Ultrastructural identification of oligodendrocyte/myelin proteins in corpus callosum of hypothyroid animals

Thyroid hormone (T3) deficiency impairs the development of the CNS, particularly myelination. We have previously described an increase in the frequency of morphological abnormalities in the central myelin sheath in a hypothyroidism model, which reinforced the hypothesis of a role for T3 in myelin compaction. However, there are no data concerning the cellular distribution of myelin proteins in hypothyroid animals. In the present work, we describe the distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), myelin basic protein (MBP) and proteolipid protein (PLP) throughout the central myelin sheath of a hypothyroidism model. We used euthyroid and hypothyroid adult rats at 90 days of age. In order to induce hypothyroid status, animals received 0.02% methimazol from the 19th gestation day onwards. After perfusion with a fixative mixture, small pieces of corpus callosum were obtained, dehydrated and embedded in LR White resin. Ultrathin sections were immunoreacted, using specific antibodies revealed by a secondary antibody coupled to colloidal gold particles of 10 nm. Gold particle density per region of myelin sheath for each one of these proteins was obtained. In normal animals, CNPase, PLP and MBP were identified in sites that had already been described in previous studies. In hypothyroid animals, CNPase was identified in the region corresponding to compact lamellae, which normally does not contain this protein, while, in this same region, PLP and MBP immunolabeling were decreased. These results suggest that thyroid hormone deficiency impairs the distribution of the major oligodendrocyte/myelin markers. This effect may justify the reduction in myelin sheath compaction previously demonstrated in a similar model of hypothyroidism.     

Selective effect of hypothyroidism on expression of myelin markers during development

Thyroid hormones are critical for maturation of the central nervous system. In a previous study, we showed a change in the pattern of mature myelinated nerve fibers by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in developing hypothyroid animals, which suggests a possible role for thyroid hormones in myelin compaction. The classical myelin markers myelin basic protein (MBP) and proteolipidic protein (PLP) are expressed later in oligodendroglial development, when myelin sheath formation is in progress. A myelin constituent designated myelin-associated/oligodendrocytic basic protein (MOBP) has been identified and related to myelin compaction. We assessed the developmental sequence of appearance of CNPase, MBP, MOPB, and PLP proteins in cerebellum (Cb) and corpus callosum (cc) in an experimental hypothyroidism model. The appearance of both MOBP isoforms occurred at postnatal day (P)25 and P30 in cc and Cb, respectively, followed by an increase with age in the control group. However, all the MOBP isoforms were weakly detectable in both regions at P30 from the hypothyroid (H) group, and the higher molecular weight isoform remains decreased in cc, even at P90. The developmental pattern of expression of CNPase, MBP, and PLP proteins was also delayed in the H group. CNPase and MBP expression was recovered in cc and Cb, whereas PLP remained below control levels at P90 in cc. Our data show that the experimental hypothyroidism affects the developmental pattern of the oligodendrocytic/myelin markers. Furthermore, thyroid hormone may modulate specific genes, as demonstrated by permanent down-regulation of MOBP and PLP expression in adulthood.     

Cognin distribution during differentiation of embryonic chick retinal cells in vitro

Differentiation of individual retina neurons is closely linked to development of retina function. This differentiation may be intrinsic to the cell or determined by the position of the cell within the developing tissue. Retina cognin, a cell-cell recognition protein, which may itself mediate positiondependent cell interactions in vivo exhibits a characteristic change in distribution during embryonic chick development. Cognin is progressively lost from the outer retina in a manner which appears position-dependent. ¹⁰ We asked if this change in cognin distribution was actually position-dependent or intrinsic to the retina cells. Neural retina cells from 8-day-old chick embryos were cultured in vitro. Continued differentiation of the cultured cells was demonstrated by neurite outgrowth and characteristic increases in choline acetyltransferase and glutamic acid decarboxylase activity. In such cultures, the characteristic developmentally related disappearance of retina cognin occurred as in vivo. This indicated that this aspect of retina neuronal differentiation was independent of position within the tissue and likely intrinsic to individual cells after 8 days of embryonic development.     

Gonadotrophin-releasing hormone and oxytocin secretion from the hypothalamus in vitro during pro-oestrus: the effects of time of day and melatonin

An accurately timed surge of luteinizing hormone (LH), during the second half of the day of pro-oestrus in rats, is a crucial part of the endocrine signal that leads to expulsion of an ovum from an ovarian follicle. LH release is partly controlled by a number of peptides, including gonadotrophin-releasing hormone (GnRH) and oxytocin, which travel from the hypothalamus to the pituitary. The profile of secretion of these peptides is poorly understood. Therefore, the amounts of GnRH and oxytocin that were secreted from hypothalamic explants were determined at several time points during the day of pro-oestrus. Basal secretion of oxytocin from hypothalami taken later in pro-oestrus was greater than from hypothalami taken earlier in the day (p < 0.02). On the other hand, basal secretion of GnRH decreased during the day of pro-oestrus (p < 0.03). The different trends of GnRH and oxytocin secretion reveal that their secretion is regulated by distinct mechanisms. GnRH secretion was higher at midpro-oestrus than late in the day (p < 0.05) consistent with a peak of GnRH having been observed by others in portal blood in the second half of the day of pro-oestrus. Responsiveness of oxytocin to stimulation by K⁺ of the hypothalami declined from the early light hours to the evening dark hours (p < 0.02). Thus, oxytocin modulation might be achieved partly by modification of intracellular processes. Melatonin, secreted during hours of darkness, is frequently involved in modulating time-dependent events in mammals, but its contribution to peptide regulation during the ovulatory cycle is unclear. Melatonin was observed to inhibit basal oxytocin secretion from hypothalami collected during light hours (p < 0.05). The investigation has, therefore, revealed the potential for melatonin to modulate peptide secretion from the hypothalamus during the day of pro-oestrus. We also observed that secretion from the hypothalamus of the two LH-regulating peptides, GnRH and oxytocin, are differently regulated during the day of pro-oestrus.     

Expression of acetylcholinesterase gene during in vitro differentiation of rabbit muscle satellite cells

We investigated the myogenic properties and the expression of acetylcholinesterase (AChE) in culture of satellite cells (SCs) isolated from slow and fast rabbit muscles. Slow SCs form myotubes more rapidly (day 9 vs day 11) than fast SCs, and differentiate further into striated and contractile fibers. AChE activity and mRNA expression are higher in SCs cultured from slow than from fast muscles, as also observed in the muscles themselves. However, the two types of SC cultures do not show obvious difference in their patterns of AChE molecular forms. Taken together, these preliminary data support the view that there might be more than one SC population in skeletal muscles.     

Regulation of cytoskeletal proteins by thyroid hormone during neuronal maturation and differentiation

Primary cultures of neurons from 16- to 17-day-old embryonic rat cerebra were maintained for 3 weeks in thyroid hormone deficient (TH_def) and thyroid hormone supplemented (TH_sup) media to investigate how TH regulates the cytoskeletal (CSK) proteins during neuronal differentiation and maturation. Two distinct phases of regulation of triton-insoluble CSK-proteins by TH were discernible – an early phase (days 4–8 of culture) when TH-deficiency resulted in down-regulation of these proteins and a late phase (days 16–20) involving up-regulation of these proteins. In contrast, the triton-soluble non-CSK proteins always remained up-regulated by TH. The two main effects of TH-deficiency were retarded neurite outgrowth and altered neuronal morphology. Of all the CSK proteins, actin was found to be predominantly sensitive. Alterations in the level of CSK actin during neuronal maturation were found to be parallel to changes in steady-state level of actin mRNA as well as actin synthesis. However, these TH-induced changes (up-regulation of actin during the early phase and down-regulation during the late phase) did not lead to parallel changes in the level of soluble G-actin which was comparable at both days 8 and 16 in TH_def and TH_sup cultures. Quantitation of different forms of intracellular actin revealed that G-actin level declined by about 50% between days 8 and 16. In the case of TH_sup neurons, this reduction in G-actin was accompanied by a parallel increase in the non-CSK F-actin, whereas TH-deficiency resulted in a corresponding increase in CSK F-actin during the terminal differentiation of neurons. Thus TH regulates the biogenesis of CSK-proteins with a predominant effect on actin and the transformation of G-actin into non-CSK F-actin appears to be the key step in neuronal maturation which is affected by hypothyroidism.     

Delayed development of GFA immunoreactivity in the parietal cortex during thyroid hormone deficiency

The influence of neonatal hypothyroidism on the development of immunoreactivity to glial fibrillary acidic protein (GFA) was studied in parietal cortex of rats treated from birth with the antithyroid agent propylthiouracil (PTU) for 3 or 8 weeks. Density of GFA immunoreactivity was evaluated in cryostate sections reacted with an antiserum specific for GFA. Three weeks postnatally, the density of GFA-immunoreactive structures in the cortical layers II-V was 70% lower in PTU-treated animals than in controls injected with the solvent. This marked difference between the groups was, however, not seen in either the molecular layer, layer VI or white matter. The inhibited development of GFA immunoreactivity was not persistent in animals treated with PTU for 8 weeks continuously. Plasma from animals treated with PTU for 1,2,3 and 8 weeks was collected and the TSH level in each group compared with samples from age-matched controls and newborn pups. The treatment with PTU resulted in a more than 10-fold increase in TSH level after 1 week of injections. In longterm groups of 8 weeks, the TSH level decreased in the PTU-treated animals, but stayed considerably higher than control values throughout the experiments.The results described in the present paper indicate a thyroid hormone dependent development of the GFA immunoreactivity in cortex cerebri astrocytes.     

Enhanced survival and differentiation in vitro of different neuronal populations by some interleukins

Data from the literature demonstrate the existence of a growing family of neuropoietic cytokines; members of this group have structural motifs in common with other members and with neurotrophic factors. In this research we studied the responses elicited in vitro by some of these molecules in two different neuronal populations: murine neuroblastoma N18TG2 and neurons from chicken dorsal root ganglia. Both IL-2 and IL-6 improve the survival of murine neuroblastoma cells in clonal density plating experiments; in addition IL-2 significantly inhibits thymidine incorporation by single cell suspension. The survival of sensory neurons, on the other hand, non-responsive to IL-2 and IL-6, was significantly supported by IL-3, which also stimulates their morphological differentiation, inducing the formation of a well-developed neural net. In conclusion, results reported here confirm the neurotrophic activity of some ILs and provide additional neuronal models for future investigations.     

Regional differences in in vitro growth of neural cell processes during development

Primary cell cultures from cerebral cortex, striatum and ventral mesencephalon obtained from rat fetal (embryonic day 17, E17) or postnatal (day 2, PN2) donors were grown either in media conditioned by subcultured astroglia from the same regions, an artificial trophic medium, normal human amniotic fluid, or in normal human cerebrospinal fluid. To estimate the presence of neuronal-like and non-neuronal cells, cell morphology and immunocytochemistry against microtubule-associated proteins and β-tubulin were taken into consideration. The percentage of emitting neural cells and length of cell processes were determined after 24 hr in culture. Growth of cell processes in neuronal and non-neuronal cells from prenatal striatum was minimal compared with that in cerebral cortex and ventral mesencephalon, regardless of the culture condition. Nerve growth factor, basic fibroblast growth factor or epidermal growth factor did not significantly modify cell growth in E17 cultures, except for epidermal growth factor, which reduced the number of emitting cells in striatal cultures and increased it in cerebral cortex ones. Cultures derived from postnatal striatum showed a significant increase in neurite length when grown in an astroglial conditioned medium as compared to cultures derived from prenatal (E17) striatum. Results suggest significant regional differences in the brain regarding growth of cell processes at age E17, and reversal of striatal ability to grow cell processes by postnatal day 2. Reduced growth of cell processes showed by E17 striatum cultures was rather independent of the culture media. This fact could suggest that such early regional differences would depend on characteristics of sublineages present at this developmental stage, which would modulate the organization of regional neuropils. The restricted growth of cell processes in cultures from E17 striatum, no longer present in postnatal striatum, suggests that inputs to the striatum may modify expression of cell lineages at later stages of development.     

Delayed development of GFA immunoreactivity in intraocular cortex cerebri grafts during thyroid hormone deficiency

Dependency of astrocytes on thyroid hormones during development was studied in intraocular cortex cerebri grafts, using antibodies to the glial fibrillary acidic protein (GFA). Fetal cortex cerebri was left in the anterior eye chamber of normal and thyroidectomized adult recipient rats 3 and 8 weeks to mature. All cortex grafts survived and became vascularized in the eye. The grafts were cryostat-sectioned, and reacted with an antiserum specific for GFA. The density of GFA immunoreactivity in the cortex graft neuropil was subjectively estimated with fluorescence microscopy. After 3 weeks in oculo, the number of positive structures was significantly lower in the thyroidectomized group as compared to the euthyroid control group. This effect of thyroidectomy on the cortex grafts was entirely counteracted by daily injections of thyroxine (100 μg/kg s.c.) to similarly thyroidectomized recipients. The distribution of GFA-positive structures was even within individual grafts of all three groups. Neither were any differences in morphology of fluorescent astroglial processes observed. The overall density of fluorescent GFA-positive structures was considerably higher than that found in age-matched normal in situ counterparts, even in the thyroidectomized group. After 8 weeks in oculo, the difference between the control and thyroidectomized groups in density of GFA-positive structures in the neuropil had disappeared, and no general increase in density of immunoreactivity was noted in the control group. These results indicate that thyroid hormones might normally play a role for the development of GFA in the early postnatal brain, as thyroid hormone deficiency causes a transient delay in development of GFA immunoreactivity in intraocular cortex cerebri grafts.     

The expression of concanavalin a binding glycoproteins during the development of cerebellar granule neurons in vitro

We present data on the expression of Concanavalin A (ConA) binding glycoproteins by granule cell enriched cultures derived from 8 day postnatal rat cerebellum. Time course studies were conducted over a 12 day culture period. ConA binding glycoproteins were localized on the cell bodies and fibres of the granule neurons using fluorescence microscopy. The fluorescence intensity increased between 4 and 12 days in vitro. Quantitative studies on the capacity of live cells to bind ¹²⁵I-iodinated ConA showed that there was a significant increase in the amount of lectin bound between 4 and 8 days in vitro. However, in contrast to previous results on the developing cerebellum in vivo [Zanetta et al. (1978) Brain Res.142, 301–319.], there was no decline in binding capacity after 8 days in culture. Glycoproteins expressed by these cells were analysed by staining SDS polyacrylamide gels with [¹²⁵I]ConA. A large number of lectin binding proteins were observed which spread over a wide range of molecular weights. Only minor changes were detected in the profile of [¹²⁵I]ConA binding glycoproteins with the maturation of the cells in culture. The comparison of the findings on granule cells developing in culture and in vivo suggested that an interaction between granule cell axons and their normal target neurons is involved in the regulation of the ConA binding protein content in the cerebellum.     

Deprenyl stimulates the efflux of monoamines from the rat hypothalamus in vitro

The direct effects of L-deprenyl, a monoamine oxidase inhibitor, on the hypothalamus of male Sprague-Dawley rats was investigated by measuring the efflux of norepinephrine (NE), dopamine (DA), serotonin (5-HT), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) using a combination of high performance liquid chromatography with electrochemical detection and an in vitro incubation system. After measuring basal efflux by incubating the hypothalami with Krebs-Ringers Henseleit (KRH) alone during the first incubation period, hypothalami were incubated either with the medium, KRH alone (0 mM), or KRH containing 0.1, 1, and 10 mM L-deprenyl. During the third incubation period, hypothalami were again incubated with KRH alone to measure the residual effects if any. During the final incubation period, the hypothalami were stimulated with high K⁺ KRH. Deprenyl produced a dose-dependent increase in the efflux of NE, DA, and 5-HT from the hypothalami. Neurotransmitter efflux returned to pretreatment levels when L-deprenyl was removed from the medium. In contrast to NE, DA, and 5-HT, the efflux of the metabolites DOPAC and 5-HIAA was inhibited in a dose-dependent fashion after incubation with L-deprenyl. Results from this study demonstrate that L-deprenyl is capable of stimulating the efflux of neurotransmitters in vitro by a direct action on the hypothalamus.     

In vitro induction and in vivo expression of bcl-2 in the hNT neurons

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.     

Neurophysiological and biochemical properties of the goldfish optic tectum maintained in vitro

The physiology and biochemistry of the in vitro goldfish optic tectum are examined. Following surgical removal of the intact optic tectum by severing its connectives, the tectal explant is maintained in a brain slice chamber in a specified medium for up to 24 hours. Electrophysiological studies show that the in vitro tectum displays the same response properties to optic nerve stimulation as does an in vivo tectum. Concurrent biochemical studies that RNA and protein synthesis is actively maintained for at least 17 hrs in vitro. Thus both biochemical and electrophysiological measurements indicate that the integrity of the tectal explant can be maintained in vitro making it a suitable model system for investigations of a variety of neurobiological questions.     

A role for the MAPK/ERK pathway in oligodendroglial differentiation in vitro: stage specific effects on cell branching

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. Oligodendroglial cells treated with MEK inhibitor were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2′3′nucleotide-cyclic 3′phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.     

Effect of growth factors on the in vitro growth and differentiation of early and late passage C6 glioma cells

The effect of different hormones and growth factors was assayed on the in vitro growth and enzymatic activities of 2′,3′-cyclic nucleotide 3′phosphohydrolase (CNP) and glutamine synthetase (GS) of rat glioma C6 cells at two different passages in culture. Young cultures (passage 26), mainly oligodendrocytic, and older cultures (passage 134), predominantly astrocytic, were treated with 10 μM dexamethasone, 20 ng/ml transforming growth factor alpha (TGFα), 10 ng/ml insulin, 20 ng/ml platelet-derived growth factor (PDGF), and 20 ng/ml, epidermal growth factor (EGF) in serum-free chemically defined media. In vitro growth rate was measured in terms of DNA content, by a fluorometric method of diaminobenzoic acid, and rate of DNA synthesis by ³H-thymidine incorporation. CNP activity (marker for in vitro oligodendrocytes) and GS activity (marker for astrocytes) were determined spectrophotometrically. Dexamethasone reversibly and significantly inhibited growth of C6 glioma in early and late passages. PDGF and insulin promoted in vitro growth only in late passage but not in early passage cells, whereas EGF and TGFα did not significantly affect growth. An increase in CNP activity was observed in early passage cells under the effect of PDGF and insulin. The increase in GS activity induced by insulin and dexamethasone suggests a differentiating role for these factors in C6 glioma cells. These results further present the C6 glioma cell line as a useful model for studies on glial cell properties and responsiveness in culture and support its use in experimental aging in vitro.     

Anti-aggregation and fibril-destabilizing effects of sex hormones on α-synuclein fibrils in vitro

The α-synuclein aggregation in the brain is the hallmark of Lewy body diseases, including Parkinson's disease and dementia with Lewy bodies, and multiple system atrophy. Some epidemiological studies have revealed that estrogen therapy reduces the risk of Parkinson's disease in females. We examined the effects of estriol, estradiol, estrone, androstenedione, and testosterone on the formation and destabilization of α-synuclein fibrils at pH 7.5 and 37 °C in vitro, using fluorescence spectroscopy with thioflavin S and electron microscopy. These sex hormones, especially estriol, significantly exert anti-aggregation and fibril-destabilizing effects; and hence, could be valuable preventive and therapeutic agents for α-synucleinopathies.     

The distribution of melanin-concentrating hormone in the monkey brain (Cebus apella)

Melanin-concentrating hormone was identified in the brain of Cebus monkey using immunohistochemical and in situ hybridization. MCH-immunoreactive neurons were found in the lateral hypothalamus and dorsolateral zona incerta. MCH-ir fibers were seen in the medial mammillary nucleus, and in the median eminence, and very few fibers in the globus pallidus. This is the first report describing the MCH-ir cell and fiber distribution in the monkey brain.