Programme Contents

Mitochondria-induced hyperpolarization in mouse locus coeruleus neurons is dependent on Ca²⁺ entry but not intracellular Ca²⁺ release

Locus coeruleus (LC) neurons are known to play a fundamental role in brain function, impacting on many physiological processes such as regulation of sleep and vigilance, learning and memory, behavioural flexibility, and a range of other functions (for review see Sara, 2009). Mitochondria are intracellular organelles that appear to be involved in vast range of different pathways, including energy production, neuronal death, oxidative stress, neurodegenerative diseases and their role in buffering intracellular Ca²⁺ and resultant impact on Ca²⁺-dependent pathways (Ishii, Hirose & Iino, 2006; Lehninger, Nelson & Cox, 2008). In rat LC neurons, it has been demonstrated that mitochondrial disruption caused an increase in intracellular Ca²⁺ and activation of Ca²⁺-activated K⁺ channels and resultant membrane hyperpolarization (Murai et al., 1997). Here, we demonstrate that the hyperpolarization caused by mitochondrial disruption in mouse LC neurons is dependent on external Ca²⁺ entry and not dependent on increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). The methods used for euthanizing mice were approved by the Animal Care and Ethics Committee at the University of Newcastle. The brain was rapidly removed with a slice containing the LC then prepared, allowed to equilibrate and placed on the stage of an upright microscope in a bath perfused with artificial cerebrospinal fluid (ACSF) at 35°C (de Oliveira et al., 2010). Recordings were made from LC neurons using patch electrodes in whole cell recording mode. Mitochondrial disruption with the protonophore CCCP (1 μM) caused hyperpolarization or outward current in current and voltage clamp modes, respectively. This outward current was likely to be dominantly generated by Ca²⁺ activated K⁺ channels of the SK type, as the conductance was largely blocked by Apamin (1 μM). The outward conductance was dependent on external Ca²⁺ entry, as determined using Ca²⁺-free (0.5 mM EGTA) ACSF and Co²⁺ ACSF (Co²⁺ substituted for Ca²⁺) solutions. This conductance was not inhibited when an internal pipette solution containing a high concentration of the Ca²⁺ buffer EGTA (15 mM) was used, suggesting that [Ca²⁺]_c was not involved in its activation. Ca²⁺ imaging demonstrated that CCCP increased intracellular Ca²⁺ in both ACSF and the Ca²⁺-free ACSF. The latter observation combined with the finding that the CCCP-generated outward conductance was not activated in Ca²⁺-free ACSF confirmed that increases in cytosolic [Ca²⁺]_c per se did not activate the outward conductance. Taken together, these results demonstrate that hyperpolarization induced by mitochondrial disruption using the protonophore CCCP causes Ca²⁺ entry and resultant Ca²⁺-activated K⁺ conductance that is independent of intracellular Ca²⁺ release from stores, but is dependent on external Ca²⁺ entry. This suggests that activation of this conductance occurs in a microdomain.

de Oliveira RB, Howlett MC, Gravina FS, Imtiaz MS, Callister RJ, Brichta AM & Helden DF. (2010). Pacemaker currents in mouse locus coeruleus neurons. Neuroscience 170(1): 166-77.

Ishii K, Hirose K & Iino M. (2006). Ca²⁺ shuttling between endoplasmic reticulum and mitochondria underlying Ca²⁺ oscillations. EMBO Reports 7: 390-396.

Lehninger AL, Nelson DL & Cox MM. (2008). Lehninger Principles of Biochemistry. W.H. Freeman, New York.

Murai Y, Ishibashi H, Koyama S & Akaike N. (1997). Ca²⁺-activated K⁺ currents in rat locus coeruleus neurons induced by experimental ischemia, anoxia, and hypoglycemia. Journal of Neurophysioloy 78: 2674-2681.

Sara SJ. (2009). The locus coeruleus and noradrenergic modulation of cognition. Nature Reviews. Neuroscience 10: 211-223.