Epilepsio, 31(Suppl. 2):S7-S19, 1990 Raven Press, Ltd., New York 0 1990 International League Against Epilepsy

Effects of Vagal Stimulation on Experimentally Induced Seizures in Rats Dixon M. Woodbury and J. Walter Woodbury The Division of Neuropharmacology and Epileptology and the Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah, U.S.A.

Summary: Repetitive stimulation of the vagus nerve inhibits chemically induced seizures in dogs. We report here the results and conclusions from studies designed to answer some of the immediate questions raised by this finding. (1) Maximal stimulation of vagal C fibers at frequencies greater than 4 Hz prevents or reduces chemically and electrically induced seizures in young male rats. (2) Antiepileptic potency is directly related to the fraction of vagal C fibers stimulated. (3) Vagal stimulation shortens but does not shut down a chemical seizure once it has begun. (4) In rats, optimal stimulus frequency is approximately 10-20 Hz; duration of stimulus, 0.5-1 ms; and stimulus strength, 0.2-0.5 d m m * of nerve crosssection. These results, when taken together with similar results obtained from dogs, monkeys, and humans, strongly suggest that periodic stimulation of the vagus

nerve using appropriate stimulation parameters is a powerful method for preventing seizures. The data from the literature suggest that the antiepileptic actions of vagal stimulation are largely mediated by widespread release of GABA and glycine in the brainstem and cerebral cortex. The probable pathway is via projections from the nucleus of the solitary tract to the reticular formation and thence by diffuse projections to the cortex and other areas. Intermittent vagal stimulation has the potentiality of reducing the number andor the intensity of seizures in patients with intractable epilepsy. These results indicate that feasibility studies in humans should be continued and expanded. Key Words: Epilepsy-Seizures-Vagus nerve-Electric stimulation-Neurological model-Pentylenetetrazol seizures-3-Mercaptopropionic acid seizures-Maximal electroshock seizures.

Zabara (1985a,b, 1987) found that repetitive stimulation of the vagus nerve inhibited chemically induced seizures in dogs. We undertook these studies as part of a program to test the feasibility of using vagal stimulation (VS)to control seizures in humans with intractable epilepsy. The approach was to treat VS as though it were an experimental antiepileptic drug. Rats were used because there are well-validated protocols for using rats to estimate the potency of antiepileptic compounds in humans (Woodbury, 1972). The adjustable parameters are different (stimulus strength and frequency vs. dose and dose interval) but the desired end result is the same: control of seizures in humans. We sought and obtained answers to the following questions but some of the results have not been adequately confirmed: (1) Can VS prevent and/or modify seizures in rats? (2) If so,

what fiber type(s)-A, B, and/or C-in the vagus nerve are responsible for the antiepileptic action? (3) What types of seizures are affected by VS? (4) What strength and pattern of stimulation produces the most potent antiepileptic action? The vagus or tenth cranial nerve is composed of somatic and visceral afferents and efferents. The vast majority of vagal nerve fibers are unmyelinated C fibers having conduction speeds of less than 1 m/ s. Small myelinated B fibers and large A fibers also run in the vagus nerve. A majority of the fibers (6580% in the cat) fibers are unmyelinated visceral afferents whose cell bodies lie in the nodose and jugular ganglia. Most of the central projections terminate in the nucleus of the solitary tract (NTS), and some continue to the medial reticular formation of the medulla, the cerebellum, the nucleus cuneatus, and elsewhere. The NTS sends fibers to many brain regions, including the hypothalamus, the amygdala, and the thalamus (Ricardo and Koh, 1978). For further details, consult the paper by Rutecki (1990).

Address correspondence and reprint requests to Dr. D. M. Woodbury at Department of Physiology University of Utah School of Medicine, Salt Lake City, UT 84108, U.S.A.

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D. M . WOODBURY AND J . W . WOODBURY

Stimulation of vagal afferents evokes detectable changes in the electroencephalogram (EEG) in all the regions listed above. The EEG changes depend on the stimulation parameters. Synchronization of the EEG results from weak stimuli that activate only the myelinated nerve fibers. Desynchronization results when the stimulus intensity is increased to a level that activates unmyelinated nerve fibers (Chase et al., 1966, 1967). A survey of the literature provides much evidence indicating that VS can produce widespread inhibitory effects. Schweitzer and Wright (1937) showed that stimulation of the cut end of the vagus produces a temporary depression or abolition of the knee-jerk reflex. Recovery was gradual when stimulation was discontinued. This inhibitory effect is not due to circulatory or respiratory changes. Zanchetti et al. (1952) showed that interictal spikes, produced by topical application of strychnine to the cerebral cortex of the cat were blocked by VS with pulses 1-2 V high, 0.5 ms in duration repeated 50 times per second. Stoica and Tudor (1967, 1968) found that low-intensity VS (1-4 V, 0.3 ms, 30 Hz) decreases the frequency of spiking activity induced by strychnine applied topically to the coronal gyrus of cats. Spread to the contralateral side was also inhibited by VS. Similar results were also obtained in the antenor sigmoid, and suprasylvian and medial marginal gyn. VS was usually more effective in inhibiting activity in the anterior than in the middle and posterior cortical areas. Stoica and Tudor (1968) concluded from these studies that the vagal afferent projections to these areas are diffuse and nonspecific, and that different numbers of fibers project to each area. The diffuse projections to the cerebral cortex probably arise from the mesencephalic and diencephalic reticular formation, which receive fibers from the NTS. Clearly, VS acts to raise the threshold for neuronal firing and to decrease the spread of seizure activity. Zabara's (1985a,b, 1987) recent findings provided the direct impetus for these studies. He found that VS (5-15 mA, 0.5-0.6 ms, 80-150 Hz) inhibited generalized seizures induced in dogs by strychnine and PTZ (pentylenetetrazol). The antiepileptic effect outlasted the stimulus. Lockard and Congdon (1986) studied the effects of VS (5 mA, 0.5 ms, frequencies from 50 to 250 Hz) on spontaneous focal and secondarily generalized seizures induced in rhesus monkeys by implantation of alumina gel in the cerebral cortex. VS was delivered at the onset of every spontaneous seizure for the duration of the seizure or every 3 h for 40 s if stimulation (initiated by a seizure) had not occurred in the preceding hour. The 6-week stimEpilepsia. Vol. 31. SUPPI.2. 1990

ulation period was preceded and followed by nonstimulation baseline periods of 2 or more weeks. In two of four monkeys, seizures were abolished. In the others, the interseizure interval was lengthened and the interval between seizures became relatively invariant. The reduction in seizure frequency carried over temporarily into the subsequent baseline period. No consistent effects on interictal spikes were observed. It'should be noted that the long, strong, high-frequency stimuli used in all these investigations were almost certainly maximal for C fibers. Most of the results described in this paper have been summarized in abstract form (Woodbury and Woodbury, 1989). METHODS

Two protocols evolved during the course of these studies: (1) Studies were performed on anesthetized rats given chemically induced convulsions. Good recordings of the compound action potential of the vagus nerve can be obtained, permitting determination of fiber group(s) involved. (2) Studies were done on unanesthetized rats with implanted vagalstimulating electrodes. Maximal tonic-clonic convulsions cannot be elicited in anesthetized rats. Studies on anesthetized rats Preparation Male Sprague-Dawley rats (100-200 g) were anesthetized with ketamine-acepromazine (ketamine 90 mg/kg; acepromazine 10 mg/kg). The scalp was exposed, and electrocorticographic (ECoG) electrodes were screwed into small holes drilled in the left and right temporal and parietal regions, and the wound was closed. The animal was laid on its back, a midline incision was made in the neck, and one of the vagus nerves (usually the left) was dissected free for about 1 cm. The nerve was lifted onto a set of five stainless steel wire-hook electrodes placed immediately above the nerve. The canoe-shaped pouch formed by the dissection was filled with mineral oil until the nerve was covered to prevent drying. Interelectrode spacing was about 1 mm. Recording system Electrocardiographic (ECG) electrodes were attached to the right front and the left hind paws; electromyographic (EMG) electrodes were inserted into the muscles of the right foreleg. Respiration (RESP) was monitored with a linear displacement transducer resting on the chest wall near the diaphragm. The ECoG, EMG, ECG, and RESP were displayed on four channels of a Grass Model 6 eight-channel electroencephalographic recorder. The paper speed in most of the records was 6 mm/s.

VAGAL STIMULATION A N D SEIZURES Convulsions were induced by intraperitoneal injection of 3-mercaptopropionate (3-MP or 3-MPA) (35 mg/kg) or pentylenetetrazol (PTZ) (50 mg/kg). This dosage usually produced a few clonic convulsions lasting about 30 s with interictal periods of 12 min. Further injections were given as needed. In a few experiments, one injection of PTZ produced a mild status epilepticus in which the EMG activity continued at a nearly constant level for many minutes. This fortuitous situation greatly simplified the process of estimating optimal stimulus parameters. Measurement of stimulus strength Quantitative studies of the excitability characteristics of the various fiber groups in the vagus require an accurate measurement of the strength of the stimulus. Probably the best measure of stimulus strength when using external stimulating electrodes is the voltage gradient halfway between the electrodes. When the nerve is bathed in oil, the voltage gradient is directly proportional to the stimulating current but the current is not always proportional to the stimulating voltage because of irreversible reactions at the metal electrode-body fluid interface. The total resistance is dependent on current density and the amount of charge transferred. Hence, it was deemed essential to measure stimulus current directly. The output of a Grass Model S4 stimulator was isolated from ground by passing it through a highfidelity pulse transformer. The output was connected to the nerve as shown in Fig. 1, lower right. Stimulus current was measured by connecting the input terminals of a high-input resistance (20 MR) differential amplifier to the ends of a 1-KR resistance in series with one of the stimulus output leads (not shown in Fig. 1). Stimulus current was taken as the average height of the current pulse, which was displayed on the lower beam of a Tektronix 502A dual-beam oscilloscope (lower trace of the lower left-hand record in Fig. 1). The sag is mostly the result of electrode polarization. Stimulus artifact It is difficult to stimulate and record from an intact nerve trunk because the stimulating current follows two paths: the usual short path between the two adjacent stimulating electrodes and another longer path through the nerve and the body fluids; that is, the two regions where the nerve reenters the body are “shorted” together by the body fluids. In the longer path, a substantial portion of the stimulating current flows past the recording electrodes and generates a huge shock artifact that totally obscures the compound action potential. This problem was largely overcome by using three stimulating elec-

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* 5 ms FIG. 1. Compound action potentials of the uncut rat vagus nerve in situ, photographed from the face of a cathode ray oscilloscope. Diagram at right: The stimulating and recording arrangement. Note that this is a bipolar recording system and all action potential recordings are biphasic. The first recording electrode is 1 mm from the nearest stimulating cathode. The potentiometer in the stimulating system is adjusted to minimize the shock artifact (see Methods). Records in left column: Compoundaction potentials. Ordinate is voltage and abscissa is time. Calibration bars are shown. Upper left record: A low amplitude stimulus (30d, 0.02 ms) was delivered slightly to the left of the second vertical line. The phasic upward deflection is the compound action potential. This stimulus reached the threshold for about half of the A (large, myelinated)fibers. The resulting action potentials conduct at a high speed as shown by the very short interval between the stimulus and the start of the upstroke of the action potential. Middle left record: Compound action potential at a stimulus strength that is about half maximal for B (small myelinated) fibers and thus supramaximalfor A fibers. The B component is the bump on the record occurring at 0.5-1 ms. Lower lefl record: MaximalC (unmyelinated)fiber compound action potential elicited by a large, long-duration (500 d, 0.25 ms) stimulus. Note changes in voltage and time bars. A and B components are not distinguishable, because of the five times slower scale and the large size of the shock artifact.

trodes connected as shown in the lower right-hand diagram in Fig. 1. The potentiometer was adjusted to minimize the stimulus artifact. The minimum occurs where the potential between the two outside electrodes is zero, the condition for no stimulus current flow past the recording electrodes. The two outside electrodes were made the stimulating cathodes, so that conduction speeds could be estimated from the latency of the action potential. Nerve recording The recording leads were connected to the input of a differential preamplifier having a gain of 100. The output was displayed on the face of a cathode ray oscilloscope and appropriate records of the compound action potential (AP) were photographed for later analysis. Points on the strength duration curve were obtained for the various fiber groups by Epilepsia. Vol. 31, Suppl. 2. 1990

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D . M . WOODBURY AND J . W . WOODBURY

adjusting stimulus strength at each stimulus duration such that the height of the action potential for that group was a fixed fraction (usually 0.5) of its maximum. Data analysis Chronaxie and rheobase were obtained in most cases from estimates from plots of the experimental data. In a few cases, the results were confirmed by fitting the data to the equation Th = Rh/[l - exp(-t/~)]

where Th = measured threshold, Rh = rheobase, t = stimulus duration, and T = membrane time constant. Chronaxie (Chr) is related to T by Chr = 0.69 7. Experiments on unanesthetized rats Preparation Male rats were given a supramaximal electroshock stimulus (SES) and the maximal electroshock seizure (MES) parameters (flexor phase duration, extensor phase duration and total duration) measured. After recovery, the rats were anesthetized, one of the vagi was exposed in the neck, and a 3or 5-wire cuff-type electrode was placed around the nerve. The wires were brought out at the back of the neck, the wounds closed, and the wires tied in a compact bundle. The animals were allowed to recover for at least 24 h. These rats totally ignored the electrode wires and engaged in normal exploratory behavior as soon as they recovered from the anesthesia. Protocol On the day of the experiment, the animal was placed in a box 6" x 6" x 4" high, ECG and EMG electrodes were attached to the rat and connected to the 8-channel recorder; the wires from the cuff electrode were connected to the stimulator and the recording amplifier. The rat was then given an SES to ascertain whether or not it had regained its ability to generate a MES. The effects of vagal stimulation were assessed by commencing vagal stimulation 30 s before an SES and continuing it for 30 s after. The stimulus parameters were: Pulse width, 0.5 ms; pulse repetition frequency, 10 or 20 Hz. Stimulus strength was set to produce excitation of all the fibers in the vagus, if possible. Shunting by body fluids greatly increased the amount of current required and sometimes the maximum available stimulus was insufficient. In most experiments, shunting reduced the size of the compound action potential to undetectable levels and it was necessary to estimate the fraction of C fibers stimulated from the change in heart rate (see below).

Epdepsia. Vol. 31. Suppl. 2, 1990

RESULTS

Control studies Fiber groups of the vagus nerve In a classic series of experiments on the compound action potentials of nerve trunks, Erlanger and Gasser (1930, 1937) established the correspondence between distinct deflections in the compound action potential and clumping of nerve fiber diameters. The correspondence arises from the direct monotonic relationship between conduction speed and fiber diameter. In their 1930 paper, they distinguished 3 major groups, A, B, and C. A fibers are large and myelinated. In mammals (cat and dog), they have conduction speeds of 90 to 30 d s corresponding to fiber diameters of about 20 to 5 pm. B fibers are small and myelinated with conduction speeds of 20 to 10 d s . C fibers are unmyelinated having conduction speeds of 1.6 to 0.3 d s . The threshold for initiating action potentials using external electrodes is approximately inversely proportional to the square of fiber diameter. As the strength of the shock applied to a nerve trunk is increased, the A fibers are recruited first, followed by B, and then C fibers. The spread is large, the thresholds of B fibers are two to three times higher than A fiber thresholds, whereas C fibers thresholds are 10 to 100 times higher. The records in Fig. 1 show the presence of at least three distinct fiber groups in the rat vagus nerve. The left-hand column shows compound action potentials recorded at small (top record), somewhat larger (middle), and very large stimulus amplitudes. The compound action potential in the top record is generated by A fibers. High conduction speeds and the short distance between the nearest stimulating cathode and the first recording electrode (about 1 mm; lower right diagram, Fig. 1) render it difficult to measure conduction speed in A fibers. In other experiments where the conduction distance was 2 mm, conduction speed estimates were of the order of 30 to 10 d s , but this is almost certainly a large underestimate, the true values could have been twice as large. The compound AP shown in the middle record was obtained when the stimulus strength was increased from 30 pa to 70 pa. The first (A fiber) component has reached its maximal amplitude and a second component having a latency to peak of about 0.75 ms has appeared. This component appears as a hump on the falling portion of the A fiber component. Conduction speed estimates for this component were in the range of 1.5-4 d s . These values are probably also underestimates. This hump is gen-

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VAGAL STIMULATION A N D SEIZURES erated by small myelinated fibers having thresholds two or three times higher than A fibers. We designate them as B fibers despite the mismatch with the conduction speeds given by Erlanger and Gasser (1930). The lower left record in Fig. 1 shows the action potential of the C fibers. This record was obtained by increasing the stimulus duration from 0.02 to 0.25 ms and increasing the stimulus strength to 300 PA. Correcting for the change in stimulus duration, the threshold for C fibers is more than 100 times that for A fibers. Note the change in the time and voltage scales. The potentiometer used to balance the shock artifact had to be readjusted to keep the trace on screen, and the A and B components are not distinguishable from the large stimulus artifact. The conduction speed of this component can be estimated from the conduction distance (1 mm) and the latency to the first deflection: speed = (1 m d 2 ms) = 0.5 d s . This deflection is generated by small, unmyelinated C fibers. The C fiber AP is large because the vast majority of the fibers in the vagus are unmyelinated; their total cross-sectional area is larger than the total for A and B fibers. It is shown below that the antiepileptic effect of VS is directly related to the fraction of C fibers stimulated. Figure 2 shows strength-duration curves for the vagus nerve. Figure 2A shows curves for A, B, and C fibers measured in anesthetized rats. Note the large differences in threshold between the fiber groups; they are close to the ratios given by Erlanger and Gasser (1930). Figure 2B shows a C-fiber curve measured in an unanesthetized rat obtained with an implanted cuff electrode. The variability is no doubt the result of changes in shunting by body fluids as the animal moved. The shape of the curve is much the same as the C-fiber curve in Fig. 2A, but the threshold currents are about 10 times larger. Table 1 summarizes the measured properties of rat vagus nerve fibers. Rheobase is converted to current density by dividing threshold current by the cross-sectional area of the nerve. Included are the properties of two distinct groups of C fibers, named C, and Cz, which were revealed by more careful analysis in an isolated nerve. Physiological effects of vs We measured the effects of VS on heart rate (HR) and RESP to obtain a rough estimate of the degree to which normal body function is altered. Figure 3 shows the effects of VS on the ECG, ECoG, and RESP in anesthetized rats. The three panels (upper, middle, lower) are portions from a continuous record obtained over a 10-min period.

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FIG. 2. Strength-duration curves of rat vagus nerve in situ. Ordinate is threshold current (pA): threshold was defined as the stimulus strength at which the height of the component (A, 8, C) action potential was half of its maximal value. Abscissa is the duration of the stimulus in ps. A: Strength-duration curves for the A, 8, and C Components. Note wide gap between the C fiber and the A and B fiber curves. 8:Strengthduration curve for the C fibers in an unanesthetized rat with an implanted cuff electrode. Threshold currents were nearly ten times those in A but the two curves have very similar shapes.

Upper panel Two closely spaced, maximal C-fiber stimuli (vertical lines on the bottom trace labeled STIM.) were delivered to the vagus shortly after the start of the record. The only noticeable effect is on RESP, which stops in inspiration for about 1 s, and then gradually returns to normal. Next, a single stimulus was given; the effect on respiration is small but distinct. Groups of three and seven closely spaced

Epilepsia, Vd.31, Suppl. 2. 1990

D . M . WOODBURYAND J . W . WOODBURY

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TABLE 1. Some electrophysiological properties of the vagus nerve measured in situ in anesthetized rats Chronaxie

group

Rheobase (pNmm2)

(PS)

Conduction speed (ds)

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APPROXIMATE C FIBER THRESHOLD

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stimuli stop respiration. Note that there are no large effects on HR. Middle panel The vagus was stimulated at 1 Hz throughout. At the start, the stimulus was maximal for A and B fibers; there are no obvious effects on HR or RESP. The stimulus strength was gradually increased to maximal for C fibers, decreased to zero and then increased to the original strength. RESP stopped only while C fibers were being stimulated. HR remained constant throughout.

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STIMULUS STRENGTH IN MICROAMPERES FIG. 4. Relationship between heart rate and strength of vagal stimulation in an unanesthetized rat with a cuff electrode implanted on the vagus nerve. The stimulation rate was 20 Hz, stimulus duration was 0.5 ms. Steady-state heart rate is plotted against stimulus strength. The arrow indicates the estimated threshold for C fibers.

Bottom panel The black band in the STIM trace indicates a 19s period during which maximal C-fiber stimuli were applied at 20 Hz. RESP stopped for 12 s and then resumed with an erratic pattern until the stimulation was terminated. Note that HR decreased dramatically and became erratic.

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FIG. 3. Effects of maximal stimulation of rat vagus C fibers on electrocardiogram (ECG), electrocorticogram (ECoG), and respiration (RESP). Vertical deflections on the STIM. line indicate the time of application of supramaximal stimuli to the nerve. Expiration is up in the RESP trace.

HR vs. stimulus strength in unanesthetized rat Figure 4 shows a plot of HR against stimulus strength in an unanesthetized rat. Stimulus frequency was 20 Hz. The approximate threshold for C fibers is indicated by the arrow. This value is not known accurately because the shock artifact largely obscured the AP. Note that the heart rate decreases from a normal of 275 down to 75 beatdmin. This curve was used for estimating actual stimulus strength from measured heart rate in most of the experiments done with implanted cuff electrodes on awake rats. Effects of VS on PTZ seizures in anesthetized rats A single intraperitoneal injection of the standard dose of PTZ (50 mg/kg) usually produces intense, brief, clonic seizures. The first one occurs within 1 min, followed by one or two more at intervals of a few minutes. The intensity and time of occurrence of each seizure are unpredictable, making it difficult to assess the effects of VS. This problem was serendipitously overcome in an experiment in which a mild, steady status was induced by the PTZ injection. EMG activity remained quite constant for a

VAGAL STIMULATION AND SEIZURES

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The bottom panel of Fig. 6 shows the effects of

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long time, so that the effects of stimulus strength and frequency on convulsive activity could be assessed rapidly and reproducibly. The results are illustrated in Fig. 5. The top trace shows the EMG activity; the bottom trace shows stimulus strength and frequency. Arrows indicate continuation of the record to the next line. The baseline level of EMG activity is shown at the beginning of the top record in the upper panel. Shortly thereafter, VS was begun at 20 Hz. The stimulus strength of 200 pA was maximal for A and B fibers. There appears to be a slight diminution in the amplitude of the EMG, but a subsequent increase shortly thereafter at the same stimulus strength indicates that this was a spontaneous variation. Stimulation at 400 pA, near the C-fiber threshold, definitely diminishes EMG activity. All C fibers are brought into activity somewhere between 600 and 800 pA, and it is seen that stimulation at these strengths nearly abolishes EMG activity. Stimulation was discontinued near the beginning of the continuation record immediately below. Thereafter, EMG activity slowly built up, reaching near control status values about 30 s later. The middle panel shows the results of an identical protocol except that the stimulus frequency was reduced to 10 Hz. The results are nearly identical. The only significant difference is that EMG activity returned to normal about 10 s after turning off the stimulation.

quencies. EMG activity slowly built up when the frequency was reduced from 5 to 4 Hz. Stimulation at 3, 2.5, and 1 Hz was nearly ineffective. The effects of VS on seizures produced by a single dose of PTZ In contrast to mild status, initiation of VS after the beginning of a full-blown PTZ seizure does not terminate it. The top panel in Fig. 6 shows a control seizure. Both the EMG and the ECoG were recorded; the EMG resembles an attenuated version of the convulsive activity in the ECoG. Although the pattern is typical, seizure duration vanes markedly from time to time and animal to animal. The middle panel of Fig. 7 indicates that maximal VS at 10 Hz was started about 10 s after the start of a seizure. Comparison with the top panel suggests that the VS had no effect on the ECoG but may have had a small to moderate effect on the EMG. The large variability in convulsion durations makes individual controlhest comparisons meaningless (see

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START OF I 2o SEC I SEIZURE FIG. 6. Effects of vagal stimulation on the electromyogram (EMG) and electrocorticogram (ECoG) of anesthetized rats with pentylenetetrazol (PT2)-induced seizures. Upper two traces: Control seizure. Middle three traces: Maximal vagal stimulation (lower trace) begun after onset of seizure. Lower three traces: Seizure development is prevented as long as maximal vagal stimulation is maintained. A seizure develops upon discontinuance of the stimulation. Resumption of stimulation does not abolish the seizure. Epilepsia. Vol. 31, Suppl. 2. 1990

D. M . WOODBURY AND J . W . WOODBURY

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TIME FROM START OF SEIZURE TO START OF VAGAL STIMULATION (SECONDS) FIG. 7 Effect of maximal vagal stimulation at 10 Hz on the duration of pentylenetetrazol (PT2)-induced seizures in anesthetized rats. Seizure duration is plotted against the time from the start of the seizure to the start of vagal stimulation. Despite the scatter, the results are statistically significant.

below). The bottom panel convincingly demonstrates that VS can prevent the initiation of seizures. VS was started after the end of a seizure and maintained longer than the expected start of the next seizure. Throughout this period, the EMG was flat except for occasional myoclonic jerks. The ECoG was abnormal, resembling the usual interictal pattern. When VS was discontinued, a very strong seizure was generated in both the ECoG and the EMG. Again, resumption of VS had no obvious effects; the marked decline in the EMG was complete before the resumption of VS and the secondary humps are larger, shorter versions of those seen in the control seizure (top panel). The mean duration of eight control seizures was 28 ? 2 s (S.E.). The mean duration of nine seizures in which VS was initiated sometime during the seizure was significantly shorter: (18 3 s);p < 0.005. Figure 7 shows a plot of seizure duration against the time from the start of the seizure to the start of VS. Although there is a large scatter in the data, the slope of the regression line (not shown) is significantly different from zero (p < 0.03). There seems little doubt that VS shortens seizhres in a time-dependent manner. Effects of maximal VS on 3-MP-induced seizures Figure 8 shows the EMG of an anesthetized rat during a long series of seizures induced by an intraperitoneal injection of 3-MP. Total elapsed time since the injection is shown at the right end of each record. Seizure duration is extremely variable, but strength and duration appear to increase gradually until about 900 s and then decline somewhat. The

vagus was stimulated maximally from 750 to 810 s and again from 910 to 970 s. During the first VS period, there is an indication that seizure duration was shortened somewhat. There was no obvious change in EMG amplitude. During the second period of VS, seizure duration was strikingly decreased; the seizure activity was reduced to a series of frequent, strong, myoclonic jerks with the exception that the last seizure during VS had already lasted 6 s when the stimulus was turned off. The seizure continued for another 15 s. The vagus was also stimulated for 1 min before and after the records shown in Fig. 8. The whole experiment is summarized in Fig. 9, where seizure duration is plotted against the time at which the seizure started. During the first period, VS was begun shortly after the start of a seizure and apparently did not shorten the seizure. The second period of VS (corresponding to the first period in Fig. 8) may have shortened seizure duration but the graph is as equivocal as the original records. The dramatic reduction in seizure duration during the third period of VS (second period, Fig. 8) is emphasized by the graph. However, this dramatic effect had largely disappeared by the time of the fourth period of VS; at best, the effects are marginal, similar to the second period. The effects of VS on FTZ-induced status (Fig. 5) and on discrete seizures (Figs. 6, 7) suggest a possible explanation for the minimal effects of VS during the first, second, and fourth periods, and the

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and wanings. VS abolishes activity during PTZ status (Fig. 5 ) and shortens discrete seizures (Fig. 6). The results shown in Figs. 8 and 9 suggest that VS has the same types of effects on 3-MPseizures. Taken altogether, these data are good evidence that VS exerts strong but not overwhelming inhibitory effects on chemically induced clonic seizures.

vs

Effects of VS on MES MES can be induced in most normal rats by a supramaximal electroshock stimulus to the brain. The seizure is characterized by an initial tonic flexion of the limbs followed shortly by tonic extension and then limb clonus. Control experiments showed that normal MESS cannot be elicited in rats during and for many hours after general anesthesia. During this period, a maximal electroshock induces a pure clonic seizure whose duration and intensity increase as the effects of the anesthetic wear off. It was thus necessary to implant the cuff electrode system at least 24 h before testing the effects of VS on MES. Figure 10 illustrates the result of one experiment. Two control electroshock stimuli were alternated

400 500 600 700 800 900 1000 1100 1200 1300 1400

TIME SINCE 3-MPA INJECTION (SECONDS)

FIG. 9. Compressed presentation of all of the data from the same experiment as that shown in Fig. 8. Seizure duration is plotted against the time the seizure began. Vertical lines marked VS show periods of vagal stimulation.

large effects in the third. Inspection of Fig. 8 shows that there was always some background EMG activity from 830 to 990 s except during the interval when the vagus was stimulated. It can be argued then that the animal was in status with large waxings

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FIG. 10. Effects of vagal stimulation on maximal electroshock seizures (MESS) in rats. The ordinate is the duration of the various components of the MES as shown by the key near the upper part of the ordinate: clonus. extension, and flexion. The plots of heart rate (beatdmin) against time after the start of the seizures (Sz) are for the bars depicting seizure parameter directly beneath them as described on the bottom row. The preoperative MES was normal but the postoperative control seizures had no tonic component, indicating incomplete recovery. After a 24-h recovery period, control and test (VS) were given alternately. It is seen that VS prevents a seizure. Epilepsia. Vol. 31. Suppl. 2. 1990

D. M . WOODBURYAND J . W . WOODBURY

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with two stimuli given at the middle of a 60-s period of VS. The lower row describes the experimental condition, the next row up shows the seizure parameters, and the upper two rows show heart rate as a function of time after the electroshock stimulus was given (“time after start of SZ”). The preoperative control MES was normal (first bar: Re-Op, MES, 0 h). The cuff electrode was implanted 24 h later and a maximal electroshock stimulus given 5 h later (second bar: MES, no VS, 29 h). The response to the maximal electroshock stimulus was entirely clonic; there was no tonus. Another electroshock stimulus was given 45 min later (third bar: MES + VS, 29.75 h) halfway through a 1-min period of VS as shown on the graph of heart rate directly above. There was no seizure response; the animal merely jumped in response to the shock. Note the fall of HR from 400 to less than 100 during the VS. Another maximal electroshock was given 20 min later (fourth bar, 30.1 h) and a prolonged clonic seizure occurred. Thirty minutes later, a final electroshock stimulus given during VS produced mild jaw chopping for about a second. These results show that VS can prevent clonic seizures in rats induced by electroshock but do not provide any evidence

about tonic-clonic (maximal) electroshock seizures. Figure 11 summarizes an experiment in which VS prevented tonic-clonic seizures. Consider first the lower row. The preoperative control seizure was within normal limits (left bar); the ratio of the duration of the extensor tonus to flexor clonus (EF) was 4.08, whereas the EF of the postoperative seizure at 24 h (second bar) was EF = 4.23, not significantly different. However, the rat’s only response to a maximal electroshock stimulus given 5.7 h later during the middle of a 1-min period of VS (third bar) was a jump during the shock. Immediately thereafter, the rat resumed normal exploratory and grooming behavior. To ascertain if the electroshock stimulus and/or VS had any residual effects on brain excitability, another electroshock stimulus was given 1 min later without vagal stimulation. The resulting MES was within normal limits. However, the extensor phase was considerably shorter than normal perhaps indicating that there was still some residual inhibitory activity from the VS. The upper row shows the results of a less satisfactory experiment. It can be seen that VS significantly reduces the severity of the electroshock sei-

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MES* MES Vogal No Vagal Stim. Stim. Stim. 24 Hr 29 Hr 29 Hr 40 min 4lmn FIG. 11. The results of two experiments testing the effects of VS on maximal electroshock seizures. See legend to Fig. 10. The only difference is that the heart rates are not given. Lower row: Maximal C-fiber stimulation of the vagus (as assayed by heart rate) prevents the occurrence of a seizure. Upper row: Less than maximal C-fiber stimulation abolishes the extensor component of the MES. Epilepsia. Vol. 31, Suppl. 2. 1990

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degree of inhibition is directly related to the fraction of C fibers stimulated.

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FIG. 12. Summary of results from experiments on 10 rats in which VS was submaximal because of technical problems. See legend to Fig. 11. * = statistically significant difference with respect to the preoperative control value.

zures but does not abolish them. Although the maximum available stimulus strength was applied to the vagus, the effects of VS on HR were quite small, presumably because of excessive shunting of the electrodes by body fluids. The conclusions from Fig. 12 are that VS can abolish the tonic phase of MESS and that the antiepileptic effect of VS depends on the fraction of C fibers fired as estimated from the change in heart rate. Figure 12 summarizes the results of several other experiments in which VS was submaximal for C fibers; there was little change in HR. VS did not change total seizure duration significantly but did significantly increase flexor and clonus durations and decrease extensor duration; both these effects are evidence of decreased seizure activity. Although the E F ratio was significantly lower than normal in postoperative controls, VS significantly reduced this lower than normal ratio. A further finding is not illustrated: During the recovery from an MES there is usually a long lasting (1 or more min) series of myoclonic jerks (every few seconds). We found that VS abolished or greatly shortened the duration of this period. These results show that VS can abolish or reduce the tonic component of the convulsive response to a supramaximal electroshock stimulus, and that the

Discussion will be limited to the reliability of the results and a general discussion of possible mechanisms of action. The relevant results are first briefly summarized to facilitate discussion. (1) Maximal stimulation of vagal C fibers at frequencies greater than 4 Hz prevents or reduces chemically and electrically induced clonic and tonic clonic seizures in young male rats. (2) Antiepileptic potency is directly related to the fraction of the vagal C fibers stimulated. (3) Vagal stimulation shortens but does not shut down a chemically induced seizure once it has begun. Reliability of the results Significant results were obtained from eight rats given chemically induced convulsions and in 10 rats given electroshock convulsions. Although these numbers are not large, all the results are consistent. There were a number of cases where it appeared that VS stirnulation had no antiepileptic effect. In all of these cases, investigation showed that the vagus nerve had not been stimulated. This happened in anesthetized animals when the convulsive activity had lifted the nerve off of the stimulating electrodes, and it occurred in awake animals when the cuff electrode was not in close contact with the nerve or when the vagus nerve had been damaged by movement of the cuff electrode during convulsions. In all cases in which negative results were obtained, autopsy showed that the stimulating electrodes were not in close proximity to the nerve. It is highly probable that we have missed some important phenomena and that some of our estimates will have to be revised. Nevertheless, our results when combined with those from the pertinent literature make it almost a certainty that VS exerts potent antiepileptic actions in mammals. Possible mechanisms of the antiepileptic action of

vs

The relevant literature provides a good basis for understanding the antiepileptic efficacy of VS. General statements about the mechanisms of seizure generation and prevention amount to truisms but may be worth stating anyway. (1) A convulsion can be initiated by increasing the excitation andlor reducing the inhibition of suitably interconnected groups of neurons in the CNS. This can be done experimentally by creating localized lesions of the cerebral cortex (alumina gel) or by administering systemic drugs that decrease the efficacy of all inEpilepsio. Vol. 31, Suppl. 2, 1990

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D . M . WOODBURYAND J . W . WOODBURY

hibitory synapses (PTZ, 3-MP, strychnine) or increase the efficacy of excitatory synapses [Nmethyl-saspartate (NMDA), kainic acid, quisqualic acid]. (2) VS could abolish seizures by increasing the level of inhibition either locally or globally. The data strongly suggest that the inhibitory effects of VS are global, as seizures induced by systemically administered convulsants are blocked. The remaining question is which inhibitory neurotransmitters are released on a global scale either directly or indirectly by maximal VS? The data in the literature point to both y-aminobutyric acid (GABA) and glycine. GABA is implicated because both PTZ and 3-MP seizures are blocked by VS. PTZ blocks activation of inhibitory chloride channels by GABA; 3-MP decreases GABA synthesis and release by inhibiting glutamic acid decarboxylase (GAD). Glycine is also implicated: Strychnine acts by competing with glycine for the glycine receptor site on the inhibitory chloride channels and VS inhibits strychnine seizures. Miller et al. (1987) used microinjections of yvinyl-y-aminobutyric acid (GVG), an irreversible inhibitor of GABA transaminase to delimit those regions of the brainstem required for generation of PTZ and MES seizures. GVG raises GABA levels in the vicinity of the injection site. PTZ seizures can be blocked by GVG injections into an extensive region involving the reticular formation, diencephalic regions in the vicinity of the anterior medial thalamus and caudal hypothalamus, and bulbar regions, which give rise to descending motor pathways to the spinal cord. Because PTZ seizures are inhibited by VS, which activates pathways that involve these same systems, it is apparent that GABA release is an important part of the antiepileptic action of VS. The anatomical substrate for MESS is much more limited. Miller et al. (1987) found that GVG microinjections block MES only when the function of the substantia nigra is affected. Because VS blocks MES seizures, the implication is that VS leads to GABA release in the substantia nigra. An increased release of both GABA and glycine could account for the massive inhibitory effect of VS on both clonic and tonic seizures. Studies by Engstrom and Woodbury (1988) of the factors determining the thresholds for clonic and for tonicclonic seizures suggest that glycine pathways are involved in regulating the mean level of brain excitability, whereas GABA pathways are involved in preventing the spread of activity that produces tonic-clonic convulsions. This fits neatly with the preceding arguments suggesting that VS causes the release of GABA and glycine in large quantities Epilepsia. Vd.31. Suppl. 2. 1990

throughout large volumes of central and cortical grey matter. The specific GABA and glycine pathways activated by VS are not known but undoubtedly involve vagal d e r e n t s that enter the nucleus of the solitary tract. The NTS is known to contain both GABAergic and glycinergic synapses (Meeley et al., 1989). There are direct connections from the NTS to the hypothalamus, amygdala, and limbic forebrain structures including the thalamus. These areas are known to be involved in seizure initiation. There are also pathways from the NTS via intermediate relays in the brain stem reticular formation to the cerebral cortex, cerebellum, and thalamus. It appears probable then that VS increases inhibitory activity in many areas of the brain that are known to be involved in preventing the initiation and spread of seizure activity. Stimulation of vagal unmyelinated (C) fibers has two effects that appear to be closely related: (1) inhibition of electrically and chemically induced seizures and (2) desynchronization of the EEG, which is known to be mediated by the reticular activating system (RAS). The obvious and probably correct conclusion is that the anticonvulsant effect of VS is exerted via the direct or indirect (via the NTS) projections of vagal afferents to the RAS. The principal neurotransmitter of the RAS is the inhibitor 5-hydroxytryptamine (5-HT), suggesting the additional possibility that VS exerts some of its anticonvulsant effects by enhancing the release of 5-HT (Browning, 1985; Puizillout et al., 1984). Overall, the case is strong for the hypothesis that vagal stimulation exerts widespread inhibitory effects that are potent enough to turn off EMG activity in status but not potent enough to turn off a discrete seizure once it has begun. More simply, VS greatly increases the threshold for seizure initiation but not enough to prevent seizure initiation by a strong stimulus. The inhibitory action of VS is strong enough to terminate the seizure early. Acknowledgment: This work was supported in part by agrant from Cyberonics, Inc., Webster, Texas. We thank Mr. Reese Terry for his continuing interest in this research and for his helpful suggestions. D.M.W. is recipient of Research Career Award ( 5 K06-NS-13838) from the National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland.

REFERENCES Browning RA. Role of the brain-stem reticular formation in tonicclonic seizures: lesion and pharmacological studies. Fed f r o c 1985;44:2424-31.

Chase MH, Nakarnura Y , Clemente CD, Sterman MB. Afferent vagal stimulation: neurographic correlates of induced

VAGAL STIMULATION AND SEIZURES EEG synchronization and desynchronization. Brain Res 1%7; 5 : 236-49.

Chase MH, Sterman MB, Clemente CD. Cortical and subcortical patterns of response to afferent vagal stimulation. Exper Neurol 1966;16:36-49. Engstrom FI, Woodbury DM. Seizure susceptibility in DBA and C57 mice: the effects of various convulsants. Epilepsia I988 ;29:389-95. Erlanger J, Gasser HS. The action potential in fibers of slow conduction in spinal roots and somatic nerves. A m J Physiol 1930;92:43-82. Erlanger J, Gasser HS. EIecrrical signs of nervous activity. Philadelphia: University of Pennsylvania Press, 1937. Lockard JS, Congdon WC. Effects of vagal stimulation on seizure rate in monkey model. Epilepsia 1986;27:626. Meeley MP, Underwood MD, Talman WT, Reis DJ. Content and in vitro release of endogenous aminoacids in the area of the nucleus of the solitary tract of the rat. J Neurochem 198933: 1807- 17. Miller JW, McKeon AC, Ferrendelli JA. Functional anatomy of pentylenetetrazol and electroshock seizures in the rat brainstem. Ann Neurol 1987;22:615-21. Puizillout JJ, Gaudin-Chazal G, Bras H. Vagal mechanisms in sleep regulation. 1984:19-38. Exp Brain Res 1984;suppl8:1938. Ricardo JA, Koh ET. Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat. Brain Res 1978;153:1-26.

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Rutecki P. Anatomical, physiological, and theoretical basis for the antiepileptic effect of vagus nerve stimulation. Epilepsia 1990;31(suppl 2):S7-19(this issue). Schweitzer A, Wright S. Effects on the knee jerk of stimulation of the central end of the vagus and of various changes in the circulation and respiration. J Physiol 1937;88:459-75. Stoica I, Tudor I. Effects of vagus afferents on strychninic focus of coronal gyrus. Rev Roum Neurol 1%7;4:287-95. Stoica 1, Tudor I. Vagal trunk stimulation influences on epileptic spiking focus. Rev Roum Neurol 1968;5:203-10. Woodbury DM. Applications to drug evaluations. In: Purpura DP, Penry JK, Tower DB, Woodbury DM, Walter RD,eds. Experimental models of epilepsy-a manual for rhe laboratory Worker. New York: Raven Press, 1972557-83. Woodbury DM, Woodbury JW. Pentylenetetrazol-inducedseizures in rats are greatly attenuated by vagal stimulation at 420 Hz. Epilepsia 1989;30:681. Zabara J. Peripheral control of hypersynchronous discharge in epilepsy. Electroencephalogr Clin Neurophysiol 1985a; 61 :S162. Zabara J. Time course of seizure control to brief repetitive stimuli. Epilepsia 19856;26:518. Zabara J. Controlling seizures by changing GABA receptor sensitivity. Epilepsia l987;28:604. Zanchetti A, Wang SC, Moruzzi G. The effect of vagal afferent stimulation on the EEG pattern of the cat. Elecrroencephalogr Clin Neurophysiol 1952;4357-61.

Epilepsia, Vol. 31. Suppl. 2. 1990

Effects of vagal stimulation on experimentally induced seizures in rats.

Repetitive stimulation of the vagus nerve inhibits chemically induced seizures in dogs. We report here the results and conclusions from studies design...
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