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First published online May 24, 2005
Journal of Experimental Biology 208, 2135-2145 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01633

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Interaction between facilitation and presynaptic inhibition at the crayfish neuromuscular junction

Colin M. DeMill and Kerry R. Delaney^*

Department of Biology, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada

View larger version (49K): [in a new window]   Fig. 1. Anatomical relationship between the excitor (Calcium Green-1) and inhibitor (Alexa 568; red) axons on the surface of the crayfish opener muscle. (A) The main Y branch of the excitor and inhibitor axons, showing how the axons project in parallel across the surface of the muscle. (B) The axons branch to form varicose terminal boutons. (C) The complex anatomical relationship between excitor and inhibitor terminals at the level of magnification used to image the Ca influx into excitor terminals. (D) Schematic of the anatomy of the excitor (green) and inhibitor (red) axons showing branching (overlap yellow). Scale bars: 200 µm (A,D), 50 µm (B) and 10 µm (C).

View larger version (32K): [in a new window]   Fig. 2. Effect of previous co-activation of excitor and inhibitor axons on the time to onset of muscle contraction. (A) The apparatus used for force transduction experiments. (B) Force measurements show that the time to onset of contraction was reduced when a period of co-activation of the excitor and inhibitor axons (1.25 s) preceded excitor stimulation alone (grey) compared with excitor stimulation from rest (black). E (arrow) indicates either the start of stimulation for the control or the first stimulus after inhibition for the test condition. The onset of contraction under the two conditions is indicated by the arrowheads. (C) The excitatory junction potentials (EJPs) produced by excitor axon stimuli alone (E,top) or by a period of co-activation of the inhibitor and excitor axons (E+I) followed by the excitor axon alone (E,bottom). (D) Data pooled from several experiments where EJP amplitude (normalized to the first EJP) is plotted versus stimulus number (mean ± S.E.M.; control, filled circles; with preceeding inhibition, open circles). After the period of co-activation of both axons (arrow), the amplitude of the first EJP is significantly larger than the previous, inhibited EJP (paired t-test, P<0.001, N=6 preparations).

View larger version (20K): [in a new window]   Fig. 3. Effect of presynaptic inhibition on Ca entry and transmitter release. (A) The relationship between Ca entry (observed as a change in fluorescence), during stimulation of the excitor alone (black) or the excitor and inhibitor (grey). Presynaptic inhibition caused a significant reduction in the fluorescence transient (average 20±1%) in excitor terminals (paired t-test, P<0.001, 122 terminals from 12 animals). (B) An example of excitatory junction potentials (EJPs) recorded from a muscle fibre when stimulating only the excitor (black) or the excitor and inhibitor together (grey). (C) Pooled data shows that, on average, the first, second and third EJPs were inhibited by 42±4%, 51±4% and 59±2%, respectively (N=27 cells from 18 animals). (D) Facilitation of EJPs resulting from stimulation of the excitor alone (black) or the excitor with the inhibitor (grey) was calculated by dividing the amplitude of the second and third EJP by the amplitude of the first. There was significantly less facilitation of the third EJP with inhibition (paired t-test, P<0.01, N=27 cells from 18 preparations).

View larger version (16K): [in a new window]   Fig. 4. Effect of presynaptic inhibition on Ca entry into terminal varicosities. (A) The complex anatomical relationship between excitor (black) and inhibitor (grey) axons and variation in the amount of inhibition between terminals. Ca entry was reduced by 0-40% at different terminals with inhibition. (B) Greater inhibition was not observed along a string of terminal boutons. (C) Greater inhibition at a terminal on a quaternary branch compared with those on tertiary branches. Inhibition at the six terminals on the tertiary branch ranged from 12 to 28%, while the terminal on the quarternary branch (Q) experienced approximately 40% inhibition. Inhibitor terminals are located in the vicinity of the bottleneck structure forming the branch of the excitor axon. Overall, there was slightly more inhibition at terminals on quaternary branches (24±2%, N=36 terminals) than on those on tertiary branches (18±1%, N=86 terminals, two-sample t-test, P<0.05). Fluorescence transients measured from the excitor axon are depicted next to the terminals that they represent (arrows). Scale bars indicate 2% F/F (vertical) and 50 µm (horizontal). The black trace resulted from stimulating the excitor alone, while the grey trace resulted from stimulating the excitor and inhibitor together. The action potential was conducted from the right to the left in all of the images.

View larger version (11K): [in a new window]   Fig. 5. Effect of increased inhibition on Ca entry and the excitatory junction potentials (EJPs). (A) More inhibitor stimuli caused slightly greater inhibition of Ca entry (excitor black, inhibitor grey). With the inhibitor stimulated in the standard manner (left-hand traces; eight inhibitor stimuli at 100 Hz), inhibition was 13±2% (mean ± S.E.M.), and with more inhibitor stimuli (right-hand traces, 24 stimuli at 100 Hz) inhibition was slightly greater: 22±2% (paired t-test, P<0.05, N=14 terminals from two preparations). Fluorescence transients measured from the excitor terminals, using Calcium Green-1, are depicted next to the terminals (arrows). Black traces resulted from stimulating the excitor alone, while grey traces resulted from stimulating the excitor and inhibitor together. The action potential was conducted from the bottom right to the top left. Scale bars indicate 5% F/F (vertical) and 50 µm (horizontal). (B) Increased stimulation of the inhibitor axon resulted in greater inhibition (10%) of the second and third EJP (paired t-test, P<0.05, N=10 cells from three preparations). Black bars represent eight inhibitor stimuli, while grey bars represent 24 inhibitor stimuli.

View larger version (33K): [in a new window]   Fig. 6. Bath application of GABA reveals cumulative effects of inhibition along branches. Examples from five experiments are shown. The most striking effect of increasing inhibition along a branch is demonstrated by A, but a general trend of increasing inhibition along extended branches or of greater inhibition for terminals at the end of a long branch far from the main axon can be seen in other preparations. In D and E, the standard inhibitor protocol of eight inhibitor stimuli and three excitor action potentials was performed for comparison.