Developmental Expression of Glutamic Acid Decarboxylase and of - Aminobutyric Acid Type B Receptors in the Ascidian Ciona intestinalis GIULIANA ZEGA, 1 * MAIRA BIGGIOGERO, 1 SILVIA GROPPELLI, 1 SIMONA CANDIANI, 2 DIANA OLIVERI, 2 MANUELA PARODI, 2 MARIO PESTARINO, 2 FIORENZA DE BERNARDI, 1 AND ROBERTA PENNATI 1 1 Dipartimento di Biologia, University of Milan, 20133 Milano, Italy 2 Department of Biology, University of Genoa, 16132 Genova, Italy ABSTRACT We describe Ciona intestinalis -aminobutyric acid (GABA)-ergic neurons during develop- ment, studying the expression pattern of Ci-GAD (glutamic acid decarboxylase: GABA synthe- sizing enzyme) by in situ hybridization. Moreover, we cloned two GABA B receptor subunits (Ci-GABA B Rs), and a phylogenetic analysis (neighbor-joining method) suggested that they clus- tered with their vertebrate counterparts. We compared Ci-GAD and Ci-GABA B Rs expression patterns in C. intestinalis embryos and larvae. At the tailbud stage, Ci-GAD expression was widely detected in central and peripheral nervous system (CNS/PNS) precursors, whereas Ci- GABA B Rs expression was evident at the level of the precursors of the visceral ganglion. GABA was localized by immunohistochemistry at the same developmental stage. In the larva, Ci-GAD transcripts and GABA immunofluorescence were also detected throughout the CNS and in some neurons of the PNS, whereas transcripts of both GABA B receptor subunits were found mainly in the CNS. The expression pattern of Ci-GABA B Rs appeared restricted to Ci-GAD-positive terri- tories in the sensory vesicle, whereas, in the visceral ganglion, Ci-GABA B Rs transcripts were found in ventral motoneurons that did not express Ci-GAD. Insofar as GABAergic neurons are widely distributed also in the CNS and PNS of vertebrates and other invertebrate chordates, it seems likely that GABA signaling was extensively present in the protochordate nervous system. Results from this work show that GABA is the most widespread inhibitory neurotransmitter in C. intestinalis nervous system and that it can signal through GABA B receptors both pre- and postsynaptically to modulate different sensory inputs and subsequent swimming activity. J. Comp. Neurol. 506:489 –505, 2008. © 2007 Wiley-Liss, Inc. Indexing terms: neurotransmitter; -aminobutiric acid; tunicate development; gene expression; metabotropic receptor; nervous system The ascidian Ciona intestinalis (Tunicata, Chordata) is a well-established model organism for studying basic mechanisms of chordate development (Satoh and Levine, 2005). Recent analysis suggests that the Tunicata repre- sent the closest living relatives of vertebrates (Delsuc et al., 2006; Vienne and Pontarotti, 2006; Can ˜ estro and Postlethwait, 2007). Thus, the ascidians are an intriguing model system in which to investigate the evolution of chordate characters; in fact phylogenetically, they are lo- cated at the protochordate– craniates transition. C. intes- tinalis develops through a swimming tadpole larva that possesses a basic chordate body plan with a dorsal neural tube and a notochord in the tail, flanked by muscle cells at both sides. Because its larva is formed by a small number of cells with an invariant lineage, C. intestinalis consti- tutes a favorable model for studying the mechanisms of cell differentiation and neural induction (Meinertzhagen et al., 2004). The central nervous system (CNS) of C. This article includes Supplementary Material available via the Internet at http://www.interscience.wiley.com/jpages/0021-9967/suppmat. The first two authors contributed equally to this work. Grant sponsor: MIUR-PRIN 2006; Grant number: 2006058952. *Correspondence to: Giuliana Zega, Dipartimento di Biologia, Sez. Zoo- logia 7B,, Universita ` di Milano, Via Celoria 26, 20133 Milano, Italy. E-mail: giuliana.zega@unimi.it Received 12 January 2007; Revised 1 June 2007; Accepted 6 October 2007 DOI 10.1002/cne.21565 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 506:489 –505 (2008) © 2007 WILEY-LISS, INC.