3rd Postnatal Week Research Articles

CHANGES IN JEJUNAL EPITHELIAL MORPHOLOGY & KINETICS DURING SUCKLING & WEANING IN THE CROWING GUINEA PIG

Weaning, the nutritional adaptation from milk to solid diet, demands changes in Cl function. We measured jejunal mucosal morphology and kinetics during suckling and weaning to determine if the marked changes seen in the altricial rat also occur in the precocial guinea pig (CP). 79 animals mere studied. Croups of at least 3 CPs aged 1,7,14,21 and 28 d were given IP colchicine at time 0, and killed at 20 to 30 min intervals. Tissue was taken 5 cm from the pylorus, fixed and bulk stained with Schiff reagent. Jejunal morphology was measured by microdissection and crypt cell production rate (CCPR) by a metaphase arrest technique. There was 40% decline in villus ht, from 986 μm to 618 μm (p<0.001), and 25% increase in crypt depth, from 148 μm to 199μm (p<0.01), during the first 2 wks. After an initial decline there was an increase in crypt: villus (C;V) ratio from 6.7 to 8.2, and in CCPR from 3.9 to 5.6 cells/crypt/hr from 14 d onwards (pz0.001). These changes represent a proliferative response of jejunal epithelium during the 3rd & 4th postnatal weeks, accounted for by increase in the depth and number of crypts, and CCPR, leading to increase in net villus influx. In contrast with the rat in which abrupt changes in epithelial morphology, function and kinetics occur around d 21 at the time of cessation of suckling, in the CP they are gradual and follow weaning by some days. The CP, which in many respects more closely resembles man than rat, is a more suitable animal model for our understanding of human Cl maturation and perinatal nutritional adaptation.

Postnatal development of β-adrenergic response in ventricular muscle of the rat heart

In adult mammalian, heart responses to beta- and alpha-adrenergic stimulation are different: the beta-type effect exhibits a larger increase of relaxation than of contraction, while the alpha-stimulation has no selective influence on relaxation. The present results show that the effect of isoprenaline (ISO) on the neonatal rat heart during the 1st postnatal week is not a typical beta-effect in that the relaxant influence of beta-stimulation is lacking. During the 2nd and 3rd postnatal weeks the typical beta-response, with improved relaxation, gradually appears. The absence of the typical beta-effect is not caused by the lack of beta-receptors or cAMP-dependent phosphorylation reactions because in other respects, the positive inotropic effect of ISO is well developed at the moment of birth. In addition to these qualitative changes, also prominent quantitative changes occurred in the ISO response. The dose-response curves were shifted to the right with advancing age, suggesting reduced beta-agonist potency of the maturing tissue. The developed tension (Tmax) abruptly increased between the 12th and 17th postnatal days and then steeply declined during the next 2 weeks. Changes in Tmax correlated fairly well with the general ability of the tissue to generate extra force, as expressed by rest-dependent potentiation of twitch. However, during the 2nd postnatal week cardiac tissue seemed to be subsensitive to ISO, since all contractile parameters except T''max were depressed. The results suggest that the postnatal changes in beta-response are primarily determined by alternations in the electromechanical coupling process of the developing tissue, and less by the proper adrenergic mechanisms.

Developmental time course of the sympathetic postganglionic innervation of the rat eye

The time course of the innervation of the rat iris by sympathetic postganglionic axons from the superior cervical ganglion (SCG) has been studied in postnatal rats. A retrogradely transported fluorescent dye. Fast blue (FB) was injected bilaterally into the anterior eye chamber of rats within 3 h of birth or at 7 or 14 days postnatal. Littermates were killed at 24 h intervals, both SCG removed, fixed and sectioned. A substantial number of sympathetic neurones were labelled retrogradely from the eye at birth. The number of labelled cells tripled over the first postnatal week to reach proportions equivalent to those labelled in 3-week-old rats. The degree of sympathetic fibre ingrowth to the iris was examined at 3 postnatal ages, using fluorescence histochemistry. This method showed a small proportion of catecholamine-containing fibres in the iris at birth with a more extensive fibre density over the iris by 7 days after birth. A further increase in fibre density had occurred by the end of the 3rd postnatal week even though the number of cells projecting to the iris at these two ages was similar. This difference in fibre density may arise from ramification of fibres which have reached the iris by 7 days postbirth.

Effect of neonatal nomifensine exposure on adult behavior and brain monoamines in rats.

The aim of the study was to examine the effects of early postnatal exposure to nomifensine, an inhibitor of catecholamine uptake, on concurrent active (REM) sleep, on later alcohol related behavior and on monoamine concentrations in various brain regions of rats. For these purposes rats were given daily injections of 10 mg/kg nomifensine s.c. between the 7th and the 18th postnatal days. During the nomifensine exposure active sleep, expressed as a percentage of total sleeping time, was reduced. At one month of age, the nomifensine rats showed increased ambulation and had lower defecation scores in the open-field than the controls. Neonatal exposure to nomifensine increased voluntary intake of 10% (v/v) alcohol when the rats were 2-3 months of age. The rats, however, did not exhibit preservation in the T-maze, and similarly to control rats suppressed drinking 0.1 M lithium chloride even when thirsty. Measurement of cerebral monoamine concentrations at the age of 3 months suggested that neonatal nomifensine treatment interferes with the noradrenergic and serotonergic systems in several regions of the brain. Concentrations of noradrenaline and 5-hydroxyindoleacetic acid (5-HIAA) were decreased in the cerebral cortex and frontal cortex, concentration of 5-HIAA was decreased in the neostriatum, and concentrations of noradrenaline, 5-hydroxytryptamine (5-HT) and 5-HIAA were elevated in the lower brain stem. Taken together, these findings show that exposure to nomifensine during the 2nd and 3rd postnatal weeks suppresses neonatal active sleep, causes changes in the adult open-field behavior, and increases voluntary alcohol intake, perhaps due to a long-lasting alteration in brain monoamines.

Stereocilia and tectorial membrane development in the rat cochlea

Maturation of the rat cochlea, from postnatal days 2 to 60, was studied using scanning electron microscopy (SEM), with emphasis on stereocilia and tectorial membrane (TM). Two days after birth, the organ of Corti was very immature. An adult appearance of its surface was observed by day 16 in the basal turn, and by the end of the 3rd postnatal week in the apex. Stereocilia started their development first on inner hair cells. By contrast, the apical pole of outer hair cells ended its maturation before that of inner hair cells. Top-links were detected very early in inner hair cell stereociliary development (postnatal day 2). Marginal pillars temporarily attached the TM to the organ of Corti; they disappeared first in the apical region. This transient attachment seems to play a role in the coupling of outer hair cells to the TM, as prints of their longest stereocilia appeared at the undersurface of the TM by the same time. Moreover, these prints were more clear and regular at the base than at the apex of the cochlea. Results are discussed in relation to ultrastructural and functional data on rat cochlea maturation.

Postnatal changes in the inotropic effect of ouabain on the rat heart ventricle.

The concentration-dependent effects of ouabain on the contractility of postnatally developing rat heart ventricles were studied. Ouabain caused a positive inotropic effect in right ventricular strips of neonatal rats up to the age of about 30 days but a negative inotropic effect in the adult cardiac tissue. When extracellular Ca concentration was lowered from 2.5 to 1.0 mmol/l and the rate of stimulation was simultaneously elevated from 0.2 to 1.0 Hz a clear positive inotropic effect was also generated in the adult rat heart. The positive inotropic effect of ouabain showed a biphasic developmental pattern: the contractile force first grew from birth to about 15 days of age but steeply declined near to the adult level during the 3rd postnatal week. The force response to ouabain occurred within two distinct dose-ranges. In the newborn only the high-dose (above 3 X 10(-6) mol/l) effect was seen but in rats older than 5 days a mixed low-dose (below 3 X 10(-6) mole/l)/high-dose effect was apparent. In both ranges the positive inotropic effect of ouabain seemed to be dependent on caffeine sensitive Ca store, perhaps the sarcoplasmic reticulum. It is suggested that during the 3rd postnatal week a transition from extracellular to intracellular Ca stores occurs in the rat heart, which is reflected as a changing inotropic effect of ouabain on the developing cardiac tissue.

Periods of sensitivity to thyroid hormone during the development of the organ of Corti.

Cochlear structures are sensitive to the morphogenetic effect of thyroid hormone during the whole duration of maturation. For each structure, there exists a period of maximal sensitivity to thyroid hormone which corresponds to the period of development during which the structure of interest undergoes its main morphological changes (6 to 13 days for the inner sulcus epithelium, 6 to 10 days for the pillars, the 2nd and a part of the 3rd postnatal week for OHCs and their efferent innervation in rats). These periods of sensitivity can be considered as critical periods because cochlear structures are maximally vulnerable to thyroid deficiency during these periods.

Incomplete maturation of brainstem auditory nuclei in genetically induced early postnatal cochlear degeneration.

In the Shaker-2 mouse mutant, a cochleo-saccular type of genetically induced inner ear degeneration occurs. Morphological signs of degeneration are evident in the 3rd postnatal week and a severe and almost total degeneration has occurred by the age of 6-9 weeks. There are no qualitative differences between the brain of the normal CBA/J mouse and that of the Shaker-2 mouse. The growth of the auditory brainstem nuclei (dorsal cochlear nucleus and ventral cochlear nucleus) in the mutant Sh-2 mouse has stopped by 14 days of age--but not in the normal CBA/J mouse. The brainstem as a whole continues to grow between 14 and 140 days of age in both strains. The early cochlear degeneration coincides with the critical period when a normal input from the peripheral receptor organ is necessary for the maturation of the central auditory pathway.

Histological development of the vomeronasal complex in the pre- and postnatal rat.

The vomeronasal complex of the rat was studied by means of different staining techniques and light microscopy between the 13th day of gestation and 42 days of postnatal life. The anlage of the vomeronasal organ in the 13-day-old embryo consisted of a cluster of cells proliferating from the olfactory placode towards the medial line. The vomeronasal organ was well developed by the end of gestation, showing the same ratio between receptor and receptor-free epithelium as in the adult animal. However, full development of the epithelia did not occur until the end of the second postnatal week. Glandular rudiments were observed within the vomeronasal area in 17-day-old fetuses, but the gland showed no sign of activity until the 7th day of postnatal life, when a slight PAS-positive reaction was observed in their cells' cytoplasm. This PAS-positive reaction intensified considerably during the second week and corresponded to the adult picture by the end of this time. The content of the vomeronasal glands contrasted with that of the glands flowing into the nasal fossa, which did not show any PAS-positive reaction but which stained positive for alcian blue. We also studied the evolution of the blood vessels such as the capsule of the complex. This capsule showed complete ossification by the beginning of the 3rd postnatal week, with the exception of certain regions which remained cartilaginous in the adult. Some capillaries were observed in the complex in 17-day-old fetuses, and by 20 days of gestation some of them could be observed penetrating the receptor epithelium. Subsequently, the vascularization of the complex became so profuse that one could consider the vomeronasal complex to consist of erectile tissue.

Activités des hydrolases rénales au cours du développement pré- et post-natal de la souris

The fetal and postnatal activity patterns of different hydrolytic enzymes (alkaline phosphatase, gamma-glutamyltransferase, trehalase, maltase, glucoamylase, lactase, and sucrase) have been examined in mouse renal homogenates. Alkaline phosphatase and gamma-glutamyltransferase activities presented approximately similar changes. They increased from 18 days of gestation up to 30 days after birth. These activities showed marked increases during the 3rd and 4th postnatal weeks. A similar important rise was observed for trehalase activity at the end of the suckling period. Maltase activity increased gradually after birth. Traces of lactase, sucrase, and glucoamylase activities were detected at each developmental stage.

Development of suckling controls in the rat: independence from thyroxine.

Until 2 weeks of age, rat pups attach to the nipples of their dam with the same avidity regardless of whether they are fed or fasted. During the 3rd postnatal week, some mode of control of suckling appears such that fasted pups attach much more rapidly than fed pups. In this study we have investigated the effects of hyper- and hypothyroidism on the maturation of suckling controls. Surprisingly, the results indicate that this aspect of behavioral development is independent of the thyroxine status of the pup. We conclude that the maturation of suckling controls is not cued by the developmental rise of serum thyroxine that occurs during the 2nd postnatal week.

A qualitative and quantitative ultrastructural study of glial cells in the developing visual cortex of the rat.

(i) This paper provides new information on the time course and fine structural features of glial cell differentiation, on the relative frequencies of glioblasts, astroblasts, astrocytes, oligodendrocytes and microglial cells, and on neuron: glia ratios in visual cortex of the rat between birth and maturity. The analyses were done on montages of electron micrographs of 75 pm wide strips extending the full depth of the cortex from animals 12 h and 4, 6, 8, 10, 12, 14, 20, 24, 90 and 180 days old (six montages from two or three animals at each age). (ii) At birth, and up to 4 days, most non-neuronal cells are poorly differentiated, irregularly shaped cells with dark nuclei (glioblasts). A few at this stage and progressively larger numbers over the next few days, can be recognized asastroblastsby the presence of a distinctive form of granular reticulum (distended cisterns with a moderately electron dense content), and some also by their position in contact with the subpial or perivascular basal laminae. Astroblasts enlarge, develop processes and transform into immature astrocytes: their nuclei become paler, the granular reticulum is no longer distended, and glial filaments begin to accumulate.Mature astrocyteswith pale nuclei, filaments and a low concentration of perikaryal organelles in a pale cytoplasmic matrix predominate at 24 days, and at 3-6 months 51 % of all glial cells are astrocytes. (iii) Concentrations of glioblasts (at 0 and 4 days) and subsequently of cells of the astrocytic lineage are apparent in the most superficial and in the deepest cortical layers, and an additional small peak is seen at the level of layer IV in the adult animals. The superficial concentration is probably associated with the subpial glia limitans and the layer IV concentration with the high density of synapses in this region; several probable explanations are considered for the concentration in layer VI. (iv) Processes ofradial glial cellsare apparent from birth to day 8 but not thereafter. No evidence was found for transformation of radial glia into astrocytes. A peak in phagocytic activity by immature microglial cells at days 6-8 suggests the possibility of loss of radial glial processes by degeneration rather than transformation. (v)Oligodendroblasts, intermediate in morphology between glioblasts and light oligodendrocytes, appear suddenly in the deep cortex and subcortical white matter at day 6 and are rapidly replaced bylight oligodendrocytes. These are large, organelle-rich cells with characteristically distended Golgi saccules, and are the only oligodendrocytes present during early myelination, which begins at day 10. Early in the 3rd postnatal week some light oligodendrocytes are replaced bymedium oligodendrocytes, which are smaller and darker, with abundant orderly stacks of granular reticulum.Dark oligodendrocytesare first apparent at the end of the 3rd week, account for about one-third of all oligodendrocytes at day 24, predominate at day 40 and constitute 90 % of all oligodendrocytes at 3 and 6 months, at which time oligodendrocytes comprise 39% of all cortical glial cells. We suggest that the progression from light to medium oligodendrocytes does not simply represent a diminution in the overall level of synthetic activity but that different components of the myelin sheath are being synthesized at the two stages. (vi)Microgliaare present from birth but are seen in significant numbers at days 6—10 and thereafter. Some are relatively mature in appearance, even in the youngest animals, and almost all are similar to the resting microglia of adult brain by day 16. At 3-6 months, 8 % of all cortical glial cells are identified as microglia and these cells are fairly evenly distributed throughout the cortical depth but are surprisingly and consistently poorly represented in layer VI. From day 6 to the end of the 2nd postnatal week, cells with poorly differentiated cytoplasm (many free polyribosomes), but containing phagocytosed products of cell degeneration, are identified asimmature microglia. However, it is possible that such cells do not mature into classical resting microglia but that they represent a different cell type. (vii) Theneuron: glia ratiois 4.54 at birth, rises to 5.09 at 4 days, and falls to approximately 2.5 at days 12-24. At 3-6 months the ratio is 2.13.

Postnatal increase in the metabolism of leucine and valine.

The catabolism of tritium-labeled leucine, valine, and glycine was determined by measuring the appearance of tritium-labeled water at various postnatal and adult ages in rats. Results for leucine and valine show a marked increase in the formation of labeled water during the 3rd postnatal week. The partial exclusion of leucine and valine from brain, as a result of the blood-brain barrier, particularly enhances observation of labeled water formation in that the product (water) and precursor (amino acid) are largely separated (supporting experiments demonstrate the prior existence of the blood-brain barrier for leucine). Results for glycine indicate its extensive metabolic degradation at all postnatal ages. These data indicate that the metabolic rate of intraperitoneally administered, radioactively labeled leucine and valine changes appreciably during early postnatal development. The early postnatal manifestation of human disorders of branched-chain amino acid metabolism is consistent with the chronology of development in the rat.

Abnormal functional development of sympathetic nerve terminal-smooth muscle complex in neonatal spontaneously hypertensive rats

In neonatal and young adult SH and WK rats, maturation of a functional unit consisting of sympathetic nerve terminals and smooth muscle (levator palpebrae) was assessed in vivo by measuring the contractile response to tyramine, an agent which releases endogenous norepinephrine from sympathetic nerve terminals. Responses in SH are comparable to WK at 5–6 days postnatally, smaller from the 8th through 19th day and larger in young adults (41–46 days old). Results indicate that functional maturation of the nerve terminal-smooth muscle complex is retarded in SH relative to WK during the 2nd and 3rd postnatal weeks. It is suggested that retardation in the neonatal SH rat is an expression of a genetic defect in the growth of the complex and that the enhanced response in young adult SH is a consequence of the neonatal abnormality.

Role of milk-borne factors in weaning and intestinal development.

To determine whether a factor appearing in the milk at the beginning of the 3rd postnatal week elicits the appearance of jejunal sucrase activity in the pups, pups were raised with dams that had been lactating for either a week less or a week more than their natural mothers. Pups were sacrificed at 15, 17, 19, 21 and 23 days of age for assay of jejunal sucrase and for measurement of the proportion of chow in the stomachs. Sucrase activity appeared at the same age in all pups (day 17); however, at days 21 and 23 the enzyme activity was higher in the pups cross-fostered to the longer-lactating dams. Correspondingly, the analysis of stomach contents showed that these pups had almost completed weaning by day 21 whereas the other pups were still taking 40-50% milk. It is concluded that neither the appearance of jejunal sucrase nor the onset of weaning is cued by the appearance of a factor in the milk at the beginning of the 3rd postnatal week. However, the lactational stage of the dam can affect the rate of completion of weaning and, secondarily, the terminal portion of the sucrase rise.

Effects of thyroid state and undernutrition on S100 protein and astroglia development in rat cerebellum.

The developmental pattern of S100 protein, a specific marker of astroglia, was studied by radioimmunoassay in the cerebellum of thyroid-deficient, thyroxine-treated and undernourished rats during development. In the control animals, the S100 protein content of the cerebellum increased maximally after the 3rd postnatal week, i.e., after cell multiplication had stopped and when the cerebellum had acquired more than 70% of its adult weight and protein content. This developmental pattern of S100 protein reflected essentially the maturation of astroglia. In the thyroxine-treated rats the total amount and the concentration of S100 protein were higher than in controls during the first 3 weeks of postnatal life and returned to normal values thereafter. In the thyroid-deficient rats both the amount and concentration were lower than in controls throughout development. In the undernourished animals the amount of S100 protein per organ was also lower than in controls during the whole experimental period studied; the S100 protein concentration was higher during the first 2 postnatal weeks and became lower thereafter. These results are discussed taking into consideration previous histological and ultrastructural observations on the effects of altered thyroid state and undernutrition on the formation and maturation of cerebellar astrocytes.

The induction of "stress" proteins in organ slices from brain, heart, and lung as a function of postnatal development

The proteins synthesized in vitro by rat brain, heart, and lung slices were compared by two-dimensional polyacrylamide gel electrophoresis. A protein, P71, which is related to one of the heat shock proteins induced in many cultured cells by hyperthermia and other harsh conditions, was the major protein synthesized by all slices from rats 3 weeks old and older. In vivo synthesis of P71 was not detected in brain, heart, or lung from these animals nor was there any detectable Coomassie brilliant blue-stained protein coinciding with P71 on the gels. P71 thus appears to be a minor protein species in normal unstressed brain, heart, and lung. While both heart and lung slices synthesized large quantities of P71 at all stages of postnatal development, brain slices synthesized little, if any, P71 until 3 weeks postnatal. There was a dramatic decrease in protein synthesis in all tissue slices during postnatal development. During this decrease, the relative abundance of newly synthesized P71 remained almost constant in heart and lung slices, but the relative abundance of P71 increased by approximately 50-fold in brain slices. The cells synthesizing P71 in brain slices were enriched in a microvascular fraction. The increase in P71 synthesis by these cells, occurring between the 3nd and 3rd postnatal week, coincides with the final maturation of brain capillaries and the blood-brain barrier.

Role of sex steroid hormones in the normal and glucocorticosteroid hormone-induced evolution of carbamoylphosphate synthase (ammonia) and arginase activity in rat liver ontogenesis.

The effects of sex steroid hormones on the development changes of carbamoylphosphate synthase and arginase activity, and DNA content in rat liver were investigated. After birth, administration of estradiol causes an increase in carbamoylphosphate synthase and arginase activity at high dosages only. Progesterone strongly inhibits or even reverses these stimulatory effects of estradiol. Administration of progesterone alone had no effects in the 1st postnatal week, a stimulatory effect in the 2nd postnatal week, and slightly inhibitory effects on enzyme activities thereafter. Testosterone hardly affected enzyme activities in the 1st postnatal weeks and slightly decreased them from the 3rd postnatal week onwards. No differences in the response of either sex to these hormone treatments were observed. During the last 4 prenatal days and during the 1st postnatal week progesterone partially inhibited the prednisolone-induced enzyme accumulation and decrease in DNA content. Testosterone had such effects during the 1st 2 postnatal week. Comparison of these and previous experimental results with the developmental profiles of carbamoylphosphate synthase (ammonia) and arginase activity and DNA content on the one hand and with the developmental profile of steroid hormone levels on the other shows that only glucocorticosteroids influence enzyme activity and DNA content profiles, while progesterone and testosterone modulate the profiles by antagonizing the effects of glucocorticosteroids. No role for estrogens could be established.

Development of gamma-glutamyltranspeptidase activity in mouse small intestine: influence of cortisone and thyroxine.

The fetal and postnatal development of gamma-glutamyltranspeptidase (gamma-GT) activity has been examined in the different parts of the mouse small intestine. At 17 days of gestation, gamma-GT is present in all intestinal thirds and a decreasing gradient of activity from the proximal to the distal segments is already established. From 17 days of gestation up to 5 days after birth, a dramatic fall of activity is observed in all segments. gamma-GT activity reaches adult level before weaning in the proximal segment while it increases only during the 3rd postnatal week in the middle and distal intestinal segments. Daily administration of cortisone acetate (25 microgram/g b.w./day) for 3 days to 8-day-old mice elicits a premature increase of gamma-GT activity in all intestinal thirds, the maximal increase being observed in the proximal segment. On the other hand, thyroxine (1 microgram/g b.w./day) has no or little effect on the normal development of gamma-GT activity.

Supra-normal sensitivity to ototoxic antibiotic of the developing rat cochlea.

A period of increased sensitivity to the ototoxic antibiotic amikacin was found in the cochlea of the developing rat. Starting either at birth, 10 or 30 days of age, rat were injected i.p. daily with 200 mg/kg of amikacine for 10 days. Only in the group injected with drug from 10 to 20 days of age was there evidence of a substantial (40-100 dB) permanent threshold shift (PTS). To characterize the period of supra-normal sensitivity more precisely, independent groups of pups were injected with 50 mg/kg of amikacin from 10 to 20, 15 to 25, 20 to 30, and 30 to 40 days of age. Only those pups injeced from 10 to 20 or 15 to 25 days of age showed PTS (30-60 dB). These data, taken together with those on rat cochlear maturation indicate that the sensitive period to ototoxicity starts at the age of onset of cochlear function (around day 10) and ends few days after the cochlea shows adult-like characteristics (3rd postnatal week).