Cyclic Nucleotide Phosphohydrolase Research Articles

Postmortem and imaging based analyses reveal CNS decreased myelination in restless legs syndrome

Restless legs syndrome (RLS) is a neurological disorder characterized by a strong urge to move the legs and has been shown in many studies with abnormally low brain iron. Iron deficiency is associated with hypomyelination in brains of animals. Therefore we hypothesized that a myelin deficit should be present in the brains of patients with RLS. We performed Western blot analysis on myelin isolated from RLS (n=11) and control (n=11) brain tissue obtained at autopsy for the expression of the integral myelin proteins, myelin basic protein (MBP), and proteolipid protein (PLP) and the oligodendrocyte specific enzyme 3'5'-cyclic nucleotide phosphohydrolase (CNPase). To expand the postmortem findings to in vivo, we analyzed the brains of RLS patients (n=23) and controls (n=23) using voxel-based morphometry (VBM). The expression of MBP, PLP and CNPase in the myelin from RLS was decreased by approximately 25% (p<0.05) compared to controls. The amounts of transferrin (Tf) and H-ferritin (H-Frt) in the myelin fraction were also significantly decreased in RLS compared to controls. The imaging analysis revealed significant small decreases in white matter volume in RLS patients compared to controls in the corpus callosum, anterior cingulum and precentral gyrus. A decrease in myelin similar to that reported in animal models of iron deficiency was found in the brains of individuals with RLS. The evidence for less myelin and loss of myelin integrity in RLS brains, coupled with decreased ferritin and transferrin in the myelin fractions, is a compelling argument for brain iron insufficiency in RLS. These data also indicate the need to look beyond the sensorimotor symptoms that typically define the syndrome and its assumed relation to the dopaminergic system. Understanding the full range of RLS pathology may help us better understand the complex, intermittent nature and diversity of the clinical features of RLS and expand our consideration of treatment options for RLS.

Pathological basis of diffusely abnormal white matter: insights from magnetic resonance imaging and histology

Background: The pathological basis of diffusely abnormal white matter (DAWM) in multiple sclerosis (MS) has not been elucidated in detail, but may be an important element in disability and clinical progression. Methods: Fifty-three subjects with MS were examined with T1, multi-echo T2 and magnetization transfer (MT). Twenty-three samples of formalin-fixed MS brain tissue were examined with multi-echo T2 and subsequently stained for myelin phospholipids using luxol fast blue, for axons using Bielschowsky, immunohistochemically for the myelin proteins myelin basic protein (MBP) and 2′,3′-cyclic nucleotide 3′ phosphohydrolase (CNP) and for astrocytes using glial fibrillary acidic protein (GFAP). Regions of interest in DAWM were compared with normal appearing white matter. Results: Fourteen of 53 subjects with MS in the in vivo study showed the presence of DAWM. Subjects with DAWM were found to have a significantly lower Expanded Disability Status Scale (EDSS) and shorter disease duration (DD) when compared with subjects without DAWM (EDSS: 1.5 versus 3.0, p = 0.031; DD: 5.4 versus 10.3 years, p = 0.045). DAWM in vivo had reduced myelin water and MT ratio, and increased T2 and water content. Histological analysis suggests DAWM, which shows a reduction of the myelin water fraction, is characterized by selective reduction of myelin phospholipids, but with a relative preservation of myelin proteins and axons. Conclusions: These findings suggest that the primary abnormality in DAWM is a reduction or perturbation of myelin phospholipids that correlates with a reduction of the myelin water fraction.

Neurogenesis from Neuroglia: Role of Epidermal Growth Factor in Neuroglial Transdifferentiation into Neuronal Precursor Cells

In the adult, neurogenesis occurs primarily in the hippocampus, olfactory bulb, and subventricular zone. Unlike neurons, neuroglia proliferate throughout life in response to brain aging and damage. We investigated the role of Epidermal Growth Factor (EGF) in transdifferentiating neuroglia into precursor cells and eventually neurons. C6 rat glioma cells, a mixture of astrocyte and oligodendrocytes, was treated with 50ng/mL EGF for 30 days. EGF increases proliferation, but reduces overtime the activity of Glutamine Synthetase (GS) and 2′3′‐cyclic nucleotide 3′ phosphohydrolase (CNP), the enzyme markers for astrocytes and oligodendrocytes, respectively. Simultaneously, immunofluoresence imaging showed that after 14 days of EGF treatment, neuroglial cells expressed the neuronal marker, Nestin. By 21 days, electrophysiological recordings show a resting membrane potential resembling that of neurons rather than that of neuroglia and some isolated action spikes. Thus, EGF treatment of neuroglial cells induces dedifferentiation of these cells and their transdifferentiation to exhibit certain neuronal characteristics. Supported by NIH Grant AG 19145‐05.

Effects of apotransferrin upon different components of the cytoskeleton in primary oligodendroglial cell cultures

Apotransferrin (aTf) produces and acceleration of oligodendroglial cell (OLGc) differentiation (Marta 2000).This effect seems to be directly mediated by an action of aTf upon the OLGc cytoskeleton (CSK) (Escobar 2000).When tissue slice cultures, obtained from 3 to 4‐day‐old‐rats, were treated with 125I‐aTf, the presence of the radioactive aTf was bound to elements of the CSK isolated from these preparations. Using the same experimental design and after the isolation of microtubules (MT) and associated proteins, the radioactive aTf was associated with MT. In primary OLGc cultures,the expression of different components of the myelin CSK such as actin, tubulin and microtubule associated proteins as well as the ‘stable tubulin only peptide’ (STOP) were increased by treatment with aTf. This increase was followed by an increase in the expression of myelin basic protein and 2′3′cyclic nucleotide phosphohydrolase. In the same cultures and using immunocytochemical techniques,we analyzed the localization of endogenous (rat) and of exogenously added (human) aTf, as well as tubulin and actin, after treating the cultures with different drugs affecting the CSK (colchicine,cytochalasine and taxol). The patterns of association of these components observed in control cultures changed significantly in the cultures treated with drugs affecting the CSK. aTf seems to be acting in close association with the CSK of the OLGc, increasing the expression of tubulin and STOP in order to stabilize the MT. It produces an increase in the number of OLGc processes resulting as a consequence, in a more differentiated cell.

Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures.

Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against beta-tubulin III, glial fibrillary acidic protein, and 2',3'-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.

Potential biomarkers of dichlorvos induced neuronal injury in rats

The present study was designed to develop suitable biochemical markers of chronic dichlorvos exposure using rat as the animal model. Animals were exposed to dichlorvos (6 mg kg-1 (body weight) day-1) for 8 weeks and the activities of five potential markers were assayed. Acetylcholinesterase, assayed as an index of cholinergic function, was found to decrease in both haemolysate and brain tissue. Cytochrome oxidase, used as a marker of impaired energy metabolism, was also seen to decrease in platelets and brains of dichlorvos-treated animals. However, acid phosphatase, a lysosomal marker of tissue injury, was increased in both serum and brains of experimental animals. Chronic dichlorvos exposure also led to a decrease in the activity of glucose-6-phosphate dehydrogenase, which was assayed in brain as an index of oxidative stress. Dichlorvos administration did not affect 2', 3'-cyclic nucleotide phosphohydrolase. The present study therefore, indicates that apart from acetylcholinesterase, which is probably a non-specific marker of dichlorvos neurotoxicity, the levels of cytochrome oxidase, acid phosphatase and glucose-6-phosphate dehydrogenase may serve as useful determinants of dichlorvosinduced neuronal injury.

Alterations in lipid composition and neuronal injury in primates following chronic aluminium exposure.

The effect of chronic aluminium exposure (25 mg/kg b.wt.) was studied on the lipid composition and various membrane-bound enzymes in different regions of monkey brain. Aluminium (Al) administration caused a significant decrease in the total lipid, glycolipid, and phospholipid content of primate brain. Cholesterol levels and the phospholipid to cholesterol ratio were, however, markedly increased as a consequence of Al administration, thereby indicating a loss of membrane integrity. This was further confirmed when Al treatment was found to have a significant effect on the various membrane-bound enzymes in terms of decreased activities of Na+ K+ ATPase and acetylcholinesterase, along with a decrease in the activity of the myelin-specific enzyme, 2' 3'-cyclic nucleotide phosphohydrolase.

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.

Signal transduction pathways in oligodendrocytes: Role of tumor necrosis factor-α

We have used a combination of electrophysiological and biochemical approaches to investigate the effects and the mechanisms of action of tumor necrosis factor-α (TNF-α) on cultured oligodendrocytes (OLGs). Our studies have led to the following conclusions: (1) prolonged exposure of mature ovine OLGs to TNF-α leads to inhibition of process extension, membrane depolarization and a decrease in the amplitudes of both inwardly rectifying and outward K+ currents; (2) brief exposure of OLGs to TNF-α does not elicit membrane depolarization or consistent changes in cytosolic Ca2+ levels; (3) incubation of OLGs with TNF-α for 1 hr results in inhibition of phosphorylation of myelin basic protein and 2′, 3′-cyclic nucleotide phosphohydrolase. Ceramides, which have been shown to be effectors of TNF-α, are ineffective in inhibiting phosphorylation, whereas sphingomyelinase mimicks TNF-α in this action. These observations suggest that other products of sphingomyelin hydrolysis may be the mediator(s) of TNF-α effect on protein phosphorylation. We have thus demonstrated that TNF-α can perturb the functions of OLGs via modulation of ion channels and of protein phosphorylation without necessarily inducing cell death. It is conceivable that modulation of ion channels and protein phosphorylation constitutes effective mechanisms for the participation of cytokines in signal transduction during myelination, demyelination and remyelination.

Effects of peroxisomal β-oxidation antagonist on 2′,3′-cyclic-nucleotide 3′-phosphohydrolase, membrane lipid compositions, and membrane fluidity in C-6 glial cells

In order to understand the relationship between peroxisomal dysfunction and clinical manifestations of peroxisomal disorders, the effect of thioridazine, a peroxisomal β-oxidation antagonist, on the differentiation, membrane lipid composition and membrane fluidity of C-6 glial cells was examined. In our study, induction of 2′,3′-cyclic-nucleotide 3′-phosphohydrolase (CNP), which was considered to be a membrane-associated enzyme closely associated with myelination, was inhibited with supplementation of thioridazine, followed by an increase in the relative concentration of longer chain fatty acids in cell membrane lipids, indicating that thioridazine causes impaired differentiation in the glial stem cell system. Membrane fluidity of C-6 glial cells was examined using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The DPH anisotropy value was decreased in the glial cells treated with thioridazine. These results indicate that the alteration of the membrane lipid composition caused by thioridazine affects the differentiation of glial cells via the changes in membrane properties.

Depolarizing agents and tumor necrosis factor-alpha modulate protein phosphorylation in oligodendrocytes.

Membrane depolarization and changes in ionic fluxes have been implicated in the signaling mechanisms between neurons and glial cells. We report here that K(+)-induced depolarization of cultured ovine oligodendrocytes (OLGs) decreases the phosphorylation of myelin basic protein (MBP) and 2'3'-cyclic nucleotide phosphohydrolase (CNPase). Membrane depolarization and decrease in phosphorylation of MBP and CNPase can also be elicited by inhibition of the inward rectifier with Ba2+ but not by inhibition of outward K+ channels with 4-aminopyridine or tetraethylammonium. These findings demonstrate that modulation of K+ currents can influence phosphorylation states of OLG proteins. Tumor necrosis factor-alpha (TNF-alpha), an immune peptide implicated in autoimmune demyelinating diseases, also inhibits the phosphorylation of these proteins. In contrast to elevated [K+]o, TNF-alpha does not decrease the stimulatory effect of protein kinase C activators or phosphatase inhibitors on MBP and CNPase phosphorylation, suggesting that depolarizing agents and TNF-alpha act via distinct mechanisms. We postulate that the presence of elevated extracellular K+ and/or cytokines under certain pathological conditions can perturb OLG function by altering the phosphorylation states of their proteins and perhaps affect myelin maintenance, contributing to demyelination.

Maintenance of glial plasticity with aging in C-6 glial cells and normal astrocytes in culture: responsiveness to opioid peptides.

In this study we used as glial cell models, early and late passage C-6 glial cells, 2B clone, and advanced passages of glial cells derived from aged mouse cerebral hemispheres (MACH) to examine responsiveness to opioids. We have previously reported that early passage C-6 glial cells, 2B clone, are bipotential and can be geared toward oligodendrocyte or astrocytic expression, whereas late passage C-6 glial cells are astrocytic. In addition, MACH cultures have been previously characterized and consist of astrocytes type 1 and 2, some oligodendrocytes, and few glial precursors. In this study, early passage (17-20) and late passage (106-108) C-6 glial cells or MACH cells of passages 16-19 were grown from plating time until harvesting, day 7 or 8, in DMEM + 10% FBS in the presence or absence of opioid peptides, Leu-enkephalin (10(-8) to 10(-10) M) or its synthetic analog, dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg; 10(-8) to 10(-10) M). We examined for the activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), enzyme markers for astrocytes and oligodendrocytes, respectively. We found that CNP activity was markedly increased in the early passage following opioid treatment, indicative of a shift to oligodendrocytic expression. In the late passage cells, already committed to astrocytic expression, opioid treatment enhanced GS activity suggesting that astrocytes respond to opioids. GS activity was markedly increased in MACH cultures grown in the presence of opioids with no changes in CNP. Thus, type 1 astrocytes, the predominant glial type in MACH cultures, responded to opioids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hypoxia on oligodendrocyte signal transduction.

We have previously established that 21-day-old postnatal rat oligodendrocytes, maintained in monolayer culture and subjected to 6 h of hypoxia, show reversible inhibition of synthesis of alpha-hydroxy fatty acid and myelin basic protein but a dramatic induction of a 22-kDa protein, suggesting that this is a good model to study the mechanism of CNS demyelination caused by hypoxic injury. We now report that hypoxia also dramatically inhibits the basal protein kinase C-mediated phosphorylation of myelin basic protein and myelin 2',3'-cyclic nucleotide phosphohydrolase by 80%, but that the inhibition of phosphorylation can be reversed by addition of a protein kinase C activator, phorbol 12-myristate 13-acetate. The mechanism of action appears to involve the uncoupling of signal transduction at a site before phospholipase C, because hypoxia did not affect protein kinase C activity or its translocation to the membrane fraction. The most potent activator of phospholipase C (as measured by inositol phosphate release) was carbachol (muscarinic M1 receptor agonist), followed by L-phenylephrine (alpha 1-adrenergic receptor agonist) in normal oligodendrocytes. Excitatory amino acids and histamine were ineffective. Hypoxia for 6 h completely inhibited both muscarinic and alpha 1-adrenergic receptor-mediated inositol monophosphate release but did not affect phospholipase D-coupled phosphatidylethanol production in response to carbachol. We therefore conclude from this and earlier work that early, reversible changes in oligodendrocyte metabolism result not simply from ATP depletion, but may specifically target GTP binding protein-mediated processes.

Influence of culture substrata on the differentiation of advanced passage glial cells in cultures from aged mouse cerebral hemispheres

We have previously reported that glial cells derived from aged mouse cerebral hemispheres (MACH) in primary cultures and after several passages consist of protoplasmic astrocytes (Type 1), differentiated stellate astrocytes (Type 2), a few oligodendrocytes and also glial precursors.4,33,34 In this study, we examined the influence of culture substrata: plastic, poly-l-lysine, laminin or collagen on the differentiation of MACH glial cells of advanced passages (P18–19) using glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) activity as biochemical markers for astrocytes and oligodendrocytes, respectively. Cultures were also examined morphologically using light microscopy. In general, GS activity was increased in cultures grown on the three chemical substrata versus plastic alone with the most striking effect being the 2-fold increase observed in those cells grown in laminin. No differences were noted in CNP activity. Morphologically, proliferation of protoplasmic (Type 1) astrocytes was enhanced by culture day 2 on polylysine substratum and stellate differentiated (Type 2) astrocytes were noted on collagen. The striking feature in cultures grown on laminin was the presence of astrocytes with markedly long processes. Thus, morphological astrocyte differentiation appears to correspond to the increased GS activity. We propose that the extracellular matrix components such as collagen and laminin may play an important role in promoting glial precursors to differentiate into astrocytes.

Myelin-associated changes in mouse brain following irradiation

The goals of this study were to quantify myelin-associated changes in the brain following single doses of radiation and to determine their relationship to the dose limits that this tissue can tolerate. Mice developed a transient loss of balance 1 month after 60 Gy doses 250 kV p X-rays to the brain and 3–4 months after 30–45 Gy radiation, but not after lower doses. The symptoms were transient and lasted ∼ 1 month. The ED 50 300 for radiation-induced brain death, which occurred largely between 200 and 240 days, was 32.4 Gy (29.1, 35.5 Gy, 95% confidence limit of mean). At the time that animals developed neurological symptoms, 3–4 months after irradiation with doses of 30–45 Gy, biochemical assays of myelin-associated proteins showed decreases in 2′,3′-cyclic nucleotide phosphohydrolase (CNPase) and myelin basic protein (MBP) levels that were not seen with lower radiation doses. By 120–180 days, further dose-dependent decreases in both CNPase and MBP levels were found after 20–45 Gy irradiation that preceded and correlated with death. The correlation of the decrease in CNPase and MBP levels with the incidence of transient neurological malfunction and animal death, together with histological evidence, suggests that demyelination is responsible for these phenomena.

Oligodendrocyte-substratum adhesion activates the synthesis of specific lipid species involved in cell signaling.

Ovine oligodendrocytes (OLGs) undergo biochemical and morphological changes following attachment to polylysine. Autoradiographs of two-dimensional thin-layer chromatograms of [14C]Gal-labeled OLG cultures revealed that attachment of OLGs to a polylysine substratum and their subsequent morphological differentiation is accompanied by an increased synthesis of multiple forms of galactosylceramide, sulfogalactosylceramide, and both sulfogalactosyl- and galactosyl-diglycerides, together with an array of complex sialoglycosphingolipids, predominantly GM2 ganglioside. As previously reported, overall lipid synthesis measured by [14C]acetate incorporation into glycerophosphatides, sphingomyelin, and neutral lipids also increased dramatically for up to 60 days (last time point examined) following OLG-substratum adhesion, reflecting membrane growth. Attachment was associated with a rapid augmentation in the synthesis of ethanolamine plasmalogen from 12 to 27% within 24 hr to reach a 35% plateau at 30 days and remain constant thereafter. In contrast, the plasmalogen content of phosphatidylcholine remained constant at 3-5%. This rapid increase in lipid synthesis (especially in the ethanolamine plasmalogen content following attachment) closely paralleled increased diacylglycerol (DAG) production and protein kinase C-dependent phosphorylation of both myelin basic protein and 2',3'-cyclic nucleotide phosphohydrolase. Labeling studies indicated that the major source of [3H]arachidonate-labeled DAG following attachment was from phosphatidylinositol turnover (and to a lesser extent phosphatidylcholine) rather than polyphosphoinositides or plasmalogens. Enhanced lipid synthesis is not only required for the production of membranes in these myelin-producing cells but is also a source of second messengers required in the posttranslational modification of key myelin and cellular proteins.

Alteration in myelin-associated proteins following spinal cord irradiation in guinea pigs

The aim of this study was to investigate the pathological and cellular basis for radiation-induced myelopathy in guinea pigs by monitoring biochemical alterations in levels of myelin basic protein and 2′,3′-cyclic nucleotide phosphohydrolase. Guinea pigs were irradiated to the lumbar region with various doses of neutrons or cobalt y irradiation. The ED 50s for paralysis were 17.2 Gy and 67.5 Gy for neutron and cobalt irradiation, respectively, and was histologically associated with demyelination. In spinal cords taken from animals at the onset of paralysis myelin basic protein levels were decreased in direct relationship to the radiation dose. The lowest doses to cause paralysis led to a 25% decrease in MBP levels. In a separate experiment, alterations in MBP were measured in the spinal cords over the time period leading up to paralysis. Surprisingly, decreases in MBP were found immediately after the end of the 4 week irradiation period. These early changes in MBP were not markedly dose dependent and occurred with nonparalyzing doses. Dose-dependent decreases were found only just before the onset of paralysis. CNPase activity measured in the same specimens showed changes that were essentially similar to those for MBP. In the CSF, MBP levels were essentially constant until onset of paralysis. This study showed that demyelination, as assessed by the levels of the myelin-associated proteins MBP and CNPase, can occur soon after spinal cord irradiation but that profound dose-dependant changes are seen only immediately preceding the onset of paralysis. Although increases in MBP in the CSF were associated with the onset of radiation-induced myelopathy, its assay is unlikely to predict this complication of irradiation.

Comparative biochemical, morphological, and immunocytochemical studies between C‐6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.

Effect of ultrasound on the neonatal rat brain

Six-day-old rat neonates were exposed to 2.25 MHz continuous wave (CW) ultrasound for 5 min at an intensity of 2.5 W/cm 2 (SATA). The temperatures on the head surface and in the mouth were measured. There was a higher average temperature elevation in the mouth (9°C) than on the head surface (7°C). Survival differs between control and exposed groups at 30 days after exposure. Ninety percent of the control group lived to 30 days, versus 59.7% for the exposed group. At differing times following exposure, the brains of the pups were removed and tested for enzymatic activities. Changes in acetylcholine esterase and in 2′,3′-cyclic nucleotide phosphohydrolase activities were not statistically significantly different from controls. There were no significant differences in brain weight and total protein between control and exposed pups.

Temporal, regional and cellular selectivity of neonatal alteration of the thyroid state on neurochemical maturation in the rat

The effects of alteration of thyroid state on neurochemical maturation have been studied in rats made hypothyroid by daily injections of methimazole or hyperthyroid by daily supplementation with thyroid hormone (T3) from postnatal days 1 to 27. Biochemical assays on seven brain regions plus the spinal cord were carried out on 14 and 28 day-old rats as well as in adult rats after at least 40 days of recovery. 2',3'cyclic nucleotide phosphohydrolase (CNPase), a specific marker for oligodendrocytes and myelination was significantly decreased in all regions except the spinal cord of hypothyroid rats. The astrocytic marker glutamine synthetase (GS) was slightly increased in the hippocampus of hypothyroid rats. Choline acetyltransferase (ChAT), a specific marker for cholinergic neurons, was decreased in the prefrontal and visual cortices, the striatum and the superior colliculus and increased in the cerebellum of hypothyroid rats; in addition, the enzyme activity was increased in the prefrontal cortex and striatum and decreased in the cerebellum of hyperthyroid rats. Acetylcholinesterase (AChE) activity was decreased in the prefrontal cortex and in the striatum of hypothyroid rats while 3H-quinuclidinyl benzilate (QNB) muscarinic binding was decreased in all cortical areas and in the hippocampus of hypothyroid rats. Glutamate decarboxylase (GAD), a specific marker for GABAergic neurons, was decreased in the cortical areas of hypothyroid rats. Aromatic amino acid decarboxylase (AAD), a general marker for monoaminergic neurons, was unaffected. Alteration of neurochemical parameters was never observed in the spinal cord. Under our experimental conditions, the effects of alteration of thyroid state appeared graded and selective with respect to temporal, regional and cellular parameters.