Localization of endothelin-A and -B receptors during the postnatal development of rat cerebellum

Intense expression of mRNA of endothelin-B receptor (ETBR) has been detected in the Bergmann glia of cerebellum by in situ hybridization, but the intracellular localization has not been reported because of the absence of a useful antibody for immunohistochemical investigations. We made polyclonal antibodies against the carboxyl terminus of human ETBR (420-442) and ETAR (403-427), and performed light- and electron-microscopic immunohistochemistry of the wild-type and ETBR-deficient (sl/sl) rat cerebella. Localization of ETBR during postnatal development was examined by double-staining immunofluorescence using antibodies against ETBR and S-100 beta. In the wild-type rats, ETBR immunoreactivity appeared from postnatal day 5 (P5) and was distributed diffusely in the processes and cell bodies of S-100 beta-positive glial cells. By P14, ETBR immunoreactivity was concentrated in the Golgi apparatus of Bergmann glial cell soma and the plasma membrane of its processes. The ETBR-positive astrocytes in the granular layer decreased in number during P7-14 and had disappeared by week 3. At 3 weeks, ETBR immunoreactivity was restricted to the Golgi apparatus of Bergmann glia. In the sl/sl rats, ETBR immunoreactivity was not observed at all. In contrast to ETBR, ETAR immunoreactivity appeared transiently in the cytoplasm of all astrocytes (Bergmann glia and astrocytes in the granular layer) in the 9- to 14-day-old wild rats and 7- to 14-day-old sl/sl rats, and disappeared within 3 weeks in both. Granule cells did not express immunoreactivity for ETBR and ETAR from the neonatal stage to adulthood. Changes in the intracellular localization of ETBR and transient expression of ETAR may be correlated with the changes of glial functions and proliferation during postnatal development of rat cerebellum.     

Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.     

Subcortical projections of the visual cortex in the adult albino rat and during postnatal development

We have investigated the subcortical projections of the rat striate cortex by using the silver-degeneration method and the HRP-technique too. Cortical lesions were made in 60 young animals (1, 4, 5, 6, 7, 10 and 14 days old) and in 6 adult rats. The terminal regions of projection occurred only ipsilateral to the lesions. After passing the internal capsule the degenerating pathway divides into 2 bundles. In the dorsal thalamus one of them runs in caudal direction. The other bundle turns ventrally, reaches the cerebral peduncle and terminates in the pons. The first fibre bundle terminates in the following structures: Nc. reticularis thalami, Nc. lateralis thalami, Nc. lateralis posterior thalami, Corpus geniculatum laterale, pars dorsalis (Cgld), Corpus geniculatum laterale, pars ventralis (Cglv), Nc. praetectalis anterior et posterior and Colliculus superior. The fibers of the second bundle innervate the Nc. lateralis pontis. Fibers from this bundle terminate probably in the Cglv and in the Zona incerta too. By using the HRP-technique it could be demonstrated that the axons terminating in the Cgld originate in layer VI of the area 17. In contrast, the projection to Cglv, Nc. lateralis posterior, Colliculus superior and Nc. lateralis pontis originates from pyramidal cells in layer V. The development of the projection in young animals indicates: Like in adults rats, terminal degeneration is present in all subcortical projection regions at postnatal day (PD) 10. At PD 4-7 we can observe heavily degenerating axons but the terminal degeneration is different. It is remarkable in the "visual" part of the reticular nucleus and iln the Cgld (decreasing from inside to outside). Only a weak terminal degeneration is visible in the pretectal region and in the superior colliculus. At PD 1 the trajectory of degenerating fibres is clearly visible. Signs of terminal degeneration can only be found in the reticular nucleus. It is discussed whether the date of generation of the cortical neurons and the time of the differentiation of the cortical layers is of importance for the time of innervation of the subcortical projection fields. The question when the axons arrive at their terminal region and form there synaptic contacts has not yet been exactly answered. To solve this problem electronmicroscopic investigations are necessary.     

Effects of deltamethrin on granule cell migration during postnatal development of rat cerebellum

Deltamethrin (DLT; 0.7mg/kg/body wt/day, i.p., dissolved in propylene glycol) administration during postnatal days 913 in Albino rat pups, resulted in a delayed appearance of radial glial fibers, that guide the migration of granule cells. Moreover, the radial glial fibers in the DLT-treated pups were disorganized, hypertrophied and heavily stained. Thus, it is being proposed that although after exposure to DLT the neuronal proliferation occurs at normal rate, the neuronal migration along the stumpy and crumpled radial fibers hamper the journey of the healthy neurons to their proper destination.     

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

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

The postnatal development of the main olfactory bulb of the rat

The postnatal development from birth to 1 year of the main olfactory bulb was examined quantitatively. The volume of the main olfactory bulb increased over seven-fold by day 30 and remained unchanged thereafter. During the same period the volume of the granular layer increased 18-fold and the mean areas of the olfactory glomeruli increased seven-fold. The mean areas of mitral cell perikarya doubled between the neonatal and juvenile periods. The total number of the mitral cells, however, declined during the first three postnatal weeks. In the internal granular layer of the main olfactory bulb, 89% of the granule cells were acquired postnatally. Much of the cellular gain occurred during the first 3 weeks, with the period of maximum acquisition between days 8 and 14. The number of subependymal cells, the precursors of granule cells, reached a peak at 12 days and gradually declined. But some primitive cells could still be found at one year of age and there was an increase in the total number of granule cells beyond day 30. The mean nuber of internal granular layer cells in a single main olfactory bulb of adult rats was about 5 X 10(6); the number of mitral cells about 4 X 10(4). In the animals injected with 3H-thymidine on day 20 and killed 2 h after injection a small but significant proportion of cells was labelled in the subependymal layer but few in the internal granular layer. In the animals killed 20 and 40 days after injection there was a 10--11-fold rise in the proportion of labelled internal granular layer cells. The proportion of labelled internal granular layer cells decreased in longer survival groups but the total number of labelled cells remained the same, even in year-old animals. However, the total number of internal granular layer cells in the sections examined increased with age.     

Neurotoxicological effects of deltamethrin on the postnatal development of cerebellum of rat

Deitamethrin (DLT) has been accepted to be 10,000 times less toxic to man than to insects. While toxicity of DLT in adult animals has been studied using biochemical and electrophysiological tools, reports on its developmental neurotoxicity are rather scanty. Wistar rat pups were exposed to DLT (0·7 mg/kg body wt/day, i.p., dissolved in propylene glycol from postnatal day 9–13. Equal number of age matched pups were used as vehicle controls. The animals were weighed and perfused intracardially on postnatal days 12,15,21, and 30 and their brains dissected out. Cerebellum along with the brainstem was separated by a transverse section at the tectal level and processed for morphometric and toxicological studies. The micro-and inter-neurons in the cerebellum are known to differentiate and mature, both morphologically and biochemically, during the postnatal life of rats. Postnatal exposure to DLT has been observed to delay the cytogenesis and morphogenesis of these neurons. In addition to this, damage to the developing vasculature has also been recorded in the form of thrombus and haemorrhage. Focal degeneration and spongy appearance of the tissue in the vicinity of the damaged blood vessels have also been recorded. The study has opened up several questions on the safety of this substance to the pregnant mothers and infants in the habitats where this substance is in use for vector control.     

Glutamate-like immunoreactivity revealed in rat olfactory bulb, hippocampus and cerebellum by monoclonal antibody and sensitive staining method

Although there is good evidence favoring L-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. --The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The crossreactivity of 'anti-glutamate' mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA.--Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.     

Glutamate-like immunoreactivity revealed in rat olfactory bulb,hippocampus and cerebellum by monoclonal antibody and sensitive staining method

Summary Although there is good evidence favoring l-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. — The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The cross-reactivity of anti-glutamate mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA. — Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.     

Effect of prenatal hypoxia during organogenesis on albino rat behavior during postnatal period

The survival rate, physical development, and spontaneous behavior has been evaluated in pups of albino rats exposed to acute hypobaric hypoxia on the 9–10th day of gestation corresponding to the onset of organogenesis. Prenatal hypoxia increased the mortality among the offspring, delayed their physical development, and affected their spontaneous behavior up to the age of 2 months. The females exposed to intrauterine hypoxia proved to be more sensitive to hypoxia than males.     

Changes in the cerebellum of the rat after actinomycin during the postnatal period]

A single dose of actinomycin was applied to young Wistar albino rats in the critical phase of their cerebellar development. The morphological alterations of the cerebellar cortex were studied by means of light and electron microscopy on several postnatal days. The cell types of the cerebellar cortex reacted in different ways toward the noxious substance according to their stage of development. The acute alterations consisted of an edematous reaction of the neuronal and glial perikarya (light degeneration) and a shrinkage of the neurons (dark degeneration). A massive intercellular edema and a rarefaction of glia cells as well as the Purkinje cell fibres proved to be the long-term damage. This pattern of the alteration was discussed regarding the chemodifferentiation of the cells of the cerebellar cortex, the onset of cerebellar function on day 14, and the establishment of a neuroglial functional unit.     

Effect of hypothyroidism on the biogenesis of free mitochondria in the cerebral hemispheres and in cerebellum of rat during postnatal development

The effect of propylthiouracil-induced neonatal hypothyroidism on some aspects of the biogenesis of free (non-synaptosomal) mitochondria in the cerebral hemispheres and in the cerebellum of developing rat has been studied. The results obtained show that in hypothyroid rats mitochondrial DNA synthesis is delayed, mitochondrial RNA synthesis is not affected and cytochrome aa₃ content of mitochondria is lower than in controls. Furthermore ultrathin sections of 14-and 21-day old hypothyroid rat cerebella show mitochondria with an altered ultrastructural organization and large intracristal spaces.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

Prenatal experience and postnatal development of the rat hippocampus: sleep deprivation of pregnant rats

The effects of sleep deprivation in pregnancy on the development of hippocampal function of the offspring have been investigated. For this purpose we compared electrophysiological characteristics in the hippocampal slices of 15-20-old-day rats of the control and two experimental groups. In the first experimental group the pups were taken from mother for weighting three times during the first postnatal week and then weekly. Another experimental group was brought up without handling. We found that CA1 population spikes developed to significantly less amplitude in experimental groups of rat pups. This phenomenon was observed at higher intensity of monosynaptic activation, although near-threshold stimuli didn't reveal any differences among groups. However, under paired-pulse stimulation (70 ms inter-pulse interval) small amplitudes in the hippocampal slices of experimental animals could facilitate up to control value, and second in pair responses didn't differ from corresponding control. Our data doesn't confirm the hypothesis about decreased connectivity in the hippocampus of experimental rats, but the efficacy of CA3-CA1 inputs seems to be lower. Besides excitatory transmission, the effectiveness of inhibition of paired-pulse facilitation at 15 ms inter-pulse interval was also significantly decreased. The observed effects of prenatal influences seem to develop under postnatal experience. We observed significant trend to more pronounced modifications upon age especially in the case of early handling and testing.