A concerted action of L- and T-type Ca2 + channels regulates locus coeruleus pacemaking
Introduction
The spontaneously active noradrenergic locus coeruleus (LC) is involved in various adaptive behaviors and its projections innervate a large number of brain regions (Benarroch, 2009). Dysfunction of the LC noradrenergic system is implied to distinct psychiatric and neurodegenerative disorders, such as depression or Parkinson's disease (PD) (Berridge and Waterhouse, 2003, Gesi et al., 2000). Loss of LC neurons correlates with prodromal, i.e. premotor symptoms in PD, that precede up to several decades the onset of motor symptoms (Lang, 2011). The mechanism that renders these neurons vulnerable to the pathogenesis of PD during aging still remains unclear. At present it is proposed that activity-dependent oxidant stress is a common feature of LC and substantia nigra pars compacta (SNpc) neurons. In both LC and SNpc mitochondrial oxidant stress is attributed to Ca2 + entry through L-type Ca2 + channels (Chan et al., 2007, Sanchez-Padilla et al., 2014).
The existence of an intrinsic pacemaker mechanism in LC neurons has been shown in various studies both in vivo (Foote et al., 1980) and in brainstem slices (Alreja and Aghajanian, 1991, Williams et al., 1984). Firing rate of LC neurons is increased in response to noxious or stressful stimuli or during periods of arousal and increased wakefulness (Aston-Jones and Cohen, 2005, Gompf et al., 2010, Takahashi et al., 2010). On the other hand, in certain stages of sleep or in situations of low vigilance or drowsiness firing rate is decreased (Foote et al., 1980). On a cellular basis firing frequency is modulated by the action of neurotransmitters, the pH or the cAMP concentration (Alreja and Aghajanian, 1991, D'Adamo et al., 2011, Murai and Akaike, 2005).
Various ionic conductances, mediating inward and outward currents, have been characterized in LC neurons. However, it is noteworthy that most of these studies were performed in the rat, while in mice, despite the predominant role of these rodents for generating transgenic animal models, LC neurons are only poorly characterized. Outward potassium currents described in LC neurons comprise a transient K+ (IA), a persistent K+ (IK), a Ca2 + activated K+ (ISK/IBK), an ATP dependent K+ (IKATP) and an inward rectifier K+ (IKir) (Murai and Akaike, 2005, Murai et al., 1997, Nieber et al., 1995, Osmanović and Shefner, 1993, Zhang et al., 2010). Inward currents include a tetrodotoxin (TTX)-sensitive and -insensitive Na+ current, an inward non-specific cation current carried primarily by Na+ and L-, N-, P- and Q-type Ca2 + conductances (Chieng and Bekkers, 1999, Murai and Akaike, 2005, van den Pol et al., 2002, Zhang et al., 2010). The speed of the spontaneous depolarization during the interspike interval of mouse LC neurons was suggested to be primarily determined by a combination of a TTX-sensitive Na+ and a TEA-sensitive K+ current (de Oliveira et al., 2010). Although low-threshold L-type and T-type Ca2 + currents are known to regulate pacemaking in distinct central neurons (Deleuze et al., 2012, Guzman et al., 2009, Wolfart and Roeper, 2002), there is a lack of studies analyzing the functional role of these channels for the spontaneous activity of LC neurons. In a recent study by Sanchez-Padilla et al. the low-threshold activated L-type Ca2 + channel Cav1.3 was shown to be expressed in LC neurons and may therefore contribute to the Ca2 + currents phase-locked to spontaneous firing. Nevertheless, a direct influence on firing frequency was not found (Sanchez-Padilla et al., 2014).
In the present study we performed RT-PCR expression analysis of collected LC neurons in combination with slice patch clamp experiments to identify low-threshold activated L-type and T-type Ca2 + channels. We furthermore elucidated the potential role of these channels in the pacemaking mechanism of spontaneously active LC neurons. We therefore provide an important novel insight for the understanding of LC neuron activity which might be ultimately coupled to the vulnerability of these neurons during degenerative diseases.
Section snippets
LC neurons are autonomous pacemakers
The region of the LC was first identified by immunohistochemistry, as an accumulation of tyrosine hydroxylase (TH) positive neurons at the border of the fourth ventricle (Fig. 1A). A typical LC neuron during a patch clamp recording is illustrated in Fig. 1B. To verify their noradrenergic nature, each neuron was filled with neurobiotin (NB) during recording and later co-stained with an anti-TH antibody (Fig. 1C). Autonomous activity of LC neurons was recorded in acute brainstem slices at room
Discussion
The analysis of ion channels underlying the electrical activity of LC neurons can lead to a better understanding of the vulnerability of these neurons in neurodegenerative diseases, such as in PD, as electrical activity of the LC is ultimately coupled to cell survival signaling pathways. Extensive studies have been performed analyzing the role of low-threshold activated Cav1.3 channels in autonomous spiking and Ca2 + influx mediated oxidative stress in SNpc and LC neurons (Chan et al., 2007,
Experimental methods
The investigation conforms to the principles outlined in the Declaration of Helsinki and to the guide for the Care and Use of laboratory Animals (NIH Publication 85-23).
Acknowledgments
This work was supported by a grant of the UKGM to L.A.M. (26/2015 MR) and by a grant of the Anneliese Pohl Stiftung to S.R. W.H. Oertel is a Hertie-Senior Research Professor supported by the Charitable Hertie Foundation, Frankfurt/Main, Germany. Work in the laboratory of T.P. Snutch is supported by an operating grant from the Canadian Institutes of Health Research (#10677) and a Canada Research Chair in Biotechnology and Genomics-Neurobiology. We are grateful to Simon Schieber, Michael Netter
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