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Mechanisms of presynaptic inhibition studied using paired-pulse facilitation
Neuroscience Letters, 126 (1991) 179-183
179
0 1991 Elsevier Scientific Publishers Ireland Ltd. 03043940/91/$03.50 ADONIS 030439409 100245H
NSL 07764
Mechanisms of presynaptic inhibition studied using paired-pulse facilitation G.J. Stuart and S.J. Redman Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra (Australia)
(Received 14 February 1991; Accepted 25 February 1991) Key worak
Presynaptic inhibition; Paired-pulse facilitation; Synaptic transmission; Spinal cord; EPSP
An investigation was made of the effect of presynaptic inhibition on paired-pulse facilitation (PPF) of group Ia afferent excitatory postsynaptic potentials (EPSPs). The main finding from this study was that PPF was enhanced during presynaptic inhibition of compound Ia EPSPs. This increase in PPF is identical to that seen at other synapses when the probability of transmitter release is decreased by lowering the extracellular calcium or raising the extracellular magnesium concentration, providing unequivocal evidence that presynaptic inhibition is associated with a decrease in the probability of transmitter release. Further, by analogy with the effects of reduced calcium influx on PPF at other synapses, the results support the idea that presynaptic inhibition is associated with reduced calcium influx into nerve terminals.
Frank and Fuortes [9] reported that in the mammalian spinal cord conditioning stimulation of certain muscle nerves could reduce the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) without causing a hyperpolarization or change in postsynaptic excitability. It was concluded that this inhibition occurred presynaptically. However, the conditioning stimulation was later shown to evoke postsynaptic inhibition [2, 51, and this led to considerable debate as to whether the reduction in EPSP amplitude occurs pre- or postsynaptically [6]. The best evidence that the amplitude of EPSPs is reduced by presynaptic rather than postsynaptic inhibition has come from a quanta1 analysis of fluctuations in amplitude of EPSPs before and after conditioning [4, 131. These results have shown that the conditioning stimulation decreased the number of quanta contributing to the inhibited EPSP without changing the quanta1 size. An alternative, simpler approach is to use paired-pulse facilitation (PPF), as this has proved to be a valuable tool for studying the mechanisms underlying synaptic transmission, particularly at the neuromuscular junction [3, 12, 16181. The aim of the present study was to investigate the effect of presynaptic inhibition on PPF of group Ia afferent EPSPs. The results indicate that PPF is enhanced during presynaptic inhibition, This increase in PPF is Correspondence: S.J. Redman, Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, 2601 Australia.
analogous to that seen at other synapses during reduced calcium influx into nerve terminals, supporting the idea that presynaptic inhibition is caused by a decrease in the probability of transmitter release due to reduced calcium influx into nerve terminals. All experiments were performed on adult cats anaesthetised with sodium pentobarbitone. Cats were initially anaesthetised by an intra-peritoneal injection (40 mg/kg) and the trachea, left common carotid artery and left cephalic vein were then cannulated and anaesthesia continued by intravenous injection (approximately 6 mg/h, or as required). The initial surgery, hindlimb dissection and laminectomy has been described previously [4]. The nerves to posterior biceps and semitendinosus (PBSt), medial gastrocnemius (MG) and lateral gastrocnemiussoleus (LGS) muscles in the left hindlimb were mounted on bipolar stimulating electrodes. Ventral roots Si, L7 and L6 on the left side of the spinal cord were cut and the Si and L7 ventral roots mounted on a stimulation electrode. Intracellular recordings were made from antidromitally identified motoneurones in the L& spinal segments using conventional glass microelectrodes filled with 2 M KCHsS04 (resting membrane potentials greater than -60 mV, spike height greater than 70 mV) and an Axoclamp 2A amplifier (Axon Instruments, U.S.A.). Compound Ia EPSPs were evoked in these motoneurones by stimulation of either the MG or LGS muscle nerves using a stimulus intensity supermaximal for activation of Ia afferents (usually 2-3 x group I
180
threshold, as determined from the cord dorsum recording). The use of supermaximal stimuli ensured that during PPF the second stimulus, evoked briefly after the first, was of sufficient strength to activate all Ia afferent fibres. In an attempt to reduce potential problems associated with nonlinear summation only small Ia EPSPs were used (225 mV in amplitude). Small Ia EPSPs were obtained by recording from motoneurones in the most rostra1 and caudal parts of the L7/St segments. PPF was evoked by recording the response to two stimuli separated by intervals of 24 ms. The amount of facilitation was expressed as the percentage increase in the peak amplitude of the second EPSP relative to the first EPSP. The protocol for evoking PPF is shown in Fig. 1. Single and paired EPSPs were evoked on alternate sweeps at 1 Hz, stored in separate buffers in a microcomputer and averaged. Offline, the response to the first stimulus alone (Fig. 1A) was digitally substracted from the response to the paired stimuli (Fig. 1B), leaving
t
B
A
I t
C
D
f
-_:::_:I::I:_, 1 mV 4
Fig.
I. Protocol used to evoke paired-pulse
recording
of the response
in a motoneurone
MG muscle nerve (arrow). muscle nerve (3 ms interval, mulus alone, obtained record
B. D: record
B: the response arrows).
following
facilitation.
second EPSP was increased
A: intracellular
to a single stimulus
of the
to paired stimuli of the MG
C: the response
digital subtraction
A and C shown
ms
together.
to the second stiof record
The amplitude
by 13% relative to the first.
A from of the
the response to the second stimulus alone (Fig. 1C). The peak amplitude of the first and second EPSPs were determined and the increase in the amplitude of the second EPSP relative to the first expressed as a percentage. The individual EPSPs evoked by the first and second stimuli during PPF have been plotted together in Fig. 1D and show that in this example the amplitude of second EPSP was increased by 13% relative to the first. Presynaptic inhibition of compound Ia EPSPs was evoked by prior conditioning stimulation of the PBSt muscle nerve. Conditioning PBSt stimulation was composed of a train of 2-14 stimuli at 300 Hz, 2 x group I threshold, initiated 50 ms prior to the first EPSP and repeated at one second intervals. The amount of presynaptic inhibition of a particular EPSP was altered by changing the number of stimuli applied to the PBSt muscle nerve [8]. Alternate records of conditioned and unconditioned EPSPs were stored in separate buffers in a microcomputer and averaged. As the conditioned EPSP was often superimposed on the repolarizing phase of an inhibitory postsynaptic potential (IPSP), a linear, sloping baseline was used in an attempt to overcome the distortion of the conditioned EPSP caused by the repolarizing IPSP. The amount of presynaptic inhibition is expressed as the percentage decrease in EPSP peak amplitude produced by the conditioning PBSt stimulation. The effect of presynaptic inhibition of PPF was determined by recording the response to alternate single and paired stimuli evoked 50 ms after the initiation of the conditioning PBSt stimulation. The response to the first stimulus was then substracted from the response during the paired stimuli. The peak amplitude of this second EPSP during presynaptic inhibition was then compared to the amplitude of the first EPSP during presynaptic inhibition. When a second EPSP was evoked briefly after the first, its amplitude was often increased. An example of this is shown in Fig. 1. Here, the amplitude of the second EPSP evoked by a second stimulus to the MG muscle nerve 3 ms after the first was increased by 13%. Facilitation of the second EPSP was not always observed and ranged from zero to 22 5”.The average percentage increase in the amplitude of the second EPSP during PPF was 1 1 + 2 % (+ S.E.M., n= 10; average amplitude of the first EPSP 3.4+ 0.3 mV). This average value for the amount of PPF of compound Ia EPSPs is identical to that obtained by Hirst, Redman and Wong [lo] for PPF of single fibre Ia EPSPs. The second EPSP usually decayed faster than the first (Fig. 1). The most likely explanation for this is that the paired stimulus activates polysynaptic inhibitory pathways which are active during the decay phase of the re-
181
sponse to the paired stimulus. If this inhibition is active during the peak of the second EPSP it could decrease the amplitude of this EPSP, decreasing the amount of PPF. However, this probably did not occur as the average amount of PPF of compound EPSPs was not significantly different from that found under similar experimental conditions using single fibre EPSPs [lo]. As single fibre EPSPs would not be expected to cause significant activation of polysynaptic inhibitory pathways, this suggests that polysynaptic inhibition has little or no effect on the amount of PPF of compound Ia EPSPs. The main observation from the present study was that during presynaptic inhibition PPF was enhanced. An example of this is shown in Fig. 2. In the absence of any presynaptic inhibition the second EPSP, evoked at a delay of 2 ms, was increased in amplitude by 11s (Fig. 2A). During presynaptic inhibition, which caused a 24% decrease in the amplitude of the first EPSP, the percentage increase in the amplitude of the second EPSP was 33% (Fig. 2B). Increasing the amount of presynaptic inhibition by increasing the number of conditioning stimuli applied to the PBSt muscle nerve caused an even greater increase in the amount of PPF of the second EPSP. Following a 44% decrease in the first EPSP the percentage increase in the second EPSP was 55% (Fig. 2C). The pooled data on the effect of presynaptic inhibition on PPF of different compound EPSPs are shown in Fig. 3. Fig. 3A shows a schematic diagram of PPF without (control) and during presynaptic inhibition, where the amplitude5 of the first and second EPSP are a and a’ in
the absence of presynaptic inhibition and b and b’ during presynaptic inhibition. The percentage change in the amplitude of the first EPSP during presynaptic inhibition is given by: (b/a-
1) x 100
Whereas the percentage increase in the second EPSP relative to the first during presynaptic inhibition is given by: (b’/b - 1) x 100
(2)
Fig. 3B plots the relationship between Eqn. 1 and Eqn. 2 and clearly shows that as the amplitude of the first EPSP is decreased during presynaptic inhibition the amount of PPF is increased, i.e. there is an inverse relationship between the level of presynaptic inhibition and the amount of PPF. At many synapses when the probability of transmitter release is decreased by lowering the extracellular calcium concentration or by raising the extracellular magnesium concentration, PPF is enhanced [7, 15-l 7, 19,2 11.Under these conditions calcium influx into presynaptic nerve terminals would be expected to be reduced. By analogy, the increase in PPF observed in the present study during presynaptic inhibition suggests that presynaptic inhibition causes a decrease in the probability of transmitter release and that this occurs following reduced calcium influx into nerve terminals. It is possible that the increase in PPF observed during
C
A
--I 4 Fig. 2. The effect of presynaptic inhibition on paired-pulse facilitation. A: in the absence of presynaptic by 11% relative to the first. B: a 24% decrease in the peak amplitude of the first EPSP during presynaptic
inhibition inhibition
in the second
presynaptic
EPSP of 33 %. C: a further
(1)
decrease
in the peak amplitude
of the first EPSP by 44% during
an even larger increase in the second EPSP of 55%.
1 mV
ms
the second EPSP is increased is associated with an increase inhibition
is associated
with
182
A PRESYNAPTIC INHIBITION
CONTROL
a’
a
[w.
_I!
,,
70 .
60.
50
tion of compound Ia EPSPs occurs infrequently and, if present, it can be removed by separating the EPSPs by only a few milliseconds [l]. A recent theoretical study also suggests that non-linear summation will have only a small effect on the amplitude of compound Ia EPSPs
t .
40 30
’
20
.
10
.
0
’ -60
.
-50
-40
-30
-20
* .
-10
0
% change in first EPSP during presynaptic inhibiiion = (b/a
- 1)x100
Fig. 3. Pooled data on the effect of presynaptic inhibition on pairedpulse facilitation (PPF). A: schematic diagram of PPF without (control) and during presynaptic inhibition. The amplitudes of the first and second EPSPs are a and a’ in the absence of presynaptic inhibition b and b’ during presynaptic inhibition. B: the relationship between the percentage change in the first EPSP during presynaptic inhibition, (/I/ a- 1) x 100, and the percentage increase in the second EPSP during PPF, (b’/b- 1) x 100.
presynaptic inhibition does not involve a direct interaction between PPF and presynaptic inhibition. For example, if the second EPSP sums non-linearly with the first EPSP during PPF, then it could be argued that a decrease in the amplitude of the first EPSP during presynaptic inhibition may reduce the amount of nonlinear summation of the second EPSP. This would result in an increase in PPF during presynaptic inhibition. However, the amount of nonlinear summation of the second EPSP is probably only small, as the voltage change that occurs in the dendrites during the first EPSP will decay rapidly back to the resting level in only a few milliseconds (i.e. its time course will be much briefer than that recorded at the soma). Under these conditions it is unlikely that nonlinear summation occurs unless the second EPSP is evoked almost simultaneously with the first EPSP [ll]. It has been previously shown [l] that nonlinear summa-
Another possible explanation for the increase in PPF during presynaptic inhibition is that it could be due to activation of polysynaptic pathways. The conditioning stimulation used to evoke presynaptic inhibition may have enhanced polysynaptic excitatory input or reduced inhibitory input to motoneurones and this could lead to an increase in the amplitude of the second EPSP during presynaptic inhibition and therefore an increase in PPF. However, this seems unlikely to have occurred as the time course of the second EPSP during presynaptic inhibition was not significantly different from the time course of the second EPSP in the absence of presynaptic inhibition. The 10 to 90% rise time and duration at half peak amplitude (half-width) of the second EPSP in the absence of presynaptic inhibition was 0.57 + 0.05 ms and 3.8 + 0.32 ms (+ S.E.M., n = lo), respectively, compared to 0.55 f 0.02 ms and 3.9 + 0.27 ms (n = 15) during different levels of presynaptic inhibition. (The percentage decrease in the first EPSP during presynaptic inhibition ranged from 8 to 53%). As the half-width of the second EPSP should be particularly sensitive to changes in the amount of polysynaptic excitation or inhibition, these results suggest that the conditioning stimulation used to evoke presynaptic inhibition does not significantly change the amount of polysynaptic input to motoneurones during the second EPSP. The evidence suggests that during PPF two opposing factors modulate the probability of transmitter release during the second response. Residual calcium increases the probability of release [12] and ‘synaptic depression’ decreases the probability of release [16, 21, 221. As the amount of synaptic depression has been shown to be directly correlated with the amount of transmitter released, synaptic depression has been attributed to depletion of the ‘immediately available store of releasable transmitter’ [14, 16, 21, 221. An increase in PPF could then occur if the probability of release is lowered during presynaptic inhibition as this would reduce synaptic depression, allowing increased facilitation of the second EPSP due to the presence of residual calcium. This explanation predicts an inverse relationship between the amount of PPF and the probability of release. An inverse relationship has been found experimentally by others as the probability of release is altered over a large range by changing the extracellular calcium or magnesium concentrations [7, 16, 17, 191. Similarly, in the present study an inverse relationship was found between the
183
amount of presynaptic inhibition and the amount of PPF (Fig. 3). In conclusion, the results from this study provide further evidence that presynaptic inhibition is associated with a reduction in the probability of transmitter release. By analogy with the effects of reduced calcium influx on PPF at other synapses, the increase in PPF observed during presynaptic inhibition supports the idea that the decrease in the probability of transmitter release that occurs during presynaptic inhibition is associated with reduced calcium influx into primary afferent nerve terminals We thank Garry Rodda for his excellent technical assistance, and Professor Peter Gage for reading the manuscript and providing useful comments.
1 Burke, R.E., Composite nature of the monosynaptic excitatory postsynaptic potential, J. Neurophysiol., 30 (1967) 11141136. 2 Carlen, P.L., Werman, R. and Yaari, Y., Post-synaptic conductance increase associated with presynaptic inhibition in cat lumbar motoneurones, J. Physiol., 298 (1980) 539-556. 3 Castillo, J. and Katz, B., Statistical factors involved in neuromuscular facilitation and depression, J. Physiol., 124 (1954) 574585. 4 Clements, J.D., Forsythe, I.D. and Redman, S.J., Presynaptic inhibition of synaptic potentials evoked in cat spinal motoneurones by impulses in single group Ia axons, J. Physiol., 383 (1987) 153-169. 5 Cook, W.A. and Cangiano, A., Presynaptic and postsynaptic inhibition of spinal motoneurones, J. Neurophysiol., 35 (1972) 389 403. 6 Davidoff, R.A. and Hackman, J.C., Spinal inhibition. In R.A. Davidoff (Ed.), Handbook of the Spinal Cord, Dekker, New York, 1984, pp. 385-460. 7 Dudel, J., Twin pulse facilitation in dependence on pulse duration and calcium concentration at motor nerve terminals of crayfish and frogs, Pfliigers Arch., 415 (1989) 3ltX-315.
8 Eccles, J.C., Eccles, R.M. and Magni, F., Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys, J. Physiol., 159 (1961) 147-166. 9 Frank, K. and Fuortes, M.G.F., Presynaptic and postsynaptic inhibition of monosynaptic reflexes, Fed. Proc., 16 (1957) 39-40. 10 Hirst, G.D.S., Redman, S.J. and Wong, K., Post-tetanic potentiation and facilitation of synaptic potentials evoked in cat spinal motoneurones, J. Physiol., 321 (1981) 97-109. 11 Iansek, R. and Redman, S.J., The amplitude, time course and charge of unitary excitatory post-synaptic potentials evoked in spinal motoneurone dendrites, J. Physiol., 234 (1973) 665-688. 12 Katz, B. and Miledi, R., The role of calcium in neuromuscular facilitation, J. Physiol., 195 (1968) 481492. 13 Kuno, M., Mechanism of facilitation and depression of the excitatory synaptic potential in spinal motoneurones, J. Physiol., 175 (1964) 10&l 12. 14 Kusano, K. and Landau, E.M., Depression and recovery of transmission at the squid giant synapse, J. Physiol., 245 (1975) 13-32. 15 Lundberg, A. and Quilisch, H., On the effect of calcium on presynaptic potentiation and depression at the neuro-muscular junction, Acta Physiol. Stand., Suppl. 111 (1953) 121-129. 16 Mallart, A. and Martin, A.R., The relation between quantum content and facilitation at the neuromuscular junction of the frog, J. Physiol., 196 (1968) 593-604. 17 Pamas, H., Dudel, J. and Parnas, I., Neurotransmitter release and its facilitation in crayfish. I. Saturation kinetics of release, and of entry and removal of calcium, Plhigers Arch., 393 (1982) l-14. 18 Parnas, H. and Segel, L.A., Facilitation as a tool to study the entry of calcium and the mechanism of neurotransmitter release, Prog. Neurobiol., 32 (1989) 1-9. 19 Rahamimoff, R., A dual effect of calcium ions on neuromuscular facilitation, J. Physiol., 195 (1968) 471-480. 20 Segev, I., Fleshman, J.W. and Burke, R.E., Computer simulation of group Ia EPSPs using morphologically realistic models of cat amotoneurones, J. Neurophysiol., 64 (1990) 648-660. 21 Thies, R.E., Neuromuscular depression and the apparent depletion of transmitter in mammalian muscle, J. Neurophysiol., 28 (1965) 42742. 22 Zucker, R.S., Short-term synaptic plasticity, Annu. Rev. Neurosci., 12 (1989) 13-31.
179
0 1991 Elsevier Scientific Publishers Ireland Ltd. 03043940/91/$03.50 ADONIS 030439409 100245H
NSL 07764
Mechanisms of presynaptic inhibition studied using paired-pulse facilitation G.J. Stuart and S.J. Redman Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra (Australia)
(Received 14 February 1991; Accepted 25 February 1991) Key worak
Presynaptic inhibition; Paired-pulse facilitation; Synaptic transmission; Spinal cord; EPSP
An investigation was made of the effect of presynaptic inhibition on paired-pulse facilitation (PPF) of group Ia afferent excitatory postsynaptic potentials (EPSPs). The main finding from this study was that PPF was enhanced during presynaptic inhibition of compound Ia EPSPs. This increase in PPF is identical to that seen at other synapses when the probability of transmitter release is decreased by lowering the extracellular calcium or raising the extracellular magnesium concentration, providing unequivocal evidence that presynaptic inhibition is associated with a decrease in the probability of transmitter release. Further, by analogy with the effects of reduced calcium influx on PPF at other synapses, the results support the idea that presynaptic inhibition is associated with reduced calcium influx into nerve terminals.
Frank and Fuortes [9] reported that in the mammalian spinal cord conditioning stimulation of certain muscle nerves could reduce the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) without causing a hyperpolarization or change in postsynaptic excitability. It was concluded that this inhibition occurred presynaptically. However, the conditioning stimulation was later shown to evoke postsynaptic inhibition [2, 51, and this led to considerable debate as to whether the reduction in EPSP amplitude occurs pre- or postsynaptically [6]. The best evidence that the amplitude of EPSPs is reduced by presynaptic rather than postsynaptic inhibition has come from a quanta1 analysis of fluctuations in amplitude of EPSPs before and after conditioning [4, 131. These results have shown that the conditioning stimulation decreased the number of quanta contributing to the inhibited EPSP without changing the quanta1 size. An alternative, simpler approach is to use paired-pulse facilitation (PPF), as this has proved to be a valuable tool for studying the mechanisms underlying synaptic transmission, particularly at the neuromuscular junction [3, 12, 16181. The aim of the present study was to investigate the effect of presynaptic inhibition on PPF of group Ia afferent EPSPs. The results indicate that PPF is enhanced during presynaptic inhibition, This increase in PPF is Correspondence: S.J. Redman, Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, 2601 Australia.
analogous to that seen at other synapses during reduced calcium influx into nerve terminals, supporting the idea that presynaptic inhibition is caused by a decrease in the probability of transmitter release due to reduced calcium influx into nerve terminals. All experiments were performed on adult cats anaesthetised with sodium pentobarbitone. Cats were initially anaesthetised by an intra-peritoneal injection (40 mg/kg) and the trachea, left common carotid artery and left cephalic vein were then cannulated and anaesthesia continued by intravenous injection (approximately 6 mg/h, or as required). The initial surgery, hindlimb dissection and laminectomy has been described previously [4]. The nerves to posterior biceps and semitendinosus (PBSt), medial gastrocnemius (MG) and lateral gastrocnemiussoleus (LGS) muscles in the left hindlimb were mounted on bipolar stimulating electrodes. Ventral roots Si, L7 and L6 on the left side of the spinal cord were cut and the Si and L7 ventral roots mounted on a stimulation electrode. Intracellular recordings were made from antidromitally identified motoneurones in the L& spinal segments using conventional glass microelectrodes filled with 2 M KCHsS04 (resting membrane potentials greater than -60 mV, spike height greater than 70 mV) and an Axoclamp 2A amplifier (Axon Instruments, U.S.A.). Compound Ia EPSPs were evoked in these motoneurones by stimulation of either the MG or LGS muscle nerves using a stimulus intensity supermaximal for activation of Ia afferents (usually 2-3 x group I
180
threshold, as determined from the cord dorsum recording). The use of supermaximal stimuli ensured that during PPF the second stimulus, evoked briefly after the first, was of sufficient strength to activate all Ia afferent fibres. In an attempt to reduce potential problems associated with nonlinear summation only small Ia EPSPs were used (225 mV in amplitude). Small Ia EPSPs were obtained by recording from motoneurones in the most rostra1 and caudal parts of the L7/St segments. PPF was evoked by recording the response to two stimuli separated by intervals of 24 ms. The amount of facilitation was expressed as the percentage increase in the peak amplitude of the second EPSP relative to the first EPSP. The protocol for evoking PPF is shown in Fig. 1. Single and paired EPSPs were evoked on alternate sweeps at 1 Hz, stored in separate buffers in a microcomputer and averaged. Offline, the response to the first stimulus alone (Fig. 1A) was digitally substracted from the response to the paired stimuli (Fig. 1B), leaving
t
B
A
I t
C
D
f
-_:::_:I::I:_, 1 mV 4
Fig.
I. Protocol used to evoke paired-pulse
recording
of the response
in a motoneurone
MG muscle nerve (arrow). muscle nerve (3 ms interval, mulus alone, obtained record
B. D: record
B: the response arrows).
following
facilitation.
second EPSP was increased
A: intracellular
to a single stimulus
of the
to paired stimuli of the MG
C: the response
digital subtraction
A and C shown
ms
together.
to the second stiof record
The amplitude
by 13% relative to the first.
A from of the
the response to the second stimulus alone (Fig. 1C). The peak amplitude of the first and second EPSPs were determined and the increase in the amplitude of the second EPSP relative to the first expressed as a percentage. The individual EPSPs evoked by the first and second stimuli during PPF have been plotted together in Fig. 1D and show that in this example the amplitude of second EPSP was increased by 13% relative to the first. Presynaptic inhibition of compound Ia EPSPs was evoked by prior conditioning stimulation of the PBSt muscle nerve. Conditioning PBSt stimulation was composed of a train of 2-14 stimuli at 300 Hz, 2 x group I threshold, initiated 50 ms prior to the first EPSP and repeated at one second intervals. The amount of presynaptic inhibition of a particular EPSP was altered by changing the number of stimuli applied to the PBSt muscle nerve [8]. Alternate records of conditioned and unconditioned EPSPs were stored in separate buffers in a microcomputer and averaged. As the conditioned EPSP was often superimposed on the repolarizing phase of an inhibitory postsynaptic potential (IPSP), a linear, sloping baseline was used in an attempt to overcome the distortion of the conditioned EPSP caused by the repolarizing IPSP. The amount of presynaptic inhibition is expressed as the percentage decrease in EPSP peak amplitude produced by the conditioning PBSt stimulation. The effect of presynaptic inhibition of PPF was determined by recording the response to alternate single and paired stimuli evoked 50 ms after the initiation of the conditioning PBSt stimulation. The response to the first stimulus was then substracted from the response during the paired stimuli. The peak amplitude of this second EPSP during presynaptic inhibition was then compared to the amplitude of the first EPSP during presynaptic inhibition. When a second EPSP was evoked briefly after the first, its amplitude was often increased. An example of this is shown in Fig. 1. Here, the amplitude of the second EPSP evoked by a second stimulus to the MG muscle nerve 3 ms after the first was increased by 13%. Facilitation of the second EPSP was not always observed and ranged from zero to 22 5”.The average percentage increase in the amplitude of the second EPSP during PPF was 1 1 + 2 % (+ S.E.M., n= 10; average amplitude of the first EPSP 3.4+ 0.3 mV). This average value for the amount of PPF of compound Ia EPSPs is identical to that obtained by Hirst, Redman and Wong [lo] for PPF of single fibre Ia EPSPs. The second EPSP usually decayed faster than the first (Fig. 1). The most likely explanation for this is that the paired stimulus activates polysynaptic inhibitory pathways which are active during the decay phase of the re-
181
sponse to the paired stimulus. If this inhibition is active during the peak of the second EPSP it could decrease the amplitude of this EPSP, decreasing the amount of PPF. However, this probably did not occur as the average amount of PPF of compound EPSPs was not significantly different from that found under similar experimental conditions using single fibre EPSPs [lo]. As single fibre EPSPs would not be expected to cause significant activation of polysynaptic inhibitory pathways, this suggests that polysynaptic inhibition has little or no effect on the amount of PPF of compound Ia EPSPs. The main observation from the present study was that during presynaptic inhibition PPF was enhanced. An example of this is shown in Fig. 2. In the absence of any presynaptic inhibition the second EPSP, evoked at a delay of 2 ms, was increased in amplitude by 11s (Fig. 2A). During presynaptic inhibition, which caused a 24% decrease in the amplitude of the first EPSP, the percentage increase in the amplitude of the second EPSP was 33% (Fig. 2B). Increasing the amount of presynaptic inhibition by increasing the number of conditioning stimuli applied to the PBSt muscle nerve caused an even greater increase in the amount of PPF of the second EPSP. Following a 44% decrease in the first EPSP the percentage increase in the second EPSP was 55% (Fig. 2C). The pooled data on the effect of presynaptic inhibition on PPF of different compound EPSPs are shown in Fig. 3. Fig. 3A shows a schematic diagram of PPF without (control) and during presynaptic inhibition, where the amplitude5 of the first and second EPSP are a and a’ in
the absence of presynaptic inhibition and b and b’ during presynaptic inhibition. The percentage change in the amplitude of the first EPSP during presynaptic inhibition is given by: (b/a-
1) x 100
Whereas the percentage increase in the second EPSP relative to the first during presynaptic inhibition is given by: (b’/b - 1) x 100
(2)
Fig. 3B plots the relationship between Eqn. 1 and Eqn. 2 and clearly shows that as the amplitude of the first EPSP is decreased during presynaptic inhibition the amount of PPF is increased, i.e. there is an inverse relationship between the level of presynaptic inhibition and the amount of PPF. At many synapses when the probability of transmitter release is decreased by lowering the extracellular calcium concentration or by raising the extracellular magnesium concentration, PPF is enhanced [7, 15-l 7, 19,2 11.Under these conditions calcium influx into presynaptic nerve terminals would be expected to be reduced. By analogy, the increase in PPF observed in the present study during presynaptic inhibition suggests that presynaptic inhibition causes a decrease in the probability of transmitter release and that this occurs following reduced calcium influx into nerve terminals. It is possible that the increase in PPF observed during
C
A
--I 4 Fig. 2. The effect of presynaptic inhibition on paired-pulse facilitation. A: in the absence of presynaptic by 11% relative to the first. B: a 24% decrease in the peak amplitude of the first EPSP during presynaptic
inhibition inhibition
in the second
presynaptic
EPSP of 33 %. C: a further
(1)
decrease
in the peak amplitude
of the first EPSP by 44% during
an even larger increase in the second EPSP of 55%.
1 mV
ms
the second EPSP is increased is associated with an increase inhibition
is associated
with
182
A PRESYNAPTIC INHIBITION
CONTROL
a’
a
[w.
_I!
,,
70 .
60.
50
tion of compound Ia EPSPs occurs infrequently and, if present, it can be removed by separating the EPSPs by only a few milliseconds [l]. A recent theoretical study also suggests that non-linear summation will have only a small effect on the amplitude of compound Ia EPSPs
t .
40 30
’
20
.
10
.
0
’ -60
.
-50
-40
-30
-20
* .
-10
0
% change in first EPSP during presynaptic inhibiiion = (b/a
- 1)x100
Fig. 3. Pooled data on the effect of presynaptic inhibition on pairedpulse facilitation (PPF). A: schematic diagram of PPF without (control) and during presynaptic inhibition. The amplitudes of the first and second EPSPs are a and a’ in the absence of presynaptic inhibition b and b’ during presynaptic inhibition. B: the relationship between the percentage change in the first EPSP during presynaptic inhibition, (/I/ a- 1) x 100, and the percentage increase in the second EPSP during PPF, (b’/b- 1) x 100.
presynaptic inhibition does not involve a direct interaction between PPF and presynaptic inhibition. For example, if the second EPSP sums non-linearly with the first EPSP during PPF, then it could be argued that a decrease in the amplitude of the first EPSP during presynaptic inhibition may reduce the amount of nonlinear summation of the second EPSP. This would result in an increase in PPF during presynaptic inhibition. However, the amount of nonlinear summation of the second EPSP is probably only small, as the voltage change that occurs in the dendrites during the first EPSP will decay rapidly back to the resting level in only a few milliseconds (i.e. its time course will be much briefer than that recorded at the soma). Under these conditions it is unlikely that nonlinear summation occurs unless the second EPSP is evoked almost simultaneously with the first EPSP [ll]. It has been previously shown [l] that nonlinear summa-
Another possible explanation for the increase in PPF during presynaptic inhibition is that it could be due to activation of polysynaptic pathways. The conditioning stimulation used to evoke presynaptic inhibition may have enhanced polysynaptic excitatory input or reduced inhibitory input to motoneurones and this could lead to an increase in the amplitude of the second EPSP during presynaptic inhibition and therefore an increase in PPF. However, this seems unlikely to have occurred as the time course of the second EPSP during presynaptic inhibition was not significantly different from the time course of the second EPSP in the absence of presynaptic inhibition. The 10 to 90% rise time and duration at half peak amplitude (half-width) of the second EPSP in the absence of presynaptic inhibition was 0.57 + 0.05 ms and 3.8 + 0.32 ms (+ S.E.M., n = lo), respectively, compared to 0.55 f 0.02 ms and 3.9 + 0.27 ms (n = 15) during different levels of presynaptic inhibition. (The percentage decrease in the first EPSP during presynaptic inhibition ranged from 8 to 53%). As the half-width of the second EPSP should be particularly sensitive to changes in the amount of polysynaptic excitation or inhibition, these results suggest that the conditioning stimulation used to evoke presynaptic inhibition does not significantly change the amount of polysynaptic input to motoneurones during the second EPSP. The evidence suggests that during PPF two opposing factors modulate the probability of transmitter release during the second response. Residual calcium increases the probability of release [12] and ‘synaptic depression’ decreases the probability of release [16, 21, 221. As the amount of synaptic depression has been shown to be directly correlated with the amount of transmitter released, synaptic depression has been attributed to depletion of the ‘immediately available store of releasable transmitter’ [14, 16, 21, 221. An increase in PPF could then occur if the probability of release is lowered during presynaptic inhibition as this would reduce synaptic depression, allowing increased facilitation of the second EPSP due to the presence of residual calcium. This explanation predicts an inverse relationship between the amount of PPF and the probability of release. An inverse relationship has been found experimentally by others as the probability of release is altered over a large range by changing the extracellular calcium or magnesium concentrations [7, 16, 17, 191. Similarly, in the present study an inverse relationship was found between the
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amount of presynaptic inhibition and the amount of PPF (Fig. 3). In conclusion, the results from this study provide further evidence that presynaptic inhibition is associated with a reduction in the probability of transmitter release. By analogy with the effects of reduced calcium influx on PPF at other synapses, the increase in PPF observed during presynaptic inhibition supports the idea that the decrease in the probability of transmitter release that occurs during presynaptic inhibition is associated with reduced calcium influx into primary afferent nerve terminals We thank Garry Rodda for his excellent technical assistance, and Professor Peter Gage for reading the manuscript and providing useful comments.
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