Brain Research 888 2001 193–202 www.elsevier.com locate bres
Research report
Differential increase in cerebral cortical glucose oxidative metabolism during rat postnatal development is greater in vivo than in vitro
a,c c
b c
Edward J. Novotny Jr. , Cristopher Ariyan , Graeme F. Mason , John O’Reilly ,
c a ,
Gabriel G. Haddad , Kevin L. Behar
a
Department of Neurology , Yale University School of Medicine, New Haven, CT 06520, USA
b
Department of Psychiatry , Yale University School of Medicine, New Haven, CT 06520, USA
c
Department of Pediatrics , Yale University School of Medicine, New Haven, CT 06520, USA
Accepted 26 September 2000
Abstract
The steady-state rate of glucose oxidation through the mitochondrial TCA cycle V was measured in acid extracts of 10- and
TCA 13
30-day-old cerebral cortex of rats receiving [1- C]glucose intravenously and in neocortical slices superfused in vitro with the same isotope. TCA cycle flux was determined for each age group based on metabolic modeling analysis of the isotopic turnover of cortical
glutamate and lactate. The sensitivity of the calculated rates to assumed parameters in the model were also assessed. Between 10 and 30
21 21
postnatal days, V increased by 4.3-fold from 0.46 to 2.0 mmol g
min in the cortex in vivo, whereas only a 2-fold from 0.17 to
TCA 21
21
0.34 mmol g min
increase was observed in neocortical slices. The much greater increase in glucose oxidative metabolism of the cortex measured in vivo over that measured in vitro as the cortex matures suggests that function-related energy demands increase during
development, a process that is deficient in the slice as a result of deafferentiation and other mechanisms.
2001 Elsevier Science B.V. All rights reserved.
Theme : Other systems of the CNS
Topic : Brain metabolism and blood flow
13
Keywords : Cerebral development; Tricarboxylic acid cycle; Oxygen consumption; Nuclear magnetic resonance spectroscopy;
C isotopes; Glutamate; Glycolysis
1. Introduction demands of synaptic activity of the neocortex during
postnatal development is not known. In adult neurons Glucose and oxygen consumption in rat brain increases
stimulation-dependent increases in glucose utilization are substantially
during the
first postnatal
month localized in synaptic regions [45,46], suggesting a similar
[4,9,14,17,32–35,48]. This period of rapid increase in location for the increase in functional glucose utilization
glucose metabolism coincides temporally with maturation observed in the developing neocortex. Although, respirato-
of synapses [1,29], receptors [28], neuronal-glial metabolic ry rates of brain slices metabolizing glucose appear lower
interactions [6,40] and development of sensory functions than those reported in the cortex in vivo under basal
and electrical activity [7,10,32]. The fraction of neocortical conditions [22], brain slices from neonatal rats show a
glucose and oxygen consumption that supports the energy strong age dependence for the capacity to increase glucose
utilization during stimulation [19,48] indicating that mech- anisms responsible for stimulation-dependent glucose utili-
zation are retained in the slice preparation. However,
Corresponding author. Department of Neurology and Magnetic Reso-
interpretation of the differences in metabolic rates between
nance Center, Yale University School of Medicine, PO Box 208024, New
brain slices and their in vivo counterpart reported in the
Haven, CT 06520-8024, USA. Tel.: 11-203-737-4121; fax: 11-203-785-
literature is hampered by differences in measurement
6643. E-mail address
: kevin.beharyale.edu K.L. Behar.
techniques and assumptions made in calculating fluxes,
0006-8993 01 – see front matter
2001 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 3 0 5 1 - 1
194 E
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animal species, brain regions measured, and the use and perfusate was replaced by [1- C]glucose 99 atom after
type of anesthetic agent. a 30-min equilibration period. Approximately 12 slices
In the present study, we have examined the influence of were prepared and incubated simultaneously. At selected
development on the difference in glucose oxidative metab- times 0, 15, 30, 60, 90, and 120 min after the additions of
olism in the cerebral cortex of 10–12- and 28–30-day-old the labeled glucose, two to three slices were quickly
rats in vivo and in brain slices prepared in vitro. These removed from the incubation chamber directly into liquid
postnatal ages border the major increases observed in N . Thus, a single animal yielded sufficient cortical tissue
2
glucose metabolism and function in the rat cortex. We to permit generation of a single turnover curve for gluta-
hypothesized that if the difference in the rates of glucose mate.
oxidation between the in vivo cortex and the brain slice is dependent on intact, functional interactions present in vivo,
2.3. Preparation of tissue and plasma extracts but absent in the slice, then a comparison of the cortices at
these two postnatal ages in vivo and in vitro would shed Frozen brain tissue was extracted in 0.1 M methanol
light on the fraction of total glucose oxidation related to HCl and 3 M perchloric acid with minor modifications
function as the brain matures. We have found that during from published methods [23]. Following centrifugation the
postnatal development the maturational increase in the supernatant was neutralized with 10 N KOH, centrifuged
basal rate of cortical glucose oxidative metabolism is again to remove perchlorate salts, and lyophilized. The
.2-fold greater in vivo than in vitro. Our results suggest powder was dissolved in 0.5 ml D O and trimethylsilyl-
2 2
that cortical metabolism associated with intact synaptic 2,2- H-propionic acid TSP was added as a chemical shift
1
inputs contributes a large fraction of the basal energy reference for
H NMR spectroscopy. Total protein was expenditure during this period of development.
measured in the acid extracted pellets after solubilization with 1 N NaOH using the ‘enhanced protocol’ of the
Pierce BCA Protein Assay.
2. Materials and methods Frozen blood plasma was extracted in 0.3 M perchloric
