Christine Huchzermeyer

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.

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.

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% O2 gas mixture. We combined recordings of local field potential and interstitial partial oxygen pressure (pO2) 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 pO2 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 pO2 as observed previously with a 95% O2 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 (pO2) and NAD(P)H fluorescence. Blockade of postsynaptic receptors reduced ion fluxes as well as pO2 and NAD(P)H transients by ~50%. Additional blockade of transmitter release further reduced Na+, K+, and pO2 transients by ~30% without altering presynaptic APs, indicating considerable contribution of Ca2+-removal, transmitter and vesicle turnover to energy consumption.

Long-range temporal correlations in the subthalamic nucleus of patients with Parkinson’s disease

Neuronal activity in the subthalamic nucleus (STN) of patients with Parkinson’s disease (PD) is characterised by excessive neuronal synchronization, particularly in the beta frequency range. However, less is known about the temporal dynamics of neuronal oscillations in PD. In this respect long‐range temporal correlations (LRTC) are of special interest as they quantify the neuronal dynamics on different timescales and have been shown to be relevant for optimal information processing in the brain. While the presence of LRTC has been demonstrated in cortical data, their existence in deep brain structures remains an open question. We investigated (i) whether LRTC are present in local field potentials (LFP) recorded bilaterally from the STN at wakeful rest in ten patients with PD after overnight withdrawal of levodopa (OFF) and (ii) whether LRTC can be modulated by levodopa treatment (ON). Detrended fluctuation analysis was utilised in order to quantify the temporal dynamics in the amplitude fluctuations of LFP oscillations. We demonstrated for the first time the presence of LRTC (extending up to 50 s) in the STN. Importantly, the ON state was characterised by significantly stronger LRTC than the OFF state, both in beta (13–35 Hz) and high‐frequency (> 200 Hz) oscillations. The existence of LRTC in subcortical structures such as STN provides further evidence for their ubiquitous nature in the brain. The weaker LRTC in the OFF state might indicate limited information processing in the dopamine‐depleted basal ganglia. The present results implicate LRTC as a potential biomarker of pathological neuronal processes in PD.

Functional and effective connectivity in subthalamic local field potential recordings of patients with Parkinson’s disease

In Parkinsons disease (PD) levodopa-associated changes in the power and long-range temporal correlations of beta oscillations have been demonstrated, yet the presence and modulation of genuine connectivity in local field potentials (LFP) recorded from the subthalamic nucleus (STN) remains an open question. The present study investigated LFP recorded bilaterally from the STN at wakeful rest in ten patients with PD after overnight withdrawal of levodopa (OFF) and after a single dose levodopa administration (ON). We utilized connectivity measures being insensitive to volume conduction (functional connectivity: non-zero imaginary part of coherency; effective connectivity: phase-slope index). We demonstrated the presence of neuronal interactions in the frequency range of 10-30 Hz in STN-LFP without a preferential directionality of interactions between different contacts along the electrode tracks. While the direction of neuronal interactions per se was preserved after levodopa administration, functional connectivity and the ventral-dorsal information flow were modulated by medication. The OFF-ON differences in functional connectivity were correlated with the levodopa-induced improvement in clinical Unified Parkinsons Disease Rating Scale scores. We hypothesize that regional neuronal interactions, as reflected in STN-LFP connectivity, might represent a basis for the intra-nuclear spatial specificity of deep brain stimulation. Moreover, our results suggest the potential use of volume conduction-insensitive measures of connectivity in STN-LFP as a marker of clinical motor symptoms in PD.

Interhemispheric functional interactions between the subthalamic nuclei of patients with Parkinson's disease

Parkinsons disease (PD) is characterized by widespread neural interactions in cortico‐basal‐ganglia networks primarily in beta oscillations (approx. 10–30 Hz), as suggested by previous findings of levodopa‐modulated interhemispheric coherence between the bilateral subthalamic nuclei (STN) in local field potential recordings (LFPs). However, due to confounding effects of volume conduction the existence of ‘genuine’ interhemispheric subcortical coherence remains an open question. To address this issue we utilized the imaginary part of coherency (iCOH) which, in contrast to the standard coherence, is not susceptible to volume conduction. LFPs were recorded from eight patients with PD during wakeful rest before and after levodopa administration. We demonstrated genuine coherence between the bilateral STN in both 10–20 and 21–30 Hz oscillations, as revealed by a non‐zero iCOH. Crucially, increased iCOH in 10–20 Hz oscillations positively correlated with the worsening of motor symptoms in the OFF medication condition across patients, which was not the case for standard coherence. Furthermore, across patients iCOH was increased after levodopa administration in 21–30 Hz oscillations. These results suggest a functional distinction between low and high beta oscillations in STN‐LFP in line with previous studies. Furthermore, the observed functional coupling between the bilateral STN might contribute to the understanding of bilateral effects of unilateral deep brain stimulation. In conclusion, the present results imply a significant contribution of time‐delayed neural interactions to interhemispheric coherence, and the clinical relevance of long‐distance neural interactions between bilateral STN for motor symptoms in PD.

Muscarinic receptor activation determines the effects of store-operated Ca2+-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.

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.