Muscle membrane preparation restores sensitivity to acetylcholine in cultured chick ciliary ganglion neurons.
Ciliary ganglion (CG) neurons grown in culture in the absence of muscle cells rapidly lose sensitivity to acetylcholine (ACh), while neurons grown in the presence of muscle or muscle cell membranes maintain sensitivity to ACh for extended periods of time. The present study examined whether exposure to muscle membrane preparation or stimulation of cAMP-dependent processes could restore sensitivity to ACh in cultured neurons 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 current responses following application of ACh (IACh) to neurons under whole-cell voltage clamp. In control cultures IACh decreased from an average of 837 pA the day of plating to 145 pA following 4 days in culture. Stimulation of cAMP-dependent processes with forskolin and 3-isobutyl-1-methylxanthine (IBMX) or 8'Br-cAMP and IBMX had variable effects on IACh. These treatments increased peak IACh in some neurons maintained in culture for less than 48 h. Treatment with these agents decreased peak IACh in cultures which were more than 48 h old. Exposure of neurons, which had lost sensitivity to ACh in culture, to muscle membranes increased IACh 2- to 3-fold over 24 to 48 h. This membrane-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 IACh. In conclusion, these results indicate that some element associated with the membranes of muscle cells has the ability to restore ACh responsiveness to CG neurons which have become insensitive to ACh in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Spitsbergen
J M
JM
Department of Neuroscience, University of Virginia Health Sciences Center, Charlottesville 22908.
Tuttle
J B
JB
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004594:Electrophysiology; D005726:Ganglia, Parasympathetic / Q000166:cytology / Q000187:drug effects*; D008566:Membranes / Q000502:physiology; D009132:Muscles / Q000502:physiology*; D009474:Neurons / Q000187:drug effects*; D014176:Protein Biosynthesis; D011950:Receptors, Cholinergic / Q000187:drug effects
D011950:Receptors, Cholinergic; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003513:Cycloheximide; D000242:Cyclic AMP; D000109:Acetylcholine; D015056:1-Methyl-3-isobutylxanthine