RIBOSOMES IN THE SQUID GIANT AXON R. BLEHER and R. MARTIN* Sektion Elektronenmikroskopie, Universita «t Ulm, Albert-Einstein-Allee 11, D 89069 Ulm, Germany AbstractöRibosome clusters, referred to as endoaxoplasmic plaques, were documented and quantitatively analyzed in the squid giant axon at the light and electron microscopic levels. The methods included nonspeci¢c high a¤nity £uorescence staining of RNA by YOYO-1, speci¢c immuno£uorescence labeling of ribosomal RNA, electron energy loss spectro- scopic mapping of ribosomal phosphorus, and conventional transmission electron microscopy. The endoaxoplasmic plaques were sharply de¢ned, oval in shape, and less than 2 Wm in diameter. While they were very numerous in the postsynaptic axonal area of the giant synapse, the frequency of occurrence was much lower in the peripheral giant axon, with a density of about 1 plaque/1000 Wm 3 . Their distribution was random within axoplasm, with no preferential local- ization near the membrane. The several thousand ribosomes in a plaque usually were not membrane bound, but vesicular structures were observed in or near plaques; plaques were often surrounded by mitochondria. We conclude that ribosomes, a requisite machinery for protein synthesis, are present in the squid giant axon in discrete con¢gurations. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: peripheral giant axon, clusters of ribosomes, distribution, frequency. As highly polarized cells, neurons have a relatively small, metabolically active cell soma, dendrites, and, in many instances, a very long extension comprising the axon, the mass of which may exceed that of the perikaryon by several orders of magnitude. How long axons are main- tained in a steady state, how degraded cytoskeletal pro- teins are replaced, how proteins in the distant synaptic endings are renewed or augmented in response to stim- ulation, or how proteins are supplied to sprouting nerve terminals, are issues that are currently not well under- stood. Are the proteins exclusively supplied by the peri- karyon and transported to the periphery by axoplasmic transport, according to a slow transport model, or are proteins locally synthesized in restricted domains distant from the perikaryon, according to a local synthesis model, or is there a combination of the two modes of supplying axoplasmic proteins (for reviews see Koenig and Giuditta, 1999; Alvarez et al., 2000)? Evidence is accumulating for sorting and fast transport of speci¢c mRNA types from the cell body into the axon (for reviews see Mohr and Richter, 1992; Olink-Coux and Hollenbeck, 1996; Bassell and Singer, 1997; Oleynikow and Singer, 1998). Data are less clear with respect to the question as to whether or not there are ribosomes in axons and synapses. According to a prevail- ing opinion based on electron microscopical investiga- tions, ribosomes are most abundant in the perikarya, but excluded from axons (Peters et al., 1970). However, a growing number of studies reported detection of ribo- somes in vertebrate (Koenig and Martin, 1996; Martin, 1997; Pannese and Ledda, 1991; Zelena, 1972; Koenig et al., 2000) and invertebrate axons (Martin et al., 1989, 1998; Giuditta et al., 1991; Van Minnen et al., 1997; Crispino et al., 1997). In the case of the squid giant axon, there is a discrep- ancy of opinions about local systems of protein synthe- sis. According to the glial^axonal protein transfer hypothesis (Gainer et al., 1977; Lasek et al., 1977; Buchheit and Tytell, 1992 ; Sheller et al., 1995), axoplas- mic proteins are synthesized locally by periaxonal glial cells and transferred to the subjacent axon, while the axon lacks the capacity to synthesize proteins. However, data from Giuditta and co-workers showed a multiplicity of components of the protein synthesis machinery in the axon, such as mRNA, tRNA, rRNA, initiation and elon- gation factors and aminoacyl tRNA synthetases (for review see Koenig and Giuditta, 1999). In addition, there are ultrastructural studies of the squid axon, which were not in agreement. Thus, while one group of studies has found a limited number of clusters of ribo- some-like particles (Martin et al., 1989, 1998 ; Giuditta et al., 1991), a recent immunocytochemical study described accumulations of ribosomes in very large number and density (Sotelo et al., 1999). In the present study we examined giant axons of Loligo vulgaris and Loligo pealei for the quantitative determination of the density of ribosomes, using four di¡erent methods of analysis. 527 *Corresponding author. Tel. : +49-731-502-3441 ; fax : +49-731-502- 3383. E-mail address : rainer.martin@medizin.uni-ulm.de (R. Martin). Abbreviations : EDTA, ethylenediaminetetra-acetate ; EGTA, ethyl- ene glycol-bis(2-aminoethyl-ether)-N,N,NP,NP-tetraacetic acid; ESI, electron spectroscopic imaging; HEPES, N-(2-hydroxy- ethyl)piperazine-NP-(2-ethanesulfonic acid) ; PBS, phosphate-bu¡- ered saline. NSC 5207 9-11-01 Cyaan Magenta Geel Zwart www.neuroscience-ibro.com Neuroscience Vol. 107, No. 3, pp. 527^534, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII:S0306-4522(01)00366-9 0306-4522 / 01 $20.00+0.00