the electrical activity of the alimentary tract

The Electrical Activity of the Alimentary Tract

E. E. DANIEL, PH.D.

T HE INTESTINE is a complex of excitable tissues, nerve, and muscle. In dis- cussing the origins and control of intestinal electrical activity, several

questions must be raised.

1. What is the characteristic electrical activity of single nerve or muscle cells as isolated cells, in arrays of similar cells, and in more or less intact tissues or organs?

2. What is the characteristic electrical activity of groups o.[ cells in tissues or organs?

3. Can the characteristic activities of groups of cells be explained as the sum of the activities of the constituent single cells?

4. What is the interrelation between cells which mutual ly influence each other's electrical activities by electrical current, chemical transmitters, or transmission of strain?

5. What is the relationship between the electrical activity of cells and groups of cells and their mechanical activity (motili ty)?

Most of the data available to answer these questions concern the isolated tenia coli of guinea pigs and other rodents. Considerable data are also available regarding the activity of the small intestine of dogs, cats, rabbits, and rats, and the antrum of dogs and cats. Limited data are available concerning the esophagus, which illustrate the activities of both smooth (cat) and striated (dog) muscle. T h e large intestine, apart from the tenia coil, when present, has also yielded information. Almost nothing is known of the electrical activity of the nervous elements of the intestine. Obviously, answers to Question 4, concerning the interrelation between groups of cells, cannot b e adequately provided until the electrical activity of intestinal intrinsic nerves is stndied. This communication will discuss only the small intestine as it exemplifies the problems of all portions. Recently the extensive data for activities of the tenia coli have been reviewed, 1,2 as have those for the esophagus. 6s

Answers to questions about tile functional interrelations by which cells influence the electrical activity of other cells depend upon knowledge of structural interrelations between these cells. However, there is a grent deal of uncertainty about intestinal structure.

T h e 6 main questions are diagramed in Fig. 1.

From the Department of Pharmacology, University of ,~lt)erta. Edmonton, Alberta.

New Ser;es; Vol. 13, No. 4, 1968 2 9 7

Belfast Symposium

1. Synaptic relations between postgangtionic sympathetic nerves and intestinal elements. Recent histochemical and physiologic evidence suggests that only the nervous elements of Auerbach 's plexus are innervated. 3-~ Repeti- tive st imulation of the nerve fibers accompanying the intestinal arteries in

I DO pest-ganglionic sympathetic fibres synapse with nerve or

muscle cells?

2 Do intrinsic parasympathetic nerve endings make intimate

contact with muscle cells?

3 What,where,how stimulated--mecbano receptors?

4 What are the synaptic relations between afferent fibres

and Auerbach's Plexus?

5 Are there n0re-adrenergic inhibitory fibres?

6 Do Some smooth muscle cells provide a conduction path

between muscle layers?

. I

/ • " ? , S ;

L . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . .

Fig. 1. Diagrammatic representation of cross section of small intestine. Dots represent longitudinal muscle cells in cross section and lines represent circular muscle cells. Nerves of Auerbach's plexus are shown beaded. Keyed questions refer to uncertainties about intestinal electrical structure,

the guinea pig tenia cell results in inhibi t ion of the gradual onset in contrast to the hyperpolarizing inhibi tory response to a single t ransmural shock. 6, 7 Th i s is logical, if we assume that the sympathetic periarterial nerves do not innervate intestinal muscle directly, whereas other inhibi tory fibers do. Thus it seems that sympathet ic inhibi t ion of electrical and mechanical events is indirect, operat ing to diminish parasympathet ic activity. Th i s is consistent with the absence of inhibi tory synaptic potentials in muscle cells during sympathetic stimulation. ~-s

2. Synaptic relations between parasympathetic intri~sic nerves and smooth muscles. No synaptic potentials have been recorded from intestinal muscle cells dur ing vagal st imulation; only gradual depolarization has been noted. 9 I t is possible that discrete, close contact between nerve endings and muscle cells does not exist, a0

3. RecepIors initiating the peristaltic reflex; effective stimulus and the role of chemicals" in their excitability. Previous studies disagree as to whether these receptors are present in the muco~sa TM ~e or elsewhere iz, ~4 and as to the im- portance of serotonin in their sensitivity to pressure al,'e, a4-a7 and activity. They have not been identified with any known sensory receptor in the intestine.

4. Synaptic relations and the transmittal of activity between the sensory

298 Arnerh;an Journal of Diges÷ive Diseases

Danle[: Alimentary Tracf

nerves for peristaltic reflex activity and the parasympathetic intrinsic nerves. Serotonin has recently been proposed as the transmitter, as but almost nothing definite is known.

5. Nervous structure mediating "nonadrenergic'" inhibition on transmural stimulation and its interconnections. Functional evidence for such structures is available, G, 7 but as yet no anatomic evidence has been presented.

6. Structural and functional interconnections between smooth muscle cells both within and between muscle layers. Few now believe that an anatomic syncytium exists, but there is much evidence for the spread of electrical activity through low resistance pathways within and between muscle layers, ~9-21 possibly via areas of close contact between membranes.

Obviously, the lack of answers for the above questions limits the number of answers available to our functional questions. Indeed, it limits drastically the precision of any functional question which may be posed.

M E T H O D S

ELECTRODES

A variety of technics have been used to record the electrical activity of the intestine. T h e electrodes used and some of their characteristics, advantages, and disadvantages are summarized in Table I. T h e types of electrodes used include glass microelectrodes, glass pressure electrodes, glass pore electrodes, wick electrodes, and metal needle electrodes. Th e y are recorded against a distant electrode, intended to be indifferent. In some cases not described in the table, two electrodes were placed on the intestine to obtain a differential record. In such cases the configuration of the record is the algebraic sum of the potentials at both electrodes and depends upon the distance between the electrodes and upon their geometric relationship to the spread of electrical activity.

No electrode is ideal for all purposes. The microelectrode provides the most readily interpretable record: the t ransmembrane potential changes, derived from a single cell. However, such records are difficult to obtain over long periods of time, especially when the intestine is contracting. There is also no independent confirmation that the potentials recorded are unaffected by the recording technic. Furthermore, microelectrodes cannot easily be used in vivo or in chronic animals. Needle or punctate electrodes are easiest to use under these conditions. Unfor tunately the results obtained are more difficult to interpret because they are derived from many cells and are affected by asynchrony between them; there is also evidence2T, z8 that the results are derived both from volume-conducted action potentials .and injury potentials under the electrode. All bu t microelectrode results are affected by asynchrony and all but results from pore and wick electrodes may be affected by variable injury potentials during motility.

New Series, Vol. 13, No. 4, 1968 299

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Daniel: Alimentary Trac÷

RECORDING CONDITIONS

ldeally, one seeks to obtain all the information possible from the electrode system used. All potential changes irrespective of direction or frequency must be recorded, so direct coupling and the widest possible band-pass should be used. In practice, particularly when recording in vivo, RC coupling is often used because of the large voltages arising from polarization, movement artifacts, and altered shunt resistance, etc. Even if the input amplifiers provide ideal conditions and retain all input informa, tion undistorted, the oscillograph used may lead to a distorted recording of the information because of inadequate frequency response and other problems related to the large inertia of some recording systems. Oscilloscopes can be used to avoid distortion of recorded potentials, but the expense of continuous filming of the oscilloscope face and the necessity for prolonged recording from mult iple electrodes limits their use. Multiple-channel tape recordings which could be rerun at any useful speed and from which data could be fed into other systems for transformation might remove many of the current problems.

RESULTS

SMALL INTESTINE

Electrical Record

T h e electrical activity of the small intestine of man, dogs, cats, rats, and several other species 9, 16. 24, _o0-~0, 32-4~ is characterized by two types of potentials. One type is periodic, is independent of the occurrence of contractions, and is propagated, usually distally, down the tipper small intestine, al though aboral propagation at similar rates (6-15 cm./sec.) is sometimes seen. This type apparently functions to increase the excitability of a ring of smooth muscle which spreads down the intestine; 2°,s~ these oscillatory potentials are called slow waves (Fig. 2, A). The other type of potential is fast spiking, associated only with contraction; 26-3~,4~ it is not propagated more than a few milli- meters,e°,29, s3-~,~9. 42 and originates from a definite phase of the slow wave.'-'°, 2~,2a-3°.32-36 With intracellular microelectrodes and pressure electrodes, spikes are positive and originate from the depolarization phase of the slow wave (Fig. 2, A). Witfi pore, wick, and punctate electrodes in contact with muscle, spikes are negative or more precisely triphasic (+ , - - , +) with the main deflection negative (Fig. 2, B).

Slow Waves

When recorded in vitro with micropressure, pore wick, or some punctate _6 .. . . 3 ~_ slow waves usually consist of sinusoidat waves (Fig. electrodeseO, 2~, ~ ,~- ~ o

3). Sometimes, however, they show an initial rapid depolarization and take on a saw-toothed appearance2, ~0 They are not, however, propagated in vitro as far or as regularly as in the intact animal. Fur thermore the velocity of

New Series, Vol. 13, No. 4, 1968 301

Belfast Symposium

conduction is less--e.g., 1 cm./sec. ''° instead of 10 cm./sec. In isolated circular muscle from cat intestine spikes, bu t no slow waves, can be recorded. 2°, e6, aT, 4G Slow waves can be recorded from isolated longitudinal muscle. '-'°, ".,6, _07 They appear to be transmit ted electrotonically into the circular muscle since they are not recorded f rom isolated circular muscle, but are recorded without any appreciable t ime delay f rom circular muscle ei ther undissected or dissected but in contact with longitudinal muscle. '-'~), -"~, 3~ T h e ampl i tude of circular- muscle slow waves decreased, apparent ly exponentially, with distance (50% in 1-2 ram.) f rom the longitudinal muscle z7 in studies on flattened slabs of the intestinal wall, intact or with longitudinal muscle dissected away, However in vivo? ~ little decrement of slow waves with distance from longitudinal muscle was observed in circular muscle. Moreover, it has proved impossible 2° regularly to trigger or drive slow waves with electrical s t imulation applied outside the cell. Indeed, the presence of slow waves in either layer of the muscle makes it less electrically excitable so that spikes can no longer be generated by external electrodes.

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Fig. 2. Electrical activity of small intest ine. T r a c i n g .4: slow wave and spike as recorded by in t racel lu lar (left) or pressure (right) electrode, cor responding to t r a n s m e m b r a n e poten- t ial changes. T rac ings B a n d C: first and second derivatives of A such as m i g h t be recorded by wick or ex t race lhdar electrode respond ing to act ion cur ren ts in vo lume conductor . T rac ing D (left) : combina t ion (wi thout spike) of A and B; (right), combina t ion of `4 and C such as m i g h t be recorded hy punc ta t e d a m a g i n g electrodes. T rac ing E: same as D bu t wi th spike.

302 American Journal of Digestive Diseases

Daniel: Alimentary Tract

T h e spread of slow waves in to circular muscle may not be an entirely passive e lectrotonic spread when cylinders (in contrast to slabs) o~ intest ine are studied. I n such cylinders T M slow waves are p ropaga ted at a rate of 8 cm./sec., with little decrement in circular muscle alongside a strip of longi tud ina l

Fig. 3. Intracellular- (A~, Ae, A~, B) and pressure-electrode (C) records from cat small intestine in vitro. Separation of electrodes in long axis (A1, A~, As) or in transverse axis (B and C) shown at right in milli- meters. Voltage calibration for A and B of 20 inv. illustrated in B. Voltage calibration in C is 2 my. Time scale in C applies to all. (From Kobayashi et al. Amer ] Physiol 211:1281, 1966. Reproduced with permission.)

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muscle. Slow waves and spikes pass in both direct ions between the muscle layers, apparen t ly by way of connec t ing muscle strands. Cu t t ing these by separat ing the two layers prevents interlayer conduct ion , bu t local anesthetics, gangl ion ic b locking drugs, cold storage, etc., do not. Conduc t ion of slow waves

New Series, Vol. 13, No. 4, 1968 303

Belfast Sympodum

in isolated longitudinal muscle both in tile longitudinal and in the transverse axes--is poor and stow. Longitudinal muscle attached to circular muscle shows nearlysynchronous slow waves in the transverse axis. tlowever, careful measurements of" the occurrence of slow waves at points distributed radially around the intact intestine show that there is a time differential, suggesting a very rapid spread of activity around the intestine. These results are interpreted to indicate that "'the slow waves may be continually reinforced around the cylinder by cycling from longitudinal to circular muscle and back, ' ' '° and that "the .near synchronization in the transverse axis of intact segments appears to be due to the combination of multiple pacemaker loci in longitudinal muscle with conduction in the circular layer. '''-'° We may tentatively conclude that slow waves originate in longitudinal intestinal muscle .and spread passively and /o r actively into circular muscle.

,Slow Wave Configuration When recorded from the clog duodenum or je junum in vivo with puno

tate'-'9, aa--a< a.~,4'_, or silver wire electrodes, a,~, 4.., the slow wave often shows an initial rapid positive deflection, followed usually by rapid partial recovery, then by a plateau of relative positivity, and finally by a further recovery phase (Fig. 4). Occasionally, however, the pattern consists of slow positive deflections followed by a rapid negative deflection and then recovery, ca, 47

When recorded with intracellular microelectrodes or with pressure electrodes (see above) from longitudinal intestinal muscle in the dog or cat jejunum. slow waves are found to consist of periodic depolarizations and repolarizations (Fig. 3). The depolarization phase corresponds .to the positive phase of the recording with punctate or silver wire electrodes. This has raised the problem of how the flow of positive current through the volume conductor and into a negative sink in the muscle under the electrode could lead to a positive potential-deflection at the electrode. This would ordinarily be expected to cause a negative deflection of the electrode when the underlying muscle became depolarized.

The explanation of this problem has not been established, but 2 promising hypotheses have been made.

1. Bortoff -08 has suggested that the punctate electrode, like the pressure electrode, damages the underlying muscle, depolarizing it and putting the recording electrode in contact with the intraeellular enviromnent throt,gh a decreased membrane resistance. The record obtained is a complex of the intracellular potential change and the volume-conducted action potential (which may be the first or second derivative of the transmembrane potential, depending upon the geometry of conduction of slow waves). Bortoff has shown that the extent to which each contributes to the final record will depend upon the magnitude of the shunt resistance, which is between the damaged and the undamaged membrane areas and in series with other resistances between the two electrodes. In the example shown in Fig. 5, only the time course of the

~0~ American dourne[ of Digestive Diseases

Dan~.ei: Alimen~'ary Trac÷

Fig. 4. Stow waves recolded in vivo horn silver wire electrodes in dog jejunum. Electrodes placed as shown above. Recording from each electrode compared in succession to recording from E~. Each record begins at slow wave positive peak from E~. Slow waves conducted aborally at velocity of 6-10 cm./sec. Spikes began 0.6-1.3 see. after slow-wave positive peak, hut individual spikes were not propagated; time of appearance was determined by slow waves. One-second intervals are marked at bottom of records.

v o l u m e conduc ted m e m b r a n e c u r r e n t ( I R or vo l tage d r o p across R,.) wi l l be r eco rded be tween Pr a n d P~ wi th e lec t rode A. T h e m a g n i t u d e of this vo l t age d r o p is p r o p o r t i o n a l to

d-OEm dt -~

wh ich is the second de r iva t ive of Em wi th respec t to t ime. W i t h e lec t rode B, d a m a g e f rom the e lec t rode e l i m i n a t e s Era ' and reduces R m ' to R m ' r a n d p a r t of the :membrane p o t e n t i a l Em appea r s across R s - - e q u i v a l e n t to

Em Rs Rs+Rm +Rm " + R i

T h e total po t en t i a l be tween P1 a n d PII becomes IR~ + E m R s / ( R s + R m +

N~w Serles, ¥o[0 13~ No. 4~ t968 30~

Belfast Symposium

R m ' ' + Ri ) . Note that Ri may also vary with damage, as may Rm and Em if there is not a sharp boundary between damaged and undamaged adjacent membrane segments.

In microelectrodes, where this shunt resistance is high because of the lipid

A, ELECTRODE

NOT DAMAGING: MUSCLE

SURFACE

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Rf Internal resistance between membrane segments

Fig. 5. Circuit diagrams redrawn from Bortoff, -"~ showing hypothetical recording arrangements with nondamaging wick or pore electrodes (4) and damaging pressure or punctate electrodes (B). Diagram at right represents arrangement with intracellular electrode if transmembrane resistance is considered to be RS.

plasma membrane, and in pressure electrodes, where it is high because of the seal between the electrode and the tissue surface, a large voltage drop will appear across this resistance, corresponding to the membrane potential or a reduced replica of it (Fig. 2). In electrode arrangements which have a low or negligible shunt resistance because the electrodes are not pressed against the tissue surface, most of the voltage drop between damaged and undamaged membrane segments will be across the membrane and internal resistances and the record will be a derivative of the membrane potential change caused by current flow in the volume conductor. This will also be the case when no membrane damage occurs. In records from punctate electrodes, Bortoff be- lieves both kinds of potential contribute. Figure 2 shows how these might combine to yield the slow wave pattern from punctate electrodes.

2. Kobayashi et a l : ~ have proposed that standing or nonpropagated posi-

3 0 6 American Journal of Dicjesfive Diseases

Danlel: Allmen÷ary Trac÷

tive slow waves may be generated when surface cells are connected to distant current sinks by low resistance pathways. They did not succeed in finding such sinks (areas of negativity) al though they did find that when steel microelectrodes (tip 1/~ in diameter) were driven 800-1000 t~ into the flattened-out intestine, slow waves were larger in comparison to their size at other depths. A depth of 800-1000 /~ corresponded to an area between circular and longitudinal muscle. Presumably, cells in this region provided more current because they were nearer to a n d / o r connected over a lower resistance pathway to the current sinks. However, variation in the potential drop across the resistance between electrodes caused by variation in IRv or IRs (Fig. 5) might produce such a result.

Bortoff's hypothesis predicts that spikes, like slow waves, will be largely positive when recorded with punctate damaging electrodes, but they are negative (Fig. 4). Kobayashi e t al . 4~ suggest that the spikes originate locally and are therefore negative, while slow waves are created in response to current flow initiated by distant current sinks. Both Bozler .~°-32 and Kobayashi e t a l . 4a

found that wick electrodes in saline recorded negatively oriented (first derivative-like) slow waves and spikes, and the latter group found a reversal of slow wave polarity in air when the surface dried. T h e failure of the spikes to reverse polarity was not explained explicitly. Th e implication is that de- hydrat ing tile intestinal surface by put t ing it in air or in oil impairs the init iation and propagation of slow waves but not of spikes at or to the surface cells. Similar changes must be assumed when needle electrodes are used. No evidence was found that surface cells between threads of the wick electrode were damaged or depolarized in air and no such evidence is available for punctate electrodes. Furthermore, the same punctate electrodes which are supposed to damage intestinal muscle record a volume-conducted triphasic initial potential (analogous to the intestinal slow wave) from the antrum and therefore apparently fail to damage antral muscle. 16, 42, 44, 45 Finally, we and others invariably record patterns with a large positive deflection followed by a pronounced negative deflection with silver wire electrodes in the dog duodenum, whereas in the je junum the same electrodes usually record a more sinusoidal pattern. 35, 36, 4'% 69 Different degrees of damage a n d / o r different shunt resistances with the same electrodes according to location would have to be postulated.

One question not yet sufficiently considered is the possible configuration of slow waves recorded with microelectrodes in vivo. Isolation or immobilization of the intestine for microelectrode recording interferes with conduction of slow potentials, reducing it from 10 cm./sec, to 1 cm./sec, or less and may therefore alter slow wave configuration. 20, 2z, 27, 29, ~-~, 39, 41-43 In other words, there is no proof that the undisturbed slow wave in vivo has the same sinusoidal configuration as recorded. In fact, both sinusoidal and saw-toothed slow waves occur 9, ~0, 4~ (Fig. 3).

New Series, Vol. 13, No. 4, 1968 ~07

Belfast Symposium

Mechanism of Slow Waves

T h e ionic conductance changes underlying intestinal slow waves are uncertain. In-vivo studies of the dog intestine ~ indicate that slow waves are insensitive to the reduction of external sodium, chloride, potassium, or freely diffusible calcium ions. They are reduced or obliterated by inhibitors of the active transport .process such as ouabain, NaF, and l i thium ion. Part of this action seems to be indirectly caused by release of norepinephrine, since it could be prevented by a pr ior dosage of reserpine, but par t is a direct action on smooth muscle. These findings were interpreted to indicate that sodium and chloride conductance changes are not involved in slow waves and that activity of an electrogenic Na p u m p may be involved. Recent studies in the longitudinal muscle of cat intestine in vitro a° have provided fur ther evidence of the relative independence of slow wave activity from sodium, potassium, and calcium conductance changes as compared to that of spikes. Slow waves persist at 20-30% of the normal Na content of tile medium (Li or sucrose replacement) while spikes are obliterated in 20% of tile normal Na content (with Li subst i tut ion) , or 5 0 ~ of the normal Na content (with sucrose re- p lacement) . They are el iminated on complete removal of Na. In contrast to these findings Bfilbring and Kur iyama ° found no difference in tenia coli in the sensitivity of slow waves and spikes to reduced external sodium concentration. However, reduction of slow wave or spike ampli tudes by lowering the external sodium does not prove that a sodium conductance change is involved. In tenia coli, spikes are reduced in low sodium media but are not affected by tetrodotoxin. 4s, 49 Te t rodotoxin exerts its electrophysiologic effects by selective inhibi t ion of increased Na conductance. 5°

Slow waves are also less dependent than spikes on a normal external Ca concentration and persist at lower membrane potentials (K depolarization) than do spikes. 40 Spikes and slow waves interact, spikes being init iated at a critical slow wave depolarization, and slow wave repolarization being speeded by a spike late in the wave. In a later study 43 longitudinal muscle cells with slow waves were found to have a lower membrane resistance than isolated circular muscle cells without them, and induction of slow waves in circular muscle cells significantly lowered their resistance. T h e authors 4n suggest that depolarization dur ing spiking involves an influx of both Na and Ca. Interpre- tation of these studies is complicated by the possible effects of ionic changes in media on release of mediators by intrinsic nerves still present (see below), and by the probabi l i ty that secondary effects may occur, such as alterations in cellular ionic contents or in metabolism.

Intestinal Nerves and Slow Waves

Slow waves recorded from dog intestine in vivo 35 with silver wire electrodes usually become larger when exposed to stimulants such as acetylcholine, and rapid negative spikes are produced along with contractions. Intracel lular



microelectrodes or pressure electrodes reveal that the slow depolarizations in cat or rabbit intestine increase in amplitude, and on reaching the firing level, initiate spikes. °, 24, 26, 27, 40, 43 (However, no junct ion potentials were observed in rabbit intestine when the vagus was stimulated. °) Intra-arterial or intra- venous injections of epinephrine, norepinephrine, and isopropylnorepinephrine usuaIly inhibit or prevent spikes and contraction. 34, 35, 44.45, 4T, 5t Th e stimulation of either alpha receptors, probably in nerves, or beta receptors, probably in muscle, leads to inhibition. 3,4 In larger concentrations, catecholamines reduce the amplitudes of slow waves recorded in this way and interfere with their propagation. 34, 35, 44.4~, 47, 51 Studies in vitro with cat intestine have not given identical results. 26,37,51 Spikes and contractions were eliminated by epinephrine, but slow wave amplitudes were not diminished. One possible explanation is that the experiments in vitro were done using small pressure- electrodes and may not have reflected a decreased propagation of slow waves produced by epinephrine, whereas the experiments in vivo did reflect such changes. However, pressure electrodes also record the activity of many cells and ough.t also to reflect any changes in the synchrony of discharge of these cells. Th e ultimate answer will depend upon records obtained with microelectrodes both in vlvo and in vitro. It seems likely on a priori grounds that if cholinergic stimulations increase the magnitude of depolarization leading to excitation of action potentials, epinephrine might produce inhibit ion by an opposite effect. As was pointed out above, both histochemical and pharmacologic evidence suggest that the sympathetic innervation of the small intestine occurs in Auerbach's plexus and not in smooth muscle cells. Sympa- thetic mh~lSxtlon would then operate by inhibit ion of cholinergic nerve activity, and the expected electrophysiologic consequences would be effects opposite from those produced by cholinergic stimulation. Gillespie s has obtained this result with sympathetic stimulation in the large intestine.

Cholinergic blocking agents, such as atropine and nicotine, and the release of serotonin by reserpine do not markedly diminish slow waves, °°, .09, 3~-86, 42-45 nor do adrenergic blocking agents such as phentolamine and propranalol affect them.44,45,47 Hence the effects of mediators from nerves seem not to be required for production of slow waves.

T h e roles of intrinsic nerves and smooth muscle in the origin and propagation of slow waves have recently been fur ther examined in two studies. 47, 52 In an initial study, Szurszewski ~ exposed a segment of dog intestine to prolonged ischemia by constant perfusion of Ringer's solution for 4 hr. This resulted in damage to 100% of the ganglion cells in Auerbach's plexus with 30% of the damaged cells recovering, and damage to 70% of the cells in Meissner's plexus with 70% recovering after 70-90 days. This was in contrast to an earlier claim by Hukuhara and co-workers 53-'~'~ that all cells were destroyed and none recovered when a related procedure was used. Slow waves of the peffused segment had a slower frequency than those in proximal or distal normal segments

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T A B L E 2. EFFECTS OF P R O C E D U R E S DESIGNED T O DESTROY I N T R I N S I C NERVES ON S L O W WAVE F R E Q U E N C I E S IN DOGS

Segment

No. of Oral co~trol Procedure dogs (jej) Perfused* A borat control*

70-90 days af ter cons tan t per fus ion (unanesthet ized) ~-~ 2 18.3 ± 0.2 13.4 ± 0.5 14.4 ± 0.4 2 wk. after cons tant pcr fus ion (anesthetized) ~7 2 14.5 ± 0.3 10.7 ± 1.0 I 1.1 ± 0.6 As above b u t s t agnan t per- tusion '~7 1.,5.5 ± 0.3 10.4 ± 0.3 I 1.8 ± 0.3 As above b u t s t agnan t per- 2 fusion with Ringer equil . wi th 95% N.. + 5% CO~ ~ 6]" 16.0 ± 0.I 9 +--- 0.1 t l .7 ± 1.0

*Before perfusion, all j e juna l segments showed identical frequencies. tVa lues for this g roup represent m e a n ± s t andard error of all m e a s u r e m e n t s f rom several

electrodes at different times. Differences were all significant when var ia t ion between dogs was e l iminated . All o ther values, wi th except ion of 2-week post per fus ion aboral controls, represent m e a n --- S.E. of m e a s u r e m e n t s f rom 3 electrodes in each segment . Differences were significant except between per fused and aboral cons tant -per fus ion segments.

(Table 2) even after 90 days. Slow waves from proximal segments were not propagated far into or within the ischemic segment (< 1 cm.), and sometimes slow waves were propagated for a short distance from a distal antral segment. Other changes in the electrical and contractile activity of postischemic segments included a decreased contractile frequency corresponding to the decreased slow wave frequency, as would be expected if slow waves determine the t ime at which spikes and contractions can occur (Fig. 6). T h e force of contractions in the peffused segments was not significantly altered. This was taken as evidence of lack of damage to smooth muscle by the procedure. Th e muscle layers had a normal histologic appearance and were continuous with proximal and distal areas. In addition in the nonperfused distal area, there was asynchrony both of the normal aboral spread of slow waves (about as many were conducted orally as aborally) and of the normally nearly simul- taneous occurrence of slow waves in the transverse axis.

Szurszewski concluded that the perfused segment generated slow waves at the inherent myogenic frequency and that the myenteric plexus was necessary to raise the excitability .of the myogenic system so that a higher frequency of slow waves occurred. Further, he postulated that the myenteric plexus produced conditions suitable for conduction of slow waves in the muscle so that proximal pacemaking activity in the duodenum could be transmitted aborally, raising the frequency still further. He suggests that continual release of some excitatory substance from the plexus might account for these results. Previous studies, 85 showing that neither reserpinization to release 5-HT, norepinephrine, atropine, hexamethonium, nor nicotine to inhibit the action of acetylcholine

310 American Journal of Dicjesflve Diseases


Fig. 6, Recordings in vivo from electrodes about 2 cm. above (Ez) and inside segment of dog jejunum made ischemic with N~-eqnilibrated Ringer's solution 2 weeks previously E~, E~, E~,, and E6, respectively: electrodes approximately 1, 3, 4, and 6 cm. below oral normal segment. E~ about 1 cm. above aboral normal segment. Note (1) slower frequency of slow waves in ischemic segment (15/rain. or every 4 sec. in normal intestine and 12/rain. or every 5 sec. in postischemic intestine); (2) aboral, slow conduction of slow waves in postischemic intestine, about 1-2 cm./sec.; (3) unusual configuration of slow waves at E~ and Ea in postischemic intestine, sharp negative deflections not accompanied by contractions. Voltage calibrations of 1 my. at left.

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interference with slow wave amplitude frequency or conduction, make the nature of such an excitatory substance problematic.

T h e most difficult aspect of the above finding is its explanation. T h e author at tr ibuted his findings to selective partial nerve damage produced by ischemia (70 3 of the ganglion cells in Auerbach's plexus were stated to be irreversibly damaged). However, no direct proof of the abolit ion of nerve function was obtained. T h e author, like Hukuhara and co-workers (the originators of the ischemia technic), relied on histologic examination of the intestine. Hukuhara and co-workers "~3-'~ also reported that reflexes elicited by stroking the mucosa or by application of HC1 or 5 -HT were abolished after ischemia.

Khin reinvestigated this problem. 47 She obtained results very nearly identical to those of Szurszewski when a constant 4-hr. perfusion of Ringer's solution was used and the animals were studied 2 weeks later (Table 2). The re was a

A B C

ql

OMPP40,UCJ/ML 5pg/M .acLxa

D E

Fig. 7. Response of strips of circular muscle from dog jejunum to stimulants (dimethyl- piperizinium [DMPP], nicotine, and hypertonic Ringer's solution) acting indirectly via nerves. A, B, and C illustrate responses of strip from postischcmic segment; D and E, responses of strip from normal segment.

oMPP 4o,u /ML

312

NICT. 5 ~ / M L NaCLX?.

American Journal of Digesflve Diseases

Daniel: Alimenfary Tracf

reduced f requency of slow waves in the per[used area and slow waves f rom proximal normal areas were often not p ropaga ted into the per[used segment or were p ropaga ted only short distances. However , these segments responded in vivo to phenyld iguanide , 5 -HT, nicotine, d ime thy lpeper i z in ium ( D M P P ) , and other drugs believed to act indirectly via nerves. I n addi t ion, isolated muscle strips f rom these segments ( longi tudinal plus circular, suspended to record longi tudinal contract ions) responded in vitro to the same drugs and to t ransnmral electrical s t imula t ion (Fig. 7 and 8). T h e r e was no remarkable quant i ta t ive difference between these responses and those to similar s t imuli in control muscle strips. Responses to submaximal t ransmura l s t imula t ion were par t ly blocked by hexame thon ium, and all responses were prevented by low

A

" "I' " - " . . . . I ' ' ~ • • • . I '

15OV C610vU~/¥L MCH AT ~ KCL-R zoP/~ o.oo5,u9/M~, o.o~,u~/,~L ~ o.oa~,ucy, L

B

150V

C o I%U~/ML NCH AT 0 02}J~/ML MCH KCL-R o.oo~y.L o.oo~yT.~

Fig. 8. Responses of strips of circular muscle from dog jejunum to transmural electrical stimulation appear at dots (150 v., 10 shocks/sec., electrodes 1 cm. apart,) A. Strip from a normal segment of intestine. B. Strip from postischemic segment. Hexamethonium (C6 10 izg./ml.) depressed responses. Atropine (0.02 gg./ml.) prevented responses to both methacholine (MCH) and transmural stimulation, but not to KC1 Ringer's solution.

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TABLE 3. NEURONE COUNTS IN PERFUSED SEGMENTS

Reversible Irreversible Normal changes changes

Constant perfusion 25 41 12 Stagnant perfusion 4 21 6 Stagnant perfusion* 14 24 13

*Means from 4 dogs.

concentrations (0.02/~g./ml.) of atropine SO4 and by 10 -7 (w/v) tetrodotoxin. Hence, functional nerves were present. Histologic examination revealed only 15% of the ganglion cells in Auerbach's plexus to be irreversibly damaged (Table 3). Reversible damage included increased granularity in the cell and peripheral dumping of the chromatin with Nissl's stain. T h e nucleus was darkly stained and had migrated to one pole of the cell, but the cell outline, nucleus, and nucleolus were clearly seen. Irreversible damage consisted of shrunken cell outlines and no discernible nucleus or nucleolus.

Th e oxygen pressure of nonaerated Ringer's solution was sufficiently high (12.6 × 10 -2 arm.) to suggest that the degree of anoxia achieved during perfusion was inadequate to destroy intrinsic nerves. Consequently the pro- cedures were varied by allowing the Ringer solution to remain stagnant in the segment, once all the blood had been replaced. This was done using both nonaerated Ringer's and Ringer's aerated with 95% N2 and 5% CO2 (subse- quently found to have an oxygen pressure o[ 10 -2 atm.). Results were similar (Tables 2 and 3) except that slow waves were much smaller in ampli tude and less regular (Fig. 6). However, responses in vivo and in vitro to drugs, and in vitro to transmural electrical stinmlation proved that functional nerves were still present (Fig. 7). Responses to KC1 and drugs believed to act directly on smooth muscle were also not affected by the procedure. Ischemia did diminish spontaneous motili ty in vivo, and there were no segmental movements in vitro. In anesthetized dogs Khin found no evidence of high tone in the ischemic segments. Finally, reflexes in vivo were abolished in the treated segment.

The re are 3 possible explanations: 1. T h e intestinal intrinsic nerves or some component required for propaga-

tion of slow waves determines their inherent frequency and amplitude, and, by affecting slow waves, diminishes segmental and other movements.

2. The activities of intrinsic nerves (e.g., the product ion of a mediator) are essential for the above functions.

3. Some other non-nervous structme or function of the intestine is involved in the observed effects. Khin's physiologic, pharmacologic, and histologic evidence of functional postganglionic cholinergic nerves along with the alterations in slow waves at tr ibuted to nerve damage by Szurszewski, raises serious questions about the first and second explanations unless it is assumed that

3 | 4 American Journal of Digesf|ve Diseases


another port ion of the intrinsic nervous system than the cholinergic postganglionic port ion is involved.

Most important , these results require that a reassessment be made o[ the studies of slow wave propagat ion in vitro. Cur ren t theories 2°, .08, 43 include no explicit role for nerves but perhaps this is because these studies were done in vitro where it is l ikely that there was anoxia at one or more parts of the intestinal wall. Perhaps the conduction and configuration of slow waves in vitro are already affected by damage to the same system as is damaged by ischemia.

Spikes

Action potent ial spikes can readily be elicited by st imulat ion of isolated intestinal circular muscle.a6, sn, 57 When slow waves are propagated into circular muscle, it becomes much less electrically excitable; the same applies to longi tudinal muscle. -00 In isolated or intact layers individual spikes are propagated only short distances (up to 2 or 3 ram.) in comparison with slow waves in vivo. 2°, 35, a6, 56-60 T h e i r velocities are 7-10 cm./sec, in the long axis and 1 cm./sec, in tile transverse axis of isolated circular muscle. 56, ~7 There is evidencO6, ~G, 5z, 61, 62 that propagat ion of action potentials requires excitation and spread of activity through a num ber o~ parallel cells. St imulating electrodes less than 75/~ in d iameter was ineffective in eliciting spikes, regardless of intensity or durat ion of stimulus, and those larger than 100 /~ were able to elicit conducted spikes. 56, ~7 In strips less than 100/~ (200-300 cells) wide conduction failed. Finally, conduction velocity and distance increased with increased stimulus strength, presumably because of the summat ion of effect over parallel and converging pathways. A num ber of authors have reported that it is difficult to st imulate intestinal smooth muscle cells by intracellularly appl ied currentsY °, 56, 57, 61-63 T h e biophysical basis for such requirements is not established 2°, 43, 56-66 and the whole prob lem of the proper interpretat ion of measurements of resistance and other m e m b r a n e parameters between an intracellular and another electrode is the subject of current discussion. 43, 56-66 Some4Z, 5z though not all 59, 63 authors find a lower resistance between two electrodes believed to be in adjacent cells than between one microelectrode in a cell and another outside. T h e amount of electrotonic interaction when microelectrodes are supposed ,to be in adjacent cells is also disputed; some authors find much interaction 43, ~ and others less. ~1, 63 Low interelectrode resistance and high electronic in, teraction are the main pieces of evidence for low resistance connections between cells, but the absolute values deduced for such "bridge" resistances and the t ransmembrane resistance have recently been seriously questioned 6~, ~6 on theoretic grounds. Recent estimates of very high specific membrane resistance are difficult to reconcile with the high values obtained for the Na fluxes. 67 Deduction of the values for membrane capacity, space constants, etc., depend upon the utilization of valid models and correct

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analysis of theln. Discuss ion of the a p p r o p r i a t e mode l s a n d the i r analysis is b e y o n d the scope of this paper . However , for d i f fe ren t reasons, a n u m b e r of au tho r s have sugges ted tha t d i f ferent pa tches of the m e m b r a n e o[ in t e s t ina l muscle cells a re r e spons ib le for slow waves a n d for spikes. "'°, 4o. ~:,, ,~

T h e ionic c o n d u c t a n c e changes r e spons ib le for spike are u n k n o w n , as was discussed above. C a l c i u m inf lux or release d u r i n g sp ik ing m a y be re spons ib le for bo th del~olar iza t ion and con t rac t ion . 4°, ~s, 49

C O N C L U S I O N S

I t is poss ible to descr ibe the charac te r i s t i c e lec t r ica l ac t iv i t ies of s ingle cells a n d g roups of i n t e s t i na l muscle cells in terms of slow waves a n d spikes, b u t these act ivi t ies vary d e p e n d i n g u p o n in t e r ac t i ons w i th o t h e r cells (nerves, muscle layers, e tc . ) . T h e m e c h a n i s m u n d e r l y i n g these ce l l u l a r in t e rac t ions is no t yet unde r s tood , b u t i t is poss ible to say tha t the act iv i t ies of cells g r o u p e d in ce r ta in a r rays canno t be d e d u c e d f rom the ac t iv i t ies of c o m p o n e n t cells in i so la t ion (e.g., l o n g i t u d i n a l a n d c i rcu la r musc le sepa ra te a n d together , c i r cu la r muscle in very fine s t r ips and in l a rge r str ips, res is tance be tween 2 cells and across a single ce l l ) . I t is poss ib le to bel ieve , a l t h o u g h p roo f is lacking, tha t most i n t e rac t ions be tween nmscle cells are e lectr ical , a l t h o u g h chemica l in ter - ac t ions be tween nerve and muscle cells are p r o b a b l e . W h e t h e r or h o w slow waves in muscles affect nerves is u n k n o w n . H o w nerves or o t h e r s t ruc tures suscept ib le to a n o x i a affect slow waves is also u n k n o w n . A c o m p l e t e discussion of the r e l a t i o n s h i p be tween e lec t r ica l a n d mec ha n i c a l ac t iv i ty is b e y o n d the scope of this pape r , as ide f rom the s t a t emen t tha t slow waves usua l ly t r igger spikes and spikes t r igger con t rac t ion . H o w slow waves exe r t con t ro l over muscle exc i t ab i l i t y to b r i n g a b o u t the c o m p l e x m o t i l i t y p a t t e r n s of the in tes t ine

was no t discussed; l i t t l e is known. Department of Pharmacology

University of Alberta Edmonton, Alberta

Canada R E F E R E N C E S

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New Sedes, Vol. 13, No. 4, 1968 ~ |~

the electrical activity of the alimentary tract

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