Oxygen consumption in the nervous system

Neuronal activity in the brain depends to a large extent on adenosine triphosphate generation and thus on the availability of oxygen. This makes oxygen a highly relevant readout for studying neuronal metabolism. To evaluate the dependency of neuronal activity on oxygen availability, semi-intact in vitro preparations of Xenopus laevis tadpoles with functional central and peripheral nervous systems were studied. Trochlear motor nerve spike discharge served as a physiological correlate for neuronal activity. O2-concentrations in the bath chamber and the brain were concurrently monitored using Clark-type oxygen microsensors during superfusion of Ringer solution with various concentration levels of oxygen. The O2-concentration was accurately set to a defined value by aeration with carbogen (95 % O2, 5 % CO2) or nitrogen. In air-saturated Ringer solutions (290 μmol/l O2), the IVth ventricle was devoid of oxygen due to consumption by adjacent brain tissue and an O2-concentration of zero was measured. At elevated oxygen bath concentrations of >290 μmol/l, the ventricular oxygen level was considerably augmented (> 0) while spontaneous burst discharge of the trochlear nerve caused a transient drop of the oxygen level within the IVth ventricle, indicating a neuronal activity-related increase in the demand for oxygen. In contrast, decreasing the concentration of oxygen in the Ringer solution below ∼40μmol/l completely ceased trochlear motor nerve activity. Oxygen delivery is limited by metabolic processes and diffusion, which are often impaired following injury in the brain, largely due to scar tissue formation or caused by associated diseases, such as lung impairments or during stroke. A good model for pathological condition are in vitro experiments, as oxygen delivery through the blood is absent. Therefore alternative delivery methods are required. Aiming at a spatially more accurate and faster means for the modulation of the oxygen level in the brain, the natural capability of algae and cyanobacteria to produce oxygen upon visible light illumination via photosynthesis was exploited. Injection of the green algae Chlamydomonas reinhardtii or the cyanobacteria Synechocystis sp. into the vascular system of Xenopus tadpoles prior to the generation of the semi-intact preparation distributed these single celled organisms throughout the vasculature of the entire brain. This new approach is termed ’Symbiotic Oxygen Supply’ (SOS). External induction of hypoxia caused an oxygen depletion within the IVth ventricle and a subsequent trochlear motor nerve activity abolishment. Illumination with visible light activated algal photosynthesis and increased the oxygen level in the brain, leading to a restart of motor nerve activity upon illumination within about 20 min. This suggests that SOS is sufficient to restore energy equivalents required for maintained neuronal activity in oxygen depleted environments. Accordingly, introduction of algae or cyanobacteria and illumination represents a promising method to augment the oxygen level in any diffusion-limited in vitro neuronal preparation devoid of a functional circulation and potentially also under in vivo conditions

Relationship between oxygen consumption and neuronal activity in a defined neural circuit

Background: Neuronal computations related to sensory and motor activity along with the maintenance of spike discharge, synaptic transmission, and associated housekeeping are energetically demanding. The most efficient metabolic process to provide large amounts of energy equivalents is oxidative phosphorylation and thus dependent on O2 consumption. Therefore, O2 levels in the brain are a critical parameter that influences neuronal function. Measurements of O2 consumption have been used to estimate the cost of neuronal activity; however, exploring these metabolic relationships in vivo and under defined experimental conditions has been limited by technical challenges. Results: We used isolated preparations of Xenopus laevis tadpoles to perform a quantitative analysis of O2 levels in the brain under in vivo-like conditions. We measured O2 concentrations in the hindbrain in relation to the spike discharge of the superior oblique eye muscle-innervating trochlear nerve as proxy for central nervous activity. In air-saturated bath Ringer solution, O2 levels in the fourth ventricle and adjacent, functionally intact hindbrain were close to zero. Inhibition of mitochondrial activity with potassium cyanide or fixation of the tissue with ethanol raised the ventricular O2 concentration to bath levels, indicating that the brain tissue consumed the available O2. Gradually increasing oxygenation of the Ringer solution caused a concurrent increase of ventricular O2 concentrations. Blocking spike discharge with the local anesthetics tricaine methanesulfonate diminished the O2 consumption by ~ 50%, illustrating the substantial O2 amount related to neuronal activity. In contrast, episodes of spontaneous trochlear nerve spike bursts were accompanied by transient increases of the O2 consumption with parameters that correlated with burst magnitude and duration. Conclusions: Controlled experimental manipulations of both the O2 level as well as the neuronal activity under in vivo-like conditions allowed to quantitatively relate spike discharge magnitudes in a particular neuronal circuitry with the O2 consumption in this area. Moreover, the possibility to distinctly manipulate various functional parameters will yield more insight in the coupling between metabolic and neuronal activity. Thus, apart from providing quantitative empiric evidence for the link between physiologically relevant spontaneous spike discharge in the brain and O2-dependent metabolism, isolated amphibian preparations are promising model systems to further dissociate the O2 dynamics in relation to neuronal computations

Transcardial injection and vascular distribution of microalgae in Xenopus laevis as means to supply the brain with photosynthetic oxygen

Oxygen in vertebrates is generally provided through respiratory organs and blood vessels. This protocol describes transcardial injection, vascular distribution, and accumulation of phototrophic microalgae in the brain of Xenopus laevis tadpoles. Following tissue isolation, oxygen dynamics and neuronal activity are recorded in semi-intact whole-head preparations. Illumination of such microalgae-filled preparations triggers the photosynthetic production of oxygen in the brain that, under hypoxic conditions, rescues neuronal activity. This technology is potentially able to ameliorate consequences of hypoxia under pathological conditions. For complete details on the use and execution of this protocol, please refer to Özugur et al. (2021)

Quantification of synapse loss in human brains with mixed Alzheimer’s and Lewy body disease

Synaptische Plastizität und hohe Synapsen Dichte werden gefördert und aufgebaut während des Lernens, während Spine- und Verbindungsverlust die Kennzeichen für Neurodegenerative Erkrankungen wie die Alzheimer Krankheit sind. Viele Faktoren könnten verantwortlich sein für den Spine Verlust, wie ein veränderter pre synaptischer Input oder externe Faktoren und es ist nicht klar ob die pre synaptische Fehlfunktion oder die Postsynaptischen Veränderungen zuerst auftreten. Dennoch ist der Synapsen Verlust nicht spezifisch um Alzheimer vorherzusagen. Viele neurodegenerative Erkrankungen zeigen diese Verluste, ebenso wie es eine Erscheinung des normalen Alterns ist. Jedoch ist es der Haupt Risikofaktor für Demenz. Synapsen Verlust ist nicht gleich verteilt in allen Gehirnregionen, was bedeutet, dass alters- abhängige Veränderungen nicht überall auftreten, aber in bestimmten Regionen, wie im Kortex und im Hippocampus. Körpereigene Kompensationsmechanismen, die den Spine Verlust entgegenwirken und synaptische Kontakte erhalten, können nur bedingt gegen die Neurodegeneration halten. Die Glutamat Freisetzung aus Vesikeln ist der Haupt Signalweg von anregenden Synapsen im Säuger und somit wichtig für die neuronale Aktivität. GABA ist der bedeutendste hemmende Neurotransmitter, bindet auf Rezeptoren der Postsynapse und leitet hemmendes Signal ein. Doch gibt es Unterschiede in den verschiedenen Synapsen-Typen? Und welche sind in Krankheit eher betroffen? Um diese Fragen zu beantworten, erregende und hemmende Synapsen wurden evaluiert um mittels typischen synaptischen Markern spezifisch markiert: GABAR- α2 für die hemmenden Synapsen und PSD-95 (DLG4) für die Postsynapse der erregenden Synapsen. Antikörper auf menschlichem Paraffin fixierten Schnitten von 15 verschiedenen Hirnregionen, in Alzheimer, Kontrolle und Lewy-Körper-Demenz, wurden mittels Konfokal Mikroskop (Zeiss LSM 780) detektiert und mit Hilfe von IgorPro7 einzelne Synapsen ausgewertet. Der stärkste Synapsen Verlust war in den hemmenden Synapsen des Entorhinal Kortex von Alzheimer Patienten zu verzeichnen. Für die hemmenden Synapsen waren alle Synapsen Rückgänge signifikant, hingegen die Zahlen bei den erregenden Synapsen wenig signifikant waren und teilweise sogar zunahmen. Für die Lewy-Körper-Demenz konnte auch keine signifikante Korrelation gefunden werden.Synapses mediate the communication between neurons and their function alters during physiological processes such as learning and memory. Cognitive decline in neurodegenerative disease has been shown to result from loss of synapses. However, previous studies only analyzed specific types of synapses in one or few specific brain regions. The aim of this study was to map the loss of excitatory and inhibitory synapses distinct brain regions in the brains of patients suffering from Alzheimer’s disease and Lewy body disease. I analyzed 67 brains from donors at the Newcastle Brain Tissue Resource, which had been neuropathologically classified as Alzheimer's disease, Lewy body disease or controls. Tissue microarrays from several brain regions were immunofluorescence labelled with antibodies against DLG4 (also known as PSD-95 scaffold protein) to stain excitatory synapses and GABRA2 (GABAA Receptor subunit α2) to stain inhibitory synapses. Images were recorded on a confocal microscope and synapse densities were quantified automatically using custom- written image analysis algorithms. I found different patterns of synapse loss, depending on the disease. Particularly in Alzheimer’s disease brains, there was a marked loss of inhibitory synapses in several brain regions, including the entorhinal cortex, temporal and parietal cortex, cingulate, visual cortex, putamen and pallidum. This loss of inhibitory synapses may cause disruption of long-range neuronal networks, which has been demonstrated in several animal studies

Green oxygen power plants in the brain rescue neuronal activity.

Neuronal activity in the brain depends on mostly aerobic generation of energy equivalents and thus on a constant O2 supply. Oxygenation of the vertebrate brain has been optimized during evolution by species-specific uptake and transport of O2 that originally derives from the phototrophic activity of prokaryotic and eukaryotic organisms in the environment. Here, we employed a concept that exploits transcardial injection and vascular distribution of unicellular green algae or cyanobacteria in the brain of Xenopus laevis tadpoles. Using oxygen measurements in the brain ventricle, we found that these microorganisms robustly produce sizable amounts of O2 upon illumination. In a severe hypoxic environment, when neuronal activity has completely ceased, the photosynthetic O2 reliably provoked a restart and rescue of neuronal activity. In the future, phototrophic microorganisms might provide a novel means to directly increase oxygen levels in the brain in a controlled manner under particular eco-physiological conditions or following pathological impairments