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.     

Mechanism of Delayed Increases in Kynurenine Pathway Metabolism in Damaged Brain Regions Following Transient Cerebral Ischemia

Abstract: Delayed increases in the levels of an endogenous N-methyl-D-aspartate receptor agonist, quinolinic acid (QUIN), have been demonstrated following transient ischemia in the gerbil and were postulated to be secondary to induction of indoleamine-2,3-dioxygenase (IDO) and other enzymes of the L-tryptophan-kynurenine pathway. In the present study, proportional increases in IDO activity and QUIN concentrations were found 4 days after 10 min of cerebral ischemia, with both responses in hippocampus > striatum > cerebral cortex > thalamus. These increases paralleled the severity of local brain injury and inflammation. IDO activity and QUIN concentrations were unchanged in the cerebellum of postischemic gerbils, which is consistent with the preservation of blood flow and resultant absence of pathology in this region. Blood QUIN and L-kynurenine concentrations were not affected by ischemia. Brain tissue QUIN levels at 4 days postischemia exceeded blood concentrations, minimizing a role for breakdown of the blood–brain barrier. Marked increases in the activity of kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase were also detected in hippocampus but not in cerebellum on day 4 of recirculation. In vivo synthesis of [¹³C₆]QUIN was demonstrated, using mass spectrometry, in hippocampus but not in cerebellum of 4-day postischemic animals 1 h after intracisternal administration of L-[¹³C₆]tryptophan. However, accumulation of QUIN was demonstrated in both cerebellum and hippocampus of control gerbils following an intracisternal injection of 3-hydroxyanthranilic acid, which verifies the availability of precursor to both regions when administered intracisternally. Notably, although IDO activity and QUIN concentrations were unchanged in the cerebellum of ischemic gerbils, both IDO activity and QUIN content were increased in cerebellum to approximately the same degree as in hippocampus, striatum, cerebral cortex, and thalamus 24 h after immune stimulation by systemic pokeweed mitogen administration, demonstrating that the cerebellum can increase IDO activity and QUIN content in response to immune activation. No changes in kynurenic acid concentrations in either hippocampus, cerebellum, or cerebrospinal fluid were observed in the postischemic gerbils compared with controls, in accordance with the unaffected activity of kynurenine aminotransferase activity. Collectively, these results support roles for IDO, kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase in accelerating the conversion of L-tryptophan and other substrates to QUIN in damaged brain regions following transient cerebral ischemia. Immunocytochemical results demonstrated the presence of macrophage infiltrates in hippocampus and other brain regions that parallel the extent of these biochemical changes. We hypothesize that increased kynurenine pathway metabolism after ischemia reflects the presence of macrophages and other reactive cell populations at sites of brain injury.     

A single peripheral injection of basic fibroblast growth factor (bFGF) stimulates granule cell production and increases cerebellar growth in newborn rats

The control of neuronal number is critical for coordinating innervation and target organ requirements. Although basic fibroblast growth factor (bFGF) is known to regulate neuron number in the developing embryonic cortex, its potential role during postnatal brain development remains undefined. To address this issue, the cerebellum, a site of postnatal neurogenesis, was used. Previously, we found that a single peripheral injection of bFGF in newborn rats elicited mitosis of neuronal precursors in the external germinal layer (EGL) 8 h after administration. We now define the sustained effects of bFGF treatment on postnatal granule cell production and cerebellar growth. Seventy-two h after a single injection of bFGF (20 ng/g) in newborn rats, the fraction of BrdU-labeled cells in the EGL increased by 46% without altering apoptotic cell number, consistent with enhanced precursor proliferation. Moreover, bFGF increased mitotically labeled cells by 100% and total cell density by 33% in the internal granular layer (IGL), the final destination of the EGL precursors. Because cerebellar volume also increased by 22%, bFGF-induced proliferation enhanced generation of total IGL neurons and increased cerebellar growth. These morphometric measures were corroborated independently by using DNA quantitation: cerebellar DNA content increased 16% after bFGF injection, consistent with increased neuron number. Furthermore, using DNA quantitation as an index, increased total cerebellar cell number elicited by bFGF injection persisted beyond the neurogenetic period, until P35. We conclude that a single postnatal injection of bFGF increases granule neuron number and enhances cerebellar growth following mitotic stimulation.     

Corticosterone regulates calbindin-D28k mRNA and protein levels in rat hippocampus

Corticosterone was administered to normal and bilaterally adrenalectomized rats (250-300 g), and hormonal regulation of brain calbindin-D28k (CaBP28k) levels was investigated by radioimmunoassay for CaBP28k protein and by slot and Northern blot analyses for CaBP28k mRNA. The specificity of the changes observed in CaBP28k mRNA levels was tested by reprobing blots with calmodulin and B-actin cDNAs. Rats were either adrenalectomized, adrenalectomized treated with corticosterone, intact, or intact treated with corticosterone. Chronic corticosterone administration (subcutaneous injection for 7 days, 10 mg/day) to normal intact rats significantly increased levels of CaBP28k immunoreactivity (43%) and mRNA (125%) in the hippocampus. Adrenalectomy (animals were killed 7 days after adrenalectomy) produced a significant decrease in hippocampal CaBP28k immunoreactivity (85%) and mRNA (80%) compared with intact controls. Immunocytochemical analysis of tissue sections inducated a marked depletion of CaBP28k immunoreactivity in the dentate gyrus of the hippocampus 2 weeks after adrenalectomy. When adrenalectomized rats were treated with corticosterone (10 mg/day for 7 days), CaBP28k protein and mRNA levels in hippocampus were restored to levels observed in intact controls. No changes in CaBP28k protein and mRNA in kidney, cerebellum, striatum, or cerebral cortex were noted in adrenalectomized rats or in intact rats treated with corticosterone when compared with controls, indicating the specificity of the effect on CaBP28k for the hippocampus. These studies present the first evidence of a regulator of CaBP28k gene expression in the brain.     

Ornithine Decarboxylase Induction by Glucocorticoids in Brain and Liver of Adrenalectomized Rats

Abstract: Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute S.C. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [³H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

3Development-Dependent Regulation of N-Acetyl-β-d-Hexosaminidase of Cerebellum and Cerebrum of Normal and Staggerer Mutant Mice

Distinctive activities of various glycosidases were expressed in the cerebellum and cerebral cortex of mice during their development. In particular, N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30) appeared to be developmentally regulated. A transient peak of enzyme activity at postnatal day 7 was characteristic for the cerebellum, whereas the activity in the cerebral cortex gradually increased through the 1st postnatal month and was maintained at a high level of activity throughout adulthood. The regulation of N-acetylhexosaminidase activity in the developing cerebellum of the staggerer mouse deviated clearly from enzyme activities in the wild-type, whereas the activity pattern in the staggerer cerebral cortex remained unaffected. In experiments mixing wild-type and staggerer cerebellum homogenates, the specific activity was additive. Thus, involvement of inhibitors or activating molecules can be excluded. This developmentally controlled regulation or disregulation in staggerer appears to be enzyme specific, sine beta-glucosidase, alpha-glucosidase, and beta-galactosidase did not exhibit such a pattern in either normal or staggerer mice. In the mutation weaver that, like staggerer, loses the majority of its cerebellar granule cells, N-acetyl-beta-hexosaminidase activity of the cerebellum was not elevated, indicating a specific defect in staggerer rather than a general effect on lysosomal enzymes due to cell death.     

Studies on the synthesis and degradation of DNA in developing and old chick cerebellum

The levels of DNA, RNA, protein and acid and alkaline DNase were studied in developing and old chick cerebellum. The in vitro synthesis of DNA, by both chick cerebrum and cerebellum was also studied, by following the incorporation of [3H]thymidine into DNA. It was observed that the increase in DNA content of chick cerebellum continued well beyond adult stages of life span. Maximal DNA synthesis, as judged by the [3H]thymidine incorporation, was noticed during the early embryonic development but decreased with advancement of age. There was, however, another peak of activity, although smaller, at about 9 months of age. Both cerebrum and cerebellum showed similar patterns. The highest specific activity of acid DNase was also found during the early period of cerebellar development, that is at a time when rapid cellular proliferation was occurring. The activity steadily declined with the aging and in 2-year-old cerebellum very little activity could be detected. Alkaline DNase, on the other hand, not only exhibited high activity during the early development but also remained at a significant level even in old cerebellum. It is concluded that acid DNase shows a positive correlation to the early embryonic DNA synthesis but not to the cell increase occurring in old age.     

Increased Uric Acid in the Developing Brain and Spinal Cord Following Cytomegalovirus Infection

Abstract: Tissue concentrations of uric acid were determined in the spinal cord, cerebellum, caudate-putamen, and cerebral cortex of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. Transient signs of neurological impairment were observed in MCMV-infected animals beginning on days 13–16 and continuing until days 19–21. At the onset of neurological impairment, uric acid concentrations in tissues from infected animals were 17–60-fold greater than in control animals. On postnatal day 70, 60 days after inoculation and 40 days after resolution of neurological signs, uric acid levels were still two- to threefold greater in infected animals. Histological examination revealed signs of focal ischemia in the cerebral and cerebellar cortices of MCMV-infected mice only at the onset of neurological impairment, with ischemic cell changes in some pyramidal neurons of the cerebral cortex. These results indicate that uric acid may be a sensitive marker of persistent vascular pathology resulting from cytomegalovirus infection of the developing nervous system     

CORRELATION OF DNA ACCUMULATION RATE WITH THYMIDYLATE SYNTHETASE AND THYMIDINE KINASE ACTIVITIES IN DEVELOPING RAT CEREBELLUM: EFFECT OF THYROXINE1

Abstract— Using the method of least squares, a logistic curve was fitted to the data points for DNA content in neonatal rat cerebellum versus postnatal age (day 0 is the day of birth). The resultant equation was differentiated to give an expression for the rate of cerebellar DNA accumulation in units of ng/h per mg wet cerebellum. The DNA accumulation rate in control rats increased from 77.0 at 2 days of age to a maximum of 108 at 7 days of age and declined thereafter to a minimum of 16.3 on day 15. Thyroxine treatment significantly (P < 0.05) increased the rate to 89.8 (117% of control) at 2 days of age, and a significant elevation was maintained to 6 days of age at which time a maximum rate of 115 (114% of control) was attained. The rate was significantly decreased below control at 9 and 12 days of age, and reached a minimum of 9.22 on day 15. The developmental pattern for the activity of cerebellar thymidylate synthetase (EC 2.1.1.6), in units of pmol/h per mg wet cerebellum, closely paralleled the pattern for DNA accumulation rate in both control and thyroxine-treated animals. In controls, thymidylate synthetase activity increased from 98.6 at 2 days of age to a maximum of 125 at 7 days of age and declined thereafter to a minimum of 30.0 at 15 days of age. In thyroxine-treated animals, the activity was significantly increased to 118 (122% of control) at 4 days of age and remained significantly elevated through 6 days of age at which time a maximum activity of 154 (115% of control) was attained; thereafter, the activity was significantly decreased below control and reached a minimum of 16.9 (56.3% of control) on day 15. The developmental pattern for the activity of cerebellar thymidine kinase (EC 2.7.1.21) did not parallel the DNA accumulation rate quite so closely, in neither treated nor control animals, as did the pattern for thymidylate synthetase activity. These data suggest that thymidylate synthetase activity in the developing rat cerebellum may be more important for maintenance of replicative DNA synthesis than is thymidine kinase activity. In addition, the thyroxine-induced acceleration of the increase and subsequent decline in rate of DNA accumulation and in the activities of thymidylate synthetase and thymidine kinase in developing rat cerebella is probably the result of alterations in the number of external granular cells undergoing replicative DNA synthesis.     

Distribution of Cyclic Nucleotide Phosphodiesterase in Mouse Brain

Seventy-one regions of mouse brain, and many subdivisions of some of these, were analyzed for cyclic nucleotide phosphodiesterase. The samples were dissected from lyophilized frozen sections. Since the average sample weighed only 25 ng (20 X 75 X 75 mu3), regions as small as the locus ceruleus could be analyzed. Activities in gray areas ranged 40-fold from a high in the pars reticulata of the substantia nigra to a low in the deep cerebellar nuclei. The activity in fiber tracts also varied about 40-fold, and on a lipid-free dry weight basis was similar to the activity in the gray matter where the fibers originated. The rank order for gray regions was basal ganglia, amygdala, hippocampus, cerebral cortex, most of the diencephalic nuclei, nuclei of the pons, cerebellum, and nuclei of the medulla.     

Influence of intermittent hypoxia and pyrimidinic nucleosides on cerebral enzymatic activities related to energy transduction

The effect of intermittent normobaric hypoxia and of biological pyrimidines (uridine and cytidine) on the specific activities of some enzymes related to cerebral energy metabolism were studied. Measurement were carried out on the following: (a) homogenate in toto; (b) purified mitochondrial fraction; (c) crude synaptosomal fraction, in different areas of rat brain: cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. Intermittent normobaric hypoxia (12 hours daily for 5 days) caused modifications of the enzyme activities in the homogenate in toto (decrease of hexokinase in cerebellum; increase of pyruvate kinase in medulla oblongata), in the purified mitochondrial fraction (increase of succinate dehydrogenase in the corpus striatum) and in the crude synaptosomal fraction (decrease of cytochrome oxidase activity in cerebral cortex, hippocampus, and cerebellum; decrease of malate dehydrogenase in hippocampus and cerebellum; decrease of lactate dehydrogenase in cerebellum). Daily treatment with cytidine or uridine altered some enzyme activities either affected or unaffected by intermittent hypoxia.     

Neonatal monosodium glutamate treatment modifies glutamic acid decarboxylase activity during rat brain postnatal development

Monosodium glutamate (MSG) produces neurodegeneration in several brain regions when it is administered to neonatal rats. From an early embryonic age to adulthood, GABA neurons appear to have functional glutamatergic receptors, which could convert them in an important target for excitotoxic neurodegeneration. Changes in the activity of the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), have been shown after different neuronal insults. Therefore, this work evaluates the effect of neonatal MSG treatment on GAD activity and kinetics in the cerebral cortex, striatum, hippocampus and cerebellum of the rat brain during postnatal development. Neonatal MSG treatment decreased GAD activity in the cerebral cortex at 21 and 60 postnatal days (PD), mainly due to a reduction in the enzyme affinity (K(m)). In striatum, the GAD activity and the enzyme maximum velocity (V(max)) were increased at PD 60 after neonatal MSG treatment. Finally, in the hippocampus and cerebellum, the GAD activity and V(max) were increased, but the K(m) was found to be lower in the experimental group. The results could be related to compensatory mechanisms from the surviving GABAergic neurons, and suggest a putative adjustment in the GAD isoform expression throughout the development of the postnatal brain, since this enzyme is regulated by the synaptic activity under physiological and/or pathophysiological conditions.     

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.     

EFFECTS OF HYPOTHYROIDISM AND UNDERNUTRITION ON DNA CONTENT AND THYMIDINE KINASE ACTIVITY DURING CEREBELLAR DEVELOPMENT IN THE RAT1,2

—The effects of hypothyroidism and several degrees of undernutrition on the development of cerebellar weight, DNA, and thymidine kinase activity were studied in young rats ranging in age from 2 to 22 days. Early propylthiouracil treatment caused a delayed cerebellar cell multiplication. The activity of cerebellar thymidine kinase was suppressed at ages 2 and 5 days and was in excess of control values on days 15 and 22, thus resulting in a delay in the developmental spectrum for thymidine kinase, and extending the time span of activity beyond that of controls. Undernutrition led to varying degrees of reduced cell proliferation at experimental ages 5, 12, and 19 days. Cerebella from the most undernourished animals showed significant differences from controls in thymidine kinase activity at ages 5 and 12 days. Comparisons between sub-groups from within the oversized litters at 5 and 12 days suggested that changes in thymidine kinase activity relate to the degree of undernutrition to which the sub-group is subjected and that during development there may be a critical degree of undernutrition at which a particular essential enzyme becomes affected. This study emphasizes the biochemical similarities and differences between neonatal hypothyroidism and undernutrition, while pointing out the difficulties which exist in biochemical separation of components of the two conditions. Further evidence is presented that thymidine kinase is responsive to hormonal stimuli during cerebellar development and may play an important role in the regulation of DNA biosynthesis in brain as well as other organs.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Influence of tryptophan on the activity of acetylcholinesterase in the brain of well-fed normal and adrenalectomized rats

Eight hours after a single tube-feeding of tryptophan the activity of acetylcholinesterase in the cerebral hemisphere of well-fed (non-fasted) normal and adrenalectomized rats was 28 and 53% higher, respectively, compared to the corresponding water-fed control. On the other hand, the enzyme activity in the cerebellum of both normal and adrenalectomized rats remained essentially unchanged following tryptophan administration. Pretreatment of adrenalectomized rats with actinomycin-D totally abolished the tryptophan-mediated stimulation of cerebral acetylcholinesterase activity. The pattern of response of cerebral acetylcholinesterase in well-fed adrenalectomized rats over a period of 24-hr following a dose of tryptophan was found to be biphasic.     

Changes in glucocorticoid receptors in different regions of brain of immature and mature male rats

Specific cytosolic binding for synthetic glucocorticoid dexamethasone was studied in several brain regions (hypothalamus, hippocampus, caudate nucleus, cerebellum, cerebral cortex) of immature (3-week) and mature (26-week) male rats, intact and adrenalectomized. A significant regional difference was observed in the concentration of in vitro [3H] dexamethasone binding in the brain of adrenalectomized rats at both ages, with the highest levels in the hippocampus. A marked decrease in specific binding was observed in all brain regions of adrenalectomized mature rats as compared to immature. The dexamethasone binding was significantly lower in all brain regions of normal intact animals as compared to adrenalectomized rats in both ages.     

RNA polymerase I and II activities in different brain regions of young, adult, and old rats

The activities of RNA polymerase I and II were assayed in nuclei isolated from different regions (cerebral cortex, cerebellum, hypothalamus, hippocampus, corpus striatum and pituitary) of brains from young (10 days), adult (6 months), and old (2 years) rats. The RNA polymerases I and II activities generally increased during maturation, i.e., from 10 days to 6 months of postnatal age and then showed a decrease from 6 months to 2 years of age in all the regions except in cerebral cortex where the RNA polymerase II activity was highest at 10 days but showed a gradual decrease through the lifespan up to 2 years.     

Distribution of neuronal nitric oxide synthase-immunoreactive neurons in the cerebral cortex and hippocampus during postnatal development

Although many reports have argued a role for nitric oxide (NO) during postnatal development, there has been no combined demonstration in the cerebral cortex and hippocampus. We have investigated the distribution and morphology of neurons and fibers expressing neuronal NO synthase (nNOS) in the cerebral cortex and hippocampal formation of rats during the postnatal development, and correlated these findings with developmental events taking place in these regions. In the cerebral cortex, the nNOS-immunoreactive cells could be divided into two classes : heavily stained neurons and lightly stained neurons. For the lightly stained nNOS-positive neurons, only the cell bodies were observed, whereas for the heavily stained neurons, the cell bodies and their dendrites were visible. During the postnatal days, heavily stained neurons reached their typical morphology in the second week and appeared in all layers except for layer I. In the hippocampus, there was a transient expression of nNOS in the pyramidal cell layer at P3â€P7, and this expression disappeared during following days. The adult pattern of staining developed gradually during the postnatal period. This study suggested that these alterations might reflect a region-specific role of NO and a potential developmental role in the postnatal cerebral cortex and hippocampus