Penetration and detection of Cu²⁺ in the postsynaptic subneural apparatus of frog skeletal muscle cells during the motor nerve stimulation.

Abstract

We have devised a new cytochemical method for detection of Cu²⁺ ions penetrated into the postsynaptic subneural apparatus of the frog skeletal muscle cells, during electrical motor nerve stimulation. CuCl₂ was added (final concentration; 1.8 mM) to the physiological solution immeadiately after the motor nerves tetanic stimulation (5 Hz for 2 min). The tetanus was disappeared within 2 min after Cu²⁺ application. After washing with an isotonic NaCl solution containing EDTA, the tissue was treated with a solution containing 2.5% glutaraldehyde (tissue fixator), 10 mM potassium ferrocyanide (precipitating agent of Cu²⁺ and donor of an oxidoreductase-like catalytic activity to the copper precipitate) and 100 mM NaCl (osmotic compensator). After permeabilizing the muscle membrane, the tissues were treated with conventional DAB-H₂O₂ procedure. The catalytic activity of copper ferrocyanide was revealed as red staining in the site of the precipitate in the level of the subneural apparatus. The subneural area was diffusively stained when direct muscle stimulation was superposed. The opposite area of the subneural apparatus in the neural region and the aneural regions of the muscle cells were devoid of the staining. d-Tubocurarine applied before CuCl₂ markedly reduced the staining and the absence of CuCl₂ provided no staining. [Jpn J Physiol 55 Suppl:S117 (2005)]