact, the choroid plexus has been estimated to utilize

1050

G . P.WILKINand J. E.

oxygen at a rate twice that of gray matter (for review, see CSAKY,1969). Thus, the finding of high levels of an enzyme of such central importance in energy metabolism in these cells is consistent with the frequent assumption, mentioned in the introduction, that metabolic needs are generally reflected in the levels of relevant enzymes. Implicit in this is the corollary that glucose represents the prime substrate for energy metabolism in the ependyma and choroid plexus. And now we would like to offer some general comments about the observations reported here. Previous studies (BENNETT et al., 1962; BIGLet al., 1971) have suggested a relatively higher lever of hexokinase in neurons than in glia. The basis for this suggestion was essentially that dissected gray regions (expected to be rich in neuronal elements) had higher hexokinase activities than did white regions (expected to contain substantial glial elements). The present results provide additional information concerning this question. Within gray regions, we were never able to see glia that were obviously deficient in hexokinase and thus their level of activity was at least comparable to that in surrounding neuronal structures. In fact, in dorsal root ganglion (Fig. 6B, C), it was evident that the hexokinase levels in the satellite glial cells actually exceeded that in the adjacent large neurons. Furthermore, the immunofluorescence procedure revealed many threadlike glial processes within myelinated fiber tracts (particularly noticeable in the cerebellum, Fig. 2B, C). And in the retina and cerebellum there was no obvious deficiency of hexokinase in the regions expected to be occupied by glial structures, e.g. the giant Muller cells in retina or the Bergmann glia of the cerebellum. In general, therefore, these results do not support the view that glia are characteristically low in hexokinase activity. Nor was the staining of neuronal cell bodies especially striking-though there clearly was staining of the neuronal cytoplasm, it was not remarkably more intense than, e.g. surrounding neuropil. In agreement with the results of KATO& LOWRY(1973u), it was quite evident that the relative hexokinase levels varied markedly among different neuronal types (cf. the large and small neurons of the dorsal root ganglion, or the neurons of the inner and outer nuclear layers of the retina). Thus, high levels of hexokinase are not universal characteristics of neurons. Clearly, the variations in hexokinase levels, and presumably in other metabolic properties, depends greatly on the specific type of neuronal or glial cell. While the classification of neural cells as neurons or glia may have a certain usefulness, it would appear that the metabolic properties of these cells cannot be so neatly compartmented. A rather marked staining was observed in several regions expected to be rich in nerve endings, e.g. the cerebellar glomeruli, the periphery of neurons in the pyramidal layer of the hippocampus, and the inner and outer plexiform layers of retina. We suggest that the relatively higher levels of hexokinase observed in rt al., 1962; BIGL et a/., 1971) gray regions (BENNETT

WlLSON

were not due to high levels of the enzyme in neuronal cell body cytoplasm but rather, due to relatively high concentrations of the hexokinase in nerve terminals also expected to be abundant in gray regions. Thus, the present studies are consistent with previous subcellular fractionation studies (WILSON,1972) in suggesting a relative concentration of hexokinase in nerve terminals. Considered in conjunction with the ‘high affinity’ transport system for glucose in nerve endings (DIAMOND& FISHMAN,1973), this suggests to us that the nerve ending is a structure well-fitted for efficiently acquiring glucose and introducing it into glycolytic metabolism. Teleologically, it seems quite reasonable that structures directly concerned with what is perhaps the most unique and critical function of the brain, neurotransmission, should also be well adapted for generation of the energy required to support this function. Neither of the procedures used here revealed observable hexokinase activity in cell nuclei-the nuclei were always remarkably noticeable by the absence of fluorescence or formazan reaction product. This is consistent with the absence of appreciable hexokinase activity in the ‘nuclear’ fraction obtained by subcellular fractionation procedures (JOHNSON, 1960; WILSON,1967). Thus, three distinct methods--conventional subcellular fractionation (JOHNSON, 1960; WILSON, 1967), immunofluorescence, and histochemistry-yield results indicating the absence of appreciable hexokinase in the nuclei of cells in rat neural tissue. LQWRYet ul. (1961) have similarly concluded that the nuclei of retinal rod cells “must be practically devoid of hexokinase.” In contrast, KATO& LDWRY (19736) reported substantial hexokinase activity in nuclei dissected from rabbit dorsal root ganglion cells. However, the data presented show exceptionally wide variations (in fact, it is difficult to reconcile the wide range of activities of the enzymes shown in Figs. 1 and 2 of KATO& WRY (1973b) with the relatively small ‘standard errors’ shown in their Table I ) and apparent inconsistency (e.g. it is concluded that the cytoplasmic levels of glucose-6-P dehydrogenase exceed nuclear levels whereas in fact, of the 14 values for cytoplasmic samples shown in their Fig. 2, almost h a l f 4 out of 14-show cytoplasmic levels equal to or less than the corresponding nuclear levels; comparably detailed data for hexokinase were not given). Considering the exceedingly delicate operations required for assay of enzyme activities in dissected nuclei and cytoplasmic samples, it is understandable that such difficulties would be encountered. Nevertheless, we do not feel that the available data convincingly demonstrates the existence of high levels of hexokinase in nuclei of rabbit dorsal root ganglion cells and certainly does not provide a basis for inferring the presence of high levels of hexokinase in nuclei of neural cells in the rat which, on the basis of the present results, are essentially devoid of hexokinase. While we obviously can not exclude the possibility that sub-detectable levels of hexokinase might indeed

Localization of hexokinase be found in the nuclei, we do conclude that the cytoplasmic levels of this enzyme must greatly exceed those of the nucleus. Ackn&vledgements-We are very grateful to Dr. SIDNEY LEIBOWITZ, Guy's Hospital Medical School, London, England, for performing the first immunofluorescence experiments, and for his interest and advice during the conduct of this work. We are also pleased to acknowledge the contributions of Sr. MARYANN SIMURDA, G . N. S. H., to the initial stages of this work. Special thanks go to Dr. REX CARROW (Anatomy Dept., Michigan State University) and his colleagues (especially D. ULMERand B. WHEATON) for generously allowing us the use of their cryostat as well as providing advice on sectioning procedures, and to Dr. ROBERT LEADER (Pathology Dept., Michigan State University) for allowing us the use of his fluorescence microscope. We also thank Dr. C. M. MANTHORPE, JR., for assistance in preparing the sections shown in Fig. 4A-D. Financial support provided by NIH Grant NS 09910 is gratefully acknowledged, as is the award of a Wellcome Research Travel Grant to G.P.W.

REFERENCES

ALLEN D. J. & Low F. N. (1973) Am. J . Anatomy 137, 483489. ALTMAN F. P. & BUTCHERR. G. (1973) Histochemie 37, 333-350. AOE H. (1974) Acta Histochem. Cytochem. 7. 184190. BACHELARD H. S. (1970) in Handbook of'Neurochemistry (LAJTHAA,, ed.) Vol. 2, pp. 1-12. Plenum Press, New York. BALAZSR. (1970) in Handbook of Neurochemistry (LAJTHA A., ed.) Vol. 3, pp. 1-36. Plenum Press, New York. J. E. (eds.) (1972) Metabolic CompartBALAZSR. & CREMER mentation in the Brain. John Wiley, New York. D. (1972) in Metabolic BALAZSR., PATELA. J. & RICHTER Compartmentation in the Brain (BALAZSR. & CREMER J. E., eds.) pp. 167-184. John Wiley, New York. BENNETT E. L., DRORIJ. B., KRECHD., ROSENZWEICM. R. & ABRAHAMS. (1962) J . bid. Chem. 237, 1758-1763. BERLS., CLARKE D. D. & SCHNEIDER D. (eds.) (1975) Metabolic Compartmentation and Neurotransmission. Plenum Press, New York. BIGLV., BIESOLDD., DOWEDOWA E. L. & PIGAREWA S. D. (1971) Acta Biol. Med. Germ. 26, 27-33. BUELLM. V., LOWRY0. H., ROBERTSN. R., CHANGM. W . & KAPPHAHN J. 1. (1958) J . bid. Chem. 232, 979-993. BUTCHER R. G. & ALTMAN F. P. (1973) Histochemie 37, 351-363. CHOUA. C. & WILSONJ. E. (1972) Archs Biochem. Biophys. 151, 48-55. CSAKYT. Z . (1969) in Handbook of Neurochemistry ( L A I T H A A,, ed.) Vol. 2, pp, 49-69. Plenum Press, New York.

1051

DIAMOND I. & FISHMAN R. A. (1973) Nature 242, 122-123. FLEISCHER S. & MCCONNELL D. G. (1966) Nature 212, 1366-1367. GARFINKEL D., ACHS M. J. & GARFINKEL L. (1972) in Metabolic Compartmentation in the Brain (BALAzs R. & CREMER J. E., eds.) pp. 353-358. John Wiley, New York. HAJOSF. & WILKING. P. (1975) in Metabolic Compartmentation and Neurotransmission (BERL S., CLARKED. D. & SCHNEIDER D., eds.) pp. 403-415. Plenum Press, New York. ISAACWN R. L. & PRIBRAM K. H. (eds.) (1975) The Hippocampus, Vol. 1. Plenum Press, New York. JOHNSONG. D. & HOLBOROW E. J. (1973) in Handbook of Experimental Immunology (WEIR D. M., ed.) Vol. 1, Ch. 18. Blackwell, Oxford. JOHNSONM. K. (1960) Biochem. J . 77, 61G618. KATOT. & ~ W R Y0. H. (1973a) J . Neurochem. 20, 151-163. KATOT. & LOWRY0. H. (19736) J . biol. Chem. 248, 20442048. KELLOCCE. W., KNULLH. R. & WILSONJ. E. (1974) J . Neurochem. 22, 461463. LOWRY0. H., ROBERTS N. R. & LEWISC. (1956) J . biol. Chem. 220, 879-892. LOWRY0. H., ROBERTS N. R., SCHULZD. W., CLOWJ. E. & CLARKJ. R. (1961) J . b i d . Chem. 236, 2813-2820. LOWRY0. H., PASSONNEAU J. V., HASSELBERGER F. X. & SCHULZD. W . (1964) J . biol. Chem. 239, 18-30. MATSCHINSKY F. M. (1970)in Biochemistry of Simple Neuronal Models (COSTA E. & GIACOBINIE., eds.) pp. 217-243. Raven Press, New York. D. B., JR., SCHULZD. W., PASSONNEAU J. V., MCDOUGAL CLARKJ. R., REYNOLDS M. A. & LQWRY0. H. (1961) J . Gen. Physiol. 44, 487498. MCLAUGHLIN B. J., WOOD J. G., SAITOK., BARBERR., VAUGHNJ. E., ROBERTS E. & WU J-Y. (1974) Brain Res. 16, 377-391. NOVIKOFF A. B., SHINW-Y. & DRUCKER J. (1960) J . Histochem. Cytochem. 8, 3740. PALAYS . L. & CHAN-PALAY V. (1974) Cerebellar Cortex, Cytology and Organization. Springer, New York. PEARSE A. G . E. (1972) Histochrmistry, pp. 88G920. Longman, New York. PETERS A. (1974) J. Neurocytol. 3, 99-108. PETERSA,, PALAY S. L. & WEBSTERH. DEF. (1976) The Fine Structure of the Nervous System. Saunders, Philadelphia. RAWOLA E. & RAVIOLA G. (1962) Z . Zellforsch. 56, 552-572. SUGDEN P. H. & NEWSHOLME E. A. (1973) Biochem. J . 134, 97-101. WACHSMUTH E. D. (1976) Histochem. J . 253-270. WILSONJ. E. (1967) Biochem. biophys. Res. Commun. 28, 123-1 27. WILSONJ. E. (1972) Archs Biochem. Biophys. 150, 96-104.