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Final Report Summary - BRAINCIROXCON (Functional and structural changes of brain circuitry in altered oxygen conditions)

Project objectives:
The aim of project is to investigate the effect of both low level (hypoxia) and high level (hyperoxia) of oxygen (O2) on brain structure and functions. We use a unique combination of various neuroanatomical tools (immunostaining, Gallyas staining etc.), electrophysiological methods (single cell recording and labelling, multichannel recording) and tissue O2 concentration measurement in order to find out which neuron types are the most sensitive to the altered O2 concentration and what is the effect of the O2 concentration. The aim of the project is to understand the underlying mechanism that modifies brain functions in altered O2 environment.
To achieve the set goal the project requires four separate methodological approaches.
1) Neuroanatomy determines the cell types that are vulnerable or resistant to O2 alterations.
2) Multichannel electrophysiology recording provides information about the network activity.
3) Single cell electrophysiology and labelling depicts the change in identified neuronal activity caused by altered O2 concentration.
4) Behavioural tests allow us to investigate the effect of O2 concentration on decision making and learning.
Task 1: The most important objective was to identify the cell populations that are the most vulnerable to the changed O2 concentration. The first aim was to characterise neuronal damage caused by either hypoxia or hyperoxia with TUNEL, silver staining, and caspase-3 double staining. The TUNEL-positive cells can be both necrotic and apoptotic, while the double-marked cells are definitely apoptotic. With the Gallyas silver staining method we investigated traumatized “compacted neurons” which undergo cytoskeletal damage. The fate of silver-labelled ‘dark neurons’ is not determined, they can either recover or go through apoptotic process. We investigated the vulnerability of various interneuron and excitatory cell populations with double histological staining using interneuron markers: calcium-binding proteins (parvalbumin, calbindin and calretinin), neuropeptides (cholecystokinin, neuropeptide Y, somatostatin) and ‘dark neuron’ labelling.
Results: We successfully completed the neuroanatomical examinations and defined the period and concentration of hypoxic and hyperoxic state when the cell damage occurs. Interestingly, even acute (few hours) of mild hyperoxia (30%) can cause neuronal damage. We mapped the damaged brain areas and identified the types of the damaged inhibitory and excitatory neuron populations. The cytosceletal damage was the most characteristic among the neuronal degenerations, found mainly in the hippocampus. We further examined neuronal damage with in vivo MRI examination. We summarised the results in a manuscript titled ‘In vivo detection of hyperacute neuronal recovery by MRI in rats’ (J Magn Reson Imaging. 2016 Oct;44(4):814-22.)).
Task 2: We recorded network activity with multichannel electrodes in the hippocampus, and measured tissue oxygenation in the brain while O2 concentration was altered in the anaesthetic mask. EEG activity was recorded in normoxic (21% O2), hyperoxic (30%, 70% and 100% O2) and hypoxic (16% O2) conditions. A 32 channel multielectrode array was inserted into the hippocampus to record spontaneous electrical network activity while the tissue O2 tension was monitored with a Clark-type O2 electrode in the close vicinity of the multichannel array.
Results: We investigated the network activity change and layer connectivity thoroughly. The analysis of the recording is in progress but the preliminary observation suggests significant change in network oscillation in hyperoxic and hypoxic conditions both in theta and non-theta states. We also observed increased activity of unidentified inhibitory neurons.
Task 3: In order to determine changes in the O2-level dependent firing pattern in various neuron types, we have used a juxtacellular single cell recording method. This technique has enabled us to record the electrical activity of a single cell, and determine the neuroanatomical (axon and dendrite arborization) and neurochemical characteristic of the cell.
The examination could not be completed due to technical difficulties but the completed tests seem to confirm the results of the neuroanatomical and acute electrophysiological tests.
Throughout the examinations Dr. Pal mastered the juxtacellular single cell recording method and therefore can complete the examinations in the electrophysiological laboratory of the Szentágothai János Research Centre in Pécs.
Based on our electrophysiology recordings, we observed no neuronal damage in the prefrontal cortex. Therefore we focused our project on two different hippocampal areas (dentate gyrus and CA3a) where most of the damages occur.
Based on previous observations, we found further acute electrophysiological tests to be necessary apart from the planned examinations (Task 4), instead of behavioural examinations. We carried out further examinations on the hippocampal areas with the most significant neuronal damage and we began the electrophysiological and molecular biological (Affymetrix gene array) description of the traumatised, regenerable neurons.
The vast amount of electrophysiological data recorded is currently being analysed.

Conclusion and impact of project results
This project has helped us to understand the neuronal mechanisms in neurodegenerative clinical pictures, and define key regulation steps and direct therapy strategies. In conclusion, examining neurodegenerative clinical pictures has its actuality all over the world because the diseases they cause have an incredible impact on human life’s length and quality and these have serious financial consequences too. It can have a serious effect on the improvement of healthcare and can help to improve the life quality of the patients. The project will strengthen Europe’s scientific viewpoint and its aim and scope is in harmony with the significant proposal called “Europe 2020 Innovative Union”. Furthermore, with the effective representation and propagation of the results and the topic we would help to improve the competence and excellence of Europe through making the topic accessible for professionals, for inexpert audiences and for the youth too.
Impact of Marie Curie Fellowship on Dr. Pal’s career
After the completion of the fellowship, Dr. Pal has returned to his home institution, where he has submitted an application for promotion. Following his successful career stages he will be promoted to a senior researcher position. Furthermore, Dr. Pal has the opportunity to develop his own laboratory for neurodegeneration studies, as part of a large national project proposal for infrastructure development. If the proposal is successful he will be the leader of the laboratory allowing him to develop his own research group.

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