Early adenosine release contributes to hypoxia-induced disruption of stimulus-induced sharp wave-ripple complexes in rat hippocampal area CA3.

We investigated the effects of hypoxia on sharp wave-ripple complex (SPW-R) activity and recurrent epileptiform discharges in rat hippocampal slices, and the mechanisms underlying block of this activity. Oxygen levels were measured using Clark-style oxygen sensor microelectrodes. In contrast to recurrent epileptiform discharges, oxygen consumption was negligible during SPW-R activity. These network activities were reversibly blocked when oxygen levels were reduced to 20% or less for 3 min. The prolongation of hypoxic periods to 6 min caused reversible block of SPW-Rs during 20% oxygen and irreversible block when 0% oxygen (anoxia) was applied. In contrast, recurrent epileptiform discharges were more resistant to prolonged anoxia and almost fully recovered after 6 min of anoxia. SPW-Rs were unaffected by the application of 1-butyl-3-(4-methylphenylsulfonyl) urea, a blocker of KATP channels, but they were blocked by activation of adenosine A1 receptors. In support of a modulatory function of adenosine, the amplitude and incidence of SPW-Rs were increased during application of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Interestingly, hypoxia decreased the frequency of miniature excitatory post-synaptic currents in CA3 pyramidal cells, an effect that was converted into increased frequency by the adenosine A1 agonist DPCPX. In addition, DPCPX also delayed the onset of hypoxia-mediated block of SPW-Rs. Our data suggest that early adenosine release during hypoxia induces a decrease in pre-synaptic glutamate release and that both might contribute to transient block of SPW-Rs during hypoxia/anoxia in area CA3.

Oxygen consumption rates during three different neuronal activity states in the hippocampal CA3 network.

The brain is an organ with high metabolic rate. However, little is known about energy utilization during different activity states of neuronal networks. We addressed this issue in area CA3 of hippocampal slice cultures under well-defined recording conditions using a 20% O(2) gas mixture. We combined recordings of local field potential and interstitial partial oxygen pressure (pO(2)) during three different activity states, namely fast network oscillations in the gamma-frequency band (30 to 100 Hz), spontaneous network activity and absence of spiking (action potentials). Oxygen consumption rates were determined by pO(2) depth profiles with high spatial resolution and a mathematical model that considers convective transport, diffusion, and activity-dependent consumption of oxygen. We show that: (1) Relative oxygen consumption rate during cholinergic gamma oscillations was 2.2-fold and 5.3-fold higher compared with spontaneous activity and absence of spiking, respectively. (2) Gamma oscillations were associated with a similar large decrease in pO(2) as observed previously with a 95% O(2) gas mixture. (3) Sufficient oxygenation during fast network oscillations in vivo is ensured by the calculated critical radius of 30 to 40 µm around a capillary. We conclude that the structural and biophysical features of brain tissue permit variations in local oxygen consumption by a factor of about five.

Energy demand of synaptic transmission at the hippocampal Schaffer-collateral synapse.

Neuroenergetic models of synaptic transmission predicted that energy demand is highest for action potentials (APs) and postsynaptic ion fluxes, whereas the presynaptic contribution is rather small. Here, we addressed the question of energy consumption at Schaffer-collateral synapses. We monitored stimulus-induced changes in extracellular potassium, sodium, and calcium concentration while recording partial oxygen pressure (pO(2)) and NAD(P)H fluorescence. Blockade of postsynaptic receptors reduced ion fluxes as well as pO(2) and NAD(P)H transients by ∼50%. Additional blockade of transmitter release further reduced Na(+), K(+), and pO(2) transients by ∼30% without altering presynaptic APs, indicating considerable contribution of Ca(2+)-removal, transmitter and vesicle turnover to energy consumption.

Muscarinic receptor activation determines the effects of store-operated Ca(2+)-entry on excitability and energy metabolism in pyramidal neurons.

In various cell types, depletion of intracellular Ca(2+)-stores results in store-operated Ca(2+)-entry (SOCE) across the cellular membrane. However, the effects of SOCE on neuronal membrane excitability and mitochondrial functions in central neurons are not well defined. We investigated such cellular downstream effects in pyramidal neurons of rat organotypic hippocampal slice cultures by applying electrophysiological and fluorescence imaging techniques. We report that SOCE is associated with (i) elevations of Ca(2+)-concentration in individual neuronal mitochondria ([Ca(2+)](m)). In addition, SOCE can result in (ii) hyperpolarizing neuronal membrane currents, (iii) increase in extracellular K(+)-concentration ([K(+)](o)), (iv) mitochondrial membrane depolarization, and (v) changes in intracellular redox state (NAD(P)H and FAD fluorescence), the latter reflecting responses of energy metabolism. These additional downstream effects of SOCE required concomitant muscarinic receptor activation by carbachol or acetylcholine, and were suppressed by agonist washout or application of antagonist, atropine. We conclude that muscarinic receptor activation determines the downstream effects of SOCE on neuronal membrane excitability and energy metabolism. This mechanism might have significant impact on information processing and neurometabolic coupling in central neurons.

Gamma oscillations in the hippocampus require high complex I gene expression and strong functional performance of mitochondria.

Fast neuronal network oscillations in the gamma range (~30-90 Hz) have been implicated in complex brain functions such as sensory processing, memory formation and, perhaps, consciousness, and appear to be exceptionally vulnerable to various pathologies. However, both energy demand and mitochondrial performance underlying gamma oscillations are unknown. We investigated the fundamental relationship between acetylcholine-induced gamma oscillations, mitochondrial gene expression and oxidative metabolism in hippocampal slice preparations of mouse and rat by applying electrophysiology, in situ hybridization, quantitative polymerase chain reaction, oxygen sensor microelectrode (interstitial partial oxygen pressure) and imaging of mitochondrial redox state [nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide fluorescence]. We show that (i) gamma oscillation power, oxygen consumption and expression of complex I (nicotinamide adenine dinucleotide:ubiquinone oxidoreductase) subunits are higher in hippocampal subfield CA3 than in CA1 and dentate gyrus; (ii) the amount of oxygen consumption of gamma oscillations reaches that of seizure-like events; (iii) gamma oscillations are exquisitely sensitive to pharmacological complex I inhibition; and (iv) gamma oscillations utilize mitochondrial oxidative capacity near limit. These data suggest that gamma oscillations are especially energy demanding and require both high complex I expression and strong functional performance of mitochondria. Our study helps to explain the exceptional vulnerability of complex brain functions in ischaemia as well as in neurodegenerative and psychiatric disorders that are associated with mitochondrial dysfunction.

Complex III-dependent superoxide production of brain mitochondria contributes to seizure-related ROS formation.

Brain seizure activity is characterised by intense activation of mitochondrial oxidative phosphorylation. This stimulation of oxidative phosphorylation is in the low magnesium model of seizure-like events accompanied by substantial increase in formation of reactive oxygen species (ROS). However, it has remained unclear which ROS-generating sites can be attributed to this phenomenon. Here, we report stimulatory effects of calcium ions and uncouplers, mimicking mitochondrial activation, on ROS generation of isolated rat and mouse brain mitochondria. Since these stimulatory effects were visible with superoxide sensitive dyes, but with hydrogen peroxide sensitive dyes only in the additional presence of SOD, we conclude that the complex redox properties of the 'Qo' center at respiratory chain complex III are very likely responsible for these observations. In accordance with this hypothesis redox titrations of the superoxide production of antimycin-inhibited submitochondrial particles with the succinate/fumarate redox couple confirmed for brain tissue a bell-shaped dependency with a maximal superoxide production rate at +10 mV (pH=7.4). This reflects the complex redox properties of a semiquinone species which is the direct electron donor for oxygen reduction in complex III-dependent superoxide production. Therefore, we conclude that under conditions of increased energy load the complex III site can contribute to superoxide production of brain mitochondria, which might be relevant for epilepsy-related seizure activity.

Gamma oscillations and spontaneous network activity in the hippocampus are highly sensitive to decreases in pO2 and concomitant changes in mitochondrial redox state.

Gamma oscillations have been implicated in higher cognitive processes and might critically depend on proper mitochondrial function. Using electrophysiology, oxygen sensor microelectrode, and imaging techniques, we investigated the interactions of neuronal activity, interstitial pO2, and mitochondrial redox state [NAD(P)H and FAD (flavin adenine dinucleotide) fluorescence] in the CA3 subfield of organotypic hippocampal slice cultures. We find that gamma oscillations and spontaneous network activity decrease significantly at pO2 levels that do not affect neuronal population responses as elicited by moderate electrical stimuli. Moreover, pO2 and mitochondrial redox states are tightly coupled, and electrical stimuli reveal transient alterations of redox responses when pO2 decreases within the normoxic range. Finally, evoked redox responses are distinct in somatic and synaptic neuronal compartments and show different sensitivity to changes in pO2. We conclude that the threshold of interstitial pO2 for robust CA3 network activities and required mitochondrial function is clearly above the "critical" value, which causes spreading depression as a result of generalized energy failure. Our study highlights the importance of a functional understanding of mitochondria and their implications on activities of individual neurons and neuronal networks.

ZENK expression in a restricted forebrain area correlates negatively with preference for an imprinted stimulus.

Sexual imprinting is an early learning process by which young birds acquire the characteristics of a potential sexual partner. The physiological basis of this learning process is an irreversible reduction of dendritic spines in two forebrain areas, the LNM (lateral nido-mesopallium) and the MNM (medial nido-mesopallium). The aim of the present study was to investigate whether these two brain areas are activated if the imprinted stimulus is presented to the adult bird after the end of the sensitive period. One group of zebra finch males was reared by their own parents. These birds, as adults, showed an exclusive preference for their own species in choice tests between a zebra finch and a Bengalese finch female. If exposed as adults to a zebra finch female, LNM and MNM showed lower activation, as measured by ZENK expression, compared to males exposed to a Bengalese finch female. A second group was reared by Bengalese finches and was exposed at day 100 to a zebra finch female for 1 week. As shown earlier, this regime leads to mixed choices, the birds are courting Bengalese and zebra finch females with a fixed ratio (preference score). If these birds were exposed to a zebra finch female as adults, the ZENK expression within LNM was much higher compared to group 1, and it showed a strong tendency to correlate negatively with the preference score: Birds with higher zebra finch preference showed lower activation compared to those with a low zebra finch and a high Bengalese finch preference. We propose that higher ZENK activation in group 2 is due to the rearing by a foster species which may result in a more complex neuronal network. The negative relation between activation and preference score may be explained by special properties of the LNM and MNM networks.