Voltage clamp study of cat spinal motoneurons during strychnine-induced seizures

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Brain Research, 204 (1981) 226-230 © Elsevier/North-Holland Biomedical Press

Voltage clamp study of cat spinal motoneurons during strychnine-induced seizures

P. C. SCHWINDT and W. E. CR1LL Seattle Veterans Administration Medical Center, Seattle, Wash. 98108 and Departments o f Physiology and Biophysics and Medicine (Neurology), University of Washington, Seattle, Wash. 98195 (U.S.A.)

(Accepted August 7th, 1980) Key words: seizures - - strychnine - - motoneurons -- voltage clamp -- synaptic currents

Cat spinal motoneurons were examined by the technique of somatic voltage clamp during strychnine-induced spinal seizures. No clear alteration of voltage-dependent ionic currents was required in order for typical strychnine-inducedparoxysmal depolarization shifts (PDSs) to develop in contrast to results previously obtained during penicillin-inducedspinal seizures.Voltage clamp of evoked and spontaneous PDSs indicates these are generated by a synchronized mixture of excitatory and inhibitory synaptic currents with excitation predominating. Application of penicillin (PCN) to the cat spinal cord results in seizures characterized by episodic sudden depolarizations of the motoneurons with superimposed repetitive firing6,10,11. The motoneuron firing is characterized by prolonged (1-3 sec) bursts of action potentials interspersed with much shorter (10-200 msec) depolarizations and bursts. In our recent voltage clamp study of PCN-induced motoneuron bursting 1~, we found that the short bursts may be caused solely by synchronized excitatory synaptic input, but the prolonged bursts depend on the existence of a persistent, net-inward, ionic current la and its relative enhancement in association with depressed outward ionic currents. The purpose of the present study was to determine if comparable alterations of voltage-dependent ionic currents were required to produce, or occurred in association with, strychnine-induced seizures. Experiments were performed on cats anesthetized with sodium pentobarbital (35 mg/kg initially with supplemental doses as required) and paralyzed with gallamine triethiodide. Lumbar motoneurons of the S1-L6 segment were identified by antidromic invasion by sciatic nerve stimulation after cutting the $2-L6 dorsal roots. One of the cut dorsal roots was placed on a bipolar nerve hook and used for orthodromic stimulation. Cells were impaled with separate current-injecting and voltage-recording microelectrodes filled with 3 M KCI and glued together so that their tips were separated by 5-10 #m. Other details of animal preparation, microelectrode fabrication, voltage clamp circuitry, and experimental procedures were identical to those previously reported in detaiP a,14. After a period of recording from normal motoneurons, seizures were induced by

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Fig. 1. Behavior of motoneuron membrane potential and membrane currents during strychnine-induced seizures. A: characteristic rhythmic paroxysmal depolarization shifts (PDSs) observed during episodes of intense seizure activity after strychnine administration. B: characteristic PDS evoked by dorsal root stimulation (at arrow) in another cell. Note subsequent afterhyperpolarization. Similar isolated PDSs occurred spontaneously in between periods of intense activity like that of A. C: superimposed voltage steps (upper) and membrane currents (lower) obtained during voltage clamp of an evoked PDS in another cell. Like-numbered current and voltage traces correspond. Also superimposed for reference on the voltage traces is the evoked PDS which was damped and a subsequent spontaneous PDS. Outward (repolarizing) current is upward in C and D. The ohmic, leakage currents and the longest capacitative charging current have been electronically subtracted before photography3,z4; only active currents are shown. Resting potential is portion of voltage records preceding steps, and zero current baseline is portion preceding outward active currents. During the voltage steps, the PDS was evoked at the time indicated by shock artefacts on the current traces. Each step was taken with and without a PDS so that the departure of the PDS current from the outward current activated by the voltage step could be clearly seen. Upward arrow indicates excitatory (inward) component of PDS current. Downward arrow indicates late, inhibitory (outward) component which is most dearly seen during depolarizing steps. Even the earliest part of the PDS current is reversed at step 6, which is to zero inV. D: voltage clamp records similar to C, but of an intermittent, spontaneous PDS in another cell. Calibrations: vertical bar in B is 20 mV for B; 40 mV for A, C (upper) and D (upper); 100 nA for C (lower); 50 nA for D (lower). Horizontal bar is 100 msec for A and D; 20 msec for B; 40 msec for C. i n t r a v e n o u s injection o f strychnine sulfate dissolved in n o r m a l saline. T h e initial dose was usually 0.5 m g / k g ; if this p r o v e d ineffective, a d d i t i o n a l injections were m a d e until seizures developed. A n a t t e m p t was m a d e to r e c o r d f r o m the same cell before a n d after seizure d e v e l o p m e n t , b u t cells were always lost as the seizure developed, p r o b a b l y because o f b l o o d pressure p u l s a t i o n s o r spinal c o r d swelling a c c o m p a n y i n g the seizure. L o n g - t e r m i m p a l e m e n t s were subsequently difficult to m a i n t a i n because i m p a l e m e n t s were a g a i n lost d u r i n g subsequent waves o f intense seizure activity. Satisfactory voltage c l a m p r e c o r d s were o b t a i n e d in 14 m o t o n e u r o n s . T y p i c a l re-

228 suits are shown in Fig. 1. After a sufficient dose of strychnine, episodes of intense seizure activity occurred (Fig. 1A). These consisted of rhythmic, relatively brief, paroxysmal depolarization shifts (PDSs) with superimposed, decrementing, action potentials. In between episodes of intense activity, or at strychnine doses too low to result in rhythmical PDSs, isolated PDSs occurred which were similar to those previously described by Fuortes and Nelson 9 (Fig. 1B). Typically, an afterhyperpolarization followed the PDS. Such isolated depolarizations occurred spontaneously or could be evoked in an all-or-none manner by dorsal root stimulation, as was done in Fig. lB. Simultaneous ventral root and intracellular recording has shown that a large number of the motoneurons of a recorded segment fire synchronously during either the spontaneous or evoked PDSs 1~. The time course of the PDSs in Fig. 1A and B is entirely typical; prolonged bursting of the type seen after PCN application was never observed. Voltage clamp analysis revealed neither grossly abnormal membrane currents compared to normal cells~,3,14, nor any apparent special requirements for typical PDS behavior. In particular, typical strychnine bursts could be observed in ceils in which the persistent inward current had deteriorated by the time records were taken in contrast to PCN-induced bursting where the persistent inward current must be present and netinward for the characteristic, prolonged bursts to occur la. Several cells were recorded which exhibited intermittent spontaneous PDSs and which were observed as the persistent inward current component deteriorated. No marked change in the nature of the PDS occurred during this time. It must be emphasized, however, that we were unable to obtain complete records for analysis of all current components from any one cell, and no one cell was studied before and after the development of PDS behavior. Our observations do not rule out the possibility of more subtle but perhaps significant changes of membrane currents as the seizures develop, The mechanism underlying the PDS seems best explained as a giant, synchronized EPSP since the currents underlying the PDS varied with membrane potential in a manner expected for synaptic currents. This is as shown in Fig. 1C for an evoked PDS. The records of Fig. 1C further indicate that the PDS is actually a mixture of EPSP and IPSP. When the cell is clamped at or below rest, only the inward (depolarizing) synaptic current is clearly seen (upward arrow in Fig. 1C). Notice that this grows in amplitude with hyperpolarization and decreases with depolarization. However, at depolarized potentials, a later, outward (repolarizing) synaptic current becomes clearly apparent (downward arrow in Fig. 1C), and it grows in amplitude with depolarization. The IPSP following the PDS and the associated outward synaptic currents during voltage clamp at resting potential are not apparent in this cell, probably because of chloride leakage from the microelectrodes and the accompanying positive shift of IPSP reversal potential. The reversal of the excitatory component (inward current near rest) was typically biphasic, as in Fig. 1C. The late part reversed at relatively small depolarizations (step 3 in Fig. IC). Most of the early part is reversed by step 4, but the earliest part is not clearly reversed until step 6, which is to zero inV. The low reversal potential of the greater part of the excitatory component is quite different than obtained for the monosynaptic EPSP s and is expected if outward (inhibitory) synaptic currents are

229 mixed with the larger inward (excitatory) currents. The records of Fig. 1C are entirely typical of each cell examined in terms of the variation of the PDS current with potential and the low reversal potential of most of the excitatory component. The currents underlying spontaneous PDSs behaved like the evoked ones, as shown in Fig. 1D. These could not be as systematically investigated because they occurred only intermittently during the applied step depolarization. It is also worth noting that, although the PDS currents are very large, the soma membrane potential is well-controlled, and there is no sign of all-or-none components on the synaptic current as might result from active, uncontrolled currents from the dendrites (i.e. dendritic spikes). In summary, the PDS observed in motoneurons during strychnine-induced spinal seizures seems best explained by simple depolarization due to synchronized synaptic input consisting of a mixed EPSP-IPSP with excitation predominating. Our data do not prove that strychnine does not alter membrane conductances in some way, but no clear, specific alteration seems required to produce characteristic strychnine bursts. Our observations are consistent with the accepted mechanism of strychnine seizure production, namely, depression of glycine-mediated IPSPs 4 to the point where their inhibitory, braking action on undefined, recurrent, excitatory pathways becomes insufficient to prevent the establishment of waves of rhythmical, synchronized excitation. A similar explanation of PCN-induced cortical seizures has been offered1 and given more credence by the recent findings that PCN can block 7-aminobutyric acid-mediated inhibition 5,7,1z. In considering this mechanism of seizure production, it is worth noting that disynaptic IPSPs in motoneurons must be reduced by 75-90 % before PDSs like those of Fig. 1B are observed 15, whereas no decrement of the same kind of IPSPs are observed during PCN-induced spinal seizures ~. The present results combined with our previous PCN study 13 show that spinal seizures may be caused by two quite different mechanisms. It seems premature to assert that cortical seizures caused by PCN or other agents involve only one of these mechanisms. Supported by the Veterans Administration and Epilepsy Foundation of America.

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230 8 Engberg, I. and Marshall, K. C., Reversal potential for Ia excitatory post synaptic potentials in spinal motoneurones of cats, Neuroscience, 4 (1979) 1583-1591. 9 Fuortes, M. G. F. and Nelson, P. G., Strychnine: its action on spinal motoneurons of cats, Science, 140 (1963) 806-808. 10 Kao, L. I. and Crill, W. E., Penicillininduced segmental myoclonus. I. Motor responses and intracellular recording from motoneurons, Arch. Neurol. (Chic.), 26 (1972) 156-161. 11 Lothman, E. W. and Somjen, G. G., Motor and electrical signs ofepileptiform activity induced by penicillin in the spinal cords of decapitate cats, Electroenceph. clin. Neurophysiol., 41 (1976) 237-252. 12 MacDonald, R. L. and Barker, J. L., Specific antagonism of GABA-mediated postsynaptic inhibition in cultured mammalian spinal cord neurons: a common mode of convulsant action, Neurology (Minneap.), 28 (1978) 325-330. 13 Schwindt, P. C. and Crill, W. E,, Role of a persistent inward current in motoneuron bursting during spinal seizures, J. NeurophysioL, 43 (1980) 1296-1318. 14 Schwindt, P. C. and Crlll, W. E., Properties of a persistent inward current in normal and TEAinjected motoneurons, J. NeurophysioL, 43 (1980) 1700-1724. 15 Tribble, G. L., Schwindt, P. C. and Crill, W. E., A quantitative study of the reduction of inhibition necessary to cause spinal seizures, NeuroscL Abstr., 5 (1979) 197.