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Mitochondrial oxidant stress in locus coeruleus is regulated by activity and nitric oxide synthase

Nature Neuroscience volume 17, pages 832–840 (2014)Cite this article

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Abstract

Loss of noradrenergic locus coeruleus (LC) neurons is a prominent feature of aging-related neurodegenerative diseases, such as Parkinson's disease (PD). The basis of this vulnerability is not understood. To explore possible physiological determinants, we studied LC neurons using electrophysiological and optical approaches in ex vivo mouse brain slices. We found that autonomous activity in LC neurons was accompanied by oscillations in dendritic Ca2+ concentration that were attributable to the opening of L-type Ca2+ channels. This oscillation elevated mitochondrial oxidant stress and was attenuated by inhibition of nitric oxide synthase. The relationship between activity and stress was malleable, as arousal and carbon dioxide increased the spike rate but differentially affected mitochondrial oxidant stress. Oxidant stress was also increased in an animal model of PD. Thus, our results point to activity-dependent Ca2+ entry and a resulting mitochondrial oxidant stress as factors contributing to the vulnerability of LC neurons.

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Figure 1: LC neurons were autonomous pacemakers with broad action potential spikes.
Figure 2: Engagement of L-type channels mediated dendritic Ca2+ oscillations during spiking activity in LC.
Figure 3: L-type Ca2+ channels were not essential to sustain autonomous spiking, but necessary under conditions of elevated spike rate.
Figure 4: ER sequestered cytosolic Ca2+ under conditions of low intrinsic Ca2+ buffering.
Figure 5: L-type channels triggered mitochondrial oxidant stress in LC.
Figure 6: Modulation of spiking and mitochondrial oxidant stress.
Figure 7: NOS contributes to oxidant stress.

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Acknowledgements

We acknowledge the technical help of S. Ulrich, Y. Chen, L. Fisher and K. Chen. We acknowledge S. Chan (Northwestern University) for supplying qPCR primer sets and J.T. Walter (Northwestern University) for supplying GP recordings. This work was supported by the JPB and IDP Foundations, US National Institutes of Health grants NS047085 (D.J.S.), NS054850 (D.J.S.), K12GM088020 (J.S.-P.), HL35440 (P.T.S.) and RR025355 (P.T.S.), and Department of Defense contracts W81XWH-07-1-0170 and W23RYX-2150-N601 (D.J.S.). The authors gratefully thank the laboratory of T. Dawson (Johns Hopkins University) for providing DJ-1−/− mice.

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Authors and Affiliations

  1. Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

    Javier Sanchez-Padilla, Jaime N Guzman, Ema Ilijic, Jyothisri Kondapalli, Daniel J Galtieri, Ben Yang, David Wokosin & D James Surmeier

  2. Department of Neurology, Philipps University, Marburg, Germany

    Simon Schieber & Wolfgang Oertel

  3. Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

    Paul T Schumacker

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Contributions

D.J.S., J.S.-P. and J.N.G. were responsible for the design and execution of experiments, as well as the analysis of results and overall direction of the experiments, analysis of data, construction of figures and communication of the results. W.O. participated in the design and the communication of the results. S.S. and D.J.G. contributed to collecting electrophysiological and relative oxidation data, respectively. B.Y. performed stereotaxic viral injections. D.W. provided expertise in optical approaches. J.S.-P. and E.I. conducted the immunocytochemical experiments. J.K. generated the AAV virus for the CMV–mito-roGFP stereotaxic injections. P.T.S. was responsible for the generation of the CMV–mito-roGFP mice and participated in the design, analysis and communication of the results. D.J.S. and J.S.-P. prepared the manuscript and the illustrations.

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Integrated supplementary information

Supplementary Figure 1 Colocalisation of LC neuronal biomarkers.

(a) Left panel shows immunoreactive LC neurons for tyrosine hydroxylase (TH). Middle panel shows immunoreactivity of LC neurons for dopamine-β-hydroxylase (DBH). Right panel displays merged immunostaining for TH and DBH showing complete overlap and confirmation of proper anatomical location for LC neurons.

Supplementary Figure 2 LC neurons have functional expression of Ca2+ channels.

(a) Somatic voltage clamp experiments were performed using the command action potential waveform obtained from a pacemaking LC neuron. Outward K+ currents were blocked using 5 mM TEA in the bath and recordings were performed under low external Na+ (50 mM), and Cs+-based internal solution supplemented with 5 mM TEA. (b) Ionic current measurements in response to the action potential waveform were sampled during normal external [Ca2+], following TTX to remove Nav1 component, and following equimolar substitution of Ca2+ with cobalt (Co2+). Co2+-sensitive currents were calculated by subtracting ionic current obtained with TTX and 2 mM Co2+ from ionic currents obtained in the presence of TTX and 2 mM Ca2+. (c) LC neurons display a subthreshold inward current during the inter-spike interval). (d) LC neurons also display a significant inward spike-associated current. (e) Box-plot quantification of charge for subthreshold and spike currents. (f) Box-plot quantification demonstrating that subthreshold inward currents carry approximately 70% of charge and the remaining 30% carried by the spike Data represents n=4 neurons from 4 mice, p=0.0143, Mann-Whitney).

Supplementary Figure 3 Ectopic spikes reset dendritic Ca2+ oscillations in LC neurons.

(a) Ectopic spikes induced by a short pulse of positive injection (2 nA, 1msec) resets the pacemaking cycle synchronized to dendritic Ca2+ transients. (b) After silencing an LC neuron with hyperpolarizing current, ectopic spikes can elicit dendritic Ca2+ transients. Data represents n=4 neurons from 4 mice.

Supplementary Figure 4 LC neurons express Nav1 and NALCN channels.

(a) Box-plot quantification of relative mRNA abundance for Nav1 channels in SNc and LC (n=5 mice). (b) Box-plot quantification of relative mRNA abundance for NALCN channels in SNc and LC (n=5 mice). (c) Sample trace of pacemaking and spikelets (with TTX). (d) LC neurons after isradipine eliminates spikelets and membrane potential remains depolarized, and sodium replacement with N-methyl-D-Glucamine (NMDG) hyperpolarizes LC neurons, suggesting a strong contribution of NALCN channels in LC neurons. (e) Box-plot quantification of membrane potential under normal Na+ and equimolar substitution with NMDG (n=4 neurons from 4 mice, p=0.0284, Mann-Whitney).

Supplementary Figure 5 Time course of Fluo4 dye loading in LC neurons synchronized to spike-evoked Ca2+ transients.

(a) Representative time course plotting Fluo4 (200 μM in the pipette) fluorescence as a function of time after whole cell break in. Fluorescence was sampled in the soma. During the time course of dye loading, action potential spikes were evoked in silenced LC neurons (shown in inset), and evoked Ca2+ transients were analyzed for buffering capacity calculations until dye reached equilibrium (∼5-10 min for soma).

Supplementary Figure 6 NOS inhibition inhibits NO production in LC neurons.

(a) DAF-FM fluorescence was measured in LC neurons every five minutes, before and after bath application of 100 μM L-NAME (10 min). The increase fluorescence is an indicator of increased NO production, calculated by linear fit, giving rise to a slope that indicates rate of NO production. L-NAME decreased the rate of NO production, shown by a decrease in the slope (middle panel). (b) Quantification of rate of NO production (slope) before and after L-NAME (n=4 neurons from 4 mice, p=0.042, one-tailed paired-t test, normal distribution was assumed but not tested formally).

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Sanchez-Padilla, J., Guzman, J., Ilijic, E. et al. Mitochondrial oxidant stress in locus coeruleus is regulated by activity and nitric oxide synthase. Nat Neurosci 17, 832–840 (2014). https://doi.org/10.1038/nn.3717

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