Ž . Brain Research 839 1999 213–220 www.elsevier.comrlocaterbres Research report Localization of L-glutamate and glutamate-like receptors at the squid giant synapse Anna Di Cosmo b , Gianna Nardi a , Carlo Di Cristo b , Amedeo De Santis a , John B Messenger c, ) a Stazione Zoologica ‘‘ Anton Dohrn’’, Naples, Italy b Dipartimento di Zoologia, UniÕersita degli Studi Federico II, Naples, Italy ` c Department of Animal and Plant Sciences, Sheffield UniÕersity, Sheffield S10 2TN, UK Accepted 4 May 1999 Abstract HPLC analysis of the amino acid contents of the second- and third-order giant fibres at the giant synapse in the stellate ganglion of the Ž . squid Loligo Õulgaris shows that there are significantly higher amounts of L-glutamate and L-aspartate in the second-order presynaptic Ž . fibre than in the third-order postsynaptic fibre. Immunocytochemical staining of sections of the ganglion with an antibody raised against L-glutamate produces specific positive staining in the synaptic region of the second-order fibre. In contrast, staining with antibodies raised Ž . against glutamate-receptors mammalian GluR1 with GluR2r3 produces positive staining in the third-order fibre at the postsynaptic region. These data provide further evidence for the hypothesis that L-glutamate is an excitatory transmitter at the giant synapse. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Squid giant synapse; Glutamate; Receptor 1. Introduction The squid giant synapse was first described 60 years w x ago by Young 28 and has since attracted the attention of w x numerous investigators 26 . Recently it has proved an especially useful system for studying the role of calcium in w x transmitter release 2,3,12,13 . Despite this, however, the Ž. nature of the endogenous transmitter s at the synapse has not been established beyond doubt and the present paper describes experiments that address this issue. The strongest candidate for the excitatory transmitter at Ž the squid giant synapse is L-glutamate for a rejection of w x. w x other possibilities see 25 . Miledi 18,19 was the first to implicate this amino acid in transmission at the synapse, but because he found that the reversal potential for the endogenous transmitter and L-glutamate were different, he was careful not to claim that it was the transmitter. Never- w x theless, subsequent experiments 1,10,11 continued to sup- AbbreÕiations: AMPA, a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-D-aspartic acid; CNQX, 6-Cyano-7- nitroquinoxaline-2,3-dione; DNQX, 6,7-Dinitroquinoxaline-2,3-dione; ŽŽ . . CPP, 3- R -2-carboxypiperazin-4-yl -propyl-1-phosphonic acid ) Corresponding author. Fax: q44-114-276-0159; E-mail: j.b.messenger@ sheffield.ac.uk port the candidature of L-glutamate. In 1989, De Santis and wx Messenger 6 found that the newly developed glutamate agonists kainate, quisqualate and AMPA, though not NMDA, were active at the synapse. They also found that CNQX and DNQX, specific antagonists of kainate and AMPA receptors, reversibly blocked transmission, al- though an NMDA antagonist, CPP, was without effect. One crucial piece of evidence in demonstrating a sub- stance to be a neurotransmitter is to show that it can activate the postsynaptic membrane when applied to it directly. Until recently, this had not been possible at the squid giant synapse because its tortuous nature and narrow Ž . w x 12 nm synaptic cleft 15,29 prevent sufficiently rapid access of a perfusate. To overcome this problem, Corrie et wx al. 4 developed a ‘‘caged’’ glutamate from which L- glutamate could be released in situ by flash photolysis. Under suitable conditions the released L-glutamate trig- gered action potentials in the third-order fibre, showing that there are indeed glutamate receptors on the post- w x synaptic membrane 16 . However, to identify a substance as a neurotransmitter, it is also necessary to show that it is actually present in the presynaptic terminal, and this paper presents evidence that Ž . L-glutamate and L-aspartate occurs in higher concentra- tions in the presynaptic fibre than in the postsynaptic. It 0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0006-8993 99 01591-7