Factors released by ciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells.
The nuclei of cultured noninnervated muscle cells are heterogeneous with respect to production of mRNA for the nicotinic acetylcholine receptor (AChR). Some nuclei actively express AChR mRNA while others have a low level of activity or are inactive. To determine if innervation, or a factor released by neurons, influences nuclear expression of AChR mRNA, we examined mRNA at a single cell level via in situ hybridization and autoradiography with an alpha-subunit AChR genomic probe. Four days after plating, we co-cultured chicken primary muscle cells with spinal cord explants, ciliary neurons, or dorsal root ganglia (DRG) cells. In situ hybridization of the spinal-cord and muscle-cell co-cultures with the AChR alpha-subunit probe revealed a high density of silver grains on muscle cells, which were within two explant diameters of the spinal cord explant, and a graded decrease in silver grain density as the distance from the explant increased, as well as the appearance of a strikingly nonhomogenous distribution of active and inactive muscle cell nuclei. When ciliary neurons were uniformly distributed over the muscle cells, a high level of AChR mRNA was induced, but no gradients appeared. Neither an increased mRNA level nor a gradient was observed when DRG cells were co-cultured with muscle cells. When ciliary neurons are cultured within Costar permeable inserts, which prevent any contact between the neurons and the underlying muscle cells, AChR messenger RNA is still induced, showing that diffusible factors are responsible. Our results indicate that molecules released by cholinergic neurons regulate the expression of AChR mRNA in the myotubes and raise the possibility that AChR expression depends on both neuronal signals and on intracellular information from the muscle cell.
Bursztajn
S
S
Department of Neurology, Baylor College of Medicine, Houston, Texas 77030.
Berman
S A
SA
Gilbert
W
W
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D002799:Cholinergic Fibers / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005726:Ganglia, Parasympathetic / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D009132:Muscles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000378:metabolism / Q000494:pharmacology*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism*
D009414:Nerve Growth Factors; D012333:RNA, Messenger; D011950:Receptors, Cholinergic; D004247:DNA