Granule cell migration in developing rat cerebellum. Influence of neonatal hypo- and hyperthyroidism.

The rate of cerebellar granule cell migration is altered by neonatal hypo- and hyperthyroidism in a manner similar to previously reported effects on the growth of granule cell axons, the parallel fibers, suggesting that the two processes may be intimately linked. Altered rates of granule cell acquisition in these experimental animals reflect changes in germinal cell proliferation in the external granular layer (EGL), movement of postmitotic cells within the EGL, as well as the rate and time course of granule cell migration. Results of this study support the hypothesis that granule cells migrate to the internal granular layer by translocation of the cell body through the descending portion of the growing parallel fiber, rather than by amoeboid-like migration of the perikaryon trailing the elongating parallel fiber behind.     

Calcitonin gene-related peptide-like immunoreactivity in the central nervous system and peripheral organs of rats

The concentrations of rat calcitonin gene-related peptide-like immunoreactivity (rCGRP-LI) in various organs of male rats as well as the molecular heterogeneity of rCGRP-LI in tissue extracts was examined using a specific radioimmunoassay (RIA) for rCGRP and gel-filtration chromatography. rCGRP-LI was high in extracts of the spinal cord (202 +/- 22.6 pg/mg wet wt. of tissue; mean +/- S.E.M.) and of the thyroid (229 +/- 62.3 pg/mg). rCGRP-LI was detectable in the brainstem, hypothalamus, stomach, duedenum, pancreas and kidney. The elution pattern of the extracts on a Sephadex G-50 column showed 3 peaks of rCGRP-LI irrespective of organs and tissues. The first peak corresponded to authentic rCGRP-(1-37). The second and third rCGRP-LI peaks probably consisted of C-terminal fragments of rCGRP, because they had a lower molecular weight than rCGRP-(1-37) and because our antiserum cross-reacts with a synthetic C-terminal fragment. The ratio of 3 rCGRP-LI molecules, however, differed between neural tissue extracts and others. The main component of rCGRP-LI in neural tissue was authentic rCGRP-(1-37), while the smaller fragments of rCGRP were chiefly contained in other tissues like the stomach, pancreas and thyroid. The relative ratio of rCGRP-LI molecules with different size in respective tissue extracts was not changed after leaving the dissected tissues for 2 h at room temperature. These findings indicate that rCGRP-LI is abundantly present in the thyroid as well as the spinal cord and it is detected in lower amounts in the alimentary tract and central nervous system. rCGRP-LI in the extracts consists of 3 different components, the proportions of which vary from one tissue to another, probably reflecting tissue-specific differences in the processing of CGRP.     

Carbonic anhydrase distributions in central and peripheral nervous system of the rat

Total and specific carbonic anhydrase activity was measured for 24 structures of the rat central and peripheral nervous system. In the CNS, white matter or regions containing largely white matter show a neuraxial distribution of enzyme activity; more cephalad structures display more activity. Gray matter regions do not show a rostrocaudal distribution and usually have lower activity than adjacent myelin-containing structures. PNS tissue shows neither the white-gray differences nor the rostrocaudal profile of CNS tissue. Subcellular fractionation of 18 regions of the CNS suggest that the predominance of membrane-bound carbonic anhydrase (60% of the total activity and independent of its magnitude) is a unique characteristic of all regions of the central nervous system.     

Membrane-Bound and cytosolic forms of heterotrimeric G proteins in young and adult rat myocardium: influence of neonatal hypo- and hyperthyroidism.

Membrane and cytosolic fractions prepared from ventricular myocardium of young (21-day-old) hypo- or hyperthyroid rats and adult (84-day-old) previously hypo- or hyperthyroid rats were analyzed by immunoblotting with specific anti-G-protein antibodies for the relative content of Gs alpha, Gi alpha/Go alpha, Gq alpha/G11 alpha, and G beta. All tested G protein subunits were present not only in myocardial membranes but were at least partially distributed in the cytosol, except for Go alpha2, and G11 alpha. Cytosolic forms of the individual G proteins represented about 5-60% of total cellular amounts of these proteins. The long (Gs alpha-L) isoform of Gs alpha prevailed over the short (Gs alpha-S) isoform in both crude myocardial membranes and cytosol. The Gs alpha-L/Gs alpha-S ratio in membranes as well as in cytosol increased during maturation due to a substantial increase in Gs alpha-L. Interestingly, whereas the amount of membrane-bound Gi alpha/Go alpha and Gq alpha/G11 alpha proteins tend to lower during postnatal development, cytosolic forms of these G proteins mostly rise. Neonatal hypothyroidism reduced the amount of myocardial Gs alpha and increased that of Gi alpha/Go alpha proteins. By contrast, neonatal hyperthyroidism increased expression of Gs alpha and decreased that of Gi alpha and G11 alpha in young myocardium. Changes in G protein content induced by neonatal hypo- and hyperthyroidism in young rat myocardium were restored in adulthood. Alterations in the membrane-cytosol balance of G protein subunits associated with maturation or induced by altered thyroid status indicate physiological importance of cytosolic forms of these proteins in the rat myocardium.     

Developmental study of tripeptidyl peptidase I activity in the mouse central nervous system and peripheral organs

Tripeptidyl peptidase I (TPPI) — a lysosomal serine protease — is encoded by the CLN2 gene, mutations that cause late-infantile neuronal ceroid lipofuscinosis (LINCL) connected with profound neuronal loss, severe clinical symptoms and early death at puberty. Developmental studies of TPPI activity levels and distribution have been done in the human and rat central nervous systems (CNS) and visceral organs. Similar studies have not been performed in mouse. In this paper, we follow up on the developmental changes in the enzyme activity and localization pattern in the CNS and visceral organs of mouse over the main periods of life — embryonic, neonate, suckling, infantile, juvenile, adult and aged — using biochemical assays and enzyme histochemistry. In the studied peripheral organs (liver, kidney, spleen, pancreas and lung) TPPI is present at birth but further its pattern is not consistent in different organs over different life periods. TPPI activity starts to be expressed in the brain at the 10th embryonic day but in most neuronal types it appears at the early infantile period, increases during infancy, reaches high activity levels in the juvenile period and is highest in adult and aged animals. Thus, in mice TPPI activity becomes crucial for the neuronal functions later in development (juvenile period) than in humans and does not decrease with aging. These results are essential as a basis for comparison between normal and pathological TPPI patterns in mice. They can be valuable in view of the use of animal models for studying LINCL and other neurodegenerative disorders.     

Immunofluorescence studies of neurofilaments in the rat and human peripheral and central nervous system

Localization of antisera to neurofilament antigens derived from rat peripheral nerve was carried out in tissues of rat and human peripheral and central nervous systems by indirect immunofluorescence. Unfixed and chloroform-methanol-fixed frozen sections of tissues were incubated in purified IgG of the experimental rabbit antisera and subsequently exposed to goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. Control studies were conducted on identical tissue preparations incubated in the same concentrations of nonspecific rabbit IgG or in experimental rabbit IgG absorbed with extracts of rat peripheral nerve containing neurofilament antigen. Extensive immunofluorescence was observed in rat and human peripheral and central nervous systems. The distribution and configuration of immunofluorescence corresponded to neurofilament-rich structural components of these tissues. Prominent immunofluorescence was also noted in neuronal cell bodies of spinal sensory ganglia, especially in perikarya of the large neuronal type. Immunofluorescence of the central nervous system was located predominantly in myelinated axons of the white matter in cerebrum, cerebellum, brain stem, and spinal cord. Less intense immunofluorescence was also seen in neuronal perikarya and in short thin linear processes of grey matter.     

Characterization of endozepine-related peptides in the central nervous system and in peripheral tissues of the rat

Endozepines represent a novel family of regulatory peptides that have been isolated by their ability to displace benzodiazepines from their binding sites. All endozepines derive from an 86 amino acid precursor polypeptide called diazepam binding inhibitor (DBI), which generates, through proteolytic cleavage, several biologically active endozepines. The aim of the present study was to compare the molecular forms of endozepines present in different regions of the rat brain and in various peripheral organs using an antiserum raised against the central (biologically active) region of DBI. Combination of HPLC analysis and RIA detection revealed the existence of two major forms (peaks I and II) of endozepine-immunoreactive peptides. The retention times of the two peaks (36 and 39 min, respectively) were identical in all tissues or organs tested. Western blotting analysis of cerebral cortex extracts confirmed the existence of two immunoreactive species with apparent molecular weights 4000 and 6000 Da, which respectively correspond to peaks I and II. Tryptic digestion of peaks I and II generated a single immunoreactive peptide that coeluted with the synthetic octadecaneuropeptide ODN [DBI(33–50)]. These results show that, in different parts of the brain and in various peripheral organs, DBI is rapidly processed to generate two peptides of apparent molecular weight of 4000 and 6000 Da, which both possess the biologically active determinant of endozepines.     

Asymmetries in the sense organs and central nervous system of the squid Histioteuthis

The visual system of Histioteuthis is markedly asymmetrical, in that the eyes and optic lobes are considerably larger on the left side, and the lens of the left eye is often yellower in colour than that of the right eye. At the histological level, the rhabdomes of the retinas of both eyes show the usual rectilinear pattern typical of cephalopods. Unlike other species described, however, the orientation of the pattern is not uniform over the retina. The optic lobes are well developed on both sides, again following the typical squid pattern, although the plexiform and inner granular layers are thicker on the left side. In life it is likely that the animals orient at an oblique angle with the arms downward, and the left eye pointing upwards and the right eye downwards, and the asymmetries of the visual system are probably related to this posture. No corresponding asymmetries in the statocysts or other parts of the central nervous system have, however, been detected     

Androgen and progesterone metabolism in the central and peripheral nervous system

This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons, in the glia, and in neuroblastoma cells. The activities of the 5α-reductase (the enzyme that converts testosterone into dihydrotestosterone, DHT), and of the 3α-hydroxysteroid dehydrogenase (the enzyme that converts DHT into 5α-androstane-3α,17β-diol, 3α-diol) have been first evaluated in primary cultures of neurons, oligodendrocytes and type-1 and -2 astrocytes, obtained from the fetal or neonatal rat brain. All the cultures were used on the fifth day. The formation of DHT or 3α-diol was evaluated incubating the different cultures with labeled testosterone or DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, type-2 astrocytes and oligodendrocytes also possess considerable 5α-reductase activity, while type-1 astrocytes show a much lower enzymatic concentration. A completely different localization was observed for 3α-hydroxysteroid dehydrogenase; the formation of 3α-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3α-diol is formed in very low yields by neurons, type-2 astrocytes and oligodendrocytes. The compartmentalization of two strictly correlated enzymes (5α-reductase and 3α-hydroxysteroid dehydrogenase) in separate central nervous system (CNS) cell populations suggests the simultaneous participation of neurons and glial cells in the 5α-reductive metabolism of testosterone. Subsequently it has been shown that, similarly to what happens when testosterone is used as the substrate, the 5α-reductase which metabolizes progesterone into 5α-pregnane-3,20-dione (DHP) shows a significantly higher activity in neurons than in glial cells; however, type-1 and -2 astrocytes as well as oligodendrocytes also possess some ability to 5α-reduce progesterone. On the other hand, 3α-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5α-pregnane-3α-ol-20-one, appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3α-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoforms of the enzyme involved in androgen and progesterone metabolism is discussed.Finally, the ability of the human neuroblastoma cell line SH-SY5Y to metabolize androgens and progesterone was studied incubating the cells in the presence of labeled testosterone or progesterone to measure, respectively, the formation of DHT or DHP (5α-reductase activity). 3α-Hydroxysteroid dehydrogenase activity was studied by evaluating the conversion of labeled DHT into 3α-diol. The results demonstrate that undifferentiated neuroblastoma cells possess a significant 5α-reductase activity, as shown by the considerable conversion of testosterone into DHT; moreover, this enzymatic activity seems to be significantly stimulated following cell differentiation induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), but not after differentiation induced by retinoic acid (RA). The 5α-reductase present in SH-SY5Y cells is also able to convert progesterone into DHP. In undifferentiated cell, this conversion is about 8 times higher than that of testosterone into DHT. Under the influences of TPA and RA, the formation of DHP follows the same pattern observed for that of DHT. SH-SY5Y cells also appear to possess the enzyme 3α-hydroxysteroid dehydrogenase, since they are able to convert DHT into 3α-diol. This enzymatic activity is not altered following TPA-induced differentiation, and appears to be decreased following treatment with RA. It is suggested that the SH-SY5Y cell line may represent a useful “in vitro” model for the study of the mechanisms involved in the control of androgen and progesterone metabolism in nervous cells.     

Distribution and molecular forms of brain natriuretic peptide in the central nervous system, heart and peripheral tissue of rat

The amino acid sequence of rat brain natriuretic peptide (rBNP) precursor has recently been deduced by the cDNA cloning method (1). In the present study, a radioimmunoassay (RIA) system for rBNP was newly established, and regional distribution of rBNP in the central nervous system, heart and other peripheral tissue of rat was investigated. In heart, especially in cardiac atrium, a high concentration of immunoreactive (ir-) rBNP was detected and identified as rBNP-45 and gamma-rBNP. No significant amount of ir-rBNP was found in other tissues examined including the central nervous system. Especially in brain, no ir-rBNP was detected, while ir-rat atrial natriuretic peptide (rANP) was observed at a relatively high concentration. These results demonstrate a sharp contrast between rat and porcine brains in ir-BNP distribution.     

The metabolism in vivo of glycine and serine in eight areas of the rat central nervous system

Abstract— Results of studies designed to estimate the rates at which glycine is derived from various possible sources in discrete areas of the rat CNS are reported. These results suggest that glycine is derived predominantly by de novo synthesis, presumably via the established pathways leading from glucose through serine to glycine. The content of glycine ranged from a low of approximately 0-6 μmol/g in the cerebellum and telencephalon to a high of 5·5 μmol/g in the spinal cord grey matter; however, based on its estimated rate of synthesis from serine, there appeared to be no correlation between the content of glycine and its rate of synthesis in the various areas studied. The flux of glycine from blood into the CNS was slower (0·03-0·15 μmol/g/h depending on the CNS structure) than that of serine (0·15-0·23 μmol/g/h) and both amino acids entered various CNS areas at rates unrelated to their respective tissue contents. These data have been discussed with regard to the putative transmitter function of glycine in the spinal cord and brainstem.