243
Journal of Physiology (1991), 435, pp. 243-256 With 6 figures Printed in Great Britain
DIFFERENTIAL CONNECTIONS BY INTRACORTICAL AXON COLLATERALS AMONG PYRAMIDAL TRACT CELLS IN THE CAT MOTOR CORTEX BY YOUNGNAM KANG, KATSUAKI ENDO AND TATSUNOSUKE ARAKI From the Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
(Received 19 June 1990) SUMMARY
1. Recurrent EPSPs were produced in fast pyramidal tract (PT) cells in the cat motor cortex by stimulation of the medullary pyramid and/or by the glutamateinduced activity of neighbouring PT cells using the spike-triggered averaging (spike-TA) method. 2. In fast PT cells located lateral to the end of the cruciate sulcus, predominantly the motor cortical representation area of the distal forelimb, two components (fast and slow) of recurrent EPSPs were produced by pyramid stimulation. 3. In response to pyramid stimulation, the appearance of the fast and slow components of recurrent EPSPs correlated with the appearance of N1 and N2 field
potentials, respectively. 4. The monosynaptic nature of both the fast and slow components of recurrent EPSPs was demonstrated by a double shock test (interstimulus interval < 5 ms) and high frequency repetitive stimulation (50-100 Hz). 5. The generation of the fast and slow components of recurrent EPSPs was attributed to the synaptic action of recurrent collaterals of fast and slow PT cells, respectively. 6. The amplitude of the slow component of recurrent EPSPs markedly increased with an increase in the stimulus frequency whereas that of the fast component did not, despite the change in stimulus frequency. 7. Selected spike-triggered averaging also revealed frequency facilitation of recurrent individual EPSPs produced in fast PT cells by the activity of single slow PT cells. 8. In fast PT cells located in the anterior and posterior lips of the cruciate sulcus, the motor cortical representation area of the proximal limb or trunk, only the slow component of recurrent EPSPs was produced by pyramid stimulation. 9. It is concluded that the pattern of recurrent connections between neighbouring PT cells differs depending on the motor cortical representation area, and that frequency facilitation of recurrent EPSPs is caused mainly by the input from axon collaterals of slow PT cells.
SIX 8577
244
Y. KANG, K. ENDO AND T. ARAKI INTRODUCTION
It has been reported that the pyramidally evoked EPSP (recurrent EPSP) of the fast PT cell in the cat motor cortex is transmitted through the axon collaterals of slow PT cells (Takahashi, Kubota & Uno, 1967). However, it has recently been shown in the cat (Kang, Endo & Araki, 1988) and monkey (Ghosh & Porter, 1988) that recurrent EPSPs are produced in fast PT cells by the activity of axon collaterals of both fast and slow PT cells. To interpret the discrepancy in the pattern of recurrent collateral connections between the two previous studies (Kang et al. 1988; Takahashi et al. 1967), it has been suggested that there may be a regional difference in the pattern of recurrent connections between the lateral and posterior parts of the cruciate sulcus of the cat motor cortex (Kang et al. 1988). On the other hand, because a group of neighbouring PT cells is thought to be linked by common and recurrent inputs (Cheney & Fetz, 1985), synchronized activation of neighbouring PT cells by common inputs may be modified ultimately by recurrent collateral feedback inputs. Since, in the natural state, PT cells are known to discharge at various frequencies in conjunction with voluntary movement (Evarts, 1968; Fromm & Evarts, 1977), it seems to be important to know how such feedback through collateral inputs may act under natural conditions. At present, it is unknown whether recurrent EPSPs produced in target PT cells are influenced by discharge frequency of the originating PT cells. In the present investigation, recurrent EPSPs were recorded from fast PT cells to study whether there is a regional difference in the pattern of recurrent axon collateral connections, and to study their frequency sensitivity. It was demonstrated that in fast PT cells located lateral to the end of the cruciate sulcus, both the fast and slow components of recurrent EPSPs were produced following activation of recurrent axon collaterals of fast and slow PT cells. However, in fast PT cells located in the anterior and posterior lips of the cruciate sulcus, only the slow component of recurrent EPSPs was produced even though axon collaterals of both fast and slow PT cells were activated. Only the slow component of recurrent EPSPs showed marked frequency facilitation. Similar frequency facilitation of recurrent individual EPSPs produced by propagation through axon collaterals of single slow PT cells was also revealed by selected spike-triggered averaging. It is concluded that PT cells are differentially inter-connected through their axon collaterals depending on their locations in the motor cortical representation area, and that frequency facilitation of recurrent EPSPs in fast PT cells results much from the input of axon collaterals of slow PT cells, but little from that of fast PT cells. METHODS
The methods are basically similar to those described in the previous study (Kang et al. 1988). The experiments were carried out on twenty cats weighing between 3-0 and 5-0 kg. The cats were anaesthetized with pentobarbitone sodium (35 mg kg-', i.P.) and the trachea and a superficial vein of the distal forelimb were cannulated. The animals were mounted in a stereotaxic instrument and immobilized with pancuronium bromide (005 mg kg-' h-1) after artificial ventilation (AR- 1, Narishige). Additional doses of pentobarbitone sodium (1-5 mg kg-' h-1) were given to maintain anaesthesia. Body temperature was maintained between 36 and 38 °C by a heating pad. A craniotomy was performed over the left motor cortex. The cortical surface was perfused with
DIFFERENTIAL CONNECTIONS BY AXON COLLATERALS
245
normal Ringer solution, automatically maintained at 36 'C. Three pairs of bipolar stimulating electrodes (diameter of 0-2 mm, interpolar distance of 1 mm), whose tips were set at three different ventro-dorsal levels separated by 1 mm, were placed stereotaxically in the medullary pyramid and in the more dorsal structures (the trapezoid body and the lemniscus medialis). Using a small silverball electrode, the electroencephalogram was monitored monopolarly at the dimple of the sensorimotor cortex to assess the general level of anaesthesia and to adjust the final position of the stimulating electrode at the pyramid to produce a and , waves (Jabbur & Towe, 1961; Takahashi, 1965). PT cells in the motor cortex were identified by antidromic spikes produced by pyramid stimulation (duration, 0-2 ms; intensity, 50-1000 ,uA). They were classified into fast and slow types if their antidromic latencies were below or above 2-3 ms (Takahashi, 1965). Recurrent composite EPSPs were recorded from fast PT cells in response to pyramid stimulation. In order to study recurrent individual EPSPs, intracellular and extracellular recordings were obtained simultaneously from two neighbouring PT cells using two glass microelectrodes. One microelectrode, filled with 1 M-Na-glutamate (DC resistance, 5-10 MQ: tip diameter, 1-2,um), was used for extracellular recording. The other, filled with 2 M-potassium citrate (DC resistance, 10-20 MQ), was used for conventional intracellular recording. The two microelectrodes were aligned at angles of 30-60 degrees relative to each other in a sagittal plane, and their tips were separated by 500-1500 ,um at the cortical surface. Then the two electrodes were inserted into the motor cortex. Extracellularly recorded spikes produced by pyramid stimulation were identified as antidromic by the following criteria: (a) constant latency of response to threshold pyramidal stimuli, (b) ability to follow high frequency stimulation up to 200 Hz, and (c) collision with spontaneous spikes. The input signal was fed into a high input impedance DC amplifier (MEZ-8201, Nihon Kohden) which was connected to a digital oscilloscope (VC-10, Nihon Kohden) equipped with an averager (DAT-i 100, Nihon Kohden). Signals were recorded on a 4-channel FM tape recorder (DFR-3515, Sony) for off-line analysis. The glutamate induced spikes of extracellularly recorded PT cells were used to trigger the averager, which summed the membrane potential changes recorded simultaneously in adjacent PT cells. The oscilloscope digitized at rates of 25 or 50 KHz and 50-1000 responses were summated by the averager. Thus, averaged recurrent individual EPSPs were recorded. Digitized signals were transferred to a computer (PC-9801M, NEC) and stored on disc for off-line analysis and for reproduction of traces using a plotter (MP 1000-01, Watanabe). Data were also photographed (RLG-6201, Nihon Kohden) on the oscilloscope. As described in the previous study (Kang et al. 1988), coupling artifacts resulting from triggering spike potentials were frequently observed before averaged EPSPs recorded intracellularly. Since the amplitude of recurrent composite EPSPs was small, 30-50 responses of recurrent composite EPSPs and extracellular field potentials were also averaged. To mark the location of intracellularly recorded PT cells on the cortical surface, the cruciate sulcus was regarded as an abscissa and an ordinate was assumed to intersect at the lateral end of the cruciate sulcus. The recording electrode was advanced in parallel with a sagittal plane to define the medio-lateral position of PT cells to the cortical surface, and was advanced as perpendicularly as possible to the cortical surface. All the PT cells investigated in the present study were recorded at a depth of 10-1lb mm from the cortical surface and the location of recording electrodes on the cortical surface was measured under a dissecting microscope equipped with a collimator and plotted on the co-ordinates described above (see Fig. 6D). To study the frequency sensitivity of recurrent individual EPSPs, extracellular spikes of reference PT cells with frequencies in a certain range were selected by a filter circuit to trigger the averager. A diagram of the filter circuit is shown in Fig. 1 A. A timing chart of the circuit is shown in Fig. lB. Extracellular spike potentials (trace 1) were fed into a window slicer (EN-601J, Nihon Kohden) to make pulses (trace 2), the rising and falling edges of which were used to make the stop (trace 3) and start (trace 4) pulses to control a stimulator (SEN-7103, Nihon Kohden). The two parameters of delay (a) and duration (b) of the output pulse (trace 5) of the stimulator were chosen to select extracellular spikes with interspike intervals in a range between (a) and (a + b). The output pulses of the stimulator and the window slicer were fed into an AND gate (trace 6) to make the final trigger pulse for the averager. An example of the interval histogram of extracellular spikes recorded from a fast PT cell is shown in Fig. 1 C. From this mother group of firing frequencies, extracellular spikes with frequencies in two ranges, 50-100 Hz (Fig. 1D) and 16 6-25 Hz (Fig. 1E), were selected by choosing the delay and duration to be 10 and 10 ms and 40 and 20 ms, respectively, to make triggering pulses. The histograms of the interval between the selected spike and its immediately
Y. KANG, K. ENDO AND T. ARAKI preceding one were identical with the latency histograms shown in Fig. ID and E which were obtained by measuring the latency from the immediately preceding start pulse to the triggering 246
pulse using a computer (ATAC 501-10, Nihon Kohden).
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20
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Journal of Physiology (1991), 435, pp. 243-256 With 6 figures Printed in Great Britain
DIFFERENTIAL CONNECTIONS BY INTRACORTICAL AXON COLLATERALS AMONG PYRAMIDAL TRACT CELLS IN THE CAT MOTOR CORTEX BY YOUNGNAM KANG, KATSUAKI ENDO AND TATSUNOSUKE ARAKI From the Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
(Received 19 June 1990) SUMMARY
1. Recurrent EPSPs were produced in fast pyramidal tract (PT) cells in the cat motor cortex by stimulation of the medullary pyramid and/or by the glutamateinduced activity of neighbouring PT cells using the spike-triggered averaging (spike-TA) method. 2. In fast PT cells located lateral to the end of the cruciate sulcus, predominantly the motor cortical representation area of the distal forelimb, two components (fast and slow) of recurrent EPSPs were produced by pyramid stimulation. 3. In response to pyramid stimulation, the appearance of the fast and slow components of recurrent EPSPs correlated with the appearance of N1 and N2 field
potentials, respectively. 4. The monosynaptic nature of both the fast and slow components of recurrent EPSPs was demonstrated by a double shock test (interstimulus interval < 5 ms) and high frequency repetitive stimulation (50-100 Hz). 5. The generation of the fast and slow components of recurrent EPSPs was attributed to the synaptic action of recurrent collaterals of fast and slow PT cells, respectively. 6. The amplitude of the slow component of recurrent EPSPs markedly increased with an increase in the stimulus frequency whereas that of the fast component did not, despite the change in stimulus frequency. 7. Selected spike-triggered averaging also revealed frequency facilitation of recurrent individual EPSPs produced in fast PT cells by the activity of single slow PT cells. 8. In fast PT cells located in the anterior and posterior lips of the cruciate sulcus, the motor cortical representation area of the proximal limb or trunk, only the slow component of recurrent EPSPs was produced by pyramid stimulation. 9. It is concluded that the pattern of recurrent connections between neighbouring PT cells differs depending on the motor cortical representation area, and that frequency facilitation of recurrent EPSPs is caused mainly by the input from axon collaterals of slow PT cells.
SIX 8577
244
Y. KANG, K. ENDO AND T. ARAKI INTRODUCTION
It has been reported that the pyramidally evoked EPSP (recurrent EPSP) of the fast PT cell in the cat motor cortex is transmitted through the axon collaterals of slow PT cells (Takahashi, Kubota & Uno, 1967). However, it has recently been shown in the cat (Kang, Endo & Araki, 1988) and monkey (Ghosh & Porter, 1988) that recurrent EPSPs are produced in fast PT cells by the activity of axon collaterals of both fast and slow PT cells. To interpret the discrepancy in the pattern of recurrent collateral connections between the two previous studies (Kang et al. 1988; Takahashi et al. 1967), it has been suggested that there may be a regional difference in the pattern of recurrent connections between the lateral and posterior parts of the cruciate sulcus of the cat motor cortex (Kang et al. 1988). On the other hand, because a group of neighbouring PT cells is thought to be linked by common and recurrent inputs (Cheney & Fetz, 1985), synchronized activation of neighbouring PT cells by common inputs may be modified ultimately by recurrent collateral feedback inputs. Since, in the natural state, PT cells are known to discharge at various frequencies in conjunction with voluntary movement (Evarts, 1968; Fromm & Evarts, 1977), it seems to be important to know how such feedback through collateral inputs may act under natural conditions. At present, it is unknown whether recurrent EPSPs produced in target PT cells are influenced by discharge frequency of the originating PT cells. In the present investigation, recurrent EPSPs were recorded from fast PT cells to study whether there is a regional difference in the pattern of recurrent axon collateral connections, and to study their frequency sensitivity. It was demonstrated that in fast PT cells located lateral to the end of the cruciate sulcus, both the fast and slow components of recurrent EPSPs were produced following activation of recurrent axon collaterals of fast and slow PT cells. However, in fast PT cells located in the anterior and posterior lips of the cruciate sulcus, only the slow component of recurrent EPSPs was produced even though axon collaterals of both fast and slow PT cells were activated. Only the slow component of recurrent EPSPs showed marked frequency facilitation. Similar frequency facilitation of recurrent individual EPSPs produced by propagation through axon collaterals of single slow PT cells was also revealed by selected spike-triggered averaging. It is concluded that PT cells are differentially inter-connected through their axon collaterals depending on their locations in the motor cortical representation area, and that frequency facilitation of recurrent EPSPs in fast PT cells results much from the input of axon collaterals of slow PT cells, but little from that of fast PT cells. METHODS
The methods are basically similar to those described in the previous study (Kang et al. 1988). The experiments were carried out on twenty cats weighing between 3-0 and 5-0 kg. The cats were anaesthetized with pentobarbitone sodium (35 mg kg-', i.P.) and the trachea and a superficial vein of the distal forelimb were cannulated. The animals were mounted in a stereotaxic instrument and immobilized with pancuronium bromide (005 mg kg-' h-1) after artificial ventilation (AR- 1, Narishige). Additional doses of pentobarbitone sodium (1-5 mg kg-' h-1) were given to maintain anaesthesia. Body temperature was maintained between 36 and 38 °C by a heating pad. A craniotomy was performed over the left motor cortex. The cortical surface was perfused with
DIFFERENTIAL CONNECTIONS BY AXON COLLATERALS
245
normal Ringer solution, automatically maintained at 36 'C. Three pairs of bipolar stimulating electrodes (diameter of 0-2 mm, interpolar distance of 1 mm), whose tips were set at three different ventro-dorsal levels separated by 1 mm, were placed stereotaxically in the medullary pyramid and in the more dorsal structures (the trapezoid body and the lemniscus medialis). Using a small silverball electrode, the electroencephalogram was monitored monopolarly at the dimple of the sensorimotor cortex to assess the general level of anaesthesia and to adjust the final position of the stimulating electrode at the pyramid to produce a and , waves (Jabbur & Towe, 1961; Takahashi, 1965). PT cells in the motor cortex were identified by antidromic spikes produced by pyramid stimulation (duration, 0-2 ms; intensity, 50-1000 ,uA). They were classified into fast and slow types if their antidromic latencies were below or above 2-3 ms (Takahashi, 1965). Recurrent composite EPSPs were recorded from fast PT cells in response to pyramid stimulation. In order to study recurrent individual EPSPs, intracellular and extracellular recordings were obtained simultaneously from two neighbouring PT cells using two glass microelectrodes. One microelectrode, filled with 1 M-Na-glutamate (DC resistance, 5-10 MQ: tip diameter, 1-2,um), was used for extracellular recording. The other, filled with 2 M-potassium citrate (DC resistance, 10-20 MQ), was used for conventional intracellular recording. The two microelectrodes were aligned at angles of 30-60 degrees relative to each other in a sagittal plane, and their tips were separated by 500-1500 ,um at the cortical surface. Then the two electrodes were inserted into the motor cortex. Extracellularly recorded spikes produced by pyramid stimulation were identified as antidromic by the following criteria: (a) constant latency of response to threshold pyramidal stimuli, (b) ability to follow high frequency stimulation up to 200 Hz, and (c) collision with spontaneous spikes. The input signal was fed into a high input impedance DC amplifier (MEZ-8201, Nihon Kohden) which was connected to a digital oscilloscope (VC-10, Nihon Kohden) equipped with an averager (DAT-i 100, Nihon Kohden). Signals were recorded on a 4-channel FM tape recorder (DFR-3515, Sony) for off-line analysis. The glutamate induced spikes of extracellularly recorded PT cells were used to trigger the averager, which summed the membrane potential changes recorded simultaneously in adjacent PT cells. The oscilloscope digitized at rates of 25 or 50 KHz and 50-1000 responses were summated by the averager. Thus, averaged recurrent individual EPSPs were recorded. Digitized signals were transferred to a computer (PC-9801M, NEC) and stored on disc for off-line analysis and for reproduction of traces using a plotter (MP 1000-01, Watanabe). Data were also photographed (RLG-6201, Nihon Kohden) on the oscilloscope. As described in the previous study (Kang et al. 1988), coupling artifacts resulting from triggering spike potentials were frequently observed before averaged EPSPs recorded intracellularly. Since the amplitude of recurrent composite EPSPs was small, 30-50 responses of recurrent composite EPSPs and extracellular field potentials were also averaged. To mark the location of intracellularly recorded PT cells on the cortical surface, the cruciate sulcus was regarded as an abscissa and an ordinate was assumed to intersect at the lateral end of the cruciate sulcus. The recording electrode was advanced in parallel with a sagittal plane to define the medio-lateral position of PT cells to the cortical surface, and was advanced as perpendicularly as possible to the cortical surface. All the PT cells investigated in the present study were recorded at a depth of 10-1lb mm from the cortical surface and the location of recording electrodes on the cortical surface was measured under a dissecting microscope equipped with a collimator and plotted on the co-ordinates described above (see Fig. 6D). To study the frequency sensitivity of recurrent individual EPSPs, extracellular spikes of reference PT cells with frequencies in a certain range were selected by a filter circuit to trigger the averager. A diagram of the filter circuit is shown in Fig. 1 A. A timing chart of the circuit is shown in Fig. lB. Extracellular spike potentials (trace 1) were fed into a window slicer (EN-601J, Nihon Kohden) to make pulses (trace 2), the rising and falling edges of which were used to make the stop (trace 3) and start (trace 4) pulses to control a stimulator (SEN-7103, Nihon Kohden). The two parameters of delay (a) and duration (b) of the output pulse (trace 5) of the stimulator were chosen to select extracellular spikes with interspike intervals in a range between (a) and (a + b). The output pulses of the stimulator and the window slicer were fed into an AND gate (trace 6) to make the final trigger pulse for the averager. An example of the interval histogram of extracellular spikes recorded from a fast PT cell is shown in Fig. 1 C. From this mother group of firing frequencies, extracellular spikes with frequencies in two ranges, 50-100 Hz (Fig. 1D) and 16 6-25 Hz (Fig. 1E), were selected by choosing the delay and duration to be 10 and 10 ms and 40 and 20 ms, respectively, to make triggering pulses. The histograms of the interval between the selected spike and its immediately
Y. KANG, K. ENDO AND T. ARAKI preceding one were identical with the latency histograms shown in Fig. ID and E which were obtained by measuring the latency from the immediately preceding start pulse to the triggering 246
pulse using a computer (ATAC 501-10, Nihon Kohden).
A
2
indow Iicer
3
L 1 1-fjj-ff-fj JImpulses
C 60-
Stp11Si a:
{3ta VDelay
40 4
AND
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.
_2
Ext. trigger
_tQAverager |
D
60 -
B 40-
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