Potentiometric monitoring of the redox state of brain structures of freely-moving rats in sleep-wake cycles

Variations of brain tissue redox state potential (E) of freely-moving white rats (300-350 g) in cycles of wakefulness (W), slow-wave sleep (SWS), and paradoxical sleep (PS) were measured by platinum electrodes symmetrically implanted into the frontal and occipital cortices and hippocampus. In addition, EMG of neck muscles and general motor activity of animals were recorded. The common reference electrode was implanted in the nasal bone. It was shown that in some brain sites (called active), episodes of W and PS were accompanied by a rise of E, and during transitions from W and PS to SWS, E dropped. The value of E varied in the range of 100 mV. It is suggested that transitions from W and PS to SWS are accompanied by shifts in the balance between the main energy sources. Oxidative phosphorylation prevails in W and PS, whereas aerobic glycolysis is the main source of energy during SWS. We think that this suggestion is supported both by a decrease in E in SWS and its oscillations typical of glucolytic processes [Aon et al., 1992]. Recent literature data [Bitter et al., 1996] suggest that astroglia is the main compartment for aerobic glycolysis.     

Changes in the rabbit brain cortex redox potential accompaning episodes of ECoG-arousal during slow-wave sleep

Local cortex E variations are well expressive indices of rate and peculiarities of energy metabolism. The brain E is determined by the ratio of processes occurring in two energy compartments in glycolysis, in whish glucose is split without oxygen utilization and in oxidative metabolism. In the present investigation, the brain cortex E changes were recorded with implanted platinum electrodes during slow wave sleep. Under such conditions, the E lowering detects acceleration in glycolytic compartment, whereas the E local rising shows acceleration in oxidative metabolism in the tissue surrounding the electrode. Earlier in rats, we have found that E significantly lowered in metabolic active cortical sites during episodes of SWS, and supposed that acceleration of glycolysis increased. Slow oscillations (a 20-40-sec prolongation of the amplitude up to several dozens millivolts) appeared at the same time. We considered these E slow oscillations to reflect changes in the rate in compartment of glycolysis. In this research, we have found the E slow oscillations to be created by regular episodes of ECoG-arousal which were accompanied by E decreases, i. e. by acceleration in glycolysis. We think the data presented show existence of functional system supporting a low level of arousal. As in any complex system with feed back connections, this system works in oscillatory regime.     

Local changes in the rabbit brain cortex redox potential accompanying the formation of a conditioned defensive reflex

The brain E is determined by ratio in rate of processes occuring in two energy compartments--in glycolysis (the more ancient one in evolution) in which glucose is splitted whithout oxygen utilization, and in oxidative metabolism which is younger in evolution than glycolysis and more effective than glycolysis. In the present investigation, the brain cortex E changes were recorded with implanted platinum electrodes. CDR was established by combination of light and electric shock applied to the left ear. It has been found that the combinations started to be accompanied by the E shift after the first 5-20 combinations. The E shifts were widely generalized over the cortex, and both increasing and decreasing E were well expressed within 50-200 combinations. As the number of combination increased, the increases in E were gradually replaced by the decreases in E. This dynamic in the balance of the major sources of the brain energy supply during the formation of CDR demonstrates, in our opinion, that subcellular structures or complexes of cells which appeared at the same stages of evolution as the compartment of oxidative metabolism make a significant contribution to the CDR acquisition when memory traces are created, while brain function during realization of well consolidated CDR are supported mainly by glycolysis.     

Oscillations in the oxidation-reduction potential of the brain tissue in rats developing during wakefulness and slow-wave sleep

Variations of the brain cortex redox state potential (E) were recorded in freely moving white rats (mass of 300-350 g) with implanted platinum electrodes (with the platinum reference electrode in the nasal bone) during sleep-wake cycles. It was found that transitions from the slow-wave sleep to wakefulness were accompanied in the number of cortical areas (metabolic-active sites) by the E rise, while the transitions from the wakefulness to slow-ware sleep were associated with a drop of E. However, the episodes of the short-term arousals during the slow-wave sleep were accompanied by the respective decreases in E thus forming the irregular E variations (1.5-3 min in duration). It was also found that the oscillations of a typical pattern (quasisinusoidal with the frequency of 10-20 osc/min and the amplitudes up to several mV) could take place in the metabolic-active cortical sites. These oscillations were defined as fast E oscillations. During the slow-wave sleep, the less regular oscillations with the lower frequency (1.2-10 osc/min) and higher amplitude were recorded in the same cortical sites. These oscillations were defined as slow. It is suggested that the fast metabolic oscillations of wakefulness are mainly controlled by the mitochondria of neuronal populations, whereas the slow metabolic oscillations which occur in the slow-wave sleep are related with glycolysis in populations of glial cells.     

The modelling of the glycolytic oscillations in the potential of the oxidative-reductive status of the brain tissue in waking and anesthetized rats]

It was found that chemical hypoxia created by intraperitoneal injection of potassium cyanide (5-7 mg/kg) induced in both waking and anaesthetized (pentobarbital, 40 mg/kg) albino rats a significant decrease in the brain redox state potential (E) monitored with platinum electrodes. This decrease could be accompanied by a generation in some brain points of local chains of gradually damped quasisinusoidal E oscillations. Such oscillations were more expressed in waking than in anaesthetized animals. The frequency range of these oscillations was 4-7 cycles/min. This is the range of overlapping frequency ranges characteristic for the high level of vigilance (5-20 cycles/min) and slow-wave sleep and drowsiness (1.5-6 cycles/min). The amplitude of the observed oscillations was close to the maximal amplitude of the brain E oscillations characteristic for the high level of vigilance (up to several mV). The obtained evidence favors our suggestion that behavior-related E oscillations are formed by the oscillations in the redox balance of glycolysis. The similarity of the normal physiological oscillations and those simulated by us under abnormal conditions suggest a certain common mechanism of their generation.     

Duration of changes in electrical characteristics of command neurons after defensive conditioning in snails

Electrical characteristics of snail command neurons were studied during and after defensive conditioning. Tapping on the shell was used as a conditioned stimulus. A light air blow into the lung cavity orifice (pneumostome) was used as an unconditioned stimulus. The conditioned defensive reflex is known to be retained for 40 days. We have shown earlier the decrease in membrane and threshold potentials of command neurons after defensive conditioning (Gainutdinov et al., 1996). In these experiments it has been found that the decreased level of membrane and threshold potentials are maintained during 40 days after defensive reflex conditioning.     

Electrographic registration of metabolic changes in the cerebral cortex evoked by sensory stimuli]

In chronic experiments on rabbits, sensory stimuli from platinum electrodes, located on pia mater in different parts of the cerebral cortex, may produce negative changes of the electrode potential or bioelectrochemical potential (BEChP) lasting from one to dozens of seconds with an amplitude from tenths of millivolts to several millivolts. Sometimes the amplitude of BEChP changes amounted to dozens of millivolts. When recorded from dura mater the changes were much smaller, and in the bone they could not be detected. The evoked BEChP changes appeared only against the background of high initial negative values of the electrode potential (2-3 mv and more) and when the electrodes were not polarized by the amplifier input. A low resistance at the amplifier input contributed to the appearance of evoked BEChP changes. The BEChP change is a complex phenomenon. On one hand, it involves a change of the electrode potential proper which proceeds from shifts in the redox systems (it is recorded both on platinum and golden electrodes) and from changes in the metabolites' content (to which platinum electrodes alone are selectively sensitive). On the other hand, the participation of polarographic processes is also probable. The BEChP changes differ by their latency and by their pattern from the shifts of the DC potential level; this possibly shows that the two kinds of nervous activity manifestaions are related to different aspects of brain tissue activity.     

Shifts in steady potential levels and changes in the cerebral bioelectrochemical potential during orientation and conditioned reflexes in rabbits]

The shifts of the steady potential level (SPL), recorded with non-polarized electrodes, and the changes in bioelectrochemical potential (BEChPs), recorded with platinum electrodes, were led from the rabbit brain surface in chronic experiments. The stimuli, that were new for the animal, caused only SPL shifts (0.1-0.3 mv), BEChPs showing no changes. BEChPs changed (by tenths to several millivolts) only in the process of the conditioned reflex formation, during the pairing of the conditioned and reinforcing stimuli, during which the shifts of SPL were also observed (up to 0.5-1 mv), different from the ones during the orienting reflex. Simultaneous recording of the SPL shifts and the changes in BEChPs showed that these phenomena are external manifestations of independent processes. It is suggested that the brain activity involved in the perception and the analysis of the informational value of a new stimulus, is connected with bioelectrical processes, rather than with the metabolic ones. During the formation of the defensive conditioned reflex, along with the enhancing of the activity connected with bioelectrical processes, other type of activity appears which is accompanied with considerable metabolic shifts.     

Effect of Dorsal and Ventral Hippocampal Lesions on Contextual Fear Conditioning and Unconditioned Defensive Behavior Induced by Electrical Stimulation of the Dorsal Periaqueductal Gray

The dorsal (DH) and ventral (VH) subregions of the hippocampus are involved in contextual fear conditioning. However, it is still unknown whether these two brain areas also play a role in defensive behavior induced by electrical stimulation of the dorsal periaqueductal gray (dPAG). In the present study, rats were implanted with electrodes into the dPAG to determine freezing and escape response thresholds after sham or bilateral electrolytic lesions of the DH or VH. The duration of freezing behavior that outlasted electrical stimulation of the dPAG was also measured. The next day, these animals were subjected to contextual fear conditioning using footshock as an unconditioned stimulus. Electrolytic lesions of the DH and VH impaired contextual fear conditioning. Only VH lesions disrupted conditioned freezing immediately after footshock and increased the thresholds of aversive freezing and escape responses to dPAG electrical stimulation. Neither DH nor VH lesions disrupted post-dPAG stimulation freezing. These results indicate that the VH but not DH plays an important role in aversively defensive behavior induced by dPAG electrical stimulation. Interpretations of these findings should be made with caution because of the fact that a non-fiber-sparing lesion method was employed.     

Energy metabolism in respiration-deficient and wild type Chinese hamster fibroblasts in culture.

This paper presents a comparison of energy metabolism in wild type and respiration-deficient Chinese hamster cells. From previous work (DeFrancesco et. al., '75) it was concluded that the mutant satisfies essentially all of its energy requirements from glycolysis and in this study we measure precisely the amount of glucose consumed and lactate produced per milligram increment of protein in exponentially growing cultures. From these measurements we calculate the amount of ATP derived from glycolysis (and hence the total energy requirement for normal proliferation) to be 105 +/- 15 mumoles ATP/delta mg protein in the mutant. It is 63 +/- 10 mumoles ATP/delta mg protein derived from glycolysis in wild type cells. We present evidence that the total energy requirement of wild type cells is similar to that of the mutant suggesting that approximately 40% of the energy requirement is derived from respiration. The oxidation of glutamine appears to be more significant than the complete oxidation of glucose to CO2 in these Chinese hamster fibroblasts. The amount of ATP required by the mutant cells per milligram increment of protein is relatively independent of pH.     

Anaerobic shift of energy metabolism in the mouse brain during the recovery period in acute radiation sickness

Three months after whole-body irradiation of mice with a sublethal dose of 5 Gy a study was made of some indices of energy metabolism like tissue respiration, oxidative phosphorylation, and formation of lactic acid in the survived brain homogenate. Revealed were the diminution of coupling of tissue respiration of oxidative phosphorylation, the rate of oxygen consumption and the level of cyano-resistant respiration being constant, the increase in the rate of glycolysis in anaerobic and particularly, in aerobic conditions, and reduction of the Pasteur and Crabtree effects. The above mentioned changes in the brain energy metabolism seem to be a manifestation of the process of the reduced metabolism formation in the nervous tissue at the remote times after irradiation.     

Regulation of Respiration and Fermentation to Control the Plant Internal Oxygen Concentration

Plant internal oxygen concentrations can drop well below ambient even when the plant grows under optimal conditions. Using pea (Pisum sativum) roots, we show how amenable respiration adapts to hypoxia to save oxygen when the oxygen availability decreases. The data cannot simply be explained by oxygen being limiting as substrate but indicate the existence of a regulatory mechanism, because the oxygen concentration at which the adaptive response is initiated is independent of the actual respiratory rate. Two phases can be discerned during the adaptive reaction: an initial linear decline of respiration is followed by a nonlinear inhibition in which the respiratory rate decreased progressively faster upon decreasing oxygen availability. In contrast to the cytochrome c pathway, the inhibition of the alternative oxidase pathway shows only the linear component of the adaptive response. Feeding pyruvate to the roots led to an increase of the oxygen consumption rate, which ultimately led to anoxia. The importance of balancing the in vivo pyruvate availability in the tissue was further investigated. Using various alcohol dehydrogenase knockout lines of Arabidopsis (Arabidopsis thaliana), it was shown that even under aerobic conditions, alcohol fermentation plays an important role in the control of the level of pyruvate in the tissue. Interestingly, alcohol fermentation appeared to be primarily induced by a drop in the energy status of the tissue rather than by a low oxygen concentration, indicating that sensing the energy status is an important component of optimizing plant metabolism to changes in the oxygen availability.Plants are obligate aerobic organisms, with oxygen being an essential substrate for mitochondrial energy production. However, the poor distribution efficiency for oxygen through root, tuber, seed, or stem tissue of various species results in steep drops of the internal oxygen concentration, ranging from values near above zero to just below 40% of air saturation (e.g. Armstrong et al., 1994; Geigenberger et al., 2000; Rolletschek et al., 2002; van Dongen et al., 2003, 2004; Vigeolas et al., 2003).These low levels of internal oxygen strongly affect plant metabolism. Several studies showed that energy-consuming metabolic pathways are adjusted to the actual oxygen availability (for review, see Geigenberger, 2003; Bailey-Serres and Voesenek, 2008). By saving energy, the plant decreases the demand for respiratory oxygen consumption that could help to postpone or even prevent the tissue from becoming anoxic. Indeed, it was observed that the metabolic flux through glycolysis slows down, respiratory oxygen consumption decreases, and adenylate levels drop in response to low internal oxygen (Geigenberger et al., 2000; Bologa et al., 2003). This inhibition of respiration is not easily explained by substrate limitation of cytochrome c oxidase (COX). First, the reduction of respiration becomes apparent already at oxygen concentrations around 20% of air saturation, whereas the K_m value for oxygen of COX lies around 0.05% of air saturation (which equals 0.14 μm under standard conditions; Drew, 1997). Second, the inhibition of respiration could be clearly distinguished from the induction of fermentation, which did not occur until oxygen fell to levels close to zero (Geigenberger, 2003).Based on these studies, it is reasonable to assume that a sensitive tuning mechanism must exist that allows the plant to regulate oxygen consumption while simultaneously preventing anoxia. However, hardly anything is known about the mechanism by which a plant induces adaptive responses to low oxygen (Bailey-Serres and Chang, 2005). Vice versa, comparably little is known about how metabolic activity affects the plant internal oxygen concentration. Previous studies focused on the effect that feeding of different sugars had on the respiration rate of tissue slices (Loef et al., 2001) or used transgenic approaches to stimulate metabolism by introducing a more energy-consuming route of Suc degradation via invertase in growing tubers (Bologa et al., 2003). In the latter case, respiration rates were increased and internal oxygen concentrations fell to very low levels that were close to zero. This shows that plant internal oxygen concentrations respond very sensitively to changes in metabolic activities. However, the underlying mechanism remains unclear.Glycolysis is part of the central backbone of primary carbohydrate metabolism and respiration. Pyruvate serves as a key metabolite linking glycolysis in the cytosol with mitochondrial respiration. Under aerobic conditions, pyruvate is transported into mitochondria and is oxidized through the tricarboxylic acid (TCA) cycle into organic acids and NADH. Moreover, pyruvate has regulatory potential, as it was shown that alternative oxidase (AOX) becomes more active in the presence of α-keto acids such as pyruvate (Millar et al., 1993, 1996; Vanlerberghe et al., 1995; Vanlerberghe and McIntosh, 1997), thus affecting the efficiency of ATP production per unit of oxygen being respired. Under oxygen-limiting conditions, pyruvate can either be converted into ethanol by pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) or to lactate by lactate dehydrogenase. Pyruvate also serves as a precursor for the synthesis of Ala via the reversible reaction catalyzed by Ala amino transferase, which was shown to play a crucial role in the rapid conversion of Ala to pyruvate during recovery from low-oxygen stress (Miyashita et al., 2007). Furthermore, pyruvate is the substrate of acetolactate synthase, which is the first enzyme committed to the biosynthesis of the branched-chain amino acids Val, Leu, and Ile. Inhibition of acetolactate synthase by the herbicide imazethapyr induces aerobic fermentation in plants (Gaston et al., 2002). Despite its central role in energy metabolism under both oxygen-rich and oxygen-depleted conditions, no investigations were made, to our knowledge, until now on the impact pyruvate has on the plant internal oxygen concentration.The aim of this study was to investigate the regulation of respiration and its relation to the plant internal oxygen concentration. To investigate this, we changed the oxygen concentration of the nutrient solution of hydroponically grown pea (Pisum sativum) plants and tested the influence of several sugars and organic acids. We measured root internal oxygen concentrations as well as the energy status of the tissue and related this to the rate of oxygen consumption by both the cytochrome c pathway and the AOX. The results are investigated in relation to the function and regulation of fermentative metabolism.     

Reexamination of Electrical Stimulation on Sarcoplasmic Reticulum Fragments In Vitro

The effect of direct electrical stimulation on suspensions of sarcoplasmic reticulum membrane fragments (SRF) was carefully re-examined using the method of Lee et al. (1966) J. Gen. Physiol. 49:689. Inhibition of Ca⁺⁺ uptake or release by electrical stimulation was observed. When platinum electrodes were used as stimulating electrodes, the effect was dependent on the total current passed through the suspension. On the contrary, when silver-silver chloride electrodes were used, no effect was observed even if voltage and current were the same as in the case of the platinum electrodes. In addition, apparent re-uptake of Ca⁺⁺ after cessation of electrical stimulation using platinum electrodes was shown to be due to a binding of Ca⁺⁺ to denatured SRF which did not require an energy supply such as ATP, although such re-uptake had been taken as strong evidence of electrical response of SRF in Lee's paper. Finally, it was concluded that the effect of electrical stimulation on SRF was attributable to the irreversible denaturation of SRF due to the oxidation caused by the chlorine generated at the platinum electrode.     

Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Sequential modification of membrane currents with classical conditioning

Pavlovian conditioning of the nudibranch mollusc Hermissenda crassicornis was previously shown to produce long-lasting reduction of two K+ currents measured across the Type B photoreceptor soma membrane (Alkon et al., 1982a; Alkon et al., 1985). Pavlovian conditioning of the rabbit was also shown to be followed by persistent K+ current reduction (Disterhoft et al., 1986). Here we report the first evidence that Ca2+ currents can also be modified by conditioning. The amplitude of the currents rather than their voltage-dependence remains reduced at least 1-2 d after conditioning (but not control procedures). Conditioning-induced changes of both K+ and Ca2+ currents increased as a function of training, the Ca2+ currents only changing substantially with greater than or equal to 250 trials. The later changes of the Ca2+ current may function to limit the magnitude of excitability increases due to associative learning.