A calcium dependent post-tetanic hyperpolarization of primary afferent terminals

Pergamon Press

Life Sciences, Vol . 25, pp . 1179-1188 Printed in the U .S .A .

A CALCIUM DEPENDENT POST-TETANIC HYPERPOLARIZATION OF PRIMARY AFFERENT TERMINALS Bhagavatula R . Sastry Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO (Received in final form August 20, 1979)

Summa In decerebrated spinal cats, the effects of iontophoretically applied calcium antagonists, cobalt, manganese and verapamil, and of strontium, which reportedly can act like a calcium agonist, were tested on post-tetanic depression of group I afferent terminal excitability . The actions of these agents on the duration of action potentials in the afferent terminals were determined by a recently described method (8) . The calcium antagonists reduced the maximum post-tetanic depression of the antidromic compound action potentials and accelerated the recovery of these potentials from the depression . Strontium, on the other hand, had the opposite effects . The duration of afferent terminal action potentials appeared to increase following a tetanic stimulation . This enhancement in the duration of the action potentials was facilitated by strontium and counteracted by calcium antagonists . These observations indicate that calcium influx into primary afferent terminals is increased following a tetanic stimulation and that post-tetanic hyperpolarization of primary afferent terminals may be, at least partly, dependent on the increased accumulation of calcium in the terminals . It is generally thought that post-tetanic potentiation of the monosynaptic reflex is due to a hyperpolarization of the presynaptic terminals (1, 2, 3) . Although the mechanisms responsible for the post-tetanic hyperpolarization are not entirely clear, an increased activity of the sodium pump was suggested to be involved (4) . Evidence in the literature indicates that a calcium dependent hyperpolarization occurs in a variety of excitable cells (5, 6, 7) . It is, therefore, possible that at least part of the post-tetanic hyperpolarization of the primary afferent terminals is calcium dependent . Since recent observations in this laboratory indicate that an influx of calcium occurs during the action potential in primary afferent terminals (8), the present investigations were undertaken 1) to examine whether the influx of calcium into primary afferent terminals increases following a tetanic stimulation and 2) to determine whether post-tetanic hyperpolarization of the afferent terminals is dependent on an increased intra-terminal accumulation of the divalent cation . 0024-3205/79(131179-09$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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Methods These experiments were carried out on 12 cats of either sex weighing 2 .5-3 .8 kg . All animals were decerebrated at mid-collicular levels using halothane anaesthesia . The trachea was cannu lated and the animal artificially respired . The expiratory CO levels were measured and maintained at 4-4 .5% . The animal's blood pressure was monitored from the left carotid artery and the mean blood pressure was not less than 95 mm Hg . The lumbosacral spinal cord was exposed by laminectomy and the ventral roots from L to S were cut bilaterally . The dorsal roots were left intact and 5the 2 peripheral nerves leading to the following muscles in the limbs were sectioned peripherally and their central ends attached to bipolar platinum electrodes : posterior biceps-semitendinosus (PBST), medial gastrocnemius (MG), lateral gastrocnemius (LG) and quadriceps (QUAD) . The spinal cord was cut at T12 -L 1 level . Following surgery anaesthesia was discontinued . A seven barrel micropipette was positioned in the spinal cord such that the tip of the pipette lay in a motor nucleus . The procedures followed were described in a recent communication (8) . The central barrel of the micropipette, which contained 4 M NaCl, was used for stimulating in the motor nucleus to antidromically activate the afferents . The antidromic stimulation was monopolar and the ground reference electrode was placed in the animal's neck muscles . The antidromic compound action potentials were recorded from the peripheral nerves and were adjusted to less than 50% of their maximum size by decreasing the stimulus intensity . A constant current unit was used to minimize fluctuations in the stimulus intensity and currents were less than 5 uA (0 .3 msec square wave pulses of negative polarity) . In all experiments, antidromic stimulations were carried out at 0 .5 Hz . To tetanically evoke the antidromic compound action potentials, the fibres were stimulated at 50 Hz for 1 min and the recovery of the compound action potential followed . Intra-axonal recordings from single primary afferents and measurements of absolute refractory periods during the afferent terminal action potential as well as of the thresholds for antidromic activation of single fibres, were made as described in a recent report (8) . Briefly, the afferents were antidromically activated with twin pulses and the action potentials were recorded with a micropipette located in the axon . Absolute refractory periods were determined from the duration of refractoriness of the terminals to suprathreshold stimulations (about 30 times the threshold) with the second pulse . The effects of tetanic stimulations (50 Hz for 1 min) on the absolute refractory period and the threshold for antidromic stimulation were also examined . Each of the outer barrels of the seven barrel micropipette contained one of the following solutions : MnC1 2 (0 .05 M) ; CoC1 2 (0 .05 M) ; verapamil HC1 (0 .05 M in 165 MM NaCl) ; SrC1 2 (0 .05 M) ; ouabain octahydrate (0 .001 M in 165 mM NaCl) ; and NaCI (4 M) . The central barrel, used for antidromic stimulation of the afferents, contained 4 M NaCl . All agents in the outer barrels were held in the pipette by passing 10 nA "backing" currents . To eject the agents iontophoretically, currents were passed through the electrode barrels, the drug barrel being positive relative to the ground . Iontophoretic applications of the agents, as well as the "backing" currents used to "retain" the drugs, were neutralized by

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automatic current balancing such that the net current flow through the tip to the ground was zero . Curent effects were checked by applying equivalent ejections of Na through the outer NaCl barrel . In accepting data from these experiments the following criteria were strictly observed : 1) the variability of the threshold for antidromic activation of single fibres as well as of the size of the antidromic compound action potentials should be less than 5% when recorded over prolonged periods of time ; and 2) only those results were accepted in which a recovery from drug effects could be noticed . Results Data from all the peripheral nerves were pooled since no differences were noted between the results . In 12 experiments, following the tetanic stimulation the size of the antidromic compound action potentials was decreased to 10-30% of the control and the potentials gradually recovered to pre-tetanic magnitudes in 30-90 sec (figures 1 and 2) . Cobalt (25-180 nA), verapamil (40-180 nA, see figure 1) and manganese (30-200 nA) decreased the reduction in the size oT +the compound action potential+during the post-tetanic 9/12 experiments) . period (Co 9/12 ; verapamil : 8/12 ; Mn These agents reduced the maximum reduction in the post-tetanic size of the compound action potential by 10-158 and accelerated the recovery of the potential to pre-tetanic levels by 10-40 sec . Figure 1 depicts the effV~ts of verapamil (80 nA) on the QUAD compound Sr (40-150 nA) potentiated the maximum reducaction potential . tion in the size of the post-tetanic antidromic compound action potential by less than 108 and slowed the recovery of the potential by 15-50 sec (8/10 experiments ; see figure 2) . Pre-T ACAP

Post -T ACAP 7-15 sec

22-30 soc

FIG . 1

Effects of verapamil 80 nA) on the post-tetanic recovery of the QUAD antiCONTROL dromic compound action potential (ACAP) . The left column shows the pre-tetanic (Pre-T) ACAP . The middle and the right columns depict the post-tetanic (Post-T) ACAPs recorded 7-15 and 2230 sec following the termination .,~ '~ VE of a 1 min tetanic stimulation at 50 Hz, respectively . A illustrates the control ACAPs . The records in B were taken 10-108 sec following the cessation `1021tß/ of a 1 min iontophoretic apThe RECO'VERY plication of verapamil . pictures in C, which show a recovery from verapamil ef~R fects, were taken 320-418 sec following the termination of the drug injection . Each record shows an averaged response of four sweeps .

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Có+, Mn++ , Sr ++ and verapamil did not significantly alter the size of the pre-tetanic antidromic compound action potentials when applied with small ejection currents (<70 nA) but decreased their These efsizes (by 10-15%) when applied in greater quantities . fects of the drugs on the control compound action potential size could not be responsible for their actions on the post-tetanic recovery of the potential, since alterations in the stimulation intensity to decrease the sizes of the control compound action potential to those during drug applications did not result in a recovery similar to that observed after the drug application .

Z O U

aQ U a

FIG . 2 Prolongation of the post-tetanic 1pression of the PBST antidromic compound action potential by Sr (68 nA) . The compound action potentials prior to the drug application were expressed as a per cent of the + ire-drug control and the potentials after the application of Sr were expressed as a per cent of the post-drug control . The recovery +V the potential before (shown as control) and after (shown as Sr 68 nA) the drug administration, are illustrated . The recording of the post-drug compound action potentials was initiaVVd 10 sec following the termination of a 1 min application of Sr The X-axis shows the post-tetanic (P-T) time . In 5 experiments, the effects of ouabain (40-200 nA) were examined on the post-tetanic recovery of the antidromic compound action potential . This agent produced a weak reduction of the re covery time by 5-15 sec . The effects of a combined application of

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this agent and manganese were found to be additive . Moreover, Sr++ was still effective in prolonging the post-tetanic recovery time in the presence of ouabain . Therefore, it appears unlikely that the actions of the divalent cations were due to alterations in the sodium pump activity .

ARP

0

++

1

2

3

4

5

GOND INTERMkL 01M~C)

FIG . 3

The effects of Sr (75 nA) and Mn ++ (68 nA) on the threshold for antidromic activation of a LG group I afferent . The fibre was stimulated in the motor nucleus with twin pulses (conditioning in terval : 0 .5-5 msec) . The first stimulation was adequate to generate an action potential in each attempt . The second pulse was adjusted to find the threshold currents . If the fibre could not be stimulated with 30 times the control threshold currents, it was considered to be due to the absolute refractory period (8) . The conditioned thresholds were expressed as a per cent of the control threshold . The figure illustrates a prolongation of the absolute refractory peri~j (ARP, the horizontal bar) following a 40 sec application of S~+ (75 nA) and an antagonism of this effect by an ejection of Mn (applied for 1 min J0 sec) whose application was initiated 1 min prior to that of Sr + In this and the next figure the inset shows the action potential duration at the recording site .

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++ The actions of Sr (10-80 nA) were examined on the threshold -for antidromic activation and on the absolute refractory period of several single group I afferents . Satisfactory recordings were possible from a total of 45 fibres . The absolute refractory period was prolonged by the agent from 0 .8-1 .7 msec to 1 .8-4 .1 msec (38/45 fibres) . The post-absolúte refractory period threshold was slightly increased by the agent in 6 afferents, but this was not observed in the remaining fibres (see figures 3 and 4) . The control thresholl+was increased by 5-108 in 22/38 afferents, by these amounts of Sr The onset and duration of action of this agent on the single fibres were siT~lar to those on th$+antidromic compound action potentials . Mn (10-85 nA), in (10-90 nA) and Co amounts that did not significantly alter the control threshold for antidromic activation of the afferents, counteracted Oeprolongation of the absolut+refractory pgiod produced by Sr in most of the experiments (Mn 14/15 ; Co 16/18 afferj!~~ts) . These results are consistent with the possibility that Sr lits like calcium agonist . Figure 3 depicts the effects of Sr and Mn on the absolute refractory period of a LG group I afferent terminal .

4

Following the antidromic stimulation of 25 afferents at 50 Hz for 1 min, the thresholds were increased by 35-608 (figure 4A, control), the absolute refractory period was prolonged by 0 .4-1 .3 msec and the post-absolute refractory period thresholds decreased by 4080% (figure 4B) . The control threshold, the absolute refractory period and the post-absolute refractory period threshold recovered their pre-tetanic sizes with similar time courses (in 30-90 sec) . All the above 25 fibres that w$~e chosen for the post-tetanic changes were those on which Sr produced an increase in the pretetanic absolute refractory period . This divalent cation (25-80 nA), in amounts that produced a slight (<108) increase in the control threshold for antidromic stimulation, potentiated the effects of the tetanic stimulation on the thresholds, the absolute refractory period and the post-absolute refractory period thresholds . As shown in figure 4, the post-tetanic enhancement in the threshold was potentiated by 15-408 and the recovery of the threshold to pretetanic levels was prolonged by 25-60 sec . The increase in the absolute refractory period was potentiated by 1-4 msec and the post absolute refractory period threshold was decreased to 45-90% of the pre-tetanic threshold . As in+~he case of the compound action potentials, these actions of Sr were persistent for 3-7 min following the termination of the ejection of the agent and, therefore, the effects cli not be due to current arti4~ts . The calcium antagonists (Mn 15-90 nA, 8/10 fibres ; Co 20-80 nA, 11/13 afferents) produced a reduction in the enhancement of the post-tetanic threshold (by 10-30%), decreased the prolongation of the posttetanic absolute refractory period (by 0 .4-0 .8 msec) and also counteracted the reduction in the post-absolute refractory period threshold produced by the tetanic stimulation (by+5-25$, ~$e figure 4 A and D) . Th$+time course of action of Mn and Co was similar to that of Sr Discussion Post-tetanic potentiation of the monosynaptic reflex was first studied in detail by Lloyd (1) who showed that this reflex could be facilitated by a prior tetanus to the same afferents that evoked the reflex . Lloyd attributed this post-tetanic potentiation to changes in the presynaptic efficacy since tetanizing one group of

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A CONTROL

Sr++45nA

Co++4OnA

ARP

CONTROL

COND INTERVAL 5npc)

++

FIG . 4

Effects of Sr (45 nA) and Co + (40 nA) on the post-tetanic (post-T) prolongation of the absolute refractory period (ARP) in a PEST group I afferent terminal region . A-D were recorded from the same afferent fibre . A depicts (1) the effects of a 50 Hz antidromic stimulation for 1 min on the threshold for antidromic activation of th$+ fibre (control) ; (2) the actions of a 50 sec application of Sr (45 nA) on the post-T thruhold (the records were ejection) ; and (3) the taken 10 sec Uter the termination of Sr effects of Co (40 nA), which was applied for 50 sec and whose ejection was terminated 10 sec prior to the initiation of the recordings . The X-axis in A shows the post-T and pre-tetanic (pre-T) time in sec . The Y-axis illustrates the thresholds expressed as a In B, C and D, the per cent of the pre-T and the pre-drug control . ARP and the post-ARP thresholds were measured using the double pulse technique (see ref . 8),prior to (pre-T) and after the termination of (post-T) the tetanic stimulation . B, C and D illustrate on the the control ARPs (both pre+ +and post-T), the effects of Sr on the ARPs, respectively . The drug ARPs and the actions of Co application schedule in B-D was same as in A . Note that following the tetanic stimulation the unconditioned threshold was increased the ARP prolonged and the post-ARP thresholl decreased . While Sr counteracted them . potentiated these post-tetanic changes, Co

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afferents caused no potentiation of the monosynaptic reflex when other afferents synapsing on the same motoneurones were stimulated following the tetanus . These findings were later supported by recordings from single motoneurones which revealed that stimulations of one peripheral nerve produced a post-tetanic enhancement in the size of the excitatory postsynaptic potentials (EPSPs) in the homonymous motoneurones . However, when a nontetanized nerve from a synergistic muscle was stimulated during the above post-tetanic period, there was no potentiation of the EPSP (9) . Lloyd (1) proposed that tetanic stimulations of primary afferents cause a hyperpolarization of the afferent terminals which results in the production of larger action potentials in the ter minals . This enhancement in the size of the action potentials is generally thought to produce an increase in the release of transmitter substances (10, 11) . Antidromic stimulations of primary afferents at high frequencies result in a post-tetanic depression of the afferent terminal excitability (2) . This post-tetanic depression of the excitability was found to be greatest in the regions of afferent terminal arborizations and reportedly followed the same time course as the posttetanic potentiation of the monosynaptic reflex (2) . Eccles and Krnjevié (3) recorded action potentials and membrane potentials from the preterminal axons in the spinal cord . These authors found that following a tetanic stimulation of these afferents, the axon membrane potential was hyperpolarized and the size of the action potential increased . The post-tetanic depression of the afferent terminal excitability, therefore, appears to be due to a hyperpolarization of the afferent terminals . Findings in the present study clearly support the evidence in the literature that the afferent terminal excitability decreases following a tetanic stimulation . Calcium is known to contribute to action potentials in the dorsal root ganglion cells (12) and in primary afferent terminals (8) . This contribution of the divalent cation appears to be more pronounced at the terminal regions rather than at nonterminal regions of the axons (8) . In this connection it is interesting that the post-tetanic depression of the primary afferent terminal excitability is greater at the terminal regions rather than at the nonterminal regions (2) . Presuming that a depression of the afferent terminal excitability reflects a hyperpolarization of the terminals, it is possible that this post-tetanic hyperpolarization can be, at least partly, dependent on the accumulation of calcium in the terminals during the tetanic stimulation . Consistent with this idea, agents which are known to inhibit the transmembrane calcium fluxes in a variety of tissues (13, 14, 15, 16, 17, 18) accelerated and strontium, which is known to be able to substitute for calcium at many sites (19, 20, 21), slowed the recovery of the afferent terminal excitability from the post-tetanic depression (figures 1, 2 and 4) . The prolongation of the absolute refractory period in the afferent terminal regions following a tetanic stimulation indicates that the duration of the action potential increases during the post-tetanic period . This increase in the duration of the action potential is likely due to an influx of calcium since strontium potentiated and calcium antagonists counteracted the post-tetanic

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prolongation of the absolute refractory period . It is possible that an increased accumulation of intra-terminal calcium can also be due to a poor sequestration of the divalent cation and an inadequate calcium pumping to cope with the total influx of the cation per unit time . In conclusion, the results of the present investigations indicate that the duration of primary afferent terminal action potential increases during the post-tetanic period . This prolongation of the action potential appears to be due to an increased influx of calcium . Post-tetanic potentiation of the monosynaptic reflex can, therefore, be due to an increased availability of calcium in the terminals . The observations also indicate that the post-tetanic hyperpolarization of the afferent terminals is, at least partly, dependent on the enhancement of the intra-terminal calcium levels . Acknowledgements Dr . E .A . Dessouki participated in some of the experiments . Miss E .M . Evoy and Miss L .M . Hansen provided technical assistance . These studies were financially supported by the Canadian Medical Research Council . References 1 . D .P .C . LLOYD, J . gen . Physiol . 33 147-170 (1949) . 2 . P .D . WALL and A.R . JOHNSON y J . Neuro h siol . 21 148-188 (1958) . 3 . J .C . ECCLES and K . KRNJEVIC, J . P ys1o . Loner) 149 274-287 (1959) . 4 . D .B . McDOUGAL and L .A . OSBORN, J . Physiol . (Lond . ) 256 41-60 (1976) . 5 . K . KRNJEVI(f and A . LISIEWICZ, J . Physiol . (Lond . ) 225 363-390 (1972) . 6 . E .F . BARRETT and J .N . BARRETT, J . Physiol . (Lond . ) 255 737-774 . (1976) . 7 . R .W . MEECH and N .B . STANDEN, J. Physiol . (Loud . ) 249 211-239 (1975) . 8 . B .R . SASTRY, Life Sci . 24 2193-2200 (1979) . 9 . D .R . CURTIS ann J .C . ECCLES, J . Physiol . (tond . ) 150 374-398 (1960) . 10 . J .J . HUBBARD and W.D . WILLIS, Nature 193 1294-1295 (1962) . 11 . A . TAKEUCHI and N . TAKEUCHI, J . gen . Physiol . 45 1181-1193 (1962) . 12 . K . DUNLAP and G .D . FISCHBACH, Nature 276 837-839 (1978) . 13 . P .F . BAKER, H . MEVES and E .B . RIDGWAY,J . Physiol . (Lond . ) 231 511-526 (1973) . 14 . E . BULBRING and T . TOMITA, Proc . Roy . Soc . B. 17 2 121-136 (1969) . 15 . R . ECKERT and H .D . LUX, J . Physiol . (Loud .) 254 129-151 (1976) . Lond.r142 516-543 16 . P . FATT and B.L . GINSBORG, J . Physiol . (1958) . 17 . S . HAGIWARA and S . NAKAJIMA, J . en . Physiol . 49 793-806 (1966) . 18 . M . KOHLHARDT, B . BAUER, H . KRAUSE an A . FLECKENSTEIN, PflU ers Arch . es . Physiol . 388 115-123 (1973) . 19 . R. MILEDI, Nature -12(1966) . 20 . B . KATZ and R. MILEDI, J . Ph siol . (Lond .) 203 459-487 (1969) . 974) . 21 . R .W . MEECH, J . Physiol . Lon . - 77