5th Postnatal Day Research Articles

Postnatal Changes in Cathepsin D in Rat Neural Tissue

Abstract:Cathepsin D, an aspartyl endopeptidase, was analyzed in cortex from forebrain and cerebellum, spinal cord, and optic and sciatic nerves, and in the liver of rats from 1 to 120 days of age. Cathepsin D was quantitated in tissue extracts by measurement of enzyme specific activity on a substrate of [methyl‐14C]‐methylated hemoglobin and by radioimmunoassay. Immunocytochemistry was used to ascertain the identity of the mixed cell types that contributed to the cathepsin D detected. As quantitated by radioimmunoassay, immunoreactive cathepsin D varied between 0.2 and 1 ng/μg of total protein. Maximum activity occurred at approximately the 15th postnatal day; the least amount of immunoreactive cathepsin D was found at 30 or 60 days of age. A subsequent increase of varying magnitude occurred at postnatal day 120. There was good correspondence between immunoreactive enzyme and enzyme specific activity, which ranged from 1 to 4 ng/μg of total protein, and the activities determined by the two methods provided similar, but not identical, developmental profiles. Cathepsin D was demonstrated by immunocytochemistry to be present in most neurons, in all choroid plexus epithelium, and in certain oligodendrocytes from the first postnatal day. Cathepsin D was present in oligodendrocytes in cord lateral funiculi and optic nerve by the first postnatal day, and by the sixth postnatal day many oligodendrocytes were abundantly stained. In contrast, oligodendrocytes in the corpus callosum and in the cerebellar white matter did not contain demonstrable cathepsin D until postnatal days 10 and 15, respectively. These results indicate a role for cathepsin D during the postnatal development of rat CNS and suggest that this proteinase may be involved in the steps of myelination.

Relation between functional maturation of cervical sympathetic innervation and ontogeny of α-noradrenergic smooth muscle contraction in the rat

The functional ontogeny of a sympathetic neuroeffector system consisting of postganglionic neuron and smooth muscle end organ was studied in vivo in the rat at selected times from birth through 75 days of age. The maximum noradrenergic contractile response of the smooth muscle (Mu¨ller's) component of the levator palpebrae was assessed with the directly acting agonist, methoxamine. The functional capacity of the nerve terminals innervating the muscle to evoke contraction was determined by displacing their endogenous noradrenaline with tyramine. The integrity of the pathway from preganglionic axon to end organ was tested by stimulating the preganglionic axons electrically. The smooth muscle contractile response to methoxamine increased in a step-wise fashion through the 20th postnatal day. On day 1, the response was 2% of the mature (70–75 day) value. The response increased to 17% on day 4, and remained at this level through the 9th day. A second increase to 32% occurred between the 10th and 12th day, and there was no further increase through the 19th day. Responses increased to 64% of the mature value by the 45–50th postnatal day. Responses to tyramine were always comparable to those evoked by methoxamine. Electrical stimulation of the preganglionic nerve failed to evoke contractions on days 1 and 4, but from day 8 on, responses to preganglionic stimulation, tyramine and methoxamine were comparable. Decentralization of the superior cervical ganglion on the 1st postnatal day decreased the response to endogenously-released nerve terminal noradrenaline by 25% at 18–19 days postnatally and the deficit persisted to maturity. In contrast, the response to direct noradrenergic stimulation was attenuated at 70–75 days postnatally, but not at 18–19 and 45–50 days. Sham operation itself significantly decreased responses to methoxamine and tyramine at 18–19 and 45–50 days of age, but not at 70–75 days. The following conclusions were reached. Functional capacity of the postganglionic sympathetic nerve terminals develops at least as rapidly as the capacity of the smooth muscle to respond directly to α-noradrenergic stimulation. The connection between central nervous system and postganglionic sympathetic neuron is not required to initiate functional maturation of the neuron, postsynaptic α-noradrenergic receptors or post-receptor smooth muscle contractile elements. After ganglionic neurotransmission is established, there is a significant, though modest, transynaptically mediated contribution to overall functional maturation of postganglionic neuron and end organ. The maturational deficit (about 25%) which follows decentralization of the postganglionic neuron on the 1st postnatal day is manifest first in the sympathetic nerve terminal; this, in turn, appears to lead to a deficit in smooth muscle response to direct α-noradrenergic stimulation. Thus, decentralization in the neonate prior to establishment of ganglionic neurotransmission leads to subsensitivity of α-noradrenergic end organ response while decentralization in mature animals is known to result in supersensitivity. Since the contribution of neural, transynaptic factors to maturation was modest in this sympathetic neuroeffector system, consideration is given to the possibility that humoral factors such as thyroid and growth hormone play major roles in development of the postganglionic neuron and end organ.

Immunohistochemical localization of alpha-mannosidase during postnatal development of the rat cerebellum.

The localization of alpha-D-mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At bith an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.

Postnatal ontogeny of catecholamine and somatostatin neuron systems in the median eminence of the rat as revealed by a colocalization technique

The ontogenetic development of catecholamine (CA) and somatostatin containing nerve terminals in the rostral, central and caudal median eminence (ME) of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. Somatostatin terminals were detected earlier in development than CA terminals and had already appeared in the lateral part of the external layer of the central ME by the 1st postnatal day. CA nerve terminals were first observed in the same region of the ME on the 7th postnatal day. At about this stage both types of terminals seemed to show early signs of a correlation in their distribution which became progressively closer as maturation proceeded. Their distribution reached a stable condition in density and pattern on the 21st postnatal day when the majority were found in the lateral part of the external layer of the central ME, in the basal part of the brain just dorsal to the tuberoinfundibular sulcus and in the upper and lower labia of the tuberoinfundibular stalk. They subsequently matured to the adult pattern of stabilized distrubition and anatomical relationship. It was also established that on the 28th postnatal day the somatostatin immunoreactive terminals reached a greater concentration than at any other stage of development including that of the adult.

Reproductive tract abnormalities in rats treated neonatally with DES.

Neonatal female Sprague-Dawley rats were given daily injections of 3 micrograms diethylstilbestrol (DES) for 5 days beginning within 24 hours of birth. Some of these rats were ovariectomized between the 15th and 18th postnatal days, and on the 60th postnatal day were given daily injections of 3 micrograms estradiol for 1, 3, or 5 weeks. Intact rats were sacrificed at 60, 95, or 130 days. The morphology of the upper vagina, cervix, and lower uterus was examined by light and scanning electron microscopy. Two abnormalities resulting from neonatal DES exposure were found. The first was squamous metaplasia observed in the uteri of rats given DES and later exposed to exogenous or endogenous estrogen. Metaplasia was seen in both the luminal and glandular epithelium. The longer the rats were exposed to exogenous or endogenous estrogen, the more extensive was the metaplasia. The second was a gross morphologic abnormality seen in all rats given DES regardless of any later treatment. That part of the cervix that protrudes into the cranial limit of the vagina was absent and, thus, a vaginal fornix was nonexistent. Previous investigations have emphasized abnormalities of the lower reproductive tract. The present study indicates that the upper reproductive tract also must be considered in investigations of the effects of hormones administered to neonates.

Ultrastructural observation on the tips of growing vascular cords in the rat cerebral cortex.

The distal ends of vascular cord in the cerebral cortex are investigated electron microscopically in rats at the 13th postnatal day. The tip of the vascular cord consists of central cuboidal cells (primitive endothelial cells) and surrounding flat cells (primitive pericytes), and has no lumen. The primitive endothelial cells (tip cells) possess several long tentacles which contain only fibrous structures and extend through the neuropile.

Development of sympathetic ganglionic neurotransmission in the neonatal rat. Pre- and postganglionic nerve response to asphyxia and 2-deoxyglucose

To determine the time course of development of neurotransmission in the sympathetic ganglion of the rat, pre- and postganglionic activity was recorded from the cervical sympathetic trunk in anesthetized neonatal and mature preparations. Tonic activity and responses to two stimuli, cellular hypoglycemia induced by 2-deoxyglucose and asphyxia, which are known to evoke CNS-mediated sympathetic activation in mature rats were measured. In 2–11-day-old neonates, tonic preganglionic activity recorded from the cervical sympathetic nerve and responses to hypoglycemia and asphyxia were comparable to or greater than that in mature rats. In 17–19-day-old neonates these variables were elevated to twice the adult value. In contrast, tonic postganglionic activity recorded from the internal carotid nerve was barely detectable through 5 days of age and there was no response to hypoglycemia. During asphyxia, maximum postganglionic impulse frequency and total number of impulses discharged were 10–20% of the mature value through the 5th postnatal day and the duration of the postganglionic response was only 25% of the preganglionic response. Tonic postganglionic activity and responses to stimuli were equivalent to those in mature rats by the 10th postnatal day. The compound action potential evoked in the postganglionic axons by direct electrical stimulation was comparable in 4–5 and 10–13 day-old rats. It is concluded that functional ganglionic neurotransmission is established in the neonatal rat between the 5th and 10th postnatal day. The relation between biochemical changes associated with maturation of the postganglionic neuron, ganglionic synaptogenisis and neurotransmission is discussed. It is concluded that synaptogenisis and onset of neurotransmission are causally associated with development of CNS regulation of postganglionic activity and end organ response rather than with maturation of the postganglionic neuron and that cholinergic excitation of the postganglionic neuron adequate to evoke action potentials is not essential to initiate maturation of the neuron.

Immunocytochemical localization of ß-endorphin (lipotropin c-fragment) in the developing rat spinal cord and hypothalamus

Immunocytochemical studies have been performed on rat spinal cord and hypothalamus during development, using an antibody to ß-endorphin. Specific immunoreactivity was demonstrated in histological sections of spinal cord, in ventral and dorsal horn cells and nerve fibres, in the meningeal layer, the ependymal lining of the central canal, and in the endothelium of the ventral spinal artery and other blood vessels. ß-Endorphin immunoreactivity was also distributed widely in neurones, central and peripheral nerve fibres, and in non-neuronal cells in cultures of spinal cord tissue explanted from rat embryos 7–10 days before birth. Immunofluorescence disappeared abruptly after the 28th postnatal day in vivo, and in the fourth week of incubation of embryonic spinal cultures. In contrast, cultures of the ventral diencephalic (hypothalamic) region of embryonic brain at the same gestational age showed a characteristically different pattern ß-endorphin immunoreactivity which persisted for more than 7 weeks. The results provide evidence for the biosynthesis of a ß-endorphin-like peptide in rat spinal cord during development. The biosynthesis terminates at an early stage both in vivo and in vitro, suggesting that control of the biosynthesis is intrinsic to spinal cord tissue and possibly to the peptide-producing cells themselves.

The dorsal lateral geniculate nucleus of the normal ferret and its postnatal development.

The anterograde transport of 3H proline and of horseradish peroxidase has been used to study the retinogeniculate pathway in normal adult ferrets and in young ferrets during postnatal development. the lateral geniculate nucleus in adults shows a characteristic "carnivore" pattern, with layers A, A1, C, C1, C2, and C3, and a medial interlaminar nucleus recognizable either cytoarchitectonically or on the basis ofth retinogeniculate innervation. In addition, there is a well-defined, rather large perigeniculate nucleus. At birth the lateral geniculate nucleus is unlaminated and essentially all parts are reached by afferents from both eyes. The crossed component is by far the larger. It extends from the optic tract medially well into the perigeniculate field, in contrast to the uncrossed component which barely reaches the perigeniculate field. During the first 3 postnatal days the uncrossed fibers restrict their arbors to a small posterior and medial region, the precursor of the biocular segment of the nucleus. The crossed fibers gradually retreat from the region within which the uncrossed fibers have concentrated. Between the fourth and eighth postnatal days the field occupied by the ipsilateral component expands again to form a major focus that will define lamina A1 and a minor focus that will define C1. At this stage the crossed and the uncrossed fibers overlap at the borders of lamina A1 and the whole region of lamina C1 is also occupied by arbors of the crossed component. The perigeniculate field becomes clearly distinguishable from the lateral geniculate nucleus and the medial interlaminar nucleus is becoming clearly recognizable between days 3 and 8. Between days 8 and 15 the cytoarchitectonic borders between layers A and A1 become clearly defined, but the retinogeniculate axons from each eye still extend across this border. These axons retreat into their appropriate lamina after the 15th postnatal day an the nucleus reaches its essentially adult structure by about the fourth postnatal week. Segregation of retinofugal axons in the C layers occurs after segregation in the A layers, but many of the cells within the C layers show signs of cytological maturity earlier than those of the A layers. The nucleus undergoes a series of migrations and changes of shape as the ipsilateral and contralateral components become segregated. Whereas in teh newborn the nucleus is roughly comma-shaped and on the lateral aspect of the dorsal thalamus, in the adult it is "L"-shaped and mainly on the posterior aspect of the dorsal thalamus.

Membrane-bound neuraminidase in the developing mouse brain

The specific activity of membrane-bound neuraminidase towards exogenously added gangliosides in the developing mouse brain was investigated. While whole brain was examined in fetuses, studies were carried out comparatively in cortex and cerebellum of postnatal stages. Considerable differences were found among brain areas. There was a rapid increase in forebrain neuraminidase activity before birth. This high level of activity was maintained throughout the first postnatal week, followed by a slow leveling-off to adult values on the 30th day. In the cerebellum a rapid increase in neuraminidase activity occurred between the 10th and 16th postnatal day. After having reached the maximum, enzyme activity declined rapidly, with adult values being reached on the 21st day. Neuraminidase activity in the adult cerebellum exceeds that in the cortex by 65.9 %. The results are discussed in reference to developmental changes in ganglioside metabolism and the possible involvement of neuraminidase in its regulation is pointed out.

Supraependymal cell clusters in the rat brain

The histological patterns of supraependymal cell clusters (CC) in rats of different ages (untreated, androgenized, and treated with monosodium glutamate) were investigated with light (LM)-, scanning- and transmission electron microscopy (SEM, TEM). These clusters were a frequent but not a constant finding. In 18 day- and older embryos, CC were always found in the recess of the olfactory bulb immediately prior to its obliteration. All other CC appear in the infundibular recess between the 3rd and the 6th postnatal day. Independent of age, all cell clusters exhibit small aggregates of subependymal tissue protruding through the ependyma. Both neurons (light cells) and neuroglia (dark cells) were found in the CC. By use of SEM, in the region of the infundibular recess it is possible to distinguish four forms of supraependymal cell clusters according to localization, size, number of cells, and presence of intraventricular axons. CC may be 1) receptors or have an additional secretory function; 2) Manifestations of a pathological type of reaction of the ventricular wall; 3) possible excrescences of the neural matrix, or 4) modifications of the ventricular wall in relation to the obliteration of the ventricular recesses. The first two interpretations are not tenable based on the present observations.

Brain carbohydrate metabolism in developing rats during hypercapnia.

Brain glucose metabolism was studied in developing rats at ages 10 and 20 days postnatal under normal and hypercapnic conditions. Brains were removed and frozen within 1 s with a freeze-blowing apparatus. Glucose utilization was measured with [2-14C]glucose and [3H]deoxyglucose as tracers. Metabolites were determined by standard enzymatic techniques. Data from [3H]deoxyglucose phosphorylation indicated that normal brain glucose utilization increased almost threefold between the 10th and 20th postnatal days. From the relative rates of utilization of the two isotopes in the 20-day-old control group, it appeared that about 25% of 14C label derived from metabolism of [2-14C]glucose was lost from brain (probably as lactate) rather than entering the Krebs cycle. Under hypercapnic conditions (20% CO2-21% O2-59% N2), rates of glucose utilization by brain were decreased by one-half at both ages and there were progressive decreases in the concentrations of many intermediary metabolites. The bases for concluding that these metabolites were used to supplement glucose as a fuel for respiration, rather than being lost by leakage into blood, are discussed. Despite the differences in brain glucose metabolism between 10-day-old and 20-day-old rats, their responses to hypercapnia are remarkably similar: Rates of glucose utilization are reduced to approximately the same proportion of the original rate by 20% CO2, and endogenous metabolites (particularly glutamate and lactate) appear to be oxidized as replacement fuels.

Pituitary response to bilateral adrenalectomy, metyrapone treatment and ether stress in the newborn rat.

ACTH level, determined by radioimmunoassay, has been used as a reliable test to investigate pituitary response to bilateral adrenalectomy, metyrapone treatment and ether stress in 8-day-old rat newborns. The pituitary gland able to enhance ACTH release significantly, first in response to the decrease in plasma corticosteroid levels induced either by bilateral adrenalectomy or by metyrapone inhibition of the corticosteroidogenesis, and second in response to 2 min of ether exposure. This latter response was greater in females than in males but smaller in neonates than in adults. These data suggest that the steroid-feedback mechanism was operating fully at the 8th postnatal day and that central mechanisms partially responded to external signals of stress. However, a relative 'stress-non-responsive period' was evident by the 8th postnatal day. The newborn rat's hypothalamic-pituitary axis has partially matured to be responsive to stress by the end of the first week after birth.

Immaturity of the enzyme activity and the response to inducers of rat liver cysteine dioxygenase during development.

Cysteine dioxygenase activity was not detected in the liver of either fetal or 2-day-old rat. The enzyme activity of neonatal rat liver gradually increased between the 4th and 12th postnatal days and then sharply increased to reach the adult level by the 28th postnatal day. No cysteine dioxygenase activity was observed in the fetal liver at the 18th day of gestation from a dam injected with hydrocortisone. Hydrocortisone-mediated induction was first observed in the livers of 4-day-old rats. The duration of hydrocortisone-mediated induction was much longer in the livers of 4- to 12-day-old rats than in those of adult rats. The enzyme half-life in livers of 10- and 20-day-old rats was 6.4 and 3.7 h, respectively. The half-life of the enzyme in 20-day-old rat liver was increased to 6.2 h by L-cysteine injection.

Ototoxicity of kanamycin in developing rats: Relationship with the onset of the auditory function

In order to test the relationship between the ototoxicity of kanamycin and the onset of the auditory function, two groups of developing rats were intoxicated with kanamycin before and after the period of onset of cochlear potentials (8th postnatal day). Kanamycin was shown to have a weak ototoxic effect before the 8th postnatal day and a strong ototoxic effect after this period. These results indicate a critical period of sensitivity to ototoxic antibiotics during auditory development.

Myelination in rabbit optic nerves is accelerated by artificial eye opening

Artificial opening of the eyes of young rabbits on the 5th postnatal day led to accelerated myelination: the myelin-specific basic and proteolipid proteins nearly doubled between the 7th and the 10th postnatal days when compared to controls; 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) activity also increased by about 60%. Conversely, lowered AChE activities presumably reflected elevated myelin/axolemma ratios. Myelination in treated animals normalized during later ontogenetic stages (> 20th postnatal day).

Harmaline-induced activation of the olivo-cerebellar system in young rabbits: Further evidence for a transient multiinnervation of purkinje cells by climbing fibres

Ontogenetic evolution of behavioural and electrophysiological responses to harmaline was studied in the maturing rabbit. As regards behavioural effects, harmaline-induced tremor could not be elicited before the second post-natal week, but as soon as it appeared, no significant difference was seen in its intensity compared with the adult animal. Electrophysiological studies of cerebellar Purkinje cell activity revealed a similar age-dependence since no rhythmic firing of climbing fibre responses was found before the 8th post-natal day. In addition, harmaline activation revealed a transient multiple innervation of Purkinje cells by climbing fibres. It is suggested that the presence of serotonergic fibres is critical for the effects of harmaline to develop.

Experimental hyperphenylalaninemia: Effect on central nervous system myelin subfractions

We examined the effects of experimental hyperphenylalaninemia on the formation of subfractions of myelin in the central nervous system of 18- to 110-day-old rats. Hyperphenylalaninemia was induced by giving rats daily subcutaneous injections of α-methylphenylalanine and phenylalanine from the 3rd to the 15th postnatal day. A mixture of [ 3H]glycine and [ 14C]glucose was injected intraventricularly into 17-, 20-, and 24-day-old control and experimental rats 18 h prior to the isolation and subfractionation of CNS myelin. Nonradioactive myelin subfractions were isolated from 43- and 110-day-old rats. Animals that had been hyperphenylalaninemic between the 3rd and 15th postnatal day exhibited a deficiency of total myelin protein throughout the study. With the exception of one age, these rats had a deficit of the classical, mature light myelin. The light and medium myelin subfractions from 18- to 43-day-old experimental rats and heavy myelin from the 25- and 43-day-old experimental rats had a deficiency of myelin basic proteins and an excess of the high molecular weight proteins. Nonetheless, previously hyperphenylalaninemic rats incorporated more [ 3H]glycine into the basic proteins of all myelin subfractions. The proportion of total brain [ 3H] glycine and [ 14C]glucose that was incorporated into combined (total) myelin subfractions was near normal in rats that were hyperphenylalaninemic between the 3rd and 15th postnatal day. However, these rats demonstrated decreased 3H and 14C incorporation into light myelin at 21 and 25 days, increased 3H and 14C incorporation into medium myelin at 25 days, and increased incorporation into heavy myelin at 21 days.

Carnitine Acetyltransferase in Developing Mammals

Carnitine acetyltransferase (CAT) activity was determined in mitochondria and microsomes of liver and brown adipose tissue in fetal and postnatal rats, rabbits and guinea pigs. In rat liver and brown adipose tissue, mitochondrial CAT activity increased perinatally. Microsomal CAT activity also increased in brown adipose tissue. In liver, however, a rise was first noted after the 20th postnatal day. The ratio of mitochondrial to microsomal activity was higher in brown fat than liver throughout the period studied. Absolute values for both were always much higher in brown adipose tissue than in liver. Catalase activity (an enzyme localized in the peroxisomes) in rat liver increased after day 20 while in brown adipose tissue it attained a peak at 7 days after birth. At all times, hepatic activity exceeded activity in brown adipose tissue. The ratio on day 30 was 1 (brown adipose tissue) to 25 (liver). In both guinea pigs and rabbits, hepatic mitochondrial CAT activity was 10- to 20-fold higher than in the rat already prenatally. Microsomal activity, on the other hand, was approximately the same in all three species. It is concluded that probably only the mitochondrial CAT is directly related to fatty acid oxidation. The role of microsomal enzyme remains unclear.

Acute Effects of Hypothalamic Ablation on Plasma Thyrotropin and Prolactin Concentrations in the Suckling Rat: Evidence That Early Postnatal Pituitary-Thyroid Regulation Is Independent of Hypothalamic Control*

Hypothalamic ablation was performed at various periods postnatally in animals previously administered propylthiouracil to raise plasma TSH concentrations. There was no significant change in plasma Tsh up to 8 h after hypothalamic ablation in pups 1--4 days old, whereas hypophysectomy of such pups produced a 60% fall in plasma TSH within 4 h. By the 5th postnatal day, hypothalamic ablation produced a 30% fall in plasma TSH within 4 h (P less than 0.05). By the 12th postnatal day and thereafter, the fall in plasma TSH after hypothalamic ablation was not significantly different from that seen in adults, except in 30-day-old rats in which there was a lesser effect of hypothalamic ablation on plasma TSH (P less than 0.01 in comparison to 23-day-old and adult groups). The greatest effect of hypothalamic ablation on plasma TSH was in 45-day-old animals (P less than 0.01 in comparison to adults). No significant change was produced in plasma PRL within 4 h postoperatively at any age. Our data indicate that regulation of TSH secretion in the rat is independent of hypothalamic control until after the 5th postnatal day and is fully developed by day 12. This corresponds temporally with the postnatal rise of plasma TSH, T4, and T3 and hypothalamic TRH to adult concentrations and indicates maturation of the hypothalamic regulation of TSH secretion.