Neuroscience Letters, 147 (1992) 49-52 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
49
NSL 09092
The effect of adenosine on spontaneous and evoked quantal secretion from different release sites of amphibian motor-nerve terminals S. K a r u n a n i t h i , N . A . L a v i d i s a n d M . R . B e n n e t t The Neurobiology Research Centre, Department of Physiology, Universityof Sydney, Sydney, NSW (Australia) (Received 12 June 1992; Revised version received 18 August 1992; Accepted 18 August 1992) Key words: Adenosine; Quanta; Motor-nerve; Synapse; Amphibia; Release site The effects of adenosine on the spontaneous quantal secretion from different release sites along terminal branches of toad (Bufo marinus) motor-nerve terminals was studied. Terminal branches were visualized using 3,3-diethyloxardicarbocyanineiodide (DiOC2(5))-fluorescenceto assist in the placement of extracellular electrodes along different release sites of terminal branches. The maximum rate of spontaneous secretions ~) observed with an extracellular electrode within any 101tin length of terminal branch declined towards the distal end of the terminal branch as does the average number of evoked quantal secretions (me).Adenosine (1-50 ltM) depressed both fe and ~e. The EDs0 of adenosine in depressing ~e was 5/aM. Adenosine (10/~M) produced on average a 43% decrease inf~ and a 63% decrease in ~e, regardless of the initial values offe and ~o. It is suggested that adenosine has qualitatively similar effectson bothf~ and ~ , regardless of their initial size, at different release sites within motor-nerve terminals.
The probability of quantal secretion at different release sites is not uniform within individual terminals of m o t o r nerves [2]. In m o t o r nerves the release sites with a high probability for secretion tend to occur in the more proximal regions of the terminal branches. There is also a non-uniform frequency of spontaneous quantal secretion at different release sites along m o t o r nerve terminals [8]. Again, the higher frequency of spontaneous secretions tends to occur for release sites in the more proximal part of terminal branches. However, it is not known if release sites with a relatively high probability for evoked quantal secretion also possess a relatively high rate of spontaneous quantal secretion, and this is determined in the present work. Adenosine, derived from ATP secreted from motornerve terminals [1], depresses the secretion of acetylcholine through autoreceptors [7]. Adenosine reduces both the mean quantal content of the endplate potential [3, 5, 7] and the rate o f secretion of spontaneous miniature endplate potentials [4]. The action of adenosine on different release sites has also been examined in order to determine if release sites with different rates of spontane-
Correspondence: M.R. Bennett, The Neurobiology Research Centre, Department of Physiology, University of Sydney, NSW 2006, Australia.
ous and evoked quantal secretion have different sensitivities to adenosine. Mature toads (Bufo marinus) ranging in weight from 30 to 40 g were used in these experiments. Animals collected from the wild in summer (December to February) and in winter (April to August) were used in the experiments within 4 weeks of collection. Animals were killed by a cervical fracture and both iliofibularis muscles with attached nerve supply dissected free from the surrounding connective tissues, tibial and pelvic tendinous insertions. Each muscle was pinned through its tendons to the base of clear Sylgard at the b o t t o m of a 3 ml capacity bath. Preparations were then continuously perfused at ambient temperature (13-16°C) with a modified Ringers solution of the following composition (mM): N a ÷ 117.0, K + 3.0, Mg z+ 2.0, C1- 103.1, HzPO4 0.64, K + 3.0, H P O ] 9.7, Ca 2+ 0.35-0.4, glucose 7.8. A reservoir of Ringers solution was used to perfuse the preparation at a rate of 3-5 ml per minute; this was gassed continuously with 95% O2 and 5% CO2 and the p H was maintained between 7.2 and 7.5, generally at 7.4. Preparations were perfused with DiOC2(5) at a concentration of 10 -7 M for 20-30 s and then washed with Ringer for 5 min. DiOC 2 was then fluoresced using a rhodamine filter set (Olympus). Nerve terminals were clearly visualized using an intensifier camera (National) and a television monitor (National). Recordings of the spontaneous secretion of quanta and
50 0.05 3
4
1
2 0.04
NE 0.4
A
0.3
IE=
T "-=
0.2
0.03
0.02
0.01 jr-
O.
~
0 0.05 I
0 0.06
L
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B 0.04
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,.-0 0 . 0 2 '..'-= 0 . 0 2 0
0
I 20
I 40
I 60 Distance
L 80
[ 100
I 120
I 140
160
0.01
(pm)
Fig. 1. A: changes in the average number of quanta secreted during 200 nerve impulses ( ~ + S.E.M.) at five different sites along the length of the terminal branch illustrated in the panel: the panel shows the point of nerve entry (NE) at the beginning of the branch (zero distance) and the numbers refer to the sequence in which the recordings were made. B: the frequency of spontaneous quantal secretions (/~), determined during the 15 min recording period at each site is also given.
the evoked secretion of quanta were taken from small groups of release sites using extracellular electrodes as described previously [2, 3]. Immediately following the recording of 200 trials, the rate of spontaneous secretions was determined by recording for a period of 10-15 min. Both the spontaneous secretion of quanta and the evoked secretion in response to a nerve impulse were recorded with extracellular electrodes placed at different sites along the length of terminal branches which were visualized using DiOC2(5)-fluorescence. The frequency of spontaneous quantal secretion (re) and the average number of evoked quanta secreted (We) at different release sites declined towards the end of terminal branches (Fig. 1). The declined inf~ tended to follow the decline in N~, so that sites with relatively high levels of evoked quantal secretion tended to have relatively high levels of spontaneous quantal secretion (Fig. 1). In order to quantify this, both spontaneous and evoked quantal secretion were recorded from 42 different groups of release sites from 5 preparations in winter and five in summer. The results show thatf~ in general increases with ~ (Fig. 2). A distinction must be made, however, between the results for animals collected in summer (Fig. 2A) and those collected in winter (Fig. 2B); whilst the slope relatingj~ to We
0
• o 0
I• 0.1
~lnm 0.2
•
l 0.3
J 0.4
I 0.5
l 0.6
I 0.7
0.8
me
Fig. 2. Relationship between the tYequencyof spontaneous quantal secretions (f~)and the average quantal content of evoked secretions (N~) recorded at different sites along the length of five visualized terminal branches on muscle fibres in summer (A) and five in winter (B). The regression lines in A and B have the same correlation coefficient(0.36) and the same slope (0.04), however only the correlation between.I;and h~ in winter is significant at the P
49
NSL 09092
The effect of adenosine on spontaneous and evoked quantal secretion from different release sites of amphibian motor-nerve terminals S. K a r u n a n i t h i , N . A . L a v i d i s a n d M . R . B e n n e t t The Neurobiology Research Centre, Department of Physiology, Universityof Sydney, Sydney, NSW (Australia) (Received 12 June 1992; Revised version received 18 August 1992; Accepted 18 August 1992) Key words: Adenosine; Quanta; Motor-nerve; Synapse; Amphibia; Release site The effects of adenosine on the spontaneous quantal secretion from different release sites along terminal branches of toad (Bufo marinus) motor-nerve terminals was studied. Terminal branches were visualized using 3,3-diethyloxardicarbocyanineiodide (DiOC2(5))-fluorescenceto assist in the placement of extracellular electrodes along different release sites of terminal branches. The maximum rate of spontaneous secretions ~) observed with an extracellular electrode within any 101tin length of terminal branch declined towards the distal end of the terminal branch as does the average number of evoked quantal secretions (me).Adenosine (1-50 ltM) depressed both fe and ~e. The EDs0 of adenosine in depressing ~e was 5/aM. Adenosine (10/~M) produced on average a 43% decrease inf~ and a 63% decrease in ~e, regardless of the initial values offe and ~o. It is suggested that adenosine has qualitatively similar effectson bothf~ and ~ , regardless of their initial size, at different release sites within motor-nerve terminals.
The probability of quantal secretion at different release sites is not uniform within individual terminals of m o t o r nerves [2]. In m o t o r nerves the release sites with a high probability for secretion tend to occur in the more proximal regions of the terminal branches. There is also a non-uniform frequency of spontaneous quantal secretion at different release sites along m o t o r nerve terminals [8]. Again, the higher frequency of spontaneous secretions tends to occur for release sites in the more proximal part of terminal branches. However, it is not known if release sites with a relatively high probability for evoked quantal secretion also possess a relatively high rate of spontaneous quantal secretion, and this is determined in the present work. Adenosine, derived from ATP secreted from motornerve terminals [1], depresses the secretion of acetylcholine through autoreceptors [7]. Adenosine reduces both the mean quantal content of the endplate potential [3, 5, 7] and the rate o f secretion of spontaneous miniature endplate potentials [4]. The action of adenosine on different release sites has also been examined in order to determine if release sites with different rates of spontane-
Correspondence: M.R. Bennett, The Neurobiology Research Centre, Department of Physiology, University of Sydney, NSW 2006, Australia.
ous and evoked quantal secretion have different sensitivities to adenosine. Mature toads (Bufo marinus) ranging in weight from 30 to 40 g were used in these experiments. Animals collected from the wild in summer (December to February) and in winter (April to August) were used in the experiments within 4 weeks of collection. Animals were killed by a cervical fracture and both iliofibularis muscles with attached nerve supply dissected free from the surrounding connective tissues, tibial and pelvic tendinous insertions. Each muscle was pinned through its tendons to the base of clear Sylgard at the b o t t o m of a 3 ml capacity bath. Preparations were then continuously perfused at ambient temperature (13-16°C) with a modified Ringers solution of the following composition (mM): N a ÷ 117.0, K + 3.0, Mg z+ 2.0, C1- 103.1, HzPO4 0.64, K + 3.0, H P O ] 9.7, Ca 2+ 0.35-0.4, glucose 7.8. A reservoir of Ringers solution was used to perfuse the preparation at a rate of 3-5 ml per minute; this was gassed continuously with 95% O2 and 5% CO2 and the p H was maintained between 7.2 and 7.5, generally at 7.4. Preparations were perfused with DiOC2(5) at a concentration of 10 -7 M for 20-30 s and then washed with Ringer for 5 min. DiOC 2 was then fluoresced using a rhodamine filter set (Olympus). Nerve terminals were clearly visualized using an intensifier camera (National) and a television monitor (National). Recordings of the spontaneous secretion of quanta and
50 0.05 3
4
1
2 0.04
NE 0.4
A
0.3
IE=
T "-=
0.2
0.03
0.02
0.01 jr-
O.
~
0 0.05 I
0 0.06
L
I
I
I
I
JJJ
•
•
[
Io
9 I
I•
L
J
B
I 0.04
B 0.04
0.03
T
•
'7
j
,.-0 0 . 0 2 '..'-= 0 . 0 2 0
0
I 20
I 40
I 60 Distance
L 80
[ 100
I 120
I 140
160
0.01
(pm)
Fig. 1. A: changes in the average number of quanta secreted during 200 nerve impulses ( ~ + S.E.M.) at five different sites along the length of the terminal branch illustrated in the panel: the panel shows the point of nerve entry (NE) at the beginning of the branch (zero distance) and the numbers refer to the sequence in which the recordings were made. B: the frequency of spontaneous quantal secretions (/~), determined during the 15 min recording period at each site is also given.
the evoked secretion of quanta were taken from small groups of release sites using extracellular electrodes as described previously [2, 3]. Immediately following the recording of 200 trials, the rate of spontaneous secretions was determined by recording for a period of 10-15 min. Both the spontaneous secretion of quanta and the evoked secretion in response to a nerve impulse were recorded with extracellular electrodes placed at different sites along the length of terminal branches which were visualized using DiOC2(5)-fluorescence. The frequency of spontaneous quantal secretion (re) and the average number of evoked quanta secreted (We) at different release sites declined towards the end of terminal branches (Fig. 1). The declined inf~ tended to follow the decline in N~, so that sites with relatively high levels of evoked quantal secretion tended to have relatively high levels of spontaneous quantal secretion (Fig. 1). In order to quantify this, both spontaneous and evoked quantal secretion were recorded from 42 different groups of release sites from 5 preparations in winter and five in summer. The results show thatf~ in general increases with ~ (Fig. 2). A distinction must be made, however, between the results for animals collected in summer (Fig. 2A) and those collected in winter (Fig. 2B); whilst the slope relatingj~ to We
0
• o 0
I• 0.1
~lnm 0.2
•
l 0.3
J 0.4
I 0.5
l 0.6
I 0.7
0.8
me
Fig. 2. Relationship between the tYequencyof spontaneous quantal secretions (f~)and the average quantal content of evoked secretions (N~) recorded at different sites along the length of five visualized terminal branches on muscle fibres in summer (A) and five in winter (B). The regression lines in A and B have the same correlation coefficient(0.36) and the same slope (0.04), however only the correlation between.I;and h~ in winter is significant at the P
