A study of the inhibitor of the crayfish neuromuscular junction by
presynaptic voltage control.
Vyshedskiy, Andrey and Jen-Wei Lin.
Department of Biology, Boston University.
APStracts 3:0223N, 1996.
The inhibitor of the crayfish opener muscle was investigated by a presynaptic
voltage control method. Two microelectrodes were inserted into the inhibitor
and the amplitude and duration of presynaptic depolarization was controlled by
a voltage clamp amplifier. Inhibitory postsynaptic potential (IPSP) was
measured from a muscle fiber located near the presynaptic voltage electrode.
Nonlinear summation of IPSP amplitudes was corrected after chloride
equilibrium potential was measured. Using 5 ms presynaptic pulses, the
depolarization-release coupling (D-R) curve constructed from IPSP peak
amplitudes (IPSP cor ) had a threshold of about -35 mV and reached its maximal
level at -5 to -10 mV. Depolarization beyond the maximum led to a suppression
of neurotransmitter release. When transmitter release during a presynaptic
pulse was completely suppressed, IPSPs activated by tail current could be
identified with an average synaptic delay of 2.5 ms. Transmitter secretion
triggered by a calcium current activated during the 5 ms pulses (IPSP on ) was
also measured on the rising phase of an IPSP, at 2.5 ms after the end of the 5
ms pulses. D-R coupling plots measured from IPSP on exhibited a more
pronounced suppression than that obtained from IPSP cor . The effect of
presynaptic pulse duration on the level of transmitter release was analyzed.
Transmitter release increased with increasing duration and was nearly
saturated by 20 ms pulses depolarized to 0 mV. The following conditions were
identified as necessary to obtain a consistent D-R curve with a clear
suppression: (1) small animals, 3.8 cm head to tail, (2) 15C, (3) 40 mM TEA
and 1 mM 4-AP, (4) an extracellular calcium concentration of 10 mM or less. In
addition, a consistent correlation was found among the branching pattern of
the inhibitor, the placement of the presynaptic electrode and the
characteristics of the D-R curves. An ideal presynaptic electrode
configuration involved placing the voltage electrode in a secondary branch,
about 100 [mu] m from the main branch point, and placing the current electrode
at the branch point. Postsynaptically, optimal recordings were obtained from
muscle fibers innervated by a single branch of the inhibitor that originated
from a point near the presynaptic voltage electrode. A cable-release model was
constructed to evaluate the relationship between the shape of the D-R coupling
curves and the space constants of the presynaptic terminals. A comparison
between the model and the D-R coupling curves suggested that the space
constant of an inhibitor branch on a muscle fiber is at least 8 times longer
than its actual length. Therefore, the upper limit estimate of the space
constant of a typical preparation is about 3 mm.
Results reported here outline morphological and physiological conditions
needed to achieve optimal control of the presynaptic branch of the crayfish
inhibitor. The cable-release model quantitatively defines the extent of
presynaptic voltage control.
Received 31 May 1996; accepted in final form 19 September 1996.
APS Manuscript Number J430-6.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996