Exp. Brain Res. 26, 443-461 (1976)
Experimental Brain Research 9 by Springer-Verlag1976
Further Study on the Excitation of Pyramidal Tract Cells by Intracortical Microstimulation H. Asanuma, A. Arnold 1 and P. Zarzecki The Rockefeller University, 1230 York Avenue, New York, N.Y. 10021, USA
Summary. The effective spread of stimulating current for pyramidal tract (PT) cells and fibers was studied using a method of cancelling the shock artifacts and the following results were obtained: 1. The excitability of PT axon collaterals was as high as that of PT cells. 2. These axon collaterals extended as far as 1.0 mm horizontally from the PT cells. 3. The low threshold area for activation of a given PT cell was as wide as 3 - 4 mm 2 on the surface of the cortex. 4. Intracortical microstimulation (ICMS) delivered to the PT cell layer produced direct (D) and indirect (I) descending volleys in the pyramidal tract, but ICMS to the superficial layer (III) produced only I-waves. 5. These I-waves grew significantly larger after 15-20 msec from the start of the train of stimuli. 6. It is concluded that either surface stimulation, or short train of ICMS is inadequate for delineating fine localization of motor function within the cortex. Longer train ( 3 0 - 4 0 m s e c ) with high frequency pulses (300-400 cy/sec) can produce muscle contraction with much smaller currents, increasing the accuracy of measuring the localization of motor function.
Key words: PT cells - ICMS - Efferent zones Introduction During the past decade, the technique of intracortical microstimulation (ICMS) has revealed many new aspects of cortical motor function (Asanuma, 1975). Because the effect of ICMS is substantially more focal than that of surface stimulation, it was possible to reveal a finer topographical localization of motor function than had been reported. Using the ICMS technique, it has been shown that there are small areas within the depth of the motor cortex, i.e., cortical efferent zones, where low intensity ICMS elicits contraction of individual 1 Present address: Department of Psychology, UCLA, Los Angeles, Calif. 90024, USA
444
H. Asanumaet al.
muscles (Asanuma et al., 1968; Asanuma and Ros6n, 1972; Murphy et al., 1975; Nieoullon and Rispal-Padel, 1976). Recently, Andersen et al. (1975) reinvestigated the localization of cortical motor function using stronger ICMS. Subsequently, Jankowska et al. (1975b) studied the same problem using surface stimulation and recording intracellularly from motoneurons. Both groups reported that the cortical areas which upon stimulation produced effects on a given muscle or a motoneuron were wider than the cortical efferent zones which had been reported previously (Asanuma and Ros6n, 1972). The difference in the interpretation of the results, however, might have been derived from an incomplete understanding of exactly which cortical elements are activated by weak or strong ICMS or surface stimuli and of the effective spread of the stimulating current. A previous study by Rosenthal et al. (1967) had demonstrated that cortical surface stimulation can activate pyramidal tract (PT) cells directly, but with some delay after the stimulation. They suggested that the stimulation excited axon collaterals which in turn activated the cell bodies and/or the initial segments. Subsequent study by Stoney et al. (1968) had suggested that ICMS can activate PT cells primarily from a small region near the cell body. Because of technical limitations, the possibility of direct activation of axons and axon collaterals of these ceils was not studied. Technical improvements since the report of Stoney et al. (1968) now allow us to reinvestigate this problem with greater ease. It will be shown that surface stimulation and ICMS may activate PT ceils as well as their axon collaterals making interpretation of the results more complicated than has been proposed previously. Another problem that has to be examined is the effectiveness of a train of 1CMS in relation to its duration. Asanuma and co-workers have demonstrated the existence of radially arranged low threshold areas in the motor cortex where ICMS with long trains (10 or more pulses) of low amplitude pulses (less than 20 ~tA) produce contraction of individual muscles in the cat (Asanuma and Ward, 1971) and the monkey (Asanuma and Rosdn, 1972). Andersen et al. (1975), using short trains (6 pulses), could not confirm the existence of such small, radially oriented efferent zones in the baboon. In particular, they reported that ICMS was ineffective in the superficial layers of the cortex. We have reported elsewhere (Asanuma and Arnold, 1975) that their failure to reproduce these results is, in part, due to noxious effects of large currents that they used. In this study, we confirm our previous report and in addition demonstrate the difference in the effect of long and short trains applied to the motor cortex. These results will be discussed in relation to the branching of the pyramidal tract fibers in the spinal cord (Shinoda et al., 1976) as well as to the results reported by Jankowska et al. (1975a, b) who stimulated the surface and also the depth of the cortex.
Methods The experiments consist of two different procedures and the methods will be described in separate series.
Excitation of PT Cells
445
Part 1: Single Pulse Stimulation The results were obtained from ten cats of either sex. T h e y were anesthetized with pentobarbital sodium (Nembutal; 35 mg/kg, i.p.). T h e medullary pyramid was exposed via a ventral approach for stimulation and the cisterna m a g n a was o p e n e d to prevent possible brain swelling during the experiments. The pericruciate cortex was exposed and a closed c h a m b e r installed over the craniotomy. A photograph was taken to m a p the locations of electrode insertions. Screws attached to the skull were clamped rigidly by a holder to prevent any m o v e m e n t of the head. A concentric stimulating electrode was placed on the surface of the medullary pyramid ipsilateral to the exposed cortex. Two tungsten microelectrodes (Stoney et al., 1968; tip size about 1 0 x 15 gm) were driven into the motor cortex at points close to each other. The angle of the two penetrations relative to each other was held constant using a closed c h a m b e r a r r a n g e m e n t similar to that shown in Figure 1 of A s a n u m a and R o s 6 n (1973). T h e lateral distance between the two electrodes was variable, to allow multiple penetrations with one electrode while recording a single cell with the other. Pyramidal tract (PT) cells were activated by 0.2 msec pulses applied to the pyramid, and the antidromic spikes were recorded in the ipsilateral m o t o r cortex with one of the two microelectrodes. Once a unitary spike was isolated a n d the cell identified as a PT cell, the other microelectrode was moved along a track in the adjacent cortical area, in the m a n n e r illustrated in Figure 2. This second microelectrode was used for both stimulation and recording. Cathodal pulses of 0.2 msec duration were delivered (with reference to an indifferent electrode in the temporal muscles) at intervals of 5 0 - 1 0 0 ~m along the track to activate the PT cell. Pulse duration of 0.2 msec was used throughout the experiments to facilitate the comparison of the results with the previous reports ( A n d e r s e n et al., 1975; A s a n u m a et al., 1968, 1972; Jankowska et al., 1975a, b). To determine whether ICMS activated the PT cell u n d e r study, the m e t h o d of Stoney et al. (1968) was used. This involved collision of the ICMS - initiated spike with the antidromic spike, which resulted in the disappearance of the antidromic spike recorded with the first electrode. W h e n the antidromic spike was abolished by the ICMS, the second microelectrode was switched to the recording m o d e to see if the same spike could also be recorded through the second electrode. Because of the stability of the closed chamber system, it was not difficult to record the same spike while m a k ing several consecutive penetrations with the second microelectrode as shown in Figure 2. Antidromic activation of PT cells was identified by a) an invariant response latency of 1.2 msec or less after stimulation of the pyramid, and b) the ability to follow 100 Hz pyramidal stimuli. Positive-going spikes were not used for the study because of their instability. Reconstruction of the electrode tracks was accomplished by making one or two small electrolytic lesions (10 ~a for 10 seconds) at m e a s u r e d depths along the tracks of both microelectrodes. T h e s e lesions were m a d e at the end of a series of examinations for a given cell, and the same area of the cortex was not used for further investigation. Distances between the several tracks of the stimulating microelectrode were m e a s u r e d from the photographic m a p of electrode insertions. Part 2: Repetitive Stimulation Seven cats were used. Five of t h e m were anesthetized with a mixture of oxygen (20 %), nitrous oxide (80%), and halothane (0.5-1.0%). T h e cisterna m a g n a was opened for fluid drainage, and a closed c h a m b e r installed over a craniotomy exposing the pericruciate cortex. T h e head was rigidly fixed with clamps attached to screws in the skull. All the w o u n d areas were infiltrated with a longlasting local anesthetic (Zyljectin, Abbott), and inhalation anesthetic was discontinued. A sedative dose of Nembutal (10 mg/kg) was injected intraperitoneally before the termination of inhalation anesthesia. Fine bipolar electrodes, insulated except at the tip, were inserted into various forearm muscles through the skin to record electromyograms (EMGs). A n o d a l surface stimulation with a silver ball electrode was used to determine the locations at which E M G s could be elicited at low threshold, then a tungsten microelectrode was inserted into such a low threshold area. Trains of cathodal pulses (relative to the indifferent electrode in the temporal muscles) were passed through the microelectrode in order to elicit E M G responses. T h e remaining two cats were used for recording pyramidal volleys elicited by cortical stimulation. This experiment was carried out u n d e r N e m b u t a l anesthesia (35 mg/kg). The motor cortex
446
H. Asanuma et al.
was exposed for stimulation and the cervical cord was exposed for recording. A tungsten electrode of larger size (exposed tip: 40 ~tmx90 ~tm) was inserted into the lateral corticospinal tract at the level of C-2 to record descending volleys elicited by the ICMS. The volleys were fed into an online averaging computor (FABRI-TEK, Model 1062) and displayed on an oscilloscope. At the end of each experiment the appropriate part of the brain was removed and fixed in 10 % formalin. The brain was then serially sectioned in a freezing microtome, and stained by the method of Kltiver and Barrera (1953).
Results Part 1: Single Pulse Stimulation In studies using stimulating and recording electrodes which are close to each other, a persistent problem is the large shock artifact which often obscures short latency responses. In a previous study in which the effect of intracortical microstimulation (ICMS) was studied (Stoney et al., 1968), this drawback was overcome by using a collision technique which placed the antidromic spikes after the test stimulation. However, there still remained difficulties in the method. When ICMS elicited synaptic activation of neurons around the recording electrode, the disappearance of the antidromic spikes of the target PT cell was obscured by the spikes of these neurons as shown in Figure 1D. H e n c e study had to be limited to the cases when ICMS produced minimal synaptic activity, i.e., when very weak ICMS excited the target PT cells from short distances. Later development of cancellation techniques by A s a n u m a and Ros6n (1973), however, succeeded in reducing the shock artifact drastically, enabling observation of the events immediately after the stimulation. Using this technique, it now becomes possible to reexamine the effect of ICMS m o r e thoroughly. Figure 1 illustrates examples of the results. As shown in Figure 1A, ICMS produces a large stimulus artifact even with a weak current of 6 ~ta, excluding the possibility of visualizing possible effects for nearly 1.0 msec after the stimulus. Stimulation of the medullary pyramid with suprathreshold intensity made it possible to determine the cortical threshold by observing the 50 % appearance of the antidromic spikes. Usage of the cancellation current, which is shown in Figure 1B, brought the base line to the original level immediately after the stimulus making possible the observation of the events following the stimulation. A question arises of whether the cancellation current changes the threshold for activating PT cells. It had been discussed that the cancellation current used with ICMS is unlikely to change the threshold for activating cortical neurons (Asanuma and Ros6n, 1973) and it had been shown that the cancellation current from the surface has little or no effect on the efficiency of activating afferent fibers in the cortex ( A s a n u m a et al., 1974). As had b e e n suggested, imposition of cancellation current to ICMS did not change the threshold for activating the PT cell as is evidenced by 50 % appearance of the same antidromic spikes (Fig. 1B). Several trials using compensation pulses of various duration (0.05-0.3 msec) revealed that within this range, the compensation pulse causes little or no change in the efficiency of the negative stimulating pulse (0.2 msec duration). This might be explained by assuming that the
Excitation of PT Cells
447
C Cx
Py
Py
B
Cx
Py
Py Cx
ii
14oov l msec
Fig. 1 A - D . Left: comparison of threshold for activation of a PT cell by ICMS with and without an anodal cancellation pulse. A Intracortical microstimulation (ICMS) without compensation current is followed by a suprathreshold shock to the pyramid. The antidromically activated spike is blocked about 50% of the time. B ICMS of the same intensity is followed by a brief anodal cancellation pulse. The firing index is changed very little if at all by the presence of the cancellation pulse. C and D illustrate the method used to establish that the ICMS activated the PT cell directly, rather than synaptically. C The suprathreshold pyramidal shock activates the cell antidromically. D The pyramidal shock is followed by the ICMS. Use of the cancellation pulse reduced the shock artifact so that the antidromic spike could be positioned within 0.8 msec after the ICMS. Note that ICMS blocked the antidromic spike about 50 % of the time. Further details are in the text
local response developed during the period of the stimulation (0.2 msec) is already powerful enough to withstand the anodal blocking effect produced by the cancellation current as has been discussed previously (Asanuma and Ros6n, 1973). Utilizing this technique, we could proceed to the following examination. Instead of delivering the pyramidal stimulation after the cortical stimulation, pyramidal stimulation was delivered prior to the cortical stimulation so that the antidromic spike was placed immediately after the ICMS as shown in Figure 1D. It has been shown that the minimum latency of the PSP's in cortical neurons following threshold ICMS is 0.8 msec (Asanuma and Ros6n, 1973). Since we always activated PT cells with threshold currents, the blockade by collision of the antidromic spikes within this latency must be produced by direct activation of the target cell. This interpretation is supported by the observation that the PT cells activated synaptically by cortical surface stimulation very rarely (one out of 26) discharged with latency of less than 0.9 msec even
448
H. Asanuma et al.
O-lO/Ja 1-25pa > 25pa
i
i
t
L
~
i
Fig. 2. Histological reconstruction of electrode tracks and sites where ICMS was delivered. The cell was recorded at the position shown by the recording electrode. Four penetrations (A-D) were made to activate the cell directly. The cell's spike could be recorded in track D at the depths at which weak ICMS (
Experimental Brain Research 9 by Springer-Verlag1976
Further Study on the Excitation of Pyramidal Tract Cells by Intracortical Microstimulation H. Asanuma, A. Arnold 1 and P. Zarzecki The Rockefeller University, 1230 York Avenue, New York, N.Y. 10021, USA
Summary. The effective spread of stimulating current for pyramidal tract (PT) cells and fibers was studied using a method of cancelling the shock artifacts and the following results were obtained: 1. The excitability of PT axon collaterals was as high as that of PT cells. 2. These axon collaterals extended as far as 1.0 mm horizontally from the PT cells. 3. The low threshold area for activation of a given PT cell was as wide as 3 - 4 mm 2 on the surface of the cortex. 4. Intracortical microstimulation (ICMS) delivered to the PT cell layer produced direct (D) and indirect (I) descending volleys in the pyramidal tract, but ICMS to the superficial layer (III) produced only I-waves. 5. These I-waves grew significantly larger after 15-20 msec from the start of the train of stimuli. 6. It is concluded that either surface stimulation, or short train of ICMS is inadequate for delineating fine localization of motor function within the cortex. Longer train ( 3 0 - 4 0 m s e c ) with high frequency pulses (300-400 cy/sec) can produce muscle contraction with much smaller currents, increasing the accuracy of measuring the localization of motor function.
Key words: PT cells - ICMS - Efferent zones Introduction During the past decade, the technique of intracortical microstimulation (ICMS) has revealed many new aspects of cortical motor function (Asanuma, 1975). Because the effect of ICMS is substantially more focal than that of surface stimulation, it was possible to reveal a finer topographical localization of motor function than had been reported. Using the ICMS technique, it has been shown that there are small areas within the depth of the motor cortex, i.e., cortical efferent zones, where low intensity ICMS elicits contraction of individual 1 Present address: Department of Psychology, UCLA, Los Angeles, Calif. 90024, USA
444
H. Asanumaet al.
muscles (Asanuma et al., 1968; Asanuma and Ros6n, 1972; Murphy et al., 1975; Nieoullon and Rispal-Padel, 1976). Recently, Andersen et al. (1975) reinvestigated the localization of cortical motor function using stronger ICMS. Subsequently, Jankowska et al. (1975b) studied the same problem using surface stimulation and recording intracellularly from motoneurons. Both groups reported that the cortical areas which upon stimulation produced effects on a given muscle or a motoneuron were wider than the cortical efferent zones which had been reported previously (Asanuma and Ros6n, 1972). The difference in the interpretation of the results, however, might have been derived from an incomplete understanding of exactly which cortical elements are activated by weak or strong ICMS or surface stimuli and of the effective spread of the stimulating current. A previous study by Rosenthal et al. (1967) had demonstrated that cortical surface stimulation can activate pyramidal tract (PT) cells directly, but with some delay after the stimulation. They suggested that the stimulation excited axon collaterals which in turn activated the cell bodies and/or the initial segments. Subsequent study by Stoney et al. (1968) had suggested that ICMS can activate PT cells primarily from a small region near the cell body. Because of technical limitations, the possibility of direct activation of axons and axon collaterals of these ceils was not studied. Technical improvements since the report of Stoney et al. (1968) now allow us to reinvestigate this problem with greater ease. It will be shown that surface stimulation and ICMS may activate PT ceils as well as their axon collaterals making interpretation of the results more complicated than has been proposed previously. Another problem that has to be examined is the effectiveness of a train of 1CMS in relation to its duration. Asanuma and co-workers have demonstrated the existence of radially arranged low threshold areas in the motor cortex where ICMS with long trains (10 or more pulses) of low amplitude pulses (less than 20 ~tA) produce contraction of individual muscles in the cat (Asanuma and Ward, 1971) and the monkey (Asanuma and Rosdn, 1972). Andersen et al. (1975), using short trains (6 pulses), could not confirm the existence of such small, radially oriented efferent zones in the baboon. In particular, they reported that ICMS was ineffective in the superficial layers of the cortex. We have reported elsewhere (Asanuma and Arnold, 1975) that their failure to reproduce these results is, in part, due to noxious effects of large currents that they used. In this study, we confirm our previous report and in addition demonstrate the difference in the effect of long and short trains applied to the motor cortex. These results will be discussed in relation to the branching of the pyramidal tract fibers in the spinal cord (Shinoda et al., 1976) as well as to the results reported by Jankowska et al. (1975a, b) who stimulated the surface and also the depth of the cortex.
Methods The experiments consist of two different procedures and the methods will be described in separate series.
Excitation of PT Cells
445
Part 1: Single Pulse Stimulation The results were obtained from ten cats of either sex. T h e y were anesthetized with pentobarbital sodium (Nembutal; 35 mg/kg, i.p.). T h e medullary pyramid was exposed via a ventral approach for stimulation and the cisterna m a g n a was o p e n e d to prevent possible brain swelling during the experiments. The pericruciate cortex was exposed and a closed c h a m b e r installed over the craniotomy. A photograph was taken to m a p the locations of electrode insertions. Screws attached to the skull were clamped rigidly by a holder to prevent any m o v e m e n t of the head. A concentric stimulating electrode was placed on the surface of the medullary pyramid ipsilateral to the exposed cortex. Two tungsten microelectrodes (Stoney et al., 1968; tip size about 1 0 x 15 gm) were driven into the motor cortex at points close to each other. The angle of the two penetrations relative to each other was held constant using a closed c h a m b e r a r r a n g e m e n t similar to that shown in Figure 1 of A s a n u m a and R o s 6 n (1973). T h e lateral distance between the two electrodes was variable, to allow multiple penetrations with one electrode while recording a single cell with the other. Pyramidal tract (PT) cells were activated by 0.2 msec pulses applied to the pyramid, and the antidromic spikes were recorded in the ipsilateral m o t o r cortex with one of the two microelectrodes. Once a unitary spike was isolated a n d the cell identified as a PT cell, the other microelectrode was moved along a track in the adjacent cortical area, in the m a n n e r illustrated in Figure 2. This second microelectrode was used for both stimulation and recording. Cathodal pulses of 0.2 msec duration were delivered (with reference to an indifferent electrode in the temporal muscles) at intervals of 5 0 - 1 0 0 ~m along the track to activate the PT cell. Pulse duration of 0.2 msec was used throughout the experiments to facilitate the comparison of the results with the previous reports ( A n d e r s e n et al., 1975; A s a n u m a et al., 1968, 1972; Jankowska et al., 1975a, b). To determine whether ICMS activated the PT cell u n d e r study, the m e t h o d of Stoney et al. (1968) was used. This involved collision of the ICMS - initiated spike with the antidromic spike, which resulted in the disappearance of the antidromic spike recorded with the first electrode. W h e n the antidromic spike was abolished by the ICMS, the second microelectrode was switched to the recording m o d e to see if the same spike could also be recorded through the second electrode. Because of the stability of the closed chamber system, it was not difficult to record the same spike while m a k ing several consecutive penetrations with the second microelectrode as shown in Figure 2. Antidromic activation of PT cells was identified by a) an invariant response latency of 1.2 msec or less after stimulation of the pyramid, and b) the ability to follow 100 Hz pyramidal stimuli. Positive-going spikes were not used for the study because of their instability. Reconstruction of the electrode tracks was accomplished by making one or two small electrolytic lesions (10 ~a for 10 seconds) at m e a s u r e d depths along the tracks of both microelectrodes. T h e s e lesions were m a d e at the end of a series of examinations for a given cell, and the same area of the cortex was not used for further investigation. Distances between the several tracks of the stimulating microelectrode were m e a s u r e d from the photographic m a p of electrode insertions. Part 2: Repetitive Stimulation Seven cats were used. Five of t h e m were anesthetized with a mixture of oxygen (20 %), nitrous oxide (80%), and halothane (0.5-1.0%). T h e cisterna m a g n a was opened for fluid drainage, and a closed c h a m b e r installed over a craniotomy exposing the pericruciate cortex. T h e head was rigidly fixed with clamps attached to screws in the skull. All the w o u n d areas were infiltrated with a longlasting local anesthetic (Zyljectin, Abbott), and inhalation anesthetic was discontinued. A sedative dose of Nembutal (10 mg/kg) was injected intraperitoneally before the termination of inhalation anesthesia. Fine bipolar electrodes, insulated except at the tip, were inserted into various forearm muscles through the skin to record electromyograms (EMGs). A n o d a l surface stimulation with a silver ball electrode was used to determine the locations at which E M G s could be elicited at low threshold, then a tungsten microelectrode was inserted into such a low threshold area. Trains of cathodal pulses (relative to the indifferent electrode in the temporal muscles) were passed through the microelectrode in order to elicit E M G responses. T h e remaining two cats were used for recording pyramidal volleys elicited by cortical stimulation. This experiment was carried out u n d e r N e m b u t a l anesthesia (35 mg/kg). The motor cortex
446
H. Asanuma et al.
was exposed for stimulation and the cervical cord was exposed for recording. A tungsten electrode of larger size (exposed tip: 40 ~tmx90 ~tm) was inserted into the lateral corticospinal tract at the level of C-2 to record descending volleys elicited by the ICMS. The volleys were fed into an online averaging computor (FABRI-TEK, Model 1062) and displayed on an oscilloscope. At the end of each experiment the appropriate part of the brain was removed and fixed in 10 % formalin. The brain was then serially sectioned in a freezing microtome, and stained by the method of Kltiver and Barrera (1953).
Results Part 1: Single Pulse Stimulation In studies using stimulating and recording electrodes which are close to each other, a persistent problem is the large shock artifact which often obscures short latency responses. In a previous study in which the effect of intracortical microstimulation (ICMS) was studied (Stoney et al., 1968), this drawback was overcome by using a collision technique which placed the antidromic spikes after the test stimulation. However, there still remained difficulties in the method. When ICMS elicited synaptic activation of neurons around the recording electrode, the disappearance of the antidromic spikes of the target PT cell was obscured by the spikes of these neurons as shown in Figure 1D. H e n c e study had to be limited to the cases when ICMS produced minimal synaptic activity, i.e., when very weak ICMS excited the target PT cells from short distances. Later development of cancellation techniques by A s a n u m a and Ros6n (1973), however, succeeded in reducing the shock artifact drastically, enabling observation of the events immediately after the stimulation. Using this technique, it now becomes possible to reexamine the effect of ICMS m o r e thoroughly. Figure 1 illustrates examples of the results. As shown in Figure 1A, ICMS produces a large stimulus artifact even with a weak current of 6 ~ta, excluding the possibility of visualizing possible effects for nearly 1.0 msec after the stimulus. Stimulation of the medullary pyramid with suprathreshold intensity made it possible to determine the cortical threshold by observing the 50 % appearance of the antidromic spikes. Usage of the cancellation current, which is shown in Figure 1B, brought the base line to the original level immediately after the stimulus making possible the observation of the events following the stimulation. A question arises of whether the cancellation current changes the threshold for activating PT cells. It had been discussed that the cancellation current used with ICMS is unlikely to change the threshold for activating cortical neurons (Asanuma and Ros6n, 1973) and it had been shown that the cancellation current from the surface has little or no effect on the efficiency of activating afferent fibers in the cortex ( A s a n u m a et al., 1974). As had b e e n suggested, imposition of cancellation current to ICMS did not change the threshold for activating the PT cell as is evidenced by 50 % appearance of the same antidromic spikes (Fig. 1B). Several trials using compensation pulses of various duration (0.05-0.3 msec) revealed that within this range, the compensation pulse causes little or no change in the efficiency of the negative stimulating pulse (0.2 msec duration). This might be explained by assuming that the
Excitation of PT Cells
447
C Cx
Py
Py
B
Cx
Py
Py Cx
ii
14oov l msec
Fig. 1 A - D . Left: comparison of threshold for activation of a PT cell by ICMS with and without an anodal cancellation pulse. A Intracortical microstimulation (ICMS) without compensation current is followed by a suprathreshold shock to the pyramid. The antidromically activated spike is blocked about 50% of the time. B ICMS of the same intensity is followed by a brief anodal cancellation pulse. The firing index is changed very little if at all by the presence of the cancellation pulse. C and D illustrate the method used to establish that the ICMS activated the PT cell directly, rather than synaptically. C The suprathreshold pyramidal shock activates the cell antidromically. D The pyramidal shock is followed by the ICMS. Use of the cancellation pulse reduced the shock artifact so that the antidromic spike could be positioned within 0.8 msec after the ICMS. Note that ICMS blocked the antidromic spike about 50 % of the time. Further details are in the text
local response developed during the period of the stimulation (0.2 msec) is already powerful enough to withstand the anodal blocking effect produced by the cancellation current as has been discussed previously (Asanuma and Ros6n, 1973). Utilizing this technique, we could proceed to the following examination. Instead of delivering the pyramidal stimulation after the cortical stimulation, pyramidal stimulation was delivered prior to the cortical stimulation so that the antidromic spike was placed immediately after the ICMS as shown in Figure 1D. It has been shown that the minimum latency of the PSP's in cortical neurons following threshold ICMS is 0.8 msec (Asanuma and Ros6n, 1973). Since we always activated PT cells with threshold currents, the blockade by collision of the antidromic spikes within this latency must be produced by direct activation of the target cell. This interpretation is supported by the observation that the PT cells activated synaptically by cortical surface stimulation very rarely (one out of 26) discharged with latency of less than 0.9 msec even
448
H. Asanuma et al.
O-lO/Ja 1-25pa > 25pa
i
i
t
L
~
i
Fig. 2. Histological reconstruction of electrode tracks and sites where ICMS was delivered. The cell was recorded at the position shown by the recording electrode. Four penetrations (A-D) were made to activate the cell directly. The cell's spike could be recorded in track D at the depths at which weak ICMS (
