Distribution and ultrastructure of the tyrosine hydroxylase-positive neurons of the central nervous system of bivalve mollusc Megangulus venulosus under the influence of increased temperature and hypoxia

Using immunocytochemistry combined with light and electron microscopy, the distribution and ultrastructure of tyrosine hydroxylase (TH)-immunoreactive neurons in the CNS of bivalve mollusc, Megangulus venulosus, have been studied under the influence of increased temperature and hypoxia. It has been established, that the stress causes changes in the amount of TH and in the structure of TH-immunopositive neurons in all ganglia. The most essential changes in CNS of M. venulosus were revealed after 60 min exposure to increased temperature and hypoxia; degenerative changes in large neurons, reduction of the synapses and reduction of TH-immunoreactivity in neurons and neuropil.     

Effect of elevated temperature and of hypoxia on activity of NO-ergic system of the central nervous system of bivalve molluscs

By light and electron microscopy methods the effect of changes of environmental conditions on the state of the nitroxidergic system has been studied in molluscs on the background of action of elevated temperature and hypoxia. Analysis is performed of biological effect of isolated and combined effects of the studied factors on dynamics of NO synthesis. A higher resistance of CNS neurons to the combined action of hyperthermia and hypoxia is revealed in molluscs with the initially high level of nitrogen oxide production. In molluscs with the initially low level of development of the nitroxidergic system, induction of NO formation in stress has been found to be accompanied by a change of morphology of nervous structures. It is suggested that nitrogen oxide participates in evolutionary established mechanisms of protection of mollusc nerve cells from hypoxia, while the initial high level of NO production reflects larger adaptational possibilities characteristic of these organisms.     

Effect of elevated temperature and of hypoxia on NO activity in the central nervous system of bivalve molluscs

By light and electron microscopy methods the effect of changes of environmental conditions on the state of the nitroxidergic system has been studied in molluscs on the background of action of elevated temperature and hypoxia. Analysis is performed of biological effect of isolated and combined effects of the studied factors on dynamics of NO synthesis. A higher resistance of CNS neurons to the combined action of hyperthermia and hypoxia is revealed in molluscs with the initially high level of nitrogen oxide production. In molluscs with the initially low level of development of the nitroxidergic system, induction of NO formation in stress has been found to be accompanied by a change of morphology of nervous structures. It is suggested that nitrogen oxide participates in evolutionary established mechanisms of protection of mollusc nerve cells from hypoxia, while the initial high level of NO production reflects larger adaptational possibilities characteristic of these organisms.     

NO-Synthase in the Central Nervous System of Freshwater Bivalve Mollusc Nodularia vladivostokensis in Norm and in Hypoxia

Using light- and electron microscopic methods, localization and activity of NO-synthase were studied in the CNS of the freshwater bivalve mollusc Nodularia vladivostokensis in norm and in acute hypoxia. Distribution peculiarities and the relative content of NO-ergic neurons were revealed in nervous ganglia. A rise of the NO-synthase activity was found in perikarya of medium-size neurons and in processes of small neurons in hypoxia. Ultrastructural localization of NO-synthase was established in cytoplasmic granules, cytosomes. In acute hypoxia an increase of the number of cytosomes and of their NO-synthase activity was revealed.     

Astrocytes Protect Neurons from Ammonia Toxicity

Ammonia is a neurotoxin that is implicated in the CNS dysfunction associated with hepatic encephalopathy, urea cycle disorders, Reye’s syndrome and other neurological conditions. While in vivo studies suggest that astrocytes are the principal target of ammonia toxicity, recent in vitro investigations suggest that neurons may also be directly affected by ammonia. To further examine the issue of neural cell sensitivity to ammonia, pure rat cortical neuronal cultures, as well as co-cultures of neurons and astrocytes, were exposed to 5 mM NH₄Cl for 48 h. Cultures were examined for morphological changes by light microscopy, measures of cell death, free radical production and changes in the mitochondrial inner membrane potential. Ammonia caused extensive degenerative changes in pure cultured neurons, while such neuronal changes were minor in the co-cultures. Similarly, processes of pure cultured neurons displayed a significant loss of the mitochondrial inner membrane potential, as compared to neurons in co-cultures. Cell death (LDH release) in ammonia-treated neuronal cultures was twice as great as untreated controls, while in co-cultures ammonia did not significantly increase cell death. Free radical production at 3 min was increased (69%, P<0.05) in pure neuronal cultures but not in co-cultures. The neuroprotective effects observed in co-cultures may have been mediated by the astrocyte’s ability to scavenge free radicals, by their detoxification of ammonia and/or by their neurotrophic actions. The neuroprotective action of astrocytes may explain the failure to detect significant pathological changes in neurons in ammonia toxicity in vivo. Special issue dedicated to Dr. Bernd Hamprecht.     

Localization of NADPH diaphorase in central nervous system of the chiton Leptochiton assimilis in normal conditions and during hypoxia

By light and electron microscope histochemical and cytochemical methods, the localization and activity of NADPH-diaphorase (NADPH-d) were studied in the central nervous system (CNS) of the chiton in control and after hypoxia. After acute hypoxia, the enzymatic activity increased in all regions of CNS. At a chronic hypoxia, the activity of NADPH-d decreased to remain, however, higher than in control. Ultrastructural studies confirmed the availability of structural changes in neurons, and shifts in the activity of NADPH-d in control and in experimental mollusks. The elevated enzymatic activity revealed in this study may be due to the fact that these mollusks have been evolutionary adapted to a periodical oxygen deficiency.     

Co-factors and co-repressors of Engrailed: expression in the central nervous system and cerci of the cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis>

In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8–A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.I acknowledge the support of NIH R01 NS45547, NIH-SCORE S06 GM0088224, and RCMI G12 RR03051.     

Calcium Signaling in <Emphasis Type="Italic">Carassius</Emphasis> Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca²⁺ ([Ca²⁺] _і ) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca²⁺, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.     

Dependence of Electric Activity of Motoneurons and Locomotor Behavior of Lymnaea stagnalis on Environmental Temperature

Experiments on serotoninergic motoneurons of the pedal A-cluster innervating the foot ciliated epithelium of Lymnaea stagnalis L. have shown that changes of the environmental temperature lead to a change of several electrophysiological parameters of these neurons. The firing rate of action potential (AP) progressively increases with elevation of the temperature up to the level of about 30°C, after that an abrupt drop of the rate occurs. The membrane potential and AP amplitude decrease as the temperature rises. The revealed changes may be explained by an increase of the membrane permeability, specifically, to sodium ions. The characteristic activity pattern of these neurons disappears in the temperature range of 34–36°C. The peculiarities observed in the neuronal activity at changes of temperature correlate with a change of characteristics of locomotor behavior of L. stagnalis.     

The role of the vagus nerve in the generation of cardiorespiratory interactions in a neotropical fish,the pacu, <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis>

The role of the vagus nerve in determining heart rate (f _H) and cardiorespiratory interactions was investigated in a neotropical fish, Piaractus mesopotamicus. During progressive hypoxia f _H initially increased, establishing a 1:1 ratio with ventilation rate (f _R). Subsequently there was a hypoxic bradycardia. Injection of atropine abolished a normoxic inhibitory tonus on the heart and the f _H adjustments during progressive hypoxia, confirming that they are imposed by efferent parasympathetic inputs via the vagus nerve. Efferent activity recorded from the cardiac vagus in lightly anesthetized normoxic fish included occasional bursts of activity related to spontaneous changes in ventilation amplitude, which increased the cardiac interval. Restricting the flow of aerated water irrigating the gills resulted in increased respiratory effort and bursts of respiration-related activity in the cardiac vagus that seemed to cause f _H to couple with f _R. Cell bodies of cardiac vagal pre-ganglionic neurons were located in two distinct groups within the dorsal vagal motor column having an overlapping distribution with respiratory motor-neurons. A small proportion of cardiac vagal pre-ganglionic neurons (2%) was in scattered positions in the ventrolateral medulla. This division of cardiac vagal pre-ganglionic neurons into distinct motor groups may relate to their functional roles in determining cardiorespiratory interactions.     

Effects of hypoxia on oxygen consumption,swimming velocity and gut evacuation in southern bluefin tuna (<Emphasis Type="Italic">Thunnus maccoyii</Emphasis>)

Of the few measurements of the behavioural and physiological responses of tuna to hypoxia, most are restricted to shallow diving tropical species. Furthermore, when wild tuna experience low dissolved oxygen, they are likely to have an increased oxygen demand associated with the metabolic cost of food digestion and assimilation (specific dynamic action). However the response of postprandial tuna to hypoxia has never been examined. This study focuses on the metabolic and behavioural responses of both fasted and postprandial southern bluefin tuna (Thunnus maccoyii) to low dissolved oxygen. Fasted T. maccoyii were exposed to dissolved oxygen levels of 4.44, 3.23, 2.49 and 1.57 mg·l⁻¹ for 20–21 h. In moderate hypoxia (4.44 and 3.23 mg·l⁻¹), swimming speed was enhanced (1.5 and 1.3 times normoxic speed, respectively) presumably to increase ventilation volume. Routine metabolic rate (R _r ) was similarly elevated (1.3 and 1.2 times normoxic R _r , respectively), most likely due to increased metabolic demand of faster swimming. At 2.49 mg·l⁻¹, swimming speed increased to over double the normoxic speed, possibly as an escape response. At 1.57 mg·l⁻¹, both swimming speed and R _r were reduced (0.8 and 0.9 times normoxic level, respectively), and tuna failed to survive the entire 20 h exposure period. This reveals that the critical oxygen level of T. maccoyii is between 1.57 and 2.49 mg·l⁻¹, demonstrating that they are remarkably well adapted to low dissolved oxygen. Feeding did not greatly influence their hypoxia tolerance with tuna surviving exposure to dissolved oxygen levels of 2.96 and 1.81 mg·l⁻¹ for 21 h, after ingesting a ration of 6.7% body weight of sardines (Sardinops sagax). In a subsequent experiment to determine the effects of hypoxia on digestion rate, T. maccoyii were fed to satiation and exposed to a dissolved oxygen level of 2.84 mg·l⁻¹ for 6.5–8 h. There was no significant difference in swimming speed, R _r and gastric evacuation rates of tuna in hypoxia compared to those in normoxia. This demonstrates that in moderate to severe hypoxia, T. maccoyii are still capable of aerobically supporting maintenance metabolism, routine swimming and specific dynamic action. It is hypothesized that adaptations which support the large metabolic scope of tuna are also likely to be beneficial for oxygen extraction and delivery in conditions of hypoxia.     

Potential Protection of Curcumin against Hypoxia-induced Decreases in Beta-III Tubulin Content in Rat Prefrontal Cortical Neurons

The central nervous system (CNS) is highly dependent on adequate supply of oxygen and is sensitive to hypoxia. It is known that hypoxia induces injuries on the brain tissue and the neuronal activity. Curcumin, a yellow pigment obtained from the rhizome of C. longa Linn., has been regarded as a multi-functional drug with antioxidative activity. In the present study, we first demonstrated a significant decrease in the content of β-III tubulin protein in rat prefrontal cortex (PFC) tissues induced by repeated hypoxia, but not in rat cerebellum tissue. These suggest a relatively higher sensitivity and probably a higher vulnerability of rat PFC tissue to hypoxia in vivo. We reconfirmed the effect of hypoxia to primary cultured neurons from rat PFC and found a significant decrease in the contents of β-III tubulin protein after chronic exposure to hypoxia. Moreover, we demonstrated that the hypoxia-induced decrease in β-III tubulin protein content could be restored by curcumin, suggesting a potential protection of curcumin against hypoxia-induced decreases in beta-III tubulin content in rat PFC neurons. Special issue article in honor of Dr. Ji-Sheng Han.     

Physiological effects of hypoxia in developing and adult honeybees <Emphasis Type="Italic">Apis mellifera</Emphasis> L.

The effect of hypoxia on activity of metabolism was studied in developing and adult honeybees. The feedback was established between the O₂ deficit in the gas medium and its consumption by honeybees. An elevation of temperature within the limits of vital diapason for development of honeybees activated the O₂ consumption regardless of the hypoxia level. A prolonged action of hypoxia on the honeybee physiological state was revealed. There was shown the existence of convergent similarity between the effect of O₂ deficit on honeybees and on homoiothermal animals.     

Ocean warming and hypoxia affect embryonic growth,fitness and survival of small-spotted catsharks,Scyliorhinus canicula

Elasmobranchs are key to a healthy marine ecosystem but are under threat from human activities, such as destructive fisheries and shark finning. Embryos of oviparous elasmobranchs may be further challenged during development by rising temperatures and falling dissolved oxygen concentrations in their intertidal environment. However, the impact of climate change on survival and growth of oviparous elasmobranchs is still poorly understood. Here, we investigate the effects of temperature and hypoxia on the growth and survival of small-spotted catshark (Scyliorhinus canicula) embryos by incubating eggs in normoxia 15°C, normoxia 20°C, hypoxia 15°C, or hypoxia 20°C. Incubation under the elevated temperature increased the embryonic growth rate, yolk consumption rate and Fulton's condition factor at hatching, whilst decreasing the total length and body mass of newly hatched sharks. Under low oxygen conditions (50% air saturation) the survival rate of S. canicula embryos dropped significantly and the temperature-induced increase in Fulton's condition factor was reversed. Together, these data demonstrate both the individual and compound effects of elevated temperature and hypoxia on the survival and growth during early ontogeny of a ubiquitous, coastal elasmobranch, S. canicula.     

Combination of elevated CO<Subscript>2</Subscript> concentration and elevated temperature and elevated temperature only promote photosynthesis of <Emphasis Type="Italic">Quercus mongolica</Emphasis> seedlings

One-year-old oak (Quercus mongolica Fisch.) seedlings were grown in growth chambers for 30 days to investigate the effects of the combination of elevated CO₂ concentration ([CO₂], 700 μmol/mol) and temperature (ambient T + 4°C) and only elevated temperature (ambient T +4°C) on leaf gas exchange, chlorophyll a fluorescence, and chlorophyll content. In the growth chambers, natural conditions of the Maoershan mountain regions of Heilongjiang Province (45–46°N, 127–128°E) of China for the average growth season were simulated. The results showed that the maximum net photosynthetic rate (P _Nmax) was ≈ 1.64 times greater at elevated temperature than at ambient temperature. The irradiance saturation point (I _s), apparent quantum yield (AQY), maximum photosystem II efficiency (F _v/F _m), and chlorophyll content significantly increased, while irradiance compensation point (I _c) was not affected by elevated temperature. The combination of elevated [CO₂] and temperature also significantly increased P _Nmax by approximately 34% but much lower than that under elevated temperature only. In the case of factor combination, dark respiration (R _d), I _c, F _v/F _m, and total chlorophyll content increased significantly, while I _s and AQY were not affected. Moreover, under elevated [CO₂] and temperature, R _d and I _c, F _v/F _m were significantly higher than under elevated temperature only. The results indicated that the combination of elevated [CO₂] and temperature expected in connection with the further global climate change may affect carbon storage of the coenotype of Q. mongolica in this region of China. This text was submitted by the authors in English.     

Octopamine-containing (OC) interneurons enhance central pattern generator activity in sucrose-induced feeding in the snail <Emphasis Type="Italic">Lymnaea</Emphasis>

In the pond snail Lymnaea stagnalis octopamine-containing (OC) interneurons trigger and reconfigure the feeding pattern in isolated CNS by excitation of the central pattern generator. In semi-intact (lip–mouth—CNS) preparations, this central pattern generator is activated by chemosensory inputs. We now test if sucrose application to the lips activates the OC neurons independently of the rest of the feeding central pattern generator, or if the OC interneuron is activated by inputs from the feeding network. In 66% of experiments, sucrose stimulated feeding rhythms and OC interneurons received regular synaptic inputs. Only rarely (14%) did the OC interneuron fire action potentials, proving that firing of OC interneurons is not necessary for the sucrose-induced feeding. Prestimulation of OC neurons increased the intensity and duration of the feeding rhythm evoked by subsequent sucrose presentations. One micromolar octopamine in the CNS bath mimicked the effect of OC interneuron stimulation, enhancing the feeding response when sucrose is applied to the lips. We conclude that the modulatory OC neurons are not independently excited by chemosensory inputs to the lips, but rather from the buccal central pattern generator network. However, when OC neurons fire, they release modulatory octopamine, which provides a positive feedback to the network to enhance the sucrose-activated central pattern generator rhythm.     

Production of reactive oxygen species and antioxidant enzymes of pea and soybean plants under hypoxia and high CO<Subscript>2</Subscript> concentration in medium

Generation of reactive oxygen species (ROS) and activities of antioxidant enzymes (catalase, peroxidase, ascorbate peroxidase) in pea (Pisum sativum L.) and soybean (Glycine max L.) under hypoxia (3–24 h) and high CO₂ concentration in medium were studied. In sensitive to hypoxia pea seedlings, hypoxia enhanced markedly production of superoxide anion-radical, hydroperoxides, and especially hydrogen peroxide. In more tolerant soybean plants, these changes were less pronounced. During first hours of hypoxia, activity of lipoxygenase in plant cells increased. This allows a suggestion that this enzyme is involved in the processes of hydroperoxide accumulation in plant tissues under oxygen deficit. In pea and soybean plants, a correlation between tolerance to hypoxia, the rate of ROS generation, and antioxidant enzyme activities was established. During the first hours of hypoxia, the catalase activity in soybean plants increased stronger than in sensitive to hypoxia pea plants. At longer exposure to hypoxia (24 h), peroxidases started to play the higher role in cell defense against hypoxia, but only in soybean plants. The medium with the higher CO₂ content induced higher changes in the processes of ROS accumulation and activities of lipoxygenase and antioxidant enzymes. This permits us to refer CO₂, accumulated as a product of respiration in the cells, to low-molecular signal molecules switching on plant adaptation to hypoxic stress.