Preparation of purified perikaryal and synaptosomal mitochondrial fractions from relatively small hypothalamic brain samples.
In order to measure the activity of neuronal mitochondria, a representative proof of neuronal processes, physiologically relevant mitochondrial samples need to be gained as simply as possible. Existing methods are, however, either for tissue samples of large size and/or homogenous microstructures only, or are not tested for mitochondrial function measurements. In the present article we describe a gradient fractionation method to isolate viable and well-coupled mitochondria from relatively heterogeneous histological microstructures such as the hypothalamus. With this new method, we are able to isolate a sufficient amount of functional mitochondria for determination of respiratory activity, in a short period of time, using affordable equipment. •Verified by electron microscopy, our method separates highly enriched and well-preserved perikaryal and synaptosomal mitochondria. Both fractions contain minimal cell debris and no myelin. Respiratory measurements (carried out by Clark-type electrode) confirmed undisturbed mitochondrial function providing well-evaluable records. The demonstrated protocol yields highly viable mitochondrial subfractions within 3 h from small brain areas for high-precision examinations. Using this procedure, brain regions with relatively heterogeneous histological microstructure (hypothalamus) can also be efficiently sampled.•Up to our present knowledge, our method is the shortest available procedure with the lowest sample size to gain debris-free, fully-viable mitochondria.
Department of Physiology and Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary.
David S
DS
Kiss
Department of Physiology and Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary.
Istvan
I
Toth
Department of Physiology and Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary.
Gergely
G
Jocsak
Department and Clinic of Internal Medicine, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary.
Agnes
A
Sterczer
Department of Physiology and Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary.
Tibor
T
Bartha
Department of Physiology and Biochemistry, Szent Istvan University Faculty of Veterinary Sciences, Budapest, Hungary.
Laszlo V
LV
Frenyo
Division of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA; Department of Animal Physiology and Animal Health, Szent Istvan University Faculty of Agricultural and Environmental Sciences, Godollo, Hungary.
Attila
A
Zsarnovszky
Clark-type electrode; Gradient fractionation; Isolation of mitochondrial fraction for oxygen-consumption measurements; Percoll; Respiration measurement