MUSCLE MEMBRANE PREPARATION RESTORES SENSITIVITY TO ACETYLCHOLINE IN CULTURED CHICK CILIARY GANGLION NEURONS

Ciliary ganglion (CG) neurons grown in culture in the absence of muscl e cells rapidly lose sensitivity to acetylcholine (ACh), while neurons grown in the presence of muscle or muscle cell membranes maintain sen sitivity to ACh for extended periods of time. The present study examin ed whether exposure to muscle membrane preparation or stimulation of c AMP-dependent processes could restore sensitivity to ACh in cultured n eurons which had lost responsiveness to ACh. CG neurons from 11- to 14 -day-old chick embryos were grown on collagen substrate in the absence of muscle cells. Sensitivity to ACh was assessed by measuring peak cu rrent responses following application of ACh (I(Ach)) to neurons under whole-cell voltage clamp. In control cultures I(ACh) decreased from a n average of 837 pA the day of plating to 145 pA following 4 days in c ulture. Stimulation of cAMP-dependent processes with forskolin and 3-i sobutyl-1-methylxanthine (IBMX) or 8'Br-cAMP and IBMX had variable eff ects on I(ACh). These treatments increased peak I(ACh) in some neurons maintained in culture for less than 48 h. Treatment with these agents decreased peak I(ACh) in cultures which were more than 48 h old. Expo sure of neurons, which had lost sensitivity to ACh in culture, to musc le membranes increased I(ACh) 2- to 3-fold over 24 to 48 h. This membr ane-induced restoration of sensitivity to ACh was blocked by exposure to the protein synthesis inhibitor cycloheximide. Stimulation of cAMP- dependent processes in neurons exposed to muscle membrane decreased I( ACh). In conclusion, these results indicate that some element associat ed with the membranes of muscle cells has the ability to restore ACh r esponsiveness to CG neurons which have become insensitive to ACh in cu lture. The ability to restore ACh responsiveness appears to be depende nt on protein synthesis as it is blocked by the protein synthesis inhi bitor cycloheximide. Restoration of ACh sensitivity by muscle cell mem brane does not appear to be associated with stimulation of cAMP-depend ent processes.