21st Postnatal Day Research Articles

Effects of chronic alcohol consumption on spatial reference and working memory tasks

The aim of this work was to determine the spatial memory impairments induced by chronic alcohol consumption in rats. The alcoholization process began on the 21st postnatal day and alcohol concentrations were gradually increased to reach a concentration of 20% that was maintained for 4 mon. Behavioral tests were performed in the Morris Water Maze (MWM). The first study assessed the effects of chronic alcohol intake on two reference memory tasks (a place learning with multiple trials and a new place learning carried out in the same experimental context). Alcohol-treated animals presented no overall impairment in their ability to process spatial information. Deficits were restricted to reduced behavioral flexibility in spatial strategies. The second study assessed working memory in two tasks in which information about platform location was only valid for one trial. In the first working memory task, the animals had to perform one trial per day and in the second task they were submitted to four trials per day. At the end of the second experiment, all animals were trained in a visual-cued task. In the second experiment, the most important deficits in alcohol-treated animals occur in spatial working memory tasks, and this impairment was independent of the intertrial interval used. In the second spatial working memory task, performance of the alcohol-treated animals in the earlier trials affected their performance in subsequent trials, suggesting that a process of proactive interference had taken place. The visual-cued task demonstrated that these behavioral impairments were produced without visuoperceptive impairments.

Effects of Perinatal Nicotine Exposure on Development of [3H]Hemicholinium-3 Binding Sites in Rat Neonate Brain

In this study, [3H]hemicholinium-3 ([3H]HC-3) binding, which labels the presynaptic high affinity-choline transport sites, was examined in two brain regions, cerebral cortex and midbrain, of nicotine-treated and -untreated rat neonates. In nicotine-untreated neonates, [3H]HC-3 binding sites of cerebral cortex increased from 64 fmol/mg protein at postnatal day 7 to 142 fmol/mg protein at postnatal day 35. In nicotine-treated neonates, the development of [3H]HC-3 binding sites in cerebral cortex was significantly retarded, compared with control neonates on the 7th, 14th and 21st postnatal days. In parallel with this, the development of muscarinic receptor in cerebral cortex, which was detected by [3H]quinuclidinyl benzylate ([3H]QNB) binding, was also retarded by nicotine treatment. However, in midbrain, neither [3H]HC-3 nor [3H]QNB binding sites at postnatal day 14 was affected by nicotine treatment. These results strongly suggest that perinatal treatment with nicotine inhibits presynaptic and postsynaptic development of the cholinergic pathway in cerebral cortex but not in midbrain of rat neonate.

Undernutrition Decreases Serine Palmitoyltransferase Activity in Developing Rat Hypothalamus

Background/Aims: Undernutrition reduces the hypothalamic ganglioside concentration. This may be attributed to some modifications in the contents of precursors of sphingolipid biosynthesis in undernourished rats. The present study evaluated the serine palmitoyl transferase activity (SPT; EC 2.3.1.50) during the development of the rat hypothalamus. This work also shows the L-[3-¹⁴C]serine metabolic labeling of hypothalamic sphingolipids in normal and undernourished rats at weaning. Methods: The SPT activity was determined in microsomal fractions obtained from the hypothalamus of normal rats (diet: 25% protein) and pre- and postnatally undernourished rats (diet: 8% protein since pregnancy) at 21 days of gestational age and at 7, 14, and 21 days of postnatal life. Results: The enzymatic activity was lower in the hypothalamus of undernourished than in the hypothalamus of control rats since the 7th postnatal day. Incorporation of the precursor L-[3-¹⁴C]serine into sphingolipid fraction was lower in the hypothalamus of undernourished rats than in the hypothalamus of control rats on the 21st postnatal day which coincided with the age of the highest difference in SPT activity between normal and undernourished rats. Conclusion: These results indicate that undernutrition reduces the biosynthesis of the main sphingolipids during the period of brain growth spurt.

Appearance of neuronal S-100β during development of the rat brain

In addition to being an astroglial protein, S-100β is localised in distinct populations of neurons in the adult rat hindbrain. We report, here, the expression of S-100β in both neurons and glia of the rat brain during development. Prenatally, S-100β immunoreactivity was confined to glial cells close to the germinal zone. After birth, S-100β positive glial cells were seen mainly in the brainstem and cerebellum, while only a few were detected in cerebral cortex and hippocampus. The number of S-100β containing glial cells increased steadily during the first 2 postnatal weeks after which the adult pattern was attained. No S-100β containing neurons were present prenatally. The first S-100β containing neurons were seen in the mesencephalic trigeminal nucleus at postnatal day 1 (P1), and in the motor trigeminal nucleus at P3. Neuronal S-100β immunoreactivity in other nuclei was mostly attained from the 10th to the 21st postnatal day. The neuronal S-100β immunoreactivity was first detected in the cell nuclei during development, then increased in the cytoplasm with ages. A nuclear staining in many immunoreactive neurons persisted until the adult. It usually took 1 to 2 weeks for neuronal S-100β to attain the adult staining pattern, i.e., heavy staining of the cytoplasm and processes, after its appearance. The forebrain never contained S-100β positive neurons. The S-100β is first expressed in glial cells, suggesting it is primarily of the glial origin. Coupled with neurotrophic effects of the protein, the time course of neuronal S-100β expression during the critical period of neuronal development implies that it may be involved in neuronal differentiation and maturation.

Effects of preweaning environmental stimulation on neuronal and behavioral impairment produced by undernutrition.

The present study investigates the effects of sensorimotor stimulation on the basal dendrogenesis of superficial cerebrocortical neurons and the accomplishment in the associated exploratory behavior in rats simultaneously exposed to nutritional deprivation. Sprague-Dawley albino rats were submitted to nutritional-environmental influences from birth to the 21st postnatal day. Exploratory behavior was assessed by the evaluation of locomotor activity in the open-field apparatus. In order to evaluate changes in neuronal morphology induced by nutritional-environmental variables, brains were stained according to the Golgi-Cox-Sholl procedure. Dendritic development was assessed under camera lucida by measuring basal dendritic branching of layer II and III pyramidal neurons, located in the dorsomedial region of the visual cortex of the rat. Morphometrical analysis revealed that both basal dendritic length and branching were significantly reduced by undernutrition. In contrast, environmental stimulation during the suckling period compensated for the neuronal impairment produced by protein-calorie deprivation. An improvement was also observed in exploratory behavior although to a lesser degree, as shown by the open field test data. In conclusion, the present results indicate that sensorimotor stimulation applied during the period of fastest rate of cortical cytodifferentiation compensates for neuronal and behavioral impairment produced by undernutrition.

Decreased Expression of the c-Fos, but Not Jun B, Transcription Factor in the Auditory Pathway of the Rat after Repetitive Acoustic Stimulation

Between the 21st and 34th postnatal days, male rats received an acoustic stimulus every second day, and the expression of c-Fos and Jun B proteins was compared with rats that received only a single acoustic stimulus at postnatal day 34. For acoustic stimulation, 8-kHz pure-tone bursts (100 ms duration at a rate of 6.3 Hz) were applied for 5 min. The numbers of neurons immunoreactive to c-Fos and Jun B were studied 2 h after the last or acute stimulus, respectively, in the ventral cochlear nucleus (VCN), dorsal cochlear nucleus (DCN) and inferior colliculus (IC). Compared with the single stimulus, repetitive stimulation significantly reduced the number of c-Fos labeled neurons in the VCN by 51%, in the DCN by 75% and in the IC by 48%. In contrast to c-Fos, repetitive stimulation did not significantly lower the number of Jun-B-expressing neurons. These findings indicate that independent intraneuronal pathways terminate on the induction of c-Fos- and Jun-B-expressing genes during the juvenile maturation of the acoustic pathway and that chronic exposure of acoustic events alters the program of intraneuronal gene expression by reducing transcriptional activities with subsequent stabilization at the genetic level.

Glucocorticoid receptor expression in the postnatal rat cochlea

The glucocorticoid receptor (GR) expression in the neonatal rat cochlea was investigated by utilization of a polyclonal antibody against GR, the immunoreactivity of which exhibited a distinct, age-dependent developmental pattern in tissues of the spiral ligament (SL). Immunostaining of GR appeared initially at the 7th postnatal day (PND), increased rapidly between the 14th and 21st PND, and reached adult-like expression levels by the 21st PND. Less pronounced, developmentally regulated expression patterns of GR were observed in cells of the spiral limbus (SLi), spiral ganglion (SG), organ of Corti (OC), and cochlear nerve (CN). For example, high expression levels of GR were observed in the SLi, SG and OC at 3 PND; subsequently, GR immunoreactivity levels decreased from 7 to 14 PND, and then GR immunoreactivity intensified in these regions by 21 PND. No remarkable changes in GR expression were observed in stria vascularis (SV). These data indicate that GR expression in the inner ear is tissue and age-specific, and that GR expression parallels both Na,K-ATPase expression and endocochlear potential development.

Preventive effects of dexamethasone on hypoxic-ischemic brain damage in the neonatal rat

To clarify the preventive effects of glucocorticoid on perinatal hypoxic-ischemic (HI) brain damage, an experiment was carried out on 4-day-old rats pretreated for 4 consecutive days with 3 different regimens; namely, a low dose dexamethasone (Dex) (0.1 mg/kg/day), a high dose Dex (0.5 mg/kg/day), and a saline administration. On the 7th postnatal day, after ligation of the left common carotid artery, the rats were exposed to 8% oxygen and decapitated on the 10th, 14th, 21st and 28th postnatal days. Ligated side brain damage was observed in 75, 7 and 3% of the rats in the saline, low and high dose Dex groups, respectively. However, a high mortality rate (42%) was noted in the high dose Dex group. The cumulative number of animals with poor outcome (death or brain damage) was 49 (80%), 13 (33%) and 24 (44%) in the saline, low and high dose Dex groups, respectively. On the 10th and 14th postnatal days, the rats in both the Dex groups showed delayed neuronal maturation and myelination in the non-ligated side motor cortex, however, these maturational differences disappeared on the 21st postnatal days. Otherwise, the number of cortical cells in both the Dex groups were significantly lower than that in the saline group on the 28th postnatal days ( P < 0.05 in each). These findings suggest that the pretreatment with Dex protects the developing brain from HI injury through the suppression of the neuronal maturation. However, a decreased number of cortical cells may give rise to psychomotor retardation later.

Hippocampal synaptogenesis in hydrocephalic HTX-rats using a monoclonal anti-synaptic vesicle protein antibody

The fluorescence intensity (FI) on immunohistochemical staining with a monoclonal antibody (Mab 171 B5) against a synaptic vesicle-associated protein (SVP-38) was measured in the CA1 sector of the hippocampus of brains obtained from hydrocephalic and non-hydrocephalic HTX-rats aged 1, 7, 14, 21 and 28 days. FI was significantly increased on the 21st and 28th postnatal days in hydrocephalic rats as compared to in non-hydrocephalic rats, despite the presence of marked morphological stretching and rotation of the dendritic arborization of Golgi-impregnated neurons in the CA1 sector. The increase in FI appeared to indicate an increase in synaptic density. As previous reports have indicated a marked reduction in SVP-38 FI in the thin cerebral cortex of 28th postnatal day hydrocephalic brains, our findings may suggest that hippocampal synaptogenesis is more resilient than that of the cerebral cortex in hydrocephalic brains.

Development of the suprachiasmatic nucleus in rats during ontogenesis: Tyrosine hydroxylase immunopositive cell bodies and fibers

This study has evaluated differentiation of tyrosine hydroxylase-immunopositive neurons in the suprachiasmatic nucleus as well as the innervation of this nucleus by tyrosine hydroxylase-immunopositive axons in rats during ontogenesis. Tyrosine hydroxylase-containing structures were detected with electron-microscopic pre-embedding immunocytochemistry at the 22nd fetal day as well as at the second, ninth and 21st postnatal days. Rare uni- and bipolar small tyrosine-hydroxylase-immunopositive neurons were observed in the suprachiasmatic nucleus both in fetuses and postnatal rats. These neurons underwent differentiation over the perinatal period that was mainly manifested in the increase of their size as well as in the development of the Golgi complex, granular endoplasmic reticulum and the onset of the dense core vesicle production. Concomitantly, tyrosine hydroxylase-immunopositive neurons, cell bodies and dendrites, became innervated by immunonegative axons first making presynapses, and, then, symmetric (Gray-type II) and asymmetric (Gray-type I) synapses. In addition to cell bodies and dendrites, tyrosine hydroxylase-immunopositive axons were regularly observed in ventral, ventrolateral and ventromedial regions of the suprachiasmatic nucleus in fetuses and postnatal rats. Tyrosine hydroxylase-immunopositive axons were observed either in simple appositions with the immunonegative neurons or making presynapses in fetuses and symmetric and asymmetric synapses in postnatal animals. The nature of the tyrosine hydroxylase-immunopositive axons and the functional significance in the suprachiasmatic nucleus in ontogenesis are discussed.

Development of the suprachiasmatic nucleus in rats during ontogenesis: Serotonin-immunopositive fibers

The innervation of the suprachiasmatic nucleus by serotoninergic fibers has been studied in rats from the 22nd embryonic until the 21st postnatal day. The serotoninergic fibers were detected with the pre-embedding immunocytochemical technique at the electron-microscopic level using antibodies to serotonin. Serotonin-immunopositive fibers were always identified as axons as they contained numerous synaptic vesicles both in fetuses and postnatal rats. Moreover, immunopositive dense core vesicles appeared in the axons after birth. From the end of fetal life onwards, the serotonin-immunopositive axons made specialized contacts with the immunonegative neurons, mainly with their dendrites and to a lesser extent with cell bodies and axons. In fetuses, only immature synapses (presynapses) were observed. After birth, presynapses were replaced by typical synapses with the thickened pre- and postsynaptic membranes, accumulations of dense materials at the pre- and postsynaptic membranes as well as with an accumulation of synaptic vesicles at the presynaptic membrane. The functional significance of the serotoninergic input to the suprachiasmatic nucleus in ontogenesis is discussed.

Calbindin-D28k gene expression in the developing mouse kidney

Calbindin-D28k appears in the metanephric kidney during embryogenesis. We studied the temporal appearance and spatial distribution of calbindin-D28k mRNA in the developing kidneys of 12-day fetal through 21-day postnatal mice by in situ hybridization. 35S-UTP-labeled antisense (cRNA) probe to calbindin-D28k mRNA hybridized to the ureteric buds of 12-day embryos, whereas adjacent metanephrogenic tissue was unlabeled. By embryonic day 13, Y-shaped bodies of "advancing" ureteric buds were labeled intensely. In 16-day embryos, ampullae of ureteric buds were located immediately beneath the renal capsule and labeled strongly, in contrast to metanephric tubules and S-shaped bodies. The former were unlabeled and the latter were labeled only at points of contact with the ampullae. Subsequently, the ampullae of the metanephric ureteric buds hybridized with the cRNA probe, and from the 18th embryonic to the 21st postnatal day, this labeling was intense. The cRNA probe did not hybridize with the renal vesicles, proximal tubules, or tubular segments of Henle's loop derived from nephrogenic blastema, but it did label distal nephron segments. By the 21st postnatal day, collecting ducts and ureter no longer were labeled. In conclusion, calbindin-D28k mRNA is present in the developing mouse kidney, and its distribution during nephrogenesis is identical to that of calbindin-D28k per se. Collectively, these findings show that the calbindin-D28k gene is transcribed and its message is translated by the cells of the ureteric bud during the initial stage of renal morphogenesis.

Early postnatal treatment with muscimol transiently alters brain GABA A receptors and open-field behavior in rat

The long-term effects of treatment with muscimol, a GABA A agonist, and with ethanol, also shown to have a GABAergic profile of action, on subsequent behavior and on the binding parameters of GABA A receptors were studied in Wistar rats. Rats were given two daily injections of muscimol (0.1 mg/kg M1 group of 0.2 mg/kg M2 group) or ethanol (1.5 g/kg) between the 1st and 21st postnatal days, with saline-treated animals serving as controls. At the age of one month, the activity of the M2 rats was decreased in the open-field arena. At the age of four months, ethanol-treated rats consumed less 10% ethanol solution in a free choice situation. No changes were seen in the elevated plus-maze test. Maximal GABA stimulation of [ 3H]flunitrazepam binding was decreased in the cerebellum and hippocampus in M2 rats at the age of one month but not four months. A significant positive correlation was found in all rats between maximal GABA-stimulated [ 3H]flunitrazepam binding in the cerebellum and ambulation in the open-field arena at the age of one month. Ethanol intake correlated negatively with maximal GABA stimulation of [ 3H]flunitrazepam binding in the cerebral cortex. None of the treatments affected the cerebellar binding of an imidazobenzodiazepine, [ 3H]Ro 15-4513. There was an age-dependent decline in the efficacy and potency (EC 50) of the GABA enhancement of [ 3H]flunitrazepam binding in the cerebellum in all treatment groups. Basal binding of [ 3H]flunitrazepam to hippocampal and cerebellar membranes as well as binding of [ 3H]Ro 15-4513 to cerebellar membranes was also decreased as a function of age. The results suggest that muscimol exposure induces a transient change in GABA A receptor sensitivity. Also, open-field behavior, ethanol consumption and GABA A receptor mechanisms may be interrelated in the rat.

Connexins and glucagon receptors during development of rat hepatic acinus.

Levels (by immunoblotting) and cell distribution (by immunofluorescence) of connexins (Cxs) and glucagon receptors (GRs) were studied during the ontogeny of the rat liver. Cx32, the main rat liver gap junction protein, was present in fetal hepatocytes, and its abundance increased between the 19th and 21st gestational days. The major increase in Cx32 levels occurred between the 7th and 21st postnatal days, after which adult levels were reached. The adult pattern of distribution of Cx26 and Cx32 was established by the 21st postnatal day. Cx43 was present in the liver capsule and perisinusoidal cells, and its levels did not show significant variations throughout development. Levels of GRs were much lower in fetal than in adult livers. After birth, GRs increased progressively, reaching adult levels at about the 28th postnatal day. In adults, GR immunoreactivity on the plasma membrane of hepatocytes was localized to the region facing the sinusoid and showed a density gradient of distribution along the hemiacinus: high in pericentral regions and decreasing toward more glucogenically active cells located in periportal regions. Because cells of the hepatic acinus communicate via gap junctions, which are permeable to second messengers, we propose that signals transduced by GRs in cells expressing high levels of receptor might spread to cells expressing low levels of receptor, thus maximizing metabolic activation.

Cortical synaptogenesis in congenitally hydrocephalic HTX-rats using monoclonal anti-synaptic vesicle protein antibody

We attempted to develop a method for investigating impairment of synaptogenesis quantitatively involving measurement of the fluorescence intensity emitted by immunohistochemically stained rat brain using a monoclonal antibody (Mab 171B5) against a synaptic vesicle protein (SVP-38). We applied this method to congenitally hydrocephalic and non-hydrocephalic brains of HTX-rats, and compared the postnatal changes in the fluorescence intensity in the molecular layer of the cerebral cortex. In non-hydrocephalic HTX-rats, the fluorescence intensity remained nearly unchanged from the 1st to 7th postnatal day and then increased at an almost linear rate until the 21st postnatal day, when it reached 4.5 times the value on the 7th postnatal day. The increase thereafter was gradual until the 28th postnatal day. In hydrocephalic HTX-rats, the fluorescence intensity increased nearly in parallel with that in non-hydrocephalic HTX-rats up to the 21st postnatal day. On the 28th postnatal day, the fluorescence intensity showed a marked reduction (P less than 0.01). This finding indicates that despite the presence of continuous pressure due to progressive ventricular dilatation after birth, synaptogenesis in the cerebral cortex may be relatively resilient.

Ontogenesis of the hypothalamic catecholaminergic system in rats: Synthesis, uptake and release of catecholamines

The development of the hypothalamic catecholaminergic system during ontogenesis in rats has been studied with glyoxylic acid histofluorescent method in vivo and with the isotopic biochemical technique in vitro. It has been demonstrated that at the 15th fetal day the catecholaminergic system was functionally inactive at least in its ability for the uptake and K +-stimulated release of catecholamines. Since the 16th fetal day, hypothalamic neuronal elements gained an ability for synthesis of catecholamines, their specific uptake and K +-evoked release. Over the subsequent two days, the intensity of the fluorescent intraneuronal product rose considerably showing the increase of either synthesis or accumulation of catecholamines. Simultaneously, the value of the uptake and K +-stimulated release of the exogenous radioactively-labelled dopamine increased significantly. The intensity of the fluorescence of the hypothalamic neuronal elements dropped from 20th fetal until the ninth postnatal day, whereas the specific uptake doubled over the same period reaching itsaadult level. By the 21st postnatal day the reaccumulation of the fluorescent product occurred.

Ontogeny of Thyrotropin-Releasing Hormone Gene Expression in the Rat Diencephalon

The development of thyrotropin-releasing hormone (TRH) gene expression in the rat diencephalon was studied using in situ hybridization histochemistry. The first neurons expressing the TRH gene were found on gestational day 14 (E14) in the lateral hypothalamus, shortly after completion of their last cell division. On E15 and E16, additional labeled cells appeared medially in the developing dorsomedial and paraventricular nuclei, respectively, followed on E17 by cells in the preoptic area. The number of labeled cells in the hypothalamus continued to increase during the last part of gestation. At birth, TRH mRNA neurons were present in all the locations seen in the adult hypothalamus. During the first week of life, the labeling intensity and number of neurons containing TRH mRNA continued to increase at the locations described above. TRH mRNA was not detected in the reticular thalamic nucleus until the 7th postnatal day when some labeled cells appeared in its dorsocaudal portion. Over the next 10 days, the number of labeled cells and the intensity of labeling in the thalamic reticular nucleus progressively increased. During the same period of time, only small changes in the number and intensity of labeled cells in the hypothalamus were seen. On the 21st postnatal day, after a decrease in labeling in the lateral hypothalamus had been noted, final adult patterns of expression were present. With the very sensitive and anatomically specific method of in situ hybridization histochemistry, the ontogeny of TRH gene expression in the rat diencephalon has been elucidated.

Cytoarchitectonic investigation of the rat spinal cord following ethylnitrosourea administration at different developmental stages

We examined the histological findings and cytoarchitectonic alterations in the rat spinal cord following matrix cell degeneration caused at different developmental stages, from neural plate formation through neuroblast generation. Ethylnitrosourea (ENU) 20 mg/kg body weight was administered transplacentally to the fetuses on the 10th embryonic day (E10) to 14th. The observations were made until the 21st postnatal day. Normally, mitoses were present scatteredly in the matrix cell layer of the neural plate or neural tube on E10 or E11, and gradually restricted to the dorsal portion of the alar plate as development occurred. The localization and number of degenerative cells as well as the site and degree of neuronal decrease in the completed dysgenetic spinal cord seemed to correlate with the topography and frequency of the mitoses in the matrix cell layer at the time of ENU administration. Disorder in the pattern of cytoarchitecture of neurons was not observed. The degree of hypoplasia of the white matter was proportional to the intensity of decrease of the spinal neurons. Aberrant myelinated fibers were not seen. No reactive gliosis, fibrosis or abnormal vascularization was observed at any time.

The level of natural antioxidant glutathione and histidine-containing dipeptides in skeletal muscles of developing chick embryos

1. 1. The levels of glutathione and histidine-containing dipeptides in skeletal muscles change in different ways during ontogenesis. 2. 2. The glutathione content in skeletal muscles increases between the 9th and 18th days of embryongenesis—from 0.5 to 2.0 μmol/g of tissue wet wt and then drops to zero in 3-week-old chickens. 3. 3. The level of histidine-containing dipeptides increases throughout the observation period beginning with their appearance on the 14th day in leg muscles and on the 15th day in breast muscles of chicken embryos up to the 21st postnatal day. 4. 4. There is a negative correlation between the antioxidative systems of glutathione and histidine-containing dipeptides in muscle tissue, i.e. dipeptide-rich tissues contain little or no glutathione and vice versa.

Cysteine and glutathione levels in developing rat kidney and liver.

Intracellular levels of cysteine (CSH) and reduced glutathione (GSH) in the kidney and liver of rats from the newborn to the adult period have been determined using a sensitive high performance liquid chromatography method. In the kidney, the intracellular level of free CSH increased 4-fold from 1 to 4 nmol/mg protein with GSH levels which ranged from 20 to 25 nmol/mg protein from the 10th to 21st postnatal day, respectively. In contrast, intracellular free hepatic CSH showed a biphasic pattern with development. Intracellular free hepatic GSH, on the other hand, increased 2-fold over the 3- to 21-day postnatal period. In adult tissues, intracellular levels of free CSH and GSH decreased as compared with levels in 21-day postnatal animals. When ratios of CSH to GSH were compared between tissues from the 3-day-old postnatal and adult rat, CSH:GSH increased approximately 4-fold in the kidney and decreased 2- to 3-fold in the liver.