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Topography of basal glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose
03o6-4522/88 $3.00 + 0.00
~euro~cim~e vol. 24, NO. 3, pp. 877-883, 1988 Printed in Great Britain
PergamonPress plc 0 1988 IBRO
TOPOGRAPHY QF BASAL GLUCOSE UTILIZATION IN THE HIPPOCAMPUS DETERMINED WITH [1J4C]GLUCOSE AND [6-14C]GLUCOSE G. E. DUNCAN,* W. E. STUMPF,* 0. BR~STLE,~ B. S. GIVENS,* G. R. BREESE*and C. PrLGRrhft *Department of Anatomy and Biological Sciences Research Center, University of North Carolina at Chapel Hill, NC 27514, U.S.A.; TAbteilung Anatomie und Zellbiologie, Universitiit Ulm, Ulm, D-7900, F.R.G.
Abstract-The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [ 1-“Qlucose and [6-‘Q3lucose and their “C-1abelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of “C occurred in the granule cell layer of the dentate gyms and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosurn-moleculare. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [I-‘4c]glucose and [C”c]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [I-“CJglucose. Although there was significantly more radioactivity at 30min than at lOmin, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1-“CJglucose utilized in different hippocampal regions is oxidized to “CO, under these conditions. Most regions of high glucose utilization in the hippocampus determined with [I-“Clglucose and [6-‘4C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature. In regions that exhibit low cytochrome oxidase activity and high [I-“C]glucose utilization, high nicotinamide adenine dinucleotide phosphate-diaphorase activity is present and high amino acid uptake occurs. These observations suggest that measurement of the uptake and retention of radioactivity from [I-“Qlucose and [6-“Qlucose provides an index of glucose utilized for biosynthetic mechanisms as well as for energy transducing processes.
Glucose is utilized ducing
processes
by the brain for energy transand for biosynthetic mechanisms.
The carbon skeletons of dietary non-essential amino acids are derived primarily from glucose molecules.‘2 Among the many other compounds which are also synthesized in part from carbon atoms of glucose in brain tissue are nucleic acids, lipids, inositol and acetylcholine. I2 Although glucose is ultimately oxidized to CO2 and H,O, the rate of evolution of 14C02 from “C-labelled glucose depends critically on the position of 14Cin the molecule.‘9J” Carbon atoms in positions C-l and C-6 are oxidized to CO, at a much slower rate than the other carbon atoms in glucose molecules.20*2’ The C-l and C-6 carbon atoms of glucose are metabolically equivalent in the glycolytic pathway and in the Krebs cycle. However, in the pentose phosphate shunt, the C-l atom is oxidized to CO2 but the C-6 atom is not.8 The functions of the pentose phosphate shunt are to generate reducing power in the form of nicotinamide adenine dinuAbbreviations: [“Cl2-DG, [“Cl2-deoxyglucose; NADPH, nicotinamide adenine dinucleotide phosphate.
cleotide phosphate (NADPH) and to produce ribose for ribonucleic acid synthesis.12 An estimate of pentose phosphate shunt activity may be obtained in vivo by comparing the uptake and retention of radioactivity after injection of [6-‘4C]glucose and [1-‘4C]glucose. 2’ Under conditions of high pentose phosphate shunt activity, less radioactivity is expected to be retained from [l-‘4C]glucose than from [6-‘4C]glucose, due to differential oxidation to 14C02. The hippocampus contains the highest activity, as measured in vitro, of glucose-6-phosphate dehydrogenase,2 a primary enzyme of the pentose phosphate shunt. In the present study, pentose phosphate shunt activity in the hippocampus was assessed in vivo, by comparing 14C incorporation into radiolabeled metabolites from [ l-‘4C]glucose and [6-‘4C]glucose. The distinct histological stratification and connectivity of the hippocampus invite analysis of functional activity within this region by means of autoradiographic determination of local glucose utilization. Previous work from our laboratory demonstrated dramatically different patterns in the 811
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uptake and retention of radioactivity within the hippocampus. 30 min after injection of [I -‘4C]glucose or [‘4C]2-deoxyglucose ([‘4C]2-DG). Much higher levels of radioactive metabolites accumulated from [I-14C]glucose, compared to [r4C]2-DC, in the granular and molecular layers of the dentate gyrus and in the CA1 pyramidal cell field.3 A number of interpretations are possible for the different hippocampal patterns of [‘4C]2-DG and [I -‘4C]glucose autoradiograms. Accumulation of high levels of r4C from [ 1-‘4C]ghtcose, relative to 14C2-DG, could result from exceptionally low rates of loss of 14C0, from [I-‘4C]glucose in certain regions compared to others, or could result from loss of [r4C]2-DG-phosphate due to locally high glucose-6phosphatase activity. If differential oxidation of [I -‘4C]glucose to 14C02 is responsible for the differences in the hippocampal labeling patterns 30 min after injection of [l-‘4C]glucose or [14C]2-DG, the relative amount of radioactivity in the various hippocampal regions should be different at shorter intervals after injection of [ 1-‘4C]glucose. Therefore, in the present study, in addition to assessing pentose phosphate shunt activity, a detailed anatomical evalautouation was made with high-resolution radiography, of the uptake and metabolic trapping of radioactivity from [ 1-‘4C]glucose in the dorsal hippocampus, 10 and 30min after i.v. injection of this compound. EXPERIMENTAL
et
al.
and [6-‘4C]glucose, as determined by quantitation of silver grain densities in autoradiograms. revealed that the relative and absolute retentions of radiolabeled metabolites from each of the “C-labeled glucose analogs were not significantly different (Fig. I). The highest levels of radioactivity from both compounds occurred in the pyramidal cell layer and in the granular cell layer of the dentate gyrus. The molecular layer of the dentate gyrus and the stratum lacunosum-moleculare contained more radioactivity than the stratum radiatum and the stratum oriens. The topography of glucose utilization in the hippocampus is best visualized by inspection of autoradiograms exposed for several months (Fig. 2). As can be seen. the relative distribution of radiolabeled metabolites is nearly identical for the two compounds. Comparison of 10-min and 30-min survival periocis for
[ 1-“C]glucose The relative topographic distribution of radioactivity from [ 1-r4C]glucose among different hippocampal regions was indistinguishable in rats killed 10 or 30min after injection. However, more radioactivity was contained in all regions in rats killed 30min after injection, compared to the IO-mm survival period (Table 1). Low-power photomicrographs of autoradiograms from the two survival periods are
PROCEDURES
Male Charles River Sprague-Dawley rats, 35 days old, were implanted with chronic jugular catheters (Microrenathane, Brain Tree) and allowed to recover for 5 days before the experiment. Rats were injected via the catheter with [I-r4C]glucose or [6-‘4C]glucose (American Radiolabeled Chemicals, specific activity 55 Ci/mol) dissolved in 0.9% saline, at a dose of 0.5 $.2/g body weight. Rats injected with [I-‘4C]glucose were killed 10 or 30 min after injection and rats given [6-‘4C]glucose were killed 30min after injection by i.v. pentobarbital. Five rats were used for each condition. Brains were mounted on tissue holders, frozen in liquid propane cooled to - 180°C with liquid nitrogen, and stored in liquid nitrogen until sectioned. The autoradiographic procedures have been described in detaiL4.“r Cryostat sections (4pm) were thaw-mounted onto Kodak NTB-3 nuclear emulsion-coated slides and stored in light-tight desiccator boxes for periods of 6 days to 4 months until photographic development. Autoradiograms were developed in Kodak D-19 at 15°C for 3 min, washed in water, and fixed for 3 min. Radioactivity in hippocampal regions was determined by quantitative analysis of silver grain densities in autoradiograms exposed for 6 days using an Artek counter.’ Seven to ten readings were obtained bilaterally for each hippocampal subregion in two to three sections per rat. Statistical comparisons were made by repeated measures analysis of variance.
RESULTS
Comparison of [ 1-‘4CJgIwose and [6-‘4C]gkrcose The topographic distribution of radioactivity in the hippocampus 30 min after injection of 11-‘4C]glucose
Fig. 1. Silver grain densities in hippocampal re*ons from rats injected with [I-“C]$ucose or [6-‘*cJ8tucose. Lined bars: [I-“C]ghrcose; white bars: [6-“C]ghtcose. Rats were killed 30min after i.v. injection of the radiolabeled compounds. Silver grain densities were determined in autoradiograms exposed for 6 days. Data are mean + S.E.M.
Topo~aphy of basal glucose in hip~mpus
Fig. 2, A~to~~~ of the ~p~p~ after injection of [I-%Jgbcose or [6-1’Q&cose.. middle: [6-“C$&wose; bottom: hippocampal fiistolagy with anatomical designation [l-” ‘Q&we; Lorente de No.‘~ Exposure time: 3 months.
880
G. E. DUNCANet al. Table 1. Silver grain densities in different hippocampal regions
Silver grain density (grains/!000 pm* f S.E.M.) Hippoeampal region Fimbria CA3 pyramidal cells CA3 stratum oriens CA3 stratum radiatum CA1 pyramidal cells CA1 stratum oriens CA1 stratum radiatum Stratum lacunosum-moleculare Dentate molecular layer Dentate granular iayer
Ratio of silver grain density to that of CA1 stratum mdiatum
1Omin survival
20-min survival*
IO-min survival
30-min survival
10.2* 1.4 23.1 f 3.7 16.1* 3.1 15.4+ 2.5 20.3 f 3.4 14.2k 2.5 15.5f 2.9 23.3 + 3.3 18.8+ 2.9 23.3 + 3.8
14.7i 1.4 33.0 f 2.0 26.9 * 2.1 25.0 * 2.2 31.7k2.1 22.9 f 3.2 24.8 f 2.7 31.9 f 2.9 30.9 f 2.0 31.9 f 2.9
0.66 1.49 1.04 0.99 1.31 0.92 1.00 1.50 I.21 1.50
0.59 1.33 1.08 1.01 1.28 0.92 1.00 1.29 1.25 1.29
*Silver grain densities in all regions were significantly greater (P < 0.05) for the 30-min survival compared
to the IO-min survival.
shown in Fig. 3. After 10 and 30 min, the greatest retention of radioacti~ty occurred in the p~~i~l cell layer and in the granular layer of the dentate gyrus. In addition, a band of relatively high radioactivity extended from the dorsal limit of the stratum
lacunosum-moleculare and through the molecular layer of the dentate gyrus at both survival times. The stratum oriens and stratum radiatum exhibited relatively low retention of radioactivity from [ 1-t4C]glucose after both 10 and 30 min.
Fig. 3. Topography of accumulated mdioactivity in the hip~mpus 10 min and 30 min after injection of fl-‘4Clglucose.l Top: IO-min survival period; bottom: 30-min survival period. Exposure time: 4 months.
Topography of basal glucose in hippocampus
881
DISCUSSION
The results comparing the metabolic trapping of radioactivity from [I-r4Cjglucose and [6-‘4C]glucose indicate that under basal conditions, very low pentose phosphate shunt activity is operative. However, intense histochemical staining for glucose-6-phosphate dehydrogenase observed in the CA1 pyramidal regionI suggests that potentially great pentose phosphate shunt activity may occur in this region. Wholebrain pentose phosphate shunt activity accounts for only 3% of the total glucolytic flux under basal conditions in rats.“’ The results of the present study also indicate that in all hippocampal regions measured, the portion of glucose utilization devoted to the pentose phosphate shunt is very low in the awake, unstressed rat. Hawkins et ~1.~ found identical glucose utilization rates in many discrete brain regions using [I-r4C]glucose and [6-‘4C]glucose, similarly implying minimal pentose phosphate shunt activity under basal conditions. Under certain experimental and pathological conditions, however, a greater amount of glucose may be processed via the pentose phosphate shunt. For example, Sacks et aI.” found that following electroconvulsive shock in rats, the percentage of brain glucose metabolized by the shunt increased to 25%. Also, in brains of patients with Alzheimer’s disease, elevated pentose phosphate shunt enzyme activity has been reported.” The relative distribution of radioactivity from [l-‘4C]glucose was nearly identical for the lo- and 30-min survival periods although there was a net increase in silver grain density at the 30-min intervals in all regions. These results indicate that a similar fraction of the [l-‘4C]glucose that is transported into different hippocampal regions is oxidized to 14C02 over a 30-min period following intravenous injection of the radiolabeled sugar. The process of glucose utilization includes both catabolic reactions devoted to generation of adenosine triphosphate, and anabolic reactions in which carbon and hydrogen atoms of glucose molecules are
NADPH-DIwhor~w h Amino acid Uptake
Fig. 4. Schematic representation of autoradiograms and histochemistry in the hippocampus. Note that superimposition of the cytochrome oxidase pattern and the amino acid uptake_NADPH-diaphorase patterns would yield what approximates the glucose utilization pattern determined with specifically labeled [r4C]ghtcose, whereas the 2-deoxyglucose pattern matches only partially the cytochrome oxidase pattern. See text and Table 2 for sources of data from which the schematics were derived. Abbreviations: PYR, pyramidal cell layer; SLM, stratum lacunosum-moleculare; DM, dentate molecular layer; DG, dentate granule cell layer.
utilized for de nouo synthesis of neurochemicals. At present, controversy exists regarding whether the and retention of radioactivity from uptake specifically labeled [r4C]glucose, or that from [r4C]2-DG, provides the best index of brain glucose utilization.7.9*‘7,‘*~22 Some basis for deciding whether the accumulation of radioactivity from [14C]2-DG or [ l-‘4C]glucose best reflects hippocampal glucose utilization may be gleaned from descriptions of the topographic distribution of enzymes in the hippocampus which participate in the metabolism of glucose. Results of various studies in this regard are summarized in Table 2 and Fig. 4. Kageyama and Wong-Riley” found very intense histochemical staining for cytochrome oxidase, indicating high oxidative phosphorylation capacity, in the CA3 pyramidal cell
Table 2. Relative distribution of metabolic enzymes and radioactivity from [‘4C]glucose and [‘%]2-deoxyglucose in the rodent hippocampus
CA3 pyramidal cell bodies CA1 pyramidal cell bodies CA1 stratum radiatum Stratum lacunosummoleculare Dentate granule cell bodies Dentate molecular
Succinate Cytochrome oxidase” dehydrogenase6,16 Fumara@ (low-high)* high
[l-14C]Glucose [6-r4C]ghrcose
[r4C]2-Deoxy glucose’
high
high
high
low
low
(low-high)*
high
high
low
low
low
low
low
low
high
high
high
high
high
high
high
low
low
low
-
high
high
low
high
high
-
low
NADPH-diaphorase’6 high
*Friede6 reports high and Meyer et a1.16reports low succinate dehydrogenase activity in CA1 and CA3 pyramidal cell bodies.
882
G. E. DUNCANel cd.
field, the stratum lacunosum-moleculare, and the molecular layer of the dentate gyrus. In those regions, relatively high amounts of 14C are detected in [6-‘4C]glucose and [l-‘4C]glucose autoradiograms. In [“Cl2-DG autoradiograms, although high amounts of radioactivity are found in the CA3 pyramidal cell field and stratum lacunosum-moleculare, very low retention of radioactivity occurs in the molecular layer of the dentate gyrus.3.4 In the CA1 pyramidal cell field and granule cell layer of the dentate gyrus, cytochrome oxidase staining is very weak.” In these regions, the retention of radioactivity is higher for [6-‘4C]glucose and [ 1-‘4C]glucose, than for [‘4C]2-DG.3,4Although the CA1 pyramidal cell bodies exhibit low cytochrome oxidase staining, histochemical staining for succinate dehydrogenase, a component of the Krebs cycle, is reported to be high in the CA1 pyramidal cell layer.6 Biochemical assessment of fumarase activity in microdissected pyramidal cells from the CA1 region also indicates high activity of this Krebs cycle enzyme.‘4 However, another histochemical studyI found low levels of succinate dehydrogenase staining in the CA1 pyramidal cell field. Histochemical studies of oxidative enzymes in the dentate gyrus have yielded consistent results. Intense staining of succinate dehydrogenase and cytochrome oxidase is found in the molecular layer of the dentate gyrus and very low staining is apparent in the granular cell layer.6,“*‘6 As noted above, in the dentate molecular layer, relatively high levels of radioactivity accumulate from [l-‘4C]glucose and [6-‘4CJglucose and low levels from [14C]2-DG. In the dentate granule cell layer, autoradiograms manifest high retention of 14C-labeled metabolites of [l-‘4Clglucose and [6-‘4C]glucose but very low accumulation of ‘“C from [‘4C]2-DG. Although there is little cytochrome oxidase staining in CA1 pyramidal cells and dentate granule cells, these cell bodies stain intensely for NADPH-dependent tetrazolium reductase (NADPHstudies with diaphorase). I6 Also, autoradiographic [35S]methionine5 and [3H]leucine’ have demonstrated very high uptake of these dietary essential amino acids in the cell bodies of hippocampal pyramidal neurons from all fields, as well as in the cell bodies of dentate granule neurons. High levels of amino
acid uptake and intense histochemical staining fo’ NADPH-diaphorase activity, in regions of low capacity for oxidative phosphorylation, imply that glucose utilized in those regions may support biosynthetic processes to a greater extent than the generation of high-energy phosphate compounds. The biochemical reaction(s) catalysed by NADPH diaphorase have not been defined, but histochemical studies have demonstrated co-localization of this enzyme with specific transmitter systems in the superior colliculusz5 and in the striatum.24 Correspondence of identified transmitter systems and NADPHdiaphorase-containing neurons in the hippocampus has not be described. Although certain hippocampal regions (pyramidal cell layer and dentate granular layer) exhibit high [6-‘4C]glucose and [ 1-‘4C]glucose uptake and high amino acid accumulation, other regions of relatively high [6-‘4C]glucose and [l-‘4C]glucose uptake (stratum lacunosum-moleculare and dentate molecular layer) exhibit low amino acid accumulation. It is unlikely that radioactivity accumulated in the brain after [6-‘4C]glucose or [ l-‘4~glucose injection results from the uptake of peripheral metabolites of glucose such as amino acids. As noted above, the brain synthesizes non-essential amino acids primarily from glucose molecules. Therefore, radiolabeled amino acids derived from [‘4C]glucose in the brain most likely result from de ~OUOsynthesis within brain cells. The overall pattern of [6-‘4C]glucose and [l -‘4C]glucose autoradiograms of the hippocampus cannot be attributed to the presence and activity of a single enzyme system. Instead, the hippocampus glucose utilization pattern appears to correspond to a composite of the histochemical distribution of cytochrome oxidase, NADPH-diaphorase and essential amino acid uptake (Fig. 4), implying that distinct histological subdivisions of this brain region utilize glucose for different purposes. It remains to be investigated what functional significance can be attributed to the correspondence of patterns of glucose utilization and enzyme localization within the hippocampus. Acknowle~e~~ts-Supported by USPHS grants NS09914 and HDO3110and NATO grant 83/0576.
REFERENCES 1.
A&man J. (1963) Regional utilization of leucine-H3 by normal rat brain: microdensitometric evaluation of autoradiograms. J. Histochem. Cytochem. 11, 741-751. 2. Bnmnemann A. and Caper H. (1964) Die Aktivitat NAD- und NADP-abhiingiger Enzyme in verschiedenen Teilen des Rattengehirns. Arch. exp. Puth. Phawwk. uri, 493-503. 3. Duncan G. E., Pilgrim C., Stumpf W. E., MeCown T. J., Breese G. R. and Mueller R. A. (1986) High resolution autoradiographic determination of the topographic distribution of radioactivity in the hippoeampal formation after injection of (I-“C)-glucose or 2-deoxy(‘Q-glu. Neufvse&nce f7, 99-106. 4. Duncan G. E., Stumpf W. E., Pilgrim C. and Breeae G. R. (1987) High resolution autoradiography at the regidnal topographic level with (“C)-2-deoxyglucose and (3H)-3-deoxyglucose.J. Neurosci. Merh. 20, 105-l 13. 5. Plan&n S., Gabrieli E. R. and MaeLean P. D, (1957) Cerebral changes revealed by radioautography with Sfi:la&led L-methionine. Archs Neurol., Chicago 77, 588-594. 6. Friede R. L. (1960) Histochemicai investigations on succinic dehydrogenase in the central nervous system--Iv. A histochemical mapping of the cerebral cortex of the guinea pig. J. Neurochem. 5, 156-171.
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7. Hawkins R. A. and Miller A. L. (1978) Loss of radioactive 2-deoxy-o-glucose-6-phosphate from brains of conscious rats: implications for quantitative autoradiographic determination of regional glucose utilization. Neuroscience 3, 251-258. 8. Hawkins R. A. (1980) Glucose utilization determined in uiuo with [2i4C]glucose. In Cerebral Metabolism and Neural Function (eds Passonneau 1. V., Hawkins R. A., Lust W. D. and Welsh F. A.), pp. 367-381. Williams & Wilkins, Baltimore. 9. Hawkins R. A., Mans A. M., Davis D. W., Vina J. R. and Hibbard L. S. (1985) Cerebral glucose use measured with (14C) glucose labeled in the 1, 2, or 6 position. Am. J. Physiol. 248, C17(H3176. 10. Hostetler K. Y. and Landau B. R. (1967) Estimation of the pentose cycle contribution to glucose metabolism in tissue in vivo. Biochemistry 6, 2961-2964.
11. Kageyama G. H. and Wong-Riley M. T. T. (1982) Histochemical localization of cytochrome oxidase in the hippocampus: correlation with specific neuronal types of afferent pathways. Neuroscience 7, 2337-2361. 12. Lajtha A. L., Maker H. S. and Clarke D. D. (1981) Metabolism and transport of carbohydrates and amino acids. In Basic Neurochemistry (eds Siegel G. J., Albers R. W., Agranoff B. W. and Ketzman R.), pp. 329-353. Little, Brown and Company, Boston. 13. Lorente de No R. (1934) Studies on the structure of the cerebra1 cortex. II. Continuation of the study of the ammonic system. J. Psychol. Neurol. 46, 113-177. 14. Lowry 0. H., Roberts H. R., Leiner K. Y., Wu M. L., Farr L. A. and Albers R. W. (1954) The quantitative histochemistry of brain. III. Ammon’s Horn. J. biol. Chem. 207, 39-49. 15. Martins R. N., Harper C. G., Stokes G. B. and Masters C. L. (1986) Increased cerebra1 glucose-bphosphate dehydrogenase activity in Alzheimer’s Disease may reflect oxidative stress. J. Neurochem. 46, 1042-1045. 16. Meyer U., Ritter J. and Wenk H. (1971) Zur Chemodifferenzierung der Hippocampusformation in the postnatalen Entwicklung the Albinoratte. I. Oxydoreduktasen. J. Hirnforsch. 13(4), 236253. 17. Nelson R., Lucignani G., Atlas S., Dienel G. A. and Sokoloff L. (1985) Reexamination of glucose-6-phosphatase activity in brain in oiuo: no evidence for a futile cycle. Science, N. Y. 229, 60-62. 18. Nelson T., Lucignani G., Goochee J., Crane A. M. and Sokoloff L. (1986) Invalidity of criticisms of the deoxyglucose method based on alleged glucose-6-phosphatase activity in brain. J. Neurochem. 46, 905-919. 19. Sacks W. (1961) Cerebral metabolism of glucose-3-‘4C, pyruvatae-I-% and lactate-l-‘% in mental disease. J. appl. Phys. 200, 175-180. 20. Sacks W. (1965) Cerebral metabolism of double labeled glucose in humans in uivo. J. appl. Phys. 20, 117-130.
21. Sacks W., Sacks S., Badalamenti A. and Fleischer A. (1982) A proposed method for the determination of cerebral regional intermediary glucose metabolism in humans in viuo using specifically labeled “C-glucose and positron emission transverse tomography (PETT). 1. An animal model with i4C-glucose and rat brain autoradiography. J. Neurosci. Res. 7, 57-69. 22. Sacks W., Sacks S. and Fleischer A. (1983) A comparison of the cerebra1 uptake and metabolism of labeled glucose and deoxyglucose in oivo in rats. Neurochem. Res. 8, 661-685. 23. Stumpf W. E. (1976) Techniques for the autoradiography of diffusible compounds. In Methods in Cell Biology (ed.
Prescott D. M.), Vol. 13, pp. 171-193. Academic Press, New York. 24. Vincent S. R., Johansson O., Hijkfelt T., Skirboll L., Elde R. P., Terenius L., Kimmel J. and Goldstein M. (1983) NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities. J. camp. Neural. 217, 252-263. 25. Walter M. N. (1986) Spatial relationship of NADPH-diaphorase and acetylcholinesterase lattices in the rat and mouse superior colliculus. Neuroscience 19, 381-391. (Accepred 20 July 1987)
~euro~cim~e vol. 24, NO. 3, pp. 877-883, 1988 Printed in Great Britain
PergamonPress plc 0 1988 IBRO
TOPOGRAPHY QF BASAL GLUCOSE UTILIZATION IN THE HIPPOCAMPUS DETERMINED WITH [1J4C]GLUCOSE AND [6-14C]GLUCOSE G. E. DUNCAN,* W. E. STUMPF,* 0. BR~STLE,~ B. S. GIVENS,* G. R. BREESE*and C. PrLGRrhft *Department of Anatomy and Biological Sciences Research Center, University of North Carolina at Chapel Hill, NC 27514, U.S.A.; TAbteilung Anatomie und Zellbiologie, Universitiit Ulm, Ulm, D-7900, F.R.G.
Abstract-The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [ 1-“Qlucose and [6-‘Q3lucose and their “C-1abelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of “C occurred in the granule cell layer of the dentate gyms and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosurn-moleculare. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [I-‘4c]glucose and [C”c]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [I-“CJglucose. Although there was significantly more radioactivity at 30min than at lOmin, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1-“CJglucose utilized in different hippocampal regions is oxidized to “CO, under these conditions. Most regions of high glucose utilization in the hippocampus determined with [I-“Clglucose and [6-‘4C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature. In regions that exhibit low cytochrome oxidase activity and high [I-“C]glucose utilization, high nicotinamide adenine dinucleotide phosphate-diaphorase activity is present and high amino acid uptake occurs. These observations suggest that measurement of the uptake and retention of radioactivity from [I-“Qlucose and [6-“Qlucose provides an index of glucose utilized for biosynthetic mechanisms as well as for energy transducing processes.
Glucose is utilized ducing
processes
by the brain for energy transand for biosynthetic mechanisms.
The carbon skeletons of dietary non-essential amino acids are derived primarily from glucose molecules.‘2 Among the many other compounds which are also synthesized in part from carbon atoms of glucose in brain tissue are nucleic acids, lipids, inositol and acetylcholine. I2 Although glucose is ultimately oxidized to CO2 and H,O, the rate of evolution of 14C02 from “C-labelled glucose depends critically on the position of 14Cin the molecule.‘9J” Carbon atoms in positions C-l and C-6 are oxidized to CO, at a much slower rate than the other carbon atoms in glucose molecules.20*2’ The C-l and C-6 carbon atoms of glucose are metabolically equivalent in the glycolytic pathway and in the Krebs cycle. However, in the pentose phosphate shunt, the C-l atom is oxidized to CO2 but the C-6 atom is not.8 The functions of the pentose phosphate shunt are to generate reducing power in the form of nicotinamide adenine dinuAbbreviations: [“Cl2-DG, [“Cl2-deoxyglucose; NADPH, nicotinamide adenine dinucleotide phosphate.
cleotide phosphate (NADPH) and to produce ribose for ribonucleic acid synthesis.12 An estimate of pentose phosphate shunt activity may be obtained in vivo by comparing the uptake and retention of radioactivity after injection of [6-‘4C]glucose and [1-‘4C]glucose. 2’ Under conditions of high pentose phosphate shunt activity, less radioactivity is expected to be retained from [l-‘4C]glucose than from [6-‘4C]glucose, due to differential oxidation to 14C02. The hippocampus contains the highest activity, as measured in vitro, of glucose-6-phosphate dehydrogenase,2 a primary enzyme of the pentose phosphate shunt. In the present study, pentose phosphate shunt activity in the hippocampus was assessed in vivo, by comparing 14C incorporation into radiolabeled metabolites from [ l-‘4C]glucose and [6-‘4C]glucose. The distinct histological stratification and connectivity of the hippocampus invite analysis of functional activity within this region by means of autoradiographic determination of local glucose utilization. Previous work from our laboratory demonstrated dramatically different patterns in the 811
878
G. E.
DUNCAN
uptake and retention of radioactivity within the hippocampus. 30 min after injection of [I -‘4C]glucose or [‘4C]2-deoxyglucose ([‘4C]2-DG). Much higher levels of radioactive metabolites accumulated from [I-14C]glucose, compared to [r4C]2-DC, in the granular and molecular layers of the dentate gyrus and in the CA1 pyramidal cell field.3 A number of interpretations are possible for the different hippocampal patterns of [‘4C]2-DG and [I -‘4C]glucose autoradiograms. Accumulation of high levels of r4C from [ 1-‘4C]ghtcose, relative to 14C2-DG, could result from exceptionally low rates of loss of 14C0, from [I-‘4C]glucose in certain regions compared to others, or could result from loss of [r4C]2-DG-phosphate due to locally high glucose-6phosphatase activity. If differential oxidation of [I -‘4C]glucose to 14C02 is responsible for the differences in the hippocampal labeling patterns 30 min after injection of [l-‘4C]glucose or [14C]2-DG, the relative amount of radioactivity in the various hippocampal regions should be different at shorter intervals after injection of [ 1-‘4C]glucose. Therefore, in the present study, in addition to assessing pentose phosphate shunt activity, a detailed anatomical evalautouation was made with high-resolution radiography, of the uptake and metabolic trapping of radioactivity from [ 1-‘4C]glucose in the dorsal hippocampus, 10 and 30min after i.v. injection of this compound. EXPERIMENTAL
et
al.
and [6-‘4C]glucose, as determined by quantitation of silver grain densities in autoradiograms. revealed that the relative and absolute retentions of radiolabeled metabolites from each of the “C-labeled glucose analogs were not significantly different (Fig. I). The highest levels of radioactivity from both compounds occurred in the pyramidal cell layer and in the granular cell layer of the dentate gyrus. The molecular layer of the dentate gyrus and the stratum lacunosum-moleculare contained more radioactivity than the stratum radiatum and the stratum oriens. The topography of glucose utilization in the hippocampus is best visualized by inspection of autoradiograms exposed for several months (Fig. 2). As can be seen. the relative distribution of radiolabeled metabolites is nearly identical for the two compounds. Comparison of 10-min and 30-min survival periocis for
[ 1-“C]glucose The relative topographic distribution of radioactivity from [ 1-r4C]glucose among different hippocampal regions was indistinguishable in rats killed 10 or 30min after injection. However, more radioactivity was contained in all regions in rats killed 30min after injection, compared to the IO-mm survival period (Table 1). Low-power photomicrographs of autoradiograms from the two survival periods are
PROCEDURES
Male Charles River Sprague-Dawley rats, 35 days old, were implanted with chronic jugular catheters (Microrenathane, Brain Tree) and allowed to recover for 5 days before the experiment. Rats were injected via the catheter with [I-r4C]glucose or [6-‘4C]glucose (American Radiolabeled Chemicals, specific activity 55 Ci/mol) dissolved in 0.9% saline, at a dose of 0.5 $.2/g body weight. Rats injected with [I-‘4C]glucose were killed 10 or 30 min after injection and rats given [6-‘4C]glucose were killed 30min after injection by i.v. pentobarbital. Five rats were used for each condition. Brains were mounted on tissue holders, frozen in liquid propane cooled to - 180°C with liquid nitrogen, and stored in liquid nitrogen until sectioned. The autoradiographic procedures have been described in detaiL4.“r Cryostat sections (4pm) were thaw-mounted onto Kodak NTB-3 nuclear emulsion-coated slides and stored in light-tight desiccator boxes for periods of 6 days to 4 months until photographic development. Autoradiograms were developed in Kodak D-19 at 15°C for 3 min, washed in water, and fixed for 3 min. Radioactivity in hippocampal regions was determined by quantitative analysis of silver grain densities in autoradiograms exposed for 6 days using an Artek counter.’ Seven to ten readings were obtained bilaterally for each hippocampal subregion in two to three sections per rat. Statistical comparisons were made by repeated measures analysis of variance.
RESULTS
Comparison of [ 1-‘4CJgIwose and [6-‘4C]gkrcose The topographic distribution of radioactivity in the hippocampus 30 min after injection of 11-‘4C]glucose
Fig. 1. Silver grain densities in hippocampal re*ons from rats injected with [I-“C]$ucose or [6-‘*cJ8tucose. Lined bars: [I-“C]ghrcose; white bars: [6-“C]ghtcose. Rats were killed 30min after i.v. injection of the radiolabeled compounds. Silver grain densities were determined in autoradiograms exposed for 6 days. Data are mean + S.E.M.
Topo~aphy of basal glucose in hip~mpus
Fig. 2, A~to~~~ of the ~p~p~ after injection of [I-%Jgbcose or [6-1’Q&cose.. middle: [6-“C$&wose; bottom: hippocampal fiistolagy with anatomical designation [l-” ‘Q&we; Lorente de No.‘~ Exposure time: 3 months.
880
G. E. DUNCANet al. Table 1. Silver grain densities in different hippocampal regions
Silver grain density (grains/!000 pm* f S.E.M.) Hippoeampal region Fimbria CA3 pyramidal cells CA3 stratum oriens CA3 stratum radiatum CA1 pyramidal cells CA1 stratum oriens CA1 stratum radiatum Stratum lacunosum-moleculare Dentate molecular layer Dentate granular iayer
Ratio of silver grain density to that of CA1 stratum mdiatum
1Omin survival
20-min survival*
IO-min survival
30-min survival
10.2* 1.4 23.1 f 3.7 16.1* 3.1 15.4+ 2.5 20.3 f 3.4 14.2k 2.5 15.5f 2.9 23.3 + 3.3 18.8+ 2.9 23.3 + 3.8
14.7i 1.4 33.0 f 2.0 26.9 * 2.1 25.0 * 2.2 31.7k2.1 22.9 f 3.2 24.8 f 2.7 31.9 f 2.9 30.9 f 2.0 31.9 f 2.9
0.66 1.49 1.04 0.99 1.31 0.92 1.00 1.50 I.21 1.50
0.59 1.33 1.08 1.01 1.28 0.92 1.00 1.29 1.25 1.29
*Silver grain densities in all regions were significantly greater (P < 0.05) for the 30-min survival compared
to the IO-min survival.
shown in Fig. 3. After 10 and 30 min, the greatest retention of radioacti~ty occurred in the p~~i~l cell layer and in the granular layer of the dentate gyrus. In addition, a band of relatively high radioactivity extended from the dorsal limit of the stratum
lacunosum-moleculare and through the molecular layer of the dentate gyrus at both survival times. The stratum oriens and stratum radiatum exhibited relatively low retention of radioactivity from [ 1-t4C]glucose after both 10 and 30 min.
Fig. 3. Topography of accumulated mdioactivity in the hip~mpus 10 min and 30 min after injection of fl-‘4Clglucose.l Top: IO-min survival period; bottom: 30-min survival period. Exposure time: 4 months.
Topography of basal glucose in hippocampus
881
DISCUSSION
The results comparing the metabolic trapping of radioactivity from [I-r4Cjglucose and [6-‘4C]glucose indicate that under basal conditions, very low pentose phosphate shunt activity is operative. However, intense histochemical staining for glucose-6-phosphate dehydrogenase observed in the CA1 pyramidal regionI suggests that potentially great pentose phosphate shunt activity may occur in this region. Wholebrain pentose phosphate shunt activity accounts for only 3% of the total glucolytic flux under basal conditions in rats.“’ The results of the present study also indicate that in all hippocampal regions measured, the portion of glucose utilization devoted to the pentose phosphate shunt is very low in the awake, unstressed rat. Hawkins et ~1.~ found identical glucose utilization rates in many discrete brain regions using [I-r4C]glucose and [6-‘4C]glucose, similarly implying minimal pentose phosphate shunt activity under basal conditions. Under certain experimental and pathological conditions, however, a greater amount of glucose may be processed via the pentose phosphate shunt. For example, Sacks et aI.” found that following electroconvulsive shock in rats, the percentage of brain glucose metabolized by the shunt increased to 25%. Also, in brains of patients with Alzheimer’s disease, elevated pentose phosphate shunt enzyme activity has been reported.” The relative distribution of radioactivity from [l-‘4C]glucose was nearly identical for the lo- and 30-min survival periods although there was a net increase in silver grain density at the 30-min intervals in all regions. These results indicate that a similar fraction of the [l-‘4C]glucose that is transported into different hippocampal regions is oxidized to 14C02 over a 30-min period following intravenous injection of the radiolabeled sugar. The process of glucose utilization includes both catabolic reactions devoted to generation of adenosine triphosphate, and anabolic reactions in which carbon and hydrogen atoms of glucose molecules are
NADPH-DIwhor~w h Amino acid Uptake
Fig. 4. Schematic representation of autoradiograms and histochemistry in the hippocampus. Note that superimposition of the cytochrome oxidase pattern and the amino acid uptake_NADPH-diaphorase patterns would yield what approximates the glucose utilization pattern determined with specifically labeled [r4C]ghtcose, whereas the 2-deoxyglucose pattern matches only partially the cytochrome oxidase pattern. See text and Table 2 for sources of data from which the schematics were derived. Abbreviations: PYR, pyramidal cell layer; SLM, stratum lacunosum-moleculare; DM, dentate molecular layer; DG, dentate granule cell layer.
utilized for de nouo synthesis of neurochemicals. At present, controversy exists regarding whether the and retention of radioactivity from uptake specifically labeled [r4C]glucose, or that from [r4C]2-DG, provides the best index of brain glucose utilization.7.9*‘7,‘*~22 Some basis for deciding whether the accumulation of radioactivity from [14C]2-DG or [ l-‘4C]glucose best reflects hippocampal glucose utilization may be gleaned from descriptions of the topographic distribution of enzymes in the hippocampus which participate in the metabolism of glucose. Results of various studies in this regard are summarized in Table 2 and Fig. 4. Kageyama and Wong-Riley” found very intense histochemical staining for cytochrome oxidase, indicating high oxidative phosphorylation capacity, in the CA3 pyramidal cell
Table 2. Relative distribution of metabolic enzymes and radioactivity from [‘4C]glucose and [‘%]2-deoxyglucose in the rodent hippocampus
CA3 pyramidal cell bodies CA1 pyramidal cell bodies CA1 stratum radiatum Stratum lacunosummoleculare Dentate granule cell bodies Dentate molecular
Succinate Cytochrome oxidase” dehydrogenase6,16 Fumara@ (low-high)* high
[l-14C]Glucose [6-r4C]ghrcose
[r4C]2-Deoxy glucose’
high
high
high
low
low
(low-high)*
high
high
low
low
low
low
low
low
high
high
high
high
high
high
high
low
low
low
-
high
high
low
high
high
-
low
NADPH-diaphorase’6 high
*Friede6 reports high and Meyer et a1.16reports low succinate dehydrogenase activity in CA1 and CA3 pyramidal cell bodies.
882
G. E. DUNCANel cd.
field, the stratum lacunosum-moleculare, and the molecular layer of the dentate gyrus. In those regions, relatively high amounts of 14C are detected in [6-‘4C]glucose and [l-‘4C]glucose autoradiograms. In [“Cl2-DG autoradiograms, although high amounts of radioactivity are found in the CA3 pyramidal cell field and stratum lacunosum-moleculare, very low retention of radioactivity occurs in the molecular layer of the dentate gyrus.3.4 In the CA1 pyramidal cell field and granule cell layer of the dentate gyrus, cytochrome oxidase staining is very weak.” In these regions, the retention of radioactivity is higher for [6-‘4C]glucose and [ 1-‘4C]glucose, than for [‘4C]2-DG.3,4Although the CA1 pyramidal cell bodies exhibit low cytochrome oxidase staining, histochemical staining for succinate dehydrogenase, a component of the Krebs cycle, is reported to be high in the CA1 pyramidal cell layer.6 Biochemical assessment of fumarase activity in microdissected pyramidal cells from the CA1 region also indicates high activity of this Krebs cycle enzyme.‘4 However, another histochemical studyI found low levels of succinate dehydrogenase staining in the CA1 pyramidal cell field. Histochemical studies of oxidative enzymes in the dentate gyrus have yielded consistent results. Intense staining of succinate dehydrogenase and cytochrome oxidase is found in the molecular layer of the dentate gyrus and very low staining is apparent in the granular cell layer.6,“*‘6 As noted above, in the dentate molecular layer, relatively high levels of radioactivity accumulate from [l-‘4C]glucose and [6-‘4CJglucose and low levels from [14C]2-DG. In the dentate granule cell layer, autoradiograms manifest high retention of 14C-labeled metabolites of [l-‘4Clglucose and [6-‘4C]glucose but very low accumulation of ‘“C from [‘4C]2-DG. Although there is little cytochrome oxidase staining in CA1 pyramidal cells and dentate granule cells, these cell bodies stain intensely for NADPH-dependent tetrazolium reductase (NADPHstudies with diaphorase). I6 Also, autoradiographic [35S]methionine5 and [3H]leucine’ have demonstrated very high uptake of these dietary essential amino acids in the cell bodies of hippocampal pyramidal neurons from all fields, as well as in the cell bodies of dentate granule neurons. High levels of amino
acid uptake and intense histochemical staining fo’ NADPH-diaphorase activity, in regions of low capacity for oxidative phosphorylation, imply that glucose utilized in those regions may support biosynthetic processes to a greater extent than the generation of high-energy phosphate compounds. The biochemical reaction(s) catalysed by NADPH diaphorase have not been defined, but histochemical studies have demonstrated co-localization of this enzyme with specific transmitter systems in the superior colliculusz5 and in the striatum.24 Correspondence of identified transmitter systems and NADPHdiaphorase-containing neurons in the hippocampus has not be described. Although certain hippocampal regions (pyramidal cell layer and dentate granular layer) exhibit high [6-‘4C]glucose and [ 1-‘4C]glucose uptake and high amino acid accumulation, other regions of relatively high [6-‘4C]glucose and [l-‘4C]glucose uptake (stratum lacunosum-moleculare and dentate molecular layer) exhibit low amino acid accumulation. It is unlikely that radioactivity accumulated in the brain after [6-‘4C]glucose or [ l-‘4~glucose injection results from the uptake of peripheral metabolites of glucose such as amino acids. As noted above, the brain synthesizes non-essential amino acids primarily from glucose molecules. Therefore, radiolabeled amino acids derived from [‘4C]glucose in the brain most likely result from de ~OUOsynthesis within brain cells. The overall pattern of [6-‘4C]glucose and [l -‘4C]glucose autoradiograms of the hippocampus cannot be attributed to the presence and activity of a single enzyme system. Instead, the hippocampus glucose utilization pattern appears to correspond to a composite of the histochemical distribution of cytochrome oxidase, NADPH-diaphorase and essential amino acid uptake (Fig. 4), implying that distinct histological subdivisions of this brain region utilize glucose for different purposes. It remains to be investigated what functional significance can be attributed to the correspondence of patterns of glucose utilization and enzyme localization within the hippocampus. Acknowle~e~~ts-Supported by USPHS grants NS09914 and HDO3110and NATO grant 83/0576.
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