- Email: [email protected]
Presynaptic modulation of transmitter release by the early outward potassium current inAplysia
Brain Research, 263 (1983) 51-56
51
Elsevier Biomedical Press
Presynaptic Modulation of Transmitter Release by the Early Outward Potassium Current in Aplysia T. SHIMAHARA Laboratoire de Neurobiologie Cellulaire, 91190 Gif sur Yvette (France)
(Accepted August 10th, 1982) Key words: transmitter release - - presynaptic modulation - - potassium current
The mechanism involved in presynaptic modulation of transmitter release was studied in an identified synapse of Aplysia californica.
Presynaptic hyperpolarization induces a decrease in the evoked postsynaptic potential amplitude zl. This is shown to be due to a reduction in the presynaptic spike amplitude during the hyperpolarization. The decreased presynaptic spike amplitude with hyperpolarization is explained as resulting from the superimposition of an early outward potassium current on the transient inward current. It is suggested that the presynaptic hyperpolarizing conditioning pulse decreases inactivation of the early outward current, which shunts the transient inward current. The superimposition of these two currents (transient inward current and the early outward current) induces a decrease in presynaptic spike amplitude, which in turn reduces the synaptic output from the terminal. INTRODUCTION The first step in the transmitter release process is an entry of Ca 2+ into the terminal during the spike potential. The synaptic efficacy (quantity of a released transmitter from the terminal) is, hence, determined by the quantity of Ca 2+ influx. At the neuromuscular junction, the synaptic efficacy as measured by the postsynaptic potential (PSP) amplitude is generally stable, since the presynaptic spike potential amplitude is constant. Thus, Ca 2+ influx is also constant. However the situation at the synapses in the central nervous system is more complicated, because a presynaptic terminal itself can receive other synaptic inputs which modify directly or indirectly its transmitter release. This presynaptic modulation of synaptic output might be an interesting basic model for a neural plasticity 11. It has been shown in a number of synapses that the synaptic output is sensitive to the presynaptic membrane potentialS,Z1, 23. In the squid giant synapse 23, presynaptic depolarization decreases the presynaptic spike potential resulting in a reduction of synaptic output, whereas presynaptic hyperpolarization has 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
the opposite effect. In the preparation, it has been suggested that the amount of transmitter released might be determined by the peak current rather than the absolute level of depolarization. In Aplysia synapses, however, presynaptic polarization has a reversed effect on synaptic output11,18, 21: a presynaptic hyperpolarization instead reduces the PSP amplitude. A similar phenomenon was also reported to occur in the leech ganglion 16. The present study concerns the paradoxical effect of presynaptic membrane potential on synaptic output. A preliminary report has been giveng, 19. MATERIALS AND METHODS All experiments were performed on the identified synapses in the cerebral ganglion and in the left pleural ganglion of Aplysia. The general properties of these synapses have already been described6, zo. The desheathed ganglion was fixed to the bottom of a lucite chamber (1 ml). The preparation was continuously perfused keeping the temperature constant at 18 °C. A double bareled microelectrode filled with 3 M KC1 was inserted into the post-
52 synaptic neuron for polarizing the cell and for recording the membrane potential. Two independent microelectrodes (filled with 3 M KC1) were inserted into the presynaptic neuron. In some cases, the presynaptic soma membrane was clamped using a conventional clamp amplifier. Total current was measured with a current to voltage converter (100 k ~ feedback resistor), which maintained the bath at ground potential. Physiological saline with the following composition was used (mM/l):460 NaCI, 10 KC1, 11 CaCle, 25 MgCI2, 28 MgSO4, 10 Tris. The p H was adjusted to 7.8 -t- 0.1. In some experiments 4-aminopyridine (4-AP) (Sigma) was added in order to block the outward potassium current.
C
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RESULTS E
Previously we have shown at an identified synapse of Aplysia that the PSP amplitude is sensitive to the presynaptic soma potential 21. A presynaptic hyperpolarization induces a marked decrease in PSP amplitude (Fig. 1A). The relationship between PSP amplitude and the presynaptic soma membrane potential shows an S-shaped curve (Fig. 1B). A similar situation has previously been reported in other Aplysia ganglia is. It should be noted that the duration of conditioning hyperpolarizing is also an important factor in this modulation. In Fig. 1C, long-lasting hyperpolarizating conditioning pulses (--10 mV) of varying duration were imposed on the presynaptic neuron. The same neuron was stimulated with a short depolarizing pulse to evoke the spike before, during and after the hyperpolarizing pulse. As is evident in Fig. 1C, the effect of the conditioning pulse on PSP amplitude is not immediate as its m a x i m u m effect is established about 300 ms after the onset of the conditioning pulse. Following the conditioning pulse, PSP amplitude recovers gradually towards its original size with a time constant of 100 ms. In order to analyze this phenomenon, the presynaptic spike potential was characterized at different presynaptic membrane potentials. The amplitude of the presynaptic spike potential was measured on high speed sweep recordings using three different methods: (1) the absolute amplitude (from foot to peak); (2) the overshoot o f spike potential, and (3) f r o m the notch of the spike potential to the
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Presynaptic soma potetnt~ (mY)
1
post
pre
~
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~
~
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:
~
Fig. 1. The effect of presynaptic membrane potential on the postsynaptic potential amplitude. A: simultaneous recording from the postsynaptic neuron (middle traces) and the presynaptic neuron (bottom traces). The top traces indicate zero potential for the presynaptic neuron. The presynaptic membrane potential was polarized to 4 3 mV (a), 4 8 mV (b), --59 mV (c) and --63 mV (d). Calibration -- 4 mV for middle traces, 40 mV for lower traces, 5 ms. B: presynaptic spike potential (©- - -©) and postsynaptic potential (A - - A) at different presynaptic membrane potentials. C: the effect of hyperpolarizing conditioning pulse duration on synaptic output. Simultaneous recording from the postsynaptic neuron (upper traces) and the presynaptic neuron (bottom traces). Superimposition of 9 sweeps. The presynaptic neuron was hyperpolarized 10 mV from the resting potential for 500 ms. A short depolarizing pulse was applied to evoke the spike potential in the presynaptic neuron before, during and after the hyperpolarizing conditioning pulse. Calibration = 4 mV for upper traces, 40 mV for lower traces, 200 ms.
53
Fig. 2. The effect of 4-AP on the early outward potassium current, a: superimposed recordings show that the test pulse to --10 mV from the holding potential (--40 mV) induces a transient inward current. The same test pulse preceeded by 200 ms hyperpolarizing conditioning pulses (to --50 mV, --60 mV and --70 mV) also induces an early outward potassium current, b: superimposed current traces in response to a test pulse (10 ms) to --10 mV at various times after the onset of a hyperpolarizing conditioning pulse. c and d: superimposed recordings under the same conditions as in a (except that the test pulse is to 0 mV in d). c: control, d: after treatment with 4-AP (10 mM). Calibration = 200 nA, 100 mV, 10 ms. peak. In all cases, it was f o u n d that the presynaptic spike potential was sensitive to the presynaptic m e m b r a n e potential. Furthermore, the relationship between the presynaptic m e m b r a n e potential and the spike overshoot amplitude follows an S-shaped curve, similar to the changes in PSP amplitude (Fig. IB). It was also observed that the spike potential was sensitive to the duration o f the conditioning pulse, as was seen with the PSP. These similar effects suggest that the changes in the presynaptic spike might be responsible for the
modulation o f synaptic output. Even t h o u g h the spike potential was measured at the soma, we can suppose that similar p h e n o m e n a occur at the axon or at the terminal. A reduction in spike amplitude would decrease Ca e+ influx into the terminal, resulting in a decrease o f synaptic output. This hypothesis raises the question why the presynaptic spike potential decreases when the m e m b r a n e potential is hyperpolarized. One possible argument is that some peculiar ionic mechanism might be involved in the spike potential o f this neuron. Thus, the ionic move-
54 ments underlying the spike potential were examined at different membrane potentials under voltage clamp. In normal sea water, a test pulse to --10 mV from the holding potential (--40 mV) induces a transient inward current followed by a delayed outward current, as seen in many other excitable cells. However, the amplitude of the transient inward current decreases if a hyperpolarizing conditioning pulse precedes the test pulse (Fig. 2a). This decrease in the inward current following conditioning hyperpolarization was first described in the Aplysia neuron by Geduldig and Gruener 7. Other detailed studies of this phenomenon have been reported in
the snail neuron by Neher15 and Standen 22. The latter authors suggested that the decrease in the inward current was not a consequence of any peculiar properties of the inward current, but rather, resulted from a superimposition of the early outward potassium current on the inward current. The kinetic and pharmacological properties of this current are quite different from those of the familiar delayed outward potassium current. This current is inactivated at resting potential (about --40 mV), while a hyperpolarizing conditioning pulse removes the inactivation 15. Therefore, a test pulse preceded by a hyperpolarizing conditioning pulse induces the early outward current. Since its time course is similar to that
A
B
i
post
f
III
pre
Fig. 3. The effect of 4-AP on the modulation of synaptic output. A: simultaneous recording from pre and postsynaptic neurons. a-d, in normal sea water. Both spike potential and PSP amplitude decrease when the presynaptic potential is hyperpolarized, e-h, after treatment with 10 m M 4-AP. PSP amplitude is independent of the presynaptic membrane potential. Calibration = 4 mV for postsynaptic neuron, 40 mV for presynaptic neuron, 10 ms. B: the effect of 4-AP (10 mM) on time-dependent PSP modulation under the same conditions as shown in Fig. 1C. a, control in normal sea water; b, after treatment with 4-AP (10 mM) in bath. Calibration 4 mV for postsynaptic neuron, 40 mV for presynaptic neuron, 200 ms.
55 of the inward current, the superimposition of these two opposite currents results in a decrease of the transient inward current amplitude. Furthermore, activation of this current also depends on the duration of the conditioning pulse. As shown in Fig. 2b, if test pulse is maintained constant while the duration of the conditioning pulse is varied, the early outward peak current increases toward a steady state value with increasing conditioning duration. Conversely, the transient inward current decreases. Such a reduction of the inward current amplitude might be responsible for the decrease in spike amplitude following conditioning hyperpolarization, and thus, also for the depression of the PSP. In order to test this hypothesis, synaptic transmission was studied under the condition where the early outward current was blocked. This current is sensitive to externally applied 4-aminopyridine (4AP), while the delayed outward current is little affected. Fig. 2b illustrates the effect of 4-AP on the early outward current and on the inward current. Under 4-AP (10 mM) treatment, the early outward current is abolished. The most significant effect of 4AP on synaptic transmission is that PSP amplitude becomes quite independent of the presynaptic membrane potential (Fig. 3A). In other words, 4-AP completely blocks the presynaptic membrane potential-dependent modulation of synaptic output. As is evident in Fig. 3B, the time-dependent PSP modulation is also suppressed under 4-AP treatment. The above results lead us to the following conclusion. The presynaptic hyperpolarizing potential removes an inactivation of the early outward potassium current. This activation of the early outward current shunts the spike potentials, resulting in a depression of synaptic output from the terminal. DISCUSSION
The early outward potassium current was discovered first in an Onchidium neuron s, where a hyperpolarizing conditioning pulse induces the anode-break response. This same current has been found in many other various preparations1-4,10,14, 17,24. The kinetic and pharmacological properties of this current have been studied in detail in gastropod neurons 15,z4. The early outward current is generally inactivated at the resting potential. Therefore, it is
necessary to hyperpolarize the membrane in order to remove the inactivation. The kinetics of this current are similar to those of the transient inward current except that it has a long time constant of decay. Also, the activation of the early outward current is time dependent (Fig. 3b). The physiological role of the early outward current has been discussed in many different preparations. Some authors~ suggested that pacemaker activity in molluscan neurons is controlled by this current. The excitatory synaptic response in snail neurons is affected by a short circuit excitatory postsynaptic potential which is due to an activation of the early outward potassium current 4. Recently Salkoff et al. 17 have shown that facilitation at the Drosophila neuromuscular junction is due to an inactivation of the early outward current during repetitive firing. It has also been reported that in the non-spiking neuron of the crustacean coxal receptor, the transient outward potassium current shunts a transient inward current lz. However, this current is apparently not present at the squid giant synapsO2 where changes in the presynaptic membrane potential have an opposite effect: presynaptic depolarization induces a decrease in PSP amplitude, while presynaptic hyperpolarization increases it. The opposite effects of presynaptic membrane potential on synaptic output in Aplysia and the squid might be due to the fact that in the former, the early outward current exists, while in the latter it is absent 12. These results reveal that the transient outward potassium current has an important role in the modulation of synaptic output in Aplysia. However, the present observations do not exclude a possible involvement of other currents in the synaptic modulation. For example, the delayed outward potassium current may be involved, as has been shown in another system of Aplysia is. In Fig. IB, it can be seen that PSP amplitude is increased when the presynaptic potential is depolarized from --40 to --30 mV, suggesting that the late outward potassium current is less inactivated. This possibility is supported by the observation (not shown) of a broadened presynaptic spike with depolarization of the holding potential. Another possible explanation may lie in the reported inactivation of calcium current with hyperpolarizatioo. These effects are now under further study.
56 REFERENCES 1 Barrett, J. N. and Crill, W. E., Voltage clamp analysis of conductances underlying cat motoneuron action potential, Fed. Proc., 31 (1972) 305. 2 Connor, J. A., Neural repetitive firing: a comparative study of membrane properties of crustacean walking leg axons, J. Neurophysiol., 38 (1975) 922-932. 3 Connor, J. A. and Stevens, C. F., Prediction of repetitive firing behaviour from voltage clamp data on an isolated neurone soma, J. Physiol. (Lond.), 213 (1971) 31-53. 4 Daut, J., Modulation of the excitatory synaptic response by fast transient K current in snail neurones, .Nature New Biol., 246 (1973) 193-196. 5 Del Castillo, J. and Katz, B., Changes in end-plate activity produced by presynaptic polarization, J. PhysioL (Lond.), 124 (1954) 586-604. 6 Fredman, S. M. and Parwar, J. B., Synaptic connections in the cerebral ganglion of Aplysia, Brain Research, 100 (1975) 209-214. 7 Geduldig, D. and Gruener, R., Voltage clamp of the Aplysia giant neurone. Early sodium and calcium currents, J. Physiol. (Lond.), 211 (1970) 217-244. 8 Hagiwara, S., Kusano, S. and Saito, N., Membrane changes of Onchidium nerve cell in potassium rich media, J. Physiol. (Lond.), 155 (1961) 470-489. 9 Israel, J. M. and Shimahara, T., Possible involvement of the early outward potassium current on transmitter release in Ap!ysia, J. PhysioL (Lond.), 307 (1980) 32-33 P. 10 Kenyon, J. L. and Gibbons, W. R., 4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers, J. gen. Physiol., 73 (1979) 139-157. 11 Klein, M., Shapiro, E. and Kandel, E. R., Synaptic plasticity and the modulation of the Ca 2+ current, J. exp. BioL, 89 (1980) 117-157. 12 Llinas, R., Steinberg, I. Z. and Walton, K., Presynaptic calcium currents in squid giant synapse, Biophys. J., 33 (1981) 289-322.
13 Mirolli, M., Fast inward and outward current channels in a non-spiking neurone, Nature (Lond.), 292 (1981) 251-253. 14 Miyazaki~ S., Ohmori, H. and Sasaki, S., Potassium rectifications of the starfish oocyte membrane and their changes during oocyte maturation, J. Physiol. (Lond.), 240 (1975) 55-78. 15 Neher, E., Two fast transient current components during voltage clamp on snail neurones, J. gen. PhysioL, 58 (1971) 36-53. 16 Nicholls, J. and Wallace, B. G., Modulation of transmission at an inhibitory synapse in the central nervous system of the leech, J. Physiol. (Lond.), 281 (1978) 157170. 17 Salkoff, L. and Wyman, R., Facilitation of membrane electrical excitability in Drosophila, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 6216-6220. 18 Shapiro, E,, Castellucci, V. F. and Kandel, E. R., Presynaptic membrane potential affects transmitter release in an identified neuron in Aplysia by modulating the Ca 2+ and K + currents, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 629-633. 19 Shimahara, T., Modulation of synaptic output by the transient outward potassium current in Aplysia. Neurosci. Lett., 24 (1981) 139-142. 20 Shimahara, T. and Tauc, L., Multiple interneuronal afferents to the giant cells in Aplysia, J. Physiol. (Lond.), 247 (1978) 229-319. 21 Shimahara, T. and Peretz, B., Soma potential of an interneurone controls transmitter release in a monosynaptic pathway in Aplysia, Nature. (Lond.), 273 (1978) 158-160. 22 Standen, N. B., Properties of calcium channel in snail neurones, Nature (Lond.), 250 (1974) 340-342. 23 Takeuchi, A. and Takeuchi, N., Electrical changes in pre- and postsynaptic axons of the giant synapse of Loligo, J. gen. Physiol., 45 (1962) 1181-1193. 24 Thompson, S. H., Three pharmacologically distinct potassium channels in molluscan neurones, J. Physiol. (Lond.), 265 (1977) 465-488.
51
Elsevier Biomedical Press
Presynaptic Modulation of Transmitter Release by the Early Outward Potassium Current in Aplysia T. SHIMAHARA Laboratoire de Neurobiologie Cellulaire, 91190 Gif sur Yvette (France)
(Accepted August 10th, 1982) Key words: transmitter release - - presynaptic modulation - - potassium current
The mechanism involved in presynaptic modulation of transmitter release was studied in an identified synapse of Aplysia californica.
Presynaptic hyperpolarization induces a decrease in the evoked postsynaptic potential amplitude zl. This is shown to be due to a reduction in the presynaptic spike amplitude during the hyperpolarization. The decreased presynaptic spike amplitude with hyperpolarization is explained as resulting from the superimposition of an early outward potassium current on the transient inward current. It is suggested that the presynaptic hyperpolarizing conditioning pulse decreases inactivation of the early outward current, which shunts the transient inward current. The superimposition of these two currents (transient inward current and the early outward current) induces a decrease in presynaptic spike amplitude, which in turn reduces the synaptic output from the terminal. INTRODUCTION The first step in the transmitter release process is an entry of Ca 2+ into the terminal during the spike potential. The synaptic efficacy (quantity of a released transmitter from the terminal) is, hence, determined by the quantity of Ca 2+ influx. At the neuromuscular junction, the synaptic efficacy as measured by the postsynaptic potential (PSP) amplitude is generally stable, since the presynaptic spike potential amplitude is constant. Thus, Ca 2+ influx is also constant. However the situation at the synapses in the central nervous system is more complicated, because a presynaptic terminal itself can receive other synaptic inputs which modify directly or indirectly its transmitter release. This presynaptic modulation of synaptic output might be an interesting basic model for a neural plasticity 11. It has been shown in a number of synapses that the synaptic output is sensitive to the presynaptic membrane potentialS,Z1, 23. In the squid giant synapse 23, presynaptic depolarization decreases the presynaptic spike potential resulting in a reduction of synaptic output, whereas presynaptic hyperpolarization has 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
the opposite effect. In the preparation, it has been suggested that the amount of transmitter released might be determined by the peak current rather than the absolute level of depolarization. In Aplysia synapses, however, presynaptic polarization has a reversed effect on synaptic output11,18, 21: a presynaptic hyperpolarization instead reduces the PSP amplitude. A similar phenomenon was also reported to occur in the leech ganglion 16. The present study concerns the paradoxical effect of presynaptic membrane potential on synaptic output. A preliminary report has been giveng, 19. MATERIALS AND METHODS All experiments were performed on the identified synapses in the cerebral ganglion and in the left pleural ganglion of Aplysia. The general properties of these synapses have already been described6, zo. The desheathed ganglion was fixed to the bottom of a lucite chamber (1 ml). The preparation was continuously perfused keeping the temperature constant at 18 °C. A double bareled microelectrode filled with 3 M KC1 was inserted into the post-
52 synaptic neuron for polarizing the cell and for recording the membrane potential. Two independent microelectrodes (filled with 3 M KC1) were inserted into the presynaptic neuron. In some cases, the presynaptic soma membrane was clamped using a conventional clamp amplifier. Total current was measured with a current to voltage converter (100 k ~ feedback resistor), which maintained the bath at ground potential. Physiological saline with the following composition was used (mM/l):460 NaCI, 10 KC1, 11 CaCle, 25 MgCI2, 28 MgSO4, 10 Tris. The p H was adjusted to 7.8 -t- 0.1. In some experiments 4-aminopyridine (4-AP) (Sigma) was added in order to block the outward potassium current.
C
-15
30 ¸
-10
RESULTS E
Previously we have shown at an identified synapse of Aplysia that the PSP amplitude is sensitive to the presynaptic soma potential 21. A presynaptic hyperpolarization induces a marked decrease in PSP amplitude (Fig. 1A). The relationship between PSP amplitude and the presynaptic soma membrane potential shows an S-shaped curve (Fig. 1B). A similar situation has previously been reported in other Aplysia ganglia is. It should be noted that the duration of conditioning hyperpolarizing is also an important factor in this modulation. In Fig. 1C, long-lasting hyperpolarizating conditioning pulses (--10 mV) of varying duration were imposed on the presynaptic neuron. The same neuron was stimulated with a short depolarizing pulse to evoke the spike before, during and after the hyperpolarizing pulse. As is evident in Fig. 1C, the effect of the conditioning pulse on PSP amplitude is not immediate as its m a x i m u m effect is established about 300 ms after the onset of the conditioning pulse. Following the conditioning pulse, PSP amplitude recovers gradually towards its original size with a time constant of 100 ms. In order to analyze this phenomenon, the presynaptic spike potential was characterized at different presynaptic membrane potentials. The amplitude of the presynaptic spike potential was measured on high speed sweep recordings using three different methods: (1) the absolute amplitude (from foot to peak); (2) the overshoot o f spike potential, and (3) f r o m the notch of the spike potential to the
=o k-
-,0o -~
-sb -rb -go -go -4b -go -~
-~
Presynaptic soma potetnt~ (mY)
1
post
pre
~
I
~
~
!
:
~
Fig. 1. The effect of presynaptic membrane potential on the postsynaptic potential amplitude. A: simultaneous recording from the postsynaptic neuron (middle traces) and the presynaptic neuron (bottom traces). The top traces indicate zero potential for the presynaptic neuron. The presynaptic membrane potential was polarized to 4 3 mV (a), 4 8 mV (b), --59 mV (c) and --63 mV (d). Calibration -- 4 mV for middle traces, 40 mV for lower traces, 5 ms. B: presynaptic spike potential (©- - -©) and postsynaptic potential (A - - A) at different presynaptic membrane potentials. C: the effect of hyperpolarizing conditioning pulse duration on synaptic output. Simultaneous recording from the postsynaptic neuron (upper traces) and the presynaptic neuron (bottom traces). Superimposition of 9 sweeps. The presynaptic neuron was hyperpolarized 10 mV from the resting potential for 500 ms. A short depolarizing pulse was applied to evoke the spike potential in the presynaptic neuron before, during and after the hyperpolarizing conditioning pulse. Calibration = 4 mV for upper traces, 40 mV for lower traces, 200 ms.
53
Fig. 2. The effect of 4-AP on the early outward potassium current, a: superimposed recordings show that the test pulse to --10 mV from the holding potential (--40 mV) induces a transient inward current. The same test pulse preceeded by 200 ms hyperpolarizing conditioning pulses (to --50 mV, --60 mV and --70 mV) also induces an early outward potassium current, b: superimposed current traces in response to a test pulse (10 ms) to --10 mV at various times after the onset of a hyperpolarizing conditioning pulse. c and d: superimposed recordings under the same conditions as in a (except that the test pulse is to 0 mV in d). c: control, d: after treatment with 4-AP (10 mM). Calibration = 200 nA, 100 mV, 10 ms. peak. In all cases, it was f o u n d that the presynaptic spike potential was sensitive to the presynaptic m e m b r a n e potential. Furthermore, the relationship between the presynaptic m e m b r a n e potential and the spike overshoot amplitude follows an S-shaped curve, similar to the changes in PSP amplitude (Fig. IB). It was also observed that the spike potential was sensitive to the duration o f the conditioning pulse, as was seen with the PSP. These similar effects suggest that the changes in the presynaptic spike might be responsible for the
modulation o f synaptic output. Even t h o u g h the spike potential was measured at the soma, we can suppose that similar p h e n o m e n a occur at the axon or at the terminal. A reduction in spike amplitude would decrease Ca e+ influx into the terminal, resulting in a decrease o f synaptic output. This hypothesis raises the question why the presynaptic spike potential decreases when the m e m b r a n e potential is hyperpolarized. One possible argument is that some peculiar ionic mechanism might be involved in the spike potential o f this neuron. Thus, the ionic move-
54 ments underlying the spike potential were examined at different membrane potentials under voltage clamp. In normal sea water, a test pulse to --10 mV from the holding potential (--40 mV) induces a transient inward current followed by a delayed outward current, as seen in many other excitable cells. However, the amplitude of the transient inward current decreases if a hyperpolarizing conditioning pulse precedes the test pulse (Fig. 2a). This decrease in the inward current following conditioning hyperpolarization was first described in the Aplysia neuron by Geduldig and Gruener 7. Other detailed studies of this phenomenon have been reported in
the snail neuron by Neher15 and Standen 22. The latter authors suggested that the decrease in the inward current was not a consequence of any peculiar properties of the inward current, but rather, resulted from a superimposition of the early outward potassium current on the inward current. The kinetic and pharmacological properties of this current are quite different from those of the familiar delayed outward potassium current. This current is inactivated at resting potential (about --40 mV), while a hyperpolarizing conditioning pulse removes the inactivation 15. Therefore, a test pulse preceded by a hyperpolarizing conditioning pulse induces the early outward current. Since its time course is similar to that
A
B
i
post
f
III
pre
Fig. 3. The effect of 4-AP on the modulation of synaptic output. A: simultaneous recording from pre and postsynaptic neurons. a-d, in normal sea water. Both spike potential and PSP amplitude decrease when the presynaptic potential is hyperpolarized, e-h, after treatment with 10 m M 4-AP. PSP amplitude is independent of the presynaptic membrane potential. Calibration = 4 mV for postsynaptic neuron, 40 mV for presynaptic neuron, 10 ms. B: the effect of 4-AP (10 mM) on time-dependent PSP modulation under the same conditions as shown in Fig. 1C. a, control in normal sea water; b, after treatment with 4-AP (10 mM) in bath. Calibration 4 mV for postsynaptic neuron, 40 mV for presynaptic neuron, 200 ms.
55 of the inward current, the superimposition of these two opposite currents results in a decrease of the transient inward current amplitude. Furthermore, activation of this current also depends on the duration of the conditioning pulse. As shown in Fig. 2b, if test pulse is maintained constant while the duration of the conditioning pulse is varied, the early outward peak current increases toward a steady state value with increasing conditioning duration. Conversely, the transient inward current decreases. Such a reduction of the inward current amplitude might be responsible for the decrease in spike amplitude following conditioning hyperpolarization, and thus, also for the depression of the PSP. In order to test this hypothesis, synaptic transmission was studied under the condition where the early outward current was blocked. This current is sensitive to externally applied 4-aminopyridine (4AP), while the delayed outward current is little affected. Fig. 2b illustrates the effect of 4-AP on the early outward current and on the inward current. Under 4-AP (10 mM) treatment, the early outward current is abolished. The most significant effect of 4AP on synaptic transmission is that PSP amplitude becomes quite independent of the presynaptic membrane potential (Fig. 3A). In other words, 4-AP completely blocks the presynaptic membrane potential-dependent modulation of synaptic output. As is evident in Fig. 3B, the time-dependent PSP modulation is also suppressed under 4-AP treatment. The above results lead us to the following conclusion. The presynaptic hyperpolarizing potential removes an inactivation of the early outward potassium current. This activation of the early outward current shunts the spike potentials, resulting in a depression of synaptic output from the terminal. DISCUSSION
The early outward potassium current was discovered first in an Onchidium neuron s, where a hyperpolarizing conditioning pulse induces the anode-break response. This same current has been found in many other various preparations1-4,10,14, 17,24. The kinetic and pharmacological properties of this current have been studied in detail in gastropod neurons 15,z4. The early outward current is generally inactivated at the resting potential. Therefore, it is
necessary to hyperpolarize the membrane in order to remove the inactivation. The kinetics of this current are similar to those of the transient inward current except that it has a long time constant of decay. Also, the activation of the early outward current is time dependent (Fig. 3b). The physiological role of the early outward current has been discussed in many different preparations. Some authors~ suggested that pacemaker activity in molluscan neurons is controlled by this current. The excitatory synaptic response in snail neurons is affected by a short circuit excitatory postsynaptic potential which is due to an activation of the early outward potassium current 4. Recently Salkoff et al. 17 have shown that facilitation at the Drosophila neuromuscular junction is due to an inactivation of the early outward current during repetitive firing. It has also been reported that in the non-spiking neuron of the crustacean coxal receptor, the transient outward potassium current shunts a transient inward current lz. However, this current is apparently not present at the squid giant synapsO2 where changes in the presynaptic membrane potential have an opposite effect: presynaptic depolarization induces a decrease in PSP amplitude, while presynaptic hyperpolarization increases it. The opposite effects of presynaptic membrane potential on synaptic output in Aplysia and the squid might be due to the fact that in the former, the early outward current exists, while in the latter it is absent 12. These results reveal that the transient outward potassium current has an important role in the modulation of synaptic output in Aplysia. However, the present observations do not exclude a possible involvement of other currents in the synaptic modulation. For example, the delayed outward potassium current may be involved, as has been shown in another system of Aplysia is. In Fig. IB, it can be seen that PSP amplitude is increased when the presynaptic potential is depolarized from --40 to --30 mV, suggesting that the late outward potassium current is less inactivated. This possibility is supported by the observation (not shown) of a broadened presynaptic spike with depolarization of the holding potential. Another possible explanation may lie in the reported inactivation of calcium current with hyperpolarizatioo. These effects are now under further study.
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