1. It has been proposed that the influence of innervation on the cholinesterase activity (ChE) of skeletal muscle and on end‐plate ChE in particular is mediated by trophic substance(s) moved by axonal transport and released from nerve. We have tested this hypothesis using rat extensor digitorum longus (e.d.l.) and diaphragm muscles denervated in vitro for several days and then maintained in organ culture to assay putative trophic substance(s). 2. The cholinesterase activity (ChE) of rat extensor digitorum longus (e.d.l.) muscles decreased dramatically after 5 days of denervation in vivo as previously reported. The ChE of rat e.d.l. muscles denervated in vivo for 3 days and then maintained in organ culture for 2 days was essentially identical to that of muscles denervated 5 days in vivo. 3. The ChE OF E.D.L. MUSCLES DENERVATED IN VIVO FOR 3 DAYS AND THEN MAINTAINED FOR 2 DAYS IN CULTURE MEDIUM SUPPLEMENTED WITH SCIATIC NERVE OR INNERVATED MUSCLE EXTRACT WAS SIGNIFICANTLY HIGHER THAN THAT OF MUSCLES DENERVATED IN VIVO FOR 5 DAYS OR DENERVATED IN VIVO FOR 3 DAYS AND THEN CULTURED FOR 2 DAYS IN CULTURE MEDIUM ALONE. Supplementing the culture medium with brain or spinal cord extract also significantly increased the ChE of organ‐cultured e.d.l. muscles. 4. Supplementing the culture medium with liver or spleen extract or with the extract of muscle denervated for 3‐‐7 days in vivo before extraction did not increase the ChE or organ‐cultured e.d.l. muscles. 5. The effect of muscle extract on the ChE of organ‐cultured e.d.l. muscles was dose dependent and occurred gradually reaching a maximum after approximately 24 h of culture. 6. Substance(s) which increased the ChE of organ‐cultured e.d.l. muscles were found to accumulate in transected sciatic nerve in the region just proximal to the site of transection where substances moved by axonal transport are known to accumulate. 7. Media conditioned with neurally stimulated e.d.l. or diaphragm muscles caused a substantial and highly significant increase in the ChE of e.d.l. or diaphragm muscles denervated in vivo and then maintained in organ culture. Media conditioned in the same way with unstimulated muscles did not increase the ChE OF ORGAN‐CULTURED MUSCLES. 8. The active substance(s) released by neural stimulation continued to be released when muscle contraction was blocked by adding D‐tubocurarine to the medium during conditioning but the release of these substance(s) was significantly reduced when magnesium (10mM) was added to the medium during conditioning. 9 The substance(s) released by neural stimulation selectively increased ChE in the end‐plate region. In diaphragm segments denervated in vivo and then maintained in medium conditioned with neurally stimulated muscle, there was a 102% increase in end‐plate ChE but no detectable increase in background ChE. 10...
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