Brain Research, 100 (1975) 205-208 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

205

Long-term synaptic facilitation with minimal calcium entry

L. E. SWENARCHUK AND H. L. ATWOOD Department of Zoology, University of Toronto, Toronto, Ont. (Canada)

(Accepted August 26th, 1975)

Long-term facilitation (LTF) at crustacean neuromuscular synapses 9 is accompanied by sodium loading of the presynaptic terminaP. It is enhanced by treatments which block sodium extrusion and diminished by procedures which reduce sodium entry during stimulation 1. Sodium loading may increase transmitter release by raising Ca 2+ in the presynaptic terminaP. However, it is possible that entry of calcium during stimulation generates LTF and that sodium loading, although occurring simultaneously, is not the important factor. To resolve this question, it is necessary to examine LTF under conditions in which sodium loading occurs, but calcium entry into the preterminal is minimized. In the present study, such experiments were performed on facilitating synapses in the opener muscle of the crayfish (Procambarus clarkii) walking leg. Procedures to minimize calcium entry were: (1) stimulation in concentrations of Mn 2+ or Co z+ known to block calcium entry into axons ~ and nerve-evoked release of transmitter3; and (2) stimulation in zero-calcium solutions containing 30 m M MgCI2 to stabilize the axonal membrane. No nerve-evoked transmitter release occurred in these solutions, so it was necessary to observe the excitatory postsynaptic potentials (EPSPs) after washout of the experimental solution to determine whether or not L T F had been induced by conditioning stimulation given during treatment with the experimental solution. Normally, LTF produces a very long-lasting enhancement of the EPSP (Fig. 1), much more prolonged than that seen after short-term facilitationT, 12. To determine whether an after-effect existed following conditioning stimulation in a synapse-blocking solution, EPSPs produced by short bursts of stimulation, 64 stimuli at 10 Hz ('test EPSPs') were measured using a signal averager, at 1 or 2 rain intervals after washout of the experimental solution. These 'test EPSPs' were compared to those evoked by the same stimulation after washout of the same solution applied for the same length of time to the preparation, but with no conditioning stimulation ('control EPSPs'). For each experiment, the protocol involved several steps. First, the preparation was perfused with Van Harreveld's solution 11 for 1 h, while the EPSPs were periodically monitored using brief bursts of stimulation. If the EPSP was stable, the experimental solution was applied for 20-30 min, with no stimulation. Then the solution

206

420 380~ 340'" 300

260r~

10G

10 20 30 40 50 60 70 80 90 Time (rain)after end of continuousstimulation

Fig. I. Effect of 6 m M MnCI~ on after-effect (decay) of LTF following conditioning stimulation

(15 Hz for 20 min). Circles and solid line: decay of LTF in Van Harreve[d's solution; triangles: decay of LTF in Van Harreveld's solution with 6 mM MnCh added. The data points are average values for 8 experiments in MnCI2 and 12 experiments in Van Harreveld's solution. Error bars represent ± S.E.M. 'Control EPSPs' were obtained after previous washout of experimental or normal Van Harreveld's solution (see text). was washed out and control EPSPs obtained at predetermined intervals. After 0.5 h, if the EPSPs had resumed near-normal amplitude and no sign of deterioration was detected, the experimental solution was re-introduced and the excitor axon was stimulated at a rate known to produce LTF (usually 10 Hz, at 15 °C). After 20 min, the experimental solution was washed out with Van Harreveld's solution and test EPSPs obtained. Comparison of test EPSPs and control EPSPs showed whether the longlasting after-effect of LTF had been induced by the conditioning stimulation. This protocol had the advantage of controlling for postsynaptic changes produced by the experimental solutions. Checks on the procedure were made by omitting stimulation during the second perfusion with experimental solution. Such checks showed that, without stimulation, control and test EPSPs usually differed by a few per cent, but these differences were very small compared with those seen when conditioning stimulation was employed during the second application of the experimental solution. Fig. 1 shows results from experiments in which 6 m M Mn 2 ~ was included in the experimental solution. For comparison, paired preparations from the same animal were run in Van Harreveld's solution with no Mn 2+. Following an initial post-washout period of partial depression, the long-lasting after-effect of LTF showed up clearly after Mn 2+ treatment. Apart from the initial depression, the effect had a similar magnitude and time course to that seen after stimulation in normal Van Harreveld's solution. Stimulation in zero Ca 2+, 30 m M Mg 2~ gave variable results. This solution produced synaptic depression which lasted at least several minutes after restoration of the normal solution. In about half the experiments, an after-effect of LTF showed up following this period of depression, while in the others no potentia-

207

1700

1500

1300

-~ .oo

!

I ,;)

I 2'o

~

~

~

Time (rain) after end of continuous stimulation

Fig. 2. Time course of the decay of LTF following conditioning stimulation (10 Hz for 20 min) applied in a solution containing 0 K +, 0 Ca 2+, 30 mM Mg ~+. Following stimulation in this solution, Van Harreveld's solution (with 0 K ÷, and equivalent Na + substituted) was introduced so that 'test EPSPs' could be measured. The 'control EPSPs' were obtained after a previous washout from the experimental solution into the 0 K + Van Harreveld's solution. Each data point is the average value for 4 experiments; error bars show 5_ S.E.M. tion was obtained. It is likely that in at least some o f the latter cases, blockage o f the axon spike m a y have occurred near the terminal region, or the axon m a y not have recovered its normal properties. It is k n o w n that crustacean axons are adversely affected by low-calcium solutions, and that addition o f M g 2+ is not a completely adequate antidoteL Treatment with zero-calcium solutions was definitely more deleterious to the preparation than addition o f M n 2+. W h e n sodium loading o f nerve terminals was p r o m o t e d in zero-calcium solutions by the use o f 3 × 10 -4 M ouabain or zero potassium 1, a very large enhancement o f the E P S P was almost always induced by stimulation (Fig. 2). W h e n stimulation was not applied, relatively little enhancement o f the E P S P occurred after the second washout. F o r example, in 4 experiments, treatment with 3 × 10-4 M ouabain in a zero-calcium solution with no conditioning stimulation resulted in a 15-30 ~ enhancement o f the EPSP at 2 min after washout, c o m p a r e d with enhancement o f 5001000 700when conditioning stimulation was applied in this solution. I n zero potassium, an even larger enhancement o f the E P S P was observed - - well over 1 0 0 0 ~ in each o f 4 repetitions (Fig. 2). F r o m these experiments, we conclude that L T F occurs under conditions which minimize entry o f Ca 2+ into the preterminal during stimulation. Since no evoked transmitter release occurred in the synapse-blocking solution, a transmitter feedback mechanism6,10 is excluded for LTF. W h e n entry o f N a + is promoted, L T F is enhanced, even when Ca z+ is not present in the external solution. Thus, agents which p r o m o t e entry o f N a ÷ are not exerting their effects by a sodium-independent effect on Ca 2+ accumulation in the preterminal during stimulation, as suggested by

208 H u b b a r d 4. A role f o r sodium a c c u m u l a t i o n in L T F is supported. The exact role o f this a c c u m u l a t i o n in p r o m o t i n g L T F remains to be determined. It is o f interest that an an al o g o u s situation, in which N a + accentuates a c a l c i u m - m e d i a t e d response, has been described in p h o t o r e c e p t o r s in the eye o f L i m u l u s 8. The w o r k was s u p p o r t e d by G r a n t s from the Muscular D y s t r o p h y Association o f C a n a d a and the N a t i o n a l Research Council o f Canada.

I ATWOOD, H. L., SWENARCHUK,L. E., AND GRUENWALD, C. R., Long-term synaptic facilitation during sodium accumulation in nerve terminals, Brain Research, 100 (1975) 198-204. 2 BAKER,P. F., MEVES, H., AND RIDGWAY,E. B., Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons, J. Physiol. (Lond.), 231 (1973) 511-526. 3 BALNAVE,R. J., AND GAGE, P. W., The inhibitory effect of manganese on transmitter release at the neuromuscular junction of the toad, Brit. J. Pharmacol., 47 (1973) 33%352. 4 HUaaARD,J. 1., Mechanism of transmitter release, Progr. Biophys. molec. Biol., 21 (1970) 33-124. 5 JULIAN, F.J., MOORE, J.W., AND GOLDMAN, D.E., Current-voltage relations in the lobster giant axon membrane under voltage clamp conditions, J. gen. Physiol., 45 (1962) 1217-1238. 6 KOELLE,G. B., Functional anatomy of synaptic transmission, Anesthesiology, 29 (1968) 643-653. 7 LINDER, T. M., Calcium and facilitation at two classes of crustacean neuromuscular synapses, J. gen. Physiol., 61 (1973) 56-73. 8 LISMAN, J. E., AND BROWN, J. E., The effects of intracellular iontophoretic injection of calcium and sodium ions on the light response of Limulus ventral photoreceptors, J. gen. Physiol., 59 (1972) 701-719. 9 SHERMAN,R. G., AND ATWOOD,H. L., Synaptic facilitation: long term neuromuscular facilitation in crustaceans, Science, 171 (1971) 1248-1250. 10 USHERWOOD,P. N. R., Insect neuromuscular mechanisms, Amer. Zool., 7 (1967) 553-582. 11 VAN HARREVELD,A., A physiological solution for freshwater crustaceans, Proc. Soc. exp. Biol. (N. Y.), 34 (1936) 428432. 12 ZUCKER, R. S., Characteristics of crayfish neuromuscular facilitation and their calcium dependence, J. Physiol. (Lond.), 241 (1974) 91-110.

Long-term synaptic facilitation with minimal calcium entry.

Brain Research, 100 (1975) 205-208 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands 205 Long-term synaptic facilitati...
205KB Sizes 0 Downloads 0 Views