J. Physiol. (1979), 286, pp. 457-477 With 10 text-fguree Printed in Great Britain
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PROLONGED CHANGES IN EXCITABILITY OF PYRAMIDAL TRACT NEURONES IN THE CAT: A POST-SYNAPTIC MECHANISM
By LYNN J. BINDMAN, 0. C. J. LIPPOLD AND ALEX R. MILNE* From the Department of Physiology, University College London, London WClE 6BT
(Received 23 February 1978) SUMMARY
1. Prolonged changes in the excitability of cortical neurones can be produced by altering their firing rates for brief periods. In the anaesthetized cat, increased firing of pyramidal tract cells induced by trains of antidromic conditioning shocks led to increases in cell excitability, as measured by the size of the mass response at the medullary pyramid to test shocks applied to the cortical surface. We have shown in two ways that post-synaptic mechanisms could be responsible. 2. In one experimental design, MgC12 solution (1 mole/l.) was applied to the cortical surface in order to block synaptic activity throughout the cortical depth. Following antidromic conditioning trains, cell excitability was increased; the size of the mass response was up to 30 % larger than the control values. This persisted undiminished for up to 3 hr. 3. In the second experimental design, synaptic activity was not blocked, but we compared the effects of antidromic plus synaptic activation of pyramidal tract cells with the effects of synaptic activation alone. Antidromic plus synaptic activation was obtained by applying conditioning trains to the pyramidal tract at the medulla ipsilateral to the cortical test shock; prolonged increases in the ipsilateral response to the test shock were produced. Synaptic activation alone was obtained by the same conditioning trains, but in those cells whose axons projected into the contralateral pyramidal tract; prolonged increases in the contralateral response to the cortical test shock were never seen. In many instances prolonged decreases in excitability were found. 4. We conclude that prolonged increases in excitability of pyramidal tract cells can occur in the absence of any synaptic input, demonstrating that the underlying mechanism is post-synaptic; this does not preclude the action of synaptic mechanisms when synaptic transmission is not blocked. INTRODUCTION
Long lasting changes in the excitability of neurones in the central nervous system are of considerable interest because of their possible connexion with the processes involved in information storage. Although such changes have been observed in the cerebral cortex, we still know little about the mechanisms involved at the cellular level. An important preliminary step in the elucidation of the underlying mechanisms * M.R.C. Scholar. Present address: Department of Physiology, School of Veterinary Science, Park Row, Bristol BS1 5LS.
458 L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE is to ascertain whether the site of prolonged changes is at presynaptic terminals, in the post-synaptic membrane or elsewhere in the neurone. Many brief experimental procedures that increase the firing rate of cortical neurones have been found to give rise to changes in excitability that last undiminished for several hours (see Lippold, 1970; Bliss & L0mo, 1973; McCabe, 1972, 1976); in all these experiments, an increase in synaptic activity could have been involved. In the present experiments we have examined the possibility that long-term changes in excitability could arise in the absence of synaptic activity. We investigated whether the post-synaptic excitability of pyramidal tract cells in the anaesthetized cat could be altered by a brief period of repetitive antidromic activation. The difficulty in using this experimental approach is that stimulation of the axons of pyramidal tract neurones, at the medullary pyramid, will not only activate the cell bodies antidromically but the impulses will also travel along recurrent collateral branches of the axons and lead to excitatory or inhibitory synaptic effects (Stefanis & Jasper, 1964; Armstrong, 1965). To overcome this problem, we devised two experimental designs. In the first, all synaptic activity in the cerebral cortex was blocked by bathing the pial surface with MgCl2 solutions of suitable concentration (Bindman & Milne, 1977). In the second experimental design, synaptic activity was not blocked, but we compared the effects of antidromic plus synaptic activation of pyramidal tract cells with the effect of synaptic activation alone. It was possible to make this comparison because some of the axons of the pyramidal tract cells cross the mid line and travel in the contralateral medullary pyramid (Fig. 1). Hence stimulation of the ipsilateral pyramidal tract affects cortical cells whose axons project into the ipsilateral tract both via synapses and antidromically, while it affects via synapses alone those cells whose axons run in the contralateral tract. We found that the excitability of pyramidal tract cells (monitored by the amplitude of mass axonal responses to 'test' shocks applied to the cortex) was increased following brief periods of antidromic 'conditioning' stimulation of the pyramidal tract in the presence of Mg, i.e. when synaptic activity was blocked. We also found prolonged increases in the excitability of pyramidal tract cells in the second experimental design, but only in the situation where cells were activated both antidromically and via the collateral branches of the axons. In the situation where no antidromic excitation occurred, namely where cells were only influenced synaptically from collaterals, no long-term increases were observed. However in the latter experiments involving only synaptic pathways, long-lasting decreases in the excitability of the pyramidal tract cell population were found. Preliminary accounts of this work have been communicated to the Physiological Society (Bindman, Lippold & Milne, 1976a, b; Bindman & Milne, 1977). METHODS
Surgical procedure Experiments were performed on 124 cats of mixed stock and either sex, anaesthetized with i.P. injections of urethane (360 g/l. solution; 1-5 g/kg body wt.). This anaesthetic was used because the cats remained fully anaesthetized at a steady level for at least 20 hr with no supplementary dosage, and cortical excitability is stable (Lippold, 1970). The trachea, femoral artery and femoral vein were routinely cannulated. Structures in the neck were dissected from the ventral side to expose the bone at the base of the brain overlying the medulla. A small hole
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was drilled in the bone to one side of the mid line, avoiding the basilar artery and its branches. The hole was enlarged to expose most of both medullary pyramidal tracts. The skull overlying the cerebral cortex was then exposed, and a trephine hole made over the parietal cortex. The bone was removed to expose part of the precruciate cortex, and much of the parietal region, making a hole of diameter approximately 2 cm. The dura mater was removed, and the cortical surface covered with cotton wool soaked in sodium chloride (9 g/l.) for protection, while a watertight cup was constructed above the cortical surface. The nasal passages were filled with dental wax. A Perspex cup was cemented to the dried bone surrounding the exposed cortex
Ipsilateral
ContralateralI
RRCl C
AC
Fig. 1. Diagram showing position of recording and stimulating electrodes. Three pyramidal tract cells with their axons extending to the medullary pyramids are shown, together with recurrent collaterals which may be either excitatory or inhibitory. Note that one axon crosses the mid line to become contralateral. The cortical electrode rests on the pre-central gyrus to deliver test shocks between it and an indifferent electrode (not shown) on the parietal cortex. On each pyramidal tract at the medulla lies a recording electrode (R), the indifferent electrodes being sited on adjacent bone. Pairs of conditioning electrodes are also shown (C), sited on the tract caudal to the recording electrodes. The cup, cemented to the bone, is watertight and kept at a pre-determined pressure using a reservoir. The electrode for recording the evoked response is not shown.
using methyl methacrylate cement; the cotton wool was then removed. Stimulating and recording electrodes were placed in position (see below) and fixed rigidly to the inner wall of the cup. Their connecting leads ran through a hole in the side wall. The top of the cup and the hole in the side were sealed and it was filled with NaCl (9 g/l.) through its side tubes (Fig. 1). The pressure of fluid in the cup was kept as close to atmospheric as possible at this stage by adjustment of the level of a reservoir of the NaCl solution. The head was then fixed rigidly into a head-holder mounted on a spindle, and the whole animal was rotated into the supine position. The dural membrane overlying the medullary pyramids was then removed, and the fluid in the reservoir adjusted to the level of the exposed
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medullary surface. This adjustment was found to be important to ensure that the blood supply of the region was not impaired. The cortical surface could be irrigated with fluids of any desired composition via the reservoir, cup and outlet tube.
Recording and stimulating electrodes Synaptic activity in the cortex was monitored by recording the responses evoked by contralateral forepaw stimulation. An insulated tungsten microelectrode of exposed tip diameter about 10 ,Am was used. The cortical surface was explored while weak electric shocks were delivered to the skin of the contralateral forepaw, once every 2 sec. The electrode tip was pushed about 1-5 nun below the pial surface in the region where the largest surface response was evoked. The deep-negative wave was recorded at intervals throughout the experiment, and sixteen or thirty-two such responses were averaged using a small computer (Biomac 1000). In some experiments glass micropipettes, tip diameter about 1 jsm, filled with NaCl (100 g/l.), were used to record spontaneously occurring spikes. Test shocks were provided by non -polarizable electrodes on the pial surface, exciting pyramidal tract neurones orthodromically. These consisted of polyethylene tubes, internal diameter 3 mm, filled with agar gel made up in NaCl (9 g/l.). Chlorided silver wire was coiled inside the tube, and at one end it was soldered to insulated wire. That end of the tube was sealed with dental wax to insulate it. One of these electrodes was placed in contact with the pial surface of the precruciate motor cortex, the other placed at the caudal end of the exposed area of cortex. Pyramidal tract responses to cortical stimulation were recorded at the medullary pyramids, using different types of electrode depending on the purpose of the experiment. Insulated tungsten micro-electrodes were employed in experiments measuring recovery cycles and the effect of MgCI2 application to the cortex. For experiments monitoring the excitability of pyramidal tract cells for long periods of time, non -penetrating wire electrodes were found to be more convenient. These consisted of a central conducting core of 120 ,m nichrome wire insulated with polyethylene, of external diameter 600 _tm. The ends of these electrodes were squared off under the microscope using a sharp cutting edge, so that an insulating ring was present round the central wire when it was pressed on the medullary surface. They were sufficiently springy to take up small movements of the medulla without either losing contact or penetrating. The stimulating electrodes were of similar construction. Up to six electrodes were positioned on the two pyramids with individual adjustments for each of them (Fig. 1). Recording electrodes were rostral, the indifferent of each pair being usually on the adjacent bone to achieve a monophasic response. A pair of stimulating electrodes was placed across the tract approximately 2 mm apart and at least 4 mm caudal to the recording electrode. Their position was adjusted with respect to the recording electrode so that the same axons were being stimulated by the pyramidal tract shock as were excited as a result of the cortical shock. This was tested by occlusion (see below). When all these electrodes had been sited correctly, the whole cavity was filled with agar gel made up in NaCl (9 g/l.) solution. This was necessary not only to prevent relative movement between the tracts and electrodes but also to stop any flow of MgCl2 solution from the cortical cup to the medulla.
Stimulus parameters Stimuli for eliciting the primary somatosensory evoked potential were delivered via two stainless-steel needles inserted under the skin of the contralateral forepaw. Shocks were given every 2 see at intervals throughout the experiment and were 0-05 msec duration and just subthreshold for producing muscle twitches before the cat was immobilized. Cortical test shocks were given every 2 see or every 5 sec, the stimulus width being 0-05 or 0-10 msec. The voltage was gradually increased until a just maximal early wave was obtained at the pyramid. This stimulus strength was used in the occlusion test. The strength was then decreased to a voltage eliciting a response of between 60 and 80% of the value eliciting the maximal response, for the remainder of the experiment. The stimuli were square waves generated by an isolated stimulator (Devices Mk IV); stimulus current as monitored by the voltage drop across an external resistor in series with the preparation did not vary throughout the experiment. Polarity was surface positive for the electrode overlying the precruciate cortex.
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Pyramidal tract conditioning shocks were usually delivered as gated frequency trains, to allow the recording of a response to the cortical test shock within the conditioning period. Parameters are given in the legends to the Figures.
Electrical recording and data proceesing Differential high impedance input circuits and direct coupled amplifiers were used. In a few experiments photographs of the responses were taken; usually averages were obtained using a Biomac 1000 and were permanently stored on punched paper tape. A Linc 8 computer was used to plot these off line. Many experiments were recorded continuously on FM magnetic tape.
Occuaion To check that the cortical test and the medullary conditioning stimuli were affecting the same, or an overlapping, population of pyramidal tract cells, a test for occlusion was carried out. Figure 2 A shows, top line, the sum of four mass responses recorded from the medullary pyramid elicited by a cortical test shock given at time Cx. When a pyramidal tract shock was given 1 msec before the cortical test shock, at time PT, the antidromic axonal wave could be seen immediately following the downward deflexion of the stimulus artifact but the response to the subsequent cortical test shock was considerably reduced (second line). When occlusion could not be obtained, electrode positions were altered until it could. In two experiments where occlusion did not occur, an after-effect due to the conditioning shocks was not obtained. In experiments in which MgCl2 was not used, and the effects of stimulating the ipsilateral pyramidal tract were compared with the effects of stimulating the contralateral one, it was necessary to ensure that the stimulating current did not spread from one side to the other. This was checked, as shown in Fig. 2B, right-hand column, by demonstrating that no occlusion occurred between the stimulus on one side and the response on the other side. A second check was to see whether current spread could directly activate axons on the contralateral side, as can be seen happening to a small extent in the lowest left-hand trace in Fig. 2B, i.e. using the 'strong' PT shock. Where either occlusion of the response in the other tract, or current spread to excite axons on the other side was observed, tha current was reduced or the stimulating electrodes were repositioned.
Stability of the recording station Many factors were controlled in order to obtain stable recordings. Movements of the cat when the pyramidal tract was repetitively stimulated were prevented by i.v. infusions of gallamine triethiodide in quantities sufficient to abolish spontaneous respiratory movements and evoked muscle twitches. Following the use of gallamine, ventilation was maintained with a positive-pressure respiration pump, at a tidal volume such that the end-expiratory Pco; remained constant. The use of I.v. gallamine and MgCl2 applied to the cerebral cortex tended to lower the blood pressure; if the systolic pressure fell below 80 mmHg the experiment was discarded. Dehydration of the animal was countered by i.v. infusions of dextrose-saline (4% and 0-18% w/v respectively). The PE cO2 was measured with a continuous sampling infra-red analyser. Arterial blood pressure was measured with a Bell & Howell transducer, and a continuous record of both and blood pressure was made throughout the experiment using Devices preamplifiers and two-channel pen recorder. Rectal temperature was measured with a mercury-in-glass thermometer, and was maintained at 37 'C by using a heating blanket, the current of which was controlled by feed-back from a thermistor in the rectum (Diete-Spiff, Ikeson & Read, 1962). Movement of the recording and stimulating electrodes in the medullary region was minimized by filling the exposed cavity with a gel of agar made up in NaCl (9 g/l.). The use of slightly flexible, individually adjustable wires surrounded by polyethylene was also of importance; in early experiments using rigid tungsten electrodes we found they tended to penetrate the medullary surface. It was necessary to keep the level of fluid in the reservoir constant throughout the experiment because any change in fluid pressure caused a change in the position of the medulla relative to the electrodes.
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Occasionally small increases in systemic blood pressure were produced by the onset of the trains of conditioning stimuli at the medulla. However these changes in blood pressure decayed by the end of the conditioning train, and wera not related to the production of after effects; after effects were seen when no rises in blood pressure were produced, conversely increases in blood pressure were not necessarily associated with the production of after effects. CX
CX
A
B
CX
PT
CX
m PT Cx
PT
Cx
m
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&5T
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Weak PT
Fig. 2. Tests for occlusion. A illustrates procedure used to ensure that the sane population of pyramidal tract cells are excited by both test and conditioning shocks. Records were taken from the pyramidal tract; each trace shown is the sum of four of these. Top trace: response to cortical test shock given at Cx. Second trace: identical cortical shock but preceded by shock delivered to ipsilateral pyramidal tract 1 msee earlier, at PT. Third trace: response to pyramidal tract shock alone. Bottom trace: repeat of cortical test shock alone. The strength and duration of the electrical stimuli were those used throughout the subsequent experiment. Voltage calibration 05 mV, time bar 1 msec. B, similar procedure to that shown in A but to check on the current spread from the conditioning electrodes directly to the opposite pyramid. In each frame the upper trace is of four superimposed ipsilateral pyramidal tract responses, and the lower trace is of the simultaneously recorded response in the contralateral pyramidal tract. Polarity of lower trace is reversed with respect to upper. Left-hand column strong, right hand weak pyramidal tract shocks. Top pair of traces: response to cortical test shock alone. Voltage calibration 02 mV, time bar 1 msec. Middle pair of traces: cortical test shock is preceded by conditioning shock given to ipsilateral pyramidal tract 1 msec earlier. Note, in left-hand column, that the ipsilateral pyramidal response shows occlusion of the earliest component, although not of later waves. The contralateral pyramidal tract response shows no obvious occlusion. Bottom pair of traces: response to pyramidal tract shock alone. Note, in left-hand column, the strong pyramidal tract shock used has resulted in some current spread to the contralateral tract. The weak shock used in the right-hand column does not spread to excite fibres in the contralateral tract, but neither does it produce complete occlusion of the earliest ipsilateral component. Same experiment as Fig. 8.
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A CF
8
*I~~~~~~~~~~~~~~. 7 9. _
Ji
-5 rr ~~~~~~~~~~~~~~~~~~~5 msec Mg C12
7.50
8.40
_~~~~~~~~~~~~~~~110.000 x 0-5
11.03 PT m 1.2a
Fig. 3. A, the effect of magnesium, topically applied to the cerebral cortex, upon the response recorded from the pyramidal tract at the medullary pyramid. The motor cortex was stimulated using 36 V; pulse width 0-05 msec (90% maximal for peak at 1 msec). Top trace: control response. Time scale 1 msec, voltage calibration 0-5 mV. Middle trace: more than 1 hr after MgCl2, 1 mole/l., applied. Lower trace: at the end of the experiment, as much as possible of the grey matter was removed, and a concentric needle electrode was used 4 mm below the surface thus exposed, to stimulate white matter. Response recorded at the pyramid. Note the presence of a prolonged response lasting for at least 6 msec after the stimulus. Stimulus parameters as before but the geometry of the excitable elements and the current density at them are not comparable. The position of the tip of the stimulating electrode was confirmed as being in the white matter histologically. Voltage calibration 0-2 mV. B, the action of MgCl2 solution on the cortical response evoked by contralateral forepaw stimulation (CF). Traces are sums of thirty-two separate responses, which were recorded at a depth of about 2 mm below the pia. Negativity upward deflexion. Trace 1, control; 2-7, cup filled with MgCl2 solution (1 mole/l.) from 6.53 p.m. Note gradual decrease in peak-to-peak amplitude. Due to difficulties with computer write-out, trace 5 is half the gain of the other traces. Numbers on the left show time course of this effect. These results are from the experiment shown in Fig. 7. Pyramidal tract stimulated at 11.20 p.m.
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L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE RESULTS
1. Monitoring the synaptic blocking action of MgCl2 Since the objective of this investigation was to see whether long lasting changes in neuronal excitability could be induced in the absence of synaptic utilization, it was of importance to be certain that in experiments when MgCl2 was applied to the cortical surface all synaptic activity within the underlying grey matter had been blocked. A series of experiments was carried out to investigate the actions of MgCl2 topically applied to the intact cortical surface in various strengths, on both cats and rats. The experiments on rats are reported elsewhere (Bindman & Milne, 1977; Milne, 1978). Waves in pyramidal tract response. Because the mass pyramidal tract response was being used to monitor the excitability of the cells in the cortex, our first concern was to investigate the effect of topically applied magnesium on this response. When a brief, submaximal, cortical shock is used (0.05 msec; 36 V) the response in the pyramidal tract consists of a major deflexion of peak latency about 1 msec, and smaller later waves, continuing for 5-10 msec (Figs. 2B, 3A top trace). The later waves are smaller in this situation than when stronger shocks of longer duration are employed. The question arises as to whether the later waves elicited by the brief weak shocks are equivalent to the 'I' waves described by Patton & Amassian (1954), which are thought to be the result of synaptic activation of pyramidal tract cells by the cortical shock. Alternatively the later waves could be the result of direct electrical stimulation of smaller cells having axons with a slower conduction velocity (Oshima, 1969), that have been shown by anatomical techniques to comprise a large proportion of the pyramidal tract fibres (van Crevel & Verhaart, 1963). The action of MgCl2 topically applied to the cortex was to reduce the over-all amplitude of the response (Fig. 3A, middle trace) but not to curtail the duration to any marked extent. The effect of stimulating cortical white matter alone (following the removal of the grey matter) can be seen in Fig. 3A (bottom trace). It is clear that late waves can result from discharges in slow conducting axons. Confirmation that late waves in the pyramidal tract following a cortical test shock can be the result of direct stimulation of cells with slowly conducting axons was obtained by simultaneous monitoring of the pyramidal tract waves and the synaptically evoked response to forelimb stimulation after asphyxiation of the animal. First the post-synaptic components, then all signs of the contralateral forepaw evoked response disappeared by 2 min, whereas the later components of the pyramidal tract response persisted, although reduced in amplitude (Fig. 4), for 6-5 min after respiration ceased (4 min after the heart stopped). Changes in excitability of pyramidal tract cells can be investigated by giving pairs of shocks separated by increasing intervals to the cortical surface. The first shock excites the cells directly and initiates synaptically mediated activity, which may be excitatory or inhibitory. As can be seen from Fig. 5A (filled circles, continuous line) the first shock leads to depression of the second response until the two shocks are 8 msec apart. Although the applied magnesium did not modify the pyramidal tract response markedly (Fig. 3 A), nevertheless the recovery cycle in the presence of Mg indicated that synaptic transmission had been blocked by it. The recovery cycle
POST-SYNAPTIC CHANGES IN PT CELLS w
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Fig. 4. The effect of asphyxia on the cortical response to forepaw stimulation and on the pyramidal tract response. A, control. Upper trace shows pyramidal tract response to shock applied to cortical surface. Negative downwards. D wave retouched. Calibration bar 0-2 mV. Lower trace shows mass potential evoked 2 sec later by stimulation of the contralateral forepaw, recorded at a depth of 1 mm in the cortex. Negative upwards. Calibration bar 1 mV. Each stimulus applied at onset of trace. B, records obtained 4 min after pump was turned off in paralysed preparation, which was 1-5 min after cardiac arrest. Lower trace shows small residual wave in evoked response, presumably due to the arrival volley in the thalamocortical afferents. Upper trace shows early and late waves in the pyramidal tract, smaller in amplitude than in A. C, records obtained 4 0 min after cardiac arrest. Gain of PT response 2 x that in B.
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L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE (Fig. 5A; open circles and dotted line) is similar in time course to that obtained by stimulating the subcortical white matter, after removal of the grey matter (Fig. 5B). This shows that the brief depression is a property of the axons, and the longer-lasting depression found before the magnesium was applied was likely to be synaptic in origin. In summary we have found the following. (1) There are late waves (up to 10 msec) in the response obtained by stimulating the cortical white matter. (2) When the cortical post-synaptic components of the somatosensory evoked response are abolished by asphyxia the later pyramidal waves are still present. These findings show that late waves can result from discharge in slowly conducting axons, and do not necessarily indicate the involvement of indirect synaptic pathways in the activation of pyramidal tract cells. (3) Correspondence was found between recovery cycles with Mg present, and those obtained by stimulating subcortical white matter; the difference between the two curves of Fig. 5A indicates that the magnesium had blocked some cortical synaptic activity of an inhibitory nature. Cortical activity. The effectiveness of topically applied MgCl2 in blocking all synaptic transmission at a particular depth depended on the strength of the solution applied to the pial surface and the length of time after the initial application. In the rat it was shown that the MgCl2 could abolish the direct cortical response, the somatosensory evoked potential, the transcallosal response, and spontaneous firing of neurones. The most resistant response was the mass potential evoked by stimulating the contralateral forepaw (Bindman & Milne, 1977); therefore it was used as an indicator of the progressive blocking action of the magnesium and was recorded at intervals throughout all the experiments. However, it was found in both the cat and the rat that all signs of the mass response were never completely obliterated; a small residual wave of up to 50TV in amplitude remained no matter how strong the magnesium solution nor for how many hours it was applied. We assumed that this was due to activity in thalamocortical fibres (Adrian, 1941). The recordings obtained during one experiment are shown in Fig. 3B. While it was found that 15 m-mole/l. solutions would in fact block activity in the superficial cortex (the direct cortical response in layer I), stronger solutions were necessary to ensure complete block at greater depths than this. We have used MgCl2 solutions varying from 250 n-mole/I. to 1 mole/l. to obtain block of synaptic activity. In all the experiments illustrated, 1 mole/l. solutions were used to ensure block throughout the sensorimotor cortex. These solutions are hypertonic at the cortical surface; however a hypertonic NaCl solution on the cortical surface does not greatly alter the somatosensory evoked response recorded below the cortical surface in the rat (Milne, 1978). A stable control record from the pyramidal tract and an after-effect following antidromic conditioning were also obtained in the cat, with hypertonic NaCl 1-5 mole/l. on the cortical surface. Criteria used to show that MgCl2 blocked synapses. The criteria routinely employed to show that the magnesium chloride solution had produced its maximum effect in the cortex beneath the test electrodes were: firstly that the test to cortical stimulation recorded from the medullary pyramidal tract had reached a minimum and constant value, secondly that the contralateral forepaw evoked response consisted only of a small residual positive wave of constant amplitude. 466
response
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AB 0 100
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. Control * > lh after MgCI2 I l S.E.M. of mean 0
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Fig. 5. Recovery cycle technique used to differentiate between excitability changes due to direct and to synaptic activation of pyramidal tract cells. Stimulating and recording situation as for Fig. 3A. Following the conditioning shock, test shocks were applied through the same electrodes at increasing intervals. The amplitudes of the responses to the test shocks were measured (waves of peak latencies of about 0-7 msec and about 2.7 msec), and were calculated as a percentage of the responses to the conditioning shock. Means and ± 1 s.E. of mean were plotted for eaoh c-t interval. A, recovery cycle of wave of peak latency 0-65-0-75 msec. Filled circles and continuous line - control. Open squares and dotted line more than 1 hr after application of MgCl2 (1 mole/l.) to the cortical surface. B, recovery cycle of wave of peak latency 0*7 msec following shock to white matter below motor cortex. Note that there is no significant difference between this curve and the dotted line on graph (A) (after MgCl2), although precisely the same axons are unlikely to be involved in the two cases.
2. After-effects with MgCl2; prolonged increases in excitability The amplitude of the mass axonal response elicited by an electrical stimulation of the cortical surface and recorded at the medullary pyramids was used as the measure of excitability of the population of pyramidal tract neurones. An increase in the amplitude or area of the mass response was taken to indicate that a larger proportion of the pyramidal tract cell population was excited by the constant test stimulus given to the cortical surface. An example of the long-lasting increase in excitability of pyramidal tract cells which follows a brief period of antidromic conditioning is shown in Fig. 6. The inset in the graph shows the change in the size of the axonal population response produced by the conditioning period. The three dotted lines are control recordings, each the average of thirty-two consecutive responses, taken at three intervals within a 40 min period. The two continuous lines are recordings made after the conditioning which consisted of 5 min of antidromic stimulation (100/sec; 0 5 msec pulse width). The graph shows the peak-to-peak amplitude of the wave plotted against time but as can be seen from the inset all components of the response are increased by about 30 %.
468 L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE In eleven animals in which MgC12 1 mole/I. was used, 13 statistically significant, long-term, maintained increases were found. There were no long-term decreases in these experiments. We have excluded four experiments in which no increase was obtained, but either supramaximal stimulation was used (two), or occlusion could not be obtained (two).
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PROLONGED CHANGES IN EXCITABILITY OF PYRAMIDAL TRACT NEURONES IN THE CAT: A POST-SYNAPTIC MECHANISM
By LYNN J. BINDMAN, 0. C. J. LIPPOLD AND ALEX R. MILNE* From the Department of Physiology, University College London, London WClE 6BT
(Received 23 February 1978) SUMMARY
1. Prolonged changes in the excitability of cortical neurones can be produced by altering their firing rates for brief periods. In the anaesthetized cat, increased firing of pyramidal tract cells induced by trains of antidromic conditioning shocks led to increases in cell excitability, as measured by the size of the mass response at the medullary pyramid to test shocks applied to the cortical surface. We have shown in two ways that post-synaptic mechanisms could be responsible. 2. In one experimental design, MgC12 solution (1 mole/l.) was applied to the cortical surface in order to block synaptic activity throughout the cortical depth. Following antidromic conditioning trains, cell excitability was increased; the size of the mass response was up to 30 % larger than the control values. This persisted undiminished for up to 3 hr. 3. In the second experimental design, synaptic activity was not blocked, but we compared the effects of antidromic plus synaptic activation of pyramidal tract cells with the effects of synaptic activation alone. Antidromic plus synaptic activation was obtained by applying conditioning trains to the pyramidal tract at the medulla ipsilateral to the cortical test shock; prolonged increases in the ipsilateral response to the test shock were produced. Synaptic activation alone was obtained by the same conditioning trains, but in those cells whose axons projected into the contralateral pyramidal tract; prolonged increases in the contralateral response to the cortical test shock were never seen. In many instances prolonged decreases in excitability were found. 4. We conclude that prolonged increases in excitability of pyramidal tract cells can occur in the absence of any synaptic input, demonstrating that the underlying mechanism is post-synaptic; this does not preclude the action of synaptic mechanisms when synaptic transmission is not blocked. INTRODUCTION
Long lasting changes in the excitability of neurones in the central nervous system are of considerable interest because of their possible connexion with the processes involved in information storage. Although such changes have been observed in the cerebral cortex, we still know little about the mechanisms involved at the cellular level. An important preliminary step in the elucidation of the underlying mechanisms * M.R.C. Scholar. Present address: Department of Physiology, School of Veterinary Science, Park Row, Bristol BS1 5LS.
458 L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE is to ascertain whether the site of prolonged changes is at presynaptic terminals, in the post-synaptic membrane or elsewhere in the neurone. Many brief experimental procedures that increase the firing rate of cortical neurones have been found to give rise to changes in excitability that last undiminished for several hours (see Lippold, 1970; Bliss & L0mo, 1973; McCabe, 1972, 1976); in all these experiments, an increase in synaptic activity could have been involved. In the present experiments we have examined the possibility that long-term changes in excitability could arise in the absence of synaptic activity. We investigated whether the post-synaptic excitability of pyramidal tract cells in the anaesthetized cat could be altered by a brief period of repetitive antidromic activation. The difficulty in using this experimental approach is that stimulation of the axons of pyramidal tract neurones, at the medullary pyramid, will not only activate the cell bodies antidromically but the impulses will also travel along recurrent collateral branches of the axons and lead to excitatory or inhibitory synaptic effects (Stefanis & Jasper, 1964; Armstrong, 1965). To overcome this problem, we devised two experimental designs. In the first, all synaptic activity in the cerebral cortex was blocked by bathing the pial surface with MgCl2 solutions of suitable concentration (Bindman & Milne, 1977). In the second experimental design, synaptic activity was not blocked, but we compared the effects of antidromic plus synaptic activation of pyramidal tract cells with the effect of synaptic activation alone. It was possible to make this comparison because some of the axons of the pyramidal tract cells cross the mid line and travel in the contralateral medullary pyramid (Fig. 1). Hence stimulation of the ipsilateral pyramidal tract affects cortical cells whose axons project into the ipsilateral tract both via synapses and antidromically, while it affects via synapses alone those cells whose axons run in the contralateral tract. We found that the excitability of pyramidal tract cells (monitored by the amplitude of mass axonal responses to 'test' shocks applied to the cortex) was increased following brief periods of antidromic 'conditioning' stimulation of the pyramidal tract in the presence of Mg, i.e. when synaptic activity was blocked. We also found prolonged increases in the excitability of pyramidal tract cells in the second experimental design, but only in the situation where cells were activated both antidromically and via the collateral branches of the axons. In the situation where no antidromic excitation occurred, namely where cells were only influenced synaptically from collaterals, no long-term increases were observed. However in the latter experiments involving only synaptic pathways, long-lasting decreases in the excitability of the pyramidal tract cell population were found. Preliminary accounts of this work have been communicated to the Physiological Society (Bindman, Lippold & Milne, 1976a, b; Bindman & Milne, 1977). METHODS
Surgical procedure Experiments were performed on 124 cats of mixed stock and either sex, anaesthetized with i.P. injections of urethane (360 g/l. solution; 1-5 g/kg body wt.). This anaesthetic was used because the cats remained fully anaesthetized at a steady level for at least 20 hr with no supplementary dosage, and cortical excitability is stable (Lippold, 1970). The trachea, femoral artery and femoral vein were routinely cannulated. Structures in the neck were dissected from the ventral side to expose the bone at the base of the brain overlying the medulla. A small hole
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was drilled in the bone to one side of the mid line, avoiding the basilar artery and its branches. The hole was enlarged to expose most of both medullary pyramidal tracts. The skull overlying the cerebral cortex was then exposed, and a trephine hole made over the parietal cortex. The bone was removed to expose part of the precruciate cortex, and much of the parietal region, making a hole of diameter approximately 2 cm. The dura mater was removed, and the cortical surface covered with cotton wool soaked in sodium chloride (9 g/l.) for protection, while a watertight cup was constructed above the cortical surface. The nasal passages were filled with dental wax. A Perspex cup was cemented to the dried bone surrounding the exposed cortex
Ipsilateral
ContralateralI
RRCl C
AC
Fig. 1. Diagram showing position of recording and stimulating electrodes. Three pyramidal tract cells with their axons extending to the medullary pyramids are shown, together with recurrent collaterals which may be either excitatory or inhibitory. Note that one axon crosses the mid line to become contralateral. The cortical electrode rests on the pre-central gyrus to deliver test shocks between it and an indifferent electrode (not shown) on the parietal cortex. On each pyramidal tract at the medulla lies a recording electrode (R), the indifferent electrodes being sited on adjacent bone. Pairs of conditioning electrodes are also shown (C), sited on the tract caudal to the recording electrodes. The cup, cemented to the bone, is watertight and kept at a pre-determined pressure using a reservoir. The electrode for recording the evoked response is not shown.
using methyl methacrylate cement; the cotton wool was then removed. Stimulating and recording electrodes were placed in position (see below) and fixed rigidly to the inner wall of the cup. Their connecting leads ran through a hole in the side wall. The top of the cup and the hole in the side were sealed and it was filled with NaCl (9 g/l.) through its side tubes (Fig. 1). The pressure of fluid in the cup was kept as close to atmospheric as possible at this stage by adjustment of the level of a reservoir of the NaCl solution. The head was then fixed rigidly into a head-holder mounted on a spindle, and the whole animal was rotated into the supine position. The dural membrane overlying the medullary pyramids was then removed, and the fluid in the reservoir adjusted to the level of the exposed
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medullary surface. This adjustment was found to be important to ensure that the blood supply of the region was not impaired. The cortical surface could be irrigated with fluids of any desired composition via the reservoir, cup and outlet tube.
Recording and stimulating electrodes Synaptic activity in the cortex was monitored by recording the responses evoked by contralateral forepaw stimulation. An insulated tungsten microelectrode of exposed tip diameter about 10 ,Am was used. The cortical surface was explored while weak electric shocks were delivered to the skin of the contralateral forepaw, once every 2 sec. The electrode tip was pushed about 1-5 nun below the pial surface in the region where the largest surface response was evoked. The deep-negative wave was recorded at intervals throughout the experiment, and sixteen or thirty-two such responses were averaged using a small computer (Biomac 1000). In some experiments glass micropipettes, tip diameter about 1 jsm, filled with NaCl (100 g/l.), were used to record spontaneously occurring spikes. Test shocks were provided by non -polarizable electrodes on the pial surface, exciting pyramidal tract neurones orthodromically. These consisted of polyethylene tubes, internal diameter 3 mm, filled with agar gel made up in NaCl (9 g/l.). Chlorided silver wire was coiled inside the tube, and at one end it was soldered to insulated wire. That end of the tube was sealed with dental wax to insulate it. One of these electrodes was placed in contact with the pial surface of the precruciate motor cortex, the other placed at the caudal end of the exposed area of cortex. Pyramidal tract responses to cortical stimulation were recorded at the medullary pyramids, using different types of electrode depending on the purpose of the experiment. Insulated tungsten micro-electrodes were employed in experiments measuring recovery cycles and the effect of MgCI2 application to the cortex. For experiments monitoring the excitability of pyramidal tract cells for long periods of time, non -penetrating wire electrodes were found to be more convenient. These consisted of a central conducting core of 120 ,m nichrome wire insulated with polyethylene, of external diameter 600 _tm. The ends of these electrodes were squared off under the microscope using a sharp cutting edge, so that an insulating ring was present round the central wire when it was pressed on the medullary surface. They were sufficiently springy to take up small movements of the medulla without either losing contact or penetrating. The stimulating electrodes were of similar construction. Up to six electrodes were positioned on the two pyramids with individual adjustments for each of them (Fig. 1). Recording electrodes were rostral, the indifferent of each pair being usually on the adjacent bone to achieve a monophasic response. A pair of stimulating electrodes was placed across the tract approximately 2 mm apart and at least 4 mm caudal to the recording electrode. Their position was adjusted with respect to the recording electrode so that the same axons were being stimulated by the pyramidal tract shock as were excited as a result of the cortical shock. This was tested by occlusion (see below). When all these electrodes had been sited correctly, the whole cavity was filled with agar gel made up in NaCl (9 g/l.) solution. This was necessary not only to prevent relative movement between the tracts and electrodes but also to stop any flow of MgCl2 solution from the cortical cup to the medulla.
Stimulus parameters Stimuli for eliciting the primary somatosensory evoked potential were delivered via two stainless-steel needles inserted under the skin of the contralateral forepaw. Shocks were given every 2 see at intervals throughout the experiment and were 0-05 msec duration and just subthreshold for producing muscle twitches before the cat was immobilized. Cortical test shocks were given every 2 see or every 5 sec, the stimulus width being 0-05 or 0-10 msec. The voltage was gradually increased until a just maximal early wave was obtained at the pyramid. This stimulus strength was used in the occlusion test. The strength was then decreased to a voltage eliciting a response of between 60 and 80% of the value eliciting the maximal response, for the remainder of the experiment. The stimuli were square waves generated by an isolated stimulator (Devices Mk IV); stimulus current as monitored by the voltage drop across an external resistor in series with the preparation did not vary throughout the experiment. Polarity was surface positive for the electrode overlying the precruciate cortex.
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Pyramidal tract conditioning shocks were usually delivered as gated frequency trains, to allow the recording of a response to the cortical test shock within the conditioning period. Parameters are given in the legends to the Figures.
Electrical recording and data proceesing Differential high impedance input circuits and direct coupled amplifiers were used. In a few experiments photographs of the responses were taken; usually averages were obtained using a Biomac 1000 and were permanently stored on punched paper tape. A Linc 8 computer was used to plot these off line. Many experiments were recorded continuously on FM magnetic tape.
Occuaion To check that the cortical test and the medullary conditioning stimuli were affecting the same, or an overlapping, population of pyramidal tract cells, a test for occlusion was carried out. Figure 2 A shows, top line, the sum of four mass responses recorded from the medullary pyramid elicited by a cortical test shock given at time Cx. When a pyramidal tract shock was given 1 msec before the cortical test shock, at time PT, the antidromic axonal wave could be seen immediately following the downward deflexion of the stimulus artifact but the response to the subsequent cortical test shock was considerably reduced (second line). When occlusion could not be obtained, electrode positions were altered until it could. In two experiments where occlusion did not occur, an after-effect due to the conditioning shocks was not obtained. In experiments in which MgCl2 was not used, and the effects of stimulating the ipsilateral pyramidal tract were compared with the effects of stimulating the contralateral one, it was necessary to ensure that the stimulating current did not spread from one side to the other. This was checked, as shown in Fig. 2B, right-hand column, by demonstrating that no occlusion occurred between the stimulus on one side and the response on the other side. A second check was to see whether current spread could directly activate axons on the contralateral side, as can be seen happening to a small extent in the lowest left-hand trace in Fig. 2B, i.e. using the 'strong' PT shock. Where either occlusion of the response in the other tract, or current spread to excite axons on the other side was observed, tha current was reduced or the stimulating electrodes were repositioned.
Stability of the recording station Many factors were controlled in order to obtain stable recordings. Movements of the cat when the pyramidal tract was repetitively stimulated were prevented by i.v. infusions of gallamine triethiodide in quantities sufficient to abolish spontaneous respiratory movements and evoked muscle twitches. Following the use of gallamine, ventilation was maintained with a positive-pressure respiration pump, at a tidal volume such that the end-expiratory Pco; remained constant. The use of I.v. gallamine and MgCl2 applied to the cerebral cortex tended to lower the blood pressure; if the systolic pressure fell below 80 mmHg the experiment was discarded. Dehydration of the animal was countered by i.v. infusions of dextrose-saline (4% and 0-18% w/v respectively). The PE cO2 was measured with a continuous sampling infra-red analyser. Arterial blood pressure was measured with a Bell & Howell transducer, and a continuous record of both and blood pressure was made throughout the experiment using Devices preamplifiers and two-channel pen recorder. Rectal temperature was measured with a mercury-in-glass thermometer, and was maintained at 37 'C by using a heating blanket, the current of which was controlled by feed-back from a thermistor in the rectum (Diete-Spiff, Ikeson & Read, 1962). Movement of the recording and stimulating electrodes in the medullary region was minimized by filling the exposed cavity with a gel of agar made up in NaCl (9 g/l.). The use of slightly flexible, individually adjustable wires surrounded by polyethylene was also of importance; in early experiments using rigid tungsten electrodes we found they tended to penetrate the medullary surface. It was necessary to keep the level of fluid in the reservoir constant throughout the experiment because any change in fluid pressure caused a change in the position of the medulla relative to the electrodes.
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Occasionally small increases in systemic blood pressure were produced by the onset of the trains of conditioning stimuli at the medulla. However these changes in blood pressure decayed by the end of the conditioning train, and wera not related to the production of after effects; after effects were seen when no rises in blood pressure were produced, conversely increases in blood pressure were not necessarily associated with the production of after effects. CX
CX
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Fig. 2. Tests for occlusion. A illustrates procedure used to ensure that the sane population of pyramidal tract cells are excited by both test and conditioning shocks. Records were taken from the pyramidal tract; each trace shown is the sum of four of these. Top trace: response to cortical test shock given at Cx. Second trace: identical cortical shock but preceded by shock delivered to ipsilateral pyramidal tract 1 msee earlier, at PT. Third trace: response to pyramidal tract shock alone. Bottom trace: repeat of cortical test shock alone. The strength and duration of the electrical stimuli were those used throughout the subsequent experiment. Voltage calibration 05 mV, time bar 1 msec. B, similar procedure to that shown in A but to check on the current spread from the conditioning electrodes directly to the opposite pyramid. In each frame the upper trace is of four superimposed ipsilateral pyramidal tract responses, and the lower trace is of the simultaneously recorded response in the contralateral pyramidal tract. Polarity of lower trace is reversed with respect to upper. Left-hand column strong, right hand weak pyramidal tract shocks. Top pair of traces: response to cortical test shock alone. Voltage calibration 02 mV, time bar 1 msec. Middle pair of traces: cortical test shock is preceded by conditioning shock given to ipsilateral pyramidal tract 1 msec earlier. Note, in left-hand column, that the ipsilateral pyramidal response shows occlusion of the earliest component, although not of later waves. The contralateral pyramidal tract response shows no obvious occlusion. Bottom pair of traces: response to pyramidal tract shock alone. Note, in left-hand column, the strong pyramidal tract shock used has resulted in some current spread to the contralateral tract. The weak shock used in the right-hand column does not spread to excite fibres in the contralateral tract, but neither does it produce complete occlusion of the earliest ipsilateral component. Same experiment as Fig. 8.
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A CF
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*I~~~~~~~~~~~~~~. 7 9. _
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Fig. 3. A, the effect of magnesium, topically applied to the cerebral cortex, upon the response recorded from the pyramidal tract at the medullary pyramid. The motor cortex was stimulated using 36 V; pulse width 0-05 msec (90% maximal for peak at 1 msec). Top trace: control response. Time scale 1 msec, voltage calibration 0-5 mV. Middle trace: more than 1 hr after MgCl2, 1 mole/l., applied. Lower trace: at the end of the experiment, as much as possible of the grey matter was removed, and a concentric needle electrode was used 4 mm below the surface thus exposed, to stimulate white matter. Response recorded at the pyramid. Note the presence of a prolonged response lasting for at least 6 msec after the stimulus. Stimulus parameters as before but the geometry of the excitable elements and the current density at them are not comparable. The position of the tip of the stimulating electrode was confirmed as being in the white matter histologically. Voltage calibration 0-2 mV. B, the action of MgCl2 solution on the cortical response evoked by contralateral forepaw stimulation (CF). Traces are sums of thirty-two separate responses, which were recorded at a depth of about 2 mm below the pia. Negativity upward deflexion. Trace 1, control; 2-7, cup filled with MgCl2 solution (1 mole/l.) from 6.53 p.m. Note gradual decrease in peak-to-peak amplitude. Due to difficulties with computer write-out, trace 5 is half the gain of the other traces. Numbers on the left show time course of this effect. These results are from the experiment shown in Fig. 7. Pyramidal tract stimulated at 11.20 p.m.
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L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE RESULTS
1. Monitoring the synaptic blocking action of MgCl2 Since the objective of this investigation was to see whether long lasting changes in neuronal excitability could be induced in the absence of synaptic utilization, it was of importance to be certain that in experiments when MgCl2 was applied to the cortical surface all synaptic activity within the underlying grey matter had been blocked. A series of experiments was carried out to investigate the actions of MgCl2 topically applied to the intact cortical surface in various strengths, on both cats and rats. The experiments on rats are reported elsewhere (Bindman & Milne, 1977; Milne, 1978). Waves in pyramidal tract response. Because the mass pyramidal tract response was being used to monitor the excitability of the cells in the cortex, our first concern was to investigate the effect of topically applied magnesium on this response. When a brief, submaximal, cortical shock is used (0.05 msec; 36 V) the response in the pyramidal tract consists of a major deflexion of peak latency about 1 msec, and smaller later waves, continuing for 5-10 msec (Figs. 2B, 3A top trace). The later waves are smaller in this situation than when stronger shocks of longer duration are employed. The question arises as to whether the later waves elicited by the brief weak shocks are equivalent to the 'I' waves described by Patton & Amassian (1954), which are thought to be the result of synaptic activation of pyramidal tract cells by the cortical shock. Alternatively the later waves could be the result of direct electrical stimulation of smaller cells having axons with a slower conduction velocity (Oshima, 1969), that have been shown by anatomical techniques to comprise a large proportion of the pyramidal tract fibres (van Crevel & Verhaart, 1963). The action of MgCl2 topically applied to the cortex was to reduce the over-all amplitude of the response (Fig. 3A, middle trace) but not to curtail the duration to any marked extent. The effect of stimulating cortical white matter alone (following the removal of the grey matter) can be seen in Fig. 3A (bottom trace). It is clear that late waves can result from discharges in slow conducting axons. Confirmation that late waves in the pyramidal tract following a cortical test shock can be the result of direct stimulation of cells with slowly conducting axons was obtained by simultaneous monitoring of the pyramidal tract waves and the synaptically evoked response to forelimb stimulation after asphyxiation of the animal. First the post-synaptic components, then all signs of the contralateral forepaw evoked response disappeared by 2 min, whereas the later components of the pyramidal tract response persisted, although reduced in amplitude (Fig. 4), for 6-5 min after respiration ceased (4 min after the heart stopped). Changes in excitability of pyramidal tract cells can be investigated by giving pairs of shocks separated by increasing intervals to the cortical surface. The first shock excites the cells directly and initiates synaptically mediated activity, which may be excitatory or inhibitory. As can be seen from Fig. 5A (filled circles, continuous line) the first shock leads to depression of the second response until the two shocks are 8 msec apart. Although the applied magnesium did not modify the pyramidal tract response markedly (Fig. 3 A), nevertheless the recovery cycle in the presence of Mg indicated that synaptic transmission had been blocked by it. The recovery cycle
POST-SYNAPTIC CHANGES IN PT CELLS w
C.r
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Fig. 4. The effect of asphyxia on the cortical response to forepaw stimulation and on the pyramidal tract response. A, control. Upper trace shows pyramidal tract response to shock applied to cortical surface. Negative downwards. D wave retouched. Calibration bar 0-2 mV. Lower trace shows mass potential evoked 2 sec later by stimulation of the contralateral forepaw, recorded at a depth of 1 mm in the cortex. Negative upwards. Calibration bar 1 mV. Each stimulus applied at onset of trace. B, records obtained 4 min after pump was turned off in paralysed preparation, which was 1-5 min after cardiac arrest. Lower trace shows small residual wave in evoked response, presumably due to the arrival volley in the thalamocortical afferents. Upper trace shows early and late waves in the pyramidal tract, smaller in amplitude than in A. C, records obtained 4 0 min after cardiac arrest. Gain of PT response 2 x that in B.
465
L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE (Fig. 5A; open circles and dotted line) is similar in time course to that obtained by stimulating the subcortical white matter, after removal of the grey matter (Fig. 5B). This shows that the brief depression is a property of the axons, and the longer-lasting depression found before the magnesium was applied was likely to be synaptic in origin. In summary we have found the following. (1) There are late waves (up to 10 msec) in the response obtained by stimulating the cortical white matter. (2) When the cortical post-synaptic components of the somatosensory evoked response are abolished by asphyxia the later pyramidal waves are still present. These findings show that late waves can result from discharge in slowly conducting axons, and do not necessarily indicate the involvement of indirect synaptic pathways in the activation of pyramidal tract cells. (3) Correspondence was found between recovery cycles with Mg present, and those obtained by stimulating subcortical white matter; the difference between the two curves of Fig. 5A indicates that the magnesium had blocked some cortical synaptic activity of an inhibitory nature. Cortical activity. The effectiveness of topically applied MgCl2 in blocking all synaptic transmission at a particular depth depended on the strength of the solution applied to the pial surface and the length of time after the initial application. In the rat it was shown that the MgCl2 could abolish the direct cortical response, the somatosensory evoked potential, the transcallosal response, and spontaneous firing of neurones. The most resistant response was the mass potential evoked by stimulating the contralateral forepaw (Bindman & Milne, 1977); therefore it was used as an indicator of the progressive blocking action of the magnesium and was recorded at intervals throughout all the experiments. However, it was found in both the cat and the rat that all signs of the mass response were never completely obliterated; a small residual wave of up to 50TV in amplitude remained no matter how strong the magnesium solution nor for how many hours it was applied. We assumed that this was due to activity in thalamocortical fibres (Adrian, 1941). The recordings obtained during one experiment are shown in Fig. 3B. While it was found that 15 m-mole/l. solutions would in fact block activity in the superficial cortex (the direct cortical response in layer I), stronger solutions were necessary to ensure complete block at greater depths than this. We have used MgCl2 solutions varying from 250 n-mole/I. to 1 mole/l. to obtain block of synaptic activity. In all the experiments illustrated, 1 mole/l. solutions were used to ensure block throughout the sensorimotor cortex. These solutions are hypertonic at the cortical surface; however a hypertonic NaCl solution on the cortical surface does not greatly alter the somatosensory evoked response recorded below the cortical surface in the rat (Milne, 1978). A stable control record from the pyramidal tract and an after-effect following antidromic conditioning were also obtained in the cat, with hypertonic NaCl 1-5 mole/l. on the cortical surface. Criteria used to show that MgCl2 blocked synapses. The criteria routinely employed to show that the magnesium chloride solution had produced its maximum effect in the cortex beneath the test electrodes were: firstly that the test to cortical stimulation recorded from the medullary pyramidal tract had reached a minimum and constant value, secondly that the contralateral forepaw evoked response consisted only of a small residual positive wave of constant amplitude. 466
response
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Fig. 5. Recovery cycle technique used to differentiate between excitability changes due to direct and to synaptic activation of pyramidal tract cells. Stimulating and recording situation as for Fig. 3A. Following the conditioning shock, test shocks were applied through the same electrodes at increasing intervals. The amplitudes of the responses to the test shocks were measured (waves of peak latencies of about 0-7 msec and about 2.7 msec), and were calculated as a percentage of the responses to the conditioning shock. Means and ± 1 s.E. of mean were plotted for eaoh c-t interval. A, recovery cycle of wave of peak latency 0-65-0-75 msec. Filled circles and continuous line - control. Open squares and dotted line more than 1 hr after application of MgCl2 (1 mole/l.) to the cortical surface. B, recovery cycle of wave of peak latency 0*7 msec following shock to white matter below motor cortex. Note that there is no significant difference between this curve and the dotted line on graph (A) (after MgCl2), although precisely the same axons are unlikely to be involved in the two cases.
2. After-effects with MgCl2; prolonged increases in excitability The amplitude of the mass axonal response elicited by an electrical stimulation of the cortical surface and recorded at the medullary pyramids was used as the measure of excitability of the population of pyramidal tract neurones. An increase in the amplitude or area of the mass response was taken to indicate that a larger proportion of the pyramidal tract cell population was excited by the constant test stimulus given to the cortical surface. An example of the long-lasting increase in excitability of pyramidal tract cells which follows a brief period of antidromic conditioning is shown in Fig. 6. The inset in the graph shows the change in the size of the axonal population response produced by the conditioning period. The three dotted lines are control recordings, each the average of thirty-two consecutive responses, taken at three intervals within a 40 min period. The two continuous lines are recordings made after the conditioning which consisted of 5 min of antidromic stimulation (100/sec; 0 5 msec pulse width). The graph shows the peak-to-peak amplitude of the wave plotted against time but as can be seen from the inset all components of the response are increased by about 30 %.
468 L. J. BINDMAN, 0. C. J. LIPPOLD AND A. R. MILNE In eleven animals in which MgC12 1 mole/I. was used, 13 statistically significant, long-term, maintained increases were found. There were no long-term decreases in these experiments. We have excluded four experiments in which no increase was obtained, but either supramaximal stimulation was used (two), or occlusion could not be obtained (two).
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