Veterinary session

The hypothalamus is one of the most important brain areas that are responsible for the regulation of homeostatic processes in the body; however, the exact physiological role of it is not clarified yet. Reproductive processes and food intake regulated by the hypothalamus are investigated by the Neuromorphological-neuroendocrin Research Group at the Department of Physiology and Biochemistry, as well. The neuronal energy consumption that is represented in mitochondrial respiration rates (mrr) are good indicators of the activity of hypothalamic centres. Thus, using this mrr method, conclusions can be made regarding to the function of certain brain areas.

In our study we examined the mitochondrial respiratory activity by a Clark-type oxygen electrode (Hanstech, Oxyterm). We measured the oxygen consumption of the mitochondria in respiration buffer solution alone and with additional respiratory modifiers. This method is well known for examination of mitochondrial activity for a long time. The examination was carried out on rats as an accepted experimental model. Regarding to the small samples that can be retrieved from rats and also considering the comparability with the previous data of literature, it became necessary to develop a fractionation method that fits our purposes. The requirements of the new method are the following: it should be possible to isolate the half of the hypothalamus or even smaller brain areas, and the sample should not be affected by uncoupling substances such as myelin and other cell debris.

Regarding to the big literature of the cellular and subcellular fractionation and after our preliminary experiments we decided to use the Percoll (GE) as our fractionation medium since it provides us a good opportunity for making a density gradient and it is not harmful on living cells and cell organelles.

The purpose of this work is to introduce the Percoll based subcellular centrifugation method which makes possible the separated examination of small brain areas or even nucleus groups. We will show that this method is good for measurement of mitochondrial activity, furthermore it is possible to separate the somal and synaptosomal mithondrial fractions from the samples obtained with this method. Thus we can get a mitochondrial fraction with a suitable quality for mitochondrial respiration measurements without special equipments in a short period of time.