Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive and familiarity memories in Drosophila.

In vertebrates, several forms of memory-relevant synaptic plasticity involve postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. Here, we provide evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). We find that the nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons for appetitive memory induction but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression and likely function downstream of α5 and the postsynaptic scaffold protein discs large (Dlg). We show that postsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative (familiarity) memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla.

Sevoflurane Effects on Neuronal Energy Metabolism Correlate with Activity States While Mitochondrial Function Remains Intact.

During general anesthesia, alterations in neuronal metabolism may induce neurotoxicity and/or neuroprotection depending on the dose and type of the applied anesthetic. In this study, we investigate the effects of clinically relevant concentrations of sevoflurane (2% and 4%, i.e., 1 and 2 MAC) on different activity states in hippocampal slices of young Wistar rats. We combine electrophysiological recordings, partial tissue oxygen (ptiO2) measurements, and flavin adenine dinucleotide (FAD) imaging with computational modeling. Sevoflurane minimally decreased the cerebral metabolic rate of oxygen (CMRO2) while decreasing synaptic transmission in naive slices. During pharmacologically induced gamma oscillations, sevoflurane impaired network activity, thereby decreasing CMRO2. During stimulus-induced neuronal activation, sevoflurane decreased CMRO2 and excitability while basal metabolism remained constant. In this line, stimulus-induced FAD transients decreased without changes in basal mitochondrial redox state. Integration of experimental data and computer modeling revealed no evidence for a direct effect of sevoflurane on key enzymes of the citric acid cycle or oxidative phosphorylation. Clinically relevant concentrations of sevoflurane generated a decent decrease in energy metabolism, which was proportional to the present neuronal activity. Mitochondrial function remained intact under sevoflurane, suggesting a better metabolic profile than isoflurane or propofol.

Low neuronal metabolism during isoflurane-induced burst suppression is related to synaptic inhibition while neurovascular coupling and mitochondrial function remain intact.

Deep anaesthesia may impair neuronal, vascular and mitochondrial function facilitating neurological complications, such as delirium and stroke. On the other hand, deep anaesthesia is performed for neuroprotection in critical brain diseases such as status epilepticus or traumatic brain injury. Since the commonly used anaesthetic propofol causes mitochondrial dysfunction, we investigated the impact of the alternative anaesthetic isoflurane on neuro-metabolism. In deeply anaesthetised Wistar rats (burst suppression pattern), we measured increased cortical tissue oxygen pressure (ptiO2), a ∼35% drop in regional cerebral blood flow (rCBF) and burst-associated neurovascular responses. In vitro, 3% isoflurane blocked synaptic transmission and impaired network oscillations, thereby decreasing the cerebral metabolic rate of oxygen (CMRO2). Concerning mitochondrial function, isoflurane induced a reductive shift in flavin adenine dinucleotide (FAD) and decreased stimulus-induced FAD transients as Ca2+ influx was reduced by ∼50%. Computer simulations based on experimental results predicted no direct effects of isoflurane on mitochondrial complexes or ATP-synthesis. We found that isoflurane-induced burst suppression is related to decreased ATP consumption due to inhibition of synaptic activity while neurovascular coupling and mitochondrial function remain intact. The neurometabolic profile of isoflurane thus appears to be superior to that of propofol which has been shown to impair the mitochondrial respiratory chain.

Flavin Adenine Dinucleotide Fluorescence as an Early Marker of Mitochondrial Impairment During Brain Hypoxia.

Multimodal continuous bedside monitoring is increasingly recognized as a promising option for early treatment stratification in patients at risk for ischemia during neurocritical care. Modalities used at present are, for example, oxygen availability and subdural electrocorticography. The assessment of mitochondrial function could be an interesting complement to these modalities. For instance, flavin adenine dinucleotide (FAD) fluorescence permits direct insight into the mitochondrial redox state. Therefore, we explored the possibility of using FAD fluorometry to monitor consequences of hypoxia in brain tissue in vitro and in vivo. By combining experimental results with computational modeling, we identified the potential source responsible for the fluorescence signal and gained insight into the hypoxia-associated metabolic changes in neuronal energy metabolism. In vitro, hypoxia was characterized by a reductive shift of FAD, impairment of synaptic transmission and increasing interstitial potassium [K+]o. Computer simulations predicted FAD changes to originate from the citric acid cycle enzyme α-ketoglutarate dehydrogenase and pyruvate dehydrogenase. In vivo, the FAD signal during early hypoxia displayed a reductive shift followed by a short oxidation associated with terminal spreading depolarization. In silico, initial tissue hypoxia followed by a transient re-oxygenation phase due to glucose depletion might explain FAD dynamics in vivo. Our work suggests that FAD fluorescence could be readily used to monitor mitochondrial function during hypoxia and represents a potential diagnostic tool to differentiate underlying metabolic processes for complementation of multimodal brain monitoring.

Possible neurotoxicity of the anesthetic propofol: evidence for the inhibition of complex II of the respiratory chain in area CA3 of rat hippocampal slices.

Propofol is the most frequently used intravenous anesthetic for induction and maintenance of anesthesia. Propofol acts first and formost as a GABAA-agonist, but effects on other neuronal receptors and voltage-gated ion channels have been described. Besides its direct effect on neurotransmission, propofol-dependent impairment of mitochondrial function in neurons has been suggested to be responsible for neurotoxicity and postoperative brain dysfunction. To clarify the potential neurotoxic effect in more detail, we investigated the effects of propofol on neuronal energy metabolism of hippocampal slices of the stratum pyramidale of area CA3 at different activity states. We combined oxygen-measurements, electrophysiology and flavin adenine dinucleotide (FAD)-imaging with computational modeling to uncover molecular targets in mitochondrial energy metabolism that are directly inhibited by propofol. We found that high concentrations of propofol (100 µM) significantly decrease population spikes, paired pulse ratio, the cerebral metabolic rate of oxygen consumption (CMRO2), frequency and power of gamma oscillations and increase FAD-oxidation. Model-based simulation of mitochondrial FAD redox state at inhibition of different respiratory chain (RC) complexes and the pyruvate-dehydrogenase show that the alterations in FAD-autofluorescence during propofol administration can be explained with a strong direct inhibition of the complex II (cxII) of the RC. While this inhibition may not affect ATP availability under normal conditions, it may have an impact at high energy demand. Our data support the notion that propofol may lead to neurotoxicity and neuronal dysfunction by directly affecting the energy metabolism in neurons.

Interleukin 6-Mediated Endothelial Barrier Disturbances Can Be Attenuated by Blockade of the IL6 Receptor Expressed in Brain Microvascular Endothelial Cells.

Compromised blood-brain barrier (BBB) by dysregulation of cellular junctions is a hallmark of many cerebrovascular disorders due to the pro-inflammatory cytokines action. Interleukin 6 (IL6) is implicated in inflammatory processes and in secondary brain injury after subarachnoid hemorrhage (SAH) but its role in the maintenance of cerebral endothelium still requires a precise elucidation. Although IL6 has been shown to exert pro-inflammatory action on brain microvascular endothelial cells (ECs), the expression of one of the IL6 receptors, the IL6R is controversially discussed. In attempt to reach more clarity in this issue, we present here an evident baseline expression of the IL6R in BBB endothelium in vivo and in an in vitro model of the BBB, the cEND cell line. A significantly increased expression of IL6R and its ligand was observed in BBB capillaries 2 days after experimental SAH in mice. In vitro, we saw IL6 administration resulting in an intracellular and extracellular elevation of IL6 protein, which was accompanied by a reduced expression of tight and adherens junctions, claudin-5, occludin, and vascular-endothelial (VE-) cadherin. By functional assays, we could demonstrate IL6-incubated brain ECs to lose their endothelial integrity that can be attenuated by inhibiting the IL6R. Blockade of the IL6R by a neutralizing antibody has reconstituted the intercellular junction expression to the control level and caused a restoration of the transendothelial electrical resistance of the cEND cell monolayer. Our findings add depth to the current understanding of the involvement of the endothelial IL6R in the loss of EC integrity implicating potential therapy options.

Event-Associated Oxygen Consumption Rate Increases ca. Five-Fold When Interictal Activity Transforms into Seizure-Like Events In Vitro.

Neuronal injury due to seizures may result from a mismatch of energy demand and adenosine triphosphate (ATP) synthesis. However, ATP demand and oxygen consumption rates have not been accurately determined, yet, for different patterns of epileptic activity, such as interictal and ictal events. We studied interictal-like and seizure-like epileptiform activity induced by the GABAA antagonist bicuculline alone, and with co-application of the M-current blocker XE-991, in rat hippocampal slices. Metabolic changes were investigated based on recording partial oxygen pressure, extracellular potassium concentration, and intracellular flavine adenine dinucleotide (FAD) redox potential. Recorded data were used to calculate oxygen consumption and relative ATP consumption rates, cellular ATP depletion, and changes in FAD/FADH2 ratio by applying a reactive-diffusion and a two compartment metabolic model. Oxygen-consumption rates were ca. five times higher during seizure activity than interictal activity. Additionally, ATP consumption was higher during seizure activity (~94% above control) than interictal activity (~15% above control). Modeling of FAD transients based on partial pressure of oxygen recordings confirmed increased energy demand during both seizure and interictal activity and predicted actual FAD autofluorescence recordings, thereby validating the model. Quantifying metabolic alterations during epileptiform activity has translational relevance as it may help to understand the contribution of energy supply and demand mismatches to seizure-induced injury.

Contribution of Intrinsic Lactate to Maintenance of Seizure Activity in Neocortical Slices from Patients with Temporal Lobe Epilepsy and in Rat Entorhinal Cortex.

Neuronal lactate uptake supports energy metabolism associated with synaptic signaling and recovery of extracellular ion gradients following neuronal activation. Altered expression of the monocarboxylate transporters (MCT) in temporal lobe epilepsy (TLE) hampers lactate removal into the bloodstream. The resulting increase in parenchymal lactate levels might exert both, anti- and pro-ictogen effects, by causing acidosis and by supplementing energy metabolism, respectively. Hence, we assessed the contribution of lactate to the maintenance of transmembrane potassium gradients, synaptic signaling and pathological network activity in chronic epileptic human tissue. Stimulus induced and spontaneous field potentials and extracellular potassium concentration changes (∆[K⁺]O) were recorded in parallel with tissue pO2 and pH in slices from TLE patients while blocking MCTs by α-cyano-4-hydroxycinnamic acid (4-CIN) or d-lactate. Intrinsic lactate contributed to the oxidative energy metabolism in chronic epileptic tissue as revealed by the changes in pO2 following blockade of lactate uptake. However, unlike the results in rat hippocampus, ∆[K⁺]O recovery kinetics and field potential amplitude did not depend on the presence of lactate. Remarkably, inhibition of lactate uptake exerted pH-independent anti-seizure effects both in healthy rat and chronic epileptic tissue and this effect was partly mediated via adenosine 1 receptor activation following decreased oxidative metabolism.

A LED-based method for monitoring NAD(P)H and FAD fluorescence in cell cultures and brain slices.

Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH2) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence.

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.