Effect of phrenic nerve stimulation on neural transmission and diaphragmatic force generation in the dog

187

Respiration Physiology (1986) 63, 187-199 Elsevier

EFFECT OF PHRENIC NERVE STIMULATION ON NEURAL TRANSMISSION AND DIAPHRAGMATIC FORCE GENERATION IN THE DOG

HYLTON BARK and STEVEN M. SCHARF Division of Lung Diseases, Soroka Medical Center, Unit of Physiology, Faculty of Health Sciences and Department of Biology, Ben Gurion University of the Negev, Beer Sheva, Israel

Abstract. The effect of supramaximal bilateral phrenic nerve stimulation on neural transmission and

diaphragmatic force generation was studied in anesthetized dogs. Different combinations of duty cycle and stimulation frequency were examined during intermittent stimulation (pacing) for 15 rain. The effect of different stimulation frequencies was also examined during continuous stimulation for 60 sec. Force declined more with increasing stimulation frequency and duty cycle with intermittent stimulation, and with increasing stimulation frequency with continuous stimulation. Neural transmission decreased with increasing stimulation frequencies. The changes were always greater with continuous than with intermittent stimulation. We found no unique relationship between changes in neural transmission and changes in force generation, suggesting that if neural transmission failure is causally responsible for fatigue, it does so by a very complex mechanism. Diaphragm Dog

Fatigue Force

Neural transmission Phrenic nerve

General acceptance of the fact that fatigue of the diaphragm can contribute to respiratory failure has led to an intensification of research on diaphragmatic fatigue in an attempt to understand its pathophysiology. A commonly used model of diaphragmatic fatigue is one in which supramaximal bilateral phrenic nerve stimulation is used to induce fatigue (Newman et al., 1984; Howell and Roussos, 1984; Scharf and Bark, 1984). Fatigue, defined as a decrease in force generation, can be due to decreased neural impulse propagation, 'neural fatigue', or failure of the contractile apparatus, 'muscle fatigue' (Edwards, 1979). Theoretically, decreased neural impulse propagation may take place in the nerve trunk, the smaller branching axons, the neuromuscular junction, the synapse or the muscle membrane (Krnjevic and Miledi, 1958; Grossman et al., 1979). Accepted for publication 26 September 1985 0034-5687/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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It has been proposed (Bigland-Ritchie et al., 1979; Krnjevic and Miledi, 1958) that when stimulation frequency is high, fatigue is more likely to result from failure of neural impulse propagation, probably at the neuromuscular junction, whereas with stimulation at low frequency, fatigue is due to failure of the contractile apparatus. In spite of the extensive use of the phrenic nerve stimulated intact canine model of diaphragmatic fatigue and the clinical and experimental use of electro-phrenic stimulation for ventilatory support (Brouillette et al., 1983; Glenn et al., 1984), little work has been done in this model to evaluate the effects of electro-phrenic stimulation on neural transmission under differing circumstances, and its relationship to force production. We therefore undertook the present study in which we examined the effects of electrophrenic stimulation on force production and neural impulse propagation in the intact anesthetized dog with different modes of stimulation, at different frequencies and duty cycles.

Methods A n i m a l preparation. Mixed breed dogs of either sex weighing 15-22kg were anesthetized with pentobarbital sodium (30 mg/kg) administered via a peripheral vein. The dogs were intubated with a cuffed endotracheal tube (I.D. 8 mm), placed in the supine position and mechanically ventilated with a volume-cycled respirator (tidal volume 15 ml/kg, 12 times/min). Body temperature was maintained at 38 + 1 °C, using an electric heating pad. During stimulation of the phrenic nerves the respirator was disconnected and a Fleisch no. 2 pneumotachograph was attached to record a flow signal which, when electrically integrated, measured tidal volume (VT). Both phrenic nerves were surgically exposed at the entrance to the thorax. Care was taken to include the C7 root. The two nerves were placed on bipolar stainless-steel electrodes and isolated from the surrounding tissue with thin rubber sheeting. The skin was closed with clips. Two 5 cm long latex balloons on the ends of polyethylene catheters were passed through the mouth. One was positioned in the stomach to record gastric pressure (Pgas), while the other was positioned in the lower third of the esophagus to record pleural pressure (Ppl). The ends of each catheter were attached to opposite sides of a differential pressure transducer to measure transdiaphragmatic pressure (Pdi). (Pdi = Pgas - Ppl). The end of the esophageal catheter was also connected via a Y-piece to another pressure transducer in order to measure Ppl independently. Ppl at end expiration was assumed to be equal to transpulmonary pressure at end expiration and to reflect changes in lung volume. In two groups (groups 1 and 2) in which the effects of changes in stimulus frequency and duty cycle were examined, a pair of linearized respiratory magnetometers (Norman Petersen, Harvard School of Public Health) was placed across the lateral thorax at the mid-chest level. A second pair was placed across the antero-postero abdominal dimension just below the level of the umbilicus. The magnetometers enabled us to record

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the rib cage (RC) and the abdominal (ABD) movements as well as the changes in end-expiratory positions of the RC and ABD. In experiments in which we examined the effects of intermittent stimulation (IS) vs continuous stimulation (CS) (group 3 below), we also placed a bipolar stainless-steel wire electrode in the muscular portion of the costal diaphragm mid-way between the ribs and the central tendon and parallel to the muscle fibers. This was performed through a midline abdominal incision. This electrode was used to record single evoked compound action potentials (CAP) in response to phrenic nerve stimulation. After closure the abdomen was placed in a firm plaster cast. The cast was applied with care in such a way that it did not apply any pressure at end-expiration.

Recordings.

VT, Pdi, Ppl, RC and ABD were monitored on an oscilloscope and recorded on paper using an Electronics for Medicine ® VR-6 simultrace recorder. Neural transmission is defined in this paper as the ability of the neural tissues to propagate impulses from the site of stimulation to the muscle fiber. It includes impulse propagation in the nerve trunk, the small branching axons, the neuromuscular junction and the muscle fiber membrane. Neural transmission was evaluated as follows: CAPs were recorded on a single sweep of a storage oscilloscope, triggered by the stimulator. The CAP signal was amplified and filtered (30 Hz to 1 kHz) before being displayed on the screen from which it was photographed. The following parameters were measured from the photograph using methods described by Bigland-Ritchie et al. (1979): (1) Conduction time (CT) - interval between the stimulus artifact and the onset of the CAP. The conduction time included the conduction time within the nerve trunk, nerve branches, the neuromuscular junction and the muscle membrane. (2) Total area of the CAP above and below the isoelectric line. These measurements were made using a magnetic digitizing board connected to a microcomputer and tracing the relevant portions of the signal.

Experimental protocol Groups 1 and 2 (14 dogs): Effects of changes in stimulus frequency and duty cycle on force generation. Thirty minutes were allowed after the preparation had been prepared for it to reach a steady state, following which the ventilator was detached, the pneumotachograph connected, and electrical stimulation of the phrenic nerves (pacing) commenced. At all times during pacing, dogs breathed 100~o oxygen to prevent hypoxia. Pacing was performed using supramaximal voltages (3-5 V) with 24 trains of stimuli per minute, each stimulus 0.5 msec in duration. In group 1 (8 dogs), the duty cycle was kept constant at 0.4 and the stimulus frequency varied (either 20, 50 or 100 Hz, order selected randomly). In group 2 (6 dogs), the stimulus frequency was kept constant at 50 Hz and the duty cycle varied (0.2, 0.4, or 0.8, selected randomly). Pacing was performed at each of the frequencies or duty cycles for 15 min followed by a 15 min recovery period during which the dog was mechanically ventilated. Pdi, VT, RC and ABD were recorded at the start of pacing (first 3 breaths), and at 5, 10 and 15 min of pacing.

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Group 3 (6 dogs) : Intermittent vs Continuous stimulation and recording of CT and CAP. In this group two modes of stimulation were studied: (1) Intermittent stimulation (IS) was performed with 24 trains of stimuli per minute, each impulse 0.5 msec in duration, at a duty cycle 0.4 and at various stimulus frequencies of 20, 50 and 100 Hz. IS was performed for 15 min at each frequency. Between pacing periods a 30 min rest period was allowed on mechanical ventilation. The order of stimulus frequency selection was randomized. (2) Continuous stimulation (CS) was performed for 60 sec at each of the stimulus frequencies mentioned above. A 30 min recovery period was allowed on mechanical ventilation between each different frequency. In the group that underwent IS we recorded Pdi, CT and CAP at the onset of stimulation (first stimulus) and at 5, 10 and 15 min of stimulation. In the group that underwent CS we recorded Pdi continuously and CT and CAP at the onset (first stimulus) and every I0 see thereafter for 60 sec. This was accomplished by resetting the oscilloscope every 10 sec and allowing it to be triggered by the next stimulus for a single sweep of the screen, the screen was not cleared, however, between each sweep the trace was lowered manually. Finally, in additional experiments, during the 60 second period of CS, we changed the stimulus frequency. In subgroup A we stimulated at low frequency (20 Hz) for 30 sec and then increased to high frequency (100 Hz) for a further 30 sec. In subgroup B we stimulated at high frequency (100 Hz) for 40 sec and then decreased to a low frequency (20 Hz) for a further 20 sec. Pdi, CT and CAP were measured as for CS. Thus, in this group we examined two modes of stimulation, IS and CS at three different stimulus frequencies as well as the effects of changing the stimulus frequency during CS. We evaluated the following indices of fatigue at different stimulus frequencies, different duty cycles, and using different modes of stimulation (IS or CS): 1. Decline in Pdi over the period of stimulation. 2. Changes in neural impulse transmission as reflected by changes in CT and CAP. All values given are the mean + SEM. Statistical significance was assessed using Student's t-test for paired or unpaired variates, or analysis of variance whichever was appropriate. The test used will be indicated in the text.

Results

Groups I and 2. Figure 1 shows the results of a typical experiment in which Pdi was measured over 15 min of pacing. In this figure we demonstrate the effect of changes in duty cycle (left panel) and the effect of changes in stimulus frequency (right panel). Note that the decrease in force is curvilinear. We, as others (Kelsen and Nochomowitz, 1982), empirically chose to describe the curves by fitting them (least squares analysis) to an exponential equation of the form Pdit/Pdi o = e - kt, where Pdi t is Pdi at time t, Pdi o is Pdi at zero time, t is time and k is a shape constant which may be thought of as

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representing the overall 'rate of decay in pressure'. The more negative the value the greater the rate of fall. Table 1 shows the initial Pdi's, the m e a n R (correlation coefficient) values and k values for the various stimulus frequencies and duty cycles. It can be seen that with increasing stimulus frequency the initial value for Pdi was higher at 50 and 100 Hz than at 20 Hz. Furthermore, Pdi fell more rapidly at 50 and 100 Hz than at 20 H z (more negative k). However, Pdi fell at essentially the same rate at 50 and 100 Hz. With increasing duty cycle, there was n o difference (group 2) in the initial Pdi values. However, Pdi fell more rapidly at a duty cycle of 0.8 than at either 0.2 or 0.4 (which were essentially similar). Magnetometry in groups 1 a n d 2 showed that the end-expiratory position of neither the A B D nor R C changed throughout the experiment. I n addition, there was no change in Ppl at end expiration. These data indicate that lung volume at end-expiration did not TABLE 1 Changes in Pdi in groups 1 and 2. Group

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H. BARK AND S.M. SCHARF

change with fatigue. The outward abdominal excursion during inspiration did not change at 20 Hz stimulus frequency, consistent with the maintained Pdi. However, it fell by 38.4 + 12.04% over 15 min pacing at 100 Hz stimulus frequency paralleling the fall in Pdi.

Group 3: Different modes of stimulation. Figure 2 demonstrates the effect of stimulus frequency and mode of stimulation on the rate of fatigue. Pdi fell both during 15 minutes of IS (left panel) and 60 seconds of CS (right panel). The fall in Pdi was greater with increasing stimulus frequencies, both with CS and with IS. The maximal Pdi values obtained at each frequency during IS were, 17.3 + 1.5 cm H 2 0 at 20 Hz, 22.1 + 2.7 cm H 2 0 at 50 Hz and 29.2 + 3.3 cm H 2 0 at 100 Hz. During CS the values were 14.8 + 1.8, 21.4 + 3.2 and 23.4 + 3.7 cm H 2 0 at 20, 50 and 100 Hz, respectively. The behavior of this group was slightly different than in group 1, in that pressure was higher and force appeared to decrease more. Furthermore Pdi in this group fell at 20 Hz stimulus frequency. It must be remembered that this group was casted, and initial force production was therefore higher. Figure 3 shows typical tracings of the CAPs at various time intervals during electrical stimulation. The left panel illustrates CS, the right panel IS. This figure demonstrates that at 20 Hz CS and IS there were only minor changes in CT, and total area. However at 50 Hz we observed an increase in the CT (slowing of the conduction velocity), and a progressive decrease in CAP area. These changes were greater with CS than with IS. At 100 Hz the changes were greater than at 50 Hz. Similarly, the changes occurring with CS were greater than those occurring with IS. (For values see fig. 4.) Note, that with CS it was impossible to measure CT and CAP area accurately from the tracing after 30 sec of stimulation at 100 Hz since the area had fallen to almost zero. Note also from the figure that there was overlap with a preceding CAP at 100 Hz stimulus frequency. These problems made it difficult to perform the measurements in the tracings from all the dogs. We chose to deal with the problem as follows. When it was not possible to measure the CAP area, we called it zero. When the CAP area was zero we could not

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measure CT. Thus, we have only a small number of measurements o f CT at 100 H z stimulus frequency during CS. Figure 4 shows the mean data o f the various parameters of action potential propagation as a function of the change in Pdi caused by either IS or CS. The left panel shows the changes in CT associated with both IS and CS at different frequencies o f stimulation. In light o f the difficulties o f measuring C T at 100 H z stimulus frequency during CS, we show the number of measurements at each point. In only 4 of 6 dogs could we obtain technically satisfactory recordings at 100 H z CS. The number o f measurable

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Fig. 4. Left panel: Percent changes in conduction time and Pdi and various stimulus frequencies (20, 50, 100 Hz) and different modes of stimulation. V1, continuous stimulation;I , intermittent stimulation.Right panel: Percent changes in compound action potential area and Pdi at various stimulus frequencies (20, 50, 100 Hz) and different modes of stimulation. [7, continuous stimulation; m, intermittent stimulation. points (N) decreased as stimulation proceeded, because of the disappearance of the CAP. The N for all other points on the graph is, of course, 6. At 20 H z there was no change in Pdi with CS, yet CT increased, whereas with IS, Pdi decreased (P < 0.025) with no change in CT. At 50 and 100 H z stimulus frequencies, CT increased more for the same decline in Pdi with CS than with IS. CT also increased with increasing frequency of stimulation for both IS and CS. The right panel shows changes in total CAP area. This graph demonstrates that at 20 H z there was no change in Pdi with CS, yet CAP area increased. With IS at 20 Hz, Pdi decreased with no change in CAP area. At 50 H z there was no significant difference in the change in CAP area for the same fall in Pdi with either IS or CS. It did appear though that area initially increased slightly with CS. However, at 100 Hz, the fall in CAP area was far greater with CS than with IS. Thus, with CS the CAP area increased slightly at 20 H z stimulus frequency, fell moderately at 50 H z stimulus frequency and fell markedly at 100 H z stimulus frequency. Figure 5, left panel, shows an example of a tracing of Pdi generated at different frequencies during CS. This tracing shows that the initial force was greater the higher the stimulus frequency. The fall in Pdi was greater and more rapid at 100 than at 50 Hz.

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Fig. 5. Exampleof a tracing from one dog. Left panel: Effectof stimulation at different stimulus frequencies (20, 50, 100 Hz) on the fall in Pdi (cm H20) during continuous stimulation. Right panel: Effects on the change in Pdi of alterations in stimulus frequency during continuous stimulation. H/L, change from high stimulus frequency( 100 Hz) to lower stimulus frequency(20 Hz) after 40 sec continuous stimulation; L/H, change from low stimulus frequency (20 Hz) to high stimulus frequency (100 Hz) after 30 sec continuous stimulation. At 20 Hz there was no change in Pdi. The right panel (fig. 5) demonstrates the change in Pdi that occurred during CS when the frequency of stimulation was changed during the stimulation period. The tracing labeled L / H shows that when the stimulus frequency is changed from 20 to 100 Hz, a sharp increase in Pdi occurred after which there was a progressive decline similar to that seen in the left panel at 100 Hz. The other tracing, labeled H/L, in the right panel shows that when the stimulus frequency was reduced from 100 to 20 Hz during the period of stimulation, there is also an increase in Pdi, but only to the level previously attained at 20 Hz stimulus frequency. Figure 6 shows a tracing of the CAP's recorded in the same dog at 10 sec intervals during CS when the stimulus frequency was changed during the stimulation period (corresponds to the right panel fig. 5). The left panel of fig. 6 shows the change that occurred when the stimulus frequency was increased from 20 to 100 Hz after 30 sec of stimulation (L/H). It can be seen that the CT increased and the total area of the CAP fell dramatically. In spite of this, force generation increased (fig. 5, right panel). In the alternate situation, when the frequency was reduced from 100 H z to 20 Hz (H/L), after 40 sec, action potential propagation improved (fig. 6, right panel). Here too, force increased. lO0-20Hz

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196

H. BARKAND S.M. SCHARF

Discussion

In this study we examined the effects of different modes of electrophrenic stimulation on neural transmission in the intact anesthetized dog. We found that neural transmission was decreased mainly at high stimulation frequencies. We attempted to relate these changes in neural transmission to the changes in force generation (fatigue) that we found. In the ensuing discussion we will evaluate these findings in the light of previously described data. We have found as have others (Kelsen and Nochomowitz, 1982; Sato et al., 1970) that the diaphragm fatigued when the phrenic nerve was electrically stimulated. Although there was impairment of neural impulse propagation during fatigue, especially at high stimulus frequencies, there was no direct or unique relationship between changes in force and changes in neural conduction. Thus, it is possible that the site at which neural impulse propagation is limited was not the same as that at which force was limited. If this were true, then limitations of neural impulse propagation need not have been associated with decline in force and vice versa. Our data certainly do not rule out the possibility that impairment of neural propagation plays a role in force decline, but would necessitate that the relationship between neural conduction and force be very complex. Any theory attempting to ascribe force failure to neural fatigue would have to account for the lack of direct and unique relationship between neural and mechanical events. It would have to explain that there were instances when neural conduction decreased, yet force remained unchanged, that the relationship between force and neural conduction varied with mode of stimulation, and there were times when force increased no matter which way neural impulse propagation changed (figs. 5 and 6). Other workers have also proposed that impairment of neural impulse propagation plays little role in force decline. Jones et al. (1979) demonstrated that changes in force with fatigue were similar whether produced by stimulation of the nerve or by direct stimulation of isolated curarized muscle. Recently, Hultman and Sjoholm (1983) have shown that EMG measurement alone is a very misleading index of fatigue and that excitation failure could only contribute partly to decline in tension in their model. B azzy and Haddad (1984), found that the integrated EMG of the diaphragm in sheep subjected to inspiratory loading fell upto 20 min before there was a change in Pdi. On the other hand, Aubier et al. (1981), found that the integrated EMG continued to rise as force fell in dogs with cardiac tamponade. These conflicting findings illustrate the difficulties with using EMG criteria for fatigue. In our studies, fatigability was increased with increasing duty cycle and stimulus frequency (from 20 to 50 Hz). These findings agree with those of others in a variety of preparations (Kelsen and Nochomowitz, 1982; Sato e t a l . , 1970). However, when stimulus frequency was increased from 50 to 100 Hz, fatigability did not increase further in the uncasted (group 1) dogs. However, with increased force generation produced by casting, fatigability did increase with increased stimulation frequency from 50 to 100 Hz. Also, in the casted animals there was fatigue at 20 Hz stimulation frequency, a finding not seen in the uncasted animals. These findings indicate that with increased initial force

PHRENIC NERVE STIMULATIONIN THE DOG

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production fatigability increased. It is unlikely that this behavior is due to changes in neural impulse propagation, since, they should be independent of force. The t'mding that fatigability was much less at low stimulus frequency (20 Hz) is similar to those of Sato et aL (1970). There are several possible explanations for this finding. (1) Force generation at 20 Hz was low and therefore energy demand was less, consequently the balance of energy demand to energy supply could be maintained. (2) The lower the stimulus frequency the fewer the total number of impulses the muscle received per unit time. Thus, less time was spent in the active state, making energy demand and accumulation of metabolites less. (3) It has now been shown conclusively by Bellemare et aL (1983), as well as by direct recording of phrenic artery flow in our own laboratory (unpublished), that blood perfusion of the diaphragm is limited during contraction as in skeletal muscle (Lind and McNichol, 1966). Since this limitation of blood flow is directly related to pressure generation and duty cycle, and since pressure generation was less at 20 Hz stimulus frequency, perhaps oxygen supply was less limited at 20 Hz than at the higher stimulus frequencies.

Changes in neural impulse propagation. Decreased neural impulse propagation could take place as the result of changes in the nerve trunk, small branching axons (Grossman et aL, 1979), neuromuscular junction or the muscle membrane. However, Krnjevic and Miledi (1958) have indicated that it is unlikely that the nerve trunk is the site of decreased impulse propagation. We and others (Bigland-Ritchie, 1978; Bigland-Ritchie et al., 1979) have observed a greater degree of impairment of impulse propagation as judged by increasing CT and decreasing CAP area with increasing stimulus frequency from 50 to 100 Hz. With CS at 20 Hz, CAP area actually increased. It has been pointed out (Bigland-Ritchie, 1978; Bigland-Ritchie etal., 1979) that with mild degrees of impairment at the level of the muscle membrane (decreased membrane excitability) an increase in CT with preservation of the CAP amplitude would lead to a widening of the action potential and therefore an increased area. Thus, the increase in CAP area seen at 20 Hz stimulus frequency probably represents a degree of impairment to neural impulse propagation along the muscle membrane. Decreased conduction along the muscle membrane could be due to the accumulation of metabolites such as lactic acid (Mortimer et aL, 1970) or changes in extracellular cation concentration (Jones et al., 1979). Bigland-Ritchie et al. (1979) claimed that such cation changes could reduce the excitability of the muscle surface membrane sufficiently to account for all aspects of high frequency fatigue, even in the total absence of neuromuscular block. What factors may be responsible for decreased CAP area? The CAP area is influenced by the number of fibers activated, the synchrony of fiber activation and the area of the action potentials for each fiber (Bigland-Ritchie et al., 1982). It is also related to the degree to which the recording electrode can record the summation of action potentials in the area of detection. We cannot rule out the possibility of drop out of muscle fibers. If this occurred, however, then changes in CAP area and those in force

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H. BARK AND S.M. SCHARF

c o u l d well be c o r r e l a t e d . H o w e v e r , we c o u l d n o t d e m o n s t r a t e any direct r e l a t i o n s h i p b e t w e e n C A P a r e a a n d force at any o f the stimulus f r e q u e n c i e s (figs. 4 - 6 ) . It is p o s s i b l e t h a t the d e c r e a s e in C A P a r e a is r e l a t e d to c h a n g e s in the d e t e c t i o n o f the p o t e n t i a l difference p r o d u c e d by i n d i v i d u a l fibers, for e x a m p l e , s o m e striated m u s c l e fibers m a y d e m o n s t r a t e a p r o l o n g e d p e r i o d o f d e p o l a r i z a t i o n f o l l o w i n g the action p o t e n t i a l , called an 'after p o t e n t i a l ' , w h i c h m a y last up to 100 m s e c . S i n c e the interstimulus i n t e r v a l at 50 a n d 100 H z stimulus f r e q u e n c y is m u c h less t h a n this, action p o t e n t i a l s c o u l d be s u p e r i m p o s e d o n the after potential. S i n c e d e p o l a r i z a t i o n w o u l d c o n t i n u e to o c c u r to the s a m e potential, this c o u l d l e a d to a d e c r e a s e d a m p l i t u d e o f the C A P as m e a s u r e d f r o m a d i s t a n t extracellular e l e c t r o d e .

Acknowledgements. This project was supported by a grant from the Chief Scientist's Office, Ministry of Health, Israel.

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