Local cerebral glucose utilization in the homozygous Brattleboro rat

Neuroscience Letters, 58 (1985) 171-176

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Elsevier Scientific Publishers Ireland Ltd. NSL 03405 L O C A L C E R E B R A L G L U C O S E U T I L I Z A T I O N IN T H E H O M O Z Y G O U S BRATTLEBORO RAT

WILLIAM J. SCHWARTZl'* and GREGORY CROSBY2 INeuroendocrinology Research Laboratory, Neurology Service, and 2Anesthesia Service, Massachusetts General Hospital and Harvard Medical School, Boston, M,4 02114 (U.S.A.)

(Received November 21st, 1984; Revised version received April 22nd, 1985; Accepted April 23rd, 1985)

Key words." Brattleboro rat - [~4C]deoxyglucose- glucose utilization - vasopressin

The [J4C]deoxyglucosetechnique was used to measure brain glucose utilization in homozygous male Brattleboro and age-matched Long-Evans control rats. Brattleboro homozygotes had significantly higher daily water intakes and plasma osmolalities and significantly lower body weights than controls. Glucose utilization for the brain as a whole and for 46 discrete brain structures was not significantly different for the two strains. Our results indicate that vasopressin is not essential for the maintenance of overall brain glucose utilization in resting, awake rats.

Brain and pituitary vasopressin is absent in h o m o z y g o u s Brattleboro rats with hereditary hypothalamic diabetes insipidus (cf. review in ref. 18). The clinical syndrome is due to defective vasopressin synthesis in the h y p o t h a l a m o - n e u r o h y p o p h y s e a l sec r e t o r y system [17]. Recent studies have revealed that this defect is associated with diverse abnormalities o f central nervous system function. Brain development is impaired [2], cortical m o r p h o l o g y is altered [8], and concentrations o f m o n o a m i n e r gic [5, 11, 22] and peptidergic [14-16] neurotransmitters are disturbed. Brattleboro h o m o z y g o t e s perform poorly in m e m o r y testing paradigms [3], perhaps due to associated sleep deficits [6], altered response to shock [23], or changed temperamental disposition [4, 24]. Since h o m o z y g o u s Brattleboro rats show this array o f functional deficits, and since brain functional activity is closely coupled to brain energy metabolism [9, 19], we wondered whether brain energy metabolism would be altered in Brattleboro h o m o z y gotes. Previous metabolic studies using the [~4C]deoxyglucose m e t h o d have specifically focused on those brain regions concerned with the regulation o f water balance. Increased glucose utilization in the h o m o z y g o u s Brattleboro neurohypophysis is now well-established [ 10, 13, 21 ], and subfornical organ metabolic activity has been specifically studied [10]. Using histochemical localization o f the respiratory enzyme c y t o c h r o m e aa3 as an index o f metabolism, other investigators have found heightened *Author for correspondence at: Neurology Research 4, Massachusetts General Hospital, Boston, MA 02114, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.

172 metabolic activity in magnocellular hypothalamic neurons in Brattleboro rats [12]. However, none of these studies has systematically addressed the metabolic status of those diverse brain regions relevant to the defective behavior of Brattleboro homozygores. Therefore, we have measured both whole and regional brain glucose utilization using the fully quantitative [~4C]deoxyglucose technique [20]. Homozygous male Brattleboro and normal control Long-Evans rats (Blue Spruce Farms, Altamount, NY), 12 weeks old at delivery, were housed for at least 2 weeks in diurnal lighting, 12 h of light per day (lights on from 07.00 to 19.00 h). Light was provided by 15 W cool white fluorescent tubes delivering an intensity of 700 lux at the middle of each cage. Purina Rat Chow and water were freely available, and the time of day that routine care was provided was randomized. Daily water consumption was recorded for at least 1 week before the deoxyglucose experiments. On the morning of the experiment, each animal was lightly anesthetized with halothane and nitrous oxide, and polyethylene catheters were inserted into a femoral vein and artery. Animals were then restrained by loosely fitting abdomino-pelvic plaster casts which were taped to lead bricks, and anesthesia was discontinued. Rats were allowed to awaken for at least 2 h before experiments began with the bolus intravenous injection of 125 itCi/kg 2-deoxy-D-[1-~4C]glucose (DG) (Amersham; spec.act. 60 Ci/mol). Free access to water was allowed at all times. During the experiment, timed arterial blood samples were collected and immediately centrifuged. Plasma concentrations of DG and glucose were determined by liquid-scintillation counting and enzymatic analysis, respectively (Beckman Instruments, Fullerton, CA). After 45 min, the animals were killed by an intravenous injection of sodium pentobarbital. Brains were removed, frozen in 2-methylbutane cooled to -40~C with dry ice, embedded with frozen section mounting medium (M-I Embedding Matrix, Lipshaw Mfg. Co., Detroit, MI) and stored at - 8 5 C . Coronal sections 20/~m thick were later cut in a cryostat at - 2 0 C and autoradiographed along with calibrated [14C]methylmethacrylate standards iAmersham) on Kodak SB-5 X-ray film. Optical density measurements of brain structures were made on a minimum of 4 autoradiographic sections with a manual microdensitometer (250-/~m aperture) (Densichron model PPD: Sargent-Welch, Skokee, IL), or the Photoscan System P1000 HS densitometer (50-4~m aperture) (Optronics International, Chelmsford, MA) coupled to the computerized image-processing system of the Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, MD. Local tissue ~4C concentrations were determined by comparing the optical densities with the calibrated standards. Local rates of glucose utilization were calculated from the local tissue concentrations of J4C, the time-courses of plasma DG and glucose concentrations, and the rate and lumped constants of normal rat brain according to the operational equation of the method [20]. Immediately prior to DG administration, arterial blood pH, pO2, and pCO2 were measured with a blood gas analyser (BM53 MK2; Radiometer, Copenhagen), and hematocrit was determined from an arterial blood sample collected in a capillary tube. Mean arterial blood pressure and rectal temperature were monitored during the experiment, and temperature was maintained with a heat lamp. The osmolality

173

of a 250-#1 sample of plasma obtained at the conclusion of the experiment was determined by freezing-point depression (Model 330 D; Fiske Assoc., Burlington, MA). Statistical comparisons were determined by either parametric or non-parametric methods as appropriate. Significance was set at P < 0.05. All rats tolerated the preparative and experimental procedures without apparent discomfort or neurological deficit. There were no significant differences in hematocrit, mean arterial blood pressure, rectal temperature, arterial blood gases or plasma glucose between the two strains (Table I). As has already been well-documented [18], homozygous Brattleboro rats had significantly higher daily water intakes and plasma osmolalities and significantly lower body weights [1] than the age-matched LongEvans controls. Autoradiographs of brains from the two rat strains appeared identical by visual inspection. The mean rate of glucose utilization for the brain as a whole, weighted for the masses of its component parts [7], was 74 + 2/zmol/100 g tissue wet wt. per min for Long-Evans and 72+2/~mol/100 g tissue wet wt. per min for Brattleboro homozygotes (not significantly different, two-tailed Student's t-test). Measurement of whole brain glucose utilization might not detect regional redistribution of metabolic activities or changes restricted to areas too small to be reflected by the whole brain average [20]. Therefore, we also measured the rates of glucose utilization in 46 discrete brain structures (Table II). Although inspection of these data shows somewhat elevated glucose utilization by anterior portions of cerebral cortex in Brattleboro homozygotes, no significant differences between the two groups were evident (two-tailed Student's t-test). Thus, both the level and the distribution

TABLE I P H Y S I O L O G I C A L VARIABLES IN L O N G - E V A N S A N D H O M O Z Y G O U S RATS

BRATTLEBORO

Values are the m e a n s + S . E . M , obtained in the number of animals indicated in parentheses. *P<0.001 (two-tailed Student's t-test). **Not significant (Wilcoxon two-sample test; a non-parametric test was used for this parameter, since the variances between the two groups were heterogeneous). Parameter

L o n g - E v a n s (6)

Homozygous Brattleboro (6)

Body weight (g) Plasma osmolality (mOsmol/liter H20) Water intake (ml/100 g body wt. per day) Hematocrit (~o) Mean arterial blood pressure (mmHg) Rectal temperature (°C) Arterial blood pH Arterial blood pO2 (mmHg) Arterial blood pCO2 (mmHg) Arterial blood glucose ( m g ~ )

416 + 13 298 ± 1 12 + 1 47 + 1 125 + 4 37.1 + 0.2 7.45+ 0.01 76 + 2 35 + 1 189 ___ 8

324 +23* 315 + 3* 86 + 9* 47 + 1 122 + 3 36.8 + 0.1 7.44_ 0.01 76 + 2 38 _+ 1 169 + 2**

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TABLE lI LOCAL CEREBRAL GLUCOSE UTILIZATION IN LONG EVANS AND HOMOZYGOU~ TLEBORO RATS Values are the means_+S.E.M, obtained in the number of animals indicated in parentheses expressed as ,umot/100 g tissue wet wt./min. Structure

Long Evans (6)

Homozygous Brattleboro (6)

Cerebral Cortex Sulcal prefrontal Frontal Piriform Sensorimotor Parietal Cingulate Auditory Visual

128_+ Ill_+ 110+ 101± 97_+ 105_+ 149_+ 116_+

9 6 7 4 3 3 9 7

145 +_ 6 118_+ 8 I10+ 4 113_+ 9 104_+ 5 107_+ 7 147_+ 10 117_+ 6

Subcortical Motor Systems Caudate-putamen Globuspallidus Lateral thalamus Ventral thalamus Substantia nigra Pontine gray Medullary gray Inferior olive

95_+ 54_+ 103_+ 78_+ 71 ± 58_+ 58_+ 67_+

4 2 3 3 2 2 I 2

98_+ 55+ 101 _+ 80+ 71 ± 55_+ 56+ 64+

5 3 5 4 6 2 3 2

Cerebellum Cerebeltar cortex Flocculus Nodulus-uvula Deep cerebellar nuclei

56_+ 2 115_+ 13 106_+ 4 95 +_ 4

54_+ 115_+ 104_+ 88_+

2 6 5 5

Limbic System Nucleus Accumbens Septum Suprachiasmatic nuclei Hypothalamus Mammitlary body Anterior thalamus Amygdala Habenula lnterpeduncular nucleus Hippocampus Dentategyrus Raphe

78_+ 64_+ 69± 58+_ 116-+ 118 + 59+_ 113-+ 100-+ 80_+ 61_+ 99_+

78-+ 66_+ 66-+ 59+ 116-+ l 17 ± 59+ 103-t99_+ 76_+ 58+ 97+

4 4 2 3 5 4 4 6 5 4 4 4

5 5 4 I 7 5 3 6 6 3 3 5

175 TABLE II (continued) Structure

Long-Evans (6)

Homozygous Brattleboro (6)

Sensory Relay Nuclei Principal sensory V Vestibular Cochlear Superior olivary Lateral lemniscus Inferior colliculus Medial geniculate Superior colliculus Lateral geniculate

62 + 4 114+ 5 90___ 9 110+ 9 99 _+10 167 _+ 6 114_+ 5 92_+ 6 102_+ 4

62___ 6 108__ 4 88 _ 9 114_+ 11 98 _+12 183 _+10 113_+ 7 88_+ 5 106_+ 7

Myelinated fiber tracts Corpuscallosum Ventral hippocampal commissure Internal capsule Middle cerebellar peduncle Cerebellar white

29+ 27-+ 31-+ 22_+ 36_+

2 2 1 3 1

32+ 32-+ 32-+ 18_+ 34_+

2 3 2 2 2

o f glucose utilization t h r o u g h o u t the b r a i n is n o r m a l in h o m o z y g o u s B r a t t l e b o r o rats. O u r results indicate that vasopressin is n o t essential for the m a i n t e n a n c e o f overall b r a i n glucose utilization in resting, a w a k e rats. It is nevertheless possible t h a t the peptide m a y p l a y a role in the r e g u l a t i o n o f energy m e t a b o l i s m in n o r m a l individuals w h o d o synthesize the h o r m o n e . A l t h o u g h b r a i n energy m e t a b o l i s m is n o r m a l in B r a t t l e b o r o h o m o z y g o t e s at rest, the p a t t e r n o f e v o k e d m e t a b o l i c activity m a y b e c o m e distinctly a b n o r m a l when the rats are required to activate defective neural circuits for the p e r f o r m a n c e o f specific b e h a v i o r a l tasks. W e t h a n k M a t t h e w M o r t o n a n d J a n e t C o h e n for expert technical assistance, Dr. Michael M a z u r e k for help with p l a s m a o s m o l a l i t y d e t e r m i n a t i o n a n d Dr. L. S o k o l o f f a n d the L a b o r a t o r y o f Cerebral M e t a b o l i s m , N . I . M . H . , for generous use o f their d e n s i t o m e t e r a p p a r a t u s . W.J.S. is s u p p o r t e d by N . I . N . C . D . S . T e a c h e r - I n v e s t i g a t o r A w a r d K07 NS00672 a n d M a r c h o f D i m e s Basil O ' C o n n o r S t a r t e r Research G r a n t 5-433, a n d G.C. by N . I . G , M . S . G r a n t G m 30502. 1 Arimura, A., Sawano, S., Redding, T.W. and Schally, A.V., Studies on retarded growth of rats with hereditary hypothalamic diabetes insipidus, Neuroendocrinology, 3 (1968) 187-192. 2 Boer, G.J., Van Rheenen-Verberg, C.M.H. and Vylings, H.B.M., Impaired brain development of the diabetes insipidus Brattleboro rat, Dev. Brain Res., 3 (1982) 557-575. 3 Bohus, B., Van Wimersma Greidanus, T.B. and DeWied, D., Behavioral and endocrine responses of rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain), Physiol. Behav., 14 (1975) 609-615.

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