- Email: [email protected]
Glutamate-operated postsynaptic channels and spontaneous excitatory postsynaptic currents in crayfish claw opener muscle
Neumsc/ence Letters. 36 (1983) 163-168 Elsevier Scientific Publishers Ireland Ltd.
163
GLUTAMATE~PERATlgn POST~YNAPTIC CHANNELS AND SPONTANEOUS EXCITATORY ~ Y N A P T I C CURRENTS IN CRAYFISH CLAW OPENER MUSCLE
WOLFGANG FINGER
Pkysiologisches lnstitut der Technischen Um'versitdt M~nchen. Biedersteiner Sir. 29, D-8000 M~nchen 40 rF.R.O )
(Received January 12th, 1983; Revised version received Janue.ry 2?th, 1983; Accepted January 28th, 1983)
Key words: crayfish - neuromuscular transmission postsynaptic currents - noise analysis
glutamate - ionic channels - excitatory
In opener muscle fibres of the crayfish claw excitatory postsynaptk currents activated by glutamate (I. 10-4-5.10 -~ tool/I) were investigated by means of the noise analysis technique. For the apparent mean open time of iglutamate-activated channels, r,,,e = I. I $ ms :t: O. I 6 (S. D.. n = 24) resulted at membrane potentials between E - - 6 0 mV and E - - 100 inV. T = ~ - 2 3 ° C . No silJnificant voltage dependence for ~'.o,~ was observed, most likely due to the pretratment of the fibres with I t~mol/I concanavalin A. For the conductance, .y, of these channels. ~ - 23.6 pS ~: $.2 (S.D., n - 24) was found. These cha:acteristics for iglutamate-operated channels differ Sillnificantly from those observed recently in opener muscle fibres of crayfish first walkinlg lelg 1131. Similarly, different characteristics were found also for the decay time constants ~(sEPSCs) of spontaneous excitatory postsynaptic currents in the respective muscles. On average, dsEPSCs) was 1.3 ms ± 0.3 (S.D.. n = I I) in the claw, In the first walkins L.'g ~(sEPSCs) was shorter by a factor of about 2.4 and in the second walking leg shorter by a factor of about 2.9, than in the claw.
Recently, we have investigated glutamate-operated postsynaptic channels in the opener muscle from the first walking leg of crayfish by means of the noise analysis technique [ 12, 13]. In this preparation glutamate is the natural transmitter mediating synaptic excitation [8]. Glutamate-sensitive channels were characterized by fast open-closed kinetics, i.e. the apparent mean open time of the channels estimated from power spectra (~no~) was about ~no~ = 0.4 ms at room temperature (T--22°C). For the channel conductance (~,) about .y = 70 pS was obtained (T = 22°C). It seemed of interest to find out whether these characteristics would apply also for ~lutamate-operated channels in other crayfish nerve-muscle preparations. In particular, it would be interesting to compare the characteristics of glutamate channels from the analogous opener muscles of the claw, of the first walking leg and of the second walking leg. Parts of the results have been communicated earlier in abstract form [51. 0304-3940/83/0000-0000/$ 03.00 @ 1983 Elsevier Scientific Publishers Ireland Ltd.
164
Opener muscles were prepared from small crayfish (0.4-2 g, Astacus astacus, Austropotamobius torrentium). Two glass capillary microelegtrodes were inserted into a muscle fibre and the membrane potential was clamped to a constant level. To prevent dmensi "ttzation of glutamate r e : e i ~ r s the muscle fibers were pretreated with I tunol/I concanavalin A (Con A) [13]. The clamp currents were measured before and after bath application of glutamate. To obtain membrane current fluctuations the currents were rdtered (high pass at 1 Hz, low pass at 500 Hz or I kl-lz), amplified and stored on a magnetic disc by means of a Nicolct Med 80 computer system. The records of current fluctuations were divided into blocks o f 512 memory ~ocations and from each block a power spectrum was calculated by a fast Fourier routine. From 30-60 spectra an average power spectrum was calculated which after being corrected for the background current noise, was represented in a double logarithmic diagram. High rates of sEPSCs were evoked by high concentrations of GABA and the average decay time constant of these sEPSCs also was estimated from power spectra [4, 5, 7]. Details of the preparations, solutions, set-up, voltage clamp and noise analysis have been described in earlier publications [6, 7, 12]. Fig. I presents a typical result from an experiment on the claw opener. Subsequent to pretreatment for 45 min with I- 10 -6 mol/I Con A, a muscle fibre was voltage-clamped at E = - 9 0 mV membrane potential. Application of 2 - l 0 - 4 mol/I glutamate gave rise to an average depolarizing membrane current of ! = - 20 ~IA (not shown). Associated with this current evoked by glutamate there was a larg: increase in curret,t noise intensity (not illustrated) reflecting statistical opening and
ld 21
S x (f)
fc:130 Hz .
",
°°1
t622
E:-gOmV T: 23°C ~:(ow opene~
f IL
1
a
•
a.a.~d
•
10
.
. aa.~.l
l
100
I
Nz
aaaa..I
1000
Fig. I. Power spectrum calculated from current fluctuations evoked by 2- 10- 4 mol/! glutamate at a fibre from claw opener muscle (E - - 9 0 mV, T = 23°C). The spectrum shown is a difference spectrum, i.e. the spectrum of background current fluctuations (no glutamate present) has been previously subtracted. A Lorentz curve was fitted to the spectrum: St(f) = S,(0)/[! + (f/fc)z], with St{O) = 2.25-10- zz A ' s and f~ = 130 Hz.
165
cJlUlOn~]le • C(x~A 'e,f,, i-
e.,/
TO '
:
/
/°
- - - - - ~ ~" = 2v 2pS
6:
"" i : L/"
o
E:-8OmV
".
T=20-22°C (cl~)
. .
.~
. . . . . . . . .
~o
..
3o
•
SOnA
.~
I!1
Fig. 2. Mean ,.'urrents I and their variance ~ obtained at different claw opener muscle fibres voltage clamped at E =- - 80 mV (T = 20--22°C). The fibres were previously treated with I ~mol/I Con A. The data were fitted by a linear regression line (r = 0.99) from which an average channel conductance "v = 21.2 pS was calculated.
closing of postsynaptic membrane channels in the presence of glutamate. The power spectrum representing this glutamate-induced current noise is shown in Fig. I. It was fitted by a Lorentz curve with a cut-off frequency fc - 130 Hz. The apparent mean open time of the channels activated by glutamate can be estimated from the power spectrum by ~noi~ - l/(2~rfc) and the conductance of these channels by 3" = Si(0)a'fJ[21(E-Es)], where St(0) is the power spectral density for f-*0, i is the mean current evoked by glutamate, and Es is the glutamate reversal potential [10, 12]. Es - + 33 mV was assumed throughout the study [2, 12, 13], thus fnoix - 1.22 ms and 3' = 18.7 pS resulted. This value of ~noi,e in crayfish claw is about 3 times larger and 3' is about 3 times smaller, than the respective figures obtained in the opener muscle of the first walking leg [12, 131. in Fig. 2 the variances ~ = Sj(0)~rfc/2 are represented for various currents 1 evoked by glutamate concentrations between 1- 10 - 4 mol/I and 5 . 1 0 - 4 mol/I at E = - 8 0 mV. The data were fitted by a linear regression fine ( r - 0.99) according to ~ -- T(E-Es)i with 3' - 21.2 pS. Therefore, it may be assumed that the probability for glutamate to open a channel is low up to concentrations of 5 . 1 0 -4 mol/I (low concentration limit [10, 13]). From n = 24 experiments in the claw opener muscle the following averages were obtained for 3' and ~noisc ( - 6 0 mV _>E_> - 100 mV, T = 20-23°C): 3' - 23.6 pS + 5.2 (S.D.) • noi~ - 1.15 ms + 0.16 (S.D.). No significant voltage dependence for Tnoi~ was found, most likely as a result of the pretreatment with Con A [13]. In order to find out whether the relatively slow kinetics of glutamate-operated channels found in the claw is also reflected in the decay time constants of sEPSCs, t
fc-325 Hz
sir),~
-
1.0!
It,,--0.~ n~l k:r-.g
01
g t"
0.01 L.
(r. =1.77ms| I
q~
E"=-bOrer f
I0
100
1000 Hz
Fig. 3. Power spectra characterizing sEPSCs obtained at a fibre of claw opener muscle and at a fibre of the opener muscle in the first walking leg. sEPSCs were evoked by addition o f 0.25 mol/I GABA to the superfusion resulting in rates for quantal release of 100 quanla/s to about 1000 quanta/s. The intensities of the spectra at f = O, S,(0), were set equal to I and the spect[a fitted by Lorcntz curves. At the claw f~ - 90 Hz (T -- 1.77 rash and at the first walking leg f, = 325 Hz Or = 0.49 ms) resulted where T = ~sEPSCs) represents the mean time constant for decay of about 5000 to 20,000 sEPSC's recorded at a single muscle fibre ( E = - 6 0 inV, T = 20°C).
release of excitatory transmitter from the nerve terminal was induced by superfusion with high doses of GABA [4, 5]. By this method release rates of about t0,000 quanta/s can be evoked resulting in current noise of large intensity which can be evaluated via power spectral densities. Fig. 3 shows results for an experiment at the claw opener (f~ = 90 Hz) and at the opener of the first walking leg (fc = 325 Hz). in these power spectra f~ represents the decay time constant of sEPSCs: r(sEPSCs)-I/(2~rf~). As found for the apparent mean open time r, oi~ of glutamate-activated channels, r(sEPSCs) was significantly larger in the claw. However. the experiment at the claw represented in Fig. 3 yielded a time constant f o r d e c a y o f s E P S C s w h i c h w a s o n e o f t h e l a r g e s t o b s e r v e d : 7 ( s E P S C s ) = 1.77 m s . T h e a v e r a g e results o b t a i n e d a r e c o n t a i n e d in T a b l e ! t o g e t h e r w i t h t h o s e o b t a i n e d f r o m o p e n e r muscles in t h e first a n d s e c o n d w a l k i n g leg. T h e d e c a y o f s E P S C s w a s TABI.E I DECAY TIME CONSTANTS FOR sEPSCs IN DIFFERENT OPENER MUSCLES OF CRAYFISH Averages ( _+. S.D.) for T = ~sEPSCs) of sEPSCs evoked by high concentrations of GABA at opener muscle fibers in the claw, the first walking leg and the second walking leg (E - - 6 0 inV, T - 20°C). The averages were calculated from T values of n different fibres obtained in experiments like those represented in Fig. 3. Lowering the temperature by 10°C increased 1"by a factor given by Qno. zo°c - 6 0 m y
Claw
First walking leg
Second walking leg
T(ms)
1.3 ± 0.3 (S.D.,n = !i)
0.55 ± 0.1 (n = II)
0.45 ± 0.14 (n = 8)
~
~
Qio
= 1.85
2.2
2.0
167
about 2.4 times shorter in the first walking leg and about 2.9 times shorter in the second walking leg than in the claw. On decreasing the temperature by 10°C, ~(sEPSCs) was increased by a factor of 1.85-2.2 (Qm in Table 1). Moreover, in preparations which were not treated with Con A results were obtained for 7(sEPSCs) at E -- - 6 0 mV which were consistent with those presented in Table I and, as expected !121, in these preparations ~(sEPSCs) decreased on hyperpolarization. If the average ~(sEPSCs) obtained at the claw is compared with the average T~oi,e of glutamate-activated channels at the claw, ~sEPSCs) appears to be larger by about 13~,. In the first walking leg the difference between ~sEPSCs) and 7noise seems to be more marked (26'V., em,~ -- 0.4 ms, 113]). However, it should be noted that ~(sEPSCs), like ~um,e, varied from fibre to fibre within an opener muscle by a factor of about 2. Furthermore, in the first walking leg some limitations for the space clamp may occur at room temperature [6, 12, 13]. These circumstances might account for the differences found between ~(sEPSCs) and 1"noisebut further experiments have to be done to compare ~(sEPSCs) and ~aoi,~at the same muscle fibre. The different characteristics found for glutamate-operated channels in opener muscles of claw, first walking leg and second walking leg, might be related to specific properties of these muscles, For frog muscle fibres it was found recently that acetylcholine-activated channels in fibres of fast type were characterized by a mean channel lifetime which was about three times shorter than that of acetylcholineactivated channels in fibres of the slow type 13, 9]. Consistent with this difference in channel lifetime it was found that the decay time constant of sEPSCs was about three times shorter in fast fibres than in slow ones, Furthermore, the conductance, "v, of acetylcholine-activated channels was about 1.5-2.1 times larger in fibres of the fast type. in view of these results it might be possible that crayfish opener muscles in the first and second walking leg belong preferentially to the class of fast muscles, whereas the opener muscle in the claw rather belongs to the slow type. However, the variation of ~ and ~,oi,, by a factor of about 2 observed within a single opener muscle might also reflect differentiation of muscle fibres and regional differences [1, I 1], This possibility will be investigated further. ! would like to thank Prof. Dr. Dudel for reading the manuscript, and Mrs. E. K&ster and Mrs. L. Bauer for technical and secretarial help. This investigation was supported by the Deutsche Forschungsgemeinschaft, Project Fi 305/1-2. I Bittner, G.D., Differentiation of nerve terminals in the crayfish opener muscle and its functional significance, J. Sen- Physiol.. 51 (1968) 731-758. 2 Dudel, J., Nonlinear voltage dependence of excitatory synaptic current in crayfish muscle, PfliJlgers Arch., 352 (1974) 227-241. 3 Fedorov, V.V., Magazanik, L.G., Snetkov, V.A. and Zefirov, A.L., Postsynaptic currents in different types of frog muscle fibre, Pfliisers Arch., 394 (1982) 202-210. 4 Finger, W., Enhanced release of inhibitory and excitatory transmitter quanta in the crayfish neuromuscular junction by glycine and GABA, Neurosci. Lett., 34 (1982) 33-38.
168
5 F _m~r, w . , Spomuams au:immy p e a s ~ q ~ : currem- ( ~ ) i n ~ Jzuromuscu~ junctions stimulmed by h i ~ ~ of GAnA, I P ~ Arch., 992, SUlpgl. (1982) ILT8/151. 6 F'ml~r, W. and Stettmei~, H., FA'Cu:acyof the two-mJcrodeczrode voltage clamp lechnkime in crayfish umsdc, Pfl~j~rs Arch., 387 (1980) 133--141. ? F J q ~ , W. and Statmeia, H., Analysis of minianue s p o a t a u a ~ inln3~ow pmsymtp~ c u n e m ($IPSC$) from current noise in crayfish opener muscle, Pfl0fgrs Arch., 392 (IgSI) 1Y7-162. 8 Kauag(~, R., Ontxlcm, K. and TsketwJti, A., On the quaatal stlmse of endojmous Illutamate from the crayfish nemomuscular junction, J. Physiol. (Loud.), 322 (1982) 529-539. 9 Miledi, R. and Uchitel, O.D., Propades of poslsynap~ chlmnds induced by acesylchoU~ in diffaent frog musde fibres, Nature (Lond.), 291 (19JJl) 162-165. tO Nehcr, E. and fdevens, C.F., Conducum~ fluctuations and ionic pores in membranes, Ann. Rev. Biophys. Bioenps., 6 (1977) 345-381. 11 Pamas, I., Parnas, H. and Dudel, J., Neurotrammitter release and its facilitation in crayfish muscle V. Basis for synapsedifferentiation of the fast and slow type in one axon, P i l f e r s Arch., $95 (1982) 261-270. 12 Stettmeier, H., Finger, W. and Dudd, J,. Glutamate activated postsynaptic channels in crayfish muscle investigated by noise analysis. Pflfiger$ Arch.. in press. t3 Stettmeier. H.. Finger. W. and Dudd. J.. Effects of concanavalin A on glutamate operated postsynaptic channels in crayfish muscle, PflfigeJ3 Arch., in press.
163
GLUTAMATE~PERATlgn POST~YNAPTIC CHANNELS AND SPONTANEOUS EXCITATORY ~ Y N A P T I C CURRENTS IN CRAYFISH CLAW OPENER MUSCLE
WOLFGANG FINGER
Pkysiologisches lnstitut der Technischen Um'versitdt M~nchen. Biedersteiner Sir. 29, D-8000 M~nchen 40 rF.R.O )
(Received January 12th, 1983; Revised version received Janue.ry 2?th, 1983; Accepted January 28th, 1983)
Key words: crayfish - neuromuscular transmission postsynaptic currents - noise analysis
glutamate - ionic channels - excitatory
In opener muscle fibres of the crayfish claw excitatory postsynaptk currents activated by glutamate (I. 10-4-5.10 -~ tool/I) were investigated by means of the noise analysis technique. For the apparent mean open time of iglutamate-activated channels, r,,,e = I. I $ ms :t: O. I 6 (S. D.. n = 24) resulted at membrane potentials between E - - 6 0 mV and E - - 100 inV. T = ~ - 2 3 ° C . No silJnificant voltage dependence for ~'.o,~ was observed, most likely due to the pretratment of the fibres with I t~mol/I concanavalin A. For the conductance, .y, of these channels. ~ - 23.6 pS ~: $.2 (S.D., n - 24) was found. These cha:acteristics for iglutamate-operated channels differ Sillnificantly from those observed recently in opener muscle fibres of crayfish first walkinlg lelg 1131. Similarly, different characteristics were found also for the decay time constants ~(sEPSCs) of spontaneous excitatory postsynaptic currents in the respective muscles. On average, dsEPSCs) was 1.3 ms ± 0.3 (S.D.. n = I I) in the claw, In the first walkins L.'g ~(sEPSCs) was shorter by a factor of about 2.4 and in the second walking leg shorter by a factor of about 2.9, than in the claw.
Recently, we have investigated glutamate-operated postsynaptic channels in the opener muscle from the first walking leg of crayfish by means of the noise analysis technique [ 12, 13]. In this preparation glutamate is the natural transmitter mediating synaptic excitation [8]. Glutamate-sensitive channels were characterized by fast open-closed kinetics, i.e. the apparent mean open time of the channels estimated from power spectra (~no~) was about ~no~ = 0.4 ms at room temperature (T--22°C). For the channel conductance (~,) about .y = 70 pS was obtained (T = 22°C). It seemed of interest to find out whether these characteristics would apply also for ~lutamate-operated channels in other crayfish nerve-muscle preparations. In particular, it would be interesting to compare the characteristics of glutamate channels from the analogous opener muscles of the claw, of the first walking leg and of the second walking leg. Parts of the results have been communicated earlier in abstract form [51. 0304-3940/83/0000-0000/$ 03.00 @ 1983 Elsevier Scientific Publishers Ireland Ltd.
164
Opener muscles were prepared from small crayfish (0.4-2 g, Astacus astacus, Austropotamobius torrentium). Two glass capillary microelegtrodes were inserted into a muscle fibre and the membrane potential was clamped to a constant level. To prevent dmensi "ttzation of glutamate r e : e i ~ r s the muscle fibers were pretreated with I tunol/I concanavalin A (Con A) [13]. The clamp currents were measured before and after bath application of glutamate. To obtain membrane current fluctuations the currents were rdtered (high pass at 1 Hz, low pass at 500 Hz or I kl-lz), amplified and stored on a magnetic disc by means of a Nicolct Med 80 computer system. The records of current fluctuations were divided into blocks o f 512 memory ~ocations and from each block a power spectrum was calculated by a fast Fourier routine. From 30-60 spectra an average power spectrum was calculated which after being corrected for the background current noise, was represented in a double logarithmic diagram. High rates of sEPSCs were evoked by high concentrations of GABA and the average decay time constant of these sEPSCs also was estimated from power spectra [4, 5, 7]. Details of the preparations, solutions, set-up, voltage clamp and noise analysis have been described in earlier publications [6, 7, 12]. Fig. I presents a typical result from an experiment on the claw opener. Subsequent to pretreatment for 45 min with I- 10 -6 mol/I Con A, a muscle fibre was voltage-clamped at E = - 9 0 mV membrane potential. Application of 2 - l 0 - 4 mol/I glutamate gave rise to an average depolarizing membrane current of ! = - 20 ~IA (not shown). Associated with this current evoked by glutamate there was a larg: increase in curret,t noise intensity (not illustrated) reflecting statistical opening and
ld 21
S x (f)
fc:130 Hz .
",
°°1
t622
E:-gOmV T: 23°C ~:(ow opene~
f IL
1
a
•
a.a.~d
•
10
.
. aa.~.l
l
100
I
Nz
aaaa..I
1000
Fig. I. Power spectrum calculated from current fluctuations evoked by 2- 10- 4 mol/! glutamate at a fibre from claw opener muscle (E - - 9 0 mV, T = 23°C). The spectrum shown is a difference spectrum, i.e. the spectrum of background current fluctuations (no glutamate present) has been previously subtracted. A Lorentz curve was fitted to the spectrum: St(f) = S,(0)/[! + (f/fc)z], with St{O) = 2.25-10- zz A ' s and f~ = 130 Hz.
165
cJlUlOn~]le • C(x~A 'e,f,, i-
e.,/
TO '
:
/
/°
- - - - - ~ ~" = 2v 2pS
6:
"" i : L/"
o
E:-8OmV
".
T=20-22°C (cl~)
. .
.~
. . . . . . . . .
~o
..
3o
•
SOnA
.~
I!1
Fig. 2. Mean ,.'urrents I and their variance ~ obtained at different claw opener muscle fibres voltage clamped at E =- - 80 mV (T = 20--22°C). The fibres were previously treated with I ~mol/I Con A. The data were fitted by a linear regression line (r = 0.99) from which an average channel conductance "v = 21.2 pS was calculated.
closing of postsynaptic membrane channels in the presence of glutamate. The power spectrum representing this glutamate-induced current noise is shown in Fig. I. It was fitted by a Lorentz curve with a cut-off frequency fc - 130 Hz. The apparent mean open time of the channels activated by glutamate can be estimated from the power spectrum by ~noi~ - l/(2~rfc) and the conductance of these channels by 3" = Si(0)a'fJ[21(E-Es)], where St(0) is the power spectral density for f-*0, i is the mean current evoked by glutamate, and Es is the glutamate reversal potential [10, 12]. Es - + 33 mV was assumed throughout the study [2, 12, 13], thus fnoix - 1.22 ms and 3' = 18.7 pS resulted. This value of ~noi,e in crayfish claw is about 3 times larger and 3' is about 3 times smaller, than the respective figures obtained in the opener muscle of the first walking leg [12, 131. in Fig. 2 the variances ~ = Sj(0)~rfc/2 are represented for various currents 1 evoked by glutamate concentrations between 1- 10 - 4 mol/I and 5 . 1 0 - 4 mol/I at E = - 8 0 mV. The data were fitted by a linear regression fine ( r - 0.99) according to ~ -- T(E-Es)i with 3' - 21.2 pS. Therefore, it may be assumed that the probability for glutamate to open a channel is low up to concentrations of 5 . 1 0 -4 mol/I (low concentration limit [10, 13]). From n = 24 experiments in the claw opener muscle the following averages were obtained for 3' and ~noisc ( - 6 0 mV _>E_> - 100 mV, T = 20-23°C): 3' - 23.6 pS + 5.2 (S.D.) • noi~ - 1.15 ms + 0.16 (S.D.). No significant voltage dependence for Tnoi~ was found, most likely as a result of the pretreatment with Con A [13]. In order to find out whether the relatively slow kinetics of glutamate-operated channels found in the claw is also reflected in the decay time constants of sEPSCs, t
fc-325 Hz
sir),~
-
1.0!
It,,--0.~ n~l k:r-.g
01
g t"
0.01 L.
(r. =1.77ms| I
q~
E"=-bOrer f
I0
100
1000 Hz
Fig. 3. Power spectra characterizing sEPSCs obtained at a fibre of claw opener muscle and at a fibre of the opener muscle in the first walking leg. sEPSCs were evoked by addition o f 0.25 mol/I GABA to the superfusion resulting in rates for quantal release of 100 quanla/s to about 1000 quanta/s. The intensities of the spectra at f = O, S,(0), were set equal to I and the spect[a fitted by Lorcntz curves. At the claw f~ - 90 Hz (T -- 1.77 rash and at the first walking leg f, = 325 Hz Or = 0.49 ms) resulted where T = ~sEPSCs) represents the mean time constant for decay of about 5000 to 20,000 sEPSC's recorded at a single muscle fibre ( E = - 6 0 inV, T = 20°C).
release of excitatory transmitter from the nerve terminal was induced by superfusion with high doses of GABA [4, 5]. By this method release rates of about t0,000 quanta/s can be evoked resulting in current noise of large intensity which can be evaluated via power spectral densities. Fig. 3 shows results for an experiment at the claw opener (f~ = 90 Hz) and at the opener of the first walking leg (fc = 325 Hz). in these power spectra f~ represents the decay time constant of sEPSCs: r(sEPSCs)-I/(2~rf~). As found for the apparent mean open time r, oi~ of glutamate-activated channels, r(sEPSCs) was significantly larger in the claw. However. the experiment at the claw represented in Fig. 3 yielded a time constant f o r d e c a y o f s E P S C s w h i c h w a s o n e o f t h e l a r g e s t o b s e r v e d : 7 ( s E P S C s ) = 1.77 m s . T h e a v e r a g e results o b t a i n e d a r e c o n t a i n e d in T a b l e ! t o g e t h e r w i t h t h o s e o b t a i n e d f r o m o p e n e r muscles in t h e first a n d s e c o n d w a l k i n g leg. T h e d e c a y o f s E P S C s w a s TABI.E I DECAY TIME CONSTANTS FOR sEPSCs IN DIFFERENT OPENER MUSCLES OF CRAYFISH Averages ( _+. S.D.) for T = ~sEPSCs) of sEPSCs evoked by high concentrations of GABA at opener muscle fibers in the claw, the first walking leg and the second walking leg (E - - 6 0 inV, T - 20°C). The averages were calculated from T values of n different fibres obtained in experiments like those represented in Fig. 3. Lowering the temperature by 10°C increased 1"by a factor given by Qno. zo°c - 6 0 m y
Claw
First walking leg
Second walking leg
T(ms)
1.3 ± 0.3 (S.D.,n = !i)
0.55 ± 0.1 (n = II)
0.45 ± 0.14 (n = 8)
~
~
Qio
= 1.85
2.2
2.0
167
about 2.4 times shorter in the first walking leg and about 2.9 times shorter in the second walking leg than in the claw. On decreasing the temperature by 10°C, ~(sEPSCs) was increased by a factor of 1.85-2.2 (Qm in Table 1). Moreover, in preparations which were not treated with Con A results were obtained for 7(sEPSCs) at E -- - 6 0 mV which were consistent with those presented in Table I and, as expected !121, in these preparations ~(sEPSCs) decreased on hyperpolarization. If the average ~(sEPSCs) obtained at the claw is compared with the average T~oi,e of glutamate-activated channels at the claw, ~sEPSCs) appears to be larger by about 13~,. In the first walking leg the difference between ~sEPSCs) and 7noise seems to be more marked (26'V., em,~ -- 0.4 ms, 113]). However, it should be noted that ~(sEPSCs), like ~um,e, varied from fibre to fibre within an opener muscle by a factor of about 2. Furthermore, in the first walking leg some limitations for the space clamp may occur at room temperature [6, 12, 13]. These circumstances might account for the differences found between ~(sEPSCs) and 1"noisebut further experiments have to be done to compare ~(sEPSCs) and ~aoi,~at the same muscle fibre. The different characteristics found for glutamate-operated channels in opener muscles of claw, first walking leg and second walking leg, might be related to specific properties of these muscles, For frog muscle fibres it was found recently that acetylcholine-activated channels in fibres of fast type were characterized by a mean channel lifetime which was about three times shorter than that of acetylcholineactivated channels in fibres of the slow type 13, 9]. Consistent with this difference in channel lifetime it was found that the decay time constant of sEPSCs was about three times shorter in fast fibres than in slow ones, Furthermore, the conductance, "v, of acetylcholine-activated channels was about 1.5-2.1 times larger in fibres of the fast type. in view of these results it might be possible that crayfish opener muscles in the first and second walking leg belong preferentially to the class of fast muscles, whereas the opener muscle in the claw rather belongs to the slow type. However, the variation of ~ and ~,oi,, by a factor of about 2 observed within a single opener muscle might also reflect differentiation of muscle fibres and regional differences [1, I 1], This possibility will be investigated further. ! would like to thank Prof. Dr. Dudel for reading the manuscript, and Mrs. E. K&ster and Mrs. L. Bauer for technical and secretarial help. This investigation was supported by the Deutsche Forschungsgemeinschaft, Project Fi 305/1-2. I Bittner, G.D., Differentiation of nerve terminals in the crayfish opener muscle and its functional significance, J. Sen- Physiol.. 51 (1968) 731-758. 2 Dudel, J., Nonlinear voltage dependence of excitatory synaptic current in crayfish muscle, PfliJlgers Arch., 352 (1974) 227-241. 3 Fedorov, V.V., Magazanik, L.G., Snetkov, V.A. and Zefirov, A.L., Postsynaptic currents in different types of frog muscle fibre, Pfliisers Arch., 394 (1982) 202-210. 4 Finger, W., Enhanced release of inhibitory and excitatory transmitter quanta in the crayfish neuromuscular junction by glycine and GABA, Neurosci. Lett., 34 (1982) 33-38.
168
5 F _m~r, w . , Spomuams au:immy p e a s ~ q ~ : currem- ( ~ ) i n ~ Jzuromuscu~ junctions stimulmed by h i ~ ~ of GAnA, I P ~ Arch., 992, SUlpgl. (1982) ILT8/151. 6 F'ml~r, W. and Stettmei~, H., FA'Cu:acyof the two-mJcrodeczrode voltage clamp lechnkime in crayfish umsdc, Pfl~j~rs Arch., 387 (1980) 133--141. ? F J q ~ , W. and Statmeia, H., Analysis of minianue s p o a t a u a ~ inln3~ow pmsymtp~ c u n e m ($IPSC$) from current noise in crayfish opener muscle, Pfl0fgrs Arch., 392 (IgSI) 1Y7-162. 8 Kauag(~, R., Ontxlcm, K. and TsketwJti, A., On the quaatal stlmse of endojmous Illutamate from the crayfish nemomuscular junction, J. Physiol. (Loud.), 322 (1982) 529-539. 9 Miledi, R. and Uchitel, O.D., Propades of poslsynap~ chlmnds induced by acesylchoU~ in diffaent frog musde fibres, Nature (Lond.), 291 (19JJl) 162-165. tO Nehcr, E. and fdevens, C.F., Conducum~ fluctuations and ionic pores in membranes, Ann. Rev. Biophys. Bioenps., 6 (1977) 345-381. 11 Pamas, I., Parnas, H. and Dudel, J., Neurotrammitter release and its facilitation in crayfish muscle V. Basis for synapsedifferentiation of the fast and slow type in one axon, P i l f e r s Arch., $95 (1982) 261-270. 12 Stettmeier, H., Finger, W. and Dudd, J,. Glutamate activated postsynaptic channels in crayfish muscle investigated by noise analysis. Pflfiger$ Arch.. in press. t3 Stettmeier. H.. Finger. W. and Dudd. J.. Effects of concanavalin A on glutamate operated postsynaptic channels in crayfish muscle, PflfigeJ3 Arch., in press.