Table 2.

Drugs targeting the mitochondrial bioenergetic machinery

Target	Drug	Concentrations (primary neurons)	Off-target effects
Complex I inhibition	Rotenone	1–2 μM	Microtubules
Complex II inhibition	3-NP	0.1–1 mM
Complex III inhibition	Antimycin A	1–3 μM	Aspecific permeabilisation of the inner mitochondrial membrane
	Stigmatellin	1–3 μM	Also inhibits Complex I at high concentrations
	Myxothiazol	1–5 μM	Also inhibits Complex I at high concentrations
Complex IV inhibition	Cyanide (NaCN, KCN)	1–5 mM	Haem-containing enzymes; formation of thiocyanate adducts
	Sodium Azide	1–5 mM	Interacts with active groups from catalase and nitrogen-based structures
F1Fo ATP Synthase inhibition	Oligomycin	1–5 μg/ml
		(1–6 μM)a
Protonophore (increases proton leak; uncouples mitochondria)	FCCP	High: 10 μM
		Low: 0.3–1 μM
	CCCP	High: 10 μM	Inhibits lysosomes and autophagy
		Low: 0.3–1 μM
	DNP	Low: 0.1–0.5 mM

^aOligomycin concentrations are often listed as μg/ml, as commercial preparations are a mixture of compounds with different individual molecular weights

Concentrations are guidelines only for primary neurons, and should be optimised for each cell type or experimental setting. Changes to the experimental buffer, such as the inclusion of bovine serum albumin, can alter some of the effective drug concentrations by more than four-fold [40, 44]. High protonophore concentrations collapse the mitochondrial membrane potential (and may also depolarise the plasma membrane potential [62]), while low concentrations induce maximal respiration (this requires titration to determine the optimal concentration for each experimental set-up [36, 37]). References for concentrations were obtained from experiments in primary neurons: [53, 64, 102, 153, 154, 192–194]

3-NP 3-nitropropionic acid, FCCP carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, CCCP carbonyl cyanide m-chlorophenylhydrazone, DNP 2,4-dinitrophenol