Neuropharmacology 62 (2012) 2472e2479
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Regulation of central noradrenergic activity by 5-HT3 receptors located in the locus coeruleus of the rat Jorge E. Ortega a, b, Aitziber Mendiguren a, Joseba Pineda a, *, J. Javier Meana a, b a b
Department of Pharmacology, Faculty of Medicine and Odontology, University of the Basque Country (UPV/EHU), Barrio de Sarriena s/n, E-48940 Leioa, Bizkaia, Spain Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 October 2011 Received in revised form 20 February 2012 Accepted 21 February 2012
A functional interaction between serotonergic and noradrenergic systems has been shown in the locus coeruleus (LC). Noradrenaline (NA) levels in the prefrontal cortex (PFC) are dependent on the ﬁring rate of LC neurons, which is controlled by a2 adrenoceptors (a2ADR). The aim of the present study was to investigate the role of 5-HT3 receptors (5HT3R) in the modulation of central noradrenergic activity. We measured extracellular NA concentrations in the LC and PFC by dual-probe microdialysis in awake rats and the ﬁring rate of LC neurons by electrophysiological techniques in vitro. Administration of the 5HT3R agonists SR57227 (1e100 mM) and m-chlorophenylbiguanide (mCPBG, 1e100 mM) into the LC increased NA in this nucleus (Emax ¼ 675 121% and Emax ¼ 5575 1371%, respectively) and decreased NA in the PFC (Emax ¼ 49 6% and Emax ¼ 25 11%, respectively). Administration of the 5HT3R antagonist Y25130 (50 mM) into LC attenuated SR57227 effect in the LC (Emax ¼ 323 28%) and PFC (Emax ¼ 37 7%). The a2ADR antagonist RS79948 (1 mM) blocked the SR57227 effect in the PFC but it did not change the effect in the LC (Emax ¼ 677 202%). In electrophysiological assays, both mCPBG (1e10 mM) and SR57227 (1e10 mM) reduced the ﬁring rate of about 50% of tested LC neurons (maximal effect ¼ 37 2% and 31 4%, respectively); this effect was partially blocked by Y25130 (50 mM). Administration of RS79948 (1 mM) reversed the inhibition induced by mCPBG. Competition radioligand assays against [3H]UK14304 and [3H]RX821002 (a2ADR selective drugs) in the rat brain cortex showed a very weak afﬁnity of SR57227 for a2ADR, whereas the afﬁnity of mCPBG for a2ADR was 17-fold higher than that of SR57227 for a2ADR. The present results suggest that 5HT3R stimulate NA release in the LC, which promotes simultaneously a decrease in the ﬁring rate of LC neurons through a2ADR and then a decrease of NA release in terminal areas such as the PFC. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Noradrenaline Microdialysis Single-unit extracellular recording Locus coeruleus Prefrontal cortex 5-HT3 receptor
Abbreviations: 2ADR, a2 adrenoceptor; 5-HT, serotonin; 5HT3R, 5-HT3 receptor; ANOVA, analysis of variance; CSF, cerebrospinal ﬂuid; HPLC, high performance liquid chromatography; IC50, inhibitory concentration 50%; LC, locus coeruleus; mCPBG, 1-(m-Chlorophenyl)-biguanide; NA, noradrenaline; PFC, prefrontal cortex; RS79948, (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-methoxy-12-(eth ylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine hydrochloride; S.E.M, standard error of the mean; SERT, 5-HT reuptake transporter; SR57227, 1-(6-chloro-2pyridinyl)-4-piperidinamine hydrochloride; Y25130, [(þ-)-N-(1-azabicyclo[2.2.2] oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxami de hydrochloride]; UK14304, 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline; RX821002, 2-(2,3-Dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1Himidazole hydrochloride. * Corresponding author. Tel.: þ34 946015577; fax: þ34946013220. E-mail address: [email protected]
(J. Pineda). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.02.018
Serotonin is an important neurotransmitter in the brain, where it plays a role in basic functions such as mood, sleep and appetite regulation (Lucki, 1998). Receptors mediating the actions of 5-HT are pharmacologically classiﬁed into seven major groups: 5-HT1 to 5-HT7 (Hoyer et al., 2002). All 5-HT receptors, except 5-HT3, represent G-protein coupled binding proteins. However, 5-HT3 receptors (5HT3R) are pentameric fast activating, ligand-gated, nonselective cation channels that act as a molecular switch at central and peripheral synapses (Lucki, 1998; Reeves and Lummis, 2002). Five 5HT3R subunits have been cloned (AeE), with the 5HT3A subunit being essential for full receptor functionality (Karnovsky et al., 2003; Niesler et al., 2003, 2007). The role of 5HT3R in general physiology and pathophysiology has been only partially established (Costall and Naylor, 2004). Activation of presynaptic 5HT3R leads to changes in synaptic neurotransmitter
J.E. Ortega et al. / Neuropharmacology 62 (2012) 2472e2479
release (Conley and Brammar, 1995), whereas activation of postsynaptic 5HT3R induces membrane depolarization and fast excitatory responses (Jackson and Yakel, 1995). The 5HT3R mediates the emetic effect of chemotherapy and anaesthetics, regulates pain, cognition and affective behaviour, and contributes to depression, anxiety, alcohol dependence and drug abuse (Barnes et al., 1992; Kelley et al., 2003; Turner et al., 2004). The locus coeruleus (LC) is the main source of noradrenergic projections in the brain and certain areas such as the prefrontal cortex (PFC) receives most of the noradrenergic innervation from this nucleus (Levitt and Moore, 1978). The homogeneous nature of this nucleus in the rat and its involvement in certain psychiatric disorders (e.g., anxiety, depression or stress) has allowed the use of this nucleus as a model to study the central noradrenergic system (Berridge and Waterhouse, 2003). A functional interaction between serotonergic and noradrenergic systems has been reported in the LC. Thus, the LC receives a dense innervation from 5-HT nuclei (Berridge and Waterhouse, 2003; Imai et al., 1986; Pickel et al., 1977), including the dorsal raphe nucleus (Pudovkina et al., 2001), expresses 5-HT reuptake transporters (SERT) (Hrdina et al., 1990) and releases 5-HT to the synaptic cleft (Singewald et al., 1997). Stimulation of brain 5-HT neurons causes reductions in the activity of noradrenergic cells in the LC (Segal, 1979), and 5-HT locally applied attenuates the excitatory responses of this nucleus to sensorial, neurochemical and electrical stimuli (Aston-Jones et al., 1991; Bobker and Williams, 1989). Furthermore, blockade of 5-HT reuptake in the LC releases noradrenaline (NA) and reduces neuronal activity in this nucleus (Mateo et al., 2000). The involvement of speciﬁc 5-HT receptor subtypes in the LC for these effects is controversial. Functional studies have suggested that 5-HT1A (and 5-HT1B) receptors may mediate the inhibitory effect of 5-HT on the LC (Bobker and Williams, 1989; Done and Sharp, 1994; Pudovkina et al., 2002). However, other authors have reported an excitatory regulation of LC neurons by 5-HT1A receptors (Engberg, 1992; Szabo and Blier, 2001). A functional role for 5-HT2A/ 2C receptors has been also proposed in the LC, although it is unclear whether these receptors are located within the LC (Done and Sharp, 1992; Haddjeri et al., 1997; Rasmussen and Aghajanian, 1986; Szabo and Blier, 2001). As for the other 5-HT receptors, their contribution has not been fully established in the LC (Engberg, 1992). Recent work has shown that 5HT3R regulate neuronal activity in other catecholaminergic areas such as the ventral tegmental area (Chenu et al., 2009). Therefore, given the contribution of 5HT3R to the regulation of neurotransmitter release, we used brain microdialysis in vivo and electrophysiological techniques in vitro to study the regulation of noradrenergic activity in the LC by 5HT3R. The prefrontal cortex and the relevance of a2 adrenoceptors (a2ADR) located in the LC were also evaluated in this study. 2. Material and methods 2.1. Animals Male Sprague-Dawley rats weighing 250e300 g at the time of microdialysis, electrophysiological recordings and binding assays were obtained from Harlan Interfauna Ibérica, S.L. (Barcelona, Spain). Three-four animals per cage were housed and maintained in a 12 h lightedark cycle in a room at 22 C with water and food ad libitum. All experimental procedures reported in this manuscript were carried out in accordance with the guidelines of animals care set by the European Communities Council Directive of 24 November 1986 (86/609/EEC), and approved by the UPV/EHU Ethical Board for Animal Welfare (CEBA). All efforts were made to minimize animal suffering and the number of animals used. 2.2. Microdialysis procedures Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and transferred to a stereotaxic frame with the incisor bar at a 15 angle. An incision into the scalp was made to reveal lambda and bregma suture points. Two concentric microdialysis
probes were implanted in the rat brain choosing coordinates from atlas Paxinos and Watson (1986); one of them in the vicinity of the right LC (AP 3.7, L 1.3, V 8.2, from lambda suture point) an the other one in the right PFC (AP þ2.8, L þ1, V 5, from bregma suture point). After that, they were ﬁxed to the skull with ﬁlled resin and protected with a plastic cover to prevent probes damage. Rats, recovered from surgery in a thermal blanquet to avoid hypothermia, were housed in microdialysis plastic bowls (CMA/Microdialysis) with water and food ad libitum. The microdialysis assays were performed between 20 and 24 h after surgery. Animals were placed in a CMA/120 microdialysis system and both inlet and outlet tubing of the probes were connected to the syringes of a CMA/102 microdialysis pump. Modiﬁed cerebrospinal ﬂuid (CSF) (NaCl 148 mM, KCl 2.7 mM, CaCl2 1.2 mM and MgCl2 0.85 mM;...