Granule cell raphes in the developing mouse cerebellum

The cerebellar cortex of many vertebrates shows a striking parasagittal compartmentation that is thought to play a role in the establishment and maintenance of functional cerebellar connectivity. Here, we demonstrate the existence of multiple parasagittal raphes of cells in the molecular layer of the developing cerebellar cortex of postnatal mouse. The histological appearance and immunostaining profile of the raphe cells suggest that they are migrating granule cells. We therefore conclude that the granule cell raphes previously described in birds also exist in a mammalian species. The raphes in mouse are visible on nuclear stains from around birth to postnatal day 6 and are frequently found at the boundaries of Purkinje cell segments that differentially express cadherins ("early-onset" parasagittal banding pattern). A similar relation between the raphe pattern and various markers for the early-onset banding pattern has been found in the chicken cerebellum. One of the cadherins mapped in the present study (OL-protocadherin) continues to be expressed in specific Purkinje cell segments until at least postnatal day 14. At this stage of development, the borders of the OL-protocadherin-positive Purkinje cell segments coincide with the borders of Purkinje cell segments that express zebrin II, a marker for the "late-onset" parasagittal banding pattern which persists in the adult cerebellum. These findings demonstrate that the early-onset banding pattern, as reflected in the complementary arrangement of raphes/Purkinje cell segments, and the late-onset pattern of zebrin II expression share at least some positional cues during development.     

Disturbed Purkinje cell migration due to reduced expression of Reelin by X-irradiation in developing rat cerebellum.

The major histogenetic events of the rat cerebellum take place in the early postnatal days. During this period, precursors of microneurons, such as granule cells, form the external granular layer (EGL), extend over the surface of the primordial cerebellum, and actively proliferate. Postmitotic granule cells leave the EOL and migrate to the internal granular layer (IGL). On the other hand, guided by radial glial fibers, immature Purkinje cells migrate from the ventricular zone of the fourth ventricle and settle in the Purkinje cell plate with thickness of several cells. Various cell adhesion molecules are involved in the interaction between the migratory immature Purkinje cells and processes of the radial glia as the basis for contact guidance. The second process is the formation of immature Purkinje cells to the monolayer. This process takes place at the first week after birth of the rat and cell adhesion molecules such as neural cell adhesion molecule (NCAM), fibronectin, tenascin and Reelin are also suggested to play an important role for the cell patterning. When rat fetuses are exposed to X-radiation in the last gestation period, abnormal foliation of the cerebellum develops with ectopic Purkinje cells. The molecular mechanism that contributes to abnormal migration of Purkinje cells and foliar malformation induced by X-irradiation in the cerebellum are not yet clear. This study was undertaken to elucidate the mechanisms of ectopic Purkinje cell formation by examining the expression of cell adhesion molecules.     

Somatotropin-releasing activity of rat hypothalamus with hypo- and hyperthyroidism

The somatotropin-releasing activity of rat hypothalamus was studied in the experiments in vitro incubating of adenohypophysis with the hypothalamic extracts or the whole hypothalamus and serotonin. The studied activity is shown to be increased in the thyroidectomized and hyperthyroid rats. Exogenic somatotropin prevents this increase. A conclusion is drawn that a link of hypothalamic adenohypophysotropic hormones of the regulatory system of the somatotropic function is not an area of the specific disturbances with hypo- and hyperthyroidism which might lead to hyposomatotropism.     

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.     

Platelet function in hypo- and hyperthyroidism

Alterations in cardiovascular hemodynamics are found in patients with hypofunction of the thyroid. Frank hypothyroidism favours the development of coronary heart disease. Platelets are thought to be important in atherosclerotic lesions initiation and progression. We studied platelet sensitivity to several agonists and to a physiological antagonist, prostacyclin, and thromboxane production in patients with hyperthyroidism and hypothyroidism. No statistically significant differences were found between platelets activities in patients and control subjects. We conclude that increased platelet function is not a direct indicator of susceptibility to cardiovascular diseases in patients with hypothyroidism whereas the hyperdynamic state, cardinal manifestation of thyrotoxicosis, is not associated with platelet activation.     

Characterization of separated cell types from developing rat cerebellum

The characteristics of [³H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [³H]GABA. In comparison with astrocytes, theV _max of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10⁶ cells) and the apparentK _t twofold greater (2 vs. 1 M). In contrast to these cell types, uptake was very low in granule cell-enriched preparations.cis-1,3-Aminocyclohexane carboxylic acid was a potent inhibitor of [³H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for -alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [³H]GABA transport was Na⁺ dependent in both cell classes. However, veratridine and ouabain selectively blocked [³H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [³H]GABA transport systems and the underlying metabolic processes.     

Direct imaging of in vivo neuronal migration in the developing cerebellum.

The upper rhombic lip (URL), a germinal zone in the dorsoanterior hindbrain, has long been known to be a source for neurons of the vertebrate cerebellum. It was thought to give rise to dorsally migrating granule cell precursors (Figure 1e); however, recent fate mapping studies have questioned the exclusive contributions of the URL to granule cells. By taking advantage of the clarity of the zebrafish embryo during the stages of brain morphogenesis, we have followed the fate of neuronal precursor cells generated within the upper rhombic lip directly. Combining a novel GFP labeling strategy with in vivo time-lapse imaging, we find, contrary to the former view, that most URL-descendants migrate anterior toward the midhindbrain boundary (MHB) and then course ventrally along the MHB (Figure 1f). As the migrating neuronal precursors reach the MHB, they form ventrally extending projections, likely axons, and continue ventral migration to settle outside of the cerebellum, in the region of the ventral brainstem. Thus, we define a new pathway for URL-derived neuronal precursor cells consistent with the recent fate maps. In addition, our results strongly suggest that the MHB plays a crucial role, not only in induction and patterning of the cerebellar anlage, but also in organizing its later morphogenesis by influencing cell migration.     

Alterations of contractility and sarcoplasmic reticulum function of rat heart in experimental hypo- and hyperthyroidism

Myocardial contractility and Ca2+-pump function of sarcoplasmic reticulum (SR) were studied on hearts of untreated, thyroidectomized and thyroxine-treated rats. In hypothyroid rats the contractile force, the maximum velocity of tension development and relaxation significantly decreased (by 73.2%, 68.2%; and 67.8%, respectively), while the time to peak tension was prolonged (by 25.9%) as compared with the control group. In hyperthyroidism opposite changes were found. Since the transport of calcium opposite changes were found. Since the transport of calcium by SR plays an important role in controlling contraction and, first of all, relaxation of muscle, function of the sarcoplasmic reticulum was also investigated under the above experimental conditions. In thyroidectomized rats the rate of Ca2+-uptake and Ca2+-activated ATPase activity of SR significantly decreased (by 31.7% and 61.0%, respectively), while Ca2+-binding remained unchanged. After thyroxine treatment both the Ca2+-uptake and binding capacity of SR were even decreased (by 25.6% and 12.9%, respectively), in spite of an increase in Ca2+-activated ATPase activity (by 67.3%). These changes in Ca2+ transport function of cardiac SR may only partially be responsible for the abnormalities in contraction and relaxation observed in hearts from hypo- and hyperthyroid rats.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Influence of hypo- and hyperthyroidism on the turnover rate of noradrenaline, dopamine and serotonin in various rat cerebral structures]

The effect of chronic treatment with tyroxine (T4) or propylthiouracile (PTU) on the turnover of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) has been studied in various areas of the rat brain (brain stem, hypothalamus, striatum and "rest of the brain"). The turnover of NE and DA was determined by the decay in endogenous levels after inhibition of tyrosine hydroxylase by alpha-methylparatyrosine and the turnover of 5-HT was evaluated by the initial accumulation of endogenous 5-HT after inhibition of monoamine oxydase by pargyline. T4 treatment accelerated the release of DA from the striatum but had no significant effects on NA release in the various cerebral areas : nevertheless the NE endogenous level was significantly reduced in the brain stem. PTU treatment delayed the release of DA and NA only from the "rest of the brain". Concerning 5-HT, the only significant variation was observed in the hypothalamus of PTU-treated rats and implied increased turnover. The possible relations between the changes in cerebral monoamines turnover and the behavioural alterations which are observed in thyroid disfunction are discussed.     

Epithelial cell proliferation and migration in the developing rat gastric mucosa

To investigate cell proliferation in developing gastric antrum and fundus, proliferating gastric epithelial cells were labeled in fetal rats by intravenously injecting mothers with [³H]thymidine. In addition 14-day postnatal (dPN) rats were given [³H]thymidine intraperitoneally. Tissue was removed 1.5 hr later, and autoradiographs were prepared to identify proliferating cells. Total epithelial labeling indices (L.I.) reached a peak at 20 days gestation (dG), coincident with the appearance of pit/glands, then declined again by 22 dG (gestation end). At 18 dG, labeled cells were distributed throughout all levels of the stratified epithelia. Between 20 and 22 dG, as pit/gland development proceeded, labeled cells became concentrated in the gland bases and were only rarely seen on the surface (L.I. of surface cells <1% at 22 dG). By 14 dPN, proliferating cells were entirely absent from the epithelial surface. Approximately 15% of the endocrine cells were labeled at 22 dG, compared to only 2% at 14 dPN; zymogen cells were labeled (～6%) at 14 dPN; parietal cells did not label at any age studied. In addition, cell migration to the epithelial surface was studied in rats labeled at 22 dG, 14 dPN, or 28 dPN, and killed 1–20 days later. Migration times were slightly shorter in the 28 dPN group and were longer in fundus than antrum in all groups.     

Tyrosine content, influx and accumulation rate, and catecholamine biosynthesis measured in vivo, in the central nervous system and in peripheral organs of the young rat. Influence of neonatal hypo- and hyperthyroidism

The influence of neonatal hypo- and hyperthyroidism on different aspects of tyrosine metabolism in the hypothalamus, striatum, brainstem, adrenal glands, heart and brown adipose tissue (BAT) were studied in 14-day old rats. The synthesis rate of catecholamines (CA) was also determined in vivo after the injection of labelled tyrosine. Hypothyroidism increases tyrosinaemia and endogenous tyrosine concentration in the hypothalamus and BAT. Hyperthyroidism decreases tyrosinaemia and endogenous tyrosine levels in the striatum, adrenals and heart. The accumulation rate of tyrosine determined 30 min after an intravenous injection of the labelled amino acid has been determined in the organs, together with the influx of the amino acid, determined within 20s. Hypothyroidism increases tyrosine accumulation rate in all the organs studied, and tyrosine clearance is decreased in the striatum and brainstem; together with an increased tyrosinaemia, this leads to a normal influx. The influx of tyrosine is increased in the hypothalamus. Hyperthyroidism decreases tyrosine accumulation rate in all the organs except the adrenals. These results indicate that the thyroid status of the young rat can influence tyrosine uptake mechanisms, without modifying an organ's tyrosine content. The fact that hypothyroidism increases tyrosine influx in the hypothalamus without modifying it in the brainstem and striatum reflects an heterogeneous reactivity to the lack of thyroid hormones in different brain structures. Neonatal hypothyroidism decreases the CA synthesis rate in the striatum, the heart and the interscapular brown adipose tissue, while synthesis was enhanced in the brainstem and the adrenals. It is likely that these variations in CA synthesis are due to thyroid hormone modulation of tyrosine hydroxylase activity, the enzyme which catalyses the rate limiting step in CA biosynthesis.     

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.     

Effect of hypo- and hyperthyroid states on phospholipid composition in developing rat heart

The aim of this study was to examine the effect of hypo- and hyperthyroidism on the phospholipid composition in developing rat heart. The hypothyroid state (PTU) was induced by 0.05% 6-n-propyl-2-thiouracil in drinking water given to nursing mothers from the postnatal day 2–21. The hyperthyroidism (T₃) was made by daily injection of 3,3,5-triiodo-L-thyronine (10 g/100 g body wt) to newborns in the same time period. Age matched intact littermates were taken as euthyroid controls. PTU decreased the concentration of total phospholipids (PL), choline phosphoglycerides (PC), ethanolamine phosphoglycerides (PE) and diphosphatidylglycerol (DPG) and increased the proportion of plasmalogen component of PE (PLPE). T₃ increased the concentration of PL, PC, PE, DPG and decreased PLPE in comparison with euthyroid controls. The ratio of saturated/unsaturated fatty acids (FA) in PE was decreased in PTU and increased in T₃ group. The ratio of n-6/n-3 polyunsaturated FA in PC, PE and phosphatidylinositol (PI) was increased in PTU due to increase of 18:2n-6 and decrease of 22:6n-3 proportion. T₃ decreased this ratio because of decline in 20:4n-6 and rise in 22:6n-3 proportion. Both hypo- and hyperthyroidism decreased the ratio of 20:4n-6/18:2n-6 in the majority of phospholipids. PTU decreased the unsaturation index in PC, PI and phosphatidylserine. It is concluded that thyroid state plays an essential role in the development of membrane phospholipid components in cardiac membranes during the early postnatal period.     

Expression of osteopontin mRNA in developing rat brainstem and cerebellum

This study was undertaken to investigate the developmental expression of osteopontin (OPN) in the rat brainstem and cerebellum by Northern blotting and in situ hybridization. The expression of OPN was noted in the mesencephalic Vth nucleus initially at embryonic day 16 (E16). At E20, the labeling extended into other brainstem nuclei including the cochlear, vestibular, facial motor, and hypoglossal nuclei. During the first week of postnatal life, the OPN signal in the brainstem increased markedly, and by P14, OPN expression was found in functionally diverse areas including motor-related areas, sensory relay nuclei, and the reticular formation. The adult labeling pattern was established in central neurons at this time. These results corresponded well with those from Northern blot analysis. On the basis of morphological and distribution criteria, the OPN signal in several nuclei appeared to be contained exclusively within neuronal soma. OPN expression in neurons occurred during the period of neuronal differentiation and increased with maturation. Our results therefore suggest that OPN contributes to developmental processes, including the differentiation and maturation of specific neuronal populations, in the rat brain.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Brown adipose tissue cell respiration in hypo- and hyperthyroidism after stimulation with selective and non selective beta-adrenergic agonists.

Brown adipocyte respiration was measured in isolated cells from hypothyroid, hyperthyroid and euthyroid Sprague-Dawley male rats. Hypothyroidism was induced by providing drinking water containing methimazole and hyperthyroidism was induced by addition of thyroid powder to the diet. Brown adipose tissue (BAT) cells were isolated by collagenase digestion and oxygen consumption (VO2) was measured by Clark type oxygen electrodes. BAT cell respiration was stimulated by selective and nonselective beta-adrenergic agonists: BRL 35135A (BRL) and Isoprenaline (ISO). Basal BAT cells respiration did not differ according to thyroid status. Maximal VO2 responses of BAT adipocytes from hypothyroid rats were significantly lower than in euthyroidism after ISO and BRL. The reduced response was more marked for ISO than for BRL. The thermogenic sensitivity was significantly greater in euthyroid than is hypothyroid cells for ISO, but not for BRL. The euthyroid-hyperthyroid differences were not significantly different. These results suggest: basal respiration of BAT cells in hypo- and hyperthyroidism does not reflect the overall changes in whole body metabolism; the decreased thermogenic response in hypothyroidism might be due to decreased beta-adrenoceptor numbers and/or decreased intracellular thyroxine-triiodothyronine conversion; changes in sensitivity to ISO and BRL in vitro reflect the changes seen in VO2 in vivo.