Feeding and a serotonergic interneuron activate an identified autoactive salivary neuron in Limax maximus

Camp. Biochem. Phvsiol. Vol. 76A, No. 1, pp. 21 to 30, 1983 Printed in Great Biitain

FEEDING ACTIVATE

0300-9629183 $3.00+0.00 ‘Q 1983 Pergamon Press Ltd.

AND A SEROTONERGIC INTERNEURON AN IDENTIFIED AUTOACTIVE SALIVARY NEURON IN LIMAX MAXIMUS JONATHAN

Department

of Zoology,

University

COPELAND

of Wisconsin-Milwaukee, U.S.A.

P.O.

Box 413, Milwaukee,

WI 53201,

and ALAN GELPERIN* Department

of Biology,

Princeton (Receiwd

University,

Princeton.

NJ 08544,

U.S.A.

14 December 1982)

Abstract-l. A single neuron is found in each buccal ganglion of the giant garden slug, Lima mu.ximus, which is autoactive and has an axon in both the ipsilateral and contralateral salivary nerve. 2. This neuron, the bilateral salivary neuron (BSN), is a slow bursting neuron and is presynaptic to some of the secretory acinar cells of the salivary gland. 3. Increases in BSN action potential frequency and saliva flow during the generation of feeding motor program are shown, as is the relationship of BSN activity to that of other salivary neurons. 4. BSN is affected synaptically by the serotonergic metacerebral giant cell.

INTRODUCTION

vertebrate and invertebrate preparations (House, 1980; Peterson, 1981). The salivary neuroeffector system in Limax has the special advantage that one can study the modulation of secretion by serotonin.

We are interested in determining the general circuit functions carried out by synaptically modulated autoactive neurons. To pursue this question, we require a set of autoactive neurons characterized as to their synaptic inputs and synaptic targets. The salivary burster (SB) neuron of Limax is one such cell (Prior and Gelperin, 1977). Recent studies of the normal reflex and synaptic events which modulate SB activity have demonstrated the existence of multiple autoactive spike initiating zones in a single neuron and of sensory responses by the SB to rhythmic proprioceptive input from the salivary duct (Beltz and Gelperin, 1980a, b). The characterization of a second autoactive neuron in Limax presented here contributes further to our understanding of the functions of synaptically modulated autoactive elements. One of the identified elements presynaptic to several autoactive neurons in Limax is the serotonergic metacerebral giant cell (MGC). Since serotonin synapses provide clear examples of neuromodulation in both vertebrates and invertebrates (Jacobs and Gelperin, 1981), it is particularly instructive to determine how synaptic input from the serotonergic MGC modulates both fast bursters such as the SB and slow bursters, such as the bilateral salivary neuron (BSN) characterized here. The bilateral salivary neuron of Limax is similar to other secretomotor neurons which have recently been described (Kater, Murphy and Rued, 1978a; Bahls, Kater and Joyner, 1980). These neurons can be used to study the synaptic events involved in exocrine secretion, a topic currently being investigated in both

MATERIALS

AND METHODS

Laboratory cultures of Limax maximus were maintained as previously described (Reingold and Gelperin, 1980). Some animals were kept at 15°C under an 8: 16 day-night light cycle. Animals weighing 3-8 g were used. All experiments were done using an in airro preparation consisting of lips, cerebral and buccal ganglia (Gelperin et al., 1978) and salivary system (bilateral salivary nerves, salivary ducts and salivary glands). Preparations were pinned in a dish lined with Sylgard to permit simultaneous recording with conventional intracellular and extracellular electrodes (Fig. l(A)). Microelectrodes were filled with 4 M potassium acetate. All experimental results were photographed directly from the oscilloscope except for Fig. 7, in which a chart recorder substituted for the oscilloscope. A saline containing 76.4 mM Na+, 2.5 mM K’, 3.42 mM Ca’+, 0.8mM Mg”, 81.94mM Cl-. 0.4mM H,PO,-, 5.0 mM HCO,-, 20 mM glucose was used for most experiments. A reduced-calcium (0.02 mM Ca”) elevated magnesium (4.19 mM Mg2+) saline, known to block peripheral and central chemical synapses in Limux (Beltz and Gelperin, 1980a) was used in some experiments. Salivary neurons were located by retrograde iontophoresis of filtered 2 M cobalt nitrate into the salivary nerve for 24-48 hr at 4 C and subsequent precipitation with ammonium sulfide. In one experiment the amount of saliva released in tttro by a salivary gland was measured by cannulating a salivary duct with a piece of polyethylene tubing secured into the salivary duct by a silk ligature. This was used as an adapter to join the duct to a longer piece of polyethylene tubing of constant and known diameter (Becton Dickinson, No. 7400). The position of the salivary meniscus was measured periodically using an ocular micrometer. From these measurements the volume of saliva produced was calculated. All experiments were done at 20-23°C.

*Present address: Department of Molecular Biophysics, Bell Laboratories, 600 Mountain Avenue, Murray Hill, NJ 07974, U.S.A. 21

22

JONATHAN COPELAND and ALAN GELPERIN cerebri 31 ganglion

n

metacwebral (MG_.j //gent cell

cannula

__-.- --- --salivary n.

salivary nerve

Fig 1. (A) Preparation for simultaneous extra- and intra-cellular recording from salivary neurons. Feeding motor program is elicited by adding standard food extracts to the lip chambers. Saliva delivery from the salivary

duct can also be measured.

RESULTS

Characterization of BSN Extracellular recordings from the two salivary nerves reveal the action potentials of six different neurons in each nerve. These neurons have synaptic targets in the salivary duct and salivary gland (Copeland et al., 1976). Spikes from three of the neurons can be recognized easily and reliably on the basis of their spontaneous firing patterns and axon distributions: the salivary burster (SB) and the two bilateral salivary neurons (BSNs) (Fig. 2(A)). The cell body of a BSN is located on the dorsal

Fig. 1. (B) Diagram of a buccal ganglion to show the location of the bilateral salivary neuron (BSN) in relation to other identified buccal neurons and the nerve roots arising from the buccat ganglion. BI: buccal neuron 1, BBC: buccobuccal connective, CBC: cerebrobuccal connective. GN: gastric nerve, Nl, 2,3: buccal nerve roots I. 2,3. SB: salivary burster. SN I, 2, 3: salivary neurons I, 2, 3.

BRl, 2: Buccal

roots,

1,2.

surface of the lateral lobe of a buccal ganglion (Fig. l(B)). BSN is found between cell 1 (the largest buccal neuron) and buccal root 2, and is white in color when epi-illuminated. Each BSN sends a process down the ipsilateral salivary nerve and a process, via the buccobuccal connective, down the contralateral salivary nerve. The axon spike of BSN is easily distinguishable from that of SB or the other salivary neurons (SNs) by the following criteria. Spontaneous BSN bursts are made up of 2-8 spikes. Instantaneous spike frequencies within a burst are low (2 Hz or less) and fairly constant. Instantaneous burst frequencies are also low (0.060.25 Hz). Occasionally BSNs do not burst but fire single spikes at regular intervals (10’s of seconds). When BSN is firing in the beating mode, it cannot be unambiguously identified on the basis of extracellular records alone. Intracellular recordings from BSN somata have voltage profiles of typical molluscan slow burster neurons (Fig. 2(B)). A pacemaker prepotential gives rise to a repetitive burst of action potentials, terminated by either a depolarizing after-potential (DAP) (Thompson and Smith, 1976) or a decay of the pacemaker potential. To determine if BSN is an autoactive neuron, two different tests were carried out. First, the preparation was bathed in reduced Caz+, elevated Mg’+ saline. This treatment blocks the junctional potentials of SB onto the salivary duct as recorded by a focal suction electrode placed on the salivary duct (Fig. 3) and also blocks central synapses (Beltz and Gelperin. 1980b).

A salivary neuron in Limax

3.8 , 20

23

set

RSB

RBSN RSN LSN

I

i0mv

2 set (B) Fig. 2. (A) Extracellular records taken from left salivary nerve (LSN) and right salivary nerve (RSN) reveal the presence of a single fast bursting neuron, the salivary burster (SB), and two slow burster neurons with bilateral axons, the bilateral salivary neurons (BSNs). Time base: 1, 3.8 set; 2, 20 sec. (B) Simultaneous intracellular recordings from the cell bodies of the right salivary burster (RSB) and right bilateral salivary neuron (RBSN) and extracellular recordings from the two salivary nerves. Note the slow depolarizing pacemaker potentials preceding each burst in SB and BSN. RSN, LSN: right and left salivary nerves.

BSN does not innervate the salivary duct, so no junctional potentials elicited by BSN spikes are evi-

dent in Fig. 3. BSN was unambiguously

identified in

normal and altered saline on the basis of its extracellular spike in the SN. BSN, like SB, continued to

burst in the altered saline. Maintenance of BSN bursting in low Ca’+ and high Mg’+ saline was found in 4 of 4 preparations. When BSN is hyperpolarized sufficiently by intracellular current injection, bursting is suppressed, no synaptic potentials are seen, and the burst rhythm is reset following release from hyperpolarization (Fig. 4). Resetting of BSN’s burst rhythm by intracellular current pulses was seen in 3 1 trials on 4 preparations. BSN produces a synaptic effect on some of the cells of the salivary gland, a tissue containing mainly secretory epithelia (Beltz and Gelperin, 1979). Simultaneous extracellular recordings from BSN and intracellular recordings from salivary gland secretory cells (acinar cells) have revealed large depolarizing poten-

tials which are time-locked with constant latency and correlated 1: 1 with BSN activity (Fig. 5). Only a small proportion of salivary cells show these potentials (less than lo%), although they are found throughout the salivary gland. Some acinar cell depolarizing potentials show complex wave forms, only a portion of which can be correlated with activity recorded from the SN. When an acinar cell is hyperpolarized by current injection, the size of the acinar cell postsynaptic potential increases (Fig. 6(A), (B)). On rare occasions, in exceptionally active preparations, the acinar cell potentials, presumably EJPs, give rise to an overshooting action potential. The autoactive bursting of BSN is synaptically modulated during the generation of feeding motor program evoked ilz vitro. The synaptic drive on BSN during FMP causes both a higher action potential frequency within a BSN burst and shorter interburst intervals, i.e., higher BSN burst frequency. Both of these effects on BSN activity during FMP are shown

JWATHAN COPELAND and ALAN GELPERIN

24

A.

LSN LSD RSN

6.

c.

2.5 set Fig. 3. Effects of low CaLi high Mg*+ saline on BSN. BSN was identified on the basis of its burst parameters, axonal distribution and action potential waveform. BSN’s axon spike, amplitude, spike frequency, and burst frequency all changed subsequent to perfusion of the bath with the altered saline (Bf. Junctional potentials recorded from the salivary duct with a focal suction electrode (middle trace of each set) are missing from record B. indicating the blockade of a peripheral synapse. A, normal saline; B, low Ca”’ high Mg’+ saline; C, normal saline. LSN: left salivary nerve. LSD: left salivary duct, RSH: right salivary nerve.

in Fig 7 which shows the transition from spontaneous BSN bursting before onset of FMP to the higher BSN spike frequency during generation of FMP. The BSN spikes are larger than the SB spikes in Fig, 7 because the frequency response of the chart recorder used to obtain this record accentuates the slower BSN action potentials.

The increase in BSN burst frequency during an entire bout of FMP is shown graphically in Fig. 8. This response pattern was observed in 6 trials on 4 preparations. The FMP response was triggered by a 30-see application of standard potato extract to the lips. During the last half of the FMP response, bite frequency and BSN burst frequency both decreased.

LBSN LSN RSN

I 40 10 Fig. 4. Hyperpohdrizdtion

of BSN by current

mv

set

injection resets the burst phase. neuron.

LBSN: left bilateral

salivary

A salivary neuron in Lintus

2s

RAC RSN LSN 140 2

tn”

set

Fig. 5. Intracellular record from a salivary gland acinar cell showing excitatory junctional potentials in the gland cell correlated with action potentials in the ipsilateral BSN. RAC: right salivary acinar cell.

In general, if the FMP response and/or slow) the BSN activation evident.

was weak (short was weak or not

Salivation during ,fkeding motor program Typically, small amounts of saliva are secreted continuously by the salivary gland into a distensible region of the salivary duct which lies on the salivary gland itself. Small amounts of saliva are usually released from this region into the tubular salivary duct continuously prior to feeding (Fig. 9). During bouts of feeding motor program evoked in t’itro the amount of saliva released from the SD increases considerably as a number of large boluses of saliva are delivered by sequential peristaltic contractions of

the SD. This large increase in the flow of saliva occurs mostly during the first minutes of the bout of feeding motor program. It never occurs except during feeding and does not occur when feeding is evoked after the SN is crushed between the en passant recording electrode and the gland.

BSN coupling to other SNs The coupling of BSN to its contralateral mate and to SB has been studied by examination of extracellular records taken simultaneously from both salivary nerves. Phase histograms were constructed for pairs of neurons from measurements of the time of occurrence of a burst in one neuron expressed as a fraction of the simultaneous interburst interval of the

A RAC RSN LSN

RSN RAC

2 set IOsec Fig. 6. (A) Intracellular record from a salivary acinar cell reveals complex EJPs correlated with BSN activity. Sometimes large EJPs occur which cannot be correlated with BSN activity. (B) EJPs recorded intracellularly from an acinar cell show an increase in amplitude when the acinar cell is hyperpolarized.

JONATHAN COPELAND and ALAN GELPERIN

26

BR2 set

WV

I

1

Fig. 7. Pattern of BSN activity during onset of a bout of feeding motor program induced by lip chemostimulation. SN: salivary nerve, BR2: buccal root 2. Additional buccal root recordings were obtained simultaneously with the records shown to verify the identity of BSN and onset of feeding motor program.

Metacerebral giant cell effects on BSN

other neuron. Before feeding, the two BSNs show no preferred phase relationship. During feeding, however, the two BSNs become relatively phase-locked to one another and burst almost synchronously (Kolmogov-Smirnov two sample test, P < 0.05). This is similar to the phase coupling shown by the two SBs before and during feeding (Prior and Gelperin, 1977). BSN shows no preferred phase relationship to the activity of SB either before or after in vitro feeding (KolmogorovSmirnov two sample test, P < 0.05). However, there is a slight tendency, although not a statistically significant one, for BSN to fire antiphasically with SB during in vitro feeding.

o”‘oO

L

I/

1

2

11 3

4

Activity of the metacerebral giant cell (MGC) inhibits some buccal neurons, such as SB, and excites others (Gelperin, 1975, 1981). To study the effects of MGC activity on BSN, the MGC was stimulated by intracellular current injection and BSN activity was monitored viu an extracellular or intracellular electrode (5 preparations). A group of MGC spikes at low frequency (< 3 spikes/set) either delays or does not affect the onset of the next BSN burst, whereas MGC activity at greater than 3 spikes/set usually advances the next BSN burst (Table I). Strong MGC activation also

11 5 TIME

6

I

7

I

6

I

9

II

I1

10

11

12

(rnlrl)

Fig. 8. Increase in BSN burst frequency during an entire bout of feeding motor program (FMP). Feeding motor program was triggered by 30 set of lip chemostimulation with a standard potato extract.

A salivary neuron in Linzux

27

Table 1. Next BSN burst delayed (%I _.__~ 63 35

Rate of stimulation of MGC @s) _-_ Single spike-3.0 3.140

TRIAL .4

.

1 . . . . . . . . . .

No effect on next BSN burst (“4 29 6

Next BSN burst advanced (%) 8 59

TRtAL

5

10

15

2

.4

TRIAL

0

N 38 17

0

20

5

4

10

15

u

u

FEEDiNG

FEEDw4G

20

TIME (MIN.1 Fig.

9.

Cumulative saliva flow measured as the amount of saliva expelled from the distal end of the salivary duct before, during and after in vitro feeding.

RBSN

RMGC 40 mv 0.5 v 40 set Fig. 10. Intracellular stimulation of the metacerebral giant cell produces a transient increase in BSN action potential frequency within a burst. RMGC: right metacerebral giant cell.

JONATHAN CDPELAND and ALAN GELWRIN

28

increases BSN spike frequency within a burst (3 animals, 10 trials) (Fig. 10). Each MGC synapses on both ipsilateral and contralateral BSN. DISCUSSION

BSN is a regular slowly bursting autoactive neuron similar in somatically recorded intracellular profile to other slowly bursting molluscan neurons. Such neurons have been described previously in a number of molluscan systems (Frazier et (11..1967; Gainer, 1972; Kater and Kaneko, 1972; Strumwasser, 1973; Willows et cd., 1973; Kupfermann and Weiss, 1976). Many of these neurons, including BSN, are white in appearance and are known, or presumed to be. neurohormonal. Action potentials in BSN are reliably followed at short and constant latency by EJP-like potentials in some acinar cells of the salivary gland. The correlation between extracellular recorded action potentials in BSN and EJPs in the acinar cells is so reliable that we interpret this as strong evidence for a direct connection between BSN and its innervated acinar cells. Final conlirmation. however, awaits simultaneous intracellular recording from the cell body of BSN and an innervated acinar cell under conditions diagnostic for monosynaptic connectivity (Berry and Pentreath. 1976; Kater, 1977). Understanding the synaptic connectivity of BSN to acinar cells is complicated by the complex electrical activity recorded in an acinar cell. These complex wave forms may have several different causes. Since electrical coupling and dye coupling have been shown between salivary acinar cells in the same and adjacent acini in both invertebrates and vertebrates (Kater et al., 1978b: Kater and Calvin. 1978; Lowenstein and Kanno, 1964; Lowenstein and Rose, 1978; Roberts et cd., 1978; Hammer and Sheridan. 1978) it is possible that some of the complex potentials recorded in Limax acinar cells were caused by activity in a nearby electrically coupled acinar cell. (Due to the complex tubulo-acinar organization of the gland and the small size of the functional units, we have not yet been able to confirm electrical coupling between Limax gland cells.) However, at least some of these complex EJPs were probably recorded from the site of a chemical synapse from BSN. Additionally, large EJPs sometimes occurred in acinar cells which could not be correlated with BSN activity. Thus, the complex synaptic activity recorded from acinar cells. or changes in this activity, could be due to electrical coupling to other active gland cells or to changes in electrical coupling between gland cells during a BSN burst. Alternatively, or additionally, some of the synapses involved in the chemical transmission could be facilitating, depressing, or failing some of the time. Finally, the activity of several thousand small (< 1 pm diameter) axons which are known to be present in the salivary nerve (Beltz and Gelperin, 1979) might go undetected by the conventional extracellular recording techniques used and, thus. their activity could be contributing to the richness of synaptic activity recorded from acinar cells. If the presence of EJPs of any size is considered a criterion for innervation by BSN. fewer than IO”,, of the acinar cells sampled are innervated by BSN.

Those cells that did not show any EJP activity were similar in resting potential (- 60 to - 90 mV) and in ability to produce action potentials when injected with current to those cells that did receive BSN evoked EJPs. Many of these same acinar cells would show subthreshold depolarization when the salivary nerve was electrically stimulated (Copeland and Gelperin. unpublished observations). It is likely, then, that the salivary gland in Limu.y is innervated by more than the paired BSN neurons. The evidence is not yet complete that BSN is a secretomotoneuron. The most convincing documentation would involve direct monitoring of primary salivary secretion subsequent to intracellular stimulation of the BSN soma, something that has not yet been done in any molluscan salivary system. However. the increase in both salivary flow and BSN activity during feeding motor program is consistent with this interpretation, as is the presence of timelocked EJPs in the secretory acinar cells. Small amounts of saliva trickle from the salivary gland and are expelled by the salivary duct prior to feeding. During feeding, however, the amount of saliva expelled from the salivary duct greatly increases. This increase in salivation has two causes: first, the increase in contraction frequency of the salivary duct, which would cause an increase in the trumport of saliva; second, the secretion of new saliva into the distal salivary duct. Although small amounts of saliva may be stored in the distal salivary duct and, thus. the initial aliquot of saliva may be made up of stored saliva, volumetric considerations require that new saliva must be secreted during feeding. Bilateral salivary neurons have been described previously in other pulmonates such as Helix pomutia (Altrup t’f ~1.. 1979) L_rmnoea stugnalis (Benjamin rt al.. 1979) and Helisoma tridt~is (Murphy and Kater, 1978). Some of these bilateral salivary neurons beat or are irregular bursters, and none of them are autoactive burster neurons (Schulze et al.. 1975; Benjamin and Rose, 1979; Kater et al., 1978a, b). Their patterns of activation differ during in vitro feeding: cell 1 in Lymnu~a is activated during protraction (Rose and Benjamin, 1979) and cell 4 in Helisomu is activated during retraction (Kater et al.. 1978b). BSN in Limux, however. shows only a slight tendency to fire in antiphase with SB during protraction (Gelperin et 01.. 1978). The homology of BSN to other known bilateral salivary neurons awaits further clarification. Cobalt backfills of the cerebrobuccal connectives have shown that the MGC axon goes down the salivary nerve and enters the salivary gland itself (Copeland and Gelperin. unpublished observations). In Limux the MGC synapses on motoneurons and has a facilitatory effect on feeding motor program (Gelperin, 1981) as it does in several other gastropod molluscs (Gillette and Davis, 1977; Granzow and Kater, 1977; Granzow and Fraser-Rowell. 1981). It will be interesting to determine the effects of MGC stimulation on acinar cell EPJs. Serotonergic activity has been shown to act as a neuromodulator in gastropods, causing presynaptic facilitation at some synapses (Kandel et d., 1981; Brunelli et rd., 1976; Shimahara and Taut, 1976, 1977), postsynaptic facilitation in some muscles (Weiss ct ul., 1975;

A salivary neuIron in Limax Kupfermann and Weiss, 1981) and reduction in contraction at other muscles [Ajimal et al., 1980). Serotonin has been implicated in mucus secretion in a number of invertebrate and vertebrate phyla (Lent, 1974) and in salivary secretion in mammals (Andersson et al., 1966; Kojima et al., 1973), so an effect in the Limax salivary glands would not be suprising. Studies of autoactive molluscan neurons are providing fundamental insights into the slow ionic conductance changes which underly autoactivity (Aldrich et al., 1979a. b; Adams et al., 1980; Carnevale and Wachtel, 1980; Culrajani and Roberge, 1978). These studies have revealed the existence of oscillating sodium and potassium conductances with cycle times of many seconds. These slow autoactive conductances. although small relative to the fast conductances underlying the action potential itself, provide a sensitive mechanism upon which synaptic inputs activity

operate to modulate (Wilson and Wachtel, 1979a, b; Pinsker. 1977).

the pattern of auto1978; Mayeri et a/.,

Acknowledgements-We thank Stephen Reingold and Barbara Beltz for critical comments on this paper. The work was supported by NIH Fellowship IF32NS05067, The Spence; -Foundat;on, and a grant from The Graduate School. Universitv of Wisconsin-Milwaukee to J.C. and bv NSF Grant BNS kOO5822 and NIH Grant NSMH I5698 to A.G. REFERENCES Adams D. J., Smith S. J. and Thompson S. H. (1980) Ionic currents in molluscan soma. A. Rec. Neurosci. 3, 141-167. Ajimal G., Shukla U. and Ram J. L. (1980) Serotonin causes opposite modulatory effects on different buccal mass muscles in Aplysia. Sot. Neurosci. Abstr. 6, 370. Aldrich R. W., Getting P. A. and Thompson S. H. (1979a) Inactivation of delayed outward current in molluscan neuron somata. J. fhysiol., Land. 291, 507-530. Aldrich R. W., Getting P. A. and Thompson S. H. (1979b) Mechanism of frequency-dependent broadening of molluscan neurone soma spikes. J. Physiol., Lond. 291, 531-544. Altrup U., Speckmann E.-J. and Caspers H. (1979) Axonal pathways and synaptic inputs of three identified neurons in the buccal ganglion of Helix pomafia. Malacologia 18, 473476. Andersson B., Jobin M. and Olsson 0. K. (1966) Serotonin and temperature control. Acta physiol. stand. 67, 50-56. Bahls F., Kater S. B. and Joyner R. W. (1980) Neuronal mechanisms for bilateral coordination of salivary gland activity in Helisomu. J. Neurobiol. 11, 365-379. Beltz B. and Gelperin A. (I 979) An ultrastructural analysis of the salivary system of the terrestrial mollusk, Limux maximus. Tissue Cell 11, 3 l-50. Beltz B. and Gelperin A. (1980a) Mechanisms of peripheral modulation of salivary and feeding neurons in Limax maximus: a presumptive sensory motor neuron. J. Newrophysiol. 44, 615-686. Beltz B. and Gelperin A. (1980b) Mechanosensory . inputs _ modulate the activity of salivary and feeding neurons in Limax maximus. J. Neuroohvsiol. 44. 656-674. Benjamin P. R. and Rose RI G. (1979) Central generation of bursting in the feeding system of the snail. Lymnaea stagnalis. J. exp. Biol. 80, 98-118. Benjamin P. R., Rose R. M., Slade C. T. and Lacy M. G. (1979) Morphology of identified neurones in the buccal ganglia of Lymnaea stugnalis. J. exp. Biol. 80, 119-135.

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Berry M. S. and Pentreath V. W. (1976) Criteria for distinguishing between monosynaptic and polysynaptic transmission. Bruin Res. 105, I-20. Brunelli M., Castellucci V. and Kandel R. R. (1976) Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP. Science 194, 1178-l 181. Carnevale N. T. and Wachtel H. (1980) Two reciprocating current components underlying slow oscillations in ApIysia bursting neurons. Brain Res. Rev. 2, 45-68. Copeland J., Symonds B. and Gelperin A. (1976) Molluscan salivary neurons: characterization and modulation during feeding. Sot. Neurosci. Abstracts 2, 319. Frazier W. T.. Kandel E. R.. KuDfermann I.. Waziri R. and Coggeshall R. E. (1967) Mo;phological and functional properties of identical neurons in the abdominal ganglion of Aplysia callfornica. J. Neurophysiol. 30, 1288- I35 I Gainer H. (I 972) Electrophysiological behavior of an endogenously active neurosecretory cell. Brain Res. 39, 403418. Gelperin A. (1975) An identified serotonergic neurons has reciprocal effects on two electrically coupled motoneurons in the terrestrial slug Limax maximus. Biol. BUN. 149, 426427. Gelperin A. (1981) Synaptic modulation by identified serotonin neurons. In Serotonin Neurotransmission and Beharior (Edited by Jacobs B. and Gelperin A.) pp. 288-304. MIT Press, Cambridge. Gelperin A., Chang J. J. and Reingold S. (1978) Feeding motor program in Limax. I. Neuromuscular correlates and control by chemosensory input. J. Neurobiol. 9, 285-300. Gillette R. and Davis W. J. (1977) The role of the metacerebral giant neuron in the feeding behavior of Pleurobranchaea. J. camp. Physiol. 116, 129-159. Granzow B. and Fraser-Rowe11 H. (1981) Further observations on the serotonergic cerebral neurons in Helisoma (Mollusca, Gastropoda): the case for homology with the metacerebral giant cells. J. exp. Biol. 91, 283-305. Granzow B. and Kater S. B. (1977) Identified higher order neurons controlling the feeding motor program of Helisoma. Neurosci. 2, 1049- 1063. Gulrajani R. M. and Roberge F. A. (1978) Possible mechanism underlying bursting pacemaker discharges in invertebrate neurons. Fedn Proc. 37, 21462152. Hammer M. E. and Sheridan J. D. (1978) Electrical coupling and dye transfer between acinar cells in rat salivary glands. J. Physiol. 275, 495-505. House C. R. (1980) Physiology of invertebrate salivary glands. Biol. Rev. 55, 417-473. Jacobs B. L. and Gelperin A., Eds. (1981) Serotonin Neurotrunsmission and Behauior. MIT Press. Cambridge. Kandel E. R.. Klein M., Bailey C. H., Hawkins R. D.. Castellucci V. F.. Lubit B. W. and Schwartz J. H. (1981) Serotonin, cyclic AMP and the modulation of the calcium current during behavior arousal. In Serotonin Neuretransmission and Behavior (Edited by Jacobs B. and Gelperin A.) DP. 21 I-254. MIT Press. Cambridge. Kater’ S. B. (1677) Calcium electroresponsiveness-and its relationship to secretion in molluscan exocrine gland cells. Neurosci. S.vmp. 2, 195-2 14. Kater S. B. and Galvin N. J. (1978) Physiological and morphological evidence for coupling in mouse salivary eland acinar cells. J. CeN Biol. 79. 2c-26. Kiter S. B. and Kaneko C. R. S. (1472) An endogenously bursting neuron in the gastropod mollusc, Helisoma trirohis: characterization of activity in uico. J. camp. Phj,.siol. 79, I 14. Kater S. B.. Murphy A. D. and Rued J. R. (1978a) Control of the salivary glands of Helisoma by identified neurons. J. exp. Biol. 72, 91-106. Kater S. B., Rued J. R. and Murphy A. D. (1978b) Propagation of action potentials through electronic junc-

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JONATHAN COPELAND and ALAN GELPERIN

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