The effect of neuronal activity on the competitive elimination of neuromuscular junctions in tissue culture.

The number of functional contacts between neurones of explanted ciliary ganglia and cultured muscle cells of the chicken, was determined electrophysiologically. After one week in culture the average muscle cell in the vicinity of a ganglion appeared to have contact with 2.5 neurones. This innervation remained unchanged during the next week. Electrophysiological measurements and a morphological study showed that the initial polyneuronal innervation was not confined to one site on the muscle cell, junctions were often found at a distance of 100 micron from one another. 'Phasic' stimulation (intermittent 2-s trains of 60 stimuli) of the neurones of a ganglion during one day or more caused most muscle cells to become mononeuronally innervated. Some muscle cells lost all functional contacts with the neurones. 'Tonic' stimulation, however (3 or 6 Hz continuously), even during one week and with the same total number of stimuli as the 'phasic' stimulation, produced no alteration in the distribution of the contacts. If two ganglia innervated the same muscle cells, after one week in culture twice as many neuronal contacts were found per muscle cell, equally divided over both ganglia. 'Phasic' stimulation of one of a pair of two ganglia eliminated nearly all functional contacts with the non-stimulated ganglion, whereas the innervation from the active ganglion became mononeuronal on most muscle cells. It is concluded that in tissue culture activity related mechanisms have a decisive influence upon the development of either polyneuronal or mononeuronal innervation of muscle cells. The results demonstrate that the signal for the elimination of supernumerary synapses in tissue culture is mediated by the postsynaptic cell, a mechanism which may also act in vivo. It is suggested that this signal may be the local depolarization around the surviving synapse.

Effects of innervation by ciliary ganglia on developing muscle in vitro.

Electrophysiological and pharmacological properties of neuromuscular junctions formed in tissue culture between chick ciliary ganglia and chick skeletal muscle cells have been studied. Functional neuromuscular junctions are formed already within 24 h. No functional acetylcholine (ACh) esterase is present at these end-plates. The neurites conduct action potentials to the neuromuscular junctions, where EPPs are generated. Tetrodotoxin (TTX) blocks this nerve conduction but in the presence of TTX MEPP-like potentials remain whose amplitudes are lowered when the Mg2+/Ca2+ ratio in the medium is raised. It is speculated that these large TTX-resistant potentials are multiquantal. The relatively high resting membrane potential in non-innervated muscle fibers was not changed by innervation. ACh-sensitivity was determined by iontophoretical application of ACh to the myotubes. Non-innervated myotubes exhibited an evenly distributed ACh-sensitivity. Local differences in ACh-sensitivity were always gradual and never exceeded a factor of 3. Innervation did not alter the overall ACh-sensitivity, but on functionally innervated muscle cells loci hypersensitive to ACh were found. Hypersensitivity was located within sharply defined areas. Apparently the parasympathetic neurons of the ciliary ganglion are able to form functional neuromuscular junctions with skeletal muscle cells in tissue culture and to induce the formation of regions of high ACh-sensitivity.

The anticholinesterase hypothermia in the rat: its practical application in the study of the central effectiveness of oximes.

Cholinesterase inhibitors that can pass the blood-brain barrier produce hypothermia when injected intravenously in just sublethal doses. From a comparison of the hypothermia-reducing effects of five cholinesterase-reactivating oximes when injected intraperitoneally or subarachnoidally into rats pretreated with DFP or soman it was possible to distinguish central and peripheral actions of the oximes. The comparative efficacy of the five oximes and the effectiveness of cholinesterase inhibitors in producing hypothermia in other animal species, including man, are discussed.