3. a polyphasic pulse excited the nerve at both sites, by the negative

Journal of Physiology (1993), 460, pp. 201-219 201With 10 figures

Printed in Great Britain

MAGNETIC COIL STIMULATION OF STRAIGHT AND BENTAMPHIBIAN AND MAMMALIAN PERIPHERAL NERVE IN VITRO:

LOCUS OF EXCITATION

BY P. J. MACCABEE, V. E. AMASSIAN*, L. P. EBERLE* AND R. Q. CRACCOFrom the Departments of Neurology and * Physiology, SUNY Health Science Center,

450 Clarkson Avenue, Brooklyn, NY 11203, USA

(Received 18 February 1992)

SUMMARY

1. According to classical cable theory, a magnetic coil (MC) should excite a linearnerve fibre in a homogeneous medium at the negative-going first spatial derivativeof the induced electric field. This prediction was tested by MC stimulation ofmammalian phrenic and amphibian sciatic nerve and branches in vitro, immersed inRinger solution within a trough, and identifying the sites of excitation by recordingresponses of similar latency to local electrical stimulation. Subsequently, theidentified sites of excitation were compared with measurements of the inducedelectric field and its calculated first spatial derivative. A special hardware device wasused to selectively reverse MC current direction and to generate predominantlymonophasic- or polyphasic-induced pulse profiles whose initial phases were identicalin polarity, shape and amplitude. When using the amphibian nerve preparation, acomplication was excitation at low threshold points related to cut branches.

2. Reversal of monophasic current resulted in latency shifts correspondingapproximately to the distance between induced cathode and anode. The location ofeach site of excitation was at, or very near, the negative-going first spatial derivativepeaks of the induced electric field measured parallel to the straight nerve.Significantly, excitation of the nerve did not occur at the peak of the induced electricfield above the centre of the 'figure of eight' MC junction.

3. A polyphasic pulse excited the nerve at both sites, by the negative-going firstphase at one location, and approximately 150 ,ts later, by the reversed negative-going second phase at the other location. Polyphasic and monophasic pulses elicitedresponses with similar latency when the induced current flowed towards therecording electrode.

4. Straddling a nerve with non-conducting solid lucite cylinders created a localizedspatial narrowing and increase in the induced electric field, resulting in a loweredthreshold of excitation. The corresponding closer spacing between first spatialderivative peaks was exhibited by a significant reduction in latency shift when MCcurrent direction was reversed.

5. When a nerve is bent and the induced current is directed along the nervetowards the bend, the threshold of excitation is reduced there. Increasing the angleof the bend from 0 deg to more than 90 deg graded the decrease in threshold.MS 1133

P. J. MACCABEE AND OTHERS

6. In a straight nerve the threshold was lowest when current was directed towardsthe cut end.

7. Optimal excitation at a low threshold point (created on the nerve by a pairof cylinders or a bend, and at a nerve ending) occurs near or within the peak of theinduced electric field, rather than at its first derivative. When adjusting MC outputintensity and moving the junction region of the MC in the long axis of the nerve 3 cmtowards or away from a low threshold point, responses were elicited at nearlyidentical latency, implying a common site of excitation. Possibly, these observationsrelate to examples in the human where a latency shift is lacking.

INTRODUCTION

Although the magnetic coil (MC) stimulator was introduced by Barker, Freeston,Jalinous, Merton & Morton in 1985, the relationship between the site of nerveexcitation and the spatial distribution of the induced electric field still requiresclarification. Intuition might suggest that a straight segment of nerve is excited ator very near the peak of the induced electric field. However, classical cable theorypredicts that this is not the case. Given that transmembrane current is proportionalto the second spatial derivative of the external voltage (e.g. Katz, 1939 and, morerecently, Rattay, 1986), it was proposed that straight axorls are excited by MCstimuli at the negative-going first spatial derivative of the induced electric fieldparallel to the axon (Durand, Ferguson & Dalbasti, 1989; Reilly, 1989; Roth &Basser, 1990). Since the theoretical distance between negative and positive spatialderivative peaks is estimated to be at least 4-6 cm for a conventional round or figure-of-eight MC, it follows that reversing current in such an MC should also reverse thepositions of the cathode and anode by a similar distance (Durand et al. 1989; Roth& Basser, 1990). Thus, over distal human peripheral nerve, a 4 cm distance betweencathode and anode should yield evoked response latency shifts of at least 0'8 ms innerve fibres conducting at 50 m s-'.The hypothesis that the spatial derivative of the induced electric field is the crucial

determinant of the site of excitation, has not been universally confirmed. Whenstimulating distal forearm nerves in the human, different laboratories were unable todemonstrate consistent significant latency shifts with reversal of current direction(c.f. Maccabee, Amassian, Cracco & Cadwell, 1988; Amassian, Maccabee & Cracco,1989b; Claus, Murray, Spitzer & Flugel 1990; Maccabee, Amassian, Cracco, Eberle &Rudell, 1990b; Nilsson, Panizza, Roth, Basser, Cohen, Caruso & Hallett, 1991). Inpart, this may reflect different experimental conditions; e.g. each laboratory variedeither the MC orientation or temporal profile of the MC pulse, and recorded either thecompound motor or sensory responses, or alternatively elicited individual motor unitresponses at threshold.

In this report, we record from mammalian and amphibian peripheral nerve invitro, i.e. in a homogeneous volume conductor at known distances from the MC, toidentify the sites of excitation. The direction of current in the MC is clearly animportant variable; conventionally, it has been reversed by flipping over the MC, butthis introduces possible errors resulting from a slight change in MC position and aphysical asymmetry of the field. These problems are avoided by using a heavy dutyswitch to reverse the direction of current in a stationary MC (Maccabee, Amassian,

202

MAGNETIC NERVE STIMULATION IN VITRO

Eberle, Rudell, Cracco. Lai & Somasundarum, 1991). The volume conductor was atrough filled with Ringer solution. Both straight and bent nerve trajectories arestudied. Another device converted the polyphasic pulse energizing the Cadwell MCinto a substantially monophasic pulse. Direct electrical stimulation is used to definethe sites of MC nerve excitation as in a study of nerve in a brain-shaped volumeconductor (Amassian, Eberle, Maccabee & Cracco, 1992 a, b). Here, the sites ofexcitation are compared to the peaks of the spatial derivative of the measured,induced electric field. A preliminary account of these experiments has previouslybeen presented (Maccabee, Amassian, Eberle, Cracco & Rudell, 1992).

METHODS

Magnetic coils and stimulatorSome of the details have previously been described in Maccabee et al. 1988, 1991; Maccabee,

Erbele, Amassian, Cracco, Rudell & Jayachandra, 1990a. A time-varying magnetic field wasgenerated by a Cadwell Laboratories (Kennewick, WA, USA) MES-10 stimulator using either a9-4 cm (outside diameter) round MC which was covered by epoxy, or a figure-of-eight MC coveredby black tape (each half coil 5 x 4-8 cm). The current profile induced in a wire by the standard MES-10 stimulator is polyphasic, with a period of 280 /is, lasting for at least 750 Its (Maccabee et al.1988).

Current reversing and phase-damping deviceThe output of the MES-10 was fed into a custom-built phase damping device (Cadwell

Laboratories, Inc.) consisting of a manual, heavy duty switch, diode and resistance which allowselective control of both the profile and the polarity of the current. Thus, the current in the MC wasreversible without a change in position of the MC.

In the in vitro experiments, the current induced in the trough volume conductor consisted ofeither the conventional MES-10 polyphasic pulse, or a highly damped predominantly 'monophasic'pulse whose major phase exactly superimposed upon the first phase of the polyphasic pulse (Fig.1E). At increments of 10%, extending from 10 to 100% output intensity, the ratio of theamplitudes of the second to the first phase induced into a wire loop were tested and found to beconstant. For the monophasic pulse, the peak amplitude of the second phase was 20% of the firstphase. For the polyphasic pulse, the second phase was 80% of the first. It is emphasized that thelow amplitude second phase of the monophasic pulse lasted nearly 10 ms while the first phaselasted 70 Its.Volume conductor modelsA monophasic pulse was employed when studying the electric field induced in volume

conductors. The direction and magnitude of the induced electric fields in relatively open volumewere measured in a transparent cylindrical tank (30 cm in diameter, wall thickness 0-3 cm, height12-4 cm (Fig. 1 C)) resting on a U-shaped wooden support. The tank was filled with isotonic salineto a depth of 7 cm.

Unless indicated otherwise, the round and figure-of-eight MCs were held beneath the trough andcylindrical tank by a clamp attached to their handles. Round MC orientations included:symmetrical-tangential and orthogonal. The figure-of-eight MC was centred beneath the cylindricaltank and orientated flat to its under-surface (Fig. 1D).

M1easurement of the electric fieldThe direction and magnitude of the electric field induced into the cylindrical tank were measured

with a vertically suspended 4-conductor miniature shielded cable (a-wire type 1122, 3 05 mm o.d.)inserted through a rigid plastic 2 cc pipette (Fig. IA; Maccabee et al. 1991). The shield and shaftof this co-axial cable were connected to short (2 cm) segments of silver wire (0-76 mm o.d.) whichprotruded from the distal end of the pipette. The wires were bent at right angles to the verticalcables and to each other. Thus, the electric field was recorded in an X-Y-plane parallel to thebottom surface of the tank. The distance between the bared tips of each bipolar wire probe was10 mm and the voltage drop recorded by each pair was fed into a differential amplifier. In 1 cm

203


steps the assembly was moved in three-dimensional space by a micromanipulator; thus, the voltagegradients (mV mm-') were recorded at 15 x 15 different locations in a given X-Y-plane. Eachmeasurement location corresponded spatially to the intersection of the X- and Y-recording axes. Asshown in previous studies, the plane of the current induced into various unrestricted flat surfaced

A EInduced pulses

C

12cm

-Y~~

30 cm

B D

02 ms

Fig. 1. The experimental probes used to record the induced electric field with the typesof induced pulse profiles and polarities used to stimulate peripheral nerve in vitro. A, theassembly consists of orthogonal probes used to record magnitude and direction ofelectrical fields parallel to the bottom surface of a cylindrical tank. -X, +X = X-record-ing axis; - Y, + Y = Y-recording axis. (For further details see Maccabee et al. 1991.)B, vertically orientated right-angle co-axial cable recording electrode used to measure theinduced electric field in the long axis of the nerve on the bottom of the trough. C,cylindrical tank filled with isotonic saline. D, view from the top of the figure-of-eight MCorientated flat to the bottom surface of the cylindrical tank. E, electrical field profiles,separate and superimposed (above) and in both directions (below), induced in normalsaline by the monophasic and polyphasic MC pulses used in this study.

as well as spherical volume conductors is predominantly parallel to the inner surface of the volumeconductor adjacent to the MC regardless of the orientation or design of the MC (Tofts, 1990; Cohen& Cuffin, 1991).Measurements by both pairs of orthogonal electrodes were used to calculate magnitude (by

Pythagorean theorem) and direction (by arctangent) of the electric field. These vector fields,illustrated as arrow diagrams in the cylindrical tank for different MCs and orientations, werepreviously illustrated in Maccabee et al. 1991. Because a homogeneous (saline) volume conductorwas used, the current density in A m-2 is directly proportional to the recorded electric field.

Recordings taken in the axis of one bipolar pair of recording electrodes were also used toconstruct topographical maps of the electric field induced in the cylindrical tank. Although an X-or Y-axis recording would have sufficed for the round MC owing to its symmetry, the Y-axisrecording was purposely selected for the figure-of-eight MC (Fig. 1D), because of the prominent

204


electric field it induces in the long axis compared to the transverse axis (Cohen, Roth, Nilsson,Dang, Panizza, Bandinelli, Friauf & Hallett, 1990; Maccabee et al. 1990a). The recorded electricfield in the Y-axis (Fig. 2A) is designated E,

Because of its potential importance in defining the site of excitation, the first spatial derivativeof the induced electric fields in the Y-axis (dE,/dY; Fig. 2B) was also calculated (for the specificalgorithm, see Maccabee et al. 1991). These are illustrated both as topographical and contour maps(Fig. 2C and D) with superimposed outlines of the MC and orientation used.

A

60

E 40

E20

0

B d Eyd Y

E 10E

E 5x0

0O

12

Y (cm)

D

0 4 8 12

X (cm)

0

4

Y (crm)8

12

0 4 8 12

X (cm)

Fig. 2. A, the topographic electric field (Ey) and its spatial derivative (dEw/dY) inducedby the figure-of-eight MC held flat to the undersurface of the cylindrical tank filled withisotonic saline. A, voltage gradients (mY mm-') recorded along the Y-axis. B, the firstspatial derivative (mV mm-2) of the topographic electric field also taken along the Y-axis(note ten times increase in amplification). Bottom, contour plots corresponding to Ey (C)and dE,/dY (D). The distance from the inner surface of the tank to the recordingelectrodes was 0 5 cm. MC stimulator output was 20% of maximum.

In-vitro studiesNerve preparation. After pithing, the sciatic nerve and branches of twelve bullfrogs (Rana

catesbeiana) and one toad (Bufo marinaris) were dissected from the level of the posterior tibial andperoneal nerves at the ankle to their rootlets at the vertebral column. Typically, nerve lengths of10-14 cm were obtained. Following dissection, the nerve was immersed in amphibian Ringersolution at ambient temperature and the ends of the nerve were ligated. Usually a sciatic nerve wasrecorded within 3-5 h of its dissection. Occasionally, after refrigeration overnight, nerves

continued to respond adequately for study.

C0

4

Y (cm)8

12

205

8 PHY 460

206 P. J. MACCABEE AND OTHERS

It was soon found that cut branches or branch points in amphibian peripheral nerves could serveas low threshold sites of excitation. As this could confound the interpretation of our results, we alsostudied monkey, pig and cat phrenic nerves, which branch only at their ends. The phrenic nervesof three Yorkshire fetal pigs (11, 19 and 28 days old) were obtained after completion of otherexperiments performed under Saffan (Pitman-Moore, UK) anaesthesia (approximately12 mg kg-' h-', i.v.). The phrenic nerves of two cats, one macaque monkey, and one sheep were alsoobtained after completion of other experiments under anaesthesia as follows: cats, 40 mg kg-'sodium pentobarbitone (i.v.) followed by 20 mg doses as needed; monkey, 100 mg ketamine (I.M.)followed by 15 mg doses of sodium pentobarbitone (i.v.) as needed; sheep, 10-15 mg kg-'thiopental (i.v.) followed by 08-1 % halothane as needed. The nerves were dissected from thediaphragm to the cervical roots; after ligation of the nerve ends, the nerve ends were immersed inmammalian Ringer solution. After excision of the nerves, all the animals were killed using anoverdose of sodium pentobarbitone. Thereafter, experiments were performed as soon as possiblebecause the duration of effective recording rarely lasted longer than 4-5 h following dissection, andnever after overnight refrigeration.Nerve in trough. The dissected peripheral nerve was immersed in Ringer solution, which filled to

a depth of 15 mm a flat-bottomed rectangular trough (19 x 29 cm) consisting of a thin (less than0 5 mm), transparent plastic. At the bottom of the trough the nerve was held down by thin threadson one or both ends (illustrated in Figs 3-7). Beyond the thread at each end, the nerve gently slopedto the surface of the Ringer solution where it emerged for monophasic recording from the proximaland distal ends. When only recording from one end, the other end was held tight to the bottom ofthe trough by a thin wooden Q-tip (2 mm in diameter) on the ligature.

In experiments on nine different nerves, a pair of plastic. non-conducting solid lucite cylinders(diameter 2-8 cm, height 6-1 cm) were placed on-end so that they straddled each nerve, separatedby a distance of 2-10 mm (illustrated in Figs 5 and 6). After removing the nerve but leaving thecylinders in place, the electrical field in a line approximating the nerve trajectory at the bottomof the trough, was also recorded with a single co-axial cable electrode (Fig. 1, bottom left). Theelectrical field was measured with a probe 4 mm in length moved in 4 mm steps. each measurementcorresponding spatially to the mid-point of the probe. Identical ineasurements were made with thecylinders removed.

In experiments on four different nerves, the nerve was bent at various angles, pivoted about asecond thread approximately half-way along the nerve (Figs 7 and 8). In these bend experiments,the proximal nerve end was affixed to the end of the Q-tip which was positioned above the troughby a movable clamp.MC and electrical stimulation of nerve in trough. The figure-of-eight MC was positioned

immediately beneath and contacting the trough with the long axis of the junction centred underand paralleling the nerve. As described above, a predominantly monophasic or a polyphasic electricfield was induced in the Ringer solution, and the direction of the current was reversed at willwithout disturbing MC position. In additional experiments on two different phrenic nerves (cat,pig), the coil was attached to a graduated mechanical clamp, and was moved beneath the troughin 0 5 cm steps with the nerve fixed in place (Figs 9 and 10). In these experiments, the distance fromMC surface to the trough was 15 mm.To determine where along the nerve it was excited by the MC, a bipolar electrode assembly was

inserted into the Ringer solution to stimulate the nerve directly. The bipolar electrode consistedof two silver wires, each 35 ,tm in diameter, which were insulated with teflon except at their tips(Amassian et al. 1992a, b). The nerve was stimulated by a 100,as duration rectangular pulse,isolated from ground by a radio-frequency unit. When the latencies of the MC and electricallyinduced compound nerve action potential responses matched, the site ofMC excitation was inferredfrom the position of the bipolar electrode.

Peripheral nerve recording. After emerging from Ringer solution, the nerve was recorded in airwith a pair of silver wire electrodes. Monophasic recordings were obtained with the ligated end onthe distal electrode. The portion of the nerve exposed to the air was coated with a petroleumjelly-mineral oil mixture to prevent drying. For both MC and electric stimulation, the earth waspositioned in the trough where stimulus artifact was minimal. Compound nerve action potentialswere amplified with a bandwidth of 0-8 Hz to 10 kHz, and displayed on a digital memoryoscilloscope for photographic recording.


RESULTS

Location of first derivative of the electric fieldThe electric fields recorded in the cylindrical tank are virtually identical to those

previously recorded by a linear co-axial cable recording probe in a rectangular trough(Maccabee et al. 1990a). It is noted that the peak electric field (Ey) over the figure-of-eight MC junction is much greater in amplitude and area than the sum of the(reversed) electric fields on either side of it (Fig. 2A and C). This discrepancy partlyreflects the fact that measurements were made only over a portion of the lateralextent of the field. The plot of the first spatial derivative (Fig. 2B) and itscorresponding contour map (D), reveal that the mid-peaks of the maxima andminima are separated by approximately 5-3 cm at both ends of the junction regionof the two half coils. The midpeaks of the maxima and minima for thesymmetrical-tangential and orthogonal round MCs were approximately 8-0 and7*3 cm apart, respectively.

In vitro nerve studiesStraight nerve in homogeneous conducting medium

Using monophasic current (Fig. 3), simultaneous recordings were obtained fromboth nerve ends at threshold and suprathreshold output intensity. At 80% output,reversing the MC current polarity shifted the response latencies in a reverse directionat the R1 and R2 recording electrodes. The shift in latency at the R1 electrode was0 90 ms and at the R2 electrode was 1 05 ms. Because the peripheral end of the nervelay at R2, it is likely that the small asymmetry in latency shift reflected a slightlyreduced conduction velocity distally. At the measured conduction velocity, the shiftin latency corresponded to a conduction distance of approximately 36-5 to 42-5 mmbetween excitation sites.

In six frog nerves, reversing the MC current similarly shifted the site of excitationby 32-3 +4'4 mm (mean +S.D.). The corresponding shifts in site of excitation inmonkey, cat, neonatal pig, and sheep were 38-8, 38-2, 30 3 and 39 0 mm respectively.The cathode closest to R1 was at, or up to 6 mm further beyond, the divergence ofthe figure-of-eight MC junction away from the handle. The cathode closest to R1 waswithin 3-7 mm of the posterior divergence of the junction (Figs 4B and 5A).Compared to the single site ofexcitation observed with one direction ofmonophasic

MC stimulus, a polyphasic MC stimulus excited the axons at both possible loci, nearlysimultaneously. In Fig. 4C, an induced monophasic cathodal pulse (top left trace)and the first phase of a polyphasic cathodal pulse (top right trace), both beingdirected towards the recording electrode, elicited responses with an identical latencyof 1 15 ms. However, while reversal of the monophasic current increased the responselatency to 1t75 ms, i.e. by 0-6 ms (bottom left), reversal of the polyphasic currentincreased the response latency to 1-30 ms, i.e. by 0-15 ms (bottom right).

Response latencies were identical or slightly reduced, e.g. by 200 ,us, when thestimulus intensity was raised above threshold (Fig. 4). Therefore, comparisons oflatencies necessarily had to be based on responses of similar amplitude.Our lowest threshold responses were often generated at the cut (and ligated) ends

8-2

207

208 P. J. MACCABEE AN\rD OTHERS

of the nerve segments when the current along the nerve was directed outward at thenerve ending. At higher MC output intensities these end responses were extinguishedby propagated nerve impulses initiated at sites on the nerve that were closer to therecording electrode. A practical consequence was to limit the length of nerve that

A BBullfrog sciatic Thread ThreadCV = 40 5 m s-'222 0C

______ R___ R2 Q RR

C~~~M

Monophasic MC 60% 70% 80%0 90 ms (_ 36-5 mm)

R2 rR2

- + -2mV1 05ms 0-5ms +

(_42-5 mm)

Fig. 3. Recordings from electrodes on proximal (R2) and distal (R1) ends of a bullfrogsciatic nerve immersed within a trough filled with Ringer solution. A, position of the MCwith respect to trough. The nerve segments emerge from the Ringer solution to rest onrecording electrodes in air. B, accurate dimensional relationship of figure-of-eight MC tonerve trajectory within the trough. In this and subsequent figures the nerves are held tothe bottom of the trough by thin threads that are weighted down by solid plastic cylinders(not shown). C, compound nerve action potentials are elicited by induced monophasiccurrent and recorded simultaneously at the R1 and R2 electrodes. In this and subsequentfigures, filled arrows indicate (+ to-) current direction. MC output intensities at 60, 70and 80%. The vertical arrow (left column) indicates a small response generated betweenthe spatial derivative loci (see text). In this and Figs 5 and 7, -indicates the calculateddisplacement between excitation sites, which is the product of the latency shift and theconduction velocity (CV).

could be studied with the coil. This indicates that the excitation threshold is lowerat a cut end than over a straight segment of nerve fibre.

Straight nerve in an inhomogeneous conducting mediumIn three experiments, the spatial distribution of the induced electric field at the

bottom of the trough was determined. In two nerves, the sites of excitation were

compared when the electrical field was induced in homogeneous and inhomogeneous

MAGNETIC NERVrE STIMULATION IN V7ITRO

conducting media. In plain Ringer solution, (Fig. 5), reversing monophasic currentpolarity resulted in a 120 ms latency shift (E). Compared to frog nerve, conductionin a sheep nerve is relatively slower at reduced temperature. Direct electricalstimulation along the nerve revealed that the sites of excitation were 35 mm apart

A Frog sciaticCV = 43 5 m s-122-25 °C

R

-50MC .;-

BThread

R

C MonophasicMC

0 50%

/- - 46+ - .0 I MC

15 74%

r ms 1 66-+ 46

k-1 75ms

R26-5 mm

PolyphasicMC

* 0

.452

+- b_ 46

MCr11s 53%

~~~~~~I+ - 2 2mVI0-. --Q15ms OS5ms

Fig. 4. Compound nerve action potentials recorded from the distal branch of a frog sciaticnerve immersed in Ringer solution. A, position of MC with respect to trough. B, accuratedimensional relationship of the figure-of-eight MC to the nerve trajectory within thetrough. Dotted lines indicate sites (indicated by E) of the bipolar electrical stimulatingelectrode that elicited responses of identical latency to those elicited by both directionsof monophasic MC-induced current. C, the responses to monophasic (left traces) andpolyphasic (right traces) current profiles are compared. At the MC excitation sites closerto and further from the recording electrodes, proposed effective excitation phases ofcorresponding induced current profiles are indicated by filled circles. Asterisks indicateproposed excitation phases at the sites furthest from the recording electrodes whoseresponses are not revealed by this experimental set-up, owing to collision. Three traces,elicited at threshold and suprathreshold MC output intensities, are superimposed.

(A). The distal cathode was located 6 mm in front of the anterior divergence of thejunction and the proximal cathode was located at the posterior central region of thejunction. A pair of (non-conducting) lucite cylinders were then placed 4 mm apartastride the nerve and approximately over the mid-junction region. With the inducedcathode closer to the recording electrodes, the latency of the compound nerve actionpotential was increased to 1-35 ms, from its value of 0 90 ms in the homogeneousmedium. Therefore, with this direction of current, the site of activation in theinhomogeneous conductor has been shifted away from the recording electrodes.Reversing the MC polarity led to a reduced latency shift of 0 30 ms (Fig. 5F); i.e. one-

209

P. J. MACCABEE ANVD OTHERS

fourth that observed (P120 ms) without the cylinders (E). Direct electrical stimulationalong the nerve now revealed that the sites of excitation were 9-5 mm apart (B). Alsothe response amplitudes were significantly increased.

In a homogeneous medium, the profile of the first spatial derivative of the electricfield consisted of a broad plateau whose peaks were 36 mm apart (C). By contrast,

A Sheep phrenicCV = 28 9 m s-25-5 OC

C

E

EXEE x

0

36 mm

E Monophasic

ThreadB

Thread

R

D

7 EEEE >> EE x0

R

12 mm

F

1 20 ms (- 34-7 mm) 030 ms (- 87 mm)05 ms

Fig. 5. MC stimulation of sheep phrenic nerve immersed in homogeneous andinhomogeneous media volume conductors. A, accurate dimensional relationship of thefigure-of-eight MC to the nerve trajectory within the trough. E indicates sites of excitationby monopolar MC pulses in both directions, obtained by matching latencies ofMC-inducedresponses with those elicited by direct electrical stimulation. B, accurate dimensionalrelationship of nerve trajectory in trough and lucite cylinders astride the nerve. C and D,the measured electric field (mV mm-') and its first spatial derivative (mV mm-2; note tentimes increase in amplification) corresponding to experimental set-ups illustrated at leftand right, respectively. E and F, nerve responses elicited by monophasic current pulses.The electrical fields were measured and the nerve responses were elicited in the same

Ringer solution, at ambient room temperature. MC immediately beneath trough.

1 mV

210

MAGNETIC -NERVE STIMULATION IN TITRO

when the cylinders were in place, the profile of the induced electric field was muchnarrower and its spatial derivative peaks were much more closely spaced (12 mmapart) moving towards the solid cylinders in the mid-junction region. In addition,the magnitude of the negative and positive-going spatial derivative peaks for the

A Pig phrenicCV = 48-8 m s30 °C Thread B Thread

R R

Gap= 2 jmGap= 2mm

C Monophasic MC 18% | ~~~~1 mV030ms 015ms +

0-5 ms

Fig. 6. MC stimulation of a segment of pig phrenic nerve in inhomogeneous conductingmedia. A and B, accurate dimensional relationships of figure-of-eight MC to nervetrajectory in trough and lucite cylinders astride nerve, respectively. C, nerve responsesrecorded from the distal end of the nerve and elicited by monophasic current pulses in twodirections.

same (20%) MC stimulus intensity were more than three times as great with thecylinders in place (I1 2 and 10 2 mV mm-2) than when the cylinders were absent (3 4and 3 4 mY mm-2). Evidently, the significant changes in the MC-induced electricfield caused by the solid cylinders are reflected in changes in the elicited neuralresponses.The magnitude of the derivatives of the electrical field required for excitation of

a number of nerve fibres can be derived by extrapolating the electrical fieldmeasurements at 20 % MC output to that required to elicit the nerve response (Fig.5). When the current flowed towards the recording electrodes in the medium withoutthe cylindrical blocks, the value was (3 4 mV mm-2) x (66%/20%) MC output,which equals 11 2 mV mm-2. With blocks, the corresponding calculation was(112 mV mm-2) x (23%/20%) MC output, which equals 12 9 mV mm-2. For aninternodal length of 2 mm, these values would be multiplied by four, yielding valuesof 448 and 516 mV (2 mm)-2.We also placed the cylinders close to the first derivative sites previously identified

in homogeneous media, and elicited responses to monophasic current of eitherpolarity (Fig. 6). When the cylinders were directly over the derivative locus closer tothe recording electrode (A), the nerve response was earlier and larger when thecurrent was directed towards the recording electrode than when the current was inthe reverse direction. By contrast, when the cylinders were placed over thederivative site further from the recording electrode (B), current directed towards the

211

212 P. J..MACCABEE AND OTHERS

recording electrode elicited an earlier but smaller response than in the reversedirection. Furthermore, reversing the current produced a much smaller latency shift(0 15 ms) as compared with the shift (0 3 ms) obtained when the cylinders were closerto the recording electrodes.

A Cat phrenic B ThreadCV = 44 9 m s-22-24 °C

38mm

0-85 ms (38-0 mm)

c Monophasic MC 0-25 ms (_ 11-5 mm)

32 % =40deg deg_

38% -00 d0 deg

28 90 deg g

05%dedegg

Electrical Icathode /

under bend * * N,,/90 deg Il1 mV

01>05 ms+ 005 ms80

Fig. 7. MC stimulation of straight and bent nerve (cat phrenic). A, position of the MC withrespect to the trough. NNhen straight, the proximal end of the nerve is held to the bottomof the trough bv a thin M-ooden dowel (i.e. stick, illustrated in B). When bent and pivotedabout a thread, the proximal end of the nerve is held to the dowel by natural adhesion,and the dowel is appropriately manipulated into position. B, accurate dimensionalrelationship between figure-of-eight MC, nerve trajectory, and pivot (left-most) thread.Dotted lines (E) indicate sites of the bipolar stimulating electrodes that elicited nerve

responses identical in latency to those elicited by both directions of monophasic MC-induced current. C, nerve responses recorded from the distal end of the nerve tomonophasic MC stimuli applied to straight (O deg) and bent (90 deg) nerve. Electricalstimulation directlv under bend as indicated.

MAGNETIC NERVE STIMULATIOA IN VITRO

Bent nerve in a homogeneous mediumThe effect of bending the nerve was tested after first identifying the sites of

excitation in a straight nerve when monophasic current in the MC was reversed. InFig. 7 the sites of excitation in a cat phrenic nerve were 38 mm apart (top two traces).

A Cat phrenic <"60 90 degCV = 449 m s1 045 60 R22-24°C Q30

MC

B Monophasic MC 125% C 90 deg

MC0 deg

ono +

45 r_ _ _ __ _ _20%?

60 _1% T

90 ~ _ _ 20% 0_+ -Poly _ ,14% - mV

05 ms

Fig. 8. MC stimulation of straight and bent nerve (cat phrenic). Experimental set-up asin Fig. 7. A, various bend angles used. B, nerve responses are elicited by monophasiccurrent, which is outward at the bend. C, nerve responses are elicited by monophasic andpolyphasic current, of both polarities, with a 90 deg bend in the nerve.

It is emphasized that excitation did not take place on the straight nerve at the sitewhere the threads transversely crossed it. After bending the nerve to 90 deg aboutthe left-most transverse thread (see Methods), current directed towards the bend (i.e.away from the recording electrodes) elicited a standard response at low stimulusintensity (28% output); the site of excitation was just before the bend (compare thethird and the fifth traces from the top). Reversing the current so that it was directedtowards the recording electrodes elicited a response nearly identical to that obtainedwhen the nerve was straight (fourth trace from the top). Thus in all bendexperiments, low threshold excitation at the bend required that the induced currentflows towards the bend.When stimulating with a monophasic current such that current was outward at

the bend, different degrees of bend resulted in different thresholds and responseamplitudes (Fig. 8). At an MC output intensity of 12-5 %, threshold was reachedbetween 30 and 45 deg, and the largest amplitude responses occurred at 90 deg (B)except when the nerve was bent back on itself. By contrast, a straight nerve wasnever excited by 12-5% MC output intensity, indicating that excitation at a bend islow threshold. When comparing monophasic with polyphasic pulses (also at a 90 degbend), powerful excitation was elicited at the bend by polyphasic pulses of eitherpolarity, but only by a monophasic pulse causing outward current at the bend (Fig.

213


8C. bottom three traces). When monophasic current was directed towards therecording electrode, i.e. away from the bend (Fig. 8C, top trace), the early responseat 20% MC output intensity was trivial. A small, very late response was alsorecorded, whose source has not yet been determined.

A Cat phrenic22 °C43-5 m s-'

Cylinders +

Gap = 2 mm

B Cat phrenic29 °C49-5 m s-'

Thread 90 deg bend - +

-3 0 +3cm

MC cm

34% 0 -

27% +3 -

12% 0 -

26% -3

Thread

R

-3 0 +3 cm

MC cm

50% 0

44% +3 -

26% 0 - 2,

50% -3 - _ 2 |1 mV

02 ms +

Fig. 9. Effect on response latency of MC stimulation at low threshold sites created bystraddling the nerve (cat phrenic) with non-conducting solid lucite cylinders (A) and bya 90 deg bend (B). With the nerve in place, the MC junction is located approximatelybeneath the cylinders or the bend, the divergence away from the handle being referred tozero. Maximal amplitude responses and those at 50% (left) and 60% (right) of maximumwere obtained at position zero. The MC was then moved 3 cm to either side and the outputintensity adjusted to obtain a response amplitude equal to the submaximal responseobtained at position zero.

A problem encountered when using an amphibian sciatic nerve preparation and itsbranches was excitation of a small response between the two derivative points; forexample vertical arrow in Fig. 3, left column. We believe that such responses arisewhere a branch bends away from the main nerve trunk which is close to the peakelectric field under the junction (see Discussion).

Effect of moving the coil when the nerve has acquired a low threshold pointThe hypothesis was tested that after a low threshold point was created on the

nerve, moving the MC junction in the long axis of the nerve would alter its responselatency very little. In the first test of the hypothesis, a low threshold point wascreated by straddling the nerve with a pair of lucite cylinders and the MC was moved

214

MAGNETIC XVER VE STIMULATION INT VITRO 215

a total distance of 6 cm (Fig. 9A). At position zero and using 12% MC outputintensity, the response amplitude was approximately 50% of maximum, which wasobtained with 34% MC output intensity. Movements of the MC to positions + 3 and-3 cm required an increase in MC output intensity to 27 and 26 %, respectively, in

Cat phrenic22 °C43-5 m s-'

A+- ThreadCylinders _ T

Gap = 2 mm

3 0 +3cm

Cat phrenic29 °C49.5 m s-'

B+ Thread90 deg bend _ r

-3 0 +3cm

CMC 14%

.%h

Position

-3 to 0cm

Oto +3cm

Normalized amplitude F

1 0 - ; AN10~~~~~~~

00

0.00 , ,-3-2-1 0 1 2 3

cm

DMC 26%

05 1 mV0-5 ms

1.0 - <

0.0-3-2-1 0 1 2 3

cm

Fig. 10. Effect on response amplitude of MC stimulation at low threshold sites createdalong the nerve (cat phrenic) by cylinders and a 90 deg bend, as in Fig. 9. At a constantoutput intensity, the MC was moved in 0 5 cm steps over a total distance of 6 cm beneaththe trough. Superimposed responses are shown when elicited at positions -3 to 0 cm, and0 to + 3 cm (C and D). E and F, normalized response amplitude as a function of MCposition.

order to elicit half-amplitude responses. Despite the difference in MC position andoutput intensity, the response latencies were nearly identical.

In the second test, a 90 deg bend in the nerve at the left-most thread (Fig. 9B)resulted in low threshold excitation with the MC at position '0'. A stimulus at 26%output intensity elicited a response that was 60% of maximal amplitude, which was

obtained at 50% output intensity. Moving the MC to positions +3 and -3 cm

E


required an increase in stimulus intensity to 44 and 50 %, respectively, to achieve the60 % amplitude response. Again, the latencies of the response were nearly identical.In Fig. 10, the intensity of the MC stimulus was held constant and the changes inresponse amplitude were studied as a function of MC position in the long axis of thenerve. The response amplitudes for both types of low threshold site peaked when theapproximate centre of the MC junction was beneath the cylinders or the bend (C andD), but symmetrically declined over an approximate 3 cm distance to either side (Eand F).

DISCUSSION

The question posed at the start of the study was whether classical membranetheory could predict the sites of excitation by an MC. The simplest system to studyis MC excitation of a linear peripheral nerve. Given that there are two derivative sitesassociated with each MC stimulus, one site should correspond to the virtual cathodeand the other to the virtual anode. Therefore, reversing MC current direction shouldelicit responses in a linear peripheral nerve at different latencies (Durand et al. 1989;Roth & Basser, 1990). In our study, when using a (5 x 10 cm) figure-of-eight MC, thespatial derivative mid-peaks in the cylindrical tank volume conductor were 53 mmapart. Therefore, in a peripheral nerve conducting at 50 m s-', MC current reversalwould be expected to elicit a latency shift as great as 1G0 ms. While our nerve modeldisplayed a significant shift in latency, the distance between excitation sites variedonly by approximately 26-42-5 mm in the homogeneous media volume conductor.This apparent discrepancy was resolved by finding that the distance between thespatial derivatives was significantly closer in the shallow trough used for the nerveexperiments than in the larger cylindrical tank where the MC was positioned furtherfrom the volume conductor (as predicted by Basser, 1993). The excitation sites arelocated approximately at the anterior and posterior divergences of the figure-of-eightMC junction. By contrast, polyphasic pulses excite axons at very short intervals atboth spatial derivatives, i.e. by the negative-going first phase at one site and,approximately 150 ,us later, by the negative-going second phase at the other site.Thus, latency shifts would be anticipated when reversing polyphasic current inhomogeneous or inhomogeneous media, but they would be small. However,monophasic and polyphasic MC pulses whose first phases are identical in form andpolarity and which induce a cathode closer to the recording electrodes (Fig. 4C, toptraces), elicit responses at the identical latency. Thus above threshold, the same firstphase of each pulse profile effectively excites the nerve at the same physical location.In Fig. 4, proposed effective excitation phases for both directions of monophasic andpolyphasic current are indicated by filled circles.

Remarkably, studies in different laboratories on nerves in the forearm have notrevealed consistent latency shifts when reversing current direction in various MCs.When using a polyphasic pulse profile, we found that current reversal minimallychanged (i.e. by 0-1 ms or less) the latency of single fibre muscle (Maccabee et al. 1988)or motor unit responses (Amassian et al. 1989b). The possibility that the secondphase of the polyphasic phase excited the nerve was considered and rejected becausethe latency measurements were considered accurate to 60 ,us and should easily haverevealed a shift of 150 ,as, corresponding to the half-period of the oscillation

216


(Amassian et al. 1989 b). Moreover, in these studies, the round coil was physicallyreversed leading to the possibility of coil displacement and/or tilt. In addition, theAmassian et al. (1989b) study elicited responses from the ulnar nerve at the elbow,where the nerve lies close to bone, which could concentrate the current locally as inthe intervertebral neuroforamina (Maccabee et al. 1991). Subsequently, using both amonophasic pulse to energize the figure-of-eight MC (phase two amplitude was 35%that of phase one) and the current reversing device described above, we again failedto detect consistently a shift in motor unit latency at threshold (Maccabee et al.1990 b). These findings imply very closely spaced derivative sites, possibly one orperhaps two nodes apart. One possible factor in explaining this is provided by theeffect of straddling a straight nerve with solid, plastic cylinders. The non-conductingcylinders create a spatial narrowing and increase in the induced electric fieldintensity resulting in a low threshold site of excitation there. Reversing themonophasic current direction with such a perturbation of the electric field resultedin a relatively smaller shift in latency owing to the closer spacing of the firstderivatives sites (Fig. 5). Significantly, the amount of latency shift for a givenseparation between cylinders varied with the position of the blocks relative to thejunction (Fig. 6). The differences in latency shift were substantial, e.g. varying from0-15 to 0 30 ms.

Another surprising finding in the human was that moving the MC distances of3-5 cm proximally and distally from the lowest threshold point over distal forearmexcited (at increasing threshold) the same motor unit at the identical latency (P. J.Maccabee, V. E. Amassian, L. P. Eberle, K. S. Lai & R. Q. Cracco, unpublishedobservations). (By contrast, when compound muscle action potentials (CMAPs) arerecorded, significant latency shifts as great as 0-5-0 6 ms are observed when flippingover the MC to reverse monophasic current direction (Claus et al. 1990; Nilsson et al.1991; Nilsson, Panizza, Roth, Basser, Cohen, Caruso & Hallett, 1992.) Factors whichmay account for conservation of latency despite large displacements of the MCinclude a concentration of current flow in the volume conductor or a bend in thenerve (Fig. 9). The problem is in identifying which, if either, of these factors, isrelevant in the human. A prior study of a segment of human cervical-thoracic spineimmersed within a saline volume conductor revealed a corresponding rise andnarrowing of the induced electric field at the neuroforamina (Maccabee et al. 1991).In the human, moving the MC over the rostral-caudal axis of the cervical spine mayfail to elicit latency shifts in motor units (Maccabee et al. 1991), or in CMAPs (Ugawa,Rothwell, Day, Thompson & Marsden, 1989; Britton, Meyer, Herdman & Benecke,1990). However, it is unclear what the comparable anatomical basis is for a lowthreshold point in distal forearm when driving motor units at threshold.Although the trajectories of axons are often straight along segments of peripheral

nerve and spinal cord tracts, they are usually curved (and branched) in the cerebrum.Our data reveal that when excitation occurs selectively at a bend in the nerve or ata cut nerve ending, it also occurs at a site within the peak electric field rather thanat the negative-going spatial derivative. These findings support the suggestion ofReilly (1989) that an effective field gradient could exist if the axon either changed itsspatial orientation with respect to a locally uniform field or abruptly terminated. Inagreement with our findings, recent simulation studies suggest that excitation of

217

218 P. J. MACCABEE AND OTHERS

short axons is lower in threshold at the terminations of the axon rather than at thefirst spatial derivative sites along the axon (Altman & Plonsey, 1990; Nagarajan,Durand, Ferguson & Warman, 1991). The importance of nerve fibre bending in MCexcitation of the corticomotor system is implied by analogous findings withperipheral nerve in a brain-shaped volume conductor used to model corticospinaltrajectories (Amassian et al. 1992 a, b). It is also significant that the round MCsuppresses perception of transient visual stimuli when the posterior coil windings aresymmetrically placed on the scalp directly overlying occipital cortex (Amassian et al.1989a). Given this MC location, the peak electrical field intensity would be close tothe midline, i.e. near the foveal representation in posterior calcarine cortex, but ata substantial distance from the laterally placed spatial derivative sites. Possibly,either curvature of input and output axons towards the midline or a termination ofthese axons at or near the midline creates low threshold points for excitation there.

The authors thank Mr Giuseppe Condemi and Mr Bruce Hundley for providing dissectedporcine phrenic nerves (supported by NIH Grant HL20864, Dr Phyllis M. Gootman, PrincipalInvestigator), Dr Devon John for providing a sheep phrenic nerve, Dr Stuart Ferguson of CaseWestern Reserve University who contributed software for topographic presentation of the firstspatial derivative and Mr Jonathan Maccabee who helped to record the electric fields.

REFERENCES

ALTMAN, K. W. & PLONSEY, R. (1990). Analysis of excitable cell activation: relative effects ofexternal electrical stimuli. Medical and Biological Engineering and Computing 28, 574-580.

AMASSIAN, V. E., CRACCO, R. Q., MACCABEE, P. J.,CRACCO, J. B., RUDELL, A. P. & EBERLE, L. P.(1989a). Suppression of visual perception by magnetic coil stimulation of human occipitalcortex. Electroencephalography and Clinical Neurophysiology 74, 458-462.

AMASSIAN, V. E., EBERLE, L. P., MACCABEE, P. J. & CRACCO, R. Q. (1992a). Factors influencingmagnetic coil excitation of amphibian and primate nerves immersed in a human brain-shapedvolume conductor. Journal of Physiology 446, 23P.

AMASSIAN, V. E., EBERLE, L. P., MACCABEE, P. J. & CRACCO, R. Q. (1992b). Modelling magneticcoil excitation in human cerebral cortex with a peripheral nerve immersed in a brain-shapedvolume conductor; the significance of fiber bending in excitation. Electroencephalography andClinical Neurophysiology (in the Press).

AMASSIAN, V. E., MACCABEE, P. J. & CRACCO, R. Q. (1989b). Focal stimulation of humanperipheral nerve with the magnetic coil: A comparison with electrical stimulation. ExperimentalNeurology 103, 282-289.

BARKER, A. T., FREESTON, I. L., JALINOUS, R., MERTON, P. A. & MORTON, H. B. (1985). Magneticstimulation of the human brain. Journal of Physiology 369, 3P.

BASSER, P. J. (1993). Focal stimulation of axon. IEEE Transactions on Biomedical Engineering (inthe Press).

BASSER, R. & ROTH, B. (1991). Stimulation of a myelinated axon by electromagnetic induction.Medical and Biological Engineering and Computing 29, 261-268.

BRITTON, T. C., MEYER, B. U., HERDMAN, J. & BENECKE, R. Clinical use of the magneticstimulator in the investigation of peripheral conduction time. M7Wuscle and Nerve 13, 396-406.

CLAUJS, D., MURRAY, N. M. F., SPITZER, A. & FLUGEL, D. (1990). The influence of stimulus type onthe magnetic excitation of nerve structures. Electroencephalography and Clinical Neurophysiology75, 342-449.

COHEN, D. & CUFFIN, B. N. (1991). Developing a more focal magnetic stimulator. Part 1: Somebasic principles. Journal of Clinical Neurophysiology 8,102-111.

COHEN, L. G., ROTH, B. J., NILSSON, J., DANG, N., PANIZZA, M., BANDINELLI, S., FRIAUF, W. &HALLETT, M. (1990). Effects of coil design on delivery of focal magnetic stimulation. Technicalconsiderations. Electroencephalography and Clinical Neurophysiology 75, 350-357.


DURAND, D., FERGUSON, A. S. & DALBASTI, T. (1989). Induced electric fields by magneticstimulation in non-homogeneous conducting media. Proceedings of the 11th Annual InternationalConference of the IEEE Engineering in Medicine and Biology Society, No. 5, 1252-1253.

KATZ, B. (1939). Electric Excitation of Nerve. Oxford University Press, London.MACCABEE, P. J., AMASSIAN, V. E., CRACCO, R. Q. & CADWELL, J. A. (1988). An analysis of

peripheral nerve motor stimulation in humans using the magnetic coil. Electroencephalographyand Clinical Neurophysiology 70, 524-533.

MACCABEE, P. J., AMASSIAN, V. E., CRACCO, R. Q., EBERLE, L.P. & RUDELL, A.P. (1990b).Effective anode and cathode are very close together when stimulating peripheral nerve with themagnetic coil. Society for Neuroscience Abstracts 16, 1261.

MACCABEE, P. J., AMASSIAN, V. E., EBERLE, L. P., CRACCO, R. Q. & RUDELL, A. P. (1992). Themagnetic coil activates amphibian and primate nerve in vitro at two sites and selectively at a

bend. Journal of Physiology 446, 228P.MACCABEE, P. J., AMASSIAN, V. E., EBERLE, L. P., RUDELL, A. P., CRACCO, R. Q., LAI, K. S. &SOMASUNDARAM, M. (1991). Measurement of the electric field induced into inhomogeneousvolume conductors by magnetic coils: Application to human spinal neurogeometry. Electro-encephalography and Clinical Neurophysiology 81, 224-237.

MACCABEE, P. J., EBERLE, L., AMASSIAN, V. E., CRACCO, R. Q., RUDELL, A. P. & JAYACHANDRA,M. (1990a). Spatial distribution of the electrical field induced in volume by round and figure '8'magnetic coils: relevance to activation of sensory nerve fibers. Electroencephalography andClinical Neurophysiology 76, 131-141.

NAGARAJAN, S. S., DURAND, D., FERGUSON, A. S. & WARMAN, E. N. (1991). Magnetic stimulationof finite neuronal structures. Proceedings of the 13th International Conference of the IEEEEngineering in Medicine and Biology Society, No. 2, 847-848.

NILSSON, J., PANIZZA, M., ROTH, B. J., BASSER, P. J., COHEN, L. G., CARuso, G. & HALLETT, M.(1991). Cathode identification in magnetic stimulation of a peripheral nerve: mathematicalmodeling supported by findings in normal volunteers. Neurology 41 suppl. 1, 251.

NILSSON, J., PANIZZA, M., ROTH, B. J., BASSER, P. J., COHEN, L. G., CARUSO, G. & HALLETT, M.(1992). Determining the site of stimulation during magnetic stimulation of a peripheral nerve.

Electroencephalography and Clinical Neurophysiology (in the Press).RATTAY, F. (1986). Analysis of models for the external stimulation of axons. IEEE Transactions inBiomedical Engineering 33, 974-977.

REILLY, J. P. (1989). Peripheral nerve stimulation by induced electric currents: Exposure to time-varying magnetic fields. Medical and Biological Engineering and Computing 27, 101-110.

ROTH, B. J. & BASSER, P. (1990). Model of the stimulation of a nerve fiber by electromagneticinduction. IEEE Transactions in Biomedical Engineering 37, 588-597.

TOFTS, P. S. (1990). The distribution of induced currents in magnetic stimulation of the nervoussystem. Physical Medicine and Biology 35, 1119-1128.

UGAWA, Y., ROTHWELL, J. C., DAY, B. L., THOMPSON, P. D. & MARSDEN, C. D. (1989). Magneticstimulation over the spinal enlargements. Journal of Neurology, Neurosurgery and Psychiatry 52,1025-1032.

219

3. a polyphasic pulse excited the nerve at both sites, by the negative

Documents