Neurochernical Research (2) 595-603 (1977)
R E G I O N A L DISTRIBUTION OF G L U C O S E IN MOUSE BRAIN MASAHISA SHIMADA, TAKASHI KIHARA, MASAHITO WATANABE, AND KIYOHISA KURIMOTO Department of Anatomy Osaka Medical College 2-7 Daigakumachi, Takatsuki City, Osaka, Japan
Accepted May 18, 1977
After rapid inactivation of the enzymes responsible for glucose metabolism by microwave irradiation, concentrations of glucose in 20 regions of the mouse brain were estimated with combined gas chromatography-mass spectrometry (GC-MS). The highest concentrations of glucose were found in the periventricular nuclei of the hypothalamus and nucleus preopticus (P< 0.05). The septum and nucleus amygdaloideus showed significantly higher glucose concentration compared with the cerebral neocortex, olfactory bulb, corpus striatum, cingulum, fornix, colliculus inferior, cerebellar cortex, corpus geniculatum laterale, substantia nigra, and nucleus ruber (P < 0.05). The glucose concentration in the substantia nigra and nucleus ruber was significantly lower than in the other regions (P < 0.01).
INTRODUCTION
Glucose is a primary substrate for the energy used by the adult mammalian brain (25). Since the brain is a highly differentiated tissue, it is generally accepted that the glucose demand is quite different in each brain region. To measure the glucose utilization in different brain regions, Reivich (16) and Sokoloff (24), estimated the glucose consumption in various brain regions indirectly by using autoradiography of radioactive 2-deoxyglucose. This paper deals with direct measurement of the glucose distribution in the brain. Accurate measurement in various regions of the brain in vivo presents great difficulty (9,30) because of the high rate of postmortem decrease of glucose. Recently, microwave irradiation, which rapidly inactivates the enzymes responsi595 This journal is copyrighted by Plenum. Each article is availablefor $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
596
SHIMADA ET AL.
ble for postmortem changes, has become available for the measurement of metabolites (1,2,14,15,20,26,27). Furthermore, since microwave irradiation applies heat evenly throughout the brain, it appears to be most suitable for regional studies of labile metabolites in brain. Chemically, the development of gas chromatographic and mass fragmentometric methods has allowed separation and quantitative determination of small amounts of low-molecular-weight compounds in biological materials (8,11-13,23). This paper presents the glucose distribution in various brain regions estimated directly by using GC-MS for the determination of glucose concentrations in small areas of brains of mice sacrificed by microwave irradiation. A correlation between glucose concentration and rate of utilization, as determined by Reivich (16) and Sokoloff (24), was found in the periventricular nuclei of the hypothalamus, nucleus preopticus, nucleus amygdaloideus, and fornix.
EXPERIMENTAL PROCEDURE
Animals and Reagents Animals used were adult male albino mice (22), weighing - 2 0 g and fed ad libitum on a normal diet. Hexamethyldisilazane (HMDS) was obtained from Ohio Valley Chemical, Inc. Methyl mannose used as an internal standard was obtained from Merck. Trimethyl chlorosilane (TMS) was purchased from Nishio, Inc., Japan. Calibration standard solution was an aqueous solution of glucose and methyl mannose at concentrations of 1 ~mol and 0.5/xmol, respectively.
Microwave Irradiation The microwave apparatus (Model Q-38, Sanyo Electric Co., Japan) was designed to rapidly elevate the temperature of the mouse head to prevent postmortem changes in glucose. The microwave power pack was a 0.6-kW 2M53 magnetron operating at a frequency of 2450 MHz. The efficiency of coupling the microwave power to the mouse head was -90%. The standing wave was controlled with the tuning stubs provided on the wave guide. The head of the mouse was precisely set on the center of the test tube coupler and heated for 3 sec, which was enough exposure time to elevate the temperature of brain by 80~ The temperature of the brain was recorded with the thermistor thermometer (Type A-700, Takara Thermistor Inc., Japan).
Preparation of Brain Regions and Blood The brains were dissected into 20 regions by using the following techniques. The brains were removed from the mice, frozen in dry-ice snow, and cut coronally in a cryostat with a microtome (-10~ 150 /zm thick, Bright Hydro-Pneumatic Sleigh, Leitz 1300). Each region on the section was removed with a punch made from an injector-needle that had a 0.70-mm i.d. Architectonic areas such as cerebral neocortex and hippocampus were cut
597
GLUCOSE IN BRAIN LOCI
into - l - m m cubes with the edge of the cutter and these were lyophilized. The areas were removed with the aid of a microscope (Objective x5, Subjective x2) in the cryostat. In order to identify the brain regions removed, the section was stained with hematoxyline, after it had been refixed with 80% methanol, and then embedded in glycerin. The preparations were suitable for histological examination (3). Blood samples (I.0 ml) collected by decapitation were deproteinized with 5% trichloroacetic acid (TCA) and centrifuged; the supernatant was washed with ether 3 times to remove TCA, lyophilized, and extracted with pyridine (2.0 ml). Methyl mannose (3.0 mmol) was added to the pyridine as internal standard. Glucose in the blood was converted to trimethylsilyl ether derivatives, as described below, and measured by gas chromatography.
Treatment o f Derivatives Each brain region thus obtained was weighed on an electric balance (PMB-I, Shimadzu Seisakusho Co., Japan), and the samples were transferred from the balance to the reaction vials (Pierce Reacti-Vials, 1 ml, Rockford Illinois). Usually, the dry weight of each region ranged from 15 to 20 /zg. After addition of 85 tzl of anhydrous pyridine and standing overnight at 20~ 15 /zl of silylating reagent (HMDS and TMS, 2:1) was placed directly into the vials with 50 pmol of methyl mannose and heated for 15 rain at 80~ (31). As there was no direct way to check recovery of glucose from the dry samples (28), glucose of the cerebral neocortex was extracted in water, lyophilized, arid then silylated (wet sample). After drying and diluting with 50 /~I anhydrous hexane, a 5-/zl aliquot was chromatographed on a 1.5-m glass column packed with 1% OV-I on 80/100 mesh chromosorb W in a GC-MS combined system (JGC-20KP, JMS-D 100, JEC-6, JEOL, Japan). To calculate relative molar response (glucose molar response/methyl mannose molar response) of m/e 204 and 217, a standard calibration solution was studied at the beginning and end of each day (10),
GC-MS Conditions Gas chromatographic conditions were as follows: column temperature, initial 170~ final 210~ raised at 3~ injection temperature, 220~ carrier flow (He), 40 ml/min. Mass spectrometer conditions were as follows: chamber temperature, 240~ ionizing voltage, 75 eV; ion multigain, 5.0; sensitivity, 0.5 V. The mass spectrometer was focused on m/e 204 and 217 (4,5).
RESULTS q-
A mass fragmentogram
+
o f m/e 204 ( T M S i - O - C H - C H - O - T M S i )
and
217 ( T M S i - O - C H - - - C H - C H - O - T M S i ) , which are common characteristic f r a g m e n t s f r o m T M S i e t h e r s o f c a r b o h y d r a t e s (4,5), is s h o w n in F i g u r e 1. C a l i b r a t i o n c u r v e s f o r g l u c o s e w i t h m e t h y l m a n n o s e as i n t e r n a l standard were linear. In order to ascertain the purification of each peak (29), m/e 217/204 r a t i o s w e r e c a l c u l a t e d in e a c h b r a i n r e g i o n a n d
598
SHIMADA ET AL.
compared with those of the calibration standard solution. The values varied between 0.42 and 0.46, and these ratios in each brain region were consistent with those of the calibration solution. Glucose concentrations in the cerebral neocortex of wet samples and of blood are reported in Table I. The data obtained from cerebral neocortex were recalculated on a dry weight basis so that they could be compared with those of the dry samples. The concentration of free water is -76% of the total brain weight (14,21). The two methods of expressing the results agreed when the concentrations were connected by this factor. Glucose concentrations in each region also are presented in Table I. The highest concentrations of glucose were found in the periventricular nuclei of the hypothalamus and nucleus preopticus (P < 0.05). The glucose concentration in the septum and nucleus amygdaloideus was significantly higher than that in the cerebral neocortex, olfactory bulb, corpus striatum, cingulum, fornix, colliculus inferior, cerebellar cortex,
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Fig. 1 Mass fragmentograms of glucose, methyl mannose (internal standard), and myoinositol. See the text for experimental details. (a, left) Calibration standard solution (glucose 1 ~tmol, methyl mannose 0.5/xmol). (b, right) Cerebral neocortex of dry sample (16.8/xg dry wt., methyl mannose 50 pmol).
G L U C O S E IN B R A I N L O C I
599
TABLE I GLUCOSE CONCENTRATIONS IN VARIOUS REGIONS OF THE MOUSE BRAIN AND BLOOD a 2.05 --_ 0.12/~mol/g wet wt. -+ S E M (8.54 • 0.46/zmol/g dry wt. • S E M 6.27 • 0.43/zmol/ml -+ S E M
Cerebral neocortex Blood (whole) Periventricular nucleus (hypothalamus) Nucleus preopticus Septum Nucleus amygdaloideus Thalamus Pyriform cortex Hippocampus Corpus geniculatum mediale Colliculus superior Cerebral neocortex Olfactory bulb Corpus striatum Cingulam Fornix Colliculus inferior CerebeUar cortex Corpus geniculatum laterale Cranial nuclei (IVth ventricle) Substantia nigra Nucleus ruber
13.1 12.5 10.7 10.1 9.42 9.39 9.00 8.98 8.29 8.28 8.23 8.22 8.21 8.06 7,90 6.99 6.95 6.89 6.09 6.08
+ 0.610,c _+ 0.77 b'c -+ 0.49 b • 0.43 ~ • 0.43 ~ • 0. I2 ~ • 0.51 b • 0.56 b • 0.54 b • 0.51 b • 0.29 b • 0.28 b • 0.51 b • 0.490 -+ 0.30 o • 0.06 ~ • 0.32 ~ • 0.23 ~ • 0.02 ~ • 0.09 b
a Data from six male mice. o Values were expressed as/zmol/g dry wt. • S E M . c Significant at P < 0.05. d Significant at P < 0.01.
corpus geniculatum laterale, substantia nigra, and nucleus ruber (P < 0.05). The glucose concentration in the substantia nigra and nucleus ruber was significantly lower than in the other regions (P < 0.01).
DISCUSSION The elevation of the brain temperature to 80~ is sufficient to inactivate all the glucose-utilizing enzymes in both superficial and deep brain structure (14,15,26). The ability to completely stop an in vivo system within at least 4 sec is essential for turnover studies (26). It was confirmed in the preliminary experiments that longer microwave irradiation (4 sec) elevated the brain temperature to 90~ giving the same
600
S H I M A D A ET A L .
glucose concentrations in whole mouse brain as those found after the brain temperature was raised to 80~ Animals were microwaved at 80~ for 3 sec in the present work. The techniques described here have allowed the measurement of glucose concentrations in small regions of mouse brain. The concentrations reported here are slightly higher than those reported by Medina et al. (14) (1.773 -+ 0.133 /xmol/g wet wt.), who used microwave irradiation to fix the tissue, and by Swaab et al. (29) (1.40 +--0.11 /xmol/g wet wt.), who used rapid freezing. Data of Nelson et al. (15) found values for glycogen and lactate in heated tissue (microwave radiation) that were significantly lower (35%) than those in frozen brain. Glucose and fructose-l,6,diphosphate concentrations were similar in the two tissues, but glucose-6-phosphate levels were increased. Since glycogen yields glucose-l-phosphate, and this can be converted to glucose (7,17,19), it is quite possible that microwave irradiation can lead to glucose formation from glycogen. In the present work, the standing wave of the microwave was used to increase the efficiency of energy transfer to the animal's brain to about 90%, two- to threefold that achieved by other workers (1,14,15,20,25). The method described in this paper for direct measurement of glucose concentrations in different brain regions complements the more convenient technique described by Sokoloff (24). It is generally accepted that the tissue glucose concentration depends on the plasma glucose, rate of blood flow, and the rate of glucose consumption (Table I) (16,18,24,25). Whole blood has a value of about 6.3 /xmol glucose per ml (Table I) (about 32.1 /xmol glucose per g of dried blood) (6), which means perivent, nuc. (hypothalamus) has a glucose content greater than one-third that of whole blood. This study shows that there is significant free glucose in mouse brain. Reivich and Sokoloff's data show that there is a remarkably close correlation between local blood flow and glucose consumption in the brains of conscious rats (16,24). The relatively high levels of glucose we found in amygdala and hypothalamus correlate well with the known relatively low rates of glucose consumption in these areas (16,24). The relatively low concentrations of glucose in the substantia nigra and nucleus ruber may be related to a high rate of metabolism in these areas, but independent measures of metabolic rates in these areas are not available. The relatively low concentrations of glucose in the fornix probably reflect the high lipid concentrations in white matter. If we correct our values for glucose for the lipid content, using the data of Folbergrowi et al. for lipids (9), the glucose concentrations in the cerebral neocortex and fornix are found to be 12.7 +__0.8 and 18.1 +-- 1.10/xmol/g of lipidfree dry weight, (mean _ SEM). The higher glucose concentrations in white
GLUCOSE IN BRAIN LOCI
601
matter (fornix) than in gray matter (cerebral neocortex) agrees with the known lower rate of glucose metabolism in white matter (16,24), and the value for fornix agrees with the value (9) for glucose concentration in lipidfree white matter. Of lesser importance, but still a factor to be considered, would be the fact that the blood in the brain at the time of microwave irradiation would be coagulated and, since different regions vary in blood content, corrections would have to be made for glucose content in this trapped blood. It would seem, therefore, that much still remains to be done to determine the true levels of free glucose in brain in vivo. The use of GC-MS provides a powerful tool to measure glucose concentrations in small areas of brain. We are currently examining the possibility of applying this method to measure the dynamics of regional brain metabolism. By measuring the specific activities of glucose (precursor) and glucose-6-phosphate (32) (product) following injection of [~C]glucose, regional brain glucose turnover can be determined (33).
ACKNOWLEDGMENTS The authors are very grateful to Mr. M. Takashima and Mr. T. Ikeda, technicians in Sanyo Electric Co., for planning the microwave power pack in the present work. The authors are also would like to thank Dr. John P. Blass and Dr. Gary E. Gibson for valuable discussions and critical reading. This work was partly supported by funds from the Education Ministry of Japan (Research for Encouragement A No. 077494).
REFERENCES ]. BALCOM, G. J., LENOX, R. H., and MYERHOFF, J. L. 1975, Regional y-aminobutyric acid levels in rat brain determined after microwave fixation. J. Neurochem. 24:609613. 2. BALCOM, G. J., LENOX, R. H., and MEYERHOFF, J. L. 1976. Regional glutamate levels in rat brain determined after microwave fixation. J. Neurochem. 26:423-425. 3. BERNARD, G. R. 1974. Microwave irradiation as a generator of heat for histological fixation. Stain Technol. 49:215-224. 4. CHIZHOV, O. S., MOLODTSOV, N. V., and KOCHETKOV, N. K. 1967. Mass spectrometry of trimethylsilyl esthers of carbohydrates. Carbohydr. Res. 4:273-276. 5. DEJONGH, D. C., RADFORD, T., HRIBAR, J. D., HANESSIAN, S.,, BIEBER, M., DAWSON, G., and SWEELEV, C. C. 1969. Analysis of trimethylsilyl derivatives of carbohydrates by gas chromatography and mass spectrometry. J. Am. Chem. Soc. 91:1728-1740. 6. DITTER, D. S. 1961. Pages 1-19, in Biological Handbooks: Blood and Other Body Fluids. Fed. Proc. Washington, D.C. 7. DODD, P. R., BRADFORD, H. F., and CHAIN, E. E. 1971. The metabolism of glucose-6phosphate by mammalian cerebral cortex in vivo. Biochem. J. 125:1027-1038.
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8. FIDONE, S. J., WEIBRAUB,S. T., and STAVINOHA,W. B. 1976. Acetylcholine content of normal and denervated cat carotid bodies measured by pyrolysis gas chromatography/mass fragmentometry. J. Neurochem. 26:1047-1049. 9. FOLBERGROV.~,J., LOWRY, O. H., and PASSONEAU,J. V. 1970. Changes in metabolites of the energy reserves in individual layers of mouse cerebral cortex and subjacent white matter during ischaemia and anaesthesia. J. Neurochem. 17:1155- 1162. 10. GEHRKE,C. W., ROACH,D., ZUNWALR,W., STALLING,D. L., and WALL, I. I. 1968. Gas-liquid chromatography of amino acids in proteins and biological substances; macro, semimicro and micromethods. Pages 27-38, in Anal. Biochem. Laboratories, in Library of Congress Catalog Card No. 68-57507. I I. JENDEN, D. J., ROCH, i . , and BOOTH, R. A. 1973. Simultaneous measurement of endogenous and deuterium-labeled tracer variants of choline and acetylcholine in subpicomole quantities by gas chromatography/mass spectrometry. Anal. Biochem. 55:438-448. 12. KAROUM, F., GILLIN, J. C., and WYATT, R. T. 1975. Mass fragmentographic determination of some acidic and alcoholic metabolites of biogenetic amines in the rat brain. J. Neurochem. 25:653-658. 13. McAooo, D. J., and COGGESHALL,R. E. 1976. Gas chromatographic-mass spectrometric analysis of biogenetic amines in identified neurons and tissues of Hirudo medicinalis. J. Neurochem. 26:163-167. 14. MEDINA,i . A., JONES, D. J., STAVINOHA,W. B., and Ross, D. H. 1975. The levels of labile intermediary metabolites in mouse brain following rapid tissue fixation with microwave irradiation. J. Neurochem. 24:223-227. 15. NELSON, S. R., and MANTZ, M. L. 1971. Metabolite levels in brain after heating (microwave radiation), the decapitated mouse head. Fed. Proc. 30:496. 16. REIVICH, M. 1974. Blood flow metabolism couple in brain. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 53: 125-140. 17. ROSEN, S. I. 1970. Glucose-6-phosphate hydrolysing activity in the cerebellum of the mouse, rat, hamster and marmoset. Acta Histochem. 36:44-53. 18. RUDEMAN,N. B., ROSS, P. S., BERGER, M., and GOODMAN,M. N. 1974. Regulation of glucose and ketone-body metabolism in brain of anaesthetized rats. Biochem. J. 138:l-10. 19. SACKS,W., and SACKS, S. 1968. Conversion of glucose phosphate-14C to glucose-14C in passage through human brain in vivo. J. Appl. Physiol. 24:817-827. 20. SCHMIDT, M. J., SCHMIDT, D. E., and ROBISON, G. A. 1971. Cyclic adenosine monophosphate in brain areas: microwave irradiation as a means of tissue fixation. Science (NY) 173:1142-1143. 21. SHIMADA,M., KURIMOTO,K., WADA, F., HIND, O., SUGINOSHITA,K., and KIHARA, T. 1971. Quantitative trial of amino acids in the mouse brain loci by gas-liquid chromatography. Acta Anat. Nippon 46:125-135. 22. SHIMADA,M., KIHARA,T., KURIMOTO,K., and WATANABE,M. 1974. Incorporation of 14C from [U-~4C]glucose into free amino acids in mouse brain regions under cyanide intoxication. J. Neurochem. 23:379-384. 23. SjSquist, B., Dailey, J., Sedvall, G., and ANGGARO,E. 1973. Mass fragmentographic assay of homovanillic acid in brain tissue. J. Neurochem. 20:729-733. 24. SOKOLOEE,L. 1975. Determination of local cerebral glucose consumption. Pages 1-8, in HAPER, A. M., JENNET, W. B., MILLER, J. A., and ROWAN, J. O. (eds.), Blood Flow and Metabolism in the Brain, Churchill Livingstone, Edinburgh. 25. SOKOLOEF,L. 1976. Circulation and energy metabolism of the brain. Pages 388-413, in SIEGEL, G. J., ALBERS, R. W., KATZMAN,R., and AGRANOFF,B. W. (eds.), Basic Neurochemistry, Little, Brown & Company, Boston.
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26. STAVINOHA,W. B., WEITRAUB, S. T., and MODAK, A. T. 1973. Use of microwave 27. 28.
29.
30. 31.
32.
33.
heating to inactivate cholinetherase in the rat brain prior to analysis for acetylcholine. J. Neurochem. 20:361-371. STAVINOHA,W. B., WEITRAUB, S. T., and MODAK, A. T. 1974. Regional concentrations of choline and acetylcholine in the rat brain. J. Neurochem. 23:885-886. STEWART, M. A., RHEE, V., KURIEN, M. M., and SHERMAN, W. R. 1969. Gas chromatographic analysis of myo-inositol in microgram samples of brain. Biochim. Biophys. Acta 192:361-363. STRONG, J. M., FIES, J. W., and ATKINSON, A. J. 1973. Quadrupole mass fragmentography in drug research. Pages 310-320, in Proceedings of the First International Conference on Stable Isotopes in Chemistry, Biology and Medicine. SWAAB,D. F. 1971. Pitfalls in the use of rapid freezing for stopping brain and spinal cord metabolism in rat and mouse. J. Neurochem. 18:2085-2092. SWEELEY, C. C., BENTLEY, R., MAKITA, M., and WELL, W. W. 1963. Chromatography of trimethylsilyl derivatives of sugers and related substances. J. Am. Chem. Soc. 85:2497-2507. WIECKO, J., and SHERMAN,W. R. 1975. Mass spectral study of cyctic alkaneboronates of sugar phosphates. Evidence of interaction between the phosphate group and boron under electron-impact conditions. Org. Mass Spectrom. 10: 1007-1020. ZILVERSMIT,D. B. 1960. The design and analysis of isotope experiments. Am. J. Med. 27(2):832-848.
R E G I O N A L DISTRIBUTION OF G L U C O S E IN MOUSE BRAIN MASAHISA SHIMADA, TAKASHI KIHARA, MASAHITO WATANABE, AND KIYOHISA KURIMOTO Department of Anatomy Osaka Medical College 2-7 Daigakumachi, Takatsuki City, Osaka, Japan
Accepted May 18, 1977
After rapid inactivation of the enzymes responsible for glucose metabolism by microwave irradiation, concentrations of glucose in 20 regions of the mouse brain were estimated with combined gas chromatography-mass spectrometry (GC-MS). The highest concentrations of glucose were found in the periventricular nuclei of the hypothalamus and nucleus preopticus (P< 0.05). The septum and nucleus amygdaloideus showed significantly higher glucose concentration compared with the cerebral neocortex, olfactory bulb, corpus striatum, cingulum, fornix, colliculus inferior, cerebellar cortex, corpus geniculatum laterale, substantia nigra, and nucleus ruber (P < 0.05). The glucose concentration in the substantia nigra and nucleus ruber was significantly lower than in the other regions (P < 0.01).
INTRODUCTION
Glucose is a primary substrate for the energy used by the adult mammalian brain (25). Since the brain is a highly differentiated tissue, it is generally accepted that the glucose demand is quite different in each brain region. To measure the glucose utilization in different brain regions, Reivich (16) and Sokoloff (24), estimated the glucose consumption in various brain regions indirectly by using autoradiography of radioactive 2-deoxyglucose. This paper deals with direct measurement of the glucose distribution in the brain. Accurate measurement in various regions of the brain in vivo presents great difficulty (9,30) because of the high rate of postmortem decrease of glucose. Recently, microwave irradiation, which rapidly inactivates the enzymes responsi595 This journal is copyrighted by Plenum. Each article is availablefor $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
596
SHIMADA ET AL.
ble for postmortem changes, has become available for the measurement of metabolites (1,2,14,15,20,26,27). Furthermore, since microwave irradiation applies heat evenly throughout the brain, it appears to be most suitable for regional studies of labile metabolites in brain. Chemically, the development of gas chromatographic and mass fragmentometric methods has allowed separation and quantitative determination of small amounts of low-molecular-weight compounds in biological materials (8,11-13,23). This paper presents the glucose distribution in various brain regions estimated directly by using GC-MS for the determination of glucose concentrations in small areas of brains of mice sacrificed by microwave irradiation. A correlation between glucose concentration and rate of utilization, as determined by Reivich (16) and Sokoloff (24), was found in the periventricular nuclei of the hypothalamus, nucleus preopticus, nucleus amygdaloideus, and fornix.
EXPERIMENTAL PROCEDURE
Animals and Reagents Animals used were adult male albino mice (22), weighing - 2 0 g and fed ad libitum on a normal diet. Hexamethyldisilazane (HMDS) was obtained from Ohio Valley Chemical, Inc. Methyl mannose used as an internal standard was obtained from Merck. Trimethyl chlorosilane (TMS) was purchased from Nishio, Inc., Japan. Calibration standard solution was an aqueous solution of glucose and methyl mannose at concentrations of 1 ~mol and 0.5/xmol, respectively.
Microwave Irradiation The microwave apparatus (Model Q-38, Sanyo Electric Co., Japan) was designed to rapidly elevate the temperature of the mouse head to prevent postmortem changes in glucose. The microwave power pack was a 0.6-kW 2M53 magnetron operating at a frequency of 2450 MHz. The efficiency of coupling the microwave power to the mouse head was -90%. The standing wave was controlled with the tuning stubs provided on the wave guide. The head of the mouse was precisely set on the center of the test tube coupler and heated for 3 sec, which was enough exposure time to elevate the temperature of brain by 80~ The temperature of the brain was recorded with the thermistor thermometer (Type A-700, Takara Thermistor Inc., Japan).
Preparation of Brain Regions and Blood The brains were dissected into 20 regions by using the following techniques. The brains were removed from the mice, frozen in dry-ice snow, and cut coronally in a cryostat with a microtome (-10~ 150 /zm thick, Bright Hydro-Pneumatic Sleigh, Leitz 1300). Each region on the section was removed with a punch made from an injector-needle that had a 0.70-mm i.d. Architectonic areas such as cerebral neocortex and hippocampus were cut
597
GLUCOSE IN BRAIN LOCI
into - l - m m cubes with the edge of the cutter and these were lyophilized. The areas were removed with the aid of a microscope (Objective x5, Subjective x2) in the cryostat. In order to identify the brain regions removed, the section was stained with hematoxyline, after it had been refixed with 80% methanol, and then embedded in glycerin. The preparations were suitable for histological examination (3). Blood samples (I.0 ml) collected by decapitation were deproteinized with 5% trichloroacetic acid (TCA) and centrifuged; the supernatant was washed with ether 3 times to remove TCA, lyophilized, and extracted with pyridine (2.0 ml). Methyl mannose (3.0 mmol) was added to the pyridine as internal standard. Glucose in the blood was converted to trimethylsilyl ether derivatives, as described below, and measured by gas chromatography.
Treatment o f Derivatives Each brain region thus obtained was weighed on an electric balance (PMB-I, Shimadzu Seisakusho Co., Japan), and the samples were transferred from the balance to the reaction vials (Pierce Reacti-Vials, 1 ml, Rockford Illinois). Usually, the dry weight of each region ranged from 15 to 20 /zg. After addition of 85 tzl of anhydrous pyridine and standing overnight at 20~ 15 /zl of silylating reagent (HMDS and TMS, 2:1) was placed directly into the vials with 50 pmol of methyl mannose and heated for 15 rain at 80~ (31). As there was no direct way to check recovery of glucose from the dry samples (28), glucose of the cerebral neocortex was extracted in water, lyophilized, arid then silylated (wet sample). After drying and diluting with 50 /~I anhydrous hexane, a 5-/zl aliquot was chromatographed on a 1.5-m glass column packed with 1% OV-I on 80/100 mesh chromosorb W in a GC-MS combined system (JGC-20KP, JMS-D 100, JEC-6, JEOL, Japan). To calculate relative molar response (glucose molar response/methyl mannose molar response) of m/e 204 and 217, a standard calibration solution was studied at the beginning and end of each day (10),
GC-MS Conditions Gas chromatographic conditions were as follows: column temperature, initial 170~ final 210~ raised at 3~ injection temperature, 220~ carrier flow (He), 40 ml/min. Mass spectrometer conditions were as follows: chamber temperature, 240~ ionizing voltage, 75 eV; ion multigain, 5.0; sensitivity, 0.5 V. The mass spectrometer was focused on m/e 204 and 217 (4,5).
RESULTS q-
A mass fragmentogram
+
o f m/e 204 ( T M S i - O - C H - C H - O - T M S i )
and
217 ( T M S i - O - C H - - - C H - C H - O - T M S i ) , which are common characteristic f r a g m e n t s f r o m T M S i e t h e r s o f c a r b o h y d r a t e s (4,5), is s h o w n in F i g u r e 1. C a l i b r a t i o n c u r v e s f o r g l u c o s e w i t h m e t h y l m a n n o s e as i n t e r n a l standard were linear. In order to ascertain the purification of each peak (29), m/e 217/204 r a t i o s w e r e c a l c u l a t e d in e a c h b r a i n r e g i o n a n d
598
SHIMADA ET AL.
compared with those of the calibration standard solution. The values varied between 0.42 and 0.46, and these ratios in each brain region were consistent with those of the calibration solution. Glucose concentrations in the cerebral neocortex of wet samples and of blood are reported in Table I. The data obtained from cerebral neocortex were recalculated on a dry weight basis so that they could be compared with those of the dry samples. The concentration of free water is -76% of the total brain weight (14,21). The two methods of expressing the results agreed when the concentrations were connected by this factor. Glucose concentrations in each region also are presented in Table I. The highest concentrations of glucose were found in the periventricular nuclei of the hypothalamus and nucleus preopticus (P < 0.05). The glucose concentration in the septum and nucleus amygdaloideus was significantly higher than that in the cerebral neocortex, olfactory bulb, corpus striatum, cingulum, fornix, colliculus inferior, cerebellar cortex,
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Fig. 1 Mass fragmentograms of glucose, methyl mannose (internal standard), and myoinositol. See the text for experimental details. (a, left) Calibration standard solution (glucose 1 ~tmol, methyl mannose 0.5/xmol). (b, right) Cerebral neocortex of dry sample (16.8/xg dry wt., methyl mannose 50 pmol).
G L U C O S E IN B R A I N L O C I
599
TABLE I GLUCOSE CONCENTRATIONS IN VARIOUS REGIONS OF THE MOUSE BRAIN AND BLOOD a 2.05 --_ 0.12/~mol/g wet wt. -+ S E M (8.54 • 0.46/zmol/g dry wt. • S E M 6.27 • 0.43/zmol/ml -+ S E M
Cerebral neocortex Blood (whole) Periventricular nucleus (hypothalamus) Nucleus preopticus Septum Nucleus amygdaloideus Thalamus Pyriform cortex Hippocampus Corpus geniculatum mediale Colliculus superior Cerebral neocortex Olfactory bulb Corpus striatum Cingulam Fornix Colliculus inferior CerebeUar cortex Corpus geniculatum laterale Cranial nuclei (IVth ventricle) Substantia nigra Nucleus ruber
13.1 12.5 10.7 10.1 9.42 9.39 9.00 8.98 8.29 8.28 8.23 8.22 8.21 8.06 7,90 6.99 6.95 6.89 6.09 6.08
+ 0.610,c _+ 0.77 b'c -+ 0.49 b • 0.43 ~ • 0.43 ~ • 0. I2 ~ • 0.51 b • 0.56 b • 0.54 b • 0.51 b • 0.29 b • 0.28 b • 0.51 b • 0.490 -+ 0.30 o • 0.06 ~ • 0.32 ~ • 0.23 ~ • 0.02 ~ • 0.09 b
a Data from six male mice. o Values were expressed as/zmol/g dry wt. • S E M . c Significant at P < 0.05. d Significant at P < 0.01.
corpus geniculatum laterale, substantia nigra, and nucleus ruber (P < 0.05). The glucose concentration in the substantia nigra and nucleus ruber was significantly lower than in the other regions (P < 0.01).
DISCUSSION The elevation of the brain temperature to 80~ is sufficient to inactivate all the glucose-utilizing enzymes in both superficial and deep brain structure (14,15,26). The ability to completely stop an in vivo system within at least 4 sec is essential for turnover studies (26). It was confirmed in the preliminary experiments that longer microwave irradiation (4 sec) elevated the brain temperature to 90~ giving the same
600
S H I M A D A ET A L .
glucose concentrations in whole mouse brain as those found after the brain temperature was raised to 80~ Animals were microwaved at 80~ for 3 sec in the present work. The techniques described here have allowed the measurement of glucose concentrations in small regions of mouse brain. The concentrations reported here are slightly higher than those reported by Medina et al. (14) (1.773 -+ 0.133 /xmol/g wet wt.), who used microwave irradiation to fix the tissue, and by Swaab et al. (29) (1.40 +--0.11 /xmol/g wet wt.), who used rapid freezing. Data of Nelson et al. (15) found values for glycogen and lactate in heated tissue (microwave radiation) that were significantly lower (35%) than those in frozen brain. Glucose and fructose-l,6,diphosphate concentrations were similar in the two tissues, but glucose-6-phosphate levels were increased. Since glycogen yields glucose-l-phosphate, and this can be converted to glucose (7,17,19), it is quite possible that microwave irradiation can lead to glucose formation from glycogen. In the present work, the standing wave of the microwave was used to increase the efficiency of energy transfer to the animal's brain to about 90%, two- to threefold that achieved by other workers (1,14,15,20,25). The method described in this paper for direct measurement of glucose concentrations in different brain regions complements the more convenient technique described by Sokoloff (24). It is generally accepted that the tissue glucose concentration depends on the plasma glucose, rate of blood flow, and the rate of glucose consumption (Table I) (16,18,24,25). Whole blood has a value of about 6.3 /xmol glucose per ml (Table I) (about 32.1 /xmol glucose per g of dried blood) (6), which means perivent, nuc. (hypothalamus) has a glucose content greater than one-third that of whole blood. This study shows that there is significant free glucose in mouse brain. Reivich and Sokoloff's data show that there is a remarkably close correlation between local blood flow and glucose consumption in the brains of conscious rats (16,24). The relatively high levels of glucose we found in amygdala and hypothalamus correlate well with the known relatively low rates of glucose consumption in these areas (16,24). The relatively low concentrations of glucose in the substantia nigra and nucleus ruber may be related to a high rate of metabolism in these areas, but independent measures of metabolic rates in these areas are not available. The relatively low concentrations of glucose in the fornix probably reflect the high lipid concentrations in white matter. If we correct our values for glucose for the lipid content, using the data of Folbergrowi et al. for lipids (9), the glucose concentrations in the cerebral neocortex and fornix are found to be 12.7 +__0.8 and 18.1 +-- 1.10/xmol/g of lipidfree dry weight, (mean _ SEM). The higher glucose concentrations in white
GLUCOSE IN BRAIN LOCI
601
matter (fornix) than in gray matter (cerebral neocortex) agrees with the known lower rate of glucose metabolism in white matter (16,24), and the value for fornix agrees with the value (9) for glucose concentration in lipidfree white matter. Of lesser importance, but still a factor to be considered, would be the fact that the blood in the brain at the time of microwave irradiation would be coagulated and, since different regions vary in blood content, corrections would have to be made for glucose content in this trapped blood. It would seem, therefore, that much still remains to be done to determine the true levels of free glucose in brain in vivo. The use of GC-MS provides a powerful tool to measure glucose concentrations in small areas of brain. We are currently examining the possibility of applying this method to measure the dynamics of regional brain metabolism. By measuring the specific activities of glucose (precursor) and glucose-6-phosphate (32) (product) following injection of [~C]glucose, regional brain glucose turnover can be determined (33).
ACKNOWLEDGMENTS The authors are very grateful to Mr. M. Takashima and Mr. T. Ikeda, technicians in Sanyo Electric Co., for planning the microwave power pack in the present work. The authors are also would like to thank Dr. John P. Blass and Dr. Gary E. Gibson for valuable discussions and critical reading. This work was partly supported by funds from the Education Ministry of Japan (Research for Encouragement A No. 077494).
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