Materials and Met: hods

IgG was prepared from plasma by the Rivanol (ethacridine)-ammonium sulfate method [17). The LEMS IgG was from a patient with no signs of carcinoma. The ability of this IgG preparation to transfer the physiological defect of LEMS to mice in vivo {8, 9) arid to bind to the active zones {I11 had been established in; previous studies. IgG from a healthy subject was used as a control. F (ab ‘) 2 and Fab fragments were prepared from the I@; preparations using pepsin and papain, respectively (181.

In previous studies, LEMS IgG was transferred to mice by injection. However, the in vivo half-life of IgG fragments is only a few hours [19,:! O). Therefore, mouse diaphragm motor terminals wer’e exposed to LEMS IgG in an organ culture system in vitro. After motor nerve is sectioned, the motor nerve terminals remain physiologically and morphologically intact for a certain period of time. The duration of this period depends can the temperature, the species, and the length of the motor nenre stump. Subsequently, the nerve terminals rapidly degenerate and then disappear from the end-plate 121, 22). For this reason the duration of the in vitro experiment had to be short enough for an adequate number of nerve termirials to remain physiologically and morphologically intact, but long enough for the antibodies to permeate the preparation and exert their pathological effects. Diaphragm muscles w1; re obtained from 15 male BKTO mice weighing 20 to 30 gm. Whole diaphragms with phrenic nerve stumps 2 to 3 cni in length were incubated for 24 hours at 22 to 24 C. in an organ-culture bath equilibrated with 95% 0 2 and 594 C02. The incubation medium contained Dulbecco’s Modified Eagle Medium with HEPES buffer, 4 mM L-glutamine, 25 mM glucose (Gibco Laboratories, Grand Island, NY), I00 units/ml penicillin, 0.1 mdml streptomycin, and 2 to 4 mglinl IgG, F (ab’) z, or Fab. After incubation, 1 hemidiaphragm of each animal was used for electrophysiological studies, as described elsewhere 1231. The other hemidiaphragrn w: s fixed with 25% glutaraldehyde in 0.1 M sodium cacodylatt:(pH 7.3) and prepared for freezefracture electron micro: icopy, as described previously [2, 10).