Shell movements associated with locomotion of Lymnaea are driven by a central pattern generator

Comp. Biochem. Physiol. Vol. 83A, No. 1, pp. 23-25, 1986

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SHELL M O V E M E N T S A S S O C I A T E D WITH L O C O M O T I O N OF L Y M N A E A A R E D R I V E N BY A C E N T R A L P A T T E R N GENERATOR P. G. HAYDON*t and W. WINLOW Department of Physiology, University of Leeds, Leeds LS2 9JT, UK. Telephone: (0532) 431 751

(Received 14 March 1985) Abstraet--l. Locomotion in Lymnaea is accompanied by rhythmic shell movements, caused by contractions of the columellar muscles. 2. Column motoneurones are located in the pedal ganglia and have ipsilateral peripheral projections to the columellar muscles. 3. The motoneurones are driven by a central pattern generator (CPG) which occasionally becomes spontaneously active in isolated brains. 4. The motoneurones are not synaptically connected to one another, nor do they usually exhibit feedback to the CPG. In 40 preparations only one motoneurone has been found to modify the output of the CPG.

preamplifiers. The fluorescent dye Lucifer Yellow CH (Stewart, 1978) was iontophoretically injected into neurones, as previously described (Haydon and Winlow, 1981).

INTRODUCTION L o c o m o t i o n of the pond snail Lymnaea stagnalis (L.) in water is caused by the rhythmic activity of locom o t o r cilia located on the sole of the foot (Kaiser, 1960). During locomotion in water cyclical movements also occur. These locomotor behaviours have been analysed and the location and activity patterns of motoneurones have been described (Winlow and Haydon, 1985). Motoneurones located in the L./R.Pe.F and D clusters (left and right pedal F and D clusters; see Slade et al., 1981) cause musculature of the body wall and column to contract and, because the column is attached to the shell, they cause the shell to move anteriorly. During aquatic locomotion excitatory synaptic inputs to these motoneurones increase their discharge rate. This in turn causes a contraction of the column and consequently an anterior shell m o v e m e n t (retraction). Subsequently the motoneuronal discharge rate decreases, there is a relaxation of the column and the shell moves posteriorly (protraction). Aquatic locomotion consists of repeated cycles of retraction/protraction. Here we attempt to determine the origin of the locomotor programme and to gain an understanding of the role that the motoneurones play in generating the motor output.

RESULTS AND DISCUSSION Motoneurones innervating the column and body wall of the snail are identifiable on the basis of their position, colour and size (Winlow and Haydon, 1985). In all cases the motoneurones injected with Lucifer Yellow had an ipsilateral axon in one of the nerve trunks down to innervate the column or bQdy wall (Fig. 1), as has already been demonstrated in reduced preparations (Winlow and Haydon, 1985). Intracellular recordings made from the motoneurones reveal that they either have a tonic discharge of action potentials or are silent. Modifications of their discharge frequency are brought about by underlying synaptic activity. In the

MATERIALS AND METHODS

The experiments reported herein were carried out on the isolated CNS of Lymnaea bathed in standard snail saline buffered with HEPES to pH 7.9 (Benjamin and Winlow, 1981). Neurone somata were penetrated with a single glass microelectrode filled with the supernatant from a saturated solution of K2SO4(electrode resistance 10-20 Mf~). Simultaneous intracellular recording and stimulating using single electrodes, were performed using bridge-balance

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Fig. 1. The structure of a putative L.Pe.F motoneurone in the isolated CNS, demonstrated with Lucifer Yellow. This cell has a single axon projection in the columellar nerve trunk which is known to innervate the column of the snail. Abbreviations: 17, columellar nerve; 18, superior pedal nerve; 21, dorsal pedal commissure; 24, cerebro-pedal connective; 25, pedo-pleural connective; L.Pe.g, left pedal ganglion.

*Emma and Leslie Reid Scholar, University of Leeds, 1979-82. tPresent address: Dept of Zoology, University of Iowa, Iowa City, Iowa 52242, USA. 23

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Fig. 2. Feedback from a presumed motoneurone of the L.Pe.F cluster to the central pattern generator (CPG) for aquatic locomotion. (a) In the isolated brain, two L.Pe.F motoneurones receive recurrent periods of excitatory synaptic input characteristic of aquatic locomotion. (b) Depolarization of the silent motoneurone causes a reversible intensification of the bursting discharge of the tonically active cell. In (c) the excitatory input to the two motoneurones can be seen to be due to summating excitatory postsynaptic potentials. Depolarization of the silent neurone (marked by the horizontal bar; spikes not seen) causes a phase advance of the subsequent phase of this synaptic input (the tonically discharging motoneurone is hyperpolarized in c--lower trace). isolated CNS the motoneurones occasionally receive recurrent periods of summating excitatory synaptic input that can cause oscillations in their discharge frequency (Fig. 2). This synaptic activity clearly resembles that seen during aquatic locomotion in motoneurones (Winlow and Haydon, 1985). The motor programme of aquatic locomotion is characterized thus: (i) there are two phases of activity (retraction followed by protraction); (ii) during retraction, locomotor motoneurones receive summating excitatory synaptic input; and (iii) during protraction the excitation subsides and little, if any, inhibitory synaptic activity is present. All of these characteristics are seen in the recordings made from motoneurones in the isolated CNS (Fig. 2a). However, it is difficult to obtain spontaneous synaptic rhythms from motoneurones of the isolated brain, or indeed from semi-isolated preparations. Evidence of spontaneous output from the central pattern generator (CPG) for aquatic locomotion has only been seen in five out of more than 40 preparations. To determine if the motoneurones are components

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Fig. 3. Lack of electrical coupling between two R.Pe.F locomotor motoneurones. Depolarizing and hyperpolarizing currents were injected (a) into the ce!'. of the upper trace and (b) into the cell of the lower trace.

of, or interact with, the CPG, the effects of experimental manipulations of motoneuronal discharge on the locomotor rhythm were investigated. Only one motoneurone was found to modify the output of the CPG. This cell is shown in the upper traces of Fig. 2. Without any artificial polarization this L.Pe.F motoneurone was silent and received sub-threshold excitatory synaptic input during retraction. The same level of excitation in a tonically active motoneurone caused bursting activity (lower traces, Fig. 2). Depolarization of the silent motoneurone to threshold for spike generation caused a reversible intensification of the bursting activity of the tonically active motoneurone (Fig. 2b). This modification did not occur by way of a direct synaptic connection between the putative motoneurones. Presumed locomotor motoneurones have never been found to make either ipsilateral or contralateral synaptic connections with one another (Fig. 3). Injection of a short duration (1.5 sec) supra-threshold depolarizing current pulse into the silent neurone can reset the locomotor rhythm (Fig. 2c), thus showing that this normally silent cell can interact with the C P G for aquatic locomotion when it is induced to spike. However, this cell is not a member of the C P G network for aquatic locomotion since this motor programme could be recorded in the absence of a discharge from the motoneurone. Under circumstances that would cause this cell to discharge (e.g. on receipt of excitation that might arise from sensory neurones) it would provide feedback to the CPG and therefore would be involved in the generation of the m o t o r programme. No other motoneurones have been found to interact with the CPG.

Acknowledgements The authors would like to thank Dr A. V. Holden for criticizing an earlier draft of this manuscript and Dr W. W. Stewart for supplying the Lucifer Yellow.

Locomotion of Lymnaea REFERENCES

Benjamin P. R. and Winlow W. (1981) The distribution of three wide-acting synaptic inputs to identified neurons in the isolated brain of Lymnaea stagnalis (L.). Comp. Biochem. Physiol. 70A, 293-307. Haydon P. G. and Winlow W. (1981) Morphology of the giant dopamine-containing neurone, R.Pe.D.1, in Lymnaea stagnalis revealed by Lucifer Yellow CH. J. exp. Biol. 94, 149 157. Kaiser P. (1960) Die leistungen des flimmerepithels bei der

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fortbewegung der basommatophoran. Z. wiss. Zool. 162, 368-393. Slade C. T., Mills J. and Winlow W. (1981) The neuronal organization of the paired pedal ganglia of Lymnaea stagnalis (L.). Comp. Bioehem. Physiol. 69A, 789-803. Stewart W. W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent napthalamide tracer. Cell 14, 741-759. Winlow W. and Haydon P. G. (1985) A behavioural and neuronal analysis of the locomotory system of Lymnaea stagnalis. Comp. Biochem. Physiol. 83A, 13-21.