J. Phy~iol. (1978), 275, pp. 303-319 With 11 text-figuree Printed in Great Britain
303
EFFECT OF REPETITIVE ACTIVATION ON THE AFTERHYPERPOLARIZATION IN DORSAL SPINOCEREBELLAR TRACT NEURONES
BY B. GUSTAFSSON AND P. ZANGGER* From the Department of Physiology, University of G6teborg, G6teborg, Sweden
(Received 30 March 1977) SUMMARY
1. The changes in the afterhyperpolarization (a.h.p.) with repetitive activation have been studied in dorsal spinocerebellar tract cells of the cat using intracellular recording techniques. 2. The a.h.p. following a single spike was conditioned at different interspike intervals by a single preceding spike. In the majority of neurones the a.h.p. following a spike added approximately linearly with that generated by a preceding spike. 3. In other cells the a.h.p. following a spike was instead depressed by a preceding spike. This depression was approximately constant at interspike intervals less than the a.h.p. duration (50-100 msec). Thereafter the a.h.p. slowly recovered during the next 100-300 msec. There was no associated decrease in the initial brief hyperpolarizing undershoot. 4. With shortlasting repetitive activation at high frequency (> 100 impulses/sec) the a.h.p. peak amplitude increased progressively with successive spikes (5-15 spikes). No change in the time constant of decay was observed. A good correspondence was found between the observed increase in peak amplitude of the a.h.p.s and that given by a theoretical linear superposition of the successive a.h.p.s. 5. Changes in the brief hyperpolarizing undershoot with repetitive activation is also described. INTRODUCTION
The afterhyperpolarization (a.h.p.) following the soma-dendritic spike has been shown to be of decisive importance for the regulation of repetitive firing in spinal motoneurones (Kernell, 1965; Kernell & Sjdholm, 1973; Baldissera & Gustafsson, 1974a, b). In a preceding paper (Gustafsson, Lindstrom & Takata, 1978a) the a.h.p. in cat dorsal spinocerebellar tract (DSCT) cells was analysed and a description of the time course of the conductance change underlying the a.h.p. following a single spike was given. For an understanding of the role played by the a.h.p. in firing regulation it is also necessary to know how the a.h.p.s of successive spikes interact. It has previously been demonstrated (Eide, Fedina, Jansen, Lundberg & Vyklicky, 1969; Kuno, Miyahara & Weakly, 1970) that the a.h.p. in DSCT neurones, at high frequency activation, increases with an increasing number of spikes. The aim of the * Present address: Institut de Physiologie, Universit6 de Fribourg, Perolles, 1700 Fribourg, Schweiz.
304 B. GUSTAFSSON AND P. ZANGGER present paper is to analyse this a.h.p. interaction in more detail. These results will be considered in a following paper (Gustafsson, Lindstrom & Zangger, 1978b) on the repetitive firing behaviour in DSCT neurones. Short preliminary reports of the present findings have been published (Gustafsson & Zangger, 1974; Gustafsson, 1974). METHODS
For description of the methods see Gustafsson, Lindstr6m & Takata (1978a). C
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200 300 Time (msec) Fig. 1. Increase in amplitude of the afterhyperpolarization (a.h.p.), conditioned by a preceding spike. A, the ratio between the peak amplitude of the conditioned and the test a.h.p. is plotted against the interspike interval. The results from two different DSCT neurones are shown (0, *). B, the distribution of the maximal ratios of a.h.p. peak amplitudes, obtained as in A, is shown for thirty-three DSCT neurones. Filled columns represent cells with an a.h.p. depression (see text). C, sample records from the cell illustrated by the filled circles in A. 100
RESULTS
I. Effects on the a.h.p. by a preceding conditioning spike In motoneurones, the a.h.p.s show summation and the time course of this summation between two spikes is not unlike that of the a.h.p. itself (Calvin & Schwindt, 1972; Baldissera & Gustafsson, 1974b). A quantitatively similar behaviour was found in the majority of the DSCT neurones where the interaction between two successive a.h.p.s was studied. This interaction between succeeding a.h.p.s was investigated by changing the interval between two antidromic volleys and measuring, from the initial baseline, the peak amplitude of the a.h.p. following the double volley relative to that following a single spike. In Fig. 1 A is shown for two DSCT neurones the ratio between the peak amplitudes (double/single) as a function of the interspike interval and in Fig. 1C a sample of the records from one of the cells is given. The a.h.p. following the double volley can be observed to be largest at short interspike intervals when the a.h.p. following the first spike is large, thereafter decreasing during its decay phase. At long interspike intervals, i.e. intervals exceeding the a.h.p. duration, the two a.h.p.s are practically the same. In twenty-four of the forty-
305 A.h.p. INTERACTION IN DSCT CELLS two neurones with this behaviour, the a.h.p. peak ratio, when measured at the shortest interspike interval tested (1-5-4 msec), varied between 1-7 and 2-6 (open squares in the histogram in B) with a mean value of 2*0 (S.D. 0.21), i.e. there was an approximate doubling of the a.h.p. amplitude. In sixteen neurones the a.h.p. interaction differed from that described above and the observations from three neurones are illustrated in Fig. 2A. It can be noted that at long interspike intervals, i.e. intervals exceeding the a.h.p. duration, there is a clear reduction in the a.h.p. following the double volley. This reaches a minimum around the end of the a.h.p. following a single spike and increases slowly thereafter A
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Fig. 2. A.h.p. depression by a preceding spike. A, the ratio between conditioned and test a.h.p. in three different DSCT neurones (O. *, x ) is plotted as in Fig. 1A. B, sample records from the cell represented by filled circles in the plot in A. Further details in the text.
with larger intervals between the two spikes. Only at short intervals is there a summation which is clearly smaller than that found in the other groups of neurone (see above). The minimal peak ratio (double/single a.h.p. peak amplitude) varied in different neurones between 0-4 and 0-85 with a mean value of 0 7. In nine of these neurones the peak ratio was also measured with an interspike interval below 4 msec. This ranged between 10 and 1 70 (filled squares in the histogram in Fig. 1 B) with a mean value of 1-35, i.e. considerably smaller than in the former group of cells. There was a clear tendency for cells with lower minimal peak ratio to have a smaller summation at short intervals and to cross the abscissa (1.0 ratio) sooner after the first spike than those with larger minimal peak ratio. It can be noted that the depressive effect of the first spike on the a.h.p. following both spikes considerably outlasts the duration of the first a.h.p. In the eight neurones where the whole recovery period was followed, the depression lasted 200-400 msec from the first spike. As a population, the neurones with this behaviour, henceforth referred to as neurones displaying a.h.p. depression, differed from the other DSCT neurones with respect to
B. GUSTAFSSON AND P. ZANGGER 306 the a.h.p. size. The a.h.p. peak amplitude in the forty-two neurones without depression varied between 0 8 and 3-1 mV (1.8 + 0 7 S.D.). On the other hand, in the sixteen neurones with depression it varied between 2-2 and 6-2 mV (4.1 + 1-3 S.D.). With respect to other parameters such as spike size, spike duration and amount of holding current (see Methods, Gustafsson et al. 1978a) there were no apparent differences between cells with and without depression. There were thus no indications that cells with depression were more damaged than the other cells. In fact, the majority of the cells with depression had spike sizes of more than 80 mV as measured from the holding potential and a spike duration of less than 0 5 msec, as measured to the peak of the undershoot (cf. Kuno & Miyahara, 1968; Eide et al. 1969). A
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Fig. 3. Comparison between depressed and control a.h.p. In A are shown superimposed tracings of a control (lower trace) and a depressed (upper trace) a.h.p. from the same cell. B, the calculated time course for the conductance process underlying the control (U) and depressed ( x ) a.h.p.s in A is plotted on 4 semilogarithmic scale. These time courses were estimated as described by Gustafsson et al. (1978 a). The resting input resistance and capacitance of the cell were 2 6 MD and 2 1 nF respectively. Note the parallel shift of the depressed curve as compared to the control, indicating a proportional reduction of the entire a.h.p. conductance. C, sample records from the cell used for the tracings in A; note in the upper record that the conditioned a.h.p. is reduced without a concomitant decrease in the initial brief hyperpolarizing undershoot; the single and double arrows in the lowermost record denote the peak of the delayed depolarization for the control and the depressed a.h.p.s respectively.
II. Depression Superimposed records of a test a.h.p. conditioned and unconditioned by a preceding spike are shown in Fig. 3A. It can be noted that in neurones with depression, the conditioned (or depressed) a.h.p. is smaller than the unconditioned one, not only at its peak level but also over the whole course from the peak of the delayed depolarization throughout the repolarizing phase. On the other hand, there were practically no changes in the peak level of the brief hyperpolarizing undershoot (Fig. 3C) or in the shape of the action potential (not illustrated). In a preceding
A.h.p. INTERACTION IN DSCT CELLS 307 paper the time course of the conductance change underlying the a.h.p. was determined by calculation from the a.h.p. voltage (Gustafsson et al. 1978a). The same procedure was adopted for five of the neurones with depression and the result from one of them is illustrated in Fig. 3B. The time course of the unconditioned a.h.p. is similar to that described for DSCT neurones in the previous paper (Gustafsson et al. 1978a), with a fast initial decay followed by a slower exponential decline. The calculated time course of the depressed a.h.p. follows roughly the same course as the unconditioned one, simply being displaced to lower values. Similar behaviour was observed in the other four neurones and this indicates that the depression is related to an overall reduction in the a.h.p. conductance. A
B
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Fig. 8. Comparison between theoretical and experimental a.h.p. amplitudes. A, the experimental peak a.h.p. amplitude, at a certain frequency of activation, is plotted on the ordinate against the a.h.p. amplitude expected on the basis of a linear summation (see text). x, cells without depression; *, cells with depression. The a.h.p. reversal level is assumed to be 15 mY below the holding potential. B, same data as in A but with the estimated reversal level 20 mV below the holding potential. The straight lines in A and B denote equivalence between the theoretical and the experimental values.
The above matching procedure was performed for fifteen neurones, ten of which had depression. Similar good correlations between the experimental and theoretical curves were found in several cells but some deviations in the course of the summation were found in others. To what extent these deviations were due to the simplifying assumptions in the theoretical calculations is not clear. However, as shown in Fig. 8A and B there was on the whole a good correspondence between the measured mnaximal a.h.p. increase and the theoretical values expected from a linear summation model. In the graphs the a.h.p. peak values attained at different frequencies are plotted against the theoretical values for each cell (cells without depression, crosses; cells with depression, closed circles). From the two graphs (Vahp = 15 and 20) it will be noted that the crosses and the filled circles are well concentrated along the line representing equivalence between the theoretical and experimental values. In other words, the maximal summated a.h.p. peak amplitude roughly corresponds to the value expected on the basis of a simple linear model. The time constant of the conductance decay during the a.h.p. following different
312 B. GUSTAFSSON AND P. ZANGGER numbers of spikes was measured in eleven neurones, either by plotting the a.h.p. voltage against time (after correction for changes in driving force) or by a calculation from the voltage. The result of this latter procedure is shown for one cell in Fig. 7 C, where the computed conductance after 1, 2, 5 and 10 spikes is plotted. The calculated a.h.p. conductance decayed exponentially with a roughly similar time constant in all cases, showing that the decay is not much affected by the number of spikes. In the other cells tested, similar results with exponential decays of roughly the same time constants, revealing no significant trend to increases or decreases with increasing spike number, were also found (spike numbers from 1-15). E
A
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100 200 300 ~~~~~1sec No. of spikes Fig. 9. A.h.p. change during a long train of spikes. A, the peak amplitude of the a.h.p. following a train of antidromic spikes with a frequency of 200/sec is plotted against the number of spikes in the train. The cell was activated by stimulation of the ipsilateral dorsal quadrant of the spinal cord at low thoracic level. B, sample records from the cell in A. C, the a.h.p. amplitude at the peak (@), at 200 msec (0) and at 400 msec ( x ) after the last spike in the train is plotted against the number of spikes as in A. The cell was activated antidromically at a frequency of 200/sec by stimulation of the anterior lobe of cerebellum. D, records from the cell in C; the baseline trace in each record was obtained by switching off the stimulator.
IV. Effect of longer jilke train A moderate increase in spike number after the a.h.p. summation has reached its plateau gave no further change in the a.h.p. in five of seven cells studied. As is illustrated in Fig. 9 from one of these neurones, up to 100 spikes could be given at a high frequency (200/sec) without any increase in a.h.p. peak amplitude or apparent change in a.h.p. time course. For the remaining two cells there were clear changes in the a.h.p. with increasing number of spikes, as illustrated in Fig. 9. Not only did the
313 A.h.p. INTERACTION IN DSCT CELLS a.h.p. peak amplitude (closed circles) continuously increase but there was also a prolongation of the a.h.p. duration. Measurements of the amplitudes 200 msec (open circles) and 400 msec (crosses) after the last spike in the train demonstrated a late component, which was absent with few spikes. This late component increased thereafter roughly in parallel with the increase of the a.h.p. peak amplitude with successive spikes. No attempts to analyse the mechanism behind this prolonged potential in these two cells were made. A
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Fig. 10. Post-tetanic depression in a DSCT neurone. A high frequency (200/sec) train of antidromic spikes, evoked by stimulation of the ipsilateral dorsal quadrant of the spinal cord, is inserted during a 'spontaneous' rhythmic discharge. A, the duration of the first interval after the train is plotted against the number of spikes in the train. B, sample records from the cell. Further details in the text. A similar prolongation of the a.h.p. associated with a peak amplitude increase was also observed in some cells activated repetitively by short constant current pulses applied intracellularly. It was, however, observed in later experiments that the longlasting phase of the a.h.p. obtained with this technique could be reversed in polarity by inversing the direction of the applied current and could also be produced with the electrode in the extracellular position.
A further indication of an increase in the a.h.p. in some cells with long spike trains was found when a high frequency train was inserted during a sustained lower frequency spontaneous discharge. In the records in Fig. 10 it can be observed that such a train causes a pause in the steady firing, the pause duration increasing with an increased duration of the train. In Fig. 10A, it is clear that this increase in duration occurs in two phases, a rapid initial one and a later slower one. The initial phase is completed within the first 10-20 spikes and is thus presumably due to the a.h.p. summation described in section III. The later, slower phase might be given by a more slowly developing hyperpolarization, as described above. Four additional cells were examined in this way with a 200 impulses/sec train with a duration up to 1 see applied during an ongoing spontaneous activity of 30-70 imp/sec. Only one of these cells showed a later slower phase (train evoked by short current pulses). Thus, as with the slowly developing hyperpolarization, this late post-tetanic depressive effect was only observed in some cells.
B. GUSTAFSSON AND P. ZANGGER
314
V. Undershoot changes during spike trains In DSCT neurones the spike is followed by a brief hyperpolarizing undershoot separated from the a.h.p. by the delayed depolarization. With repetitive activation there are changes not only in the a.h.p. amplitude but also in the amplitude of this initial undershoot. As earlier described by Kuno et al. (1970), with high frequency activation there is a decrease of the undershoot (Fig. 11 A). This decrease was frequency dependent, being large at the highest frequencies (400 and 200/sec) and small or even absent with lower frequencies (100 and 50/sec) of activation. At these lower frequencies there was often a slight increase in the undershoot from the first to the second spike (cf. Fig. I1 A3-A4), as if the undershoot summated with the a.h.p. A2
Al
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400/sec
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Fig. 11. Changes in the brief hyperpolarizing undershoot with repetitive activation and current injection. A, changes in the peak amplitude of the undershoot at different stimulation frequencies. There is a gradual decrease in amplitude of the undershoot in A, and A, but hardly any change at the lower frequencies in A3 and A4. In fact there is a small increase of the undershoot initially in A, and A,. In A. is illustrated the slow gradual decline following a longer spike train. B and C, postspike potentials during a train of antidromic impulses in two other DSCT neurones; C1 and C2 show the same response taken with two different sweep speeds. Observe in C2 that the peak of the undershoot decreases continuously during the train (arrows) and that the emerging hyperpolarizing peak is caused by the descending phase of the a.h.p., interrupted by the rising phase of the succeeding spike. D and E, effect of current injection on the membrane potential and the peak of the undershoot. D, a train of antidromic spikes were evoked during the displacement of the membrane potential in depolarizing (dpol.) and hyperpolarizing (hpol.) direction by a constant current pulse. E, the displacement of the membrane potential (@) and the peak of the undershoot, following the first (0) and the last spikes ( x ) in the train, is plotted on the ordinate against the amount of injected current on the abscissa. Note that the displacement of the undershoot is about 40 % of the membrane potential change.
A.h.p. INTERACTION IN DSCT CELLS 315 Kuno et al. (1970) described that the initial decrease of the undershoot with high frequency activation, after a short number of spikes, was replaced by an increase, so that the peak level eventually even exceeded the amplitude of the undershoot after the first spike. Such a behaviour was never observed in the present study. In some cells, however, changes in the potentials occurring during the train could give the illusion of such behaviour (Fig. 11 B-C). In these cases it was clear from records taken with a faster time base that the undershoot was continuously decreasing (Fig. 11C2, arrows) while the increasing hyperpolarizing peak was created by the descending phase of the a.h.p. interrupted by the rising phase of the succeeding spike. In motoneurones the absolute voltage level of the peak of the undershoot is practically unaffected by current injection, indicating a very high conductance at this point (Nelson & Burke, 1967). As illustrated from one DSCT neurone in Fig. liD, the undershoot peak could be substantially affected by current injected through the recording microelectrode. In five of nine neurones tested the displacement of the undershoot peak was 30-50% of the membrane potential change (giving an 'input resistance' of 1 2-2 5 MQ at the peak of the undershoot). In the remaining neurones the displacement was considerably smaller, being less than 10% of the membrane potential change ('input resistance' less than 0-2 MQ). This displacement of the undershoot peak with hyperpolarizing current seems incompatible with the notion that the undershoot is polarizing the membrane down to the potassium equilibrium potential (Kuno et al. 1970, see also Magherini, Precht & Schwindt, (1976). DISCUSSION
In many central neurones, including the DSCT cells, the afterhyperpolarization following the soma-dendritic spike increases in amplitude when preceded at short intervals by one or more spikes. This property of the a.h.p. has long been considered to be of decisive importance for the initial decrease in frequency of the rhythmic firing evoked by injection of depolarizing current into the cell. Direct experimental support for the idea that the a.h.p. increase is responsible for the initial neuronal adaptation has recently been obtained for spinal motoneurones (Kernell, 1972; Baldissera & Gustafsson, 1974b; Baldissera, Gustafsson, & Parmiggiani, 1977). As shown by the present study, the increase in the a.h.p. amplitude with successive spikes in DSCT neurones is well described as a linear superposition of 'identical' a.h.p.s evoked by each spike. This means that the a.h.p. kinetics in DSCT neurones can be represented by a relatively simple mathematical expression, consisting of an equation for the time course of the a.h.p. conductance following a single spike (Gustafsson et al. 1978a), combined with a linear summation. This expression will be used in a succeeding paper to model the firing behaviour of DSCT neurones (Gustafsson et al. 1978b). In these computations it is assumed that during its entire time course, including the initial fast decay phase, the a.h.p. is identical for each spike (or, in cells with a.h.p. depression, a constant fraction of the first a.h.p., see below). However, several factors (e.g. the possible influence of the neuronal geometry on the early phase (Gustafsson et al. 1978a), or changes in the delayed depolarization current during repetitive activation), restrained us from pursuing an experimental analysis on this early phase of the a.h.p. It should be pointed out that the linear
B. GUSTAFSSON AND P. ZANGGER superposition model for DSCT neurones differs from an earlier theoretical DSCT model (Wall0e, Jansen & Nygaard, 1969) in which the refractory increase with successive spikes was given by a prolongation of the time constant for the decay of the postspike refractory period. The linear superposition model of a.h.p. interaction is entirely descriptive, and we do not imply that this a.h.p. behaviour casts any light on the mechanism for the increase of the a.h.p. with repetitive activation. There are two principally different mechanisms that have been suggested to explain the a.h.p. increase, both of which have until recently lacked experimental support. Firstly, an a.h.p. enhancement could be due to an increase in the membrane area giving rise to the a.h.p. Such an effect could be caused by a more extensive dendritic invasion of the soma-dendritic spike with successive spikes (Calvin & Schwindt, 1972). Secondly, the a.h.p. potassium conductance in the affected membrane areas could be increased by succeeding spikes, thus giving larger a.h.p. amplitudes. This could happen if the a.h.p. conductance increases slowly with time so that only a fraction of the potassium conductance underlying the a.h.p. is activated by a single spike (Kernell & Sj6holm, 1972; Baldissera, Gustafsson & Parmiggiani, 1976). This latter mechanism has now been demonstrated in frog spinal motoneurones (Barret & Barret, 1976). For a step depolarization, the 'a.h.p.' amplitude in these neurones continued to increase with pulse duration, the maximal value not reached even after 50-100 msec. The characteristics of the a.h.p. summation with such a mechanism depend mainly on how the a.h.p. potassium conductance increases with time after a step depolarization, and how large a fraction of the maximal conductance increase is evoked by each spike. The linearity observed in DSCT neurones could be accounted for by operation on a roughly linear part of the relation between conductance and time. In cat spinal motoneurones the a.h.p. summation can deviate considerably from linearity, showing for instance a clear saturation with increasing spike number, i.e. a successive decrease in the net a.h.p. conductance contribution from each spike (Baldissera & Gustafsson, 1974b; Baldissera et al. 1977). This feature may well be related to operation on a nonlinear part of the relationship between the a.h.p. conductance increase and time, with a large fraction of the maximal a.h.p. conductance activated by the first few spikes. In DSCT neurones there were also some deviations from linearity suggesting a more complex mechanism. Moreover, due to the small a.h.p. amplitude, no detailed examination of the summation at longer intervals could be made. Deviations at these intervals, similar to those demonstrated in cat motoneurones could also be present in DSCT cells. An additional feature of the a.h.p. interaction in DSCT cells is the a.h.p. depression, i.e. the fact that the first a.h.p. can give rise to a decrease in the amplitude of the succeeding a.h.p.s. A similar depression is also seen in ventral spinocerebellar tract neurones and unidentified spinal neurones, possibly interneurones (Gustafsson, B., unpubl.). A decrease in the peak amplitude of the second a.h.p. has been reported in vestibulospinal neurones (Ito, Hongo, Yoshida, Okada & Obata, 1964) and rubrospinal neurones (Tsukahara, Toyama & Kosaka, 1967), and may also be present in pyramidal tract cells (cf. Calvin & Sypert, 1976, Fig. 5). In the rubrospinal neurones this decrease of the second a.h.p. is associated with a decreased membrane conductance, both as calculated from the a.h.p. voltage, and as measured with short 316
A.h.p. INTERACTION IN DSCT CELLS
317
current pulses applied through the intracellular micro-electrode (Hultborn, H., Murakami, F. & Tsukahara, N., in preparation). This latter finding supports the view that the depression is related to a decrease in the potassium conductance underlying the a.h.p. Descriptively, the depression seems to be a resetting of the a.h.p. amplitude following each spike to a new constant value. The resetting starts immediately after the first spike, is roughly constant throughout the duration of the first a.h.p. and disappears slowly thereafter if no new spikes appear. From the computed conductance curves the resetting involves the whole time course, including the initial fast decay and the later slower decline. The resetting did not involve the falling phase of the spike nor the overshoot. This selectivity shows that the potassium conductance underlying the a.h.p. in DSCT neurones is, as in frog spinal motoneurones (Barret & Barret, 1976), a separate conductance from the potassium conductance giving rise to the falling phase of the spike. The present study gives no clue regarding the possible mechanism underlying the depression. In motoneurones the a.h.p. is mediated by an influx of Ca2+ ions (Krnjevic, Puil & Werman, 1975; Barret & Barret, 1976). If this is also the case for DSCT neurones then the depression may be due to some factor affecting the kinetics of the calcium conductance, for example a longlasting Ca2+ conductance inactivation. Summation of the a.h.p. will give rise to an initial adaptation of an impulse train generated by a neurone in response to a steady depolarizing current (Kernell, 1972; Baldissera & Gustafsson, 1974b; Gustafsson et al. 1978b). This initial adaptation seems to be functionally appropriate in spinal motoneurones, matching the mechanical properties of the muscle cells (cf. Gustafsson et al. 1978 b). An a.h.p. summation with linear characteristics, in combination with an exponentially decaying a.h.p. conductance, leads to a linear steady state frequency-current relationship (see Gustafsson et al. 1978b). This would seem appropriate for a relay cell such as the DSCT neurone (Eide et al. 1969). The depression will cause a small or even negative initial adaptation (Gustafsson et al. 1978b), which would presumably be disadvantageous for the muscle contraction. A depression has never been observed in spinal or brain stem motoneurones (Ito & Oshima, 1962; Baker & Precht, 1972; Kitai, Tanaka, Tsukahara & Yu, 1972; Baldissera & Gustafsson, 1974b) whilst almost all other central neurones, studied in this respect, have displayed an a.h.p. depression (see above). This selectivity indicates that the depression is a physiological phenomenon and not an experimental artifact caused for example by excessive cell damage (cf. section II). A direct functional role for the depression is not easily envisaged. However, by reducing the adaptation, the depression will give a steeper steady state frequency-current relationship with a higher sensitivity of the neurones to incoming activity. Moreover, the initial negative adaptation might be important for the efficiency in the synaptic transmission to the target cells of neurones displaying a.h.p. depression. This work was supported by the Swedish Medical Research Council (Project no. 00094) and the Swiss National Foundation.
318
B. GUSTAFSSON AND P. ZANGGER REFERENCES
BAEER, R. & PRECHT, W. (1972). Electrophysiological properties of trochlear motoneurons as revealed by IVth nerve stimulation. Expl Brain Bee. 14, 127-157. BALDISSERA, F. & GUsTAFSSON, B. (1974a). Firing behaviour of a neurone model based on the afterhyperpolarization conductance time course. First interval firing. Acta physiol. 8cand. 91, 528-544.
BALDISSERA, F. & GUSTAFSSON, B. (1974b). Firing behaviour of a neurone model based on the afterhypolarization conductance time course and algebraic summation. Adaptation and steady state firing. Acta phyeiol. scand. 92, 27-47. BALDISSERA, F., GUSTAFSSON, B. & PARMIGGIANI, F. (1976). A model for refractoriness accumulation and 'secondary range' firing in spinal motoneurones. Biol. Cyberneticw. 24, 61-65. BALDISSERA, F., GUsTAFSSON, B. & PARMIGGIANI, F. (1977). Saturation of a.h.p. conductance summation as a mechanism for 'secondary range' firing in spinal motoneurones. Brain Res. (In the Press.) BARRET, E. F. & BARRET, J. N. (1976). Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones. J. Physiol. 255, 737-775. CALVIN, W. H. & SCHWINDT, P. C. (1972). Steps in production of motoneuron spikes during rhythmic firing. J. Neurophysiol. 35, 297-310. CALVIN, W. H. & SYPERT, G. W. (1976). Fast and slow pyramidal tract neurons: an intracellular analysis of their contrasting repetitive firing properties in the cat. J. Neurophyeiol. 39, 420-434. EIDE, E., FEDINA, L., JANSEN, J., LUNDBERG, A. & VYKLICKY, L. (1969). Properties of Clarke's column neurones. Aca physiol. scand. 77, 125-144. GUSTAFSSON, B. (1974). Afterhyperpolarization and the control of repetitive firing in spinal neurones of the cat. Acta physiol. scand. supply. 416. GusTAFssoN, B., LINDSTROM, S. & TAKATA, M. (1978a). Afterhyperpolarization mechanism in the dorsal spinocerebellar tract cells of the cat. J. Phyeiol. 275, 283-301. GusTAFssoN, B., LINDSTROM, S. & ZANGaER, P. (1978b). Firing behaviour of dorsal spinocerebellar tract neurones. J. Physiol. 275, 321-343. GusTAFssON, B. & ZANGGER, P. (1974). Depression of the afterhyperpolarization in dorsal spinocerebellar tract neurones. Brain Re8. 72, 320-322. ITO, M., HONGO, T., YOSHIDA, M., OKADA, Y. & OBATA, K. (1964). Antidromic and transsynaptic activation of Deiters' neurones induced from the spinal cord. Jap. J. Phyeiol 14, 638658.
ITO, M. & OSHIMA, T. (1962). Temporal summation of after-hyperpolarization following a motoneurone spike. Nature, Lond. 195, 910-911. KERNELL, D. (1965). The limits of firing frequency in cat lumbosacral motoneurones possessing different time course of afterhyperpolarization. Acta physiol. 8cand. 65, 87-100. KERNELL, D. (1972). The early phase of adaptation in repetitive impulse discharges of cat spinal motoneurones. Brain Me&. 41, 184-186. KERNELL, D. & SJOHOLM, H. (1973). Repetitive impulse firing: comparison between neurone models based on 'voltage clamp equations' and spinal motoneurones. Acta physiol. 8cand. 87, 40-56.
KITAI, S. T., TANAKA, T., TSUKAHARA, N. & Yu, H. (1972). The facial nucleus of the cat: antidromic and synaptic activation and peripheral nerve representation. Expl Brain Ree. 16, 161-183.
KRNJEVI6, K., PUIL, E. & WERMAN, R. (1975). Evidence for Ca2+-activated K+ conductance in cat spinal motoneurons from intracellular EGTA injections. Can. J. Physiol. Pharmac. 53, 1214-1218.
KUNO, M. & MIYAHARA, J. T. (1968). Factors responsible for multiple discharge of neurons in Clarke's column. J. Neurophyeiol. 31, 624-638. KUNO, M., MIYAHARA, J. T. & WEAKLY, J. N. (1970). Post-tetanic hyperpolarization produced by an electrogenic pump in dorsal spinocerebellar tract neurones of the cat. J. Physiol. 210, 839-855. MAGHERINI, P. C., PRECHT, W. & SCHWINDT, P. C. (1976). Electrical properties of frog motoneurons in the in situ spinal cord. J. Neurophysiol. 39, 459-473.
A.h.p. INTERACTION IN DSCT CELLS
319
NELSON, P. G. & BuIRKE, R. E. (1967). Delayed depolarization in cat spinal motoneurons. Expl Neurol. 17, 16-26. TsuKAHARA, N., TOYAMA, K. & KoSACA, K. (1967). Electrical activity of red nucleus neurones investigated with intracellular microelectrodes. Expl Brain Rea. 4, 18-33. WALLT0E, L., JANsEN, J. K. S. & NYGAARD, K. (1969). A computer simulated model of a second order sensory neurone. Kybernetik 6, 130-140.
303
EFFECT OF REPETITIVE ACTIVATION ON THE AFTERHYPERPOLARIZATION IN DORSAL SPINOCEREBELLAR TRACT NEURONES
BY B. GUSTAFSSON AND P. ZANGGER* From the Department of Physiology, University of G6teborg, G6teborg, Sweden
(Received 30 March 1977) SUMMARY
1. The changes in the afterhyperpolarization (a.h.p.) with repetitive activation have been studied in dorsal spinocerebellar tract cells of the cat using intracellular recording techniques. 2. The a.h.p. following a single spike was conditioned at different interspike intervals by a single preceding spike. In the majority of neurones the a.h.p. following a spike added approximately linearly with that generated by a preceding spike. 3. In other cells the a.h.p. following a spike was instead depressed by a preceding spike. This depression was approximately constant at interspike intervals less than the a.h.p. duration (50-100 msec). Thereafter the a.h.p. slowly recovered during the next 100-300 msec. There was no associated decrease in the initial brief hyperpolarizing undershoot. 4. With shortlasting repetitive activation at high frequency (> 100 impulses/sec) the a.h.p. peak amplitude increased progressively with successive spikes (5-15 spikes). No change in the time constant of decay was observed. A good correspondence was found between the observed increase in peak amplitude of the a.h.p.s and that given by a theoretical linear superposition of the successive a.h.p.s. 5. Changes in the brief hyperpolarizing undershoot with repetitive activation is also described. INTRODUCTION
The afterhyperpolarization (a.h.p.) following the soma-dendritic spike has been shown to be of decisive importance for the regulation of repetitive firing in spinal motoneurones (Kernell, 1965; Kernell & Sjdholm, 1973; Baldissera & Gustafsson, 1974a, b). In a preceding paper (Gustafsson, Lindstrom & Takata, 1978a) the a.h.p. in cat dorsal spinocerebellar tract (DSCT) cells was analysed and a description of the time course of the conductance change underlying the a.h.p. following a single spike was given. For an understanding of the role played by the a.h.p. in firing regulation it is also necessary to know how the a.h.p.s of successive spikes interact. It has previously been demonstrated (Eide, Fedina, Jansen, Lundberg & Vyklicky, 1969; Kuno, Miyahara & Weakly, 1970) that the a.h.p. in DSCT neurones, at high frequency activation, increases with an increasing number of spikes. The aim of the * Present address: Institut de Physiologie, Universit6 de Fribourg, Perolles, 1700 Fribourg, Schweiz.
304 B. GUSTAFSSON AND P. ZANGGER present paper is to analyse this a.h.p. interaction in more detail. These results will be considered in a following paper (Gustafsson, Lindstrom & Zangger, 1978b) on the repetitive firing behaviour in DSCT neurones. Short preliminary reports of the present findings have been published (Gustafsson & Zangger, 1974; Gustafsson, 1974). METHODS
For description of the methods see Gustafsson, Lindstr6m & Takata (1978a). C
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200 300 Time (msec) Fig. 1. Increase in amplitude of the afterhyperpolarization (a.h.p.), conditioned by a preceding spike. A, the ratio between the peak amplitude of the conditioned and the test a.h.p. is plotted against the interspike interval. The results from two different DSCT neurones are shown (0, *). B, the distribution of the maximal ratios of a.h.p. peak amplitudes, obtained as in A, is shown for thirty-three DSCT neurones. Filled columns represent cells with an a.h.p. depression (see text). C, sample records from the cell illustrated by the filled circles in A. 100
RESULTS
I. Effects on the a.h.p. by a preceding conditioning spike In motoneurones, the a.h.p.s show summation and the time course of this summation between two spikes is not unlike that of the a.h.p. itself (Calvin & Schwindt, 1972; Baldissera & Gustafsson, 1974b). A quantitatively similar behaviour was found in the majority of the DSCT neurones where the interaction between two successive a.h.p.s was studied. This interaction between succeeding a.h.p.s was investigated by changing the interval between two antidromic volleys and measuring, from the initial baseline, the peak amplitude of the a.h.p. following the double volley relative to that following a single spike. In Fig. 1 A is shown for two DSCT neurones the ratio between the peak amplitudes (double/single) as a function of the interspike interval and in Fig. 1C a sample of the records from one of the cells is given. The a.h.p. following the double volley can be observed to be largest at short interspike intervals when the a.h.p. following the first spike is large, thereafter decreasing during its decay phase. At long interspike intervals, i.e. intervals exceeding the a.h.p. duration, the two a.h.p.s are practically the same. In twenty-four of the forty-
305 A.h.p. INTERACTION IN DSCT CELLS two neurones with this behaviour, the a.h.p. peak ratio, when measured at the shortest interspike interval tested (1-5-4 msec), varied between 1-7 and 2-6 (open squares in the histogram in B) with a mean value of 2*0 (S.D. 0.21), i.e. there was an approximate doubling of the a.h.p. amplitude. In sixteen neurones the a.h.p. interaction differed from that described above and the observations from three neurones are illustrated in Fig. 2A. It can be noted that at long interspike intervals, i.e. intervals exceeding the a.h.p. duration, there is a clear reduction in the a.h.p. following the double volley. This reaches a minimum around the end of the a.h.p. following a single spike and increases slowly thereafter A
1-5 cvv 0)
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-10
I
200
100
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i
50 msec 400 msec
300
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0
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0
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00
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Fig. 2. A.h.p. depression by a preceding spike. A, the ratio between conditioned and test a.h.p. in three different DSCT neurones (O. *, x ) is plotted as in Fig. 1A. B, sample records from the cell represented by filled circles in the plot in A. Further details in the text.
with larger intervals between the two spikes. Only at short intervals is there a summation which is clearly smaller than that found in the other groups of neurone (see above). The minimal peak ratio (double/single a.h.p. peak amplitude) varied in different neurones between 0-4 and 0-85 with a mean value of 0 7. In nine of these neurones the peak ratio was also measured with an interspike interval below 4 msec. This ranged between 10 and 1 70 (filled squares in the histogram in Fig. 1 B) with a mean value of 1-35, i.e. considerably smaller than in the former group of cells. There was a clear tendency for cells with lower minimal peak ratio to have a smaller summation at short intervals and to cross the abscissa (1.0 ratio) sooner after the first spike than those with larger minimal peak ratio. It can be noted that the depressive effect of the first spike on the a.h.p. following both spikes considerably outlasts the duration of the first a.h.p. In the eight neurones where the whole recovery period was followed, the depression lasted 200-400 msec from the first spike. As a population, the neurones with this behaviour, henceforth referred to as neurones displaying a.h.p. depression, differed from the other DSCT neurones with respect to
B. GUSTAFSSON AND P. ZANGGER 306 the a.h.p. size. The a.h.p. peak amplitude in the forty-two neurones without depression varied between 0 8 and 3-1 mV (1.8 + 0 7 S.D.). On the other hand, in the sixteen neurones with depression it varied between 2-2 and 6-2 mV (4.1 + 1-3 S.D.). With respect to other parameters such as spike size, spike duration and amount of holding current (see Methods, Gustafsson et al. 1978a) there were no apparent differences between cells with and without depression. There were thus no indications that cells with depression were more damaged than the other cells. In fact, the majority of the cells with depression had spike sizes of more than 80 mV as measured from the holding potential and a spike duration of less than 0 5 msec, as measured to the peak of the undershoot (cf. Kuno & Miyahara, 1968; Eide et al. 1969). A
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Fig. 3. Comparison between depressed and control a.h.p. In A are shown superimposed tracings of a control (lower trace) and a depressed (upper trace) a.h.p. from the same cell. B, the calculated time course for the conductance process underlying the control (U) and depressed ( x ) a.h.p.s in A is plotted on 4 semilogarithmic scale. These time courses were estimated as described by Gustafsson et al. (1978 a). The resting input resistance and capacitance of the cell were 2 6 MD and 2 1 nF respectively. Note the parallel shift of the depressed curve as compared to the control, indicating a proportional reduction of the entire a.h.p. conductance. C, sample records from the cell used for the tracings in A; note in the upper record that the conditioned a.h.p. is reduced without a concomitant decrease in the initial brief hyperpolarizing undershoot; the single and double arrows in the lowermost record denote the peak of the delayed depolarization for the control and the depressed a.h.p.s respectively.
II. Depression Superimposed records of a test a.h.p. conditioned and unconditioned by a preceding spike are shown in Fig. 3A. It can be noted that in neurones with depression, the conditioned (or depressed) a.h.p. is smaller than the unconditioned one, not only at its peak level but also over the whole course from the peak of the delayed depolarization throughout the repolarizing phase. On the other hand, there were practically no changes in the peak level of the brief hyperpolarizing undershoot (Fig. 3C) or in the shape of the action potential (not illustrated). In a preceding
A.h.p. INTERACTION IN DSCT CELLS 307 paper the time course of the conductance change underlying the a.h.p. was determined by calculation from the a.h.p. voltage (Gustafsson et al. 1978a). The same procedure was adopted for five of the neurones with depression and the result from one of them is illustrated in Fig. 3B. The time course of the unconditioned a.h.p. is similar to that described for DSCT neurones in the previous paper (Gustafsson et al. 1978a), with a fast initial decay followed by a slower exponential decline. The calculated time course of the depressed a.h.p. follows roughly the same course as the unconditioned one, simply being displaced to lower values. Similar behaviour was observed in the other four neurones and this indicates that the depression is related to an overall reduction in the a.h.p. conductance. A
B
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Fig. 8. Comparison between theoretical and experimental a.h.p. amplitudes. A, the experimental peak a.h.p. amplitude, at a certain frequency of activation, is plotted on the ordinate against the a.h.p. amplitude expected on the basis of a linear summation (see text). x, cells without depression; *, cells with depression. The a.h.p. reversal level is assumed to be 15 mY below the holding potential. B, same data as in A but with the estimated reversal level 20 mV below the holding potential. The straight lines in A and B denote equivalence between the theoretical and the experimental values.
The above matching procedure was performed for fifteen neurones, ten of which had depression. Similar good correlations between the experimental and theoretical curves were found in several cells but some deviations in the course of the summation were found in others. To what extent these deviations were due to the simplifying assumptions in the theoretical calculations is not clear. However, as shown in Fig. 8A and B there was on the whole a good correspondence between the measured mnaximal a.h.p. increase and the theoretical values expected from a linear summation model. In the graphs the a.h.p. peak values attained at different frequencies are plotted against the theoretical values for each cell (cells without depression, crosses; cells with depression, closed circles). From the two graphs (Vahp = 15 and 20) it will be noted that the crosses and the filled circles are well concentrated along the line representing equivalence between the theoretical and experimental values. In other words, the maximal summated a.h.p. peak amplitude roughly corresponds to the value expected on the basis of a simple linear model. The time constant of the conductance decay during the a.h.p. following different
312 B. GUSTAFSSON AND P. ZANGGER numbers of spikes was measured in eleven neurones, either by plotting the a.h.p. voltage against time (after correction for changes in driving force) or by a calculation from the voltage. The result of this latter procedure is shown for one cell in Fig. 7 C, where the computed conductance after 1, 2, 5 and 10 spikes is plotted. The calculated a.h.p. conductance decayed exponentially with a roughly similar time constant in all cases, showing that the decay is not much affected by the number of spikes. In the other cells tested, similar results with exponential decays of roughly the same time constants, revealing no significant trend to increases or decreases with increasing spike number, were also found (spike numbers from 1-15). E
A
B
.
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100 200 300 ~~~~~1sec No. of spikes Fig. 9. A.h.p. change during a long train of spikes. A, the peak amplitude of the a.h.p. following a train of antidromic spikes with a frequency of 200/sec is plotted against the number of spikes in the train. The cell was activated by stimulation of the ipsilateral dorsal quadrant of the spinal cord at low thoracic level. B, sample records from the cell in A. C, the a.h.p. amplitude at the peak (@), at 200 msec (0) and at 400 msec ( x ) after the last spike in the train is plotted against the number of spikes as in A. The cell was activated antidromically at a frequency of 200/sec by stimulation of the anterior lobe of cerebellum. D, records from the cell in C; the baseline trace in each record was obtained by switching off the stimulator.
IV. Effect of longer jilke train A moderate increase in spike number after the a.h.p. summation has reached its plateau gave no further change in the a.h.p. in five of seven cells studied. As is illustrated in Fig. 9 from one of these neurones, up to 100 spikes could be given at a high frequency (200/sec) without any increase in a.h.p. peak amplitude or apparent change in a.h.p. time course. For the remaining two cells there were clear changes in the a.h.p. with increasing number of spikes, as illustrated in Fig. 9. Not only did the
313 A.h.p. INTERACTION IN DSCT CELLS a.h.p. peak amplitude (closed circles) continuously increase but there was also a prolongation of the a.h.p. duration. Measurements of the amplitudes 200 msec (open circles) and 400 msec (crosses) after the last spike in the train demonstrated a late component, which was absent with few spikes. This late component increased thereafter roughly in parallel with the increase of the a.h.p. peak amplitude with successive spikes. No attempts to analyse the mechanism behind this prolonged potential in these two cells were made. A
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Fig. 10. Post-tetanic depression in a DSCT neurone. A high frequency (200/sec) train of antidromic spikes, evoked by stimulation of the ipsilateral dorsal quadrant of the spinal cord, is inserted during a 'spontaneous' rhythmic discharge. A, the duration of the first interval after the train is plotted against the number of spikes in the train. B, sample records from the cell. Further details in the text. A similar prolongation of the a.h.p. associated with a peak amplitude increase was also observed in some cells activated repetitively by short constant current pulses applied intracellularly. It was, however, observed in later experiments that the longlasting phase of the a.h.p. obtained with this technique could be reversed in polarity by inversing the direction of the applied current and could also be produced with the electrode in the extracellular position.
A further indication of an increase in the a.h.p. in some cells with long spike trains was found when a high frequency train was inserted during a sustained lower frequency spontaneous discharge. In the records in Fig. 10 it can be observed that such a train causes a pause in the steady firing, the pause duration increasing with an increased duration of the train. In Fig. 10A, it is clear that this increase in duration occurs in two phases, a rapid initial one and a later slower one. The initial phase is completed within the first 10-20 spikes and is thus presumably due to the a.h.p. summation described in section III. The later, slower phase might be given by a more slowly developing hyperpolarization, as described above. Four additional cells were examined in this way with a 200 impulses/sec train with a duration up to 1 see applied during an ongoing spontaneous activity of 30-70 imp/sec. Only one of these cells showed a later slower phase (train evoked by short current pulses). Thus, as with the slowly developing hyperpolarization, this late post-tetanic depressive effect was only observed in some cells.
B. GUSTAFSSON AND P. ZANGGER
314
V. Undershoot changes during spike trains In DSCT neurones the spike is followed by a brief hyperpolarizing undershoot separated from the a.h.p. by the delayed depolarization. With repetitive activation there are changes not only in the a.h.p. amplitude but also in the amplitude of this initial undershoot. As earlier described by Kuno et al. (1970), with high frequency activation there is a decrease of the undershoot (Fig. 11 A). This decrease was frequency dependent, being large at the highest frequencies (400 and 200/sec) and small or even absent with lower frequencies (100 and 50/sec) of activation. At these lower frequencies there was often a slight increase in the undershoot from the first to the second spike (cf. Fig. I1 A3-A4), as if the undershoot summated with the a.h.p. A2
Al
A3
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400/sec
A4
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Fig. 11. Changes in the brief hyperpolarizing undershoot with repetitive activation and current injection. A, changes in the peak amplitude of the undershoot at different stimulation frequencies. There is a gradual decrease in amplitude of the undershoot in A, and A, but hardly any change at the lower frequencies in A3 and A4. In fact there is a small increase of the undershoot initially in A, and A,. In A. is illustrated the slow gradual decline following a longer spike train. B and C, postspike potentials during a train of antidromic impulses in two other DSCT neurones; C1 and C2 show the same response taken with two different sweep speeds. Observe in C2 that the peak of the undershoot decreases continuously during the train (arrows) and that the emerging hyperpolarizing peak is caused by the descending phase of the a.h.p., interrupted by the rising phase of the succeeding spike. D and E, effect of current injection on the membrane potential and the peak of the undershoot. D, a train of antidromic spikes were evoked during the displacement of the membrane potential in depolarizing (dpol.) and hyperpolarizing (hpol.) direction by a constant current pulse. E, the displacement of the membrane potential (@) and the peak of the undershoot, following the first (0) and the last spikes ( x ) in the train, is plotted on the ordinate against the amount of injected current on the abscissa. Note that the displacement of the undershoot is about 40 % of the membrane potential change.
A.h.p. INTERACTION IN DSCT CELLS 315 Kuno et al. (1970) described that the initial decrease of the undershoot with high frequency activation, after a short number of spikes, was replaced by an increase, so that the peak level eventually even exceeded the amplitude of the undershoot after the first spike. Such a behaviour was never observed in the present study. In some cells, however, changes in the potentials occurring during the train could give the illusion of such behaviour (Fig. 11 B-C). In these cases it was clear from records taken with a faster time base that the undershoot was continuously decreasing (Fig. 11C2, arrows) while the increasing hyperpolarizing peak was created by the descending phase of the a.h.p. interrupted by the rising phase of the succeeding spike. In motoneurones the absolute voltage level of the peak of the undershoot is practically unaffected by current injection, indicating a very high conductance at this point (Nelson & Burke, 1967). As illustrated from one DSCT neurone in Fig. liD, the undershoot peak could be substantially affected by current injected through the recording microelectrode. In five of nine neurones tested the displacement of the undershoot peak was 30-50% of the membrane potential change (giving an 'input resistance' of 1 2-2 5 MQ at the peak of the undershoot). In the remaining neurones the displacement was considerably smaller, being less than 10% of the membrane potential change ('input resistance' less than 0-2 MQ). This displacement of the undershoot peak with hyperpolarizing current seems incompatible with the notion that the undershoot is polarizing the membrane down to the potassium equilibrium potential (Kuno et al. 1970, see also Magherini, Precht & Schwindt, (1976). DISCUSSION
In many central neurones, including the DSCT cells, the afterhyperpolarization following the soma-dendritic spike increases in amplitude when preceded at short intervals by one or more spikes. This property of the a.h.p. has long been considered to be of decisive importance for the initial decrease in frequency of the rhythmic firing evoked by injection of depolarizing current into the cell. Direct experimental support for the idea that the a.h.p. increase is responsible for the initial neuronal adaptation has recently been obtained for spinal motoneurones (Kernell, 1972; Baldissera & Gustafsson, 1974b; Baldissera, Gustafsson, & Parmiggiani, 1977). As shown by the present study, the increase in the a.h.p. amplitude with successive spikes in DSCT neurones is well described as a linear superposition of 'identical' a.h.p.s evoked by each spike. This means that the a.h.p. kinetics in DSCT neurones can be represented by a relatively simple mathematical expression, consisting of an equation for the time course of the a.h.p. conductance following a single spike (Gustafsson et al. 1978a), combined with a linear summation. This expression will be used in a succeeding paper to model the firing behaviour of DSCT neurones (Gustafsson et al. 1978b). In these computations it is assumed that during its entire time course, including the initial fast decay phase, the a.h.p. is identical for each spike (or, in cells with a.h.p. depression, a constant fraction of the first a.h.p., see below). However, several factors (e.g. the possible influence of the neuronal geometry on the early phase (Gustafsson et al. 1978a), or changes in the delayed depolarization current during repetitive activation), restrained us from pursuing an experimental analysis on this early phase of the a.h.p. It should be pointed out that the linear
B. GUSTAFSSON AND P. ZANGGER superposition model for DSCT neurones differs from an earlier theoretical DSCT model (Wall0e, Jansen & Nygaard, 1969) in which the refractory increase with successive spikes was given by a prolongation of the time constant for the decay of the postspike refractory period. The linear superposition model of a.h.p. interaction is entirely descriptive, and we do not imply that this a.h.p. behaviour casts any light on the mechanism for the increase of the a.h.p. with repetitive activation. There are two principally different mechanisms that have been suggested to explain the a.h.p. increase, both of which have until recently lacked experimental support. Firstly, an a.h.p. enhancement could be due to an increase in the membrane area giving rise to the a.h.p. Such an effect could be caused by a more extensive dendritic invasion of the soma-dendritic spike with successive spikes (Calvin & Schwindt, 1972). Secondly, the a.h.p. potassium conductance in the affected membrane areas could be increased by succeeding spikes, thus giving larger a.h.p. amplitudes. This could happen if the a.h.p. conductance increases slowly with time so that only a fraction of the potassium conductance underlying the a.h.p. is activated by a single spike (Kernell & Sj6holm, 1972; Baldissera, Gustafsson & Parmiggiani, 1976). This latter mechanism has now been demonstrated in frog spinal motoneurones (Barret & Barret, 1976). For a step depolarization, the 'a.h.p.' amplitude in these neurones continued to increase with pulse duration, the maximal value not reached even after 50-100 msec. The characteristics of the a.h.p. summation with such a mechanism depend mainly on how the a.h.p. potassium conductance increases with time after a step depolarization, and how large a fraction of the maximal conductance increase is evoked by each spike. The linearity observed in DSCT neurones could be accounted for by operation on a roughly linear part of the relation between conductance and time. In cat spinal motoneurones the a.h.p. summation can deviate considerably from linearity, showing for instance a clear saturation with increasing spike number, i.e. a successive decrease in the net a.h.p. conductance contribution from each spike (Baldissera & Gustafsson, 1974b; Baldissera et al. 1977). This feature may well be related to operation on a nonlinear part of the relationship between the a.h.p. conductance increase and time, with a large fraction of the maximal a.h.p. conductance activated by the first few spikes. In DSCT neurones there were also some deviations from linearity suggesting a more complex mechanism. Moreover, due to the small a.h.p. amplitude, no detailed examination of the summation at longer intervals could be made. Deviations at these intervals, similar to those demonstrated in cat motoneurones could also be present in DSCT cells. An additional feature of the a.h.p. interaction in DSCT cells is the a.h.p. depression, i.e. the fact that the first a.h.p. can give rise to a decrease in the amplitude of the succeeding a.h.p.s. A similar depression is also seen in ventral spinocerebellar tract neurones and unidentified spinal neurones, possibly interneurones (Gustafsson, B., unpubl.). A decrease in the peak amplitude of the second a.h.p. has been reported in vestibulospinal neurones (Ito, Hongo, Yoshida, Okada & Obata, 1964) and rubrospinal neurones (Tsukahara, Toyama & Kosaka, 1967), and may also be present in pyramidal tract cells (cf. Calvin & Sypert, 1976, Fig. 5). In the rubrospinal neurones this decrease of the second a.h.p. is associated with a decreased membrane conductance, both as calculated from the a.h.p. voltage, and as measured with short 316
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current pulses applied through the intracellular micro-electrode (Hultborn, H., Murakami, F. & Tsukahara, N., in preparation). This latter finding supports the view that the depression is related to a decrease in the potassium conductance underlying the a.h.p. Descriptively, the depression seems to be a resetting of the a.h.p. amplitude following each spike to a new constant value. The resetting starts immediately after the first spike, is roughly constant throughout the duration of the first a.h.p. and disappears slowly thereafter if no new spikes appear. From the computed conductance curves the resetting involves the whole time course, including the initial fast decay and the later slower decline. The resetting did not involve the falling phase of the spike nor the overshoot. This selectivity shows that the potassium conductance underlying the a.h.p. in DSCT neurones is, as in frog spinal motoneurones (Barret & Barret, 1976), a separate conductance from the potassium conductance giving rise to the falling phase of the spike. The present study gives no clue regarding the possible mechanism underlying the depression. In motoneurones the a.h.p. is mediated by an influx of Ca2+ ions (Krnjevic, Puil & Werman, 1975; Barret & Barret, 1976). If this is also the case for DSCT neurones then the depression may be due to some factor affecting the kinetics of the calcium conductance, for example a longlasting Ca2+ conductance inactivation. Summation of the a.h.p. will give rise to an initial adaptation of an impulse train generated by a neurone in response to a steady depolarizing current (Kernell, 1972; Baldissera & Gustafsson, 1974b; Gustafsson et al. 1978b). This initial adaptation seems to be functionally appropriate in spinal motoneurones, matching the mechanical properties of the muscle cells (cf. Gustafsson et al. 1978 b). An a.h.p. summation with linear characteristics, in combination with an exponentially decaying a.h.p. conductance, leads to a linear steady state frequency-current relationship (see Gustafsson et al. 1978b). This would seem appropriate for a relay cell such as the DSCT neurone (Eide et al. 1969). The depression will cause a small or even negative initial adaptation (Gustafsson et al. 1978b), which would presumably be disadvantageous for the muscle contraction. A depression has never been observed in spinal or brain stem motoneurones (Ito & Oshima, 1962; Baker & Precht, 1972; Kitai, Tanaka, Tsukahara & Yu, 1972; Baldissera & Gustafsson, 1974b) whilst almost all other central neurones, studied in this respect, have displayed an a.h.p. depression (see above). This selectivity indicates that the depression is a physiological phenomenon and not an experimental artifact caused for example by excessive cell damage (cf. section II). A direct functional role for the depression is not easily envisaged. However, by reducing the adaptation, the depression will give a steeper steady state frequency-current relationship with a higher sensitivity of the neurones to incoming activity. Moreover, the initial negative adaptation might be important for the efficiency in the synaptic transmission to the target cells of neurones displaying a.h.p. depression. This work was supported by the Swedish Medical Research Council (Project no. 00094) and the Swiss National Foundation.
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B. GUSTAFSSON AND P. ZANGGER REFERENCES
BAEER, R. & PRECHT, W. (1972). Electrophysiological properties of trochlear motoneurons as revealed by IVth nerve stimulation. Expl Brain Bee. 14, 127-157. BALDISSERA, F. & GUsTAFSSON, B. (1974a). Firing behaviour of a neurone model based on the afterhyperpolarization conductance time course. First interval firing. Acta physiol. 8cand. 91, 528-544.
BALDISSERA, F. & GUSTAFSSON, B. (1974b). Firing behaviour of a neurone model based on the afterhypolarization conductance time course and algebraic summation. Adaptation and steady state firing. Acta phyeiol. scand. 92, 27-47. BALDISSERA, F., GUSTAFSSON, B. & PARMIGGIANI, F. (1976). A model for refractoriness accumulation and 'secondary range' firing in spinal motoneurones. Biol. Cyberneticw. 24, 61-65. BALDISSERA, F., GUsTAFSSON, B. & PARMIGGIANI, F. (1977). Saturation of a.h.p. conductance summation as a mechanism for 'secondary range' firing in spinal motoneurones. Brain Res. (In the Press.) BARRET, E. F. & BARRET, J. N. (1976). Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones. J. Physiol. 255, 737-775. CALVIN, W. H. & SCHWINDT, P. C. (1972). Steps in production of motoneuron spikes during rhythmic firing. J. Neurophysiol. 35, 297-310. CALVIN, W. H. & SYPERT, G. W. (1976). Fast and slow pyramidal tract neurons: an intracellular analysis of their contrasting repetitive firing properties in the cat. J. Neurophyeiol. 39, 420-434. EIDE, E., FEDINA, L., JANSEN, J., LUNDBERG, A. & VYKLICKY, L. (1969). Properties of Clarke's column neurones. Aca physiol. scand. 77, 125-144. GUSTAFSSON, B. (1974). Afterhyperpolarization and the control of repetitive firing in spinal neurones of the cat. Acta physiol. scand. supply. 416. GusTAFssoN, B., LINDSTROM, S. & TAKATA, M. (1978a). Afterhyperpolarization mechanism in the dorsal spinocerebellar tract cells of the cat. J. Phyeiol. 275, 283-301. GusTAFssoN, B., LINDSTROM, S. & ZANGaER, P. (1978b). Firing behaviour of dorsal spinocerebellar tract neurones. J. Physiol. 275, 321-343. GusTAFssON, B. & ZANGGER, P. (1974). Depression of the afterhyperpolarization in dorsal spinocerebellar tract neurones. Brain Re8. 72, 320-322. ITO, M., HONGO, T., YOSHIDA, M., OKADA, Y. & OBATA, K. (1964). Antidromic and transsynaptic activation of Deiters' neurones induced from the spinal cord. Jap. J. Phyeiol 14, 638658.
ITO, M. & OSHIMA, T. (1962). Temporal summation of after-hyperpolarization following a motoneurone spike. Nature, Lond. 195, 910-911. KERNELL, D. (1965). The limits of firing frequency in cat lumbosacral motoneurones possessing different time course of afterhyperpolarization. Acta physiol. 8cand. 65, 87-100. KERNELL, D. (1972). The early phase of adaptation in repetitive impulse discharges of cat spinal motoneurones. Brain Me&. 41, 184-186. KERNELL, D. & SJOHOLM, H. (1973). Repetitive impulse firing: comparison between neurone models based on 'voltage clamp equations' and spinal motoneurones. Acta physiol. 8cand. 87, 40-56.
KITAI, S. T., TANAKA, T., TSUKAHARA, N. & Yu, H. (1972). The facial nucleus of the cat: antidromic and synaptic activation and peripheral nerve representation. Expl Brain Ree. 16, 161-183.
KRNJEVI6, K., PUIL, E. & WERMAN, R. (1975). Evidence for Ca2+-activated K+ conductance in cat spinal motoneurons from intracellular EGTA injections. Can. J. Physiol. Pharmac. 53, 1214-1218.
KUNO, M. & MIYAHARA, J. T. (1968). Factors responsible for multiple discharge of neurons in Clarke's column. J. Neurophyeiol. 31, 624-638. KUNO, M., MIYAHARA, J. T. & WEAKLY, J. N. (1970). Post-tetanic hyperpolarization produced by an electrogenic pump in dorsal spinocerebellar tract neurones of the cat. J. Physiol. 210, 839-855. MAGHERINI, P. C., PRECHT, W. & SCHWINDT, P. C. (1976). Electrical properties of frog motoneurons in the in situ spinal cord. J. Neurophysiol. 39, 459-473.
A.h.p. INTERACTION IN DSCT CELLS
319
NELSON, P. G. & BuIRKE, R. E. (1967). Delayed depolarization in cat spinal motoneurons. Expl Neurol. 17, 16-26. TsuKAHARA, N., TOYAMA, K. & KoSACA, K. (1967). Electrical activity of red nucleus neurones investigated with intracellular microelectrodes. Expl Brain Rea. 4, 18-33. WALLT0E, L., JANsEN, J. K. S. & NYGAARD, K. (1969). A computer simulated model of a second order sensory neurone. Kybernetik 6, 130-140.
