Functional activity of ganglion neurons from the pond snail upon temperature variations

Changes in the functional state of the synthetic apparatus of neurons of the peripharyngeal ring of the mollusk Lymnaea stagnalis upon elevating the temperature of the habitat from 4 to 18-22 degrees C were studied. In the first series of experiments, it was found that the synthetic activity of neurons of the great and small parietal, visceral, and pleural ganglia increases on the average by 70%. In the second series of experiments, changes in the average synthetic activity for all ganglia at 4.0, 6.5, 13.0, 16.8, 20.5, 21.5, 21.9, and 22.0 degrees C were studied. The increase in the synthetic activity of neuron cytoplasm (35% higher than the control values at 4.0 degrees C) was maximal at 13.0 degrees C; then the synthetic activity decreased to reach the level 20% higher than the control. The physiological range of temperature-dependent changes in the functional state of neurons was determined.     

Sleep-related c-Fos protein expression in the preoptic hypothalamus: effects of ambient warming

Preoptic area (POA) neuronal activity promotes sleep, but the localization of critical sleep-active neurons is not completely known. Thermal stimulation of the POA also facilitates sleep. This study used the c-Fos protein immunostaining method to localize POA sleep-active neurons at control (22 degrees C) and mildly elevated (31.5 degrees C) ambient temperatures. At 22 degrees C, after sleep, but not after waking, we found increased numbers of c-Fos immunoreactive neurons (IRNs) in both rostral and caudal parts of the median preoptic nucleus (MnPN) and in the ventrolateral preoptic area (VLPO). In animals sleeping at 31.5 degrees C, significantly more Fos IRNs were found in the rostral MnPN compared with animals sleeping at 22 degrees C. In VLPO, Fos IRN counts were no longer increased over waking levels after sleep at the elevated ambient temperature. Sleep-associated Fos IRNs were also found diffusely in the POA, but counts were lower than those made after waking. This study supports a hypothesis that the MnPN, as well as the VLPO, is part of the POA sleep-facilitating system and that the rostral MnPN may facilitate sleep, particularly at elevated ambient temperatures.     

Changes in protein metabolism in brain neurons during the withdrawal syndrome in chronically alcoholized rats (histo-autoradiographic and interferometric research)

The incorporation of 3H-leucine into the neuronal proteins and cytochemistry of nuclear and cytoplasmic protein levels in pyramidal neurons in layer III, V of the sensomotor cortex and hippocampus of the brain were studied in rats which were exposed to chronic ethanol consumption at different stages of "the abolish syndrome". There were significant heterogeneous changes of the neuronal protein metabolism at all studied structures in the experimental group as compared to control group. It was shown that after abolishment of ethanol consumption the neuronal protein synthesis decreased and content of protein increased in the nucleus and cytoplasm of neurons. After 12 h there was elevated synthetic activity of protein system and tendency to reduction of content of protein in the nucleus. After 24 h reduction of synthetic activity and tendency to normalization of the content of the neuronal protein were found. The functional importance of the results obtained is discussed.     

Transition temperatures of the electrical activity of ion channels in the nerve membrane

The temperature dependence of some of the electrical characteristics of neuronal membranes from Aplysia giant neurons and crustacean and cuttlefish giant axons has been analyzed. Arrhenius plots for the maximum rate of depolarization of (V+max) or repolarization (V-max) of the action potential, for the resting membrane conductance, and for the speed of propagation of the action potential, exhibited clear breaks at characteristic temperatures between 17 and 20 degrees C. Lobster giant axons and frog nodes of Ranvier were voltage-clamped at different temperatures between 5 and 30 degrees C. Arrhenius plots for relaxation times related to the opening and closing processes affecting the Na+ and K+ channels were linear. No 'transition' temperature was detected. However, clear-cut changes in (Formula: see text) Na+ and K+ currents, were consistantly observed around 18 degrees C. Values for (Formula: see text) plateaued above 18 degrees C, then decreased gradually as a function of reduced temperature. Variations in temperature between 1 and 30 degrees C did not alter the binding properties of [3H]tetrodotoxin to a purified crab axonal membrane. Pharmacological properties of the Na+ channel are sensitive to temperature. The temperature-dependent effect of veratridine has been studied and indicates a change in properties of the Na+ channel below 20 degrees C. These results support the possibility that the fluidity of membrane lipids in the ionic channel microenvironment may influence the degree to which the channel can open.     

Trace changes in the quantity of RNA in neurons and neuroglia of the hypothalamic nuclei of the brain following short-term intermittent exposure to cold]

Adult albino rats were kept for 2 minutes at --20 degrees C, then for 5 minutes at 25 degrees C, this intermittent cooling being repeated 15 times. One hour after the end of the cooling, the RNA quantity per cell was determined by means of visible cytospectrophotometry to find the increase in the cytoplasm of medial preoptic area neurons and in the nuclei of their glial satellite cells as well as in the nuclei of the perineuronal glial cells of mammillary bodies. Two days after the rats had stayed at the room temperature, these RNA changey bodies the RNA content decreased. 3 days later, the RNA content in the whole neuron--neuroglia unit of the preoptic area returned to normal, while in the neurons and neuroglia of mammillary bodies augmented markedly: this augmentation was found as late as 15 days after the cessation of the cooling. 30 days after the end of the cooling, the RNA quantity in the mammillary body neurons returned to normal, whiel in the neuroglia of this hypothalamic nucleus decreased even lower than the normal level. The authors discuss the problem of a so-called "vegetative memory" and of the involvement of hypothalamic neuron--neuroglia units in the metabolism tranformation dealing with a consolidation of this memory.     

Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.     

Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis.

Heat shock protein (HSP)105 is a testis-specific and HSP90-related protein. The aim of this study was to explore the functions of HSP105 in the rat testis. Signals of HSP105 were detected immunohistochemically in the germ cells and translocated from the cytoplasm to the nucleus at 2 days after experimental induction of cryptorchidism. In cultured testicular germ cells, a significant increase in the expression of HSP105 in response to heat stress (37 degrees C) was detected in the insoluble protein fractions. Several binding proteins were isolated from rat testis using a HSP105 antibody immunoaffinity column, and p53, the tumor suppressor gene product, was copurified with these. Furthermore, immunoprecipitation using antibodies to p53 led to coprecipitation of HSP105 together with p53 after culturing germ cells at 32.5 degrees C, but not at 37 or 42 degrees C. In conclusion, HSP105 is specifically localized in the germ cells and may translocate into the nucleus after heat shock. HSP105 is suggested to form a complex with p53 at the scrotal temperature, and dissociate from it at suprascrotal temperatures. At scrotal temperature, HSP105 may thus contribute to the stabilization of p53 proteins in the cytoplasm of the germ cells, preventing the potential induction of apoptosis by p53.     

Effect of stimulating the medial and lateral zones of the respiratory center on the electrical activity of the diaphragm, intercostal muscles and phrenic neurons

Experiments on cats showed that the nucleus of the solitary tract displayed zones whose stimulation provoked separately stimulation or inhibition of the electrical activity of the phrenic neurons and the diaphragm. Stimulation in the nucleus ambiguus of such zones caused stimulation and inhibition of electrical activity of the intercostal inspiratory muscles. In stimulation of the corresponding zone in the giant cell nucleus the electrical activity of both groups of the inspiratory muscles proved to change. It is suggested that the action of stimulation of the giant cell nucleus zones on both groups of inspiratory muscles is mediated through the neurons of the solitary tract and the nucleus ambiguus.     

Intracellular monosaccharide and amino acid concentrations and activities and the mechanisms of insulin action.

Current amino acid and monosaccharide transport models are based on an assumption which equates the intracellular chemical activity of a solute with its concentration. This assumption was tested for alpha-aminoisobutyric acid and 3-O-methylglucose in a giant cell, the amphibian oocyte, by using recently developed cryomicrodissection and internal reference phase techniques. We found the following. (i) alpha-Aminoisobutyric acid and 3-O-methylglucose activities were much greater in cytoplasm than was suggested by concentration data; i.e., activity coefficients were higher than in ordinary water solutions. This is attributable to the inaccessibility of considerable water as solvent (solute exclusion). (ii) Solute concentrations varied regionally as follows: nucleus > > animal cytoplasm > vegetal cytoplasm. Insulin increased the nucleus/cytoplasm concentration asymmetry, apparently by increasing cytoplasmic solute exclusion. (iii) Nuclear activity coefficients more closely resembled those of ordinary saline solutions so that nucleus/ extracellular concentration ratios reflected transmembrane activity gradients better than did cytoplasm (or whole cell)/extracellular ratios. (iv) Mediated passive alpha-aminoisobutyric acid and 3-O-methylglucose transport were constituent oocyte membrane properties. Membrane active transport was initiated with time (in the presence of substrate) and by insulin. (v) Increased temperature mimicked insulin in enhancing transmembrane alpha-aminoisobutyric acid activity gradients and increasing the nucleus/cytoplasm concentration asymmetry. These results indicated that concentration data are a misleading measure of cellular amino acid and monosaccharide activity; some consequences of this observation were explored. A model is proposed in which cell water has reduced solvent capacity or is compartmentalized (considered less likely) and is susceptible to physiological modulation. The model accounts for many observations in small cells, suggesting generality of the exclusion phenomenon and a previously unrecognized metabolic control mechanism.     

兔脑内Orexin B免疫阳性神经元的分布定位

采用免疫组织化学方法研究了10只青紫蓝兔脑内Orexin B免疫阳性神经元的分布定位。结果显示,Orexin B免疫阳性神经元分布于下丘脑的室旁核、背内侧核、穹隆周核、外侧区和后区以及底丘脑的未定带。以下丘脑背内侧核、穹隆周核和外侧区的阳性神经元数量较多,下丘脑室旁核、后区和未定带较少。表明了兔脑内Orexin B免疫阳性神经元的分布与Orexin A免疫阳性神经元的分布存在一些差异,提示两种Orexin的产生部位和生理功能可能也存在差异。     

Coexpression of cytokeratin and vimentin in mice trophoblastic giant cells

Trophoblastic giant cells reach their maximum size and exhibit a conspicuous synthetic and invasive activity during mouse placentation. The cytoskeleton, given the complex functions of the cells, shows a well-developed network of intermediate filament proteins. Immunohistochemistry combined with confocal and conventional immunofluorescence studies of intermediate filaments proteins cytokeratin and vimentin were performed in mice trophoblastic giant cells on days 9-11 of pregnancy. Specimens were fixed in phosphate-buffered formaldehyde and tissues were processed for routine paraffin embedding. Trophoblastic giant cells from antimesometrial, lateral or mesometrial uterine regions, through days 9-11 of pregnancy, expressed the same staining with both immunoperoxidase and immunofluorescent techniques. Cytokeratin filamentous structures were intensely immunoreactive and were detected throughout the cells cytoplasm; a few cells exhibited strongest fluorescence in the peripheral cytoplasm. Vimentin-positive staining was often distributed throughout the cells cytoplasm, most frequently and more intensely in the peripheral region; in some cells, it was present only in the peripheral regions. It is probable that expression of vimentin in midpregnancy trophoblastic giant cells may be associated with the rapid and conspicuous increase in size and synthetic activity of the cells and also with phagocytosis of degraded materials and invasion of decidual tissue.     

Dynamics of the ultrastructure of fibroblasts during pre- and postnatal development of the dermis in the rat according to electronic morphometry

Fibroblasts have been investigated in 18-day-old embryos, in newborns, in 2-week-old and in 2-month-old rats of postnatal development. Comparison of fibroblasts population is specific for each stage. Average section area of the fibroblasts nucleus is progressively decreasing during ontogenesis. Average section area of cytoplasm reaches its maximum by the end of the second week of the life and then decreases sharply, and therefore the nucleus/cytoplasm ratio in the fibroblasts is minimal at the age of two weeks. The Golgi complex section area decreases significantly in mature animals as compared to the young ones. Summational extent of the granular endoplasmic reticulum membranes in the fibroblast section in the 2-week-old animals is 2.5 times as great as the corresponding parameters in the fetuses, newborns and mature animals. This parameter is greatly informative for determining the fibroblast type in accordance with its synthetic activity.     

Electron-cytochemical study of the changes in the structure of hyperchromic neurons shown after chlorpromazine administration

By means of Bernhard's electron cytochemical method for revealing DNP and RNP, the hyperchromic neurons in the cerebral cortex have been investigated 3, 6, 24 and 48 hours after chlorpromazine hydrochloride administration (15 mg/kg). Development of hyperchromia in the neurons has been followed beginning from minimal changes in the nucleus and cytoplasm up to their maximal reconstruction and a subsequent restoration to the normal stage. The data obtained demonstrate that hyperchromia of the neuron during various phases of its development can be characterized both by a decreasing synthetic activity and by restorative processes during the last period when the functional state is normalized.     

Morpho-chemical characteristics of the brain of rats genetically predisposed to catalepsy

Cell organization of the cerebral cortex and striatum has been studied by+light optics and enzymatic activity of neuromediators catabolism--histochemically. In layers III and V of the sensomotor cortex and in the nucleus caudatus the number of neurons per unit area is increased, and their size is decreased. Volume of cytoplasm and nuclei of neurons in both layers is decreased, the latter--to less extent. The total amount of the perineuronal glia in the nucleus caudatus is also decreased at the expense of astroglia. In the cerebral cortex it is equal, but the ratio between astro-++- and oligodendroglia is changed. Acetylcholinesterase activity in the layers III and V of the sensomotor cortex is lower than in the control, while monoamine oxidase activity is kept at the control level. The genetically determined anomalies++ of growth and development of the cerebral cells is supposed to be considered as a structural base of the cataleptic state.     

Segregation of saturated chain lipids in pulmonary surfactant films and bilayers

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.     

Synthetic heat at mild temperatures

"Synthetic heat", also known as the heat grill illusion, occurs when contact with spatially adjacent warm and cold stimuli produce a sensation of "heat". This phenomenon has been explained as a painful perception that occurs when warm stimulation inhibits cold-sensitive neurons in the spinothalamic tract (STT), which in turn unmasks activity in the pain pathway caused by stimulation of C-polymodal nociceptors (CPNs). The "unmasking model" was tested in experiment 1 by combining warm (35-40 degrees C) and cool (> or = 27 degrees C) stimuli that were too mild to stimulate CPNs. After discovering that these temperatures produced nonpainful heat, experiment 2 was designed to determine whether heat could be induced when near-threshold cooling was paired with mild warmth, and whether lowering the base temperature for cooling would increase the noxious (burning, stinging) components of heat for fixed cooling steps of 1-3 degrees C. Cooling by just 1 degrees C from a base temperature of 33 degrees C led to reports of heat on more than 1/3 of trials, and cooling by just 3 degrees C evoked heat on 75% of trials. Lowering the base temperature to 31 or 29 degrees C increased reports of heat and burning but did not produce significant reports of pain. Perception of nonpainful heat at such mild temperatures indicates either that cold-sensitive nociceptors with thresholds very similar to cold fibers innervate hairy skin in humans, or that heat can result from integration of warm fiber and cold fiber activity, perhaps via convergence on nonspecific (e.g., WDR) neurons in the STT.     

Splicing thermotolerance maintains pre-mRNA transcripts in the splicing pathway during severe heat shock

Thermotolerance, the ability of cells and organisms to withstand severe elevated temperatures after brief exposure to mild elevated temperatures, has been studied in numerous laboratories. Survival thermotolerance is defined as the increase in cell or organism survival at severe elevated temperatures after a pretreatment at mild elevated temperatures. This study examines splicing thermotolerance in Drosophila melanogaster, the ability to splice pre-mRNAs made at the severe temperature (38 degrees C) after a brief pretreatment at a milder temperature (35 degrees C). It is probably one of a number of mechanisms by which cells adapt to heat shock. These experiments demonstrate that pre-mRNAs synthesized at the severe temperatures in splicing thermotolerant cells, although protected in splicing-competent complexes, are not actually processed to mature mRNAs until the cells are returned to their normal temperature. We have also studied the kinetics of acquisition and loss of splicing thermotolerance. As little as 10 min of pretreatment at 35 degrees C was sufficient to provide full splicing thermotolerance to a 30-min severe heat shock of 38 degrees C. Pretreatments of less than 10 min provide partial splicing thermotolerance for a 30-min severe heat shock. Full splicing thermotolerance activity begins to decay about 4 h after the cessation of the 35 degrees C incubation and is completely lost by 8 h after the pretreatment. The kinetics experiments of pre-mRNAs synthesized during the 38 degrees C treatment in splicing thermotolerant cells indicate that one or more splicing thermotolerance factors are synthesized during the 35 degrees C pretreatment which interact with pre-mRNA-containing complexes to keep them in a splicing-competent state. These kinetic experiments also indicate that in cells which are partially splicing thermotolerant, the pre-mRNAs synthesized early during the 38 degrees C incubation are protected, whereas those synthesized late are not. In the absence of splicing thermotolerant factors, the pre-mRNA-containing complexes leave the normal splicing pathway and are allowed to exit to the cytoplasm.     

Effect of temperature and pH on the ribulose-1,5-diphosphate carboxylase activity of the thermophilic hydrogen bacterium, Pseudomonas thermophila

The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completely loses its activity at 80 degrees C. The activity of RDP carboxylase at 60 degrees C increases by 40% during the first 10 min of heating in the presence of Mg2+ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50--55 degrees C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 kcal-mole-1, but 11.6 kcal-mole-1 in frozen preparations. The optimal pH is 7.0--7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps. thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temperature (the enzyme activity is negligible at 30 degrees C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.     

Temperature dependent plasticity of habituation in the crayfish

To determine the effects of thermal preconditioning on a simple form of learning and memory, habituation, we preconditioned crayfish with extreme temperatures and subsequently analysed their effects on mechanosensory input that evokes a response in the lateral giant interneurons, within the normal temperature range of the animal. We found that repetitive stimulation with a 1 s interstimulus interval led to habituation of the response the lateral giant in control animals at 22°C. Neither heat nor cold preconditioning had any effect on the probability of evoking a response in the lateral giant nor on the rate at which habituation occurred. With a 1 min interstimulus interval, however, the rate of habituation of the lateral giant in the heat-preconditioned group was less than either the control or cold-preconditioned animals. The effect of heat or cold pre-exposure was specific to the input to the lateral giant at control temperatures. For example, at 22°C prior heat and cold preconditioning had no effect on spontaneous reductor motor neurone activity. They did, however, provide thermoprotection at extreme temperatures, with the probability of spontaneous activity higher in the cold-preconditioned group at low temperatures but higher in the heat-preconditioned group at higher temperatures.     

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.