Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content

Oxidative Medicine and Cellular Longevity, Apr 2012

The present study investigated the effects of the antioxidants trolox and dithiothreitol (DTT) on mouse Locus coeruleus (LC) neurons. Electrophysiological measurement of action potential discharge and whole cell current responses in the presence of each antioxidant suggested that there are three neuronal subpopulations within the LC. In current clamp experiments, most neurons (55%; 6/11) did not respond to the antioxidants. The remaining neurons exhibited either hyperpolarization and decreased firing rate (27%; 3/11) or depolarization and increased firing rate (18%; 2/11). Calcium and JC-1 imaging demonstrated that these effects did not change intracellular Ca2

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Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content

Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content Ramatis B. de Oliveira, Fernanda S. Gravina, Rebecca Lim, Alan M. Brichta, Robert J. Callister, and Dirk F. van Helden School of Biomedical Sciences and Pharmacy, The University of Newcastle and Hunter Medical Research Institute, University Drive, Newcastle, NSW 2308, Australia Received 13 January 2012; Revised 24 January 2012; Accepted 8 February 2012 Academic Editor: Daniel Pens Gelain Copyright © 2012 Ramatis B. de Oliveira et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The present study investigated the effects of the antioxidants trolox and dithiothreitol (DTT) on mouse Locus coeruleus (LC) neurons. Electrophysiological measurement of action potential discharge and whole cell current responses in the presence of each antioxidant suggested that there are three neuronal subpopulations within the LC. In current clamp experiments, most neurons (55%; 6/11) did not respond to the antioxidants. The remaining neurons exhibited either hyperpolarization and decreased firing rate (27%; 3/11) or depolarization and increased firing rate (18%; 2/11). Calcium and JC-1 imaging demonstrated that these effects did not change intracellular Ca2+ concentration but may influence mitochondrial function as both antioxidant treatments modulated mitochondrial membrane potential. These suggest that the antioxidant-sensitive subpopulations of LC neurons may be more susceptible to oxidative stress (e.g., due to ATP depletion and/or overactivation of Ca2+-dependent pathways). Indeed it may be that this subpopulation of LC neurons is preferentially destroyed in neurological pathologies such as Parkinson’s disease. If this is the case, there may be a protective role for antioxidant therapies. 1. Introduction Oxidative stress in neurons arises because of an imbalance between free radical production and antioxidant control. This process leads to cell damage and, when severe, can trigger apoptosis or necrosis [1]. Indeed, such oxidative stress may initiate certain neuropathologies such as Alzheimer’s and Parkinson’s disease whereby oxidative damage to biomolecules causes cellular dysfunction and neuronal death [1, 2]. The exact mechanisms whereby healthy neurons become sensitive to oxidative stress are unknown, and it is also unclear whether antioxidant treatments limit the spread of oxidative damage. For example, it has been reported that varying levels of oxidants can modulate ion channels and consequently effect important neuronal functions such as pacemaking [3–5]. Thus, antioxidant treatments have been developed and used and have indeed had beneficial effects in preventing or slowing the onset of neurological disease [6]. Antioxidants may modulate transcription factors that ultimately lead to oxidative stress [7]. On the other hand, it has been demonstrated that molecules that were thought to be antioxidants due to their antioxidant capacity in vitro (such as vitamin A and retinoids) have different effects in living organisms (in vivo) as they can increase oxidative damage to biomolecules and generate oxidative stress [8–10]. In fact free radicals are crucial components of many intracellular signalling pathways (for a review see [11]), and an exaggerated decrease in their levels could lead to undesired cellular events. Thus, many factors contribute to the varied effectiveness of antioxidants in both human and animal trials including differences in dose and timing of antioxidant administration. The present paper explores the effect of two different antioxidants (Trolox and DTT) on pacemaking in Locus coeruleus (LC) neurons. These antioxidants can chelate different reactive species. Both compounds are membrane permeable, so they can easily access intracellular compartments. Trolox is a water-soluble vitamin E analogue with a broad antioxidant spectrum [12], whereas DTT is a reducing agent that acts on thiol (–SH) groups [13]. Here, we demonstrate that LC neurons exhibit three different types of electrophysiological responses to these antioxidants. We believe these responses may represent three neuronal populations that have differential sensitivity to free radicals and are involved in the massive loss of LC neurons observed in pathologies such as Parkinson’s disease. 2. Experimental Procedures2.1. Preparation of Brain Slices All procedures used in this study were approved by the University of Newcastle Animal Care and Ethics Committee. Brain slices containing the LC were prepared from Swiss mice (P7–12, both sexes) rendered unconscious with Ketamine (100 mg/kg i.p.) according to a previous established protocol [14]. Mice were decapitated, and the brain was rapidly removed and immersed in ice-cold “m (...truncated)


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Ramatis B. de Oliveira, Fernanda S. Gravina, Rebecca Lim, Alan M. Brichta, Robert J. Callister, Dirk F. van Helden. Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content, Oxidative Medicine and Cellular Longevity, 2012, 2012, DOI: 10.1155/2012/820285