A.J. Patel

Manifestation of metabolic compartmentation during the maturation of the rat brain.

—(1) The fate of [U‐14C]leucine was studied in rat brain in vivo from birth to five weeks of age. The major route of leucine metabolism at all ages was conversion into protein. The rate of protein synthesis was low in the newborn; it reached a peak at about 15 days and slowed down moderately later. Incorporation into brain lipids was relatively low under the experimental conditions (less than 2 per cent of the total tissue 14C).

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of 14C incorporation into brain DNA at 30 min after administration of [2‐14C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 106 and 18 × 106 cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.

Effect of thyroid deficiency on cell acquisition in the postnatal rat brain: A quantitative histological study

The mechanisms underlying transient reduction of cell number in the developing cerebellum of thyroid-deficient rats have been studied by quantitative histological methods. Thyroid deficiency has no significant effect on the generation cycle of dividing cells in either the subependymal layer of the lateral ventricular walls or the external granulay layer of the cerebellum: the length of the cell cycle and the duration of the different phases of the cycle, including the DNA synthesis time appears to be normal. However, the external granular layer of the cerebellum contains fewer cells than in control at 12 days. Pyknotic nuclei are prominent in the granule cell layer of the hypothyroid cerebellum at this age. These amount to an estimated loss of about 1% of the total cerebellar cell population in 24 h. It is suggested that death of granule cells is for the most part a consequence of reduced Purkinje cell dendritic arborization, with failure of parallel fibre/Purkinje cell synaptogenesis. In the second postnatal week, granule cell death and reduced numbers of cells in the germinal zone can account to a great extent for the observed shortfall in cerebellar DNA content. The eventual attainment of normal cell numbers in the cerebellum of hypothyroid rats is related to a persistent external granular layer in the forth and fifth postnatal weeks.

Effect of thyroid deficiency on postnatal cell formation in the rat brain: A biochemical investigation

In thyroid deficiency, interference with postnatal cell formation seems to be confined to those regions of the brain where postnatal neurogenesis is significant. In comparison with controls the increase in cell number in the cerebellum is retarded in the second week of life, but a normal number is reached by 35 days. In contrast the DNA content of the olfactory bulbs is apparently irreversibly depressed. Mitotic activity, in terms of incorporation of [2-14C]thymidine into DNA, is mainly affected in the cerebellum: in thyroid deficient rats, it is depressed below control levels at day 12, but it is about 4 times higher than in controls at day 21 when, under normal conditions, cell proliferation has virtually ceased. The time course (15-240 min) of [14C]thymidine metabolism at day 14 shows regional differences in control rats. The rate of conversion of [14C]thymidine to [14C]thymidine nucleotides, and of these in turn to [14C]DNA is slower in the forebrain - where cell proliferation occurs on a smaller scale - than in the cerebellum. Consequently, in the forebrain nearly linear DNA synthesis rate is maintained for a longer time than in the cerebellum (1 h vs. 0.5 h), and since less 14C is conserved in DNA a significant efflux of unconverted [14C]thymidine is evident during the experimental period. The effect of thyroid deficiency on [14C]thymidine metabolism in the brain is only slight, and is due to an abnormally large supply of [14C]thymidine consequent to depressed systemic utilization of this precursor.

Effect of reserpine on cell proliferation in the developing rat brain: A quantitative histological study

Rats aged 11 days were injected with reserpine (2.5 mg/kg body wt.) and the rate of [3H]thymidine incorporation into brain DNA was followed over a period of 36h. In the forebrain this was significantly depressed by 2h, and it reached a nadir of about 30% of the control level at 4h, at which it remained for another 26h. A partial recovery occurred by 36h. The effect was less pronounced in the cerebellum. On the basis of this information brains of rats were examined histologically and by autoradiography between 7 and 36 h after reserpine to obtain estimates of cell cycle parameters and of rates of cell proliferation and cell loss. In the forebrain lateral ventricular subependymal layer the labelling index was markedly reduced in comparison withe controls. Cell cycle time was prolonged by 50% and turnover time increased by 60%. In the cerebellar external granular layer, the mitotic index was reduced and increased numbers of degenerate postmitotic nuclei were found, notably in the latter part of the experimental period. These effects are potentially of functional and clinical significance.

Effect of reserpine on cell proliferation in the developing rat brain: A biochemical study

Reserpine, a well-known CNS depressant which depletes central monoamine stores, was found to produce in the brains of 11-day-old rats a severe depression in cell proliferation in terms of the rate of [3H]thymidine incorporation into DNA. The effect was studied in detail 12 h after ther administration of the drug (2.5 mg/kg, s.c.) when the rate of in vivo DNA synthesis in the forebrain was about one-third of control: the decrease was less marked in the cerebellum (rate about two-thirds of control). It was possible to exclude side effects of the drug, such as restricted food intake, hypothermia and an elevation of the level of blood corticosteroids being responsible for the reduction of [3H]thymidine incorporation into DNA. Kinetic studies showed that reserpine had no marked effect on the entry of [3H]thymidine from blood to brain, but it caused some retardation in the rate of [3H]thymidine conversion into [3H]thymidine nucleotides. Nevertheless, the severe depression of DNA labelling was evident even after correcting the values on the basis of [3H]thymidine nucleotide concentrations. In contrast to these effects, thymidine kinase activity was normal in the brain of reserpine-treated animals.

DO DRUGS ACTING ON THE NERVOUS SYSTEM AFFECT CELL PROLIFERATION IN THE DEVELOPING BRAIN

Drugs which alter the balance of neurotransmitter activity may, while failing to cause gross structural malformations of the brain, produce long-lasting functional disturbances if given when the brain is developing. Subtle anatomical changes may underlie such disturbances; reserpine has been shown to interfere with cell proliferation in the brain of suckling rats, and long-term alterations in behaviour reported after treatment with reserpine may be related to this effect of the drug. Neurotransmitters, apart from their conventional role, may also function as neurohumours and be involved in the regulation of cell proliferation in the nervous system. Thus drugs influencing central neurotransmitter activity, such as phenothiazines and adrenergic agonists and antagonists, which in clinical practice are often given to pregnant mothers, may affect the developing brain through mechanisms similar to those reported for reserpine. More experimental information is needed about the influence of such drugs on cell proliferation in the brain, and about their behavioural teratogenicity.

Effect of chlorpromazine on cell proliferation in the developing rat brain. A combined biochemical and morphological study

Chlorpromazine, a widely used drug in current clinical practice, produced a severe reduction of the rate of [3H]thymidine incorporation into brain DNA of 11-day-old rats. The depression of in vivo synthesis rate was detectable by 6 h after chlorpromazine administration (50 mg/kg, s.c.) and the rate was less than 40% and 60% of controls during period 14-30 h in forebrain and 6-30 h in cerebellum respectively. The depression was dose-dependent and half maximal effect was produced with about 10 mg/kg chlorpromazine. The drug caused some retardation in the rate of conversion of [3H]thymidine to [3H]thymine nucleotides in the brain, but the severe depression in DNA labelling was also evident after correcting the values on the basis of [3H]thymine nucleotides concentration. Mitotic activity was significantly reduced in the cerebellar external granular layer. Increased numbers of cell degenerations, shown by Feulgen cytophotometry to be postmitotic, were seen in both layers 12 and 32 h after chlorpromazine. Analysis of cell cycle parameters showed no significant changes. However, the labelling index in subependymal cells was reduced, indicating an increase in turnover time of about 40%. The results are consistent with an action of chlorpromazine on cell proliferation, either by direct effects on the generation and survival of cells, or via its major pharmacological actions on neurotransmitter balance. These effects are potentially of functional and clinical significance.

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4‐14C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [14C]succinate was rapid, as shown by the incorporation of 14C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue ‐“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [14C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4‐14C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points.

METABOLIC COMPARTMENTATION IN THE BRAIN: EFFECTS OF A CENTRAL NERVOUS SYSTEM DEPRESSANT, 1-HYDROXY-3-AMINO-PYRROLIDONE-2

—The effect of 1‐hydroxy‐3‐aminopyrrolidone‐2(HA‐966), a CNS depressant, was studied on the metabolism of [14C]glucose and [3H]acetate in the brain in mice. HA‐966 had a marked effect on glucose metabolism. The conversion of glucose carbon into amino acids associated with the tricarboxylic acid cycle (‘cycle’) was severely reduced, while the concentration of brain glucose was approximately doubled. Relative to the specific radioactivity of glucose in the brain, the specific radioactivity of alanine was 60–70 per cent of the control, indicating a reduction in the rate of glycolysis, and those of the‘cycle’amino acids were also lowered. A reduction in‘cycle’flux of 30–35 per cent was estimated. It was established that the depressed glucose utilization flux was not due to either impaired uptake of glucose from blood to brain or to hypothermia. In contrast to [14C]glucose, there was no change in the labelling of the amino acid fraction from [3H]acetate, which is preferentially metabolized in the small’compartment believed to be associated with glia. Thus it seems that CNS depression caused by HA‐966 resulted in a selective decrease in energy production in the‘large’metabolic compartment where glucose is oxidized preferentially and which is believed to be associated with neuronal structures.