ANDERSEDSTROM. From the Department of Zoophysiology, University of Goteborg, Goteborg,SwedenINTRODUCTIONRapid axonal transport is likely to be dependentupon the integrity of microtubular systems (seereviews- by Samson, 1971; Ochs, 1972). Ac-tomyosin-like protein has been isolated from thebrain (Bert et al., 1973). In a hypothesis for fastaxonal transport, Ochs(1972) suggested the exist-ence of axonal transport filaments, which wouldslide along microtubules by means of cross-bridgesactivated by ATP. In analogy with muscle con-tractility the importance of Ca was suggested.In these circumstances it seemed of interest tostudy the effects of Ca and Mg` on fast axonaltransport.MATERIALS AND METHODSThe Transport SystemAn in vitro system from the frog Rana temporaria wasused. The preparation, which consists of the dorsalganglia (nos. 8 and 9), the sciatic nerve, and thegastrocnemius muscle, was placed in frog Ringer solutionin an incubation chamber with three compartments (A,B, C). The parts were separated from each other withsilicone grease barriers. [3H]Leucine was added to theganglionic compartment (compartment A), which madeit possible to follow the transport of labeled proteinsfrom the ganglia, along the sciatic nerve (compartmentB) towards the muscle (compartment C). Application ofthe silicone grease barrier between the nerve and themuscle compartments was not necessary for the experi-ments described in this paper. The preparation andincubation of the system has earlier been described indetail (Edstrom and Mattsson, 1972). It was demon-strated that proteins synthesized in the ganglia weretransported within the axons in anterograde direction ata rate of 127 t 10 mm/day at 18°C (Edstrom andMattsson, 1972; Edstrom and Hanson, 1973). The twopaired preparations from the same animal were used ineach experiment. One preparation served as a controland was incubated in a standard Ringer solution whilethe contralateral preparation was incubated in a modifiedRinger's. A ligature was placed at the middle level of thenerve and the preparation was incubated for 17 h at18°C. After treatment with trichloracetic acid (TCA) thedistribution of TCA-insoluble labeled components in theganglia and along the nerve was determined as describedpreviously (Edstrom and Mattsson, 1972; Anderson et812al., 1972). Soluene (Packard Instrument Co., Inc.,Downers Grove, 111.) dissolved samples in a 0.55%Permablend III (Packard Instrument Co.) solution intoluene were analyzed for radioactivity with a PackardTriCarb (model 3375) liquid scintillation spectrometer .Atomic-Absorption SpectrophotometryNerves exposed to elevated levels of Ca were washedtwice, 20 s each time, with standard Ringer's before wetashing. After wet ashing of nerves in concentrated H202,calcium was determined in a LaCI, solution by the use ofa Perkin-Elmer model 303 atomic-absorption spectro-photometer (Perkin-Elmer Corp., Instrument Div., Nor-walk, Conn.). Contents were referred to initial wetweights of nerves.Electron MicroscopyPreparations which had been incubated in modifiedRinger's were rinsed twice, 30 s each time, with standardRinger's immediately before fixation. The fixation solu-tion consisted of purified glutaraldehyde and freshlyprepared formaldehyde in a cacodylate buffer (Kar-novsky, 1965) containing 0.5% (vol/vol) dimethylsulfox-ide. The specimens were postfixed in cacodylate-bufferedosmium teroxide, dehydrated in a graded series ofethanol, and embedded in Epon. Sections, 1 µm thick,were prepared on an LKB Pyramitome or an LKBUltrotome III ultramicrotome and examined in a lightmicroscope. Thin sections were prepared from selectedareas and double stained with uranyl acetate and leadcitrate before the examination in aSiemens I A electronmicroscope.ChemicalsAqueous solutions of t .--[4, 5-2H]leucine (36-50 Ci/mmol, I mCi/ml) were purchased from The Radiochem-ical Centre, Amersham, England. The standard Ringersolution had the following millimolar composition:NaCl, 111.2; KC1, 1 .9 ; MgCl2, 1.6: CaCl2 , 1 .1 ;NaHCO3 , 2.4; and glucose, 5.5 . Ca-high and Mg'-high Ringer's were obtained by isotonic substitution of10-30 mM CaCl2 and of 20 mM MgCl2 for NaCl (78.2mM CaCl2 = 111.2 mM NaCl, 78.7 mM MgCl2 = 111.2mM NaCI). The pH of the various Ringer solutions wasadjusted to 7.4 by adding small amounts of HCI. Thesolutions were gassed with 02 before use. Ethyleneglycol-bis(s-aminoethyl ether)-N, N'-tetraacetic acid(EGTA) was obtained from Sigma Chemical Co., St.Louis, Mo.