an electrophysiological and neurochemical study on GIRK2 mutant

International Journal of Neuropsychopharmacology (2013), 16, 1093–1104. f CINP 2012 doi:10.1017/S1461145712000971

ARTICLE

Role of GIRK channels on the noradrenergic transmission in vivo: an electrophysiological and neurochemical study on GIRK2 mutant mice Marı´a Torrecilla1, Irrintzi Ferna´ndez-Aedo1, Aurora Arrue2, Mercedes Zumarraga2 and Luisa Ugedo1 1 2

Department of Pharmacology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa, Vizcaya, Spain Department of Neurochemical Research, Psychiatric Hospital of Zamudio, Zamudio, Vizcaya, Spain

Abstract Dysfunctional noradrenergic transmission is related to several neuropsychiatric conditions, such as depression. Nowadays, the role of G protein-coupled inwardly rectifying potassium (GIRK)2 subunit containing GIRK channels controlling neuronal intrinsic excitability in vitro is well known. The aim of this study was to investigate the impact of GIRK2 subunit mutation on the central noradrenergic transmission in vivo. For that purpose, single-unit extracellular activity of locus coeruleus (LC) noradrenergic neurons and brain monoamine levels using the HPLC technique were measured in wild-type and GIRK2 mutant mice. Girk2 gene mutation induced significant differences among genotypes regarding burst activity of LC neurons. In fact, the proportion of neurons displaying burst firing was increased in GIRK2 heterozygous mice as compared to that recorded from wild-type mice. Furthermore, this augmentation was even greater in the homozygous genotype. However, neither the basal firing rate nor the coefficient of variation of LC neurons was different among genotypes. Noradrenaline and serotonin basal levels were altered in the dorsal raphe nucleus from GIRK2 heterozygous and homozygous mice, respectively. Furthermore, noradrenaline levels were increased in LC projecting areas such as the hippocampus and amygdale from homozygous mice, although not in the prefrontal cortex. Finally, potency of clonidine and morphine inhibiting LC activity was reduced in GIRK2 mutant mice, although the efficacy remained unchanged. Altogether, the present study supports the role of GIRK2 subunit-containing GIRK channels on the maintenance of tonic noradrenergic activity in vivo. Electric and neurochemical consequences derived from an altered GIRK2-dependent signalling could facilitate the understanding of the neurobiological basis of pathologies related to a dysfunctional monoaminergic transmission. Received 8 March 2012 ; Reviewed 11 April 2012 ; Revised 23 July 2012 ; Accepted 2 August 2012 ; First published online 8 October 2012 Key words : Dorsal raphe, HPLC, locus coeruleus, noradrenaline, single-unit extracellular recording.

Introduction Noradrenergic neurotransmission alteration underlies the aetiopathology of multiple psychiatric diseases, such as attention deficit/hyperactivity disorder, anxiety, depression and drug addiction (Itoi & Sugimoto, 2010 ; Sara, 2009). Medications that target the noradrenergic system are considered as valuable therapeutic tools for the treatment of these pathologies, as for example the selective noradrenaline (NA) reuptake inhibitors for depression, atomoxetine for deficit/hyperactivity disorder and clonidine for opiate withdrawal syndrome and detoxification (Cipriani et al. 2009 ; Hanwella et al. 2011 ; Papakostas et al. 2008 ; Soyka et al. 2011). Address for correspondence : Dr Marı´a Torrecilla, Department of Pharmacology, Faculty of Medicine and Dentistry, University of the Basque Country UPV/EHU, 48940 Leioa, Vizcaya, Spain. Tel : +34 94 601 3401 Fax : +34 94 601 3220 Email : [email protected]

Nevertheless, the treatment efficacy is in many cases unsatisfactory, as for example in depressive patients (Blier, 2008 ; Lieberman et al. 2005 ; Turner et al. 2008). Studies of locus coeruleus (LC) noradrenergic neurons have had a key role in understanding the aetiology of some psychiatric illnesses related to dysfunction of this neuromodulatory system (Itoi & Sugimoto, 2010). This nucleus contains the largest population of central noradrenergic neurons and innervates almost the entire neuroaxis (Dahlstrom & Fuxe, 1964 ; Swanson & Hartman, 1975). In LC neurons, activation of m opioid receptors and a2 adrenoceptors leads to a decreased excitability (North & Williams, 1983 ; Pepper & Henderson, 1980 ; Williams et al. 1982), mainly through activation of G protein-coupled inwardly rectifying potassium (GIRK ; also known as Kir3) channels (Aghajanian & Wang, 1986 ; Travagli et al. 1995, 1996 ; Williams et al. 1988) and, in particular, those formed by GIRK2 and GIRK3 subunits (Cruz et al. 2008 ; Torrecilla et al. 2002, 2008). Additionally,

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constitutive GIRK channel activity contributes to the resting membrane potential of LC neurons in vitro (Torrecilla et al. 2002 ; Velimirovic et al. 1995). Moreover, in mice lacking both GIRK2 and GIRK3 subunits, the spontaneous firing rate of LC neurons from brain slices and cortical NA concentrations are augmented (Cruz et al. 2008). However, the role of GIRK channels controlling LC-noradrenergic function in vivo has not yet been investigated. Dysfunction of GIRK channels can lead to excessive or deficient neuronal excitability, which is related to several pathologies (Lu¨scher & Slesinger, 2010). In this context, the use of GIRK knockout mice has been a key tool to look into the role of specific GIRK channel composition on neuronal physiology and animal behaviour. Thus, GIRK2 subunit-containing GIRK channels functional relevance has been linked to increased susceptibility to seizures (Signorini et al. 1997), hyperalgesia and analgesia (Blednov et al. 2003 ; Marker et al. 2004, 2005), drug addiction and alcohol-induced behaviours (Blednov et al. 2001a ; Cruz et al. 2008 ; Kobayashi et al. 1999 ; Morgan et al 2003), motor activity and coordination and reward and anxiety-related behaviours (Blednov et al. 2001b ; Pravetoni & Wickman, 2008). In this way, electrophysiological studies using Xenopus oocyte expression assays have suggested that GIRK channel modulators might have therapeutic benefits in the treatment of several neurological disorders and cardiac arrhythmias (Hashimoto et al. 2006, Kobayashi et al. 2004, 2010). Thus, GIRK channels have been proposed as new pharmacological targets that could be effective for the treatment of several illnesses related to an altered central neurotransmission. Therefore, the electrophysiological and neurochemical study of GIRK2 mutant mice could highlight the involvement of GIRK2 subunit-containing GIRK channels on noradrenergic transmission in vivo, which might be a key point for a better understanding of the neurobiological basis of the psychiatric diseases related to improper noradrenergic signalling.

Materials and method Animals Wild-type and GIRK2 adult mutant mice (aged 12–15 wk) derived from heterozygote crossing (Signorini et al. 1997) were used in electrophysiological and neurochemical experiments. Animals were housed 4–5 per cage in a colony room at 22 xC, under 12 : 12 h light :dark cycle (lights on 08 : 00 hours) with food and water provided ad libitum. Every effort was made to minimize animal suffering and to use the minimum possible number of animals. The procedures were approved by the Local Committee for Animal Experimentation at the University of the Basque Country. All the experiments were performed in compliance with the European Community

Council Directive on ‘The Protection of Animals Used for Experimental and Other Scientific Purposes’ (86/609/ EEC) Spanish Law (RD 1201/2005) for the care and use of laboratory animals. Electrophysiological procedures Mice were anaesthetized with chloral hydrate (400 mg/ kg i.p.). After cannulating the trachea, the mouse was placed in the stereotaxic frame with the skull positioned horizontally. A burr hole was drilled and the recording electrode was placed 1.5 mm posterior to lambda and 0.2–1.2 mm from the midline and lowered into the LC usually encountered at a depth of between 2.7 and 4.0 mm from the brain surface (Gobbi et al. 2007). A catheter (Terumo Surflo1 ; Teruma Medical Products, USA) was then inserted in the peritoneo for additional administrations of anaesthetic and systemic drug. The body temperature was maintained at y37 xC for the entire experiment using a heating pad. Single-unit extracellular recordings of mouse LC neurons were performed as previously described by Gobbi et al. (2007). The recording electrode was filled with 2 % solution of Pontamine Sky Blue in 0.5 % sodium acetate and broken back to a tip diameter of 1–2 mM. The electrode was lowered into the brain by means of a hydraulic microdrive (model 640 ; David Kopf1 Instruments, USA). LC neurons were identified by standard criteria, which included spontaneous activity displaying a regular rhythm and firing rate between 0.5 and 5 Hz, characteristic spikes with a long-lasting (>2 ms), positive–negative waveform action potentials and the biphasic excitation–inhibition response to pressure applied on contralateral hind paw (paw pinch), as previously described in mice (Gobbi et al. 2007) and rats (Cedarbaum & Aghajanian, 1976). The extracellular signal from the electrode was preamplified and amplified later with a high-input impedance amplifier and then monitored on an oscilloscope and on an audio monitor. This activity was processed using computer software (Spike2 software ; Cambridge Electronic Design, UK) and the following patterns were calculated : firing rate ; the coefficient of variation (percentage ratio of standard deviation to the mean interval value of an interspike time-interval histogram) ; percentage of spikes in burst ; mean spikes/burst ; percentage of cells exhibiting burst firing ; response to drug administration. Basal firing rate and other electrophysiological parameters were measured for 3 min. Changes in firing rate were expressed as percentages of the basal firing rate (mean firing rate for 3 min prior to drug injection) and were measured after each dose of drug. Only one cell was studied in each animal when any drug was administered. A burst was defined according to Gartside et al. (2000) as a train of at least two spikes with the first interspike interval of f20 ms and a termination interval o160 ms. Burst-firing of LC neurons was detected as a train of

Regulation of LC activity by GIRK2 channels at least two spikes with the first interspike interval 160 ms, as previously described in publications from our group (Miguelez et al. 2011a, b ; Pineda et al. 1997). All active neurons recorded from each mouse were analysed according to the above defined criteria using the computer software Spike2 (script w_burst.s2s). Basal firing period (3 min) was used to evaluate whether the neuron displayed a burst-firing pattern or not. If electrical activity fitted burst criteria, percentage of spikes in burst and mean interspike interval of the burst train were also provided by the script. Neurochemical procedures The mice were anaesthetized with chloral hydrate (400 mg/kg i.p.) and decapitated and their brains were removed. The LC, dorsal raphe nucleus (DRN) prefrontal cortex (PFC), hippocampus (HPC) and amygdala (AM) were rapidly dissected on ice-cooled plate. The tissue was homogenized in 0.1 M perchloric acid, centrifuged (30 min, 12 500 g) and the supernatant was spin filtered through a 0.2 mM filter. Finally, one aliquot corresponding to 2 mg tissue was assessed by HPLC with amperometrical detection. The detector was operated at 0.6 V potential between the working electrode and the Ag/AgCl reference electrode with a sensitivity of 2 nA full-scale deflection. The mobile phase consisted of phosphoric–citric buffer (6.7 mM disodium phosphate, 13.3 mM citric acid, 0.05 mM EDTA, 3.64 mM heptanosulfonic acid, pH 3.1) and 15 % methanol (v/v). The column was a 250r4.6 mm Symetry C18, 5 mM particle size (Waters Corporation, USA). All separations were achieved isocratically at room temperature. Flow rate was 1 ml/min. A calibration curve was performed daily within a concentration range of 0.15–2.4 ng of each standard per injection. The concentrations of NA, serotonin (5-HT) and its metabolite 5-hidroxyindolacetic acid (5-HIAA) in the tissue samples were calculated by extrapolation of the sample peak heights to the calibration curve. Drugs Chloral hydrate, clonidine hydrochloride and morphine hydrochloride (Sigma Aldrich, USA) were prepared in 0.9 % saline. Drugs were prepared on the day of the experiment, except for chloral hydrate. Statistical analysis of data Compiled data are expressed as the mean¡S.E.M., except for the percentage of neurons with burst firing. The sample size (n) represents the number of recorded neurons in electrophysiological experiments and the number of animals in neurochemical experiments. Dose–response curves for the inhibitory effect of clonidine or morphine were constructed by systemic administration of the drug

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at cumulative doses until a maximal response was reached. The inhibitory effect on LC cells induced by each dose was quantified as the percentage reduction from the basal firing rate. Experimental data regarding dose–response curves from each animal were analysed for the best simple nonlinear fit to the three-parameter logistic equation (Parker & Waud, 1971) using GraphPad Prism Software (v. 5.01 ; GraphPad Software Inc., USA) as described previously (Miguelez et al. 2009). The following equation was used : E=Emax [A]n =(ECn50 +[A]n ), where [A] is the concentration of the drug, E is the effect on the firing rate induced by A, Emax is the maximal effect, EC50 is the effective concentration for eliciting 50 % of Emax and n is the slope factor of the concentration–effect curve. Statistical comparison between dose–response curves parameters was done using the extra sum-of-squares F test (GraphPad Prism 5.01). Differences in the percentage of neurons presenting burst firing were statistically evaluated by two-tailed x2 analysis of contingency tables. Parameters derived from burst pattern were analysed by a non-parametric test, Kruskal–Wallis test followed by Dunn’s post-hoc test. Spontaneous firing rate, coefficient of variation and neurochemical data were compared through genotypes by one-way analysis of variance (ANOVA) followed by Newman–Keuls post-hoc test. Unpaired t test was used for selected pair comparisons. The level of significance was considered as p2 ms) and positive–negative waveform (Fig. 1 a). These neurons also displayed a biphasic excitation–inhibition response to a pinch of the contralateral paw (Fig. 1 b) as that previously described in mice (Gobbi et al. 2007) and rats (Cedarbaum & Aghajanian, 1976). Thus, the firing frequency of LC neurons from homozygous (GIRK2x/x) mice was not significantly different from that measured in WT or heterozygous (GIRK2+/x) mice (WT: 2.60¡0.16 Hz, n=109; GIRK2+/x : 2.13¡0.14 Hz, n=91 ; GIRK2x/x : 2.08¡0.16 Hz, n=47, p>0.05 ; Fig. 2a). The coefficient of variation did not differ among genotypes (WT : 50.77¡1.41 %, n=109 ; GIRK2+/x : 45.82¡2.09 %, n=91 ; GIRK2x/x : 50.79¡2.06 %, n=47, p>0.05 ; Fig. 2b). However, the number of neurons discharging in bursts was significantly higher not only in GIRK2x/x mice [55 % of total

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Fig. 1. In vivo extracellular signal of action potential recorded from locus coeruleus (LC) neurons in G protein-coupled inwardly rectifying potassium (GIRK)2x/x mice. (a) Example of a single spike from a mouse LC neuron recorded in vivo. The shape and duration of the action potential signal recorded from GIRK2x/x mouse LC were similar to those previously reported in the rat and wild-type (WT) mouse (Cedarbaum & Aghajanian, 1976 ; Gobbi et al. 2007). (b) LC neurons from GIRK2x/x mutant mice also responded to a pinch of the contralateral paw with a brisk increase in firing rate followed by a short pause (so-called Korf’s response). Arrow indicates pinch of paw.

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recorded cells, x2(1)=46.15, p