Quantitative histochemical changes in enzymes involved in energy metabolism in the rat brain during postnatal development. II. Glucose-6-phosphate dehydrogenase and beta-hydroxybutyrate dehydrogenase.

The postnatal maturation of glucose-6-phosphate and beta-hydroxybutyrate dehydrogenase activity was assessed by histochemistry in rats at eight postnatal stages, P0, P5, P10, P14, P17, P21, P35 and the adult stage. Enzyme activities were revealed on cryostat brain sections with nitroblue tetrazolium. Both enzyme activities were low and homogeneous at birth, and increased to reach a peak in all areas studied, at P17 for beta-hydroxybutyrate dehydrogenase and at P21 for glucose-6-phosphate dehydrogenase. Then, glucose-6-phosphate dehydrogenase activity decreased regularly by 20-49% from P21 to adult stage, except in cerebellar white matter where activity did not change after P21. beta-hydroxybutyrate dehydrogenase activity decreased regularly from P17 to adult stage in globus pallidus, hippocampus, thalamus, brainstem, genu of corpus callosum and cerebellar white matter. It sensorimotor cortex, medial geniculate body, caudate nucleus, hypothalamus and inferior colliculus, beta-hydroxybutyrate dehydrogenase activity stayed stable between P17 and P35 and decreased thereafter to adult levels. Finally, in parietal, auditory and cerebellar cortices, beta-hydroxybutyrate dehydrogenase activity either stayed stable or slightly increased after P17. The present study shows that there is a quite good correlation between postnatal changes in cerebral glucose-6-phosphate and beta-hydroxybutyrate dehydrogenase activities and the importance of pentose phosphate pathway and ketone body utilization in the developing brain. Our results also reflect the regional heterogeneity of beta-hydroxybutyrate utilization in the adult rat brain, translating into a remaining high activity of beta-hydroxybutyrate dehydrogenase in cerebral cortex.     

Quantitative autoradiographic measurement of local cerebral glucose utilization in freely moving rats during postnatal development

The quantitative 2-14C-deoxyglucose autoradiographic method of Sokoloff et al. (1977) was used to measure local cerebral glucose utilization in freely moving developing rats. The animals were studied at 10, 14, 17, 21, and 35 d and at the adult stage. Glucose utilization was very low and quite uniform in 10- and 14-d-old rats, ranging from 20 to 30 mumol/100 gm/min, except in a few posterior areas. Between these 2 stages, rates of glucose utilization significantly increased in 6 areas, among which 4 were belonging to the auditory system. Between 14 and 17 d, glucose utilization significantly changed in 9 structures out of the 68 studied, mainly auditory, visual, parietal, and thalamic areas. Between the stages of 17 and 21 d, glucose utilization was increased by 50 or 100% in all brain structures studied, except in the medial habenula and white matter areas. After weaning time, rates of glucose utilization still significantly changed in 50 areas, widely distributed through all studied systems. Between 35 d and the adult stage, the average rate of glucose utilization did not change and rates of energy metabolism significantly increased in 13 brain areas. In one structure, the medial habenula, glucose utilization was already high 10 d after birth and did not change over the whole studied period. These increases in the rates of glucose utilization are consistent with the behavioral, anatomical, and functional changes known to occur during this period of development in the rat.     

Consequences of chronic phenobarbital treatment on local cerebral glucose utilization in the developing rat

The influence of a chronic phenobarbital (PhB) treatment on postnatal evolution of local cerebral metabolic rates for glucose (LCMRglc) was studied in 58 cerebral structures of freely moving rats. The animals received a daily subcutaneous injection of PhB at a dose of 50 mg/kg between days 2 and 35 or an equivalent volume of saline for controls and were studied at 5 postnatal stages, i.e. 10, 14, 17, 21 and 35 days, and at the adult stage. Body and brain weights were both reduced by 6-21% over the whole period studied. PhB exposure induced significant decreases in LCMRglc during the period of pharmacological treatment, i.e. until 35 days, except at the stage of 17 days as well as long-term reductions in LCMRglc of adult rats in 36 out of the 58 brain regions studied. These decreases affected all systems studied, sensory systems as well as limbic, hypothalamic, motor and white matter areas. In addition to a growth retardation, PhB also seemed to be able to induce a delay in the acquisition of auditory function which matures early during postnatal life. The long-term deficits in cerebral energy metabolism due to PhB in the adult rat also confirm the behavioral deficits which have been shown previously after early PhB exposure.     

Quantitative histochemical changes in enzymes involved in energy metabolism in the rat brain during postnatal Development. I. Cytochrome oxidase and lactate dehydrogenase

The postnatal maturation of cytochrome oxidase and lactate dehydrogenase activity was assessed by histochemistry in rats at 8 postnatal stages, P0, P5, P10, P14, P17, P21, P35 and the adult stage. Enzyme activities were revealed on cryostat brain sections with diaminobenzidine for cytochrome oxidase and nitroblue tetrazolium for lactate dehydrogenase. Lactate dehydrogenase activity remained unchanged between P0 and P10, significantly increased in 8 areas of the 14 studied between P10 and P14 and in 6 structures from P14 to P17. These were mainly parietal, auditory and cerebellar cortices, hippocampus, thalamus, hypothalamus and medial geniculate body. There was no further change until P35 and lactate dehydrogenase activity increased then significantly to reach higher adult levels in hippocampus and medial geniculate body. Cytochrome oxidase activity was low from P0 to P10 and increased in 8 regions between P10 and P14. These were all cortices, caudate nucleus, hippocampus, inferior colliculus and genu. Enzyme activity further increased between P14 and P17 in auditory cortex, medial geniculate body and brainstem, did not vary from P17 to P21 but increased by 92 to 371% in all areas between P21 and P35. Cytochrome oxidase activity rose further from P35 to adult stage in hippocampus and medial geniculate body. From birth to adulthood, cytochrome oxidase activity increased 5 to 19 fold and lactate dehydrogenase activity 1.8 to 3.0. The present study shows that there is a quite good correlation between postnatal changes in regional cerebral glucose utilization and activity of enzymes involved in glycolytic and oxidative glucose metabolism in the rat.     

Metabolic maturation of the brain: a study of local cerebral protein synthesis in the developing cat

We used quantitative L-[1-(14)C]leucine autoradiography to study the maturation of cerebral protein synthesis metabolism in kittens, starting at birth and through postnatal age (P) 180 days as well as in adult cats. We found that at birth most brain structures show protein synthesis (nmol/min/g; lCPS(leu)) rates already within the range of adult values (with some exceptions; e.g., the hippocampus and putamen). Likewise, most structures show a transient developmental peak during which the rates climb to levels higher than in adulthood. This peak often occurred at P60, but in some regions lasted from P30 to P90. Therefore, there is some regional heterogeneity in the maturation of brain protein synthesis. These results are compared with our previous findings on the maturation of cerebral glucose utilization and oxidative metabolism. We discuss the meaning of these maturational profiles in terms of time course of morphological development and of maturation of behavior in the cat. Correlations with findings in other mammalian species are also discussed.     

Effects of early chronic phenobarbital treatment on the maturation of energy metabolism in the developing rat brain. II. Incorporation of beta-hydroxybutyrate into amino acids

The influence of phenobarbital (PhB) on the utilization of beta-hydroxybutyrate by the cerebral cortex and the cerebellum was studied in rats during postnatal maturation. The animals were treated from day 2 to day 35 after birth either by a daily injection of 50 mg/kg PhB or by saline. The rats were studied at 5 postnatal stages: 7, 10, 14, 21 and 35 days. Plasma beta-hydroxybutyrate and acetoacetate levels reached their peak values between 10 and 14 days after birth. The concentration of both ketone bodies was significantly higher in PhB- than in saline-treated rats between 10 and 35 days after birth. The total incorporation of [3-14C]beta-hydroxybutyrate into amino acids reached a peak value at 14 days after birth and was down to very low values at 35 days. It was higher in PhB- than in saline-treated rats. The specific radioactivity values of glutamate, glutamine, aspartate and GABA were significantly higher in PhB- than in saline-treated especially at 10 days after birth. These results demonstrate that a PhB treatment induces an increase in brain ketone body utilization in neonate rats, which is likely to balance the decrease in brain glucose utilization induced by this pharmacological treatment.     

Effects of early chronic phenobarbital treatment on the maturation of energy metabolism in the developing rat brain. I. Incorporation of glucose carbon into amino acids

The influence of phenobarbital (PhB) on the utilization of glucose by the cerebral cortex and the cerebellum was studied in rats during postnatal development. The animals were treated from day 2 to day 35 after birth by a daily injection of 50 mg/kg PhB or by saline. The rats were studied at 5 postnatal stages: 7, 10, 14, 21 and 35 days. PhB treatment induced a 10% decrease in body and brain weight over the whole period studied and a transient significant decrease in circulating thyroxin levels at 14 days after birth. Amino acid levels in the cerebral cortex and particularly in the cerebellum were not greatly affected by the pharmacological treatment. The conversion of [2-14C]glucose into amino acids was significantly decreased in both cerebral structures between day 7 and day 14 after birth. The distribution of radioactivity between amino acids was not affected in the cerebral cortex but was significantly changed by PhB treatment in the cerebellum. Specific radioactivity values of amino acids were lower in PhB- than in saline-treated animals in both studied structures. The results of the present study show that glucose utilization is reduced in the brain of PhB-treated animals as compared to the controls and that the cerebellum seems to be more affected than the cerebral cortex.     

Effects of a xanthine derivative, propentofylline, on local cerebral blood flow and glucose utilization in the rat

The effects of the xanthine derivative propentofylline [3-methyl-1-(5′-oxohexyl)-7-propylxanthinel were measured on local cerebral blood flow and glucose utilization in the rat using quantitative autoradiographic techniques. A dose of 0.5 mg/kg/min i.v. produced increases in local cerebral blood flow and minimal effects on glucose utilization in the majority of cerebral structures measured. A higher dose of propentofylline (1.5 mg/kg/min) produced an overall increase in local cerebral blood flow and a marked reduction in glucose utilization. Furthermore, propentofylline increased the average ratio of blood flow per unit glucose utilization and thus is capable of increasing cerebral blood flow in excess of metabolic demand. While the mechanism of action of this compound has not been fully defined. it is possible that its cerebrovascular and cerebral metabolic effects can at least partially be explained by a blockade of adenosine uptake. These actions of propentofylline on cerebral blood flow and metabolism may play a role in protecting neuronal tissue under hypoxic/ischemic conditions in the brain.     

Age-dependent pathways of brain energy metabolism: the suckling rat, a natural model of the ketogenic diet

As a consequence of the high fat content of maternal milk, the suckling rat may be viewed as a 'natural model' of the ketogenic diet. Changes in energy metabolism during this period of development may give us some clues into the antiepileptic properties of the ketogenic diet. We have, therefore studied the postnatal evolution of local cerebral metabolic rates for glucose (LCMRglcs) and of regional rates of cerebral uptake of beta-hydroxybutyrate (betaHB) in the developing rat between postnatal day (PN) 10 and 35. LCMRglcs were low and homogeneous at PN10. They increased significantly in four auditory regions between PN10 and PN14, at the time of maturation of auditory function. Between PN14 and PN17, they increased further in two auditory regions, one visual area (the lateral geniculate nucleus), three limbic and three motor areas. These increases occurred simultaneously with the maturation of vision and the development of locomotion and general exploratory behavior. Between PN17 and PN21, LCMRglcs increased by 28-97% (depending on brain area) and by a mean value of 25% in all areas studied. In contrast to the function-related increases in LCMRglcs, regional rates of cerebral betaHB uptake underwent a generalized non-specific increase between PN1O and PN14, and stayed at a high level until PN17. Between PN17 and PN21, rates of cerebral betaHB uptake decreased significantly in all brain regions studied, and reached very low levels by PN35. Thus, even in the suckling rat, whose cerebral metabolic activity depends upon both glucose and ketone bodies, it is the postnatal increases in LCMRglcs that appear to be critical for the acquisition of new functions and neurological competence. Conversely, the homogeneous increase in cerebral betaHB uptake occurring between PN10 and PN17 at a period of active brain growth may rather reflect non-specific mechanisms of cell growth.     

Effects of glycerol on local cerebral glucose utilization and local cerebral blood flow of hypoxic rats

Effects of a continuous infusion of glycerol on the hypoxic rat were assessed by measurements of local cerebral glucose utilization and local cerebral blood flow using the quantitative autoradiographic 1-[C-14]-2-deoxyglucose and [C-14]-iodoantipyrine techniques, respectively. Local cerebral glucose utilization of the hypoxic rat was decreased in most of cerebral structures except for the amygdala, septal nucleus and the white matter. Local cerebral glucose utilization of the hypoxic rat treated with glycerol was recovered to the pattern of normal control, although glucose utilization of the nucleus raphe and locus ceruleus was accelerated. Cerebral blood flow of the hypoxic rat was elevated in almost of all measured structures. In particular, blood flow in the substantia nigra and locus ceruleus was increased significantly. The pattern of local cerebral blood flow of the hypoxic rat treated with glycerol was more analogous to that of normal control than of hypoxic rat without treatment. It is suggested that glycerol has beneficial effects by elevating local cerebral glucose utilization inhibited and decreasing local cerebral blood flow overshot on the hypoxic condition.     

Local cerebral blood flow during lithium-pilocarpine seizures in the developing and adult rat: role of coupling between blood flow and metabolism in the genesis of neuronal damage.

Coupling between local cerebral blood flow and local cerebral metabolic rate for glucose is involved in the pathogenesis of epilepsy-related neuronal damage in the adult brain; however, its role in the immature brain is unknown. Lithium-pilocarpine-induced status epilepticus is associated with extended damage in adult rats, mostly in the forebrain limbic areas and thalamus, whereas damage was moderate in 21-day-old rats (P21) or absent in P10 rats. The quantitative autoradiographic [14C]iodoantipyrine technique was applied to measure the consequences of lithium-pilocarpine status epilepticus on local cerebral blood flow. In adult and P21 rats, local cerebral blood flow rates increased by 50% to 400%; the highest increases were recorded in regions showing damage in adults. At P10, local cerebral blood flow rates decreased by 40% to 60% in most areas, except in some forebrain regions showing no change during status epilepticus. In areas injured when status epilepticus was induced in adults, a strong hypermetabolism (Fernandes et al., 1999) not matched by comparable local cerebral blood flow increases was present in rats of all ages, whereas in damage-resistant areas, local cerebral metabolic rate for glucose and local cerebral blood flow remained coupled in the three age groups. Thus, the level of coupling between blood flow supply and metabolism is not involved in seizure-related brain damage in the developing brain, which appears to be resistant to the consequences of such a mismatch.     

Effects of pentylenetetrazol-induced seizures on dopamine and norepinephrine levels and on glucose utilization in various brain regions of the developing rat

Levels of dopamine and norepinephrine were measured in seven brain areas after 60 min of sustained seizure activity induced by intraperitoneal repetitive timed administrations of pentylenetetrazol in rats at 10, 14, 17 and 21 days of postnatal life. The tissue levels of norepinephrine were markedly reduced in the majority of brain structures, except for striatum at 10 and 14 days. Conversely, dopamine concentrations increased in many areas and at various ages, except in cerebral cortex at 10 and 14 days and in midbrain between 14 and 21 days. PTZ seizures induced marked increases over control levels in the rates of glucose utilization, measured by the quantitative autoradiographic [14C]2-deoxyglucose method, in all dopamine- and norepinephrine-innervated areas studied at 10 and 14 days, except in cerebellar cortex at both ages and in frontal cortex and anteroventral thalamus at 14 days. At 17 and 21 days, glucose utilization remained increased over control levels in some areas, mainly in catecholaminergic cell groupings such as substantia nigra, ventral tegmental area and locus coeruleus, but was significantly reduced in cortex, caudate nucleus and thalamus, and similar to control rates in other regions. The present results suggest that pentylenetetrazol-induced seizures lead to a simultaneous increase in functional activity of norepinephrine neurons and an inhibition of dopaminergic-mediated neurons. They also confirm the maturation of connections, of metabolic activity and of neurotransmitter interaction within the brain, occurring mainly during the third week of postnatal life, paralleled by an increased selective vulnerability of some regions to this kind of insult.     

An experimental model of generalized seizures for the measurement of local cerebral glucose utilization in the immature rat. I. Behavioral characterization and determination of lumped constant.

An experimental model of status epilepticus has been developed in the immature rat by administration of pentylenetetrazol (PTZ) using repetitive, timed intraperitoneal injections of subconvulsive doses. The pattern of behavioral signs has been well characterized in each age group, i.e. 10 (P10), 14 (P14), 17 (P17) and 21 postnatal days (P21). In this model, the dose of convulsant could be adjusted as a function of interindividual sensitivity and status epilepticus lated for quite a long duration to allow the measurement of local cerebral metabolic rates for glucose (LCMRglc) by means of the [14C]2-deoxyglucose method [J. Neurochem., 28 (1977) 897-916]. To estimate LCMRglc during status epilepticus, the lumped constant (LC) was re-calculated in controls and PTZ-treated rats. The control LC was 0.54 at P10 and 0.50-0.51 at the three older ages studied (P14, P17 and P21). During status epilepticus, it increased to 0.64 in P10 rats and decreased to 0.42 and 0.40, respectively, in P17 and P21 animals. At P14, LC was not affected by seizures. The measurements of brain lactate levels showed a large 4.5-10-fold increase in PTZ-treated rats as compared to controls at all ages. The results of the present study show that the immature brain responds to sustained seizure activity in a specific way according to its postnatal age. Moreover, our results underscore the necessity of re-calculation of LC to the quantification of LCMRglc in such pathological states, particularly in immature animals.     

Changes in local cerebral glucose utilization after chronic ethanol in rats

Local rates of cerebral glucose utilization were investigated in ethanol-treated rats using the 2-deoxy[14C]glucose method. Rats received ethanol in drinking water for 3 h each day. After 4 days of exposure to 2 or 4% ethanol (v/v), there was a general tendency toward increased local cerebral glucose utilization. In contrast, after 28 days of exposure to concentrations to 10% ethanol a general depression of cerebral glucose metabolism was observed. Brain regions showing significantly reduced glucose utilization rates in the 28-day group included components of the extrapyramidal system, several thalamic and hypothalamic nuclei, and forebrain limbic structures. A group of animals tested 7 days after discontinuation of ethanol showed essentially normal rates of local cerebral glucose utilization, indicating that alterations in regional brain glucose metabolism induced by this particular regimen and duration of ethanol administration were largely reversible.     

Ketamine effects on local cerebral blood flow and metabolism in the rat

The effects of an anesthetic dose (100 mg/kg) of ketamine, a phencyclidine derivative, on local rates of cerebral glucose utilization (LCGU) and CBF (LCBF) have been investigated by the quantitative [14C]2-deoxy-glucose and [14C]iodoantipyrine techniques in the unparalyzed, spontaneously breathing rat. In ketamine-injected animals, LCGU was significantly increased in some limbic structures and decreased in inferior colliculus, vestibular, and cerebellar nuclei. The degree and spatial distribution of drug-induced changes was similar for local blood flow rates, LCBF being increased in limbic regions and decreased in the inferior colliculus. Although Paco2 values were higher in anesthetized animals, the pattern of LCBF/LCGU ratios was not significantly affected by ketamine in the 36 brain regions examined in this study. So, at least in the rat and at the anesthetic level studied here, a net vasodilatory in vivo effect was not observed. These results support the hypothesis that CBF changes induced by the drug in animals and man are primarily related to the metabolic effects exerted by ketamine on cerebral structures.     

Preprotachykinin A mRNA expression in the rat brain during development

Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.     

The effects of the GABAergic agonist muscimol upon the relationship between local cerebral blood flow and glucose utilization

The relationship between cerebral blood flow and glucose utilization was studied in conscious rats using the [14C]iodoantipyrine and [14C]2-deoxyglucose quantitative autoradiographic techniques respectively. Both blood flow and glucose use decreased in parallel in the 38 areas of the brain analyzed following administration of the GABA agonist muscimol, thus maintaining the relationship observed in control rats. The present study offers no evidence for a direct vasodilatatory action for muscimol in the conscious rat, but points to underlying regional metabolic activity as the primary determinant of cerebral blood flow.     

Fructose 2,6-bisphosphate in developing rat brain.

Fructose 2,6-bisphosphate (Fru-2,6-P2) levels and 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase activities have been studied in rat brain during development from embryonal to adult state. Fru-2,6-P2 increases slightly from day 16 of gestation, reaching a maximum 24 h after birth, remaining quite constant during postnatal development. In contrast with 6-phosphofructo-1-kinase, which increases progressively after the first week of age, 6-phosphofructo-2-kinase remains unaltered throughout the period studied. The role of Fru-2,6-P2 in controlling cerebral glycolysis is discussed.     

Quantitative autoradiographic study of the postnatal development of benzodiazepine binding sites and their coupling to GABA receptors in the rat brain.

The postnatal development of benzodiazepine binding sites in the rat brain was studied by quantitative receptor autoradiography using [3H]flunitrazepam. The coupling of these sites to GABA receptors was assessed in 43 cerebral structures by examining the effects of in vitro addition of GABA on flunitrazepam specific binding. Benzodiazepine-specific binding was relatively high at birth and exhibited an heterogeneous distribution pattern, anatomically different from the adult one. Data showed a sequential development of benzodiazepine receptors in relation to the time course of maturation of cerebral structures. A proliferation peak which paralleled rapid brain growth was noticed. High levels of benzodiazepine sites were transiently observed in some areas, e.g. thalamus and hypothalamus, and might be related to maturational events. In every brain structure examined, benzodiazepine binding sites were linked to GABA receptors. However, enhancement of flunitrazepam specific binding by exogenous GABA differed according to the structures studied and decreased during development, suggesting some changes in the control of GABA/benzodiazepine regulation during postnatal maturation.     

Ontogenesis of neurons immunoreactive for nitric oxide synthase in rat forebrain and midbrain.

We studied the immunohistochemical localization of neuronal nitric oxide synthase (nNOS) in the developing rat brain on embryonic days 13 (E13), 15 (E15), 17 (E17) and 19 (E19) and postnatal days 0 (P0), 7 (P7) and 14 (P14). A few neurons positive for nNOS were first detected at E15 in the hypothalamus and pons. At E17, many positive cells became detectable in the thalamus. At E19, the positive cells in these three regions were rapidly increased in number, and a few positive neurons were also observed in such regions as the cerebral cortex and striatum. Positive cells in the hypothalamus tended to locate ventrolaterally. Positive neurons, stained very intensely as in adult rats, were seen in the pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parafascicular nucleus. Two weeks after birth, positive neurons of larger somata with many processes were distributed widely in the cerebral cortex and hippocampus. The present study indicates that, in the forebrain and midbrain, the distribution pattern of nNOS-containing neurons is fundamentally completed by E19.