Critical Effects on Akt Signaling in Adult Zebrafish Brain Following Alterations in Light Exposure.

Evidence from human and animal studies indicate that disrupted light cycles leads to alterations of the sleep state, poor cognition, and the risk of developing neuroinflammatory and generalized health disorders. Zebrafish exhibit a diurnal circadian rhythm and are an increasingly popular model in studies of neurophysiology and neuropathophysiology. Here, we investigate the effect of alterations in light cycle on the adult zebrafish brain: we measured the effect of altered, unpredictable light exposure in adult zebrafish telencephalon, homologous to mammalian hippocampus, and the optic tectum, a significant visual processing center with extensive telencephalon connections. The expression of heat shock protein-70 (HSP70), an important cell stress mediator, was significantly decreased in optic tectum of adult zebrafish brain following four days of altered light exposure. Further, pSer473-Akt (protein kinase B) was significantly reduced in telencephalon following light cycle alteration, and pSer9-GSK3ß (glycogen synthase kinase-3ß) was significantly reduced in both the telencephalon and optic tectum of light-altered fish. Animals exposed to five minutes of environmental enrichment showed significant increase in pSer473Akt, which was significantly attenuated by four days of altered light exposure. These data show for the first time that unpredictable light exposure alters HSP70 expression and dysregulates Akt-GSK3ß signaling in the adult zebrafish brain.

Variation of radiation-sensitivity of neural stem and progenitor cell populations within the developing mouse brain.

PURPOSE: We investigated the DNA damage response (DDR) of fetal neural stem and progenitor cells (NSPC), since exposure to ionizing radiation can severely impair the brain development. MATERIAL AND METHODS: We compared apoptosis induction in the dorsal telencephalon and the lateral ganglionic eminences (LGE) of mouse embryos after an in utero irradiation. We used two thymidine analogs, together with the physical position of nuclei within brain structures, to determine the fate of irradiated NSPC. RESULTS: NSPC did not activate an apparent protein 21(p21)- dependent G1/S checkpoint within the LGE as their counterparts within the dorsal telencephalon. However, the levels of radiation-induced apoptosis differed between the two telencephalic regions, due to the high radiation sensitivity of intermediate progenitors of the LGE. Besides radial glia cells, that function as neural stem cells, were more resistant and were reoriented toward self-renewing within hours following irradiation. CONCLUSIONS: The lack of the p21-dependent-cell cycle arrest at the G1/S transition appears to be a general feature of NSPC in the developing brain. However, we found variation of radiation-response in function of the types of NSPC. Factors involved in DDR and those involved in the regulation of neurogenesis are intricately linked in determining the cell fate after irradiations.

Radiation-induced H2AX phosphorylation and neural precursor apoptosis in the developing brain of mice.

We showed that gamma irradiation of the developing mouse brain with 2 Gy induced a massive apoptosis of neural precursors but not of neurons within 24 h. Successive phosphorylation and dephosphorylation of histone H2AX have been linked to DNA breaks and repair. Similar numbers of nuclear foci of phosphorylated H2AX (gamma-H2AX) were found 1 h postirradiation in neural precursors and in neurons, suggesting that differences in radiosensitivity were not related to variations in the numbers of DNA double-strand breaks induced by radiation. Surviving neural precursors like neurons totally lost gamma-H2AX within 24 h after irradiation, but they had a slower kinetics of loss of gamma-H2AX foci. This suggests that the DNA repair machinery processed damage more slowly in these neural precursors in relation to their greater radiosensitivity. We also found a bright and diffuse gamma-H2AX staining of nuclei of cells at an early stage of apoptosis, whereas cells at later stages of apoptosis were unstained. This was probably related to phosphorylation and subsequent degradation of H2AX in the course of DNA fragmentation during apoptosis. Detection of gamma-H2AX-bright nuclei may thus be a useful marker of neural cells at an early stage of apoptosis.

Telefonía móvil. ¿Una apuesta con nuestra salud? / Mobile telephones: playing with our health?

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Prevención y tratamiento de la toxicidad en SNC / No disponible

Las complicaciones agudas en SNC suelen ser transitoriasy reversibles. Sin embargo la toxicidad tardía tiene una granrelevancia por la sintomatología muchas veces incapacitanteo letal. Más que prevenir, ya que no disponemos de fármacoseficaces que pudieran evitar los efectos secundarios, podemosreducir el riesgo de los mismos. La indicación de latécnica y dosis de RT más adecuada en cada caso, con especialcuidado en minimizar el volumen de tejido sano fundamentalmenteen los niños, utilizar dosis por fracción igual omenor a 2 Gy y la localización de los órganos de riesgo convaloración del volumen y dosis recibida por los mismos, sonlas mejores armas para minimizar la morbilidad sin reducirel control. El tratamiento varía en relación a la secuela específicaque se produzca, siendo efectivo en algunos casos, comoen las alteraciones endocrinológicas con la administraciónde la hormona deficitaria o en el caso de algunas radionecrosisque requieren esteroides y/o cirugía. Sin embargo,en otras ocasiones no disponemos de armas efectivasutilizando medidas de soporte. En cualquier caso el diagnósticoes fundamental y como el tiempo de aparición de losefectos tardíos varía entre 6 meses y muchos años se requierede seguimientos muy prolongados

Afferent subcortical connections into the motor cortical larynx area in the rhesus monkey.

In three rhesus monkeys (Macaca mulatta), the inferior motor cortex was explored by electrical stimulation for sites yielding vocal fold adduction. The retrograde tracer wheat germ-agglutinin-conjugated horseradish peroxidase was injected into the effective sites. Within the forebrain, retrogradely labeled cells were found in the claustrum, basal nucleus of Meynert, substantia innominata, extended amygdala, lateral and posterior hypothalamic area, field H of Forel, and a number of thalamic nuclei with the strongest labeling in the nuclei ventralis lateralis, ventralis posteromedialis, including its parvocellular part, medialis dorsalis and centrum medianum, and weaker labeling in the nuclei ventralis anterior, ventralis posterolateralis, intermediodorsalis, paracentralis, parafascicularis and pulvinaris anterior. In the midbrain, labeling was found in the deep mesencephalic nucleus, ventral tegmental area, and substantia nigra. In the lower brainstem, labeled cells were found in the pontine reticular formation, median and dorsal raphe nuclei, medial parabrachial nucleus, and locus coeruleus. The findings are discussed in terms of the possible role of these structures in voluntary vocal control.

NMDA receptor-dependent long-term potentiation in the telencephalon of the zebrafish.

In Mg2+ -free aCSF, bursting discharges were induced in the posterior telencephalon of zebrafish following an electrical stimulation of the anterior telencephalon. The bursting discharges were partially reduced by CNQX (10 microM), an AMPA receptor antagonist, and the remaining activity was completely blocked by an additional treatment of APV (50 microM), an NMDA receptor antagonist. Long-term potentiation that lasted more than 1 h was also induced after 20 min of perfusion with KCl (10 mM). The degree of KCl-induced long-term potentiation (K-LTP) was reduced when a concomitant electrical stimulation was not delivered during a KCl perfusion. K-LTP was blocked by APV (50 microM) but not by nifedipine (1 microM), an L-type Ca2+ channel blocker. Furthermore, K-LTP was not induced in the presence of a broad spectrum inhibitor for protein kinases, H-7 (10 microM). These results suggest that NMDA receptors and protein kinases play important roles in the synaptic plasticity of the zebrafish brain.

Atm-null mice exhibit enhanced radiation-induced birth defects and a hybrid form of embryonic programmed cell death indicating a teratological suppressor function for ATM.

ATM (ataxia-telangiectasia mutated) is a genotoxic stress transducer. In this first report of Atm-dependent birth defects, Atm-null embryos were uniquely susceptible to low-dose (0.5 Gy) radiation, exhibiting severe runting, tail anomalies, and lethality, independent of cell cycle arrest or insulin-like growth factor 1. This treatment enhanced levels of p53 protein and central nervous system (CNS) apoptosis in wild-type mice, but not Atm-null mutants, at 6 h postirradiation. At 48 h, however, this pattern was reversed, with Atm-null mice exhibiting high levels of a hybrid form of programmed cell death within the CNS. Even heterozygous Atm-deficient embryos were radiosensitive to a higher radiation dose of 2 Gy. These results show that Atm is a novel teratologic suppressor gene protecting embryos from pathological cell death and teratogenesis initiated by even mild DNA damage.

Caspase regulation of genotoxin-induced neural precursor cell death.

Neural precursor cells (NPCs) critically regulate brain morphogenesis and recent studies have revealed an unexpectedly high frequency of NPC chromosomal abnormalities and apoptosis in the developing brain. We have shown previously that the apoptotic response of NPCs to genotoxic agents is dependent on p53 and caspase-9, but not Bax or caspase-3 expression. In this study, we found that NPCs deficient in Apaf-1, or both the pro-apoptotic multidomain Bcl-2 family members Bax and Bak, were resistant to cytosine arabinoside and gamma-irradiation-induced apoptosis. Inhibitors of gene transcription, protein translation, and caspase activity also blocked genotoxin-induced NPC apoptosis. Although caspase-3 and caspase-6 were both cleaved in response to DNA damage, neither of these effector caspases was critical for apoptosis. Genotoxin-induced NPC death was accompanied by the generation of reactive oxygen species and could be inhibited by several known antioxidants. Conversely, DNA damage-induced reactive oxygen species generation was inhibited significantly by gene disruption of p53, Apaf-1, or caspase-9, and combined deficiency of Bax and Bak, but not by caspase-3 or caspase-6 deficiency. These studies suggest that caspase-9 activation is both necessary and sufficient for genotoxin-induced neural precursor cell reactive oxygen species generation and death.

Light-dependent development of asymmetry in the ipsilateral and contralateral thalamofugal visual projections of the chick.

Light-exposure of the chick embryo induces development of asymmetry in the thalamofugal visual projections to the Wulst regions of the forebrain since the embryo is turned so that it occludes its left and not its right eye. This asymmetry can be reversed by occluding the embryo's right eye and exposing its left eye to light. Here we show that three sub-regions of the thalamus (two in the dorsolateral anterior thalami (DLA) and one more caudal) have differing asymmetries of contralateral and/or ipsilateral projections. Hence the effect of asymmetrical light stimulation is regionally specific within the thalamus. Lateralised light stimulation appears to promote the development of ipsilateral projections from DLA pars dorsolateralis pars anterioris and contralateral projections from the caudal regions (the nucleus superficialis parvocellularis especially) but it may suppress the development of contralateral projections from the nucleus dorsolateralis anterior thalami pars lateralis rostralis. We also show that the light stimulation causes lateralised expression of c-fos and receptors for neurotransmitters.

Responses of neurons to an amplitude modulated microwave stimulus.

In this study we investigated the effects of a pulsed radio frequency signal similar to the signal produced by global system for mobile communication telephones (900 MHz carrier, modulated at 217 Hz) on neurons of the avian brain. We found that such stimulation resulted in changes in the amount of neural activity by more than half of the brain cells. Most (76%) of the responding cells increased their rates of firing by an average 3.5-fold. The other responding cells exhibited a decrease in their rates of spontaneous activity Such responses indicate potential effects on humans using hand-held cellular phones.

Proteinase-activated receptor-2 expression on cerebral neurones after radiation damage: immunohistochemical observation in Wistar rats.

Radiation damage results in blood-brain barrier damage followed by blood plasma transfer into the neuropil. The transferred liquid contains high amounts of biologically active substances/proteinases including factor Xa and a free pool of serum trypsin, which is not bound to antiproteases (alpha1 AT, alpha2-macroglobulin). The aim of this study was to follow up expression of proteinase-activated receptor-2 (PAR-2) in the brains of Wistar rats after single exposure to radiation at 26 Gy (60Co, 23 min, 15 sec). After irradiation, the animals were sacrificed on days 10, 20, 30 and 40. Control rat brains served as negative control. Coronal sections of caudal diencephalons were investigated using histology and immunohistochemistry. Polyclonal goat specified antibody against the NH-end of murine and rat PAR-2. Significant PAR-2 membrane positivity of scattered swollen neurons in deeper cortical layers was found in irradiated animals compared with controls. Although this membrane positivity was noticed in all irradiated animals, the most prominent occurred on day 30. Diffuse cytoplasmic positivity was also demonstrated on shrunken neurons in the cortex and hippocampus. Increased cytoplasmic and polarized membrane positivity was also noticed on the neurons of hypothalamic nuclei The causal relationship between blood-brain barrier damage, PAR-2 activation and neurodegeneration has not yet been verified. However, the present findings indicate that PAR-2 mediates a certain cellular response. It remains to be demonstrated whether this is a response to higher concentrations of factor Xa, a free pool of trypsin or other unknown possible proteinases in brain tissue; whether changes in PAR-2 expression are consequences of direct radiation damage to neuronal cells; whether this reaction is protective; and whether primary PAR-2 activation results in neuronal damage.

Effects of X-irradiation on glial cells in the developing rat brain.

Sprague-Dawley rats were given a single dose of 2 Gy X-rays when 1 or 3 days of age. Dying cells in the germinal layer of the telencephalon reached peak values 6 h after irradiation; dead cells were cleared 48 h later. These effects were almost abolished with the injection of cycloheximide (1 microgram/g body weight) given at the time of irradiation. PCNA-immunoreactive cells (cells in late G1 and S phases of the cell cycle) and PCNA-negative cells were sensitive to X-rays. Long-term effects on glial cell populations in the subcortical white matter of the cingulum were examined in irradiated rats, killed at postnatal day 30 (P30), by means of glial fibrillary acidic protein, vimentin and S-100 immunohistochemistry, as well as with anti-TGF-alpha (transformerly growth factor) antibodies that are used as putative oligodendroglial cell markers in the white matter of rat. The subcortical white matter was reduced in irradiated animals, mainly in rat irradiated at P3, as revealed with myelin basic protein immunohistochemistry. Quantitative studies showed no significant differences in the number of glial cells in animals irradiated at P1 when compared with age-matched controls. However, reduced numbers of vimentin-, S-100-, and TGF-alpha-immunoreactive cells were found in animals irradiated at P3. These features indicate a limited capacity of surviving germinal cells, in animals irradiated at P3, to give rise to normal values of glial cells in the white matter in rat aged 30 days. This limitation mainly affects putative oligodendrocytes and glial cell precursors.

Characterisation of an allosteric modulatory protein associated with alpha-[3H]amino-3-hydroxy-5-methylisoxazolepropionate binding sites in chick telencephalon: effects of high-energy radiation and detergent solubilisation.

alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) binds to 1-day-old chick telencephalon membranes with KD and Bmax values of 138 nM and 2.56 pmol/mg of protein, respectively. High-energy radiation bombardment of intact frozen telencephalon resulted in a biphasic inactivation curve for [3H]AMPA binding. At a 5.8-Mrad radiation dose, the affinity of [3H]AMPA binding was increased (54 nM), but there was no apparent alteration in the Bmax value (2.76 pmol/mg of protein). We attribute this phenomenon to the inactivation of a high molecular weight modulatory protein that down-regulates the affinity of [3H]AMPA binding. The estimated molecular masses of the AMPA binding site and of the modulatory component were 59 and 108 kDa, respectively. Solubilisation with n-octyl-beta-glucopyranoside resulted in an increase in the Bmax (4.7 pmol/mg of protein) with no pronounced alteration in the affinity (109 nM) of [3H]AMPA binding. However, the solubilisation-induced increase in Bmax did not occur in telencephalon irradiated before solubilisation. In contrast, the increase in affinity induced by radiation treatment was still detected in solubilised extracts. These results suggest that the number and affinity of [3H]AMPA sites in chick telencephalon are closely regulated and that the modulatory systems involved are affected by both irradiation and solubilisation.

Acute response of the fetal telencephalon to short-term maternal exposure to ethanol in the rat.

Pregnant rats were exposed to either ethanol (total dose 18 g/kg) on gestational days 14 and 15 or whole-body ionizing radiation (0.5 Gy) on gestational day 15. On gestational day 16, 24 h following the last dose of ethanol or exposure to ionizing radiation, the developing cerebral cortex of the fetus was examined histologically. Ionizing radiation caused extensive cell death within the fetal cerebral cortex whereas ethanol caused more subtle morphological changes such as cortical thinning and petechial intraventricular hemorrhages. These findings suggest that ethanol, unlike ionizing radiation, acts by some mechanism other than cell death to cause cortical thinning and cortical malformations. The pathogenesis of ethanol-induced cortical dysgenesis may include fetal hypoxia and inhibition of neuroblast proliferation within the developing cerebral cortex.

Developmental-stage-dependent radiosensitivity of neural cells in the ventricular zone of telencephalon in mouse and rat fetuses.

Pregnant ICR mice were treated with single whole-body X-radiation at a dose of 0.24 Gy on day 10, 13, or 15 of gestation. Fetuses were obtained from mothers during 1 and 24 hours after irradiation. Pyknotic cells in the ventricular zone of telencephalon were counted in serial histological sections. Incidence of pyknotic cells peaked during 6 and 9 hours after irradiation in each gestation day group. Then, dose-response curves were obtained 6 hours after 0-0.48 Gy of irradiation. All three dose-response curves showed clear linearity in the dose range lower than 0.24 Gy. Ratios of radiosensitivity estimated from the slopes of dose-response curves in day 10, 13, and 15 groups were 1, 1.4, and 0.4, respectively. These demonstrated that ventricular cells in the day 13 fetal telencephalon were the most radiosensitive among the three different age groups. In order to confirm the presence of the highly radiosensitive stage common to mammalian cerebral cortical histogenesis, pregnant F344 rats were treated with single whole-body gamma-irradiation at a dose of 0.48 Gy on day 13, 14, 15, 17, or 19 of gestation. The incidence of pyknotic cells in the ventricular zone of telencephalon was examined microscopically during 1 and 24 hours after irradiation. The peak incidence was shown 6 hours after irradiation in all the treated groups, and the highest peak incidence was shown in day-15-treated group. The developmental stage of telencephalon of day 15 rat fetuses was comparable to that of day 13 mouse fetuses. Thus, the highest radiosensitivity in terms of acute cell death was shown in the same developmental stage of brain development, i.e., the beginning phase of cerebral cortical histogenesis, in both mice and rats.

Kinetics of telencephalic neural cell proliferation during the fetal regeneration period following a single X-irradiation at the late organogenesis stage. II. Cycle times and the size of the functional compartment of neural epithelial cells of distinct lesion districts.

Autoradiographic studies were conducted at the cerebral hemispheres of mouse embryos X-irradiated on day 12 of gestation and of normal litter mates during the subsequent developmental period. By counting the percentage of labeled mitoses the generation time, the potential doubling time, the growth fraction, as well as the length of the individual cell cycle stages of the neuroblast cells were determined. A continuous increase of generation time was found in the normal brains, concomitant with a latero-medial gradient in telencephalic wall differentiation progress. After X-irradiation this normal differentiation pattern still prevails, but with some marked topographical peculiarrities. The most important finding was a significant lengthening of the generation time at the medially situated rudiments of the ventricular zone and, similarly at the heterotopic cell islets located within the intermediary zone. Concomitant with this effect, which was seen mainly on days 15 and 17 of gestation, there was a marked increase of mitotic time of these special neuroblasts. The latter finding was regarded as a random event only, which has no causal relationship to the pathogenesis of the heterotopic islets or similar overgrowth anomalies after X-irradiation. In spite of the long generation time of these histological peculiarities, they make a considerable contribution to the regeneration of the injured telencephalic wall: Up to day 15 of gestation the heterotopias had a growth fraction of nearly 1.0 (= 100%), whereas the percentage of proliferating cells within the orthotopic remainders of the ventricular zone was only 44%.

X-ray induced dysplasia in the developing telencephalic choroid plexus of mice exposed in utero.

Pregnant NMRI-mice were X-irradiated with single doses of 0.95 Gy (100 R) and 1.9 Gy (200 R) on day of gestation (dg) 12. For sampling, anesthetized animals were perfused with buffered glutaraldehyde solution or fixed by immersion in Karnovsky solution. LM, SEM, and TEM studies were carried out on brains prenatally and up to the age of 20 months to follow the radiation effects on the developing lateral choroid plexus. Radiation-induced changes were found using all three methods and at all stages studied. The normally sickle-shaped and stretched choroid plexus is shortened and irregular, and the dome-shaped plexus cells are flattened. Their superficial fine structures, i.e., the microvilli and cilia, are altered. Three stages of severity can be distinguished and the internal hydromicrocephalus increases from stage I to III. Intercellular spaces of the treated plexus epithelium are often dilated, but the tight junctions at the ventricular surface seem to be intact. The interstitium shows large dilations in comparison with the controls. Thus, gross changes and alterations in the fine structure can be induced in the choroid plexus by doses of 0.95 Gy and 1.9 Gy, which persist throughout postnatal life.

Two types of congenital hydrocephalus induced in rats by X-irradiation in utero: electron microscopic study on the telencephalic wall.

Stage-specific incidences of congenital hydrocephalus induced by X-irradiation of pregnant rats showed a bimodal distribution. At a dose level of 200 R, 100% hydrocephalic offspring were obtained by irradiation at embryonic days 11 and 14. When pregnant rats were subjected to 200 R X-irradiation at embryonic day 11, numerous ventricular cells of the telencephalic wall of the embryo became necrotic during the first 2 hours post-irradiation, but the paraventricular cell-to-cell interconnexions made up of zonulae adhaerentes were less affected. Mitosis took place in the surviving paraventricular surface cells throughout subsequent development. The full-term fetus exhibited little change in the cytoarchitectural arrangement of neural cells and neuropils, although it was only about half the thickness of the untreated control. After 200 R X-irradiation at embryonic day 14, most of the ventricular cells became necrotic within 6 hours. The paraventricular cell-to-cell interconnexions were completely destroyed, and never repaired in subsequent development. Mitosis took place either freely in cell clusters, or in rosettes which formed randomly in the telencephalic wall between 48 and 72 hours post-irradiation. The resulting telencephalic wall of the full-term fetus was also about half the thickness of the control. In the outer part of the tissue, the cortical plate made up of differentiating neuroblasts was hypoplastic, but the inner half was filled with numerous heterotopic masses of pleomorphic cells and bundles of primitive axons. The ependymal layer at the paraventricular surface was never formed. Whether the paraventricular zonulae adhaerentes were destroyed or not by X-irradiation was considered to be an important factor in the determination of the subsequent cytoarchitectural organization of the telencephalic wall.