Electrophysiological and freeze‐fracture studies of changes following denervation at frog neuromuscular junctions.

Abstract

1. Changes which occur at frog neuromuscular junctions following denervation have been studied by combining intracellular recording and freeze‐fracture electron microscopy. 2. Shortly after nerve section, both neuromuscular transmission and intramembrane structures of neuromuscular junctions remain normal. 3. Later, neuromuscular transmission fails, beginning with the disappearance of end‐plate potentials (e.p.p.s) and followed by the disappearance of miniature end‐plate potentials (m.e.p.p.s). The frequency of m.e.p.p.s which persist after cessation of e.p.p.s is not increased dramatically in K+‐rich or hypertonic solutions. 4. Concomitant with the changes of transmission are changes in intramembrane structures. The first sign of these changes in disruption of active zones, which become disorganized, fragmented or vanish. Nerve terminals then disintegrate and eventually are engulfed by Schwann cells. 5. When neuromuscular transmission has failed completely, former sites of the neuromuscular junction are occupied by Schwann cells. These cells develop transverse ridges which lie opposed to junctional folds, just like active zones of nerve terminals. However, the ridges on Schwann cells do not contain organized rows of particles or clusters of any synaptic organelles, even at later stages when Schwann cell m.e.p.p.s commence. 6. It is suggested that the failure of e.p.p.s involves at least an impairment of the transmitter release mechanism at the nerve terminal, which is probably associated with the disruption of active zones. The cessation of m.e.p.p.s is thought to be caused by the engulfment of terminals by Schwann cells.

Precision of Reinnervation and Synaptic Remodeling Observed in Neuromuscular Junctions of Living Frogs

“…Therefore, anatomical control experiments also were necessary. Previous studies have shown that the terminals of axotomized motoneurons degenerate over a 2 d period (Ko, 1981) and that the earliest regenerating terminals appear at denervated junctional sites ϳ7 d after nerve injury (DeCino, 1981;Ding, 1982). We therefore examined regenerated terminals in vivo, then 2-5 d later used the NBT histological method to reexamine the same terminals.…”

Acetylcholine release at identified nerve terminals in the organ‐cultured frog neuromuscular preparation.

“…We found that the 'transient stage' (stage II, according to Ko, 1981) consists of at least three distinct periods each characterized by a unique trend of change in synaptic activity. The initial change involved a simultaneous decay of both spontaneous and nerve stimulation-evoked release of acetylcholine (sub-stage II1).…”

“…The deterioration may also be due, in part, to the exposure of the preparation to the relatively high room temperature (24 'C) during the recording session (Birks et al 1960;Ko, 1981;Cull-Candy et al 1982). When both spontaneous and evoked release were examined repeatedly, only two sessions of stimulation were imposed on each preparation and no more than 800 stimuli were applied to the nerve during each session.…”

“…In this stage some of the examined endplates retain their spontaneous activity but fail to respond to nerve stimulation: 'non-transmitting junctions' (Usherwood, 1963;Miledi & Slater, 1970;Ko, 1981). Other endplates respond to nerve stimulation with subthreshold endplate potentials (EPPs) or with delayed action potentials (Birks et al 1960; Miledi & Slater, 1970).…”

“…However, at that final stage acetylcholine was most probably released from Schwann cells (Katz & Miledi. 1959; Miledi & Slater, 1968; Cull-Candy, Miledi & Uchitel, 1981). Recently Ko (1981) has divided the time course of denervation changes into four stages: normal (I), transient (II), silent (III) and Schwann miniature endplate potential (MEPP) stage (IV).…”