Method for in situ detection of the mitochondrial function in neurons.
Conventional studies of neuronal mitochondria have been limited to the use of purified preparations of isolated mitochondria, neural cell homogenates, living neurons, or brain slices. However, each technique has several drawbacks. Here, we demonstrate that the neuronal cell's membrane can be effectively permeabilized by saponin-treatment and that these permeabilized neurons can be used for qualitative and quantitative assessments of oxygen consumption in combination with registration of mitochondrial membrane potential and free [Ca2+] in the matrix. Under these conditions, the mitochondrial function can be studied without removing the mitochondria from their natural milieu thus avoiding the damage of the associated cytoskeleton and outer membrane. At the same time, the method allows the estimation of the mitochondrial function independently of other processes in the cell, and the easy manipulation of the milieu surrounding the mitochondria. Thus, the presented method offers the opportunity to study the neuronal mitochondrial function in situ and can also be applied to examine the mitochondrial function by other commonly used methods.
Safiulina
Dzhamilja
D
Department of Pharmacology, Faculty of Medicine, Centre of Excellence for Molecular and Clinical Medicine, University of Tartu, Ravila 19, 51014 Tartu, Estonia.
Kaasik
Allen
A
Seppet
Evelin
E
Peet
Nadezhda
N
Zharkovsky
Alexander
A
Seppet
Enn
E
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D019069:Cell Respiration / Q000502:physiology*; D002478:Cells, Cultured; D007425:Intracellular Membranes / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000502:physiology*; D009448:Neurochemistry / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D009482:Neurophysiology / Q000295:instrumentation / Q000379:methods*; D010101:Oxygen Consumption / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012503:Saponins / Q000494:pharmacology
D012503:Saponins; D002118:Calcium