Detached, purified nerve terminals from skate electric organ for biochemical and physiological studies.

Abstract

Electric organs of skate (Raja species) dissociate to form populations of individual electrocytes when incubated in saline solutions containing collagenase. The rate of dissociation was highly temperature dependent, with an apparent Q10 of > 6 in the range of 6 degrees-26 degrees C. The number of electrocytes per organ was relatively constant and independent of electric organ size, whereas mean cell diameters increased with organ size. The activities of two cholinergic marker enzymes, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in extracts of whole fresh organs were much less than those reported for the electric ray Torpedo, suggesting a lower volume of terminals in the organ. Electrocytes prepared from collagenase-treated organs had good resting potentials and generated postsynaptic evoked potentials. Spontaneous and electrode pressure-evoked miniature endplate potentials (MEPPs) were readily recorded from isolated electrocytes. Incubation periods of more than 4 days in collagenase at 6 degrees C produced electrocytes with good resting potentials and very low MEPP frequencies, indicating denervation. Detachment of terminals and decreased MEPP frequencies were concurrent. The time course of denervation was followed with the appearance of ChAT and AChE activities in a small particulate fraction derived from washed electrocytes. Peak activities of both enzymes were seen at 4 days of incubation at 16 degrees C, but after 20 h at 16 degrees C. Electrocytes from 4-day, 6 degrees C incubations showed detached, mitochondria-rich nerve terminals and dissociated Schwann cells. In unfixed preparations examined with Nomarski optics, isolated nerve terminals were recognized and distinguished from nucleated Schwann cells. Electron micrographs show that isolated terminals were similar to attached terminals just before they dissociated. The MEPP frequencies and evoked potentials were normal at terminals just before dissociation. We conclude that the transmitter release process was normal in detached terminals and in terminals free of Schwann cells.