J. Phyeiol. (1978), 285, pp. 25-34 With 3 text-ftsee Printed in Great Britain
25
VARIABILITY OF TRANSMITTER QUANTA RELEASED DURING INCORPORATION OF A FALSE TRANSMITTER INTO CHOLINERGIC NERVE TERMINALS
By W. A. LARGE AND H. P. RANG From the Department of Pharmacology, St George's Hospital Medical School, University of London, Cranmer Terrace, London, SW17 ORE
(Received 15 May 1978) SUMMARY
1. Electrophysiological techniques have been used to investigate the variability of quanta released during various stages of the incorporation of acetylmonoethylcholine (AMECh) into the transmitter store at the neuromuscular junction. 2. Stimulation of the rat phrenic nerve-diaphragm preparation at 3 Hz for 1525 min reduced the time constant of decay Tm e pjc of miniature end-plate currents from 1-61 + 0-08 (mean + s.E. of mean) msec to 1-32 + 0-09 msec; this represents 45% replacement of acetylcholine (ACh) by AMECh. Total replacement of ACh was achieved by a further 30-60 min stimulation which reduced Tm.e.p.c. to 0-93 + 0-06msec. 3. At the intermediate stage of exchange, the coefficient of variation c.v., of Tm.e.p-c. increased from the control value of 0-094 + 0-005 to 0-154 + 0-012. The latter value was less than the value (0-29) calculated for two distinct populations of quanta (i.e. pure ACh and pure AMECh quanta). Moreover, frequency distribution histograms of Tm.e.p.c. did not reveal a bimodal distribution. Total replacement of ACh by AMECh achieved by the second period of stimulation decreased c.v., towards the control value. 4. Pre-incubation of muscles with MECh (to replace any stored choline) or stimulation in the presence of a cholinesterase inhibitor (to prevent ACh destruction and hence re-uptake of choline) did not produce any greater degree of heterogeneity than was seen in the absence of these procedures. 5. The increase in c.v.T observed at the intermediate stage of replacement disappeared spontaneously with a half-time of about 1 hr at 20 'C. 6. These results are discussed in terms of the vesicle hypothesis. INTRODUCTION
When transmitter release is evoked at motor nerve terminals in the presence of the choline analogue, monoethylcholine (MECh), the store of acetylcholine (ACh) is gradually replaced by the false transmitter acetylmonoethylcholine (AMECh). The false transmitter can be distinguished from ACh by the fact that its post-synaptic action is briefer, as revealed by a shortening of the time constant of decay (Tm.e.p.c.) of miniature end-plate currents (m.e.p.c.s) in rat muscle recorded by the voltage
W. A. LARGE AND H. P. RANG
26 clamp
reported
progressively
quite simple
was
maximally
the
which
to
this
is
value
quanta
is
value
bring the
intermediate
at
question
of
stages
Tm.e.p-c-
ACh)
and with exchange, the
The
AMECh).
entirely
work
Tm.ep.c.
mean
(representing entirely
value
The
of
released, and it
transmitter
stimulation to
nerve
whether,
of
amount
(representing
concerned
Rang, 1978b).
showed that the
normal
the
between
Large &
1977;
paper,
judge the period of
reduced
paper
this
transmitter
Rang,
function of the
intermediate
value
a
Large &
latter, preceding
the
in
decreased
to
(Colquhoun,
method
population two disAMECh, mixture of residual populations discernible, representing quanta and newly-formed quanta. On simple interpretation of the vesicle hypothesis the pre-existing expected quanta would be released and respontaneously form
quantum containing
each
with
released
tinct
a
would
was
from
cytoplasmic pool
In
variability supposed
we
of
investigated
stages
of
of
The
the
vesicles
the
are
are
consistent
filled
before
of
on
and
ACh
distribution of values We have found
no
AMECh. of
Tm.e.p.c.
evidence
for the
with the vesicle hypothesis if
it is
is
store
is
discernible
a
in
form
the
released,
being
would,
1977)
in
there
transmitter
the directly the other until
of transmitter
mixture
a
exchange.
quanta, though
of
release
Tauc,
1977;
populations
distinct
two
to
frequency
the
transmitter
results
that
which
rise
favouring
Marchbanks,
(see
have
TmepC.
giving
so
hypothesis
quantum would consist of
populations
either
from
An
each
intermediate
distinct
pool
that
study
this
during two
complete.
predict
hand,
ACh
ACh
quanta,
change a
whether
or
a
that
AMECh
and
a
be
placed by
single homogeneous
a
ACh
are
AMECh
it
of
mixture
or
increase
of
cytoplasmic
a
there
that
rapid
is
exchange of transmitter between individual vesicles. A
preliminary
account
of
this work has
already appeared (Large &
Rang, 1978a).
METHODS Rang, 1978b).
described in the preceding paper (Large experiments
omohyoid Sterz,
1976).
gave
muscle was
the
previous -80
clamped
mV;
variability tape.
&
r
Rang,
Tm.e.p.c. values deviation of
was expressed , T
=
mean
(0-1
rmTepecC
mm)
min,
by
Recordings
min. 30-40
as a
was
a
Most of the
For a few
experi-
(Dreyer, Muller, the m.e.p.c amplitude
its motor nerve
with
diaphragm, but slightly more difficult.
the
Peper
The
same
recording
of
50-150
were done at
spontaneously occurring
as
histograms.
St/7),
(c.v.T
.
m.e.p.c.s
experiments
most
In
coefficient of variation
series of control
tech-
mV. For measurerem.e.p.c.s were
=
the
variability
made
nerve
a
as
was first recorded in each of six different
sufficient in most preparations to bring about a reduction 45
replacement of
ACh
by
was then stimulated for a further
was
made.
30-60
min at
at
of
AMECh).
quickly as possible from another four to six cells; this
final series of recordings
of
where St = standard
then added to the bathing solution and the nerve was stimulated
was
&
For most of the experiments the membrane potential
(corresponding to approximately
the exchange, after which
'C.
measured by the template method as described previously
plotted
T
which
%
were
value of
experiments
15-25
'runs
m.e.p.c.'
and
T
paper.
nerve preparations at
some of the later experiments
of
values
1978b)
as
recording
made
niques
used,
results
same
smaller,
usually
(Large
diaphragm-phrenic
rat
usually
Hz to complete
VARIABILITY OF TRANSMITTER QUANTA
27
RESULTS
Control measurement Nerve stimulation in the absence of MECh did not affect the variability of m.e.p.c.s. A single experiment is shown in histogram form in Fig. 1 (A, B and C) and the results of five control experiments are summarized in Table 1. Two of the experiments were carried out on the mouse omohyoid muscle but have been grouped with the experiments on the rat diaphragm to obtain over-all mean values as there appeared to be no important differences between the two preparations. In these five experiments the mean c.v, did not change after the first 15 min period of 3 Hz nerve stimulation 40
A
D
-
20
G
3 30
15
20
20
10
10
10
5-
030
1
0
0
20
1 5
2-5
40
n
40
3-0 CB
20
20
10
10
0
"II0 20 25
15
40 -c 30 -30
30
1.5 20 2-5
05 1 0 40 30 -30 -15
E
15
2-0 H
5
A.0
15
05 10 40 -F
20
25
05 20 15
10
15
1-0
1-5
20
5
10 r
1-0
10 F
10 1-5
0 05 20
H
10
-
20 -20
0
-
J0 1
2-0
2-5
0.5
1.0
1-5
0
0.5
(msec) Fig. 1. Frequency distribution of the time constants of decay of m.e.p.c.s, histograms A, D and G are from three different cells in control conditions and B, E and H show the distribution of Tm.e.p.c. values after the nerve had been stimulated at 3 Hz for 15 min in normal Krebs solution (B) or in the presence of MECh (E and H); C, F and I illustrate the m.e.p.c. distribution after a further 60 min stimulation either in normal Krebs solution (C) or MECh (F and I). Note that 15 min stimulation in the presence of MECh produces a reduction in the meant value (E and H) which is associated with an increase in the scatter of the individual values and a further 60 min stimulation decreases the mean -T further while the scatter decreases towards the control level. In the control muscle (A, B, C) neither the mean r nor the degree of scatter changes appreciably. r
or after a further 30-60 min stimulation. The mean value of Tm.e.p.c. was slightly increased after stimulation (by an average of 10%) but the effect was rather inconsistent and we did correct the values of -rm.e.p.c. obtained in the MECh experiments to allow for it. Consequently we may have tended to underestimate slightly the
28
~ ='O.0r
W. A. LARGE AND H. P. BANG 6te 6 6ott 6 06666
.o.+ + + + + 4i N5
+ + + ++
41
~~~_oooo______
6
25
VARIABILITY OF TRANSMITTER QUANTA RELEASED DURING INCORPORATION OF A FALSE TRANSMITTER INTO CHOLINERGIC NERVE TERMINALS
By W. A. LARGE AND H. P. RANG From the Department of Pharmacology, St George's Hospital Medical School, University of London, Cranmer Terrace, London, SW17 ORE
(Received 15 May 1978) SUMMARY
1. Electrophysiological techniques have been used to investigate the variability of quanta released during various stages of the incorporation of acetylmonoethylcholine (AMECh) into the transmitter store at the neuromuscular junction. 2. Stimulation of the rat phrenic nerve-diaphragm preparation at 3 Hz for 1525 min reduced the time constant of decay Tm e pjc of miniature end-plate currents from 1-61 + 0-08 (mean + s.E. of mean) msec to 1-32 + 0-09 msec; this represents 45% replacement of acetylcholine (ACh) by AMECh. Total replacement of ACh was achieved by a further 30-60 min stimulation which reduced Tm.e.p.c. to 0-93 + 0-06msec. 3. At the intermediate stage of exchange, the coefficient of variation c.v., of Tm.e.p-c. increased from the control value of 0-094 + 0-005 to 0-154 + 0-012. The latter value was less than the value (0-29) calculated for two distinct populations of quanta (i.e. pure ACh and pure AMECh quanta). Moreover, frequency distribution histograms of Tm.e.p.c. did not reveal a bimodal distribution. Total replacement of ACh by AMECh achieved by the second period of stimulation decreased c.v., towards the control value. 4. Pre-incubation of muscles with MECh (to replace any stored choline) or stimulation in the presence of a cholinesterase inhibitor (to prevent ACh destruction and hence re-uptake of choline) did not produce any greater degree of heterogeneity than was seen in the absence of these procedures. 5. The increase in c.v.T observed at the intermediate stage of replacement disappeared spontaneously with a half-time of about 1 hr at 20 'C. 6. These results are discussed in terms of the vesicle hypothesis. INTRODUCTION
When transmitter release is evoked at motor nerve terminals in the presence of the choline analogue, monoethylcholine (MECh), the store of acetylcholine (ACh) is gradually replaced by the false transmitter acetylmonoethylcholine (AMECh). The false transmitter can be distinguished from ACh by the fact that its post-synaptic action is briefer, as revealed by a shortening of the time constant of decay (Tm.e.p.c.) of miniature end-plate currents (m.e.p.c.s) in rat muscle recorded by the voltage
W. A. LARGE AND H. P. RANG
26 clamp
reported
progressively
quite simple
was
maximally
the
which
to
this
is
value
quanta
is
value
bring the
intermediate
at
question
of
stages
Tm.e.p-c-
ACh)
and with exchange, the
The
AMECh).
entirely
work
Tm.ep.c.
mean
(representing entirely
value
The
of
released, and it
transmitter
stimulation to
nerve
whether,
of
amount
(representing
concerned
Rang, 1978b).
showed that the
normal
the
between
Large &
1977;
paper,
judge the period of
reduced
paper
this
transmitter
Rang,
function of the
intermediate
value
a
Large &
latter, preceding
the
in
decreased
to
(Colquhoun,
method
population two disAMECh, mixture of residual populations discernible, representing quanta and newly-formed quanta. On simple interpretation of the vesicle hypothesis the pre-existing expected quanta would be released and respontaneously form
quantum containing
each
with
released
tinct
a
would
was
from
cytoplasmic pool
In
variability supposed
we
of
investigated
stages
of
of
The
the
vesicles
the
are
are
consistent
filled
before
of
on
and
ACh
distribution of values We have found
no
AMECh. of
Tm.e.p.c.
evidence
for the
with the vesicle hypothesis if
it is
is
store
is
discernible
a
in
form
the
released,
being
would,
1977)
in
there
transmitter
the directly the other until
of transmitter
mixture
a
exchange.
quanta, though
of
release
Tauc,
1977;
populations
distinct
two
to
frequency
the
transmitter
results
that
which
rise
favouring
Marchbanks,
(see
have
TmepC.
giving
so
hypothesis
quantum would consist of
populations
either
from
An
each
intermediate
distinct
pool
that
study
this
during two
complete.
predict
hand,
ACh
ACh
quanta,
change a
whether
or
a
that
AMECh
and
a
be
placed by
single homogeneous
a
ACh
are
AMECh
it
of
mixture
or
increase
of
cytoplasmic
a
there
that
rapid
is
exchange of transmitter between individual vesicles. A
preliminary
account
of
this work has
already appeared (Large &
Rang, 1978a).
METHODS Rang, 1978b).
described in the preceding paper (Large experiments
omohyoid Sterz,
1976).
gave
muscle was
the
previous -80
clamped
mV;
variability tape.
&
r
Rang,
Tm.e.p.c. values deviation of
was expressed , T
=
mean
(0-1
rmTepecC
mm)
min,
by
Recordings
min. 30-40
as a
was
a
Most of the
For a few
experi-
(Dreyer, Muller, the m.e.p.c amplitude
its motor nerve
with
diaphragm, but slightly more difficult.
the
Peper
The
same
recording
of
50-150
were done at
spontaneously occurring
as
histograms.
St/7),
(c.v.T
.
m.e.p.c.s
experiments
most
In
coefficient of variation
series of control
tech-
mV. For measurerem.e.p.c.s were
=
the
variability
made
nerve
a
as
was first recorded in each of six different
sufficient in most preparations to bring about a reduction 45
replacement of
ACh
by
was then stimulated for a further
was
made.
30-60
min at
at
of
AMECh).
quickly as possible from another four to six cells; this
final series of recordings
of
where St = standard
then added to the bathing solution and the nerve was stimulated
was
&
For most of the experiments the membrane potential
(corresponding to approximately
the exchange, after which
'C.
measured by the template method as described previously
plotted
T
which
%
were
value of
experiments
15-25
'runs
m.e.p.c.'
and
T
paper.
nerve preparations at
some of the later experiments
of
values
1978b)
as
recording
made
niques
used,
results
same
smaller,
usually
(Large
diaphragm-phrenic
rat
usually
Hz to complete
VARIABILITY OF TRANSMITTER QUANTA
27
RESULTS
Control measurement Nerve stimulation in the absence of MECh did not affect the variability of m.e.p.c.s. A single experiment is shown in histogram form in Fig. 1 (A, B and C) and the results of five control experiments are summarized in Table 1. Two of the experiments were carried out on the mouse omohyoid muscle but have been grouped with the experiments on the rat diaphragm to obtain over-all mean values as there appeared to be no important differences between the two preparations. In these five experiments the mean c.v, did not change after the first 15 min period of 3 Hz nerve stimulation 40
A
D
-
20
G
3 30
15
20
20
10
10
10
5-
030
1
0
0
20
1 5
2-5
40
n
40
3-0 CB
20
20
10
10
0
"II0 20 25
15
40 -c 30 -30
30
1.5 20 2-5
05 1 0 40 30 -30 -15
E
15
2-0 H
5
A.0
15
05 10 40 -F
20
25
05 20 15
10
15
1-0
1-5
20
5
10 r
1-0
10 F
10 1-5
0 05 20
H
10
-
20 -20
0
-
J0 1
2-0
2-5
0.5
1.0
1-5
0
0.5
(msec) Fig. 1. Frequency distribution of the time constants of decay of m.e.p.c.s, histograms A, D and G are from three different cells in control conditions and B, E and H show the distribution of Tm.e.p.c. values after the nerve had been stimulated at 3 Hz for 15 min in normal Krebs solution (B) or in the presence of MECh (E and H); C, F and I illustrate the m.e.p.c. distribution after a further 60 min stimulation either in normal Krebs solution (C) or MECh (F and I). Note that 15 min stimulation in the presence of MECh produces a reduction in the meant value (E and H) which is associated with an increase in the scatter of the individual values and a further 60 min stimulation decreases the mean -T further while the scatter decreases towards the control level. In the control muscle (A, B, C) neither the mean r nor the degree of scatter changes appreciably. r
or after a further 30-60 min stimulation. The mean value of Tm.e.p.c. was slightly increased after stimulation (by an average of 10%) but the effect was rather inconsistent and we did correct the values of -rm.e.p.c. obtained in the MECh experiments to allow for it. Consequently we may have tended to underestimate slightly the
28
~ ='O.0r
W. A. LARGE AND H. P. BANG 6te 6 6ott 6 06666
.o.+ + + + + 4i N5
+ + + ++
41
~~~_oooo______
6
