Recently, we established methods in the laboratory that allow primary culture of differentiated cell types derived from isolated C. elegans blastomeres. The availability of robust cell culture models provides experimental opportunities heretofore unavailable in the field. We have begun to characterize the physiological properties of cultured body muscles and cholinergic neurons.
myo-3 and
unc-54 encode specific myosin heavy chain isoforms that are co-expressed in body muscles and muscle precursor cells. The PD4251 worm strain expresses two GFPs with peptide signals that target them to either the nucleus or mitochondria of all body wall and vulval muscles . Cultures derived from PD4251 worms exhibit bright GFP fluorescence in vitro . GFP expression is detected in 8-10% of undifferentiated, freshly isolated blastomeres. Twenty-four hours after isolation, GFP fluorescence was detected exclusively in spindle-shaped cells with 1 or 2 well-formed processes. Approximately 20% of cells in culture express
myo-3 ::GFP, which is similar to that observed in the newly hatched L1 larva. GFP was localized to both the nucleus and elongated intracellular structures that are likely mitochondria. All
myo-3 ::GFP-positive muscle cells in culture also exhibited immunofluorescence localization of UNC-54 myosin.
unc-4 encodes a homeodomain transcription factor that is expressed in the 13 embryonic cholinergic motor neurons, which represent 2.4% of the 550 cells that comprise the newly hatched L1 larva. GFP-positive cells are present at a frequency of 2-4% in cultures produced from
unc-4 ::GFP transgenic worms. All GFP-expressing cells in culture have well-developed, neuron-like morphology. Cholinergic motor neurons form neuromuscular junctions with striated body wall muscles in vivo . A specific synaptic vesicle protein, synaptotagmin (SNT-1), that functions at these neuromuscular synapses co-localized to
unc-4 ::GFP neurons in vitro . In
unc-4 ::GFP-expressing cultures, we observed rare examples of cholinergic motor neurons sending out processes that made physical contact with spindle-shaped muscle cells. Furthermore, we observed muscle cells undergoing what appeared to be rhythmic contractions. Removal of bath Cl - , which may depolarize the muscle cell membrane, increased the number of cells exhibiting contractile-like activity and the frequency of apparent contractions. These intriguing observations suggest that co-cultures enriched in muscle cells and motor neurons may be useful for investigating neuromuscular synapse formation and physiological processes associated with muscle contraction. Muscle cells were readily patch clamped in the whole-cell mode. When dialyzed with a high K + pipette solution, slowly inactivating, strongly outwardly rectifying K + currents were detected. These currents were inhibited ~40% by 20 mM TEA and ~85% by 20 mM TEA plus 3 mM 4-aminopyridine (4-AP). Recently, Richmond and Jorgensen reported elegant in situ patch clamp studies of C. elegans body wall muscles ( Nat. Neurosci. 2: 791-797, 1999). The electrophysiological properties of C. elegans muscle cells in culture are remarkably similar to those that they described. The similarity between in vivo and in vitro patch clamp recordings argues that primary cultures of body wall muscles recapitulate at least some of their native functional properties.