430 reported to be a neurogenic region comprising neuronal stem cells [4]. Recent studies have shown that the proliferating cells of the PVZ of the lateral ventricles in larval and adult X. laevis are homologous to neural stem cells of higher vertebrates [3]. In mammals, the cells of the embryonic and adult PVZ of many brain regions have been thoroughly characterized [5–8]. In lower vertebrates such as amphibians, comparable detailed studies are lacking. Extracellular nucleotides act via purinergic receptors, which have been divided in two main families: adenosine or P1 receptors and P2 receptors [9]. The P1 receptors all couple to G proteins and have been further subdivided into four subtypes (A1, A2A, A2B, and A3). The P2 receptors have been split into ionotropic P2X receptors and G protein coupled P2Y receptors. To date, seven subtypes of P2X receptors (P2X1–P2X7) and eight subtypes of P2Y recep- tors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been described [10–12]. An additional P2Y receptor, named P2Y8, with an extremely broad agonist selectivity has been cloned from X. laevis embryos [13]. Activation of purinergic receptors has been shown to play an important role in many physiological processes [14], includ- ing embryonic and adult neurogenesis [14, 15] and neuron– glia interactions in the OB [16]. It has been shown, mainly by means of immunohistochemical studies, that purinergic receptors and associated nucleotide-degrading enzymes are expressed in the murine OB and PVZ [14, 15]. However, a thorough functional study of the purinergic system of these two brain regions is as yet missing, and to our knowledge in non-mammalian vertebrates there are no studies at all. In the present study, we described the ultrastructural organization of the PVZ of larval X. laevis, and determined the proliferation rates of its neuronal stem cells. We then identified and investigated the purinergic system of the OB and its neurogenic PVZ and demonstrate that activation of purinergic receptors initiates stereotypic Ca2+ signaling in cells of both brain areas. Thereby, we found that cells of the two brain areas express different purinergic receptor subtypes. Also, we obtained evidence that the purinergic receptors expressed in the OB are not evidently involved in the immediate processing of olfactory information. Possible physiological roles of the purinergic system in the OB and the PVZ are discussed. Materials and methods Slices of the anterior telencephalon and nose–brain preparations Larval X. laevis (stages 51 to 54; staged after [17]) were cooled to produce complete immobility, and then killed by transection of the brain at its transition to the spinal cord. Purinergic Signalling (2010) 6:429–445 All procedures for animal handling and tissue dissections were carried out according to the guidelines of the Göttingen University Committee for Ethics in Animal Experimentation. For slices of the anterior telencephalon, a block of tissue containing the terminal part of the olfactory nerves and the anterior brain was cut out. Then, the tissue was glued onto the stage of a vibroslicer (VT 1000S, Leica, Bensheim, Germany), cut horizontally into one 120–130 μm thick slice and kept in bath solution. For nose–brain preparations a block of tissue containing the olfactory epithelia, the olfactory nerves and the anterior part of the brain was cut out and kept in bath solution. The tissue was then glued onto the stage of the vibroslicer and only the dorsal surface of the OB was sliced off. The olfactory epithelia were left intact. For a more detailed description of these preparations see earlier work of our lab [18, 19] and Figs. 3 and 8. Solutions, staining protocol, and stimulus application Standard bath solution consisted of (in mM): 98 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 5 glucose, 5 Na-pyruvate, 10 HEPES, 230 mOsmol/l, pH 7.8. Ca2+-free bath solution consisted of (in mM): 98 NaCl, 2 KCl, 2 MgCl2, 5 glucose, 5 Na-pyruvate, 10 HEPES, 2 EGTA, 230 mOsmol/l, pH 7.8. Tissue preparations were transferred to a recording chamber, and bath solution containing 50 μM Fura-2/AM (Molecular Probes, Leiden, The Netherlands) was added. Fura-2/AM was dissolved in DMSO (Sigma, Deisenhofen, Germany) and Pluronic F-127 (Molecular Probes). The final concentrations of DMSO and Pluronic F-127 did not exceed 0.5% and 0.1%, respectively. Cells of the nervous system of larval X. laevis express multidrug transporters [20, 21] with a wide substrate spectrum, including calcium- indicator dyes. To avoid transporter-mediated destaining of the slices, 50 μM MK571 (Alexis Biochemicals, Grünberg, Germany), an inhibitor of multidrug transporters was added to the incubation solution. The preparations were incubated on a shaker at room temperature for 35 min. 5-Bromo-2′-deoxyuridine (BrdU); adenosine; adenosine-5′- diphosphate (ADP); adenosine-5′-triphosphate (ATP); uridine; uridine-5′-triphosphate (UTP); α,β-methylene-ATP (α,β- MeATP); β,γ-methylene-ATP (β,γ-MeATP); 2-methylthio- ATP (2MeSATP); 3′-O-(4-benzoyl)benzoyl ATP (BzATP); suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic ac- id (PPADS), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D(−)-2-amino-5-phosphonopentanoic acid (APV) and bath solution chemicals were purchased from Sigma. As odorants, we used a mixture of 15 L-amino acids (glycine, alanine, serine, threonine, cysteine, valine, leu- cine, isoleucine, methionine, proline, arginine, lysine, histidine, phenylalanine, and tryptophan, all from Sigma, each at a concentration of 100 μM).