Low concentrations of hydrogen sulphide alter monoamine levels in the developing rat central nervous system.

The central nervous system is one of the primary target organs for hydrogen sulphide (H2S) toxicity; however, there are limited data on the neurotoxic effects of low-dose chronic exposure on the developing nervous system. Levels of serotonin and norepinephrine in the developing rat cerebellum and frontal cortex were determined following chronic exposure to 20 and 75 ppm H2S during perinatal development. Both monoamines were altered in rats exposed to 75 ppm H2S compared with controls; serotonin levels were significantly increased at days 14 and 21 postnatal in both brain regions, and norepinephrine levels were significantly increased at days 7, 14, and 21 postnatal in cerebellum and at day 21 in the frontal cortex. Exposure to 20 ppm H2S significantly increased the levels of serotonin in the frontal cortex at day 21, whereas levels of norepinephrine were significantly reduced in the frontal cortex at days 14 and 21, and at day 14 in the cerebellum.     

Thyroxine effects on polyamine metabolism in rat cerebellum and brain cortex during postnatal development

Polyamine content, ODC and SAMDC activities have been assayed in cerebellum and brain cortex of hyperthyroid rats during postnatal development. Daily thyroxine treatment induced ODC and SAMDC biosynthesis in early periods of postnatal life in both cerebellum and brain cortex. In addition, in comparison to controls an increase in spermidine and spermine content was shown to occur in hyperthyroid rats. A functional correlation between polyamine content and nucleic acids could explain a correlation between polyamine biosynthesis and morphofunctional maturative processes in the brain.     

Excess of taurine supplementation in rat: effects on GABA-related amino acids in developing nervous tissues.

The effects of taurine supplementation on GABA-related amino acid homeostasis in developing nervous tissues of suckling rats were studied. In the first two weeks of postnatal growth, cerebral cortex and cerebellum appear more accessible to taurine supplementation in comparison to retina; in addition, different changes in excitatory/inhibitory amino acids were observed. After the 5th day of life, in the retina and cerebellum of taurine-supplemented pups a decrease in GABA levels was found; in contrast, in cerebral cortex GABA content significantly increased throughout 20 days of postnatal growth. In all nervous tissues studied (except for cerebellum) glutamine concentration increased at the 5th day; then in cerebellum and in retina, but not in cerebral cortex, a significant decrease until the 20th day occurred. Furthermore, in cerebellum and retina taurine supplementation decreased glutamate levels, in comparison to controls, at the 10th and until the 20th day of postnatal life, respectively, whereas in cerebral cortex an increase in glutamate level was observed only at the 5th day. In conclusion, taurine supplementation, in excess to the usual amount from the mother's milk, affected the glutamate compartments in various cell types. The changes in GABA-related amino acid concentrations in cerebral cortex, cerebellum, and retina may depend on the different pattern of the metabolic processes at different maturative stages.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

Glial fibrillary acidic protein in the brain of prenatally stressed rats

The content of glial fibrillary acidic protein (GFAP) was studied in the brain structures of rats borne by intact females and females that underwent stress. In the offspring of stressed rats, the GFAP content in the brain gray and white matter on the 15th postnatal day noticeably dropped. On the 30th postnatal day, the GFAP content in the cortex and pons increased, while it somewhat decreased in the striatum and cerebellum. The results suggest that formation of the intermediate astrocyte filaments in the animals subjected to prenatal stress is markedly disturbed.     

Effects of methylazoxymethanol given at different stages of postnatal life on development of the rat brain. Comparison with those of thyroid deficiency

Newborn rats were treated at different stages of their development with low doses of methylazoxymethanol acetate. The postnatal increase of the DNA content of the cerebrum did not differ from that of controls. In the cerebellum, the DNA content was transitorily reduced, but later, the external granular layer became thicker and DNA deposition increased in comparison with controls; finally, the cerebellar DNA returned to a normal value. Morphological abnormalities of the cerebellum, abnormal orientation of migrating cells, scattering of Purkinje cell bodies within the internal granule cells and specially striking abnormalities of the morphology and orientation of Purkinje cell dendrites were noted in rats treated with MAM from birth to day 3. The effects on the Purkinje cell morphogenesis persisted but were much less marked when MAM was given from 4 to 7 or from 8 to 11 days. Neonatal thyroid deficiency, as MAM-treatment between days 0 and 3, leads to an abnormal position of Purkinje cell bodies within the cerebellar cortex; it also leads to morphological abnormalities of their dendritic arborization which closely resemble those observed after MAM-treatment during the second postnatal week. It also alters the cell formation in the cerebellum. Thyroid deficiency probably exerts its effect on cell formation earlier than previous biochemical studies have shown. On another hand, the morphological abnormalities of Purkinje cell arborizations in the thyroid-deficient animals may be partly due to the perturbations of cell formation which persist later in the cerebellum.     

Critical periods for noradrenergic regeneration in rat brain regions following neonatal subcutaneous 6-hydroxydopamine

The critical period for 60HDA-induced noradrenergic hypertrophy was assessed in several subregions of the cerebellum by treating rat pups at various ages over the first postnatal week. Noradrenergic recovery was greatest closest to birth and declined gradually over this period. Within the cerebellum recovery appeared to decline most rapidly in the middle region. In contrast, in the cerebral cortex noradrenergic fibers apparently increased their resistance and survivability over the first postnatal week. The results are discussed in terms of a hypothesized target-elicited retrograde control of locus coeruleus growth.     

Stimulation of ornithine decarboxylase by histamine or norepinephrine in brain regions of the developing rat: Evidence for biogenic amines as trophic agents in neonatal brain development

Ornithine decarboxylase (ODC) initiates the synthesis of polyamines which play key roles in regulation of cellular development. Intracisternal administration of histamine or norepinephrine to developing rats produced age-dependent stimulation of ODC in brain. In cerebral cortex and pons-medulla, stimulation by norepinephrine was demonstrable at postnatal day 7 and maximum stimulation occured at about day 9. In contrast, cerebellum showed no initial reactivity to norepinephrine but still developed a large peak of response capability by day 9. In all 3 regions, the response declined rapidly thereafter during the period of major synaptogenesis of noradrenergic pathways. With histamine, none of the regions displayed ODC reactivity at 7 days postnatally; stimulation appeared by day 9, peaked at about day 11 and then declined rapidly. Thus, the trophic effect of histamine or norepinephrine toward ODC activity is present or develops postnatally and appears to terminate with synaptogenesis and onset of neurotransmitter properties of the amines.     

The Influence of Bicuculline-Induced Seizures on Free Fatty Acid Concentrations in Cerebral Cortex, Hippocampus, and Cerebellum

Abstract: Using ventilated rats maintained on N₂O-O₂ (70:30, vol/vol) we induced continuous seizures with i.v. bicuculline and analysed free fatty acids (FFA) in cerebral cortex, hippocampus, and cerebellum after seizure durations of 1–120 min. In the cerebral cortex, peak FFA concentrations were observed after 5 min, with a threefold increase in total FFA content. The values then remained unchanged for the next 15-20 min, but decreased thereafter. At 60 and 120 min, total FFA contents were only moderately increased above control. In the initial period, arachidonic acid increased about 10-fold and stearic acid 2- to 3-fold, with little change in palmitic acid and linoleic acid concentrations. At all times, the docosahexenoic acid concentration was markedly increased. Following its massive accumulation at 1 min, arachidonic acid gradually decreased in concentration. Pretreatment of animals with indomethacin did not alter this behaviour. After 20 and 120 min of seizure activity, changes in total and individual FFA concentrations in the hippocampus were similar to those observed in the cerebral cortex. The cerebellum behaved differently. Thus, at 20 min the only significant change was a 5- to 10-fold increase in arachidonic acid concentration and, after 120 min, total and individual FFA concentrations were similar to control values. Furthermore, since the control values for arachidonic acid were much lower in the cerebellum, the 20-min values were only about 20% of those observed in the cerebral cortex and the hippocampus.     

A neurochemical study of developmental impairment of the brain caused by the administration of cytosine arabinoside during the fetal or neonatal period of rats

Injection of pregnant rats with cytosine arabinoside (ara-C) (280 mg/kg) on day 15 of gestation caused a significant rise (about two times the control value) in monoamine concentrations (norepinephrine, dopamine, and serotonin) accompanied by a decrease (about 60% of the control) in the brain weight and DNA content in the cerebrum of the offspring at 60 days of age. When neonatal rats were injected with ara-C (30 mg/kg/day) for four consecutive days from the fourth to seventh days after birth, a decrease of DNA content per cerebellum and an elevation of monoamine concentrations in the cerebellum were found. However, the total content of each monoamine per cerebrum or cerebellum showed no difference from the control. These results suggest that monoaminergic neurons may remain intact, with normal monoaminergic synapses compressed into a small brain volume. The neonatal administration of ara-C caused an elevation of 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) (EC 3.1.4.37) activity and myelin protein content in the cerebellum, suggesting a relative increase in myelin concentration as a result of hypoplasia of granule cells.     

Alterations in monoamine levels in discrete regions of rat brain after chronic administration of carbamazepine

Carbamazepine (25 mg/kg body weight) was administered intraperitoneally to adult male Wistar rats for 45 days and norepinephrine (NE), dopamine (DA) and serotonin (5-HT) levels were simultaneously assayed in discrete brain regions by high performance liquid chromatographic (HPLC) method. Experimental rats displayed no behavioral abnormalities. Body and brain weights were not significantly different from control group of rats. After exposure it was observed that norepinephrine levels were elevated in motor cortex (P<0.01) and cerebellum (P<0.05), while dopamine levels were decreased in these two regions (P<0.001, P<0.05). However, dopamine levels were increased in hippocampus (P<0.01). Serotonin levels were significantly decreased in motor cortex (P<0.001) and hypothalamus (P<0.001) but increased in striatum-accumbens (P<0.001) and brainstem (P<0.001). These results suggest that carbamazepine may mediate its anticonvulsant effect by differential alterations of monoamine levels in discrete brain regions particularly in motor cortex and cerebellum.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

Utilization in vivo of glucose carbon in the optic lobes and the cerebellum of the chick during postnatal growth.

1. The incorporation of glucose carbon in vivo into amino acids was studied in the chick optic lobes and cerebellum during postnatal growth after subcutaneous injection of [U-14C]glucose. 2. The rapid incorporation of glucose carbon into free amino acids appears between the 1st and the 2nd day of postnatal growth in the optic lobes and between the 1st and the 4th day after hatching in the cerebellum. 3. The period during which the properties of mature brain metabolism are obtained is characterized in both structures during the first 48 hr of postnatal growth by changes in the specific radioactivity of some amino acids such as aspartate and alpha-alanine, and also by transient increases of glucose and glutamine concentrations. 4. The gamma-aminobutyrate content in the optic lobes is very high; the cerebellum on the contrary is characterized by its low gamma-aminobutyrate concentration linked to a very fast metabolism of this amino acid.     

Norepinephrine content of the rat kidney during development: Alterations induced by perinatal methadone

The concentration and the total content of norepinephrine (NE) in the kidney were measured in Sprague-Dawley rats from 3 to 120 days after birth. Renal NE concentration was relatively low until the end of the second week, when it rose abruptly to adult levels; total NE content per kidney increased steadily throughout development. The effects of perinatal methadone treatment on renal NE development were examined by administering the drug either directly to the pups from 1 to 19 days after birth, or to the mother from 10 days of gestation to 20 days after birth. Both treatments resulted in significant deficits of body weight and kidney weight. Maternal methadone caused a significant deficit in renal NE which was most pronounced at two weeks of postnatal age; direct methadone had less effect on renal NE. These results suggest that renal sympathetic neurotransmission may become mature two weeks after birth and indicate further that maternal methadone interfares with this maturation.     

Postnatal Development of Thiamine Metabolism in Rat Brain

The activities of thiamine diphosphatase (TDPase), thiamine triphosphatase (TTPase), and thiamine pyrophosphokinase and the contents of thiamine and its phosphate esters were determined in rat brain cortex, cerebellum, and liver from birth to adulthood. Microsomal TTPase activity in the cerebral cortex and cerebellum increased from birth to 3 weeks, whereas that in the liver did not change during postnatal development. Microsomal TDPase activity in the cerebral cortex showed a transient increase at 1-2 weeks, but that in the cerebellum did not change during development. In contrast to the activity of the brain enzyme, that of liver microsomal TDPase increased stepwise after birth. Thiamine pyrophosphokinase activity in the cerebellum increased from birth to 3 weeks and then decreased, whereas that in the cerebral cortex and liver showed less change during development. TDP and thiamine monophosphate (TMP) levels increased after birth and plateaued at 3 weeks whereas TTP and thiamine levels showed little change during development in the cerebral cortex and cerebellum. The contents of thiamine and its phosphate esters in the liver showed more complicated changes during development. It is concluded that thiamine metabolism in the brain changes during postnatal development in a different way from that in the liver and that the development of thiamine metabolism differs among brain regions.     

Enhanced Brain Cell Proliferation Following Early Adrenalectomy in Rats

We have previously demonstrated an increase in adult brain DNA content in rats adrenalectomized on postnatal day 11. The present studies examined cell proliferation in cerebral cortex, cerebellum, hippocampus, and midbrain-diencephalon following adrenalectomy at this age. Compared to sham-operated controls, adrenalectomized animals showed increased [3H]thymidine incorporation into DNA (measured at 1 h following a pulse injection) in all brain regions at 7 and 14 days postsurgery. In some areas, the effect was already present as early as 2 days following adrenalectomy. Chronic replacement with corticosterone prevented this increase in DNA labelling in a dose-dependent manner. When cell proliferation in the cerebral cortex and cerebellum was independently assessed by measuring changes in thymidine kinase activity, enzyme activity was significantly elevated in both areas at 7 and 14 days postsurgery. Finally, histological examination of the cerebellar cortex suggested a delayed disappearance of the external granular layer in several cerebellar lobules of adrenalectomized animals. Overall, these findings indicate that day-11 adrenalectomy leads to a prolonged stimulation of mitotic activity in areas where cell formation at this time is exclusively glial (i.e., cerebral cortex and mid-brain-diencephalon) as well as in areas where postnatal neurogenesis is also occurring (cerebellum and hippocampus). It is hypothesized that this stimulation results from the removal of a tonic inhibitory effect exerted by circulating glucocorticoids in the normal intact animal.     

The effect of chronically ingested inorganic lead on brain levels of Fe,Zn, Cu,and Mn of 25 day old rat

Lead poisoning was produced in suckling rats by lead fed to lactating mother rats being transmitted to newborns $\text{via}$ maternal milk. The brain of 25 day old lead poisoned rats contained 6.0 to 12.5 ppm lead. Zinc was depressed 56% (cerebral cortex), 45% in cerebellum and 43% (caudate nucleus) whereas copper was depressed 52% in the cerebellum. Inhibition of monoamine oxidase (pargyline) or partial depletion of serotonin and norepinephrine did not alter the concentration of Fe, Cu, Zn, or Mn in brain.     

Developmental regulation of somatostatin gene expression in the brain is region specific

Developmental regulation of somatostatin (SRIF) gene expression was studied in five regions of rat brain and in rat stomach. Total RNA was isolated from hypothalamus, cortex, brainstem, cerebellum, and olfactory bulb, as well as stomach at eight stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF cDNA probe to Northern blots of total RNA from the above tissues during development demonstrated a single hybridizing band approximately 670 base pairs in length. When SRIF mRNA levels from each stage of development were quantified and normalized by the amount of poly (A)+ RNA present at that stage of development, a unique pattern of SRIF gene expression was seen in each region. In brainstem and cerebellum, SRIF mRNA levels peaked early in development between prenatal day 21 and postnatal day 8 and then declined until postnatal day 82. Hypothalamus and cortex, on the other hand, showed a progressive increase during development with peak levels occurring between postnatal days 13 and 82. In contrast, stomach and olfactory bulb showed SRIF mRNA levels which were low during early development and which rose late in development (postnatal days 13 to 82). Marked differences in the amount of SRIF mRNA within each region were present as well. These data suggest that there is differential expression of the SRIF gene in different regions of the brain and in the stomach during development. Further study of this phenomenon may provide insight into the in vivo control of SRIF gene expression and the role of SRIF in the developing brain.     

Ontogeny of Neurochemical Markers for Noradrenergic, GABAergic and Cholinergic Neurons in Neocortex Lesioned with Methylazoxymethanol Acetate

Abstract: Methylazoxymethanol acetate (MAM), a potent, rapidly eliminated nucleic acid alkylating agent, produces microencephaly in rat pups when injected into their dams on day 15 of gestation. In the adult microencephalic rats, neuronal loss is largely confined to telencephalic structures, such as the superficial neocortical laminae, whose neuroepithelial progenitor cells were undergoing vigorous replication during the chemical exposure. Histological examination of the forebrain 2 days after injection revealed early selective damage to the ventricular geminal zone with relative sparing of cortical plate neurons generated on earlier days. The degree of specificity of MAM's action on neurochemically defined neuronal populations was examined by measuring presynaptic markers for GABAergic, noradrenergic and cholinergic neurons in atrophic lateral cortex from 20 days gestation to adulthood. Although treatment reduced GABAergic markers (GABA, its synthetic enzyme and synaptosomal uptake process) in proportion to loss of cortex mass (-67%), the maturational pattern for remaining GABAergic neurons was virtually normal. Although the maturational sequence of noradrenergic markers was similar to control, the concentration of endogenous norepinephrine, [³H]norepinephrine uptake and tyrosine hydroxylase specific activity were two- to fourfold higher than control at each time. However, total noradrenergic markers per cortex section were nearly identical to control throughout development, indicating that development of the noradrenergic axonal arbor in neocortex was insensitive to loss of neurons in the terminal field. Maturation of cholinergic markers (endogenous acetylcholine, its synthetic enzyme and [³H]choline uptake) in the atrophic cortex was biphasic: concentrations were similar to control values for the first 12 postnatal days, but gradually rose to levels twofold higher than control. These results indicate that neurochemical alterations observed in cortex from prenatally MAM-treated rats are primarily the result of early selective elimination of neuronal subpopulations. Fetal MAM exposure appeared to have minimal effects on biochemical differentiation of neurons remaining intact in the atrophic cortex. MAM appears to be a useful toxin for producing selective loss of neuronal groups based on their time of generation in the fetus.