We describe Ciona intestinalis gamma-aminobutyric acid (GABA)-ergic neurons during development, studying the expression pattern of Ci-GAD (glutamic acid decarboxylase: GABA synthesizing 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 clustered 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 territories 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.
Neurotransmitters play an important role in larval metamorphosis in different groups of marine invertebrates. In this work, the role of dopamine and serotonin during metamorphosis of the ascidian Phallusia mammillata larvae was examined. By immunofluorescence experiments, dopamine was localized in some neurons of the central nervous system and in the adhesive papillae of the larvae. Dopamine and serotonin signaling was inhibited by means of antagonists of these neurotransmitters receptors (R(+)-SCH-23390, a D(1) antagonist; clozapine, a D(4) antagonist; WAY-100635, a 5-HT(1A) antagonist) and by sequestering the neurotransmitters with specific antibodies. Moreover, dopamine synthesis was inhibited by exposing 2-cell embryos to alpha-methyl-l-tyrosine. Dopamine depletion, obtained by these different approaches, caused early metamorphosis, while serotonin depletion delayed the onset of metamorphosis. The opposite effects were obtained using agonists of the neurotransmitters: lisuride, a D(2) agonist, inhibited metamorphosis, while DOI hydrochloride and 8-OH-DPAT HBr, two serotonin agonists, promoted it. So, it is possible to suppose that dopamine signaling delayed metamorphosis while serotonin signaling triggers it. We propose a mechanism by which these neurotransmitters may modulate the timing of metamorphosis in larvae.
The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an important role in a wide range of non-neural processes. Using immunofluorescence with an antiserotonin antibody, 5-HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5-HT on development, we treated embryos with two different 5-HT receptor subtype antagonists. Treatment at the gastrula stage with 10 microM ondansetron, an antagonist of the 5-HT(3) receptor, induced anterior truncation and a short tail. At 10 microM, ritanserin, a 5-HT(2B) receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5-HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5-HT is suggested.
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