CB1 cannabinoid receptors inhibit the glutamatergic component of KCl-evoked excitation of locus coeruleus neurons in rat brain slices

CB(1) cannabinoid receptors located at presynaptic sites suppress synaptic transmission in the rat brain.
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Neuropharmacology 52 (2007) 617e625 www.elsevier.com/locate/neuropharm

CB1 cannabinoid receptors inhibit the glutamatergic component of KCl-evoked excitation of locus coeruleus neurons in rat brain slices Aitziber Mendiguren, Joseba Pineda* Department of Pharmacology, Faculty of Medicine, University of the Basque Country, Barrio Sarriena s/n, E-48940 Leioa, Bizkaia, Spain Received 31 May 2006; received in revised form 12 September 2006; accepted 13 September 2006

Abstract CB1 cannabinoid receptors located at presynaptic sites suppress synaptic transmission in the rat brain. The aim of this work was to examine by single-unit extracellular techniques the effect of the synthetic cannabinoid receptor agonist WIN 55212-2 on KCl-evoked excitation of locus coeruleus neurons in rat brain slices. Short applications of KCl (30 mM) increased by 9-fold the firing rate of locus coeruleus cells. Perfusion with the GABAA receptor antagonist picrotoxin (100 mM) increased KCl-evoked effect, whereas NMDA and non-NMDA glutamate receptor antagonists (D-AP5 100 mM and CNQX 30 mM, respectively) were able to decrease KCl-evoked effect only in the presence of picrotoxin (100 mM). Bath application of WIN 55212-2 (10 mM) inhibited KCl-evoked effect; this inhibition was blocked by the CB1 receptor antagonist AM 251 (1 mM). However, a lower concentration of WIN 55212-2 (1 mM) did not significantly change KCl effect. In the presence of picrotoxin (100 mM), perfusion with D-AP5 (100 mM) or CNQX (30 mM) blocked WIN 55212-2-induced inhibition, although picrotoxin (100 mM) itself failed to affect cannabinoid effect. In conclusion, GABAergic and glutamatergic components are both involved in KCl-evoked excitation of LC neurons, although CB1 receptors only seem to inhibit the glutamatergic component of KCl effect in the locus coeruleus. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Locus coeruleus; KCl; WIN 55212-2; Single-unit extracellular recording; D-AP5; CNQX

1. Introduction A cannabinoid system has been described to include specific cannabinoid receptors, which belong to the Gi/Go protein-coupled receptor family (Howlett et al., 2002), and several endogenous cannabimimetic lipids. Until now, two cannabinoid receptor types have been cloned: the CB1, which is mainly found in the central nervous system and the CB2, expressed primarily by immune cells (Howlett et al., 2002). In the central nervous system, the CB1 cannabinoid receptor is highly expressed in the basal ganglia, hippocampus, cerebellum and cerebral cortex. These regions regulate brain functions that are strongly influenced by cannabinoid administration such as motor activity, memory and perception

* Corresponding author. Tel.: þ34 94 601 5577; fax: þ34 94 601 3220. E-mail address: [email protected] (J. Pineda). 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.09.004

(Herkenham et al., 1991; Tsou et al., 1998). Several lines of evidence in vitro indicate that CB1 receptors are predominantly located at presynaptic level in many brain regions of the rat, where they suppress excitatory and inhibitory synaptic transmission by modulation of calcium and potassium channels (for reviews, see Freund et al., 2003; Szabo and Schlicker, 2005). Thus, immunohistochemical studies have revealed the existence of CB1 receptors on nerve terminals throughout the rat brain (Tsou et al., 1998). Moreover, in slice or synaptosome preparations CB1 receptor agonists inhibit neurotransmitter release from axon terminals in the striatum (Kofalvi et al., 2005), hippocampus (Katona et al., 1999; Cannizzaro et al., 2006) or cerebral cortex (Godino et al., 2005). Finally, electrophysiological studies have demonstrated that cannabinoid agonists decrease excitatory or inhibitory postsynaptic currents in rodent brain areas such as the periaqueductal gray (Vaughan et al., 2000), striatum (Szabo et al., 1998), ventral tegmental area (Szabo et al., 2002), nucleus accumbens


A. Mendiguren, J. Pineda / Neuropharmacology 52 (2007) 617e625

(Hoffman and Lupica, 2001) or hippocampus (Hajos et al., 2000; Hoffman and Lupica, 2000). The locus coeruleus (LC) is the main source of noradrenergic innervation of the brain. This nucleus participates in triggering different pathophysiological reactions of the brain including stress, alertness, pain and drug addiction (Margalit and Segal, 1979; Nestler and Aghajanian, 1997; Sved et al., 2002; Berridge and Waterhouse, 2003). Several findings have suggested a cannabinoid regulation of central noradrenergic system. Thus, binding radioautographic studies have shown the existence of an intermediate density of CB1 receptors in the LC (Herkenham et al., 1991). Furthermore, cannabinoid receptor activation increases the number of Fos protein immunoreactive noradrenergic cells (Patel and Hillard, 2003; Oropeza et al., 2005) and the firing activity of LC neurons (Gobbi et al., 2005; Mendiguren and Pineda, 2006; Muntoni et al., 2006), and they modulate noradrenaline release in projection areas (Moranta et al., 2004; Oropeza et al., 2005). Several behavioral effects of cannabinoids such as catalepsy, nociception and hypothermia have been postulated to occur through regulation of central noradrenergic systems (Singh and Das, 1976; Kataoka et al., 1987; Gutierrez et al., 2003). Previous assays have also demonstrated that CB1 receptors modulate NMDA-induced responses in the LC (Mendiguren and Pineda, 2004). The exact nature of cannabinoid regulation of the LC is unclear, because this nucleus has scarce CB1 receptor mRNA labeling (Matsuda et al., 1993). Since LC neurons are targeted by GABAergic and glutamatergic afferents (Aston-Jones et al., 1986), which play an important role in drug addiction (Berridge and Waterhouse, 2003), we considered of interest to evaluate the effect of a synthetic cannabinoid on the GABAergic and glutamatergic neurotransmission present in this nucleus. For this purpose, we characterized by extracellular electrophysiological techniques in vitro the importance of GABAergic and glutamatergic components in KCl-evoked excitation of LC neurons and then the role of CB1 receptors in the regulation of KCl-evoked effect. 2. Materials and methods 2.1. Animals Male SpragueeDawley rats weighing 200e300 g were housed under standard laboratory conditions (22  C, 12:12-h light/dark cycles) with free access to food and water. All experimental procedures reported in this manuscript were carried out in accordance with the European Community Council Directive on Protection of Animals Used in Experimental and Other Scientific Purposes of 24 November 1986 (86/609/EEC), and all efforts were made to minimize animal suffering and to reduce the number of animals used.

2.2. Brain slice preparation Animals were anesthetized with chloral hydrate (400 mg/kg i.p.) and decapitated. The brain was rapidly extracted after death and placed in icecold artificial cerebrospinal fluid (ACSF), where NaCl was substituted by sucrose to improve neuronal viability. Coronal brainstem sections of 600 mm thickness containing the LC were cut using a vibratome. The slice was allowed to recover from the slicing for 90 min in a modified Hass-type interface chamber maintained at 33  C and continuously perfused with ACSF at a flow rate of 1.5 ml/min. The ACSF contained (in mM): NaCl 129 mM, KCl 3 mM,

NaH2PO4 1.25 mM, MgSO4 2 mM, CaCl2 2 mM, NaHCO3 21 mM and D-glucose 10 mM, and was bubbled with 95% O2 plus 5% CO2 (pH 7.34).

2.3. Extracellular recordings Single-unit extracellular recordings of LC noradrenergic neurons were made as previously described (Pineda et al., 1996). The recording electrode, which consisted of an Omegadot glass micropipette, was pulled and filled with NaCl (0.05 M). The tip was broken back to a size of 2e5 mm (3e 5 MU). The electrode was positioned in the LC, which was identified visually in the rostral pons as a dark oval area on the lateral borders of the central gray and the IVth ventricle, just anterior to the genu of the facial nerve. The extracellular signal from the electrode was passed through a high-input impedance amplifier and monitored on an oscilloscope and also with an audio unit. Individual neuronal spikes were isolated from the background noise with a window discriminator and the firing rate was analyzed by means of a PC-based custommade software, which generated consecutive 10-s bin rate histograms (HFCPÒ, Cibertec SA, Madrid, Spain). Noradrenergic cells were identified by their spontaneous and regular discharge activity, the slow firing rate and the longlasting biphasic positiveenegative waveform (Andrade et al., 1983).

2.4. Pharmacological procedures To explore GABAergic and glutamatergic neurotransmission in the LC, we evaluated the excitation of LC neurons evoked by KCl in the presence or absence of GABA/glutamate receptor antagonists. To assess the effect of KCl on the firing activity of noradrenergic neurons, we perfused the slice for 30e60 s with ACSF containing a lower concentration of NaCl equiosmotically substituted for a high concentration of KCl (30 mM). The duration of this perfusion was adjusted at the beginning of each experiment to obtain a reproducible excitatory effect of KCl on LC neurons, which was tested every 10 min to allow recovery from previous application. To characterize the GABAergic and glutamatergic components of KCl-evoked excitation of LC neurons in each cell we calculated the effect of KCl application before (baseline effect) and several minutes after perfusion with the GABAA receptor antagonist picrotoxin (100 mM), the NMDA glutamate receptor antagonist D-AP5 (100 mM) or the non-NMDA glutamate receptor antagonist CNQX (30 mM). To study whether cannabinoids modulate KCl-evoked excitation, we tested the effect of KCl in the absence or presence of the synthetic CB1/CB2 receptor agonist WIN 55212-2. Finally, to analyze the mechanism involved in the putative effect of WIN 55212-2 on K...

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