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Morphology and electrophysiology of neurons innervating the ciliated locomotor epithelium in Lymnaea stagnalis (L.)
Comp. Biochem. Physiol.Vol. 93A,No. 3, pp. 633--644, 1989 Printed in Great Britain
0300-9629/89 $3.00 + 0.00 © 1989 MaxwellPergamon Macmillanpie
MORPHOLOGY A N D ELECTROPHYSIOLOGY OF N E U R O N S I N N E R V A T I N G THE CILIATED LOCOMOTOR EPITHELIUM IN LYMNAEA STAGNALIS (L.) N. I. SYED* and W. WINLOWt
Department of Physiology, University of Leeds, Leeds LS2 9NQ, UK. Telephone: (0532) 334247
(Received 22 December 1988) AMtract--1. The pedal A-cluster neurons of Lymnaea stagnalis (L.) may either modulate the activities of the locomotor cilia or act as ciliomotoneurons. Their morphology has been gtudied using a modified technique for injection of the fluorescent marker substance Lucifer Yellow and their electrophysiological characteristics have been studied using conventional intracellular recording techniques. 2. The paired pedal A-clusters are each composed of 30 bilaterally symmetrical pairs of neurons lying on the medial faces of the paired pedal ganglia. 3. With respect to the foot, all the A-cluster neurons have unipolar axons which are partially somatotopically organized. 4. Small A-cluster (40-50 #m soma diameter) neurons project ipsilaterally and are weakly electrically coupled to adjacent cells. The medium-sized (50-60#m) and large (60-70#m) cells project both ipsilaterally and contralaterally and make electrical connections with their contralateral homologues as well as their smaller near neighbours. 5. The A-cluster neurons have similar action potential shapes and receive several identifiable synaptic inputs whose sources are unknown. Some of these inputs have widespread synaptic actions in several ganglia whilst the "A-cluster rhythm" (ACR) is entirely restricted to the A-cluster neurons. 6. The identified interneuron VD4 (= the visceral white interneuron, VWI) is known to be involved in respiratory behaviour and also has excitatory actions on all the A-cluster neurons.
INTRODUCTION
In gastropod molluscs such as Lymnaea stagnalis, the locomotory cilia provide the main propulsive force (Kaiser, 1960; Jones, 1975), whilst the animal changes direction by contractions of the musculature of the foot and body wall on contact with objects placed in its path. Rhythmical shell movements are usually associated with locomotion (Winlow and Haydon, 1986), but may play a part in the cardiorespiratory system of the animal rather than being directly linked to locomotion itself. In Lymnaea the pedal ganglia contain many pairs of giant neurons and neuronal clusters (Slade et al., 1981), and a number of locomotor motoneurons have been identified (Winlow and Haydon, 1986). Detailed morphological and electrophysiological studies of the neurons of the pedal ganglia are published elsewhere (Slade et al., 1981; Kyriakides et aL, submitted). In Mytilus edulis pharmacological, histofluorescent and biochemical evidence is consistent with physiological studies which suggest that the lateral cilia of the gill can be excited or depressed by electrical stimulation of the branchial nerve which innervates it (Takahashi, 1971). There is strong evidence suggesting that serotonin (excitatory) and dopamine (inhibitory) are the peripheral neurotransmitters mediating ciliary activity in the Mytilus gill (Stefano et al., *Present address: Department of Medical Physiology, University of Calgary, Health Sciences Centre, 3330 Hospital Drive, NW, Calgary, Alberta, Canada T2N 4NI. tAuthor to whom all correspondence should be addressed.
1988). In Tritonia, paired, serotonin-containing, electrically coupled neurons are known to provide tonic excitation of the pedal cilia (Audesirk, 1978; Audesirk et al., 1979). In contrast, central neurons causing inhibition of stigmatal cilia in the branchial basket of the ascidian Chelyosoma productum have recently been identified and mediate ciliary arrest via direct chemical connections (Arkett, 1987). It is difficult to observe ciliary beating directly in a semi-intact preparation of Lymnaea, but iron filings or carbon particles placed on the foot sole are observed to move, in the absence of muscular contractions, following stimulation of the pedal A-cluster neurons (Syed, unpublished observations). These neurons are known to contain serotonin (Audesirk, 1985; Casey and Winlow, 1985) and injection of serotonin into the foot excites previously quiescent pedal cilia or increases the rate of beat of active cilia (Syed et aL, 1988). The presence of serotonin in axon tracts and varicosities at the base of the ciliated epithelium of the foot of Lymnaea has recently been demonstrated by immunocytochemistry (Caunce et al., 1988; McKenzie et aL, 1987a). Furthermore, electron microscopic studies reveal that axon terminals, containing dense-cored vesicles, make intimate contact with both ciliated cells and mucus-secreting cells of the foot (Caunce et al., 1988; McKenzie et al., 1987a, b). Here we describe the morphological and electrophysiological characteristics of the serotonergic pedal A-cluster neurons of Lymnaea, which innervate the ciliated epithelium of the foot sole.
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N.I. S"~D and W. WI~Low MATERIALS AND METHODS
Specimens of Lymnaea stagnalis (L.) were obtained from animal suppliers or from the Leeds-Liverpool canal. These snails were kept at 14-18°C in water obtained from local ponds and fed on lettuce. Isolated brains of Lymnaea were prepared for electrophysiological and morphological studies and maintained in snail saline buffered to pH 7.8 or 7.9, as previously described (Benjamin and Winlow, 1981). Individual neurons were impaled with 10-20Mf~ glass microelectrodes filled with saturated K2SO4 and recordings of neuronal activity were made using conventional techniques. Electrophysiological signals were amplified, displayed and recorded by conventional means (Benjamin and Winlow, 1981). The morphology of identified neurons was studied using the dye Lucifer Yellow CH (Stewart, 1978), with technical modifications which improved both the "fills" and the clearing of the tissue. Projections of Lucifer-filled axons into the tissues of the foot were traced using frozen serial sections prepared on a cryotome.
An improved techniquefor intraneuronal injection of Lucifer Yellow (CH) The injection of neural marker substances can be problematical and Lucifer Yellow is no exception. The difficulties include: an inability to inject sufficient dye; leakage around the point of microelectrode penetration (Fig. la) and poor clearing of the tissue (Fig. lb). A number of small modifications of the technique greatly improved the quality of Lucifer "fills" in the neurons of L. stagnalis (Fig. lc, d). Microelectrodes were filled with 10% Lucifer Yellow in double-distilled and deionized water, which gave a tip resistance of 20-80 Mf~. During impalement a constant holding current of +2 nA was applied to the electrodes through the preamplifier, in order to prevent the leakage of the dye from the microelectrode tip. Upon successful penetration this current was switched off. Dye was then delivered into the cell body by applying a constant - 2 n A current through the preamplifier. Since constant currents often caused a change in the resistance of liquid-filled microelectrodes, - 2 to - 1 0 n A current pulses of 500 ms duration at a frequency of 0.5-1.5 Hz, were superimposed on this constant current for 20-40 min. Microelectrode blocking was further minimized by reversing the constant hyperpolarizing ( - 2 nA) current to a depolarizing current (+2 nA) for 1 or 2 min, once or twice during the course of dye injection. The cells being filled were regularly observed using a mercury lamp and light guide, but not at sufficient intensity to kill the neurons (Miller and Selverston, 1979). When the cells appeared to be adequately filled, dye injection was terminated. During withdrawal of microelectrodes from the cell a +2 nA constant current was again applied. The preparations were left overnight in a refrigerator at 4°C, and were fixed in buffered formalin fixative (formaldehyde, 4%, in 0. ! M sodium phosphate buffered to pH 7.4), for 3 hr. These fixed brains were then dehydrated, defatted and cleared according to the schedule below. Dehydration 1. 50% ethanol 2. 50% ethanol 3. 70% ethanol 4. 70% ethanol 5. 90% ethanol 6. 90% ethanol 7. absolute ethanol 8. absolute ethanol 9. 100% chloroform 10. 100% xylene Tissue clearing 1. Dimethyl sulphoxide 30 rain 2. Methyl salicylate 10 min 3. Methyl methacrylate 10 rain
30 rain 30 min 30 rain 30 min 30 min 30 min 30 min 30 min 30 min 30 min
For reasons that are not apparent, omission of either the dimethyl sulphoxide or methyl salicylate diminished the quality of clearing whilst omission of methyl methacrylate increased the background fluorescence of the tissue. Cleared tissues were mounted in fluorolite on depression slides and observed using a Leitz Diahix 20EB incident fluorescence microscope. Successfully injected neurons were photographed using 400 ASA Kodak Ektachrome slide film and drawn using a camera lucida. Our modified techniques for Lucifer injection and for clearing tissue aided us greatly in resolving fine branches and dendrites, but of course the very finest branches were still beyond the limits of resolution of the light microscope. RESULTS
Morphology Location, nomenclature and projections of A-cluster neurons. The paired, symmetrical, pedal A-cluster neurons o f Lymnaea are located on the medial faces of the largely symmetrical left and right pedal ganglia (Fig. 2 and see Kyriakides et al., in press, Slade et al., 1981). There are 30 individually identifiable neurons in each cluster as shown in Fig. 2a and b). These cells are numbered from posterior to anterior in the sequence 1-30. Cells 1-12 lie on the dorsal surfaces, whilst 13-30 lie on the ventral surfaces of the pedal ganglia (Fig. 2). Within the pedal A-clusters there are three pairs of giant cells, which Slade et al. (1981) designated as L.Pe.D.4, R.Pe.D.4, L.Pe.D.8, R.Pe.D.8, L.Pe.V.2 and R.Pe.V.2. These giant cells are included in the A-cluster, since they have similar morphological and elcctrophysiologlcal properties to them. Their numbers have been retained within the present maps so that the A-cluster cells labelled L.Pe.A4 and R.Pe.A4 correspond to L.Pe.D.4/R.Pe.D.4 etc. (see Fig. 2 for full details). The majority of A-cluster neurons project ipsilaterally through the pedal, cervical and columellar nerve trunks (Table 1) and all these neurons have unipolar axons which undergo complex arborizations in the neuropile. On leaving the neuropile the axons undergo second order branching sending a major projection into one o f the pedal nerves and other major projections into nerve trunks emanating from the pedal ganglia (Table 1). Multiple branching often occurs in Lymnaea nerve trunks and has also been described in Aplysia (Winlow and Kandel, 1976). The seven pairs of larger neurons within the clusters (L./R.Pe.A2, 4, 8, 10, 26, 30), including those previously identified as giant cells, make contralateral projections (Fig. 3a, b), and are electrically coupled to their contralateral homologues. The 23 smaller pairs of cells only project ipsilaterally and are electrically coupled to their ipsilateral near neighbours. To explore the hypothesis that one o f the targets o f the serotonergic A-cluster neurons is the ciliatezi pedal epithelium, large numbers of A-clustor neurons were filled with Lucifer Yellow in several semi-intact preparations. Frozen serial sections o f the h e a d - f o o t complex revealed fine branches of Lucifer-filled axons beneath the ciliated epithelium (Fig. 4a, b).
A-cluster neurons exhibit partial somatotopy and are individually identifiable. The pedal ganglia are clearly bilaterally organized and the A-cluster neurons are, in addition, partially somatotopically
Innervation of a ciliated locomotor epithelium
635
Fig. 1. The effects of modification of the technique for Lucifer Yellow injection on the quality of the preparation. (a) An example of visceral M group cell where dye has leaked into the extracellular spaces (arrows) prior to or just after injection. (b) A-cluster neurons in poorly cleared tissue (arrows). Chloroform and DMSO were not used in processing. (c) Conspicuous dendrites and varicosities around the soma of the giant dopamine-containing cell R.Pe.D.1, prepared according to our improved schedule. Note that there is no leakage of the dye (large arrows), very little background fluorescence and good clearing of the tissue revealing varicosities on the neurites (small arrows). (d) Photomontage of a dorsal view of R.Pe.D. 1, showing its extensive dendritic tree in the visceral and right parietal ganglia (open arrows). The fine dendritic branches are conspicuous as a result of the modifications of the Lucifer technique to give good clearing of ganglia (solid arrows). organized with respect to the locomotor epithelium. Table 1 demonstrates that the positions of A-cluster neurons are somatotopically related to their ipsilateral projections to the foot of Lymnaea, via the three ventrally positioned pedal nerve trunks: anterior, CBP(A) 93/3--J
median and inferior pedal nerves. Neurons of the anterior group of the cluster mainly project out of posterior pedal nerve trunks, whilst those of the posterior group have a higher proportion of branches down the anterior pedal nerve trunks (Fig. 5). The
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P Fig. 2. Location of individually identifiable pedal A-duster neurons on the medial faces of the pedal ganglia. Dorsal (upper) and ventral (lower) views of the ganglia are presented. Diagrams modified from Slade et al. (1981). The pairs of giant neurons originally identified within the clusters as L./R.Pe.V.2, L./R.Pe.D.4 and L./R.Pe.D.8 are now considered to be an integral part of each duster and have been renamed L./R.Pe.A2, L./R.Pe.A4 and L./R.Pe.AS, respectively. There are 30 individually identifiable A-cluster neurons present on the medial faces of the right and left pedal ganglia. Cells 1-12 are present on the dorsal surface while cells 13-30 are situated on the ventral surfaces. Abbreviations are as follows: 18, superior pedal nerve; 19, inferior pedal nerve; 20, medial pedal nerve; 24, cerebropedal connective; 25, pedal-Neural connective; L.Pe.G, left pedal ganglion; R.Pe.G, right pedal ganglion; A, anterior; P, posterior; L, left; R, right. axons of the neurons in the middle of the duster project in similar numbers down all three pairs of ventral nerve trunks. For further details see the legend to Table 1. Axons from posteriorly positioned neuron somata tend to run in tracts towards the anterior nerve trunks and vice versa (Fig. 5) and large numbers of axonal projections also pass to the superior cervical, inferior cervical and columellar nerves. According to de Vlieger (1968), Janse (1974) and Dorsett (1986), the main innervation areas of the nerve trunks of the pedal ganglia of Lymnaea are, from anterior to posterior: superior pedal nerves - medial pedal nerves -inferior pedal nerves - superior cervical nerves-inferior cervical nerves - columellar nerves
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Thus, the superior, medial and inferior pedal nerves innervate the ventral surface of the animal whilst the superior and inferior cervical nerves innervate the dorsal body surface and the A-cluster
neurons exhibit no obvious somatotopic organization in these areas. Because of their exact location, size, colour, precise projections and electrophysiological properties it is possible to identify A-cluster neurons individually and to separate them from neurons of the neighbouring B- and E-clusters. Despite the fact that some larger cells of the B- and E-clusters appear similar to A-cluster neurons, their branch patterns and electrophysiological properties are clearly different from those of the pedal A-clusters (Kyriakides et al., in press). The axon projections of individual A-cluster neurons are summarized by Syed (1988). All the A-cluster neurons have extensive fine neurites, particularly within the ipsilateral pedal ganglion and the neuropile. Those cells that have contralateral projections as well, show similar fine varicosities in the contralateral ganglion. Another area of extensive branching is in the neuropile below the somata of putative and identified locomotor motoneurons (L. and R. Pc.D, F, G, clusters) (Winlow and Haydon, 1986; for further details see Kyriakides et aL, in press). A few A-cluster neurons also have fine neurites
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in the left and right cerebral ganglia and left and right pleural ganglia. Electrophysiology
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Neurons of the pedal A-clusters fire irregularly, but often with a patterned discharge (Kyriakides et al., in press). A|l the neurons of the cluster have type 2 action potentials (Winlow et al., 1982), with a pronounced pseudoplateau on the falling phase. The action potential trajectories of these neurons and their responses to injected currents are described in detail elsewhere (Winlow et al., 1982). The majority of A-cluster neurons (L./E.Pe.A1, 3, 5-7, 11-25, 27 and 29) are 4 0 - 5 0 # m in soma diameter, project ipsilaterally and are weakly electrically coupled to adjacent cells (Fig. 6). The medium-sized cells (50-60#m) known as L./R.Pe. Al0, 26, 28 and 30 have contralateral projections and make electrical connections with their contralateral homologues as well as their smaller near neighbours and one another. The giant neurons (60-70 pm) of the cluster also project contralaterally and again make electrical connections with their contralateral homologues, one another and their smaller near neighbours (Fig. 6d, e). The smaller cells appear to be more weakly coupled than the larger ones, but an accurate determination of coupling coefficients has not been carried out. The A-cluster neurons receive a number of identifiable synaptic inputs, some of whose sources are unknown. All cells in the pedal A-clusters receive a regular, common, excitatory input (Fig. 7), which we have termed the "A-cluster rhythm" (ACR). We have used this terminology because this synaptic input, when it occurs, is restricted to the A-cluster neurons and may be expected to have excitatory effects on the pedal cilia. The ACR occurs in both isolated brains and in semi-intact preparations. Input 3 is a wide-acting synaptic input (Winlow and Benjamin, 1976; Benjamin and Winlow, 1981; Syed et al., 1988), which is thought to be involved in respiration (Syed and Winiow, 1988). It has powerful effects on follower cells of the giant dopaminecontaining neuron R.Pe.D. 1 (Winlow et al., 1981). In Fig. 8 we demonstrate that input 3 also has widespread effects and acts on neurons of the pedal and visceral ganglia, including visceral white interneuron (VWI of Benjamin, 1984), best described as visceral dorsal 4 (VD4 of Janse et al., 1985). This cell has widespread actions and is implicated in the control of the pneumostome (Syed and Winlow, 1988). The A-cluster neurons receive delayed inhibition immediately following the effects of input 3 on other neurons (Fig. 8). Input 4 is a compound post-synaptic potential which has actions on visceral, right parietal, and pedal neurons (Winlow and Benjamin, 1976; Winlow, 1979; Winlow et al., in preparation). It occurs irregularly and can produce inhibition for several seconds on all the A-cluster neurons. It has inhibitory effects on the pedal B-, D-, F- and G-clusters and excites the pedal E-clusters (Fig. 9a). Many of these clusters contain locomotor motoneurons (Winlow and Haydon, 1986). In addition input 4 inhibits the giant dopamine-containing neuron R.Pe.D.I (Winlow et al., in preparation). Furthermore, the ring neuron of
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Fig. 3. Camera lucida drawings showing both ipsilateral and contralateral projections of "giant" A-cluster neurons. (a) L.R.Pe.A4. Both these identical cells have similar ipsilateral and contralateral projections. (b) L./R.Pe.A8 neurons projecting both ipsilaterally and contralaterally. Abbreviations as in Fig. 2. the cerebral ganglia (Jansen and ter Maat, 1985), which is known to inhibit the A-cluster neurons, is also excited by input 4 and may further increase A-cluster neuron inhibition. All the A-cluster neurons receive synaptic inputs in common with the motoneurons of the body wall, column and foot. These inputs normally occur during the acquatic and/or terrestrial phases of locomotion (Winlow and Haydon, 1986). Work on semi-intact preparations indicates that, during the aquatic phase of locomotion, which is primarily dependent on ciliary beating, only A-cluster neurons are strongly excited and the neurons of both clusters discharge synchronous bursts of action potentials. The A-cluster neurons are also strongly excited, for a short time when the shell is pulled forward, at the end of each locomotory cycle. During shell movements, contractions of the foot musculature are not usually observed. These inputs during shell movements are also received by body wall and column motoneurons and are often observed in the isolated brain (Fig. 9b and see Syed et al., 1988). In Fig. 8, VD4 of the visceral ganglion was shown to have excitatory actions on the A-cluster neurons (Syed et al., 1988) and is believed to be involved in the respiratory system of Lymnaea (Janse et aL, 1985; Syed and Winlow, 1988). The A-cluster neurons and
the identified cells L./R.Pe.D.2 are strongly excited by VD4 (Fig. 10a), and we believe that both these connections and the inhibitory connections to neurons of the pedal F-cluster and pedal E-cluster are monosynaptic (Fig. 10b) on the bases of constancy of delay and one for one occurrence of spike and p.s.p. at all frequencies so far used. More rigorous evidence for this supposition is not yet available. DISCUSSION
Some of the cells within the pedal A-clusters of Lymnaea were previously identified as giant neurons (Slade et al., 1981), but, in common with other A-cluster neurons, they had no role to play in the muscular contractions associated with locomotion (Winlow and Haydon, 1986). Recent light and electron-microscopical observation (McKenzie et aL, 1987a, b; Caunce et aL, 1988), coupled with neuroethological studies (Syed, 1988; Syed and Winlow, unpublished observations) indicate that these neurons form direct neuroeiliated cell junctions (n.c.c.j.s.) with the eiliated epithelium of the foot sole. The A-cluster neurons are bilaterally symmetrical (Fig. 2), each pair of neurons within the cluster is individually identifiable and all contain serotonin (Casey and Winlow, 1985). The
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Fig. 4. Lucifer-filled axons in the foot of Lymnaea. (a) Frozen serial section of the head-foot complex, 24 hr after the injection of Lucifer Yellow. The dye was injected iontophoretically into several A-cluster neurons and was allowed to travel overnight. Lucifer-filled axons were found in pedal nerves and were tracked deep down into the foot (arrows). The fine branches of the axons filled with dye can easily be seen. (b) A photograph of the frozen section of the foot revealing Lucifer Yellow-filled axon (small arrow) terminating beneath the ciliated epithelium (large arrow). Unfortunately, Lucifer fades rapidly on exposure to UV light and it is particularly difficult to photograph fine axon branches as they approach the ciliated epithelium.
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Fig. 5. Somatotopic organization of ciliomotoneurons. Photomicrograph of ciliomotoneurons showing their somatotopic organization. Cells present at the anterior end of the cluster exit via the posterior pedal nerve trunks and vice versa. Broad arrows: axon projections in anterior (right) and posterior nerve trunks (left). Fine arrows: neurites.
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Fig. 7. The "A-cluster rhythm", a strong excitatory synaptic input which is received only by ciliomotoneurons. (a) Synchronous spontaneous patterned activity of two small A-cluster neurons on opposite sides of the isolated brain. These neurons are not directly connected to one another. (b) The A-cluster rhythm recorded in L./R.Pe.A4 and L./R.Pe.A8 in an isolated brain. The contralateral homologues make direct electrial connections with one another. role of the branches of the A-cluster neurons passing to the cervical and columellar nerves is both puzzling and unclear. We do not know whether they pass to the musculature or to the body surface of the animal and although the A-clusters appear to modulate ciliary activity, their possible involvement in other functions cannot be ruled out. Despite the fact that all the A-cluster neurons receive inputs in c o m m o n with other locomotor
motoneurons, there are a few inputs which are solely confined to the A-cluster neurons. One of these inputs is the "A-cluster rhythm" (Fig. 4) which synchronizes the activities of the A-cluster neurons, presumably aided by the electrical junctions between these cells. Perhaps a central pattern generator underlies this rhythm, but whether any or all of the A-cluster neurons participate in its circuitry is unknown. The A-cluster neurons are excited during both the aquatic
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and terrestrial phases of locomotion, and are active during the initiation, maintenance and modulation of locomotor behaviour. A-cluster neurons are in receipt of wide-acting input 3 which has widespread effects in the brain of Lymnaea (Winlow and Benjamin, 1981; Syed et al., 1988). This input is believed to be involved in respiration (Syed and Winlow, 1988) and has delayed inhibitory effects on the putative ciliomotoneurons. It also inhibits other locomotor motoneurons. On the other hand, the interneuron VD4 is also involved in respiratory behaviour and has strong excitatory effects on the putative ciliomotoneurons. During the initial phase of respiratory behaviour, which we believe is caused by input 3 (i.e. opening of the pneumostome and expiration), the locomotor and withdrawal systems are temporarily inhibited, especially the foot and body wall motoneurons and the putative ciliomotoneurons, in order to facilitate the opening of the pneumostome.
VD4 plays an important role in the second phase of respiration by (i) closing the pneumostome; (ii) inhibiting foot and body wall motoneurons to minimize interference with pneumostome movements and (iii) exciting A-cluster neurons presumably in order to resume locomotion. VD4 and input 3 (driving inspiration and expiration, respectively) have antagonistic actions on one another as might be expected (Syed and Winlow, 1988). From these studies we conclude that the serotonergic A-cluster neurons are quite distinct from other pedal neurons on the bases of their morphological and electrophysiological characteristics. On the other hand, they receive inputs in common with other locomotor motoneurons and may play an integral part in several complex behaviours such as locomotion, respiration and egg-laying behaviour. Immunocytochemical and ultrastructural evidence supports our contention that the A-cluster neurons innervate the ciliated, locomotor epithelium of the foot sole, but further evidence is required to
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Fig. 10. The widespread actions of interneuron VD4 ( = V.W.I) on neurons of the pedal ganglia. (a) The excitatory effects of VD4 (second trace) on a ciliomotoneuron (L.Pe,A4, bottom trace) and the identified neurons R.Pe.D.2 (top trace) and L.Pe.D.2 (third trace). Bursts o f spikes in VD4 elicit strong bursts in each of these neurons (arrows). (b) Inhibitory actions of VD4 (second trace) on putative locomotor motoneurons. The L.Pe.F. neurons (top trace and third trace) are at their normal membrane potentials and the L.Pe.E. cell (bottom trace) is hyperpolarized. Clear one-to-one p.s.p.s, in the upper L.Pe.F. neuron and the L.Pe.E. neuron are associated with action potentials in VD4.
644
N.I. SY~D and W. WINLOW
substantiate our hypothesis motoneurons.
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Acknowledgements--We thank Walter Stewart for supplies of Lucifer Yellow CH, Mr D. Johanson for photographic assistance and Mr D. Harrison for technical assistance. WW was in receipt of an SERC project grant. N.I.S. was supported on an O.R.S. award. REFERENCES
Arkett S. A. (1987) Ciliary arrest controlled by identified central neurons in a urochordate (Ascidiacea). J. comp. Physiol. 161, 837-847. Audesirk G. (1978) Properties of central motor neurones exciting locomotory cilia in Tritonia diomedia. J. comp. Physiol. 128, 259-267. Audesirk G. (1985) Amine-containing neurones in the brain of Lymnaea stagnalis: distribution and effects of precursors. Comp. Biochem. Physiol. glA, 359-365. Audesirk G., McCaman R. E. and Willows A. O. D. (1979) The role of serotonin in the control of pedal ciliary activity by identified neurones in Tritonia diomedta. Comp. Biochem. Physiol. 62C, 87-91. Benjamin P. R. (1984) Interneuronal network acting on snail neurosecretory neurones (Yellow cells and YellowGreen cells of Lymnaea). J. exp. Biol. 113, 165-185. Benjamin P. R. and Winlow W. (1981) The distribution of three wide-acting synaptic inputs to identified neurones in the isolated brain of Lymnaea stagnalis (L.). Comp. Biochem. Physiol. 70A, 293-307. Casey C. and Winlow W. (1985) Cellular localisation of serotonin by glycoxylic acid condensation in Lymnaea. J. Physiol., Lond. 369, 169P. Caunce M., McKenzie J. D., Tripp J. and Winlow W. (1988) Serotonerglc innervation of the pedal epidermis of Lymnaea. In Neurobiology of Invertebrates: Transmitters, Modulators and Receptors (Edited by Salanki J. and Rosza K. S.). Syrup. Biol. Hungarica 36, 691-692. de Vlieger T. A. (1968) An experimental study of the tactile system of Lymnaea stagnalis. Neth. J. Zool. 18, 105-154. Dorsett D. A. (1986) Brains to cells: the neuroanatomy of selected gastropod species. In The Mollusca, Vol. 9, Neurobiology and Behaviour, Part 2, pp. 101-187. Academic Press, New York. Janse C. (1974) A neurophysiological study of the peripheral and tactile system of the Pond Snail Lymnaea stagnalis. Neth. J. Zool. 24, 93-161. Janse C., Van der Wilt C. J., Van der Plas J. and Van der Roest M. (1985) Central and peripheral neurones involved in oxygen perception in the pulmonate Snail Lymnaea Stagnalis, Mollusca, Gastropoda. Comp. Biochem. Physiol. g2A, 459-469. Jansen R. F. and ter Maat A. (1985) Ring neurone control of columellar motor neurones during egg-laying behaviour in the pond snail. J. Neurobiol. 16, !-14. Jones H. D. (1975) Locomotion. In Pulmonates, Vol. 1 (Edited by Seller V. F. and Peake J.), pp. 1-32. Academic Press, London. Kaiser P. (1960) Die Leistungen des Flimmerepithels bei der Fortbewegung der Basommatophoran. Z. wiss. Zool. 162, 368-393.
Kyriakides M., McCrohan C. R., Slade C. T., Syed N. I. and Winlow W. (1989) The morphology and electrophysiology of the neurones of the paired pedal ganglia of Lymnaea stagnalis (L.). Comp. Biochem. Physiol. (in press). McKenzie J. D., Syed N. I., Tripp J. and Winlow W. (1987a) Are pedal cilia under neural control? In Neurobiology, Molluscan Models (Edited by Boer H. H., Geraerts W. P. M. and Joose J.), pp. 26-30. North Holland Publ. Co., Amsterdam. McKenzie J. D., Syed N. I., Tripp J. and Winlow W. (1987b) Innervation of ciliated cells and mucus-containing cells in Lymnaea. J. Physiol., Lond. 386, 32P. Miller J. D. and Selverston A. I. (1979) Rapid killing of single neurones by irradiation of intracellularly injected dyes. Science 206, 702-704. Slade C. T., Mills J. and Winlow W. (1981) The neuronal organisation of the paired pedal ganglia of Lymnaea stagnalis (L.). Comp. Bioehem. Physiol. 69A, 789403. Stefano G. B., Braham E., Sinisterra J. I., Bentvena L., DeFiglia J. and Leung H. K. (1988) The effects of cocaine on peripheral monoaminergic regulatory mechanisms in the gill of Mytilus edulis. In Neurobiology of Invertebrates: Transmitters, Modulators and Receptors (Edited by Salanki J. and Rosza K. S.). Syrup. Biol. Hungarica 36. Stewart W. W. (1978) Functional connections between cells are revealed by dye coupling with a highly fluorescent Naphthalimide tracer. Cell 14, 741-759. Syed N. I. (1988) The neural control of locomotion in Lymnaea. PhD Thesis, University of Leeds, U.K. Syed N. I. and Winlow W. (1988) The role of central neurones in respiratory behaviour in Lymnaea. J. Physiol., Lond. 403, 62P. Syed N. I., Harrison D. and Winlow W. (1988) Locomotion in Lymnaea: role of serotonergic motoneurones controlling the pedal cilia. A-cluster neurones. In Neurobiology of Invertebrates: Transmitters, Modulators and Receptors (Edited by Salanki J. and Rosza K. S.). Syrup. Biol. Hungarica 36, 387-402. Takahashi K. (1971) Abrupt stoppage of Mytilus cilia caused by chemical stimulation. J. Fac. Sei. Univ. Tokyo (Sect. IV, Zool). 12, 219-228. Winlow W. (1979) Wide-acting synaptic inputs in the brain of the pond snail. J. Physiol., Lond. 310, 31P. Winlow W. and Benjamin P. R. (1976) Neuronal mapping of the brain of the pond snail, Lyrrmaea stagnalis (L.). In Neurobiology of Invertebrates, Gastropoda Brain (Edited by Salanki J.), pp. 41-59. Akademiai Kiado, Budapest. Winlow W. and Kandel E. R. (1976) The morphology of identified neurones in the abdominal ganglion of Aplysia californica. Brain Res. 112, 221-249. Winlow W. and Haydon P. G. (1986) A behavioural and neuronal analysis of the locomotory system of Lymnaea stagnalis. Comp. Biochem. Physiol. 83A, 13-21. Winlow W., Haydon P. G. and Benjamin P. R. (1981) Multiple postsynaptic actions of the giant dopamine-containing neurones, R.Pe.D. 1, of Lymnaea stagnalis (L.). J. exp. Biol. 94, 137-148. Winlow W., Holden A. V. and Haydon P. G. (1982) Characterisation of Lymnaea neurones by determination of action potential trajectories. J. exp. Biol. 99, 207-221.
0300-9629/89 $3.00 + 0.00 © 1989 MaxwellPergamon Macmillanpie
MORPHOLOGY A N D ELECTROPHYSIOLOGY OF N E U R O N S I N N E R V A T I N G THE CILIATED LOCOMOTOR EPITHELIUM IN LYMNAEA STAGNALIS (L.) N. I. SYED* and W. WINLOWt
Department of Physiology, University of Leeds, Leeds LS2 9NQ, UK. Telephone: (0532) 334247
(Received 22 December 1988) AMtract--1. The pedal A-cluster neurons of Lymnaea stagnalis (L.) may either modulate the activities of the locomotor cilia or act as ciliomotoneurons. Their morphology has been gtudied using a modified technique for injection of the fluorescent marker substance Lucifer Yellow and their electrophysiological characteristics have been studied using conventional intracellular recording techniques. 2. The paired pedal A-clusters are each composed of 30 bilaterally symmetrical pairs of neurons lying on the medial faces of the paired pedal ganglia. 3. With respect to the foot, all the A-cluster neurons have unipolar axons which are partially somatotopically organized. 4. Small A-cluster (40-50 #m soma diameter) neurons project ipsilaterally and are weakly electrically coupled to adjacent cells. The medium-sized (50-60#m) and large (60-70#m) cells project both ipsilaterally and contralaterally and make electrical connections with their contralateral homologues as well as their smaller near neighbours. 5. The A-cluster neurons have similar action potential shapes and receive several identifiable synaptic inputs whose sources are unknown. Some of these inputs have widespread synaptic actions in several ganglia whilst the "A-cluster rhythm" (ACR) is entirely restricted to the A-cluster neurons. 6. The identified interneuron VD4 (= the visceral white interneuron, VWI) is known to be involved in respiratory behaviour and also has excitatory actions on all the A-cluster neurons.
INTRODUCTION
In gastropod molluscs such as Lymnaea stagnalis, the locomotory cilia provide the main propulsive force (Kaiser, 1960; Jones, 1975), whilst the animal changes direction by contractions of the musculature of the foot and body wall on contact with objects placed in its path. Rhythmical shell movements are usually associated with locomotion (Winlow and Haydon, 1986), but may play a part in the cardiorespiratory system of the animal rather than being directly linked to locomotion itself. In Lymnaea the pedal ganglia contain many pairs of giant neurons and neuronal clusters (Slade et al., 1981), and a number of locomotor motoneurons have been identified (Winlow and Haydon, 1986). Detailed morphological and electrophysiological studies of the neurons of the pedal ganglia are published elsewhere (Slade et al., 1981; Kyriakides et aL, submitted). In Mytilus edulis pharmacological, histofluorescent and biochemical evidence is consistent with physiological studies which suggest that the lateral cilia of the gill can be excited or depressed by electrical stimulation of the branchial nerve which innervates it (Takahashi, 1971). There is strong evidence suggesting that serotonin (excitatory) and dopamine (inhibitory) are the peripheral neurotransmitters mediating ciliary activity in the Mytilus gill (Stefano et al., *Present address: Department of Medical Physiology, University of Calgary, Health Sciences Centre, 3330 Hospital Drive, NW, Calgary, Alberta, Canada T2N 4NI. tAuthor to whom all correspondence should be addressed.
1988). In Tritonia, paired, serotonin-containing, electrically coupled neurons are known to provide tonic excitation of the pedal cilia (Audesirk, 1978; Audesirk et al., 1979). In contrast, central neurons causing inhibition of stigmatal cilia in the branchial basket of the ascidian Chelyosoma productum have recently been identified and mediate ciliary arrest via direct chemical connections (Arkett, 1987). It is difficult to observe ciliary beating directly in a semi-intact preparation of Lymnaea, but iron filings or carbon particles placed on the foot sole are observed to move, in the absence of muscular contractions, following stimulation of the pedal A-cluster neurons (Syed, unpublished observations). These neurons are known to contain serotonin (Audesirk, 1985; Casey and Winlow, 1985) and injection of serotonin into the foot excites previously quiescent pedal cilia or increases the rate of beat of active cilia (Syed et aL, 1988). The presence of serotonin in axon tracts and varicosities at the base of the ciliated epithelium of the foot of Lymnaea has recently been demonstrated by immunocytochemistry (Caunce et al., 1988; McKenzie et aL, 1987a). Furthermore, electron microscopic studies reveal that axon terminals, containing dense-cored vesicles, make intimate contact with both ciliated cells and mucus-secreting cells of the foot (Caunce et al., 1988; McKenzie et al., 1987a, b). Here we describe the morphological and electrophysiological characteristics of the serotonergic pedal A-cluster neurons of Lymnaea, which innervate the ciliated epithelium of the foot sole.
633
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N.I. S"~D and W. WI~Low MATERIALS AND METHODS
Specimens of Lymnaea stagnalis (L.) were obtained from animal suppliers or from the Leeds-Liverpool canal. These snails were kept at 14-18°C in water obtained from local ponds and fed on lettuce. Isolated brains of Lymnaea were prepared for electrophysiological and morphological studies and maintained in snail saline buffered to pH 7.8 or 7.9, as previously described (Benjamin and Winlow, 1981). Individual neurons were impaled with 10-20Mf~ glass microelectrodes filled with saturated K2SO4 and recordings of neuronal activity were made using conventional techniques. Electrophysiological signals were amplified, displayed and recorded by conventional means (Benjamin and Winlow, 1981). The morphology of identified neurons was studied using the dye Lucifer Yellow CH (Stewart, 1978), with technical modifications which improved both the "fills" and the clearing of the tissue. Projections of Lucifer-filled axons into the tissues of the foot were traced using frozen serial sections prepared on a cryotome.
An improved techniquefor intraneuronal injection of Lucifer Yellow (CH) The injection of neural marker substances can be problematical and Lucifer Yellow is no exception. The difficulties include: an inability to inject sufficient dye; leakage around the point of microelectrode penetration (Fig. la) and poor clearing of the tissue (Fig. lb). A number of small modifications of the technique greatly improved the quality of Lucifer "fills" in the neurons of L. stagnalis (Fig. lc, d). Microelectrodes were filled with 10% Lucifer Yellow in double-distilled and deionized water, which gave a tip resistance of 20-80 Mf~. During impalement a constant holding current of +2 nA was applied to the electrodes through the preamplifier, in order to prevent the leakage of the dye from the microelectrode tip. Upon successful penetration this current was switched off. Dye was then delivered into the cell body by applying a constant - 2 n A current through the preamplifier. Since constant currents often caused a change in the resistance of liquid-filled microelectrodes, - 2 to - 1 0 n A current pulses of 500 ms duration at a frequency of 0.5-1.5 Hz, were superimposed on this constant current for 20-40 min. Microelectrode blocking was further minimized by reversing the constant hyperpolarizing ( - 2 nA) current to a depolarizing current (+2 nA) for 1 or 2 min, once or twice during the course of dye injection. The cells being filled were regularly observed using a mercury lamp and light guide, but not at sufficient intensity to kill the neurons (Miller and Selverston, 1979). When the cells appeared to be adequately filled, dye injection was terminated. During withdrawal of microelectrodes from the cell a +2 nA constant current was again applied. The preparations were left overnight in a refrigerator at 4°C, and were fixed in buffered formalin fixative (formaldehyde, 4%, in 0. ! M sodium phosphate buffered to pH 7.4), for 3 hr. These fixed brains were then dehydrated, defatted and cleared according to the schedule below. Dehydration 1. 50% ethanol 2. 50% ethanol 3. 70% ethanol 4. 70% ethanol 5. 90% ethanol 6. 90% ethanol 7. absolute ethanol 8. absolute ethanol 9. 100% chloroform 10. 100% xylene Tissue clearing 1. Dimethyl sulphoxide 30 rain 2. Methyl salicylate 10 min 3. Methyl methacrylate 10 rain
30 rain 30 min 30 rain 30 min 30 min 30 min 30 min 30 min 30 min 30 min
For reasons that are not apparent, omission of either the dimethyl sulphoxide or methyl salicylate diminished the quality of clearing whilst omission of methyl methacrylate increased the background fluorescence of the tissue. Cleared tissues were mounted in fluorolite on depression slides and observed using a Leitz Diahix 20EB incident fluorescence microscope. Successfully injected neurons were photographed using 400 ASA Kodak Ektachrome slide film and drawn using a camera lucida. Our modified techniques for Lucifer injection and for clearing tissue aided us greatly in resolving fine branches and dendrites, but of course the very finest branches were still beyond the limits of resolution of the light microscope. RESULTS
Morphology Location, nomenclature and projections of A-cluster neurons. The paired, symmetrical, pedal A-cluster neurons o f Lymnaea are located on the medial faces of the largely symmetrical left and right pedal ganglia (Fig. 2 and see Kyriakides et al., in press, Slade et al., 1981). There are 30 individually identifiable neurons in each cluster as shown in Fig. 2a and b). These cells are numbered from posterior to anterior in the sequence 1-30. Cells 1-12 lie on the dorsal surfaces, whilst 13-30 lie on the ventral surfaces of the pedal ganglia (Fig. 2). Within the pedal A-clusters there are three pairs of giant cells, which Slade et al. (1981) designated as L.Pe.D.4, R.Pe.D.4, L.Pe.D.8, R.Pe.D.8, L.Pe.V.2 and R.Pe.V.2. These giant cells are included in the A-cluster, since they have similar morphological and elcctrophysiologlcal properties to them. Their numbers have been retained within the present maps so that the A-cluster cells labelled L.Pe.A4 and R.Pe.A4 correspond to L.Pe.D.4/R.Pe.D.4 etc. (see Fig. 2 for full details). The majority of A-cluster neurons project ipsilaterally through the pedal, cervical and columellar nerve trunks (Table 1) and all these neurons have unipolar axons which undergo complex arborizations in the neuropile. On leaving the neuropile the axons undergo second order branching sending a major projection into one o f the pedal nerves and other major projections into nerve trunks emanating from the pedal ganglia (Table 1). Multiple branching often occurs in Lymnaea nerve trunks and has also been described in Aplysia (Winlow and Kandel, 1976). The seven pairs of larger neurons within the clusters (L./R.Pe.A2, 4, 8, 10, 26, 30), including those previously identified as giant cells, make contralateral projections (Fig. 3a, b), and are electrically coupled to their contralateral homologues. The 23 smaller pairs of cells only project ipsilaterally and are electrically coupled to their ipsilateral near neighbours. To explore the hypothesis that one o f the targets o f the serotonergic A-cluster neurons is the ciliatezi pedal epithelium, large numbers of A-clustor neurons were filled with Lucifer Yellow in several semi-intact preparations. Frozen serial sections o f the h e a d - f o o t complex revealed fine branches of Lucifer-filled axons beneath the ciliated epithelium (Fig. 4a, b).
A-cluster neurons exhibit partial somatotopy and are individually identifiable. The pedal ganglia are clearly bilaterally organized and the A-cluster neurons are, in addition, partially somatotopically
Innervation of a ciliated locomotor epithelium
635
Fig. 1. The effects of modification of the technique for Lucifer Yellow injection on the quality of the preparation. (a) An example of visceral M group cell where dye has leaked into the extracellular spaces (arrows) prior to or just after injection. (b) A-cluster neurons in poorly cleared tissue (arrows). Chloroform and DMSO were not used in processing. (c) Conspicuous dendrites and varicosities around the soma of the giant dopamine-containing cell R.Pe.D.1, prepared according to our improved schedule. Note that there is no leakage of the dye (large arrows), very little background fluorescence and good clearing of the tissue revealing varicosities on the neurites (small arrows). (d) Photomontage of a dorsal view of R.Pe.D. 1, showing its extensive dendritic tree in the visceral and right parietal ganglia (open arrows). The fine dendritic branches are conspicuous as a result of the modifications of the Lucifer technique to give good clearing of ganglia (solid arrows). organized with respect to the locomotor epithelium. Table 1 demonstrates that the positions of A-cluster neurons are somatotopically related to their ipsilateral projections to the foot of Lymnaea, via the three ventrally positioned pedal nerve trunks: anterior, CBP(A) 93/3--J
median and inferior pedal nerves. Neurons of the anterior group of the cluster mainly project out of posterior pedal nerve trunks, whilst those of the posterior group have a higher proportion of branches down the anterior pedal nerve trunks (Fig. 5). The
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P Fig. 2. Location of individually identifiable pedal A-duster neurons on the medial faces of the pedal ganglia. Dorsal (upper) and ventral (lower) views of the ganglia are presented. Diagrams modified from Slade et al. (1981). The pairs of giant neurons originally identified within the clusters as L./R.Pe.V.2, L./R.Pe.D.4 and L./R.Pe.D.8 are now considered to be an integral part of each duster and have been renamed L./R.Pe.A2, L./R.Pe.A4 and L./R.Pe.AS, respectively. There are 30 individually identifiable A-cluster neurons present on the medial faces of the right and left pedal ganglia. Cells 1-12 are present on the dorsal surface while cells 13-30 are situated on the ventral surfaces. Abbreviations are as follows: 18, superior pedal nerve; 19, inferior pedal nerve; 20, medial pedal nerve; 24, cerebropedal connective; 25, pedal-Neural connective; L.Pe.G, left pedal ganglion; R.Pe.G, right pedal ganglion; A, anterior; P, posterior; L, left; R, right. axons of the neurons in the middle of the duster project in similar numbers down all three pairs of ventral nerve trunks. For further details see the legend to Table 1. Axons from posteriorly positioned neuron somata tend to run in tracts towards the anterior nerve trunks and vice versa (Fig. 5) and large numbers of axonal projections also pass to the superior cervical, inferior cervical and columellar nerves. According to de Vlieger (1968), Janse (1974) and Dorsett (1986), the main innervation areas of the nerve trunks of the pedal ganglia of Lymnaea are, from anterior to posterior: superior pedal nerves - medial pedal nerves -inferior pedal nerves - superior cervical nerves-inferior cervical nerves - columellar nerves
nterior foot edge middle of foot posterior of foot tentacle and lips column, skin behind tentacle - - columellar region
Thus, the superior, medial and inferior pedal nerves innervate the ventral surface of the animal whilst the superior and inferior cervical nerves innervate the dorsal body surface and the A-cluster
neurons exhibit no obvious somatotopic organization in these areas. Because of their exact location, size, colour, precise projections and electrophysiological properties it is possible to identify A-cluster neurons individually and to separate them from neurons of the neighbouring B- and E-clusters. Despite the fact that some larger cells of the B- and E-clusters appear similar to A-cluster neurons, their branch patterns and electrophysiological properties are clearly different from those of the pedal A-clusters (Kyriakides et al., in press). The axon projections of individual A-cluster neurons are summarized by Syed (1988). All the A-cluster neurons have extensive fine neurites, particularly within the ipsilateral pedal ganglion and the neuropile. Those cells that have contralateral projections as well, show similar fine varicosities in the contralateral ganglion. Another area of extensive branching is in the neuropile below the somata of putative and identified locomotor motoneurons (L. and R. Pc.D, F, G, clusters) (Winlow and Haydon, 1986; for further details see Kyriakides et aL, in press). A few A-cluster neurons also have fine neurites
Innervation of a ciliated locomotor epithelium
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Neurons of the pedal A-clusters fire irregularly, but often with a patterned discharge (Kyriakides et al., in press). A|l the neurons of the cluster have type 2 action potentials (Winlow et al., 1982), with a pronounced pseudoplateau on the falling phase. The action potential trajectories of these neurons and their responses to injected currents are described in detail elsewhere (Winlow et al., 1982). The majority of A-cluster neurons (L./E.Pe.A1, 3, 5-7, 11-25, 27 and 29) are 4 0 - 5 0 # m in soma diameter, project ipsilaterally and are weakly electrically coupled to adjacent cells (Fig. 6). The medium-sized cells (50-60#m) known as L./R.Pe. Al0, 26, 28 and 30 have contralateral projections and make electrical connections with their contralateral homologues as well as their smaller near neighbours and one another. The giant neurons (60-70 pm) of the cluster also project contralaterally and again make electrical connections with their contralateral homologues, one another and their smaller near neighbours (Fig. 6d, e). The smaller cells appear to be more weakly coupled than the larger ones, but an accurate determination of coupling coefficients has not been carried out. The A-cluster neurons receive a number of identifiable synaptic inputs, some of whose sources are unknown. All cells in the pedal A-clusters receive a regular, common, excitatory input (Fig. 7), which we have termed the "A-cluster rhythm" (ACR). We have used this terminology because this synaptic input, when it occurs, is restricted to the A-cluster neurons and may be expected to have excitatory effects on the pedal cilia. The ACR occurs in both isolated brains and in semi-intact preparations. Input 3 is a wide-acting synaptic input (Winlow and Benjamin, 1976; Benjamin and Winlow, 1981; Syed et al., 1988), which is thought to be involved in respiration (Syed and Winiow, 1988). It has powerful effects on follower cells of the giant dopaminecontaining neuron R.Pe.D. 1 (Winlow et al., 1981). In Fig. 8 we demonstrate that input 3 also has widespread effects and acts on neurons of the pedal and visceral ganglia, including visceral white interneuron (VWI of Benjamin, 1984), best described as visceral dorsal 4 (VD4 of Janse et al., 1985). This cell has widespread actions and is implicated in the control of the pneumostome (Syed and Winlow, 1988). The A-cluster neurons receive delayed inhibition immediately following the effects of input 3 on other neurons (Fig. 8). Input 4 is a compound post-synaptic potential which has actions on visceral, right parietal, and pedal neurons (Winlow and Benjamin, 1976; Winlow, 1979; Winlow et al., in preparation). It occurs irregularly and can produce inhibition for several seconds on all the A-cluster neurons. It has inhibitory effects on the pedal B-, D-, F- and G-clusters and excites the pedal E-clusters (Fig. 9a). Many of these clusters contain locomotor motoneurons (Winlow and Haydon, 1986). In addition input 4 inhibits the giant dopamine-containing neuron R.Pe.D.I (Winlow et al., in preparation). Furthermore, the ring neuron of
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Fig. 3. Camera lucida drawings showing both ipsilateral and contralateral projections of "giant" A-cluster neurons. (a) L.R.Pe.A4. Both these identical cells have similar ipsilateral and contralateral projections. (b) L./R.Pe.A8 neurons projecting both ipsilaterally and contralaterally. Abbreviations as in Fig. 2. the cerebral ganglia (Jansen and ter Maat, 1985), which is known to inhibit the A-cluster neurons, is also excited by input 4 and may further increase A-cluster neuron inhibition. All the A-cluster neurons receive synaptic inputs in common with the motoneurons of the body wall, column and foot. These inputs normally occur during the acquatic and/or terrestrial phases of locomotion (Winlow and Haydon, 1986). Work on semi-intact preparations indicates that, during the aquatic phase of locomotion, which is primarily dependent on ciliary beating, only A-cluster neurons are strongly excited and the neurons of both clusters discharge synchronous bursts of action potentials. The A-cluster neurons are also strongly excited, for a short time when the shell is pulled forward, at the end of each locomotory cycle. During shell movements, contractions of the foot musculature are not usually observed. These inputs during shell movements are also received by body wall and column motoneurons and are often observed in the isolated brain (Fig. 9b and see Syed et al., 1988). In Fig. 8, VD4 of the visceral ganglion was shown to have excitatory actions on the A-cluster neurons (Syed et al., 1988) and is believed to be involved in the respiratory system of Lymnaea (Janse et aL, 1985; Syed and Winlow, 1988). The A-cluster neurons and
the identified cells L./R.Pe.D.2 are strongly excited by VD4 (Fig. 10a), and we believe that both these connections and the inhibitory connections to neurons of the pedal F-cluster and pedal E-cluster are monosynaptic (Fig. 10b) on the bases of constancy of delay and one for one occurrence of spike and p.s.p. at all frequencies so far used. More rigorous evidence for this supposition is not yet available. DISCUSSION
Some of the cells within the pedal A-clusters of Lymnaea were previously identified as giant neurons (Slade et al., 1981), but, in common with other A-cluster neurons, they had no role to play in the muscular contractions associated with locomotion (Winlow and Haydon, 1986). Recent light and electron-microscopical observation (McKenzie et aL, 1987a, b; Caunce et aL, 1988), coupled with neuroethological studies (Syed, 1988; Syed and Winlow, unpublished observations) indicate that these neurons form direct neuroeiliated cell junctions (n.c.c.j.s.) with the eiliated epithelium of the foot sole. The A-cluster neurons are bilaterally symmetrical (Fig. 2), each pair of neurons within the cluster is individually identifiable and all contain serotonin (Casey and Winlow, 1985). The
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Fig. 4. Lucifer-filled axons in the foot of Lymnaea. (a) Frozen serial section of the head-foot complex, 24 hr after the injection of Lucifer Yellow. The dye was injected iontophoretically into several A-cluster neurons and was allowed to travel overnight. Lucifer-filled axons were found in pedal nerves and were tracked deep down into the foot (arrows). The fine branches of the axons filled with dye can easily be seen. (b) A photograph of the frozen section of the foot revealing Lucifer Yellow-filled axon (small arrow) terminating beneath the ciliated epithelium (large arrow). Unfortunately, Lucifer fades rapidly on exposure to UV light and it is particularly difficult to photograph fine axon branches as they approach the ciliated epithelium.
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Fig. 5. Somatotopic organization of ciliomotoneurons. Photomicrograph of ciliomotoneurons showing their somatotopic organization. Cells present at the anterior end of the cluster exit via the posterior pedal nerve trunks and vice versa. Broad arrows: axon projections in anterior (right) and posterior nerve trunks (left). Fine arrows: neurites.
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Fig. 7. The "A-cluster rhythm", a strong excitatory synaptic input which is received only by ciliomotoneurons. (a) Synchronous spontaneous patterned activity of two small A-cluster neurons on opposite sides of the isolated brain. These neurons are not directly connected to one another. (b) The A-cluster rhythm recorded in L./R.Pe.A4 and L./R.Pe.A8 in an isolated brain. The contralateral homologues make direct electrial connections with one another. role of the branches of the A-cluster neurons passing to the cervical and columellar nerves is both puzzling and unclear. We do not know whether they pass to the musculature or to the body surface of the animal and although the A-clusters appear to modulate ciliary activity, their possible involvement in other functions cannot be ruled out. Despite the fact that all the A-cluster neurons receive inputs in c o m m o n with other locomotor
motoneurons, there are a few inputs which are solely confined to the A-cluster neurons. One of these inputs is the "A-cluster rhythm" (Fig. 4) which synchronizes the activities of the A-cluster neurons, presumably aided by the electrical junctions between these cells. Perhaps a central pattern generator underlies this rhythm, but whether any or all of the A-cluster neurons participate in its circuitry is unknown. The A-cluster neurons are excited during both the aquatic
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642
N. I. SYEOand W. WINLOW
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Fig. 9. Synaptic inputs to A-cluster cells and other locomotor motoneurons. (a) The widespread actions of input 4 (IP.4) recorded from isolated brains. Synchronous inhibition (arrows) of R.Pe.A4 (top trace), LPe.A8 (bottom trace) and a R.Pe.F. cell (second trace) coincident with excitation of an L.Pe.E. neuron. The R.Pe.F. and L.Pe.E. neurons are putative locomotor motoneurons (Winlow and Haydon, 1986). (b) Strong excitatory synaptic inputs to a putative ciliomotoneuron (top trace) and putative locomotor motoneurons (traces 2 and 3) of the pedal B- and F-clusters. The F-cluster neuron on the bottom trace receives coincident inhibition (large arrow). Data from semi-intact preparations reveals that these synaptic inputs are received during shell movements. Here, they are recorded from isolated brains and occur in the absence of sensory inputs. The small arrows indicate the occurrence of input 4.
and terrestrial phases of locomotion, and are active during the initiation, maintenance and modulation of locomotor behaviour. A-cluster neurons are in receipt of wide-acting input 3 which has widespread effects in the brain of Lymnaea (Winlow and Benjamin, 1981; Syed et al., 1988). This input is believed to be involved in respiration (Syed and Winlow, 1988) and has delayed inhibitory effects on the putative ciliomotoneurons. It also inhibits other locomotor motoneurons. On the other hand, the interneuron VD4 is also involved in respiratory behaviour and has strong excitatory effects on the putative ciliomotoneurons. During the initial phase of respiratory behaviour, which we believe is caused by input 3 (i.e. opening of the pneumostome and expiration), the locomotor and withdrawal systems are temporarily inhibited, especially the foot and body wall motoneurons and the putative ciliomotoneurons, in order to facilitate the opening of the pneumostome.
VD4 plays an important role in the second phase of respiration by (i) closing the pneumostome; (ii) inhibiting foot and body wall motoneurons to minimize interference with pneumostome movements and (iii) exciting A-cluster neurons presumably in order to resume locomotion. VD4 and input 3 (driving inspiration and expiration, respectively) have antagonistic actions on one another as might be expected (Syed and Winlow, 1988). From these studies we conclude that the serotonergic A-cluster neurons are quite distinct from other pedal neurons on the bases of their morphological and electrophysiological characteristics. On the other hand, they receive inputs in common with other locomotor motoneurons and may play an integral part in several complex behaviours such as locomotion, respiration and egg-laying behaviour. Immunocytochemical and ultrastructural evidence supports our contention that the A-cluster neurons innervate the ciliated, locomotor epithelium of the foot sole, but further evidence is required to
Innervation of a ciliated locomotor epithelium
643
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Fig. 10. The widespread actions of interneuron VD4 ( = V.W.I) on neurons of the pedal ganglia. (a) The excitatory effects of VD4 (second trace) on a ciliomotoneuron (L.Pe,A4, bottom trace) and the identified neurons R.Pe.D.2 (top trace) and L.Pe.D.2 (third trace). Bursts o f spikes in VD4 elicit strong bursts in each of these neurons (arrows). (b) Inhibitory actions of VD4 (second trace) on putative locomotor motoneurons. The L.Pe.F. neurons (top trace and third trace) are at their normal membrane potentials and the L.Pe.E. cell (bottom trace) is hyperpolarized. Clear one-to-one p.s.p.s, in the upper L.Pe.F. neuron and the L.Pe.E. neuron are associated with action potentials in VD4.
644
N.I. SY~D and W. WINLOW
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Acknowledgements--We thank Walter Stewart for supplies of Lucifer Yellow CH, Mr D. Johanson for photographic assistance and Mr D. Harrison for technical assistance. WW was in receipt of an SERC project grant. N.I.S. was supported on an O.R.S. award. REFERENCES
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