Proceedings of The Physiological Society

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, C19

Oral Communications

Mitochondrial metabolism regulates GABAergic neurotransmission in cerebellar granule cells

M. V. Accardi1, B. A. Orser2, D. Bowie1

1. Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada. 2. Physiology, University of Toronto, Toronto, Ontario, Canada.

Reactive oxygen species (ROS) are ubiquitous signaling molecules in the CNS primarily formed as by-products of mitochondrial metabolism. Involved in both physiological and pathophysiological processes, ROS are important in neurotransmission with recent evidence demonstrating that mitochondrial-derived ROS (mROS) regulate the strength of GABAergic synapses in cerebellar stellate cells (Accardi et al. 2014). However, it remains unclear whether this regulation is found at other GABAergic synapses. To test this, C57BL/6 mice (Wild-type and δ-knock out [δ-KO], male/female, P15-28) were anaesthetized (isoflurane, inhalation), decapitated and parasagittal cerebellar slices were obtained, using a vibratome, so that inhibitory events from cerebellar granule cells could be recorded. GABAergic miniature IPSCs (mIPSCs) were pharmacologically isolated using glutamate receptor blockers and tetrodotoxin and mIPSCs were recorded using whole-cell patch-clamp electrophysiology. The mitochondrial poison, antimycin-A (2 µM), was used to elevate mROS by including it in the recording pipette solution. Antimycin-A significantly increased the frequency of mIPSCs in granule cells in a time dependent manner reaching a maximal effect after 20 minutes (normalized to the first minute, antimycin-A: 187.6 ± 30.7% at 20 min vs. normalized 100% at 1 min; paired t-Test, p = 0.041; n = 7; all values are means ± S.E.M). In the absence of antimycin-A, mIPSC frequency was unaffected over 25 minutes demonstrating the stability and health of granule cell recordings (Control: 112.23 ± 25.7% at 25 min vs. normalized 100% at 1 min; paired t-Test, p = 0.734; n = 7). The increase in mIPSC frequency was blocked by the presence of 1 mM N-acetylcysteine (NAC, n = 6), an antioxidant which acts through its thiol group to reduce ROS by electron donation (antimycin-A: 187.6 ± 30.7% at 20 min vs. antimycin-A + NAC: 102.9 ± 5.4% at 20 min; unpaired t-Test, p = 0.0291). To assess the impact on tonic GABA responses, we applied 10 µM bicuculline at the end of each recording. Using this approach, we observed that antimycin-A (n = 6) significantly reduced the baseline current compared to control cells (n = 5) (Control: 20.46 ± 5.6 pA vs. antimycin-A: 3.46 ± 0.93 pA, unpaired t-Test, p = 0.0147). The presence of the δ-subunit, which is found in extrasynaptic GABAA receptors, was essential for the increase in mIPSC frequency since δ-KO mice lacked an mROS-induced increase in mIPSC frequency (δ-KO Control: 92.5 ± 10.6% at 25 min vs. δ-KO + antimycin-A: 85.5 ± 9.9% at 25 min; unpaired t-Test, p = 0.639; n = 6 and 6 respectively; values were normalized to their respective first minute). Taken together, our data suggests a more global role for mROS in regulating GABAA receptor signaling at both synaptic and extrasynaptic locations.

Where applicable, experiments conform with Society ethical requirements