Neuropsychopharmacology (2007) 32, 1242–1250 & 2007 Nature Publishing Group All rights reserved 0893-133X/07 $30.00 www.neuropsychopharmacology.org The Effect of Intrahippocampal Injection of Group II and III Metobotropic Glutamate Receptor Agonists on Anxiety; the Role of Neuropeptide Y ´ ,1 1 1 1 Maria Smiałowska* , Joanna M Wieron´ska , Helena Domin and Barbara Zie˛ba 1Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland Earlier studies conducted by our group and by other authors indicated that metabotropic glutamatergic receptor (mGluR) ligands might have anxiolytic activity and that amygdalar neuropeptide Y (NPY) neurons were engaged in that effect. Apart from the amygdala, the hippocampus, another limbic structure, also seems to be engaged in regulation of anxiety. It is rich in mGluRs and contains numerous NPY interneurons. In the present study, we investigated the anxiolytic activity of group II and III mGluR agonists after injection into the hippocampus, and attempted to establish whether hippocampal NPY neurons and receptors were engaged in the observed effects. Male Wistar rats were bilaterally microinjected with the group II mGluR agonist (2S,10S,20S)-2-(carboxycyclopropyl)glycine (L-CCG-I), group III mGluR agonist O-Phospho-L-serine (L-SOP), NPY, the Y1 receptor antagonist BIBO 3304, and the Y2 receptor antagonist BIIE 0246 into the CA1 or dentate area (DG). The effect of those compounds on anxiety was tested in the elevated plus-maze. Moreover, the effects of L-CCG-I and L-SOP on the expression of NPYmRNA in the hippocampus were studied using in situ hybridization method. It was found that a significant anxiolytic effect was induced by L-SOP injection into the CA1 region or by L-CCG-I injection into the DG. The former effect was inhibited by BIBO 3304, the latter by BIIE 0246. NPY itself showed antianxiety action after injection into both structures. In the CA1 area, the effect of NPY was prevented by BIBO 3304, whereas in the DG by BIIE 0246. Both the mGluR agonists L-CCG-I and L- SOP induced a potent increase in NPYmRNA expression in the DG region of the hippocampal formation. The obtained results indicate that group II and III mGluR agonists, L-CCG-I and L-SOP, as well as NPY display anxiolytic activity in the hippocampus, but act differently in the CA1 and DG. It was observed that group III mGluRs and Y1 receptors were engaged in the response in the CA1 area, whereas group II mGluRs and Y2 receptors played a pivotal role in the DG region. Neuropsychopharmacology (2007) 32, 1242–1250. doi:10.1038/sj.npp.1301258; published online 29 November 2006 Keywords: metabotropic glutamate receptors; hippocampus; anxiety; neuropeptide Y INTRODUCTION Duvoisin, 1995). Ionotropic glutamatergic receptors show similar glutamate-binding properties. Antianxiety-like ef- Anxiety-related disorders are a common disease imposing a fects were observed after the mGluR5 antagonist MPEP or significant social burden. At present, pharmacotherapy of the mGluR group II agonist LY354740, injected systemically these disorders is based mainly on benzodiazepines (BZDs). to rats (Pilc et al, 2002; Chojnacka-Wo´jcik et al, 2001), after However, BZDs are not free of side effects, such as sedation, the intrahippocampal injection of others group I mGluR motor disturbances or nausea. Moreover, dependence and antagonists (Chojnacka-Wo´jcik et al, 1997) and after group tolerance develop after a prolonged treatment (Chojnacka- II and III mGluR agonists (Tatarczynska et al, 2001, 2002; Wo´jcik et al, 2001). Recent experimental studies have Palucha et al, 2004). shown that ligands of metabotropic glutamatergic receptors Our earlier studies showed that the amygdala was an (mGluRs) may also regulate anxiety level. Metabotropic important structure participating in the antianxiety action GluRs are a group of receptors that bind glutamate, the of MPEP and of group II and III mGluR agonists. Moreover, most abundant excitatory neurotransmitter (Pin and neuropeptide Y (NPY) neurons and receptors took part in that effect (Wieron´ska et al, 2003, 2005). NPY in the amygdala has been postulated to be a potent, endogenous *Correspondence: Dr M S´miałowska, Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Sme˛tna Street anxiolytic (Heilig et al, 1993), as anxiolytic-like effects were 12, 31-343 Krako´w, Poland, Tel: + 48 12 66 23 288, Fax: + 48 12 637 observed after intraamygdalar NPY microinjection. Our 45 00, E-mail: [email protected] previous results indicated that NPY neurons in the Received 3 February 2006; revised 25 September 2006; accepted 25 amygdala were regulated by Glu transmission, and that September 2006 mGluRs were engaged in this regulation, as mGluR ligands Anxiolytic effect of intrahippocampal mGluR ligands and NPY MS´miałowska et al 1243 changed the expression of NPY and/or NPYmRNA in that nyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2- structure (Wieron´ska et al, 2003, 2005). [4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piper- The hippocampal formation is a limbic structure, which is azinyl]-2-oxoethyl]-cyclopentaneacetamide (Tocris), a Y2 also engaged in the regulation of anxiety (Gray, 1982; receptor antagonist; (6) control rats received similar Eichenbaum et al, 1992; Vianna et al, 2004; Kalisch et al, volumes of solvent. 2006). It contains numerous NPY interneurons and is rich All the compounds were dissolved in distilled water, their in mGluRs (Kohler et al, 1986; Redrobe et al, 1999; Dumont pH was adjusted to 7.4 with NaOH or HCl when necessary. et al, 1998). It was revealed that the enhancement of All drugs were injected bilaterally into the hippocampus. glutamatergic transmission strongly stimulated NPY synth- esis and/or increased the content of that peptide in the Cannulae Implantation and Histology hippocampus (S´miałowska et al, 2003; Bellmann et al, 1991; Schwarzer et al, 1996). Thus, it seemed of interest to find The rats were anesthetized with Equitesin and were out whether anxiolytically active mGluR ligands could stereotaxically bilaterally implanted with chronic guide regulate NPY neurons in the hippocampal formation and cannulae aimed at the dorsal hippocampus CA1 or dentate whether such an interaction was engaged in their anti- area (DG) (dentate gyrus) region, 2 mm above the injection anxiety effects. Our earlier findings indicated that group II sites. The guide cannulae (23-gauge stainless steel tubing), and III mGluR agonists ((2S,10S,20S)-2-(carboxycyclopro- secured by dental cement, were anchored to the skull by pyl)glycine (L-CCG-I) and O-Phospho-L-serine (L-SOP)) three stainless steel screws. In order to prevent clogging, induced an increase in the expression of NPY-immuno- stainless steel stylets were placed in the guide cannulae and reactivity in the hippocampus, as demonstrated by a left until the animals were microinjected 5–7 days later. The radioimmunoassay (RIA) method (S´miałowska et al, rats were adapted to handling, and on the test day the stylets 2002). Therefore, in the present study, we examined the were withdrawn and replaced with injection cannulae effects of two mGluR agonists, L-CCG-I (group II agonist) ending 2 mm below the tip of the guide cannulae. The and L-SOP (group III agonist), after intrahippocampal coordinates for injection sites were: A + 5.7, L72.1, H + 7.2 administration, on anxiety and NPYmRNA expression in (for injections into the CA1), and A + 5.7, L71.6, H + 6.3 that structure. Moreover, we tried to find out whether the (for injections into the DG) measured from the interaural anxiolytic effects of those compounds were dependent on line according to Paxinos and Watson stereotaxic atlas NPY transmission. Studies with mGluR ligands aimed to (Paxinos and Watson, 1986). On completion of each assess their potential, as future anxiolytic drugs seem to be experiment, the rats were killed, their brains were removed, important, as it is anticipated that they may not elicit fixed in formalin, and injection sites were verified in tolerance and dependence, unlike BZDs. This is because coronal sections. Only animals with the correct injection some previous studies concerned with mGluR5 antagonists sites were taken for a further analysis. with anxiolytic activity (MPEP and MTEP) showed that those compounds did not engage BZD receptors, acted via Behavioral Studies: Plus-Maze Procedure NPY neurons, and at least MPEP did not induce tolerance after prolonged treatment (Kłodzin´ska et al, 2004; Plus-maze test was carried out according to the method Wieron´ska et al, 2004; Pilc et al, 2002). described by Pellow (Pellow and File, 1986) and similarly as in our previous study (Wieron´ska et al, 2003). Briefly, the plus-maze apparatus was made of wood and consisted MATERIALS AND METHODS of two open arms, 50 Â 10 cm, and two closed arms, Animals 50 Â 10 Â 40 cm, and an open roof. It was painted black but the wooden walls of the closed arms maintained their Male Wistar rats weighing 200–230 g at the beginning of natural color. The apparatus was elevated 50 cm above the experiments were used. The animals were obtained from a floor. Two open arms were opposite to each other and were local breeding farm. The rats were age-matched, and were illuminated with a 40 W bulb positioned 20 cm above each housed six to a cage under a 12:12 light–dark cycle, with free of them. access to food and tap water. The rats after cannulae The design of experiment is presented in Table 1. implantation were housed singly. During the experiment, all NPY and mGluR ligands were given 20 min before the efforts were made to minimize the animals’ suffering and to plus-maze test, whereas BIBO 3304 or BIIE 0246 was reduce the number of animals used, in accordance with the injected 10 min before those drugs.