Accommodation of cat spinal motoneurones to linearly rising currents before and during long-term changes of membrane potential

BraOt Research, 76 (1974) 213-221

213

~,~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Research Reports

A C C O M M O D A T I O N OF CAT SPINAL M O T O N E U R O N E S TO L I N E A R L Y R I S I N G C U R R E N T S BEFORE A N D D U R I N G L O N G - T E R M C H A N G E S OF MEMBRANE POTENTIAL

W. R. SCHLUE*, D. W. RICHTER**, K. H. MAURITZ*** ANDA. C. NACIM1ENTO§ Physiological Institute, Central Nervous System Research Unit, Saarland University, 665 HomburgSaar (G.F.R.)

(Accepted February 21st, 1974)

SUMMARY The rates of accommodation of slowly accommodating a-motoneurones of cats remain unchanged as long as the membrane potential (MP) remains constant. The rates increase during long-term artificially imposed MP depolarisation and decrease during hyperpolarisation. The decrease of the maximum rate of rise of the action potential (VAB) with change in the action potential latency is greater during a depolarising and less during a hyperpolarising shift of the MP, as compared with the relationship found at the resting MP. The current-voltage curves are linear. Linearity is preserved and the slopes of the curves are unchanged during all MP displacements. The relationships between VaB and membrane potential before and during long-term changes in membrane potential are unchanged ; they are not shifted along the potential axis. The results suggest that different states of the sodium-carrying system alone account for the dependence of rate of accommodation upon MP.

INTRODUCTION The accommodative properties of a single motoneurone in the cat spinal cord, as a rule, remain unchanged as long as there are no spontaneous or artificially imposed changes in the membrane potential az. However, when the membrane potential Present addresses: * Fachbereich Biologie, Universit/it Konstanz, G.F.R. ** Physiologisches lnstitut der Universitfit Miinchen, G.F.R. *** Abt. Physiol. Psychologie Universit/it Konstanz, G.F.R. § To whom reprint requests should be sent.

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is artificially shifted, the rates of accommodation are altered 1~. A similar dependence of the rate of accommodation upon membrane potential is also found in pyramidal tract ceils'), ``}. It has been suggested that changes in sodium and potassium conductance may be responsible for this dependence ",~. The present paper describes experiments designed to determine, for slowly accommodating motoneurones, whether one or both of these conductance changes underly the dependence of accommodation rate upon membrane potential. MATERIALS AND METHODS

Preparation Adult cats were anaesthetised with pentobarbital (Nembutalf(t) 35 mg/kg, i.p.); further injections (2.5 mg/kg) were given as necessary. They were spinalised at the Th 12 level, the cord was exposed from S 1 to L V, and the dorsal roots were sectioned from S I to L VI. The lateral and medial Nn. gastrocnemii were exposed on one side. The animals were immobilised with gallamine triethiodide (Flaxedil:B:) and respirated artificially. Bilateral pneumothorax was performed and the blood pressure was measured continuously. The CO2 concentration in the expired air was monitored (3-4~;, end-tidal volume). Body temperature was kept at a constant level (38 2} 0.5 °C). Stimulation and recording The exposed nerves were stimulated in order to excite the motoneurones antidromically. Only a-motoneurones, were studied in detail. Intracellular recordings were obtained with single glass microelectrodes filled with 2M potassium citrate (5-10 M~2 in 0 . 9 ~ NaCI solution). Potentials were measured with a feedback amplifierlL Intracellular square-wave or linearly rising (ramp) currents were injected through the recording electrode and an appropriate bridge circuit. These electrodes were selected for a linear current-voltage relationship in the current range required for the measurements. Constant current stimulation was maintained by the feedback amplifier. Action potentials were differentiated by an operational amplifier (time constant 0.1 msec). Measurements began with the recording of an antidromic action potential and its time derivative. This measurement was repeated regularly throughout the experiment. Next, several determinations of the rheobase were made using intracellularly applied square-wave currents. Finally, the threshold-latency (TL) cur~,e was measured by applying ramp currents with various rates of rise. Measurement of complete TL curves required only a few minutes. Following these measurements, rheobases were determined again. The membrane potential was altered by intracellularly applied DC. For spikes resulting from current ramps, the maximum rate o f rise o f the ABspike 5 will be termed "~aB. To measure the dependence of 9AB upon membrane potential {~)ABcurve), the membrane potential was changed prior to the test stimulus

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by depolarising or hyperpolarising square-wave c o n d i t i o n i n g currents (duration I00 msec). Each of these c o n d i t i o n i n g currents was followed immediately by a test pulse of 50/~sec duration, which caused an action potential. ~/AB of this action potential was determined and plotted against the m e m b r a n e potential existing at the t e r m i n a t i o n of the c o n d i t i o n i n g current. In order to determine c u r r e n t - v o l t a g e curves, long (50-60 msec) square-wave currents were applied. The electrotonic potentials were measured at the plateau and plotted as a function o f the polarising current. RESULTS

The it~uence of the membrane potential upon the threshold-latency curve in a previous investigation o f the a c c o m m o d a t i v e properties o f phasic ~z-motoneurones in cats it was shown that there is no m i n i m u m ramp rate below which an action potential is not eventually elicited. A characterisation o f the m o t o n e u r o n e a c c o m m o d a t i o n rates was given by the t h r e s h o l d - l a t e n c y (TL) curvest-a,H, la and the ratio of threshold current (the final current of a I-sec r a m p with a rate of rise just sufficient to produce an action potential) to the rheobase current. This ratio

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0 100 200 300' 400 500' 61~0 t [msecI Fig. 1. Threshold-latency curves of a single motoneurone with current ramps at different times following microelectrode penetration, the membrane potential remaining approximately unchanged. The rate of rise of the stimulating current was decreased in a stepwise manner during the measurements: at every step, threshold current and latency of the elicited action potential were determined. Ordinate : threshold current (1) in hA. Abscissa: action potential latency (t) in msec. Circles: first measurement, 20 rain after the microelectrode penetrated the cell, Triangles and squares: subsequent measurements, 15 and 30 min later, respectively. The inset table shows membrane potential (MP, in mV) during these measurements and rheobases, in nA, at the beginning (I~) and the end (lf;) of the series of measurements.

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or hyperpolarising shifts of the membrane potential by intracellularly applied DC current. The membrane potentials (MP) in each case, as well as the direction of the displacements (arrows), are indicated above the curves. The remaining experimental procedure was as for Fig. 1. Data taken before and during the shifts are distinguished by different symbols (circles and triangles, respectively). Ordinates: ratio of threshold current to rheobase current, I/Io; abscissae: action potential latency (t) in msec. The membrane input resistance of both motoneurones remained unchanged during the entire course of the measurements.

was termed the a c c o m m o d a t i o n coefficient, (l/I0)see. in this study, (l/10)see lay between 1.1 and 3.3, values between 1.1 and 1.6 being most c o m m o n . The a c c o m m o d a t i o n coefficients, (l/Io)see and complete T L curves were also measured in the present experiments, but only motoneurones with (l/lo)see ~ 1.8 ('slowly a c c o m m o d a t i n g m o t o n e u r o n e s ' ) were investigated before and during longterm m e m b r a n e potential shifts. For each r a m p the current at the time an action potential was elicited was plotted as a function o f the time after the beginning o f the r a m p at which an action potential occurred. This T L curve is reproducible for as long as 30 min provided that the m e m b r a n e potential remains approximately unchanged (Fig. 1). The T L curve is affected, however, if the resting potential is altered by applying a steady D C current. W h e n the m e m b r a n e potential is shifted in a depolarising direction, the threshold values are increased above the control (Fig. 2A). (ln Fig. 2 and in subsequent figures the threshold is expressed as multiples o f the rheobase current, I/Io. A value o f one for 1/Io implies no a c c o m m o d a t i o n . ) Hyperpolarising shifts o f the resting potential have an opposite effect in motoneurones with a higher rate o f a c c o m m o d a t i o n ; the threshold changes with increasing latency considerably less than at the resting potential (Fig. 2B).

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Fig. 3. Threshold and maximum rate of rise of the action potential (~/AB)as a function of latency in a motoneurone before (A) and during (B) a depolarisation of the membrane potential by 12 inV. The rate of rise of the current ramp was decreased in a stepwise manner during the measurement. Circles: thresholds at different action potential (AP) latencies. Squares: 9AB. Left ordinates: ratio of threshold current to rheobase current, l/lo. Right ordinates: maximal rate of rise of the AP in V/sec. Abscissae in A and B: AP latency (t) in msec. The squares in A and B at the beginningand end of the sequence (q,, ce) are control measurements to check the condition of the motoneurone during the course of the experiment. These measurements were obtained with square-wave currents (duration, 0. l msec); ¢¢A]~of the elicited AP was then determined. The membrane input resistance before and during the displacement of the MP was 1.4 ME2.

Changes of (/AB during membrane potential shifts When a m o t o n e u r o n e accommodates d u r i n g a c u r r e n t ramp, the m a x i m u m rate o f rise of the action potential (VAB) decreases. The degree o f decrease of V~B is correlated with the accommodative properties of the m o t o n e u r o n e s . In m o t o n e u tones with higher rates o f a c c o m m o d a t i o n , VAB decreases distinctly more during a c u r r e n t r a m p than in those with lower rates of a c c o m m o d a t i o n . A similar difference in the behaviour Of VAB during a current r a m p is also f o u n d when the accommodative properties are changed by long-term shifts of the m e m b r a n e potential. Fig. 3 shows the dependence o f the threshold (1/I0) and VAB upon the action potential latency in a m o t o n e u r o n e with the m e m b r a n e potential m a i n t a i n e d at different levels. A t the resting potential, 1/10 increases only slightly as a function of latency (Fig. 3A) and the change in the VAB values is slight. D u r i n g a depolarising shift o f the m e m b r a n e potential, the n e u r o n e shows an increase in a c c o m m o d a t i o n as indicated by l/Io and a

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Fig. 4. Current-voltage (l-V) curves of 3 different motoneurones. Ordinates, polarising current (1) in nA; abscissae, change of membrane potential (V) in inV. A: I-V curves of two motoneurones (the same as in Fig. 2A, B) with different rates of accommodation. These rates are indicated by the TL curves in Fig. 2 before the long-term shifts of the membrane potential, Circles, motoneurone with a lower rate of accommodation (TL curve in Fig. 2A). Triangles, motoneurone with a higher rate of accommodation (TL curve in Fig. 2B). B : I-V curve of a single motoneurone (the same as in Fig. 5) before and during a long-term hyperpolarising shift of the membrane potential by 10 mV (circles and triangles, respectively). The shift of the membrane potential reduced the accommodation rate of this motoneurone as indicated by the TL curves in Fig. 5.

corresponding decrease in "V'AB(Fig. 3B). O n the other hand, hyperpolarising shifts in m o t o n e u r o n e s with a higher rate o f a c c o m m o d a t i o n at the resting potential lead to a decrease in a c c o m m o d a t i o n and an increase in VARY.

Current-voltage curves Two processes in particular deserve consideration as possible causes o f the effect o f m e m b r a n e potential on a c c o m m o d a t i o n : c h a n g e s o f the sodium conductance and/or the potassium conductance. If the potassium conductance is altered, changes o f the m o t o n e u r o n e m e m b r a n e input resistance are to be expected. These would be reflected in the shape o f the current-voltage curve. In the motoneurones investigated here, the measured current-voltage curves are linear in both the depolarising and hyperpolarising regions (Fig. 4A). The slopes o f the curves are the same prior to and during the shifts o f the m e m b r a n e potential (Fig. 4B). Only in motoneurones with higher rates o f a c c o m m o d a t i o n - - (I/Io)see >~ 2.5 - - do the current-voltage curves have a greater slope in the depolarising than in the hyperpolarising region. The properties o f a c c o m m o d a t i o n in these neurones have n o t thus far been investigated during longterm changes o f the m e m b r a n e potential.

(IAB curve before and during membrane potential s h ~ s As described in the methods, the dependence o f VAB u p o n the m e m b r a n e potential (~rAB curve) was measured by means o f depolarising or hyperpolarising squarewave conditioning currents which were followed by a test pulse just sufficient to

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Fig. 5. Threshold-latency (TL) curves and the dependence of the maximal rate of rise of the action potential (gin0 of a single motoneurone before and during a long-term hyperpolarising shift on the membrane potential (the same motoneurone as in Fig. 4B). A: TL curves. Other details as in Fig. 2. B: dependence of 9~B upon membrane potential (the same symbols as in A). For measuring the 9m~ curve the membrane potential was altered by square-wave conditioning currents (duration 100 msec) in both the depolarising and the hyperpolarising directions. Immediately following the conditioning current a rectangular test pulse of 50/tsec duration was given. The ordinate represents 9AB of the action potential thus produced, in percent of the maximal 9,~ observed.

elicit an action potential. Fig. 5B shows an e x a m p l e o f such a "~a~ curve, where V~B o f the test action potential is p l o t t e d against the m e m b r a n e potential measured at the t e r m i n a t i o n o f the c o n d i t i o n i n g current. A l t h o u g h the ~¢a~ curve does n o t reflect precisely the relationship between sodium current a n d m e m b r a n e potential, it does permit inferences a b o u t the degree o f the sodium inactivation 1°,t4. 9m~ a n d T L curves were m e a s u r e d for each m o t o n e u r o n e before a n d during long-term shifts o f the m e m b r a n e potential. Fig. 5A shows the T L curves a n d Fig. 5B the ~/A~ curves o f such an experiment. During a hyperpolarising shift o f the m e m b r a n e potential, the m o t o n e u r o n e shows a decrease o f a c c o m m o d a t i o n rate as indicated by 1/Io. Despite the considerable change in a c c o m m o d a t i o n rate, the curve o f V a ~ as a function o f m e m b r a n e potential remains unchanged; there is no shift a l o n g the

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potential axis. Comparable results were obtained before and during hmg-term depolarising shifts of the membrane potential. DISCUSSION

The results in the present paper indicate that the rates of accommodation increase with depolarising and decrease with hyperpolarising changes of the membrane potential. On the assumption that the ionic theory of excitations can be applied without reservation to spinal motoneurones, the rates of accommodation could increase because of an increased sodium inactivation and/or potassium activation during depotarisation of the membrane potential. These processes, individually or together, would be suppressed by hyperpolarisation, and thus cause accommodation rates to decrease. In the motoneurones investigated here the membrane input resistance did not change during long-term shifts of the membrane potential (Fig. 4B). The currentvoltage curves are linear (Fig. 4A, B), even during strong depolarisations. Thus the potassium conductance must remain unchanged during the long-term shifts of the membrane potential. Consequently, the differences in accommodation could be based entirely on changes in the degree of sodium inactivation. The wmations in amount of decrease of ~/ABin the TL curves before and during changes of the membrane potential support this hypothesis. When accommodation is increased by depolarising the membrane potential, VABis correspondingly decreased. On the other hand, lowering the level of accommodation by hyperpolarising the membrane potential leads to an increase in VAB. These changes in ~rA~should be a reflection of the degree of sodium inactivation. In the Loligo axon and in myelinated nerve fibres the maximal rate of rise of the action potential ('¢) is proportional to the sodium current<6. The relationship between ~raB and membrane potential before and during longterm changes in membrane potential is also consistent with this hypothesis. The curves remain unchanged during depolarisation and hyperpolarisation (Fig. 5B), rather than being shifted along the potential axis. Because "qAB permits inferences about h6, ~4, it is most probable that the inactivation curve remains unaltered during long-term shifts of the membrane potential. Thus, at normal membrane potential the sodium inactivation increases relatively slowly during a current ramp. When steady depolarising current is applied, the membrane potential is shifted into a region in which the inactivation increases rapidly when a depolarising current ramp is applied. The result is a faster rate of accommodation. On the other hand, a hyperpolarising membrane-potential shift drives the system away from this region, suppresses the sodium inactivation, and thus decreases the motoneurone accommodation rate. ACKNOWLEDGEMENTS

This investigation and the participation of K. H. M., D. W. R. and W, R. S. were supported by the Sonderforschungsbereich 38 'Membranforschung' of the Deutsche Forschungsgemeinschaft.

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M r s . M . E r a s e r , M r s . H. M o r a t z k i a n d M r s . P. R o e d e r p r o v i d e d efficient t e c h nical a s s i s t a n c e .

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