Localization of Glutamate and Glutamate Transporters in the Sensory Neurons of Aplysia JONATHAN LEVENSON, 1 DAVID M. SHERRY, 2 LAURENCE DRYER, 1 JEANNIE CHIN, 3 JOHN H. BYRNE, 3 AND ARNOLD ESKIN 1 * 1 Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5513 2 College of Optometry, University of Houston, Houston, Texas 77204 3 Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, Houston, Texas 77225 ABSTRACT The sensorimotor synapse of Aplysia has been used extensively to study the cellular and molecular basis for learning and memory. Recent physiologic studies suggest that glutamate may be the excitatory neurotransmitter used by the sensory neurons (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163–7167; Armitage and Siegelbaum [1998] J Neurosci. 18: 8770 – 8779). We further investigated the hypothesis that glutamate is the excitatory neuro- transmitter at this synapse. The somata of sensory neurons in the pleural ganglia showed strong glutamate immunoreactivity. Very intense glutamate immunoreactivity was present in fibers within the neuropil and pleural-pedal connective. Localization of amino acids metabolically related to glutamate was also investigated. Moderate aspartate and glutamine immunoreactivity was present in somata of sensory neurons, but only weak labeling for aspartate and glutamine was present in the neuropil or pleural-pedal connective. In cultured sensory neurons, glutamate immunoreactivity was strong in the somata and processes and was very intense in varicosities; consistent with localization of glutamate in sensory neurons in the intact pleural-pedal ganglion. Cultured sensory neurons showed only weak labeling for aspartate and glutamine. Little or no -aminobutyric acid or glycine immunoreactivity was observed in the pleural-pedal ganglia or in cultured sensory neurons. To further test the hypothesis that the sensory neurons use glutamate as a transmitter, in situ hybridization was performed by using a partial cDNA clone of a putative Aplysia high-affinity glutamate transporter. The sensory neurons, as well as a subset of glia, expressed this mRNA. Known glutamatergic motor neurons B3 and B6 of the buccal ganglion also appeared to express this mRNA. These results, in addition to previous physiological studies (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163–7167; Trudeau and Castellucci [1993] J Neurophysiol. 70:1221–1230; Armitage and Siegelbaum [1998] J Neurosci. 18:8770 – 8779)) establish glu- tamate as an excitatory neurotransmitter of the sensorimotor synapse. J. Comp. Neurol. 423: 121–131, 2000. © 2000 Wiley-Liss, Inc. Indexing terms: mollusc; aspartate; glutamine; glycine; GABA The synapse between the ventral-caudal (VC) cluster of sensory neurons in the pleural ganglion and their associ- ated motor neurons in the pedal ganglion of Aplysia cali- fornica has been used extensively to study the cellular and molecular basis of learning and memory (reviewed in By- rne et al., 1991; Bailey et al., 1996; Abel et al., 1998). Although a great deal is known about regulatory mecha- nisms affecting transmission at this synapse, the identity of the excitatory neurotransmitter of this synapse remains equivocal. Consequently, it is important to identify the Grant sponsor: Basic Research And Initiatives In Neuroscience (BRAIN) program at the University of Houston; Grant sponsor: National Institutes of Health; Grant numbers: NS28462, NS32748, and NS19895. *Correspondence to: Arnold Eskin, Department of Biology and Biochem- istry, University of Houston, 4800 Calhoun Rd, SRII, Houston, TX 77204- 5513. E-mail: eskin@uh.edu Received 9 December 1999; Revised 6 March 2000; Accepted 14 March 2000 THE JOURNAL OF COMPARATIVE NEUROLOGY 423:121–131 (2000) © 2000 WILEY-LISS, INC.