Penha C. Barradas

Alterations in metabolism and gap junction expression may determine the role of astrocytes as “good samaritans” or executioners

Our knowledge of astroglia and their physiological and pathophysiological role(s) in the central nervous system (CNS) has grown during the past decade, revealing a complex picture. It is becoming increasingly clear that glia play a significant role in the homeostasis and function of the CNS and that neurons should no longer be considered the only cell type that responds, both rapidly and slowly, to electrochemical activity. We discuss recent advances in the field with an emphasis on the impact of hypoxia and ischemia on astrocytic metabolism and the functional relationship between glucose metabolism and gap junctions in astrocytes. We also address the controversy over whether astrocytic gap junctions mediate protection or killing of neurons during or after hypoxic or ischemic insults.

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.

Thyroid hormone deficiency changes the distribution of oligodendrocyte/myelin markers during oligodendroglial differentiation in vitro

Myelination depends on the proper differentiation of oligodendrocytes and several factors may influence this event. For instance, thyroid hormone (T3) affects the timing of differentiation and regulates the expression of several enzymes involved in the synthesis of complex lipids and in the expression of some myelin structural proteins. We investigated the effect of T3 deficiency on oligodendroglial differentiation and in the distribution of oligodendrocyte/myelin proteins 2′3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and myelin basic protein (MBP). Oligodendroglial‐enriched cultures were obtained from cerebra of neonate rats grown in a modified medium. The T3‐deficient status was induced by using medium devoid of T3. We observed a delay, in T3‐deficient cultures, in oligodendroglial maturation characterized by less extensive processes and membrane vellum than in controls. In control cultures, CNPase immunoreactivity was punctated, showing cell bodies and processes at earlier stages and redistribution to cytoskeleton vein‐like structures in later stages. In T3‐deficient cultures, CNPase remained in a punctated pattern and only at 10 days in vitro we observed CNPase redistribution to the presumptive cytoskeleton vein‐like structures. MBP in control cultures was distributed through the whole cell body and processes whereas in T3‐deficient cultures, MBP immunoreactivity was concentrated in the perinuclear region. These results reinforce the hypothesis that T3 is an important factor in oligodendrocyte differentiation, particularly regarding the distribution of myelin proteins.

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. J. Neurosci. Res. 66:254–261, 2001.

Effects of experimental hypothyroidism on myelin sheath structural organization

A previous study using the 2′3′cyclic nucleotide 3′phosphodiesterase (CNPase), an oligodendroglial marker that also stain ensheathed fibers, showed a decrease in the number of immunoreactive fibers and a change in the pattern of CNPase immunoreactivity (CNPase+) in hypothyroid animals. CNPase+ fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls. As CNPase, in adult animals, can be found only in regions in which oligodendrocyte cytoplasm remains as internal, external and paranodal loops, it was suggested that the reduction of thyroid hormone levels leads to a failure in myelin compaction. Previous data showed a higher frequency of some abnormalities in myelin sheath as multiple cytoplasmic loops and redundant myelin profiles in mutant animals that present a failure in myelin compaction. The increase in the frequency of these abnormalities (multiple internal and external loops and redundant myelin) indicates a failure in the interrelations between the axons and the oligodendroglial processes. To verify if the thyroid hormone deficiency during CNS development disturbs these interrelations, we evaluated the frequency of the morphological abnormalities (multiple internal and external loops and redundant myelin) in myelin sheath of corpus callosum (cc) in experimental hypothyroidism. Randomic fields were kept by electron microscopy and the analysis of the frequency of morphological abnormalities showed a significant difference in hypothyroid animals at 60-day-old (PND60), with no significant differences at 90-day-old (PND90) animals. The frequency of multiple internal loops is higher in hypothyroid animals at PND60 that indicates a disturbance in the wrapping by the oligodendroglial process. These findings showed that thyroid hormone might modulate the axon-oligodendroglial relationships that are important for the adequate temporal sequence of events that occur during myelinogenesis, with possible consequences on myelin compaction.

2'3'Cyclic nucleotide 3'phosphodiesterase immunohistochemistry shows an impairment on myelin compaction in hypothyroid rats.

The effects of hypothyroidism on oligodendroglial differentiation and myelination are for the first time studied by immunohistochemical localization of an early oligodendroglial marker, the 2′3′cyclic nucleotide 3′phosphodiesterase (E.C. 3.1.4.37‐CNPase), in developing rats. Two groups received methimazol; one during gestation (H) and another postnatally (PN). One H sub‐group received thyroxine after birth (T). We observed a delay in CNPase expression followed by a decrease in the number of CNPase immunoreactive fibers in both H and PN groups. The T sub‐group was not different from controls. Furthermore, the immunoreactive fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls.

Undernutrition during early life alters neuropeptide Y distribution along the arcuate/paraventricular pathway.

Perinatal nutrient restriction exerts profound influences on brain development. Animals that suffer undernutrition during lactation also display impaired weight gain. Feeding behavior is mainly modulated by neural and hormonal inputs to the hypothalamus. The arcuate-paraventricular neuropeptidergic Y pathway has a prominent role in appetite regulation. The aim of this work was to study the effects of protein undernutrition during lactation on this hypothalamic pathway. We used rats from 5 to 60 postnatal (P) days whose dams were fed a 0% protein diet (PFG) or a normoprotein diet (CG) from P1 to P10. To reproduce the same amount of calorie ingested by the PFG we used an underfed group (UFG). Immunohistochemistry was performed to assess neuropeptide Y (NPY) distribution in the arcuate, periventricular and paraventricular nuclei. Our results showed a NPY immunostaining peak at P10 in all nuclei in CG animals. In UFG animals this peak was observed by P15, while, in the PFG animals only by P20. Our results suggest that the neuropeptidergic arcuate-paraventricular pathway suffered a delay in NPY distribution in undernourished animals, particularly those fed a 0% protein diet, reflecting an effect on this pathway maturation that could explain previously reported alterations on feeding behavior in these animals.

Myelin basic protein accumulation is impaired in a model of protein deficiency during development

Abstract During the development of the central nervous system (CNS) there is a great possibility of permanent effects in consequence of environmental disturbances. Nutritional deficiency is one of the factors that impair the normal CNS formation. In general, the protein deficiency evokes, beyond the damages in the maturation of nervous system, several consequences in body growth, biochemical maturation, motor function and the major cognitive functions. These effects were observed in undernourished children all over the world. Even in a restricted period, the malnutrition status may evoke permanent impairments in feeding behavior and in metabolism. Rats submitted to malnutrition during development, showed a marked decrease in the number of myelinated fibers. This condition may reflect a failure in the beginning of the wrapping of axons by oligodendroglial processes and/or a delay in the myelin synthesis. Myelin basic protein (MBP) is an intracellular oligodendrocyte protein that is directly related to the formation of the myelin sheath. In this study we verified the temporal pattern of MBP expression, by immunohistochemical and immunoblotting analyses, in a model of protein malnutrition induced during the first half of the lactation period. We showed that MBP expression was impaired in our malnutrition model and that some of the effects were maintained in adulthood, with possible consequences in the maturation of myelin sheath.

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.

Expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in the developing olfactory bulb and subventricular zone rostral extension.

The olfactory bulb (OB) presents a unique pattern of permanent acquisition of primary afferents and interneurons, but not much detail is known about the differentiation of its oligodendroglial cells. We studied the expression of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase), a protein related to axonal ensheathment by myelinating cells. Expression of CNPase in OB follows a general caudorostral gradient, with the exception of the glomerular layer (GL). At postnatal day 5–6 (P5–P6), the first CNPase+ profiles appeared in the dorsal lateral olfactory tract adjacent to the accessory OB (AOB), followed by rare cell bodies and processes in AOB internal plexiform layer at P7. At P9, the main OB (MOB) granular cell layer (GrCL) already showed intensely stained CNPase+ processes. From P5 to P12, small numbers of CNPase+ cells were found in the subventricular zone (SVZ), throughout its rostral extension (SVZ‐RE), and in the intrabulbar subependymal layer. The appearance of CNPase+ profiles delimiting glomeruli started in the GL rostralmost region at P12, extending to all GL levels, but glomeruli remained open caudally at P15. At P18, oligodendroglial glomeruli were evident throughout OB, but the adult pattern was established only after P30. There was no age‐related loss of CNPase immunoreactivity in glial cell bodies, possibly indicating de novo ensheathment of neurites. Our results show an earlier onset of oligodendroglial differentiation in OB than previously reported and a rostrocaudal gradient of formation of oligodendroglial glomeruli. They also raise the possibility that a minor fraction of OB oligodendrocytes might derive from the SVZ‐RE.