CONTROL OF THE SALIVARY GLANDS OF …J. exp. Biol. (1978), 7a, 91-106 01 With 1 $ figures Printed in Great Britain CONTROL OF THE SALIVARY GLANDS OF HELISOMA BY IDENTIFIED NEURONES

J. exp. Biol. (1978), 7a, 91-106 0 1With 1 $ figures

Printed in Great Britain

CONTROL OF THE SALIVARY GLANDS OF HELISOMABY IDENTIFIED NEURONES

BY S. B. KATER, A. D. MURPHY, AND J. R. RUED

Department of Zoology, University of Iowa, Iowa City, Iowa 52242

{Received 24 May 1977)

SUMMARY

The neural regulation of an exocrine gland was investigated at the levelof identified effector neurones. The salivary gland neuroeffector system ofHelisoma consists of a pair of acinous glands innervated by two sym-metrically located, identified buccal ganglion neurones (4R and 4L). Neurones4R and 4L usually are electrically coupled and display synchronous activity.Action potentials in these neurones elicit EPSPs and action potentials inepithelial cells of the salivary glands. Spontaneous miniature potentialssimilar to those seen at neuromuscular junctions can be recorded from manyof the glandular cells. Neurones 4R and 4L, and thus also salivary glandcells, can display bursts of action potentials phase-locked with those seen inbuccal mass motoneurones during feeding.

INTRODUCTION

Significant progress has been made towards our understanding of the neural basesunderlying muscle-mediated behaviour such as locomotion (e.g. Stein et al. 1973),feeding (e.g. Kater, Heyer & Hegmann, 1971; Kater & Rowell, 1973; Kater, 1974;Siegler, Mpitsos & Davis, 1974) and cardiac responses (e.g. Mayeri et al. 1974;Thompson & Stent 1976a, b, c). Much of this progress is a direct consequence ofinvestigations utilizing invertebrate preparations which are highly amenable for theintracellular study of identified neurones (cf. Kater, Heyer & Kaneko, 1975; Kandel,1976). However, the neuronal control of glandular effector systems has not beeninvestigated so extensively, even though many complex behavioural acts encompassa number of effector processes involving glandular secretion as well as muscularcontractions.

Studies of neural effects on, for instance, the mammalian pancreas (Woods &Porte, 1974) and insect (e.g. House, 1973; Ginsborg & House, 1976) and mammalian(e.g. Thulin, i974;Emmelin&Gjorstrup, 1975) salivary glands have been documented,but such studies, in general, have been restricted for technical reasons to examiningthe effects of nerve trunks rather than individual neurones. One point which doesemerge from these studies is that secretory systems under direct neural control canrespond with quite short latencies to neuronal commands, allowing secretion to beintegrated into a more global behavioural output. By exploiting the well-known

•advantages of large, gastropod molluscan neurones it should be possible to analyse

92 S. B. KATER, A. D. MURPHY AND J. R. RUED

integration of glandular neuroeffector systems by the same methods commonly usedto study neuromuscular systems.

We have discovered a highly tractable preparation for the investigation of theneural control of a gland whose secretory cells are electrically excitable and extensivelyelectrically coupled to one another (Kater, Rued & Murphy, 1977). The preparationconsists of the buccal ganglia and salivary glands of the snail, Hetisoma trivolvis. Thiscommunication demonstrates that the electrophysiological responses of the salivaryglands are regulated by a pair of large neurones in the buccal ganglia. These neuronesare readily recorded from and identified in different animals on the basis of their sizeand location. The activity of this glandular effector system is integrated into the morecomplex behavioural act of feeding in Hetisoma (Kater, 1974).

MATERIALS AND METHODS

Dissection procedures for the Hetisoma salivary effector system, as well as theelectrophysiological techniques and physiological saline have been described pre-viously (Kater et al. 1977). Although no calcium was included in the 'low' calcium,high magnesium saline (27-6 mM-MgSO4 was substituted for the CaCl,), previousexperience with molluscan glandular preparations has shown that some calciumremains in the bath (Kater, 1977). For the high calcium saline 24/6 mM-CaClj wasused.

Possible regional differences were examined by arbitrarily visually dividing thesalivary glands into four equal linear regions. These began with the first proximalacinus next to the duct and ended at the distal end where the paired glands areoften joined by connective tissue (Fig. 4). Fast rise-time miniature potentials wereoperationally defined as those which had an essentially vertical rise-time at an oscil-loscope sweep of 50 ms/cm.

RESULTS

Spontaneous transient membrane potentials

The salivary gland cells of Hetisoma trivolvis display regenerative, propagatingaction potentials (Kater et al. 1977). Two additional forms of membrane potentialtransients suggested that these gland cell action potentials might be mediated byneural input (Fig. 1). Transient depolarizations resembling classical neural excitatorypost8ynaptic potentials (EPSPs) were frequently recorded. In some cells the amplitudeof these EPSPs could be augmented by hyperpolarization of the cells, as would beexpected if they were chemically mediated. In other cells, however, hyperpolarizationfailed to augment the amplitude of EPSPs as might be expected of electrotonicpotentials arriving from other glandular cells. Extensive electrical couplings in thesalivary glands previously has been described, and electrotonic EPSPs have beenobserved in response to action potentials evoked in distant cells (Kater et al. 1977,Fig.9C).

A second type of spontaneous voltage fluctuation further suggests chemical synaptictransmission (arrowheads, Fig. 1). These are miniature membrane potentials similarin form and amplitude to the classical miniature endplate potentials occurring at theneuromuscular junction (Fatt & Katz, 1952). These miniature potentials occur wity

Neural control of salivary glands 93

Fig. i. Spontaneous activity recorded from salivary gland cells at high gain (upper traces) andlow gain (lower traces). In addition to overshooting action potentials, there are excitatorypost-synaptic potential* (arrow*) and miniature potentials (arrowheads) iimilar to those seenat neuromusculax junctions. Calibrations: a mV upper traces, 10 mV lower traces, aoo ms.

various rise-times and amplitudes (Fig. 2A). A large portion of this variation apparentlyresults from the electrotonic decay of miniature potentials originating in neighbouringelectrically coupled salivary gland cells. This conclusion is based upon experimentswith simultaneous intracellular recording of miniature potentials from two glandularcells of a single acinus (see Kater et al. igTJ, for the morphology of the glands). Thefirst asterisk of Fig. 2 B marks a miniature potential apparently initiated in a cell closerto cell ' P ' than cell 'D ' . The second asterisk marks a miniature apparently initiatedin a cell closer to cell ' D ' than to cell 'P ' . As recording electrodes are moved fartherapart the correlation of occurrence of miniature potentials decreases and eventuallydisappears as does the actual electrical coupling as directly measured with d.c. currentpulses (Kater et al. 1977). Such electrical coupling effects have precluded an effectivestatistical analysis of miniature potentials initiated in a given salivary gland cell.

The amplitude and frequency of occurrence of classical miniature postsynapticpotentials can be modulated by external concentrations of calcium and magnesiumions (Hubbard, 1961; Katz & Miledi, 1969a; Kriebel & Gross, 1964; Matthews &Wickelgren, 1977). Effects of Ca*+ and Mg*+ concentrations on miniature potentialsin a salivary gland cell are illustrated in Fig. 3. The first trace depicts miniaturepotentials recorded in normal saline. Low Ca24^ high Mg2"1" saline (middle record)nearly eliminates miniature potentials, and completely eliminates spontaneous EPSPsand action potentials. Conversely, high calcium Ringer (lower record) greatly increasesthe frequency of the miniature potentials, as expected for such potentials originatingfrom a chemically mediated synapse. The amplitudes of the miniature potentials arealso greatly increased in the high Ca2+ Ringer, possibly as a result of an increase ininput impedance of the glandular cells (Cole, 1949; Katz & Thesleff, 1957).

Not all salivary gland cells display miniature potentials. Many of those which dofcpve miniatures only display lower amplitudes and slower rise-times, suggesting

4 EXB 72


Fig. a. Salivary gland miniature potentials. (A). Consecutive sweeps photographed from anoscilloscope demonstrate variability of the amplitude and rise-time of the miniature potentials.(B) Simultaneous recordings from two salivary gland cells within a single acinus suggest thatmuch of the variability of amplitude and rise-time is due to electrotonic decay of miniaturepotentials arising in other cells. The first asterisk indicates a miniature potential initiated ina cell apparently more closely coupled to cell 'P' (the proximal cell), from which the lowertrace was recorded. The second asterisk depicts a miniature potential initiated in a cell closerto cell 'D' (the more distal cell), from which the upper trace was recorded. Calibrations:(A) 175 mV, l o o m i ; (B) i mV, 50 ms.

electrotonic decay from other cells. An attempt was made to use 'fast rise-time'miniature potentials as an assay for cells which were more directly innervated. Thesedata were used in an attempt to examine patterns of innervation of these glands.

A survey was made to determine the percentage of cells in each of the predefinedregions of the gland (see Methods) which display fast rise-time miniature potentials.Considerable regional differentiation was observed within any single gland. Regionallocalization of miniatures seems to be a constant feature of any particular gland andwas not found to vary even in surveys lasting as long as 12 h. However, the relativefrequencies of fast miniature potentials recorded in the various regions differ indifferent preparations. Fig. 4 includes a histogram of the frequency recorded in eachregion of two exemplary preparations. A consistent feature revealed by these surveysis the existence of relative ' hot spots' displaying high frequencies of fast miniaturepotentials. Frequently such 'hot spots' were found near the proximal duct.

The confounding effect of the extensive electrical coupling between these cellsis emphasized when miniature potentials are surveyed without the criterion of fa^a


Fig. 3. The effect of calcium upon miniature potentials. All three traces are recorded from thesame salivary gland cell. Upper trace, a representative sample of miniature activity recordedin normal saline. Middle trace, the effect of low Ca1+, high Mg*+ Ringer. Lower trace, miniatureactivity recorded in high Ca*+ Ringer. Calibrations: a mV, 1 s.

lOO-i

Ea 0•3. 100 1

5 0 -

N=9

N=\2

N=\\ N=\0

N=2Q

#=24

_ " = 2 0 N=24

1 2 3 4Linear region no.

Fig 4. The distribution of miniatures along the salivary glands. Horizontal axis represents thearbitrary spatial division of the acinar portion of a gland into four equal linear regions delineatedby the dotted lines. Vertical axis represents the percentage of cells recorded from in eachregion which displayed 'fast miniature potentials'. The distributions for two representativeglands are displayed (preparation A, upper graph; preparation B, lower graph).

rise-time. Under these conditions innervation may appear more diffuse and pervasivethan is actually the case.

Neurally evoked transient potentials

The major sources of salivary gland innervation are the salivary nerves which arisefrom the paired oesophageal trunks originating at the buccal ganglia (Kater et al. 1977,Fig. 1). We have stimulated these trunks extracellularly while recording intracellularlyfrom salivary gland cells. Brief single shocks may evoke short latency (< 10 ms)EPSPs which typically fail with repetitive firing frequencies in excess of 10/s. SuchPSPs may evoke directly an overshooting action potential. Also observed are casesflfcere a slower rise-time shoulder - presumably due to electrotonic decay of action

S. B. KATER, A. D. MURPHY AND J. R. RUED

Fig. 5. Neurally evoked salivary gland action potentials. Superimposed intracellular recordsof a salivary gland cell triggered by several successive extracellular stimuli to the oesophagealtrunk (from which arises the salivary nerve). Such stimuli could elicit PSPs or overshootingaction potentials. The dotted line indicates zero potential. Calibrations: ao mV by 50 ms.

potentials in adjacent coupled cells (cf. Joyner, Ramon & Moore, 1975) - may beinterposed between the PSP and the overshooting action potential (Fig. 5).

To examine whether more than one axon could effect activity in the salivary glandcells, a series of stimuli of increasing intensity was applied to an oesophageal trunk.Fig. 6 shows that once threshold is reached, further increases in stimulus intensity,over a wide range, fail to alter the waveform or latency of salivary gland actionpotentials. This suggests that only one axon in the oesophageal trunk affects theelectrical activity of glandular cells.

If simultaneous intracellular recordings are made from a cell in each of themorphologically separate right and left salivary glands, and either oesophageal trunkis stimulated, an action potential can be recorded in both of the salivary gland cells(Fig. 7). If an oesophageal trunk is severed and the distal stump is stimulated,a response occurs only in the ipsilateral gland. If the proximal stump is stimulated,action potentials are recorded only in the cell of the contralateral salivary gland. Thus,stimulation of an oesophageal trunk must evoke activity in the contralateral glandvia a pathway through the buccal ganglia.

Identification of the effector neurones

Possible salivary gland effector neurones were identified morphologically byincubating the proximal stump of a severed oesophageal trunk in a cobalt chloridesolution. This allowed retrograde movement of cobalt chloride up the axons and cobaltwas precipitated by standard procedures (Kater, Nicholson & Davis, 1973; Mulloney,1973; Pitman, Tweedle & Cohen, 1973). In every instance the somata of a symmetridfl


c

LFig. 6. Sequential action potentials evoked in a salivary gland cell while gradually increasingthe amplitude (top to bottom) of the stimulus applied to the oesophagesl trunk. Once thresholdfor generating an action potential was reached (C), further increases in stimulus intensityproduced no observable change in waveform or latency of the salivary gland action potential(D, E). Calibration: 50 mV, 50 ma.

pair of large neurones corresponding to 4R and 4L in the map of Kater & Rowell (1973)were stained (Fig. 8).

Simultaneous intracellular recordings from neurone 4 and salivary gland cellsdemonstrate that there is usually a one-to-one correspondence between spontaneousaction potentials observed in neurone 4 and those observed in glandular cells (Fig. 9).For every action potential recorded from neurone 4 there is a concomitant actionpotential or PSP in the salivary gland cells, but on rare occasions action potentials areseen in the gland cells without a corresponding action potential in neurone 4. Theorigin of such salivary gland action potentials unassociated with activity in neurone 4remains unknown.

Fig. 10 demonstrates that the correlation of activity in neurone 4 and the salivaryJ i n d cells has a causal basis. Intracellularly evoked action potentials in neurone 4

S. B. KATER, A. D. MURPHY AND J. R. RUED

JFig. 7. Stimuli applied to either oesophageal trunk elicited action potentials in both ipsilateraland contralateral salivary glands when the buccal ganglia were left intact Upper traces, record-ings from a cell in right salivary gland; lower traces, recordings from a cell in left salivarygland. The vertical deflection at the beginning of each trace is the stimulus artifact. Thedotted lines indicate the time when peak amplitude is reached in the cell ipsilateral to thestimulated oesophageal trunk. Calibration: 50 mV, 100 ms.

effect action potentials and EPSPs in salivary gland cells. No other neurones of thebuccal ganglia have ever been found to produce such driving effects on salivary glandcells.

Interactions between neurones 4J? and 4L

Simultaneous intracellular recordings from neurones 4R and 4L reveal synchronousaction potentials and EPSPs (Fig. 11) in them. This synchrony of activity probablyresults from common inputs to these neurones and from tight electrotonic couplingbetween neurones 4R and 4L. Electrotonic coupling of neurones 4R and 4L is readilydemonstrable by passage of current from one cell to the other (Fig. 12). The couplingcoefficient (VJV^ see Bennett, 1966) between neurones 4R and 4L can be quitevariable from preparation to preparation; it is often as much as 0*5 but may be zero.Under the latter conditions little correspondence of spontaneous activity is observedin either the two neurones or the two bilateral salivary glands. We have not yetattempted to make a rigorous quantitative survey of the differences in couplingbetween preparations nor to ascertain the underlying basis of such differences.

Fig. 13 further demonstrates the magnitude of electrical coupling that can existbetween neurones 4R and 4L. This experiment entailed simultaneous intracellularrecording from neurones 4R and 4L and extracellular stimulation of the leftoesophageal trunk to produce antidromic action potentials. Antidromically evokedaction potentials in both 4R and 4L were blocked simultaneously at a discrete level ofhyperpolarization produced by current injection into the soma of either neurone(Fig. 13). In this preparation we were unable to obtain a level of current injectionthat blocked antidromic action potentials in only one of the somata.

Journal of Experimental Biology, Vol. 72 Fig. 8

Fig. 8. Cobalt backfill of oesophageal trunk (o.T.). Note that when one o.T. is backfilled thesomata of both neurones 4R and 4L (arrows) are filled. Calibration: 200 mm.

S. B. KATER, A. D. MURPHY AND J. R. RUED {Facing p. 98I


.JOUMVJ VIVJ

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Fig. 9. Spontaneous bursting activity in a salivary gland cell and in neurone 4. (A) Spontaneousbursts of action potentials recorded from a salivary gland cell. (B) Hyperpolarization ofa salivary gland cell reveals EPSPs underlying the bursts of action potentials. (C) Simultaneousrecordings of a salivary gland cell (upper) and neurone 4 (lower) display a one-to-one cor-respondence of action potentials. Calibrations: 20 mV, 2 s.

Fig. 10. Intracellularly evoked action potentials in neurone 4 (lower traces) elicit actionpotentials (A) or EPSPs (B) in salivary gland cells (upper traces). Calibrations: (A), aomV,20 mi; (B), 5 mV (upper trace), 14 mV (lower trace), and 20 ms.

IOO S. B. KATER, A. D. MURPHY AND J. R. RUED

L L

Fig. I I . Synchrony of spontaneous action potentials and PSPs recorded simultaneously inneurone 4L (upper traces) and 4R (lower traces). Calibrations: 50mV; zooms (A), and40 mi (B) and (C).

Integration of the salivary gland neuroeffector system into the feeding motor programme

It seems likely that salivary secretion is not a randomly occurring function, butrather, in some way is integrated with food ingestion. Neurone 19 is the motoneuroneto one of the major muscles involved in mastication (Kater & Rowell, 1973; Kater,1974). We have made simultaneous intracellular recordings from neurones 19 and 4 todetermine whether these systems can be co-ordinated. A wide variety of relationshipshas been observed, the most striking of which is when neurone 19 produces cyclicalbursts characteristic of the feeding motor programme (Kater & Rowell, 1973) andneurone 4 produces alternating bursts of 1-5 action potentials in antiphase with thisactivity (Fig. 14), and thus in phase with retractor motoneurones (Kater, 1974). Thisis not, however, an obligate relationship. Neurone 4 can produce action potentials ina regular, non-bursting manner when motoneurones are producing rhythmic burstsof action potentials characteristic of feeding motor output.

DISCUSSION

Exocrine glands of gastropod molluscs are highly amenable to studies of stimulus-secretion coupling, neuronal regulation of glandular secretion, and integration ofglandular effector systems into complex behavioural acts. The common mediator of


I

VFig. ia. Demonstration of electrical coupling by passage of depolarizing and hyperpolarizingcurrent pulses (upper traces) from neurone 4L (middle traces) to neurone 4R flower traces).Calibrations: 1 nA, 50 mV, and 200 mi.

stimulus-secretion coupling appears to be calcium (Douglas, 1974; 1976). The salivaryand pedal glands of gastropods apparently provide Ca2* for the release process byemploying regenerative action potentials with Ca*"1" currents (Kater, 1977; Kater et al.1977) as in presynaptic nerve terminals (Katz & Miledi, 1965; 1969a, b; 1970; Llinas,Blinks & Nicholson, 1972; Llinas & Nicholson, 1975) and the mammalian endocrinepancreas (Matthews & Sakamoto, 1975). Although we have yet to quantify the releaseprocess in the salivary glands of HeUsoma, we have shown that the secretion of mucusfrom the pedal glands of the slug ArioUmax is a function of the amount of stimulation. Inaddition, the calcium component of the action potential is necessary for secretion(Kater, 1977). Preliminary unpublished results indicate that there is a significantcalcium component in the action potential of HeUsoma salivary glands, but details ofpiis await a complete treatment of the ionic currents.


Fig. 13. Antidromic action potentials recorded in neurones 4R (upper trace) and 4L (lowertrace) following stimulus to left oesophageal trunk. When d.c hyperpolariiing current waspassed into the soma of either neurone 4L or 4R (arrows) blockage of the antidromic actionpotential occurred in both cells (traces not continuous). No level of current injection was foundwhich would allow an antidromic spike to occur in only one of the cells. Calibrations: 50 mV,500 ms.

Fig. 14. Integration of the activity of neurone 4 within the feeding motor programme. Simul-taneous recordings from protractor motoneurone 19 (upper traces), and neurone 4 (lower traces).(A) Cyclical activity characteristic of the feeding motor output of Heliioma. Neurone 4 tendsto fire action potentials just prior to the large inhibitory postsynaptic potentials generated inmotoneurone 19. (B) Neurone 4 was hyperpolarized and recorded from at higher gain todemonstrate the cyclical excitatory postsynaptic potentials. (C) Recordings as in (B) on anexpanded time base. Calibrations: (A) 10 raV, 5 s; (B) 10 mV, upper trace; 5 mV, lower trace,and 58; (C) 10 mV, upper trace; 5 mV, lower trace; and 1 s.

It is extremely difficult to define rigorously the precise nature of subthresholdactivity recorded from gland cells. In large part this is due to the extensive electricalcoupling. Frequently an action potential arises out of an EPSP which could be eitherchemical or electrical in origin. We have already shown that action potentials inelectrically coupled gland cells can decay and appear as EPSPs which may or may notevoke an action potential in distant cells (Kater et al. 1977, Fig. 9C). Chemicalsynaptic transmission between neurone 4 and selected salivary gland cells is suggestedon the basis of: the failure of 'following' with repetitive stimulation, presence ol


4R

I

[

4L

SG '-Ti

n - w j U A T ^wj L-J

SG

A/V

Fig. 15. A summary of the salivary gland neuroeffector system. Two bilateral, symmetricallylocated, identified buccal ganglion (BG) neurones (4R and 4L) innervate the paired salivaryglands (SG). These neurone* are electrically coupled and their activity can be integrated withthat of motoneurones during the feeding cycle. Chemical excitatory synapses are made betweenthese neurones and some of the salivary gland acinar cells. Miniature potentials can be recordedfrom these salivary gland cells. A single EPSP usually evokes an all-or-none, overshootingaction potential in these acinar cells. Salivary gland cells show a very high level of electricalcoupling with their neighbours (Kater et al. 1977). Action potentials generated in proximalacinar cells propagate throughout each salivary gland via the electrotonic junctions and resultin the co-ordinated activation of the entire salivary gland.

miniature potentials, augmentation of EPSP amplitudes by hyperpolarization ofsalivary gland cells, and dependence of EPSPs upon external calcium. The combinationof electrical and chemical inputs possible on any particular salivary gland cell probablyaccounts for the extremely complex waveforms routinely observed in these cells (e.g.Fig. 1 and Figs. 4, 5 and 9 in Kater et al. 1977).

The effect of neurone 4 on gland cells is reproducible and is as constant a featureof this effector complex as is a motoneurone's effect on muscle. It is tempting to relyon the motor control literature and consider synaptic transmission to the gland cellsto be chemically mediated and monosynaptic. While this at present seems likely, it isemphasized that the data now available are only circumstantial on this point. Thepresence of miniature potentials could be interpreted as evidence for chemical synaptictransmission; however, there is no proof that such miniatures represent transmitterrelease from neurone 4. The tentative maps of 'innervation' provided by analyses offast rise-time miniature locations are not meant as a proof of specific innervationpatterns, but rather could be used in more direct testing of the nature of transmissionbetween neurone 4 and selected salivary gland cells. A particular question which nowcan be addressed is whether neurone 4 provides monosynaptic input to particulargland cells, a possibility which seems likely but must be proven. Another aspect offuture studies will be to carefully analyse whether other neural and/or humoral agentscan effect salivary gland activity, a possibility which has not yet been ruled out.

Exocrine secretion involves two distinct processes: (1) the release of material fromindividual secretory cells, and (2) the propulsion of secretory material through the


duct of the gland to be emptied at its orifice. Both of these secretomotor processes canbe under neural control and integrated within more complex behavioral outputs. Forinstance, Thulin (1974) has been able to distinguish between secretory and motoreffects of autonomic nerves upon salivary glands of the rat. Earlier Lent (1973) demon-strated that the large, well-characterized Retzius cells of the leech are neuroeffectorswhich control mucus release. He did not investigate the glandular cells.

Gastropod molluscs have provided the opportunity to examine the regulation of bothaspects of exocrine secretion (referred to above), at the level of identified effectorneurones, and also to investigate the integration of such processes into more complexbehaviour. Prior and Gelperin (1977) have recently identified a pair of buccal ganglionmotoneurones in the slug, Limax maximus, which elicit contractions of the salivaryduct, presumably effecting the release of saliva from the duct. Like cells 4R and 4Lin Helisoma the activity of these motoneurones in Limax can be phase-locked to theprotraction-retraction cycle during feeding.

In this communication we have described the activation of individual salivary glandcells by the identified neurones 4R and 4L and have shown that their activity can bean integral part of feeding behaviour. Various elements of the feeding motor pro-gramme may sometimes be activated independently (cf. Kater, 1974). The neuralbasis of higher order control of feeding in Helisoma is a current topic of investigation(e.g. Granzow & Kater, 1977), but a rigorous treatment of the factors determining therelative activities of neurone 4 and feeding motoneurones awaits further studies.

Fig. 15 provides a schematic representation of our current understanding of thesalivary neuroeffector system in Helisoma. Among the interesting features exhibitedby this system is the use of similar cellular communication mechanisms by the neuraland glandular elements to co-ordinate and integrate the activities of specialized cellularensembles. These mechanisms include: (1) synchronization of cellular activity byelectrical coupling; (2) utilization of regenerative action potentials for propagationof information over distance (Kater et al. 1977); and (3) employment of action poten-tials for stimulation of secretion - of neurotransmitter and of saliva.

Electrical coupling is essentially ubiquitous in epithelia but its function in suchtissues has remained more obscure than in neural tissues (Bennett, 1973; Staehelin,1974). Information transfer by the propagation of action potentials has previouslybeen described in at least two widely disparate types of epithelia (Roberts & Stirling,1974; Mackie, 1976). In addition to certain molluscan exocrine gland cells (Kater,1977; Kater et al. 1977), at least three secretory cell types of non-neural derivationhave been reported to produce regenerative action potentials (Matthews & Saffran,1973; Matthews & Sakamoto, 1975; Mackie, 1976). These data, combined with thedata obtained from the Helisoma salivary neuroeffector system, suggest that many ofour concepts of cellular communication which arose initially from investigations inneurobiology may have fundamental applications in non-neural systems.

We thank B. Granzow for technical assistance in cobalt staining, J. Kater for art-work and assistance in preparation of the manuscript and R. Hadley and A. Frost forinstructive comments during the course of this investigation. This work was sup-ported by Public Health Service research grant 1 Roi NS09696 and in part1 Roi AM19858. D.M. was supported by training grant no. H.D.-00152.


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