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Electrophysiological characteristics of peripheral neurons and their synaptic connections in the intestinal nerve of Helix pomatia L.
Camp. Biochem. Physiol. Vol. 82A, No. 2, pp. 345-353, 1985
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1985 Pergamon Press Ltd
ELECTROPHYSIOLOGICAL CHARACTERISTICS OF PERIPHERAL NEURONS AND THEIR SYNAPTIC CONNECTIONS IN THE INTESTINAL NERVE OF HELIX POMATIA L. AGNES VEHOVSZKY
and
KAROLY
ELEKES
Balaton Limnological Research Institute of the Hungarian Academy of Sciences, H-8237 Tihany, Hungary. Telephone: 80-44-082 (Received
21 January 1985)
Nerve cells in the intestinal nerve of Helix pomutiu were studied, with respect to their localization, light microscopic morphology and electrophysiological properties, in a semi-intact prepara-
Abstract-l.
tion consisting of the ganglionic ring, intestinal nerve trunk and heart-kidney complex. 2. After retrograde labelling with Co 2+ through the cardiac nerve, a population of nerve cell bodies, 30-40 lm in diameter, can be observed around the first bifurcation of the intestinal nerve trunk and along the finer nerve branches. In addition, a few large elongated neuronal perikarya, 80-90 pm in length, are present at the base of the branching point of the intestinal nerve trunk. 3. On the basis of synaptic responses evoked either by the electrical stimulation of the peripheral nerves running to the central ganglia or by the tactile stimulation of the heart and kidney, the nerve cells could be divided into three groups. 4. Blockage of the synaptic transmission in the central nervous system with a high Mg2+, low Ca*+-containing medium decreases or blocks the responses of the peripheral neurons evoked by the stimulation of peripheral nerves and peripheral organs. This observation suggests that the neuronal elements of the CNS are in a presynaptic position to, and may have a facilitatory influence on, the neurons located in the intestinal nerve. 5. The present results support our previous suggestion that the peripheral neurons located in the intestinal nerve trunk may participate in the integrative processes contributing to the control of visceral functions.
INTRODUCTION
The presence of nerve cells in Peripheral nerve trunks and plexuses of molluscs is well known (Bullock and Horridge, 1965; Gorman and Mirolli, 1969; Blackshaw, 1976; Prior and Lipton, 1977). In the siphon nerve of Aplysia three types of neurons have been described by Bailey et al. (1979) based on light and electron microscopy and electrophysiology. These neurons may contribute to local reflex activity at the periphery, similarly to those found by Prior (1972) in Spisula siphon nerve. In addition, an integrative role can also be attributed to the peripheral neurons located in the nerve trunks or in different peripheral organs, partly realizing the regulatory role of the central nervous system and partly co-ordinating the function of the peripheral and central nervous systems (Lukowiak and Peretz, 1977; Lukowiak, 1979; Perlman, 1979). In the central nervous system of Helix several nerve cells have been identified on the basis of electrophysiological and morphological criteria (Sakharov and SalBnki, 1969; Pusztai et al., 1976; Kerkut et al., 1975; Johansen et al., 1982). A neuronal network responsible for the regulation of the activity of visceral organs has also been described (S.-R6zsa and SalBnki, 1973; S.-R6zsa, 1976; Van Wilgenburg and Milligan, 1976; S.-R6zsa and Zhuravlev, 1981). In contrast, there is little information on the possible role of the neurons located in the intestinal nerve innervating the visceral organs (Schlote, 1957; Bull-
ock and Horridge, 1965; Bagust et al., 1979). The participation of these peripheral nerve cells in the electrical activity of the intestinal nerve, partly independent from the CNS, has been suggested by electrophysiological experiments. In the case of isolated heart-intestinal nerve preparations (i.e. disconnected from the CNS), the selective change of some components of the extracellular activity of the nerve can be observed following the stimulation of the heart (S.-R6zsa, 1972). After the stimulation of the mantle surface, a M$+-sensitive action potential pattern could be recorded from the nerve trunk isolated from the CNS (Bagust et al., 1979). On the basis, it was concluded that these extracellularly recorded action potentials were due to the activity of neurons localized in the intestinal nerve. A detailed ultrastructural study (Elekes et al., 1985) has revealed different types of neurons as well as synaptic contacts, axo-axonic and axo-somatic synapses in the intestinal nerve of Helix pomatia.
The aim of the present study was to analyse and describe the electrophysiological characteristics of the nerve cells and their synaptic connections in the intestinal nerve of Helix pomatia, using semi-intact preparations. These neurons could be divided into distinct populations, according to their input from the central nervous system and peripheral organs (heart and kidney). In order to prove the chemical nature of the synaptic input, experiments were also carried out in a high Mg*+, low Ca*+-containing 345
CBP 8212.4
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AGNFSVEHOVSZKY and K~OLY ELEKE~
Fig. 1. Schematic representation of the experimental set-up showing the position of the central nervous system, the intestinal nerve and the heart-kidney complex, indicating the intracellular recordings from one of the central and one of the peripheral nerve cells. S.G., suboesophageal ganglia; i.n., intestinal nerve; c.n., cardiac nerve; a.n., apical nerve; k., kidney; a., heart auricle; v., heart ventricle; DCA I, DCA II, amplifiers for the intracellular recordings; S I, S II, suction electrodes on the anal (I) and the apical (II) nerve.
ing solutions in both parts of the chamber to be applied locally. The composition of the physiological saline used in our experiments was: NaCl 3.5 g, KC1 0.3 g, MgCl,.6H,O 2.4 g, CaCl,. 2H,O 1.47g, TrisCl 0.6 g, in 1000 ml of distilled water, pH = 7.6. For the inhibition of the synaptic transmission a modified physiological solution containing four times the Mg’+ and half the Ca2+ content of the standard medium was applied by perfusion. For the electrophysiological characterization of the nerve cells intracellular recordings were performed by the routine electrophysiological technique. During these experiments the activity of one of the centrally located identified giant neurons (Sakharov and Salanki, 1969; S.-Rozsa, 1976) and one of the peripheral cells located in the intestinal nerve were simultaneously recorded. The central connections of neurons were activated by electrical stimulation applied by a suction electrode to the cut end of one of the (mostly the anal) nerve trunks running to the central ganglia. The duration of each single 4-8 V pulse was 2-4 msec. (These values were about one and a half that of the threshold, depending on the preparation.) In some experiments the apical branch of the intestinal nerve was also stimulated as mentioned above. For the analysis of the peripheral afferent connections of the nerve cells in the intestinal nerve, tactile stimuli were applied to the heart and kidney, by touching their surface with a fine brush. RESULTS
medium, uncoupling the synaptic transmission. The location and light microscopic morphology of the nerve cells in the intestinal nerve was also determined by the retrograde Co’+ tracing method. METHODS Retrograde labeiiing
Adult specimens of Helix pomatia were collected in their natural surroundings during spring and summer, and were used throughout the investigation. Following the isolation of the intestinal nerve trunk with its peripheral branches, three types of experiments were carried out. The distal end of the heart nerve (Experiment 1) and of the apical nerve (Experiment 2) or the proximal end (i.e. which joins to the CNS) of the intestinal nerve trunk (Experiment 3) was placed in a Vaseline ball containing 10% of CoCI, solution in distilled water, and then incubated at 10°C for 48 hr. Development was carried out according to Quicke and Brace (1979) in 75% rubeanic acid in 70% alcohol. After dehydration the preparations were cleared in methylbenzoate and mounted in Canada balsam. Whole mount preparations were examined under the light microscope. Electrophysiological experiments
For the electrophysiological investigations semi-intact preparations (S.-Rozsa, 1976) comprising the circumoesophageal ganglionic ring and the intestinal nerve attached to the heart-kidney complex were used (Fig. 1). The preparation was placed in an experimental chamber having two compartments isolated from each other by a Vaseline layer, so as to allow a connection only through the intestinal nerve. This experimental set-up enabled the bath-
Morphology
of the nerve cells of the peripheral neurons labelled retrogradely through the heart nerve were situated around the first bifurcation of the intestinal nerve (Fig. 2). A group of labelled cell bodies was also found along the heart nerve (Fig. 2c) and some along the apical nerve (Fig. 2a). After labelling through the apical nerve, peripheral cell bodies occurred mainly in the apical nerve and only a few of them were seen in the region of the first bifurcation of the intestinal nerve. Along the finer branches of the intestinal nerve, nerve cells with ovoid or spheroid perikaryon of about 30-40 pm in diameter were Seen (Fig. 2a, e). A cluster of similar neurons was also found to be labelled at the primary bifurcation of the intestinal nerve and at the origin of the heart nerve; some had an axonal process projecting toward the heart (Fig. 2d). In the intestinal nerve a few large neuronal perikarya with a diameter of 80-90 pm along their longer axis were observed. These cells differed markedly from the above-mentioned type by exhibiting a large nucleus and an elongated shape and giving off several axon-like processes. The primary axon of these nerve cells was seen to run along the axis of the nerve trunk, whereas other axon-like processes projected to the surface of the nerve covered by a thick perineurium (Fig. 2b, c). When Co2+ retrograde labelling was performed from the proximal end of the intestinal nerve, labelled
Most
Fig. 2. Distribution and morphology of peripheral neurons near the first bifurcation of the intestinal nerve. a-e, nerve cells labelled by retrograde Co 2+-transport through the cardiac nerve. Scale: 50 pm in each cases. (a) Neurons with ovoid cell bodies in the apical nerve. (b) Large elongated neuron sending off an axonal process (arrow) toward the perineurium. (c) Multipolar neuron in the intestinal nerve giving rise to one of its processes to the perineurium (arrow), the other axon process runs parallel to the longitudinal axis of the intestinal nerve (small arrows). (d) Neurons with elongated or spheroid perikarya projecting with their axon toward the heart (arrow). (e) Group of labelled neuronal perikarya in the distal part of the heart nerve.
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AGNEW VEHOVSZKY and KIROLY ELEKE~
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Fig. 3. Types of peripheral neurons located in the intestinal nerve, classified according to their synaptic input. Recordings after the electrical stimulation (dot) of a central (A,C,D,E,F) or the apical (B) nerve, as well as following the tactile stimulation (arrow) of the heart and kidney. Scale: 20 mV, 10 sec. (A) Response of a silent neuron of Pl type, firingatIerthe stimulation of both the anal nerve and the kidney. (B) Action potentials evoked by the stimulation of the apical nerve or the heart. Q A neuron of Pl type responds with a series of action potentials to the stimulation of the anal nerve and to the touching of the heart. (D) Silent neuron of P2 type responds with an inhibitory postsynaptic potential (IPSP) to the stimulation of the anal nerve, whereas tactile stimulation of the kidney (arrow) evokes increased frequency of action potentials. (E) Response of a pacemaker neuron of type P3: the stimulation of the anal nerve
evokes an action potential and a subsequenttemporalinhibition, whereasthe tactile stimulation interrups the activity of the cell. (F) A neuron of P3 type displays an EPSP atIer stimutatiagthe anal nerve and a long-lasting IPSP (arrow)after stimulating the heart. nerve cells were only occasionally observed. These cell bodies were situated along the marginal part of the nerve beneath the neural sheath. Electrophysiology of the nerve cells (a) General characteristics of the peripheral neurons. Most of the investigated neurons were silent,
displaying a stable resting potential ranging from 25 to 4OmV, or a regular beating rhythm (with 0.2-0.8seC’) without any apparent synaptic input. In only a few of our experiments were irregular firing patterns or spontaneously appearing post-synaptic potentials characteristic for the synaptically driven neurons observed.
A
Fig. 4. Synchronously appearing activity patterns of central and peripheral neurons. Simultaneous recordings on two channels. Scale: 10 mV, 5 sec. (A) In the activity of a neuron locat&! in the right parietal ganglion, periodicaliy appearing depolarizations (upper mgistration) are foliowed by groups of action potentials in the perip@ral .aeur~m (lower registration). (B) EPSPs (upper trace: 1.2,3, . . .) of the RPa2 central neuron are followed by sihgfe action potentials (lower trace: 1’,2’,3’, . . .) of ~tx&mt delay in the activity of a peripheral neuron.
Peripheral neurons in Helix nerve
Fig. 5. Effect of the uncoupling of the central synaptic connections on the synaptic input of the nerve cells. Simultaneous recordings from the central BPa2 neuron (upper trace) and from a peripherally located nerve cell of type Pl (lower trace). Scale: 20 mV, 5 sec. The anatomical position of both neurons is schematically shown in the upper right part of the figure. (A) Control responses following the stimulation of the anal nerve (1, dot), of the heart (2, arrow) and of the kidney (3, arrow). (B) When the ganglionic ring is maintained in a high Mg’+ and low Ca*+containing solution for 10 min, the response of the central neuron decreases, whereas that of the peripheral neuron disappears after the stimulation of the anal nerve (1). Tactile stimulation of the peripheral organs (2,3) evokes a weaker response in the central neuron as compared to the control but the response of the peripheral nerve cell remains unchanged. (C) Rinsing in physiological solution. All the responses given to the central (1) and peripheral (2,3) stimulations reappear; moreover, some facilitation can be seen on both neurons.
When stimulating the peripheral nerve trunk running to the central ganglia, a synaptic response with a delay of 20@-5OOmsec could be evoked from the peripheral cells analysed. Very rarely the latency of the synaptic responses reached l-2 sec. Tactile stimuli on the heart or kidney caused a long-lasting (up to 30 set) change in the activity pattern with a delay of 3-5 sec. This long latency of the synaptic responses after central or peripheral stimuli was found to be characteristic for the peripheral neurons examined. During simultaneous recording from central and peripheral neurons, it could be observed that the response evoked by the stimulation of a central (anal) nerve or a peripheral organ appeared first in the central neuron and then in the peripheral one with a few seconds of delay. On the basis of the input received from the central ganglia and the peripheral organs (heart or kidney), respectively, the neurons in the intestinal nerve could be devided into three populations. Type Pl neurons: Excitatory input both from the central ganglia and the periphery. The majority of the nerve cells analysed were found to belong to this population of neurons. When stim-
ulating the anal nerve, the silent or irregularly firing neurons displayed action potentials (Fig. 3A). A similar reaction could be recorded after the stimulation of the apical nerve at the periphery (Fig. 3B). Tactile stimulation of the heart or kidney evoked a burst-like firing pattern consisting of 10-25 action potentials lasting for 15-20 set (Fig. 3A). Nerve cells firing with a regular frequency responded to the electrical stimulation of the anal nerve trunk with 2-4 action potentials, followed mostly by the inhibition of activity (Fig. 3C). Following the tactile stimulation of the peripheral organs, the frequency of the potential generation increased, and sometimes this phenomenon lasted for more than 1Osec (Fig. 3C). Type P2 neurons: Inhibitory input from the central ganglia and excitatory input from the peripheral organs. From some of the silent or irregularly firing neurons, an inhibitory postsynaptic potential (IPSP) could be recorded after the stimulation of the anal nerve, while the tactile stimulation of the heart and kidney was followed by a series of action potentials (Fig. 3D). In regularly beating neurons the electrical stimulation of the anal nerve inhibited the spontaneous
350
&WFS VEHOVSZKY and KAROLY ELEKES
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A n Fig. 6. Effect of the uncoupling of the central synaptic connections on the synaptic input of the nerve cells. Simultaneous recordings from the central RPal neuron (upper trace) and from a peripherally located nerve cell of type PI (lower trace). Scale: 10 mV, 5 sec. (A) Control response evoked by heart (1) or kidney (2) stimulation (arrow), consisting of a long-lasting hyperpolarixation of the central neuron, and a burst-like group of action potentials displayed by the peripheral neuron. (B) When the centra1 ganglion is maintained in a high Mg’+ and low Ca*+-containing medium for 5 min, the response of the RPal neuron is unchanged, whereas the peripheral neuron fails to show any response following the stimulation of the
kidney. Note the occasional appearance of spontaneous hyperpolarization (2) in the activity of the central neuron. (C) After 15 min in the same experimental medium, the response of both nerve cells becomes similar to the control, when stimulating the kidney. Furthermore, the spontaneous hyperpoiarization recorded from the central neuron is followed by a burst-iike firing of the peripheral neuron (2).
activity. These neurons displayed an increased tiring rate, lasting from 15 to 20 set, in response to the peripheral stimulus from the heart and kidney. Type P3 neurons: Excitatory input from the central ganglia and inhibitory input from the periphery. The spontaneously active neurons responded to the central input (electrical stimulation of the anal nerve) with 2-3 action potentials followed by an inhibition (Fig. 3E), while in the case of silent neurons the response was the appearance of an excitatory postsynaptic potential (EPSP) (Fig. 3F). Tactile stimulation of peripheral organs inhibited the spontaneous firing of the neurons (Fig. 3E) or produced a long lasting (up to 25 set) IPSP (Fig. 3F). (b) Connections between centrat ~per~~er~ MUrons. To clarify whether there is any functional connection between the central nervous system and peripheral neurons located in the intestinal nerve, simultaneous recordings were performed from certain central (mostly previously identified) and peripheral neurons belonging to one of the groups described above. Direct synaptic connections between the central and peripheral neurons were not observed; however, synchronous firing patterns indicative of indirect connections were in some case recorded. Ckcassionally, the postsynaptic potentials recorded from the central neuron were followed by the action
potentials of the peripheral cells with a constant delay (Fig. 4A, B, 6C2). These synchronous activity patterns appeared only temporarly and they also disappeared together (Fig. 4B). In some cases, this correlation of activity could reversibly be blocked by the inhibition of the synaptic transmission in a high Mg’+, low Ca*+-containing medium (Fig. 6B2, C2). (c) Blockage of the synaptic transmission. in order to decide whether the peripheral neurons in the intestinal nerve trunk possess a direct contact with the central nervous system and peripheral organs, or their functional connections to both directions are realized through several synapses interposed, we ap plied a high Mg+, low Ca*+-containing medium to block the chemical synaptic ~ans~ssion. When applying this saline on to central ganglia, the central connections of the peripheral neurons were partially or totally blocked, A decrease or inhibition of responses evoked by the electrical stimulation of the anal nerve was observed in both the central and the peripheral nerve c&s (Figs 5-7). Sometimes, the cessation of the central connections also modulated the efficacy of the response of nerve cells evoked by the tactile stimulation of the peripheral organs; namely, the response of certain central (Fig. 5) and peripheral (Figs 6 and 7) neurons became weaker (Fig. 7) or disappeared (Fig. 6). When applying normal physiological solution
Peripheral neurons in Helix nerve
351
2
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01
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Fig. 7. Simultaneous uncoupling of the synaptic connections in the central nervous system and in the periphery. Simultaneous recordings from the central RPal neuron (upper trace.) and from a peripheral neuron of type P2 (lower trace). Scale: 20mV, 10 sec. (A) Control response following the electrical stimulation of the anal nerve (1, dot) and after the tactile stimulation of the kidney (2, arrow). (B) The central ganglia are placed in a high Mg*+ and low Ca*+-containing solution for 12 min. The response of the central neuron disappears, and that of the peripheral one decreases, after the stimulation of the anal nerve (I); the response of both neurons decreases after stimulation of the kidney (2). (C) Both compartments of the experimental chamber contained the uncoupling solution for 12 min. Following the stimulation of the anal nerve (1) the synaptic response of both neurons (IPSPs) disappears almost entirely. The stimulation of the kidney (2) evokes a response (hyperpolarization) only in the central neuron. (D) Rinsing the central ganglia with the normal saline for 17 min, an IPSP with increased amplitude appears in the peripheral neuron after the stimulation of the anal nerve (1); the hyperpolarization of the central nerve cell and the burst-like series of action potentials in the peripheral neuron are increased (2). again in the experimental chamber containing the ganglionic ring, both the firing rate and the amplitude of the synaptic responses of the central and the peripheral neurons increased (Figs 5 and 7). Upon replacement of the normal physiological solution with a high Mg*+, low Ca*+-containing solution in the compartment of the experimental chamber with the peripheral organs, the abovementioned changes of responses were also observed, however, at a minor and rather temporary scale (Fig. 7). The inhibitory effect of the uncoupling solution
was much stronger when both compartments of the experimental chamber were perfused with it; i.e. chemical transmission was blocked both in the CNS and the periphery (Fig. 7C). The evoked synaptic responses reappeared, sometimes with an increased amplitude, after washing out the central ganglia with the normal physiological solution. DISCUSSION According phologically
to our present findings, a morand electrophysiologically heterogenous
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AGNES
VEHOVSZKY and K~OLY ELEKES
neuronal population occurs in the intestinal nerve trunk of Helix pomatia. Even among nerve cells giving off axonal processes to the same peripheral organ, different types can be distinguished on the basis of their shape and size. The nerve cells with a diameter of about 304Opm located around the primary bifurcation of the intestinal nerve and in the finer branches are similar to those described by Bagust et al. (1979) in the intestinal nerve of Helix aspersa. The large multipolar neurons with elongated somata are comparable to those described as “type I”, whereas the smaller sized neurons with spheroid cell bodies to those described as “type II” in the siphon nerve of Aplysia (Bailey et al., 1979). According to electron microscopic observations (Elekes et al., 1985), the peripheral neurons in the intestinal nerve of Helix pomatia can be devided into at least three different groups. The peripheral neurons investigated also ’ differ from each other as to their electrophysiological characteristics, activity pattern and input from both the central nervous system and peripheral organs. Most of the nerve cells are silent or exhibit regular firing. The series of intracellular action potentials appeared after stimulating the peripheral organs are similar to those recorded extracellularly by S.-Rozsa (1972) from the intestinal nerve in Helix pomatia in an isolated heart-nerve preparation. Burst-like firing patterns evoked by the stimulation of the mantle surface have also been reported by Bagust et al. (1979) in the intestinal nerve trunk of Helix aspersa. After stimulation of the heart and kidney, most of the nerve cells show similar change of activity and appear to receive a similar type of synaptic input from both organs. An extensive convergence of synaptic input on the sensory neurons have been described in the body wall of Lymnaea (Janse, 1974), and in motoneurons in the siphon nerve of Aplysia (Bailey et al., 1979). A convergence of several peripheral inputs of different origin has also been found on the identified sensory neurons of the CNS in Helix pomatia (S-Rbzsa, 1979; S.-Rozsa and Zhuravlev, 198 l), suggesting the overlapping of functionally different networks. For the operation of peripheral input of the neurons in the intestinal nerve the maintenance of the central connections proved, in many cases, to be essential. When the ganglionic ring is placed into a solution with high Mg2+ and low Ca2+ content uncoupling the chemical synaptic transmission, the response of the nerve cells is reduced or disappears even after the stimulation of the peripheral organs. In contrast, the inhibition of the responses is moderate and temporary when only the peripheral organs were bathed in the high Mg2+, low Ca2+-containing medium. As to the time relation of the spontaneously appearing patterns and the synaptically evoked responses, it is remarkable that the potentials appear in the central neuron prior to the activity of the peripheral neuronal counterpart. This observation suggests that the neurons of the central nervous system are in a presynaptic position to the nerve cells located in the intestinal nerve. They might mediate a general facilitation toward the peripheral neurons as described in Aplysia (Perlman, 1979). Nor can direct mono-
synaptic connections between the central and peripheral neurons be ruled out, similarly to the situation between peripheral and central neurons in Spisula (Prior, 1972) and Aplysia (Bailey et al., 1979). In Anisodoris, central neurons are also in a presynaptic position, compared to those located in the peripheral nerve plexus (Gorman and Mirolli, 1969). On the basis of our observations, it is suggested that the peripheral neurons located in the intestinal nerve of Helix pomatia may constitute the efferent part of a neuronal network regulating the function of the heart and kidney. An afferent motor function has been attributed to neurons in the peripheral nerves of Spisula (Prior, 1972), Aplysia (Bailey et al., 1979) and Helix aspersa (Bagust et al., 1979). The connections for integrative processes in the intestinal nerve of Helix pomatia have also been established by electron microscopy (Elekes et al., 1985). Both axo-axonic and axo-somatic synapses, some of them connected to the axon processes of identified central neurons, have been demonstrated. Our present results support the previous suggestion (Elekes et al., 1985) that in analysing the network regulating visceral functions one has to pay attention to the possible integrative role of neuronal elements situated between the central nervous system and peripheral target organs. REFERENCES
Bailey C. H., Castellucci V. F., Koester J. and Kandel E. R. (1979) Cellular studies of peripheral neurons in siphon skin of Aplysia californica. J. Neurophysiol. 42, 530-557. Bagust J., Fitzsimons J. T. R. and Kerkut G. A. (1975) Evidence for integrating activity in the isolated intestinal nerve of Helix aspersa. Comp. Biochem. Physiol. 62A, 397-408. Blackshaw S. E. (1976) Dye injection and electrophysiological mapping of giant neurons in the brain of krchido&. Proc.‘R. .?oc. iond. Ser. B. 142, 393-419. Bullock T. H. and Horridae G. A. (1965) Structure and Function in the Nervous System of bwertebrates. W. H. Freeman, San Francisco. Elekes K., S.-Rozsa K., Vehovszky A., Hernldi L. and Salanki J. (1985) Nerve cells and synaptic connections in the intestinal nerve of the snail, Helix pomotiu L. An ultrastructural and HRP study. Cell Tiss. Res. 239, 61 l-620. Gorman A. L. F. and Mirolli M. (1969) The input-output organization of a pair of giant neurons in the mollusc, Anisodoris nobilis (McFarland). J. exp. Biol. 51, 615-634. Janse C. (1974) A neurophysiological study of the peripheral tactile system of the pond snail Lymnaea stagnuhs (L.). Neth. J. 2001. 24, 93-161. Johansen J., Jensen L. H. and Holm Chr. (1982) Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganglia of Helix pomatia. Comp. Biochem. Physiol. 71A, 283-291. Kerkut G. A., Lambert J. D. C., Gayton R. J., Laker J. E. and Walker R. J. (1975) Mapping of nerve cells in the suboesophageal ganglia of Helix ospersa. Comp. Biochem. Physiol. 5OA, l-25. Lukowiak K. (1979) The development of central nervous system control of the gill withdrawal reflex evoked by siphon stimulation in Aplysia californica. Con. J. Physiol. Pharmacol. 57, 987-997. Lukowiak K. and Peretz B. (1977) The interaction between the central and peripheral nervous systems in the mediation of gill withdrawal reflex behavior in Aplysta. J. camp. Physiol. A, 157, 219-244. Perlman A. J. (1979) Central and peripheral control of
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Prior D. J. (1972) Electronhvsiolonical analysis of nerinhera1 neurones and their possible ;ole in the local reflexes of a mollusc. J. exp. Bioi. 57, 133-145. Prior D. J. and Lipton B. H. (1977) An ultrastructural study of peripheral neurons and associated non-neural structures in the bivalve mollusc, Spisula solidissima. Tissue Cell 9, 233-240.
Pusztai J., Detari L. and Sz.enLsi G. (1976) Electrophysiological and morphological characteristics of neurones of the snail, Helix oomatia L. In Neurobiology of Invertebrates Gastropoda *Brain (Edited by Salanki- J.), pp. 597613. Akademiai Kiado, Budapest. Quicke D. L. J. and Brace R. C. (1979) Differential staining of cobalt- and nickel-filled neurons using rubeanic acid. J. Microsc. 115, 161. Sakharov D. A. and Salanki J. (1969) Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia L. Acta physiol. Acad. Sci. hung. 35, 19-30.
Schlote F.-W. (1957) Submikroskopische Morphologic van Gastropoden-nerven. Z. Zelvorsch. 45, 543-568.
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S.-R&a K. (1972) Characterization of the feed-back system in the heart of Helix pomatia L. Annul. Biol. Tihany 39, 39-48.
S.-R&a K. (1976) Neuronal nerwork underlying the regulation of heart beat in Helix pomatia L. In Neurobiology of Invertebrates, Gastropoda Brain (Edited by Salanki J.), pp. 597613. Akademiai Kiado, Budapest. S.-R&a K. (1979) Analysis of the neural network regulating the cardio-renal system in the central nervous system of Helix pomatia L. Am. Zool. 19, 117-128. S.-R&a K. and Salanki J. (1973) Single neurone responses to tactile stimulation of the heart in the snail, Helix pomatia L. J. camp. Physiol. 84, 267-279. S.-R&a K. and Zhuravlev V. (1981) Central regulation and coordination of the activity of cardio-renal system and pneumostoma in the subodsophageal ganglion of Helix oomatia L. Comn. Biochem. Phvsiol. 69A. 85-98. Van Wilgenburg H. and Milligan- J. V. (1976) Neurons in the suboesophageal ganglion complex of the snail, Helix pomatia, having a functional relation to cardiorespiratory activity. In Neurobiology of Invertebrates, Gastropoda Brain (Edited by Sallnki J.), pp. 615-627. Akadbmiai Kiadb, Budapest.
0300-9629/85 $3.00 + 0.00 0
Printed in Great Britain
1985 Pergamon Press Ltd
ELECTROPHYSIOLOGICAL CHARACTERISTICS OF PERIPHERAL NEURONS AND THEIR SYNAPTIC CONNECTIONS IN THE INTESTINAL NERVE OF HELIX POMATIA L. AGNES VEHOVSZKY
and
KAROLY
ELEKES
Balaton Limnological Research Institute of the Hungarian Academy of Sciences, H-8237 Tihany, Hungary. Telephone: 80-44-082 (Received
21 January 1985)
Nerve cells in the intestinal nerve of Helix pomutiu were studied, with respect to their localization, light microscopic morphology and electrophysiological properties, in a semi-intact prepara-
Abstract-l.
tion consisting of the ganglionic ring, intestinal nerve trunk and heart-kidney complex. 2. After retrograde labelling with Co 2+ through the cardiac nerve, a population of nerve cell bodies, 30-40 lm in diameter, can be observed around the first bifurcation of the intestinal nerve trunk and along the finer nerve branches. In addition, a few large elongated neuronal perikarya, 80-90 pm in length, are present at the base of the branching point of the intestinal nerve trunk. 3. On the basis of synaptic responses evoked either by the electrical stimulation of the peripheral nerves running to the central ganglia or by the tactile stimulation of the heart and kidney, the nerve cells could be divided into three groups. 4. Blockage of the synaptic transmission in the central nervous system with a high Mg2+, low Ca*+-containing medium decreases or blocks the responses of the peripheral neurons evoked by the stimulation of peripheral nerves and peripheral organs. This observation suggests that the neuronal elements of the CNS are in a presynaptic position to, and may have a facilitatory influence on, the neurons located in the intestinal nerve. 5. The present results support our previous suggestion that the peripheral neurons located in the intestinal nerve trunk may participate in the integrative processes contributing to the control of visceral functions.
INTRODUCTION
The presence of nerve cells in Peripheral nerve trunks and plexuses of molluscs is well known (Bullock and Horridge, 1965; Gorman and Mirolli, 1969; Blackshaw, 1976; Prior and Lipton, 1977). In the siphon nerve of Aplysia three types of neurons have been described by Bailey et al. (1979) based on light and electron microscopy and electrophysiology. These neurons may contribute to local reflex activity at the periphery, similarly to those found by Prior (1972) in Spisula siphon nerve. In addition, an integrative role can also be attributed to the peripheral neurons located in the nerve trunks or in different peripheral organs, partly realizing the regulatory role of the central nervous system and partly co-ordinating the function of the peripheral and central nervous systems (Lukowiak and Peretz, 1977; Lukowiak, 1979; Perlman, 1979). In the central nervous system of Helix several nerve cells have been identified on the basis of electrophysiological and morphological criteria (Sakharov and SalBnki, 1969; Pusztai et al., 1976; Kerkut et al., 1975; Johansen et al., 1982). A neuronal network responsible for the regulation of the activity of visceral organs has also been described (S.-R6zsa and SalBnki, 1973; S.-R6zsa, 1976; Van Wilgenburg and Milligan, 1976; S.-R6zsa and Zhuravlev, 1981). In contrast, there is little information on the possible role of the neurons located in the intestinal nerve innervating the visceral organs (Schlote, 1957; Bull-
ock and Horridge, 1965; Bagust et al., 1979). The participation of these peripheral nerve cells in the electrical activity of the intestinal nerve, partly independent from the CNS, has been suggested by electrophysiological experiments. In the case of isolated heart-intestinal nerve preparations (i.e. disconnected from the CNS), the selective change of some components of the extracellular activity of the nerve can be observed following the stimulation of the heart (S.-R6zsa, 1972). After the stimulation of the mantle surface, a M$+-sensitive action potential pattern could be recorded from the nerve trunk isolated from the CNS (Bagust et al., 1979). On the basis, it was concluded that these extracellularly recorded action potentials were due to the activity of neurons localized in the intestinal nerve. A detailed ultrastructural study (Elekes et al., 1985) has revealed different types of neurons as well as synaptic contacts, axo-axonic and axo-somatic synapses in the intestinal nerve of Helix pomatia.
The aim of the present study was to analyse and describe the electrophysiological characteristics of the nerve cells and their synaptic connections in the intestinal nerve of Helix pomatia, using semi-intact preparations. These neurons could be divided into distinct populations, according to their input from the central nervous system and peripheral organs (heart and kidney). In order to prove the chemical nature of the synaptic input, experiments were also carried out in a high Mg*+, low Ca*+-containing 345
CBP 8212.4
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AGNFSVEHOVSZKY and K~OLY ELEKE~
Fig. 1. Schematic representation of the experimental set-up showing the position of the central nervous system, the intestinal nerve and the heart-kidney complex, indicating the intracellular recordings from one of the central and one of the peripheral nerve cells. S.G., suboesophageal ganglia; i.n., intestinal nerve; c.n., cardiac nerve; a.n., apical nerve; k., kidney; a., heart auricle; v., heart ventricle; DCA I, DCA II, amplifiers for the intracellular recordings; S I, S II, suction electrodes on the anal (I) and the apical (II) nerve.
ing solutions in both parts of the chamber to be applied locally. The composition of the physiological saline used in our experiments was: NaCl 3.5 g, KC1 0.3 g, MgCl,.6H,O 2.4 g, CaCl,. 2H,O 1.47g, TrisCl 0.6 g, in 1000 ml of distilled water, pH = 7.6. For the inhibition of the synaptic transmission a modified physiological solution containing four times the Mg’+ and half the Ca2+ content of the standard medium was applied by perfusion. For the electrophysiological characterization of the nerve cells intracellular recordings were performed by the routine electrophysiological technique. During these experiments the activity of one of the centrally located identified giant neurons (Sakharov and Salanki, 1969; S.-Rozsa, 1976) and one of the peripheral cells located in the intestinal nerve were simultaneously recorded. The central connections of neurons were activated by electrical stimulation applied by a suction electrode to the cut end of one of the (mostly the anal) nerve trunks running to the central ganglia. The duration of each single 4-8 V pulse was 2-4 msec. (These values were about one and a half that of the threshold, depending on the preparation.) In some experiments the apical branch of the intestinal nerve was also stimulated as mentioned above. For the analysis of the peripheral afferent connections of the nerve cells in the intestinal nerve, tactile stimuli were applied to the heart and kidney, by touching their surface with a fine brush. RESULTS
medium, uncoupling the synaptic transmission. The location and light microscopic morphology of the nerve cells in the intestinal nerve was also determined by the retrograde Co’+ tracing method. METHODS Retrograde labeiiing
Adult specimens of Helix pomatia were collected in their natural surroundings during spring and summer, and were used throughout the investigation. Following the isolation of the intestinal nerve trunk with its peripheral branches, three types of experiments were carried out. The distal end of the heart nerve (Experiment 1) and of the apical nerve (Experiment 2) or the proximal end (i.e. which joins to the CNS) of the intestinal nerve trunk (Experiment 3) was placed in a Vaseline ball containing 10% of CoCI, solution in distilled water, and then incubated at 10°C for 48 hr. Development was carried out according to Quicke and Brace (1979) in 75% rubeanic acid in 70% alcohol. After dehydration the preparations were cleared in methylbenzoate and mounted in Canada balsam. Whole mount preparations were examined under the light microscope. Electrophysiological experiments
For the electrophysiological investigations semi-intact preparations (S.-Rozsa, 1976) comprising the circumoesophageal ganglionic ring and the intestinal nerve attached to the heart-kidney complex were used (Fig. 1). The preparation was placed in an experimental chamber having two compartments isolated from each other by a Vaseline layer, so as to allow a connection only through the intestinal nerve. This experimental set-up enabled the bath-
Morphology
of the nerve cells of the peripheral neurons labelled retrogradely through the heart nerve were situated around the first bifurcation of the intestinal nerve (Fig. 2). A group of labelled cell bodies was also found along the heart nerve (Fig. 2c) and some along the apical nerve (Fig. 2a). After labelling through the apical nerve, peripheral cell bodies occurred mainly in the apical nerve and only a few of them were seen in the region of the first bifurcation of the intestinal nerve. Along the finer branches of the intestinal nerve, nerve cells with ovoid or spheroid perikaryon of about 30-40 pm in diameter were Seen (Fig. 2a, e). A cluster of similar neurons was also found to be labelled at the primary bifurcation of the intestinal nerve and at the origin of the heart nerve; some had an axonal process projecting toward the heart (Fig. 2d). In the intestinal nerve a few large neuronal perikarya with a diameter of 80-90 pm along their longer axis were observed. These cells differed markedly from the above-mentioned type by exhibiting a large nucleus and an elongated shape and giving off several axon-like processes. The primary axon of these nerve cells was seen to run along the axis of the nerve trunk, whereas other axon-like processes projected to the surface of the nerve covered by a thick perineurium (Fig. 2b, c). When Co2+ retrograde labelling was performed from the proximal end of the intestinal nerve, labelled
Most
Fig. 2. Distribution and morphology of peripheral neurons near the first bifurcation of the intestinal nerve. a-e, nerve cells labelled by retrograde Co 2+-transport through the cardiac nerve. Scale: 50 pm in each cases. (a) Neurons with ovoid cell bodies in the apical nerve. (b) Large elongated neuron sending off an axonal process (arrow) toward the perineurium. (c) Multipolar neuron in the intestinal nerve giving rise to one of its processes to the perineurium (arrow), the other axon process runs parallel to the longitudinal axis of the intestinal nerve (small arrows). (d) Neurons with elongated or spheroid perikarya projecting with their axon toward the heart (arrow). (e) Group of labelled neuronal perikarya in the distal part of the heart nerve.
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AGNEW VEHOVSZKY and KIROLY ELEKE~
348
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..
Fig. 3. Types of peripheral neurons located in the intestinal nerve, classified according to their synaptic input. Recordings after the electrical stimulation (dot) of a central (A,C,D,E,F) or the apical (B) nerve, as well as following the tactile stimulation (arrow) of the heart and kidney. Scale: 20 mV, 10 sec. (A) Response of a silent neuron of Pl type, firingatIerthe stimulation of both the anal nerve and the kidney. (B) Action potentials evoked by the stimulation of the apical nerve or the heart. Q A neuron of Pl type responds with a series of action potentials to the stimulation of the anal nerve and to the touching of the heart. (D) Silent neuron of P2 type responds with an inhibitory postsynaptic potential (IPSP) to the stimulation of the anal nerve, whereas tactile stimulation of the kidney (arrow) evokes increased frequency of action potentials. (E) Response of a pacemaker neuron of type P3: the stimulation of the anal nerve
evokes an action potential and a subsequenttemporalinhibition, whereasthe tactile stimulation interrups the activity of the cell. (F) A neuron of P3 type displays an EPSP atIer stimutatiagthe anal nerve and a long-lasting IPSP (arrow)after stimulating the heart. nerve cells were only occasionally observed. These cell bodies were situated along the marginal part of the nerve beneath the neural sheath. Electrophysiology of the nerve cells (a) General characteristics of the peripheral neurons. Most of the investigated neurons were silent,
displaying a stable resting potential ranging from 25 to 4OmV, or a regular beating rhythm (with 0.2-0.8seC’) without any apparent synaptic input. In only a few of our experiments were irregular firing patterns or spontaneously appearing post-synaptic potentials characteristic for the synaptically driven neurons observed.
A
Fig. 4. Synchronously appearing activity patterns of central and peripheral neurons. Simultaneous recordings on two channels. Scale: 10 mV, 5 sec. (A) In the activity of a neuron locat&! in the right parietal ganglion, periodicaliy appearing depolarizations (upper mgistration) are foliowed by groups of action potentials in the perip@ral .aeur~m (lower registration). (B) EPSPs (upper trace: 1.2,3, . . .) of the RPa2 central neuron are followed by sihgfe action potentials (lower trace: 1’,2’,3’, . . .) of ~tx&mt delay in the activity of a peripheral neuron.
Peripheral neurons in Helix nerve
Fig. 5. Effect of the uncoupling of the central synaptic connections on the synaptic input of the nerve cells. Simultaneous recordings from the central BPa2 neuron (upper trace) and from a peripherally located nerve cell of type Pl (lower trace). Scale: 20 mV, 5 sec. The anatomical position of both neurons is schematically shown in the upper right part of the figure. (A) Control responses following the stimulation of the anal nerve (1, dot), of the heart (2, arrow) and of the kidney (3, arrow). (B) When the ganglionic ring is maintained in a high Mg’+ and low Ca*+containing solution for 10 min, the response of the central neuron decreases, whereas that of the peripheral neuron disappears after the stimulation of the anal nerve (1). Tactile stimulation of the peripheral organs (2,3) evokes a weaker response in the central neuron as compared to the control but the response of the peripheral nerve cell remains unchanged. (C) Rinsing in physiological solution. All the responses given to the central (1) and peripheral (2,3) stimulations reappear; moreover, some facilitation can be seen on both neurons.
When stimulating the peripheral nerve trunk running to the central ganglia, a synaptic response with a delay of 20@-5OOmsec could be evoked from the peripheral cells analysed. Very rarely the latency of the synaptic responses reached l-2 sec. Tactile stimuli on the heart or kidney caused a long-lasting (up to 30 set) change in the activity pattern with a delay of 3-5 sec. This long latency of the synaptic responses after central or peripheral stimuli was found to be characteristic for the peripheral neurons examined. During simultaneous recording from central and peripheral neurons, it could be observed that the response evoked by the stimulation of a central (anal) nerve or a peripheral organ appeared first in the central neuron and then in the peripheral one with a few seconds of delay. On the basis of the input received from the central ganglia and the peripheral organs (heart or kidney), respectively, the neurons in the intestinal nerve could be devided into three populations. Type Pl neurons: Excitatory input both from the central ganglia and the periphery. The majority of the nerve cells analysed were found to belong to this population of neurons. When stim-
ulating the anal nerve, the silent or irregularly firing neurons displayed action potentials (Fig. 3A). A similar reaction could be recorded after the stimulation of the apical nerve at the periphery (Fig. 3B). Tactile stimulation of the heart or kidney evoked a burst-like firing pattern consisting of 10-25 action potentials lasting for 15-20 set (Fig. 3A). Nerve cells firing with a regular frequency responded to the electrical stimulation of the anal nerve trunk with 2-4 action potentials, followed mostly by the inhibition of activity (Fig. 3C). Following the tactile stimulation of the peripheral organs, the frequency of the potential generation increased, and sometimes this phenomenon lasted for more than 1Osec (Fig. 3C). Type P2 neurons: Inhibitory input from the central ganglia and excitatory input from the peripheral organs. From some of the silent or irregularly firing neurons, an inhibitory postsynaptic potential (IPSP) could be recorded after the stimulation of the anal nerve, while the tactile stimulation of the heart and kidney was followed by a series of action potentials (Fig. 3D). In regularly beating neurons the electrical stimulation of the anal nerve inhibited the spontaneous
350
&WFS VEHOVSZKY and KAROLY ELEKES
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A n Fig. 6. Effect of the uncoupling of the central synaptic connections on the synaptic input of the nerve cells. Simultaneous recordings from the central RPal neuron (upper trace) and from a peripherally located nerve cell of type PI (lower trace). Scale: 10 mV, 5 sec. (A) Control response evoked by heart (1) or kidney (2) stimulation (arrow), consisting of a long-lasting hyperpolarixation of the central neuron, and a burst-like group of action potentials displayed by the peripheral neuron. (B) When the centra1 ganglion is maintained in a high Mg’+ and low Ca*+-containing medium for 5 min, the response of the RPal neuron is unchanged, whereas the peripheral neuron fails to show any response following the stimulation of the
kidney. Note the occasional appearance of spontaneous hyperpolarization (2) in the activity of the central neuron. (C) After 15 min in the same experimental medium, the response of both nerve cells becomes similar to the control, when stimulating the kidney. Furthermore, the spontaneous hyperpoiarization recorded from the central neuron is followed by a burst-iike firing of the peripheral neuron (2).
activity. These neurons displayed an increased tiring rate, lasting from 15 to 20 set, in response to the peripheral stimulus from the heart and kidney. Type P3 neurons: Excitatory input from the central ganglia and inhibitory input from the periphery. The spontaneously active neurons responded to the central input (electrical stimulation of the anal nerve) with 2-3 action potentials followed by an inhibition (Fig. 3E), while in the case of silent neurons the response was the appearance of an excitatory postsynaptic potential (EPSP) (Fig. 3F). Tactile stimulation of peripheral organs inhibited the spontaneous firing of the neurons (Fig. 3E) or produced a long lasting (up to 25 set) IPSP (Fig. 3F). (b) Connections between centrat ~per~~er~ MUrons. To clarify whether there is any functional connection between the central nervous system and peripheral neurons located in the intestinal nerve, simultaneous recordings were performed from certain central (mostly previously identified) and peripheral neurons belonging to one of the groups described above. Direct synaptic connections between the central and peripheral neurons were not observed; however, synchronous firing patterns indicative of indirect connections were in some case recorded. Ckcassionally, the postsynaptic potentials recorded from the central neuron were followed by the action
potentials of the peripheral cells with a constant delay (Fig. 4A, B, 6C2). These synchronous activity patterns appeared only temporarly and they also disappeared together (Fig. 4B). In some cases, this correlation of activity could reversibly be blocked by the inhibition of the synaptic transmission in a high Mg’+, low Ca*+-containing medium (Fig. 6B2, C2). (c) Blockage of the synaptic transmission. in order to decide whether the peripheral neurons in the intestinal nerve trunk possess a direct contact with the central nervous system and peripheral organs, or their functional connections to both directions are realized through several synapses interposed, we ap plied a high Mg+, low Ca*+-containing medium to block the chemical synaptic ~ans~ssion. When applying this saline on to central ganglia, the central connections of the peripheral neurons were partially or totally blocked, A decrease or inhibition of responses evoked by the electrical stimulation of the anal nerve was observed in both the central and the peripheral nerve c&s (Figs 5-7). Sometimes, the cessation of the central connections also modulated the efficacy of the response of nerve cells evoked by the tactile stimulation of the peripheral organs; namely, the response of certain central (Fig. 5) and peripheral (Figs 6 and 7) neurons became weaker (Fig. 7) or disappeared (Fig. 6). When applying normal physiological solution
Peripheral neurons in Helix nerve
351
2
.
01
?
2
Fig. 7. Simultaneous uncoupling of the synaptic connections in the central nervous system and in the periphery. Simultaneous recordings from the central RPal neuron (upper trace.) and from a peripheral neuron of type P2 (lower trace). Scale: 20mV, 10 sec. (A) Control response following the electrical stimulation of the anal nerve (1, dot) and after the tactile stimulation of the kidney (2, arrow). (B) The central ganglia are placed in a high Mg*+ and low Ca*+-containing solution for 12 min. The response of the central neuron disappears, and that of the peripheral one decreases, after the stimulation of the anal nerve (I); the response of both neurons decreases after stimulation of the kidney (2). (C) Both compartments of the experimental chamber contained the uncoupling solution for 12 min. Following the stimulation of the anal nerve (1) the synaptic response of both neurons (IPSPs) disappears almost entirely. The stimulation of the kidney (2) evokes a response (hyperpolarization) only in the central neuron. (D) Rinsing the central ganglia with the normal saline for 17 min, an IPSP with increased amplitude appears in the peripheral neuron after the stimulation of the anal nerve (1); the hyperpolarization of the central nerve cell and the burst-like series of action potentials in the peripheral neuron are increased (2). again in the experimental chamber containing the ganglionic ring, both the firing rate and the amplitude of the synaptic responses of the central and the peripheral neurons increased (Figs 5 and 7). Upon replacement of the normal physiological solution with a high Mg*+, low Ca*+-containing solution in the compartment of the experimental chamber with the peripheral organs, the abovementioned changes of responses were also observed, however, at a minor and rather temporary scale (Fig. 7). The inhibitory effect of the uncoupling solution
was much stronger when both compartments of the experimental chamber were perfused with it; i.e. chemical transmission was blocked both in the CNS and the periphery (Fig. 7C). The evoked synaptic responses reappeared, sometimes with an increased amplitude, after washing out the central ganglia with the normal physiological solution. DISCUSSION According phologically
to our present findings, a morand electrophysiologically heterogenous
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VEHOVSZKY and K~OLY ELEKES
neuronal population occurs in the intestinal nerve trunk of Helix pomatia. Even among nerve cells giving off axonal processes to the same peripheral organ, different types can be distinguished on the basis of their shape and size. The nerve cells with a diameter of about 304Opm located around the primary bifurcation of the intestinal nerve and in the finer branches are similar to those described by Bagust et al. (1979) in the intestinal nerve of Helix aspersa. The large multipolar neurons with elongated somata are comparable to those described as “type I”, whereas the smaller sized neurons with spheroid cell bodies to those described as “type II” in the siphon nerve of Aplysia (Bailey et al., 1979). According to electron microscopic observations (Elekes et al., 1985), the peripheral neurons in the intestinal nerve of Helix pomatia can be devided into at least three different groups. The peripheral neurons investigated also ’ differ from each other as to their electrophysiological characteristics, activity pattern and input from both the central nervous system and peripheral organs. Most of the nerve cells are silent or exhibit regular firing. The series of intracellular action potentials appeared after stimulating the peripheral organs are similar to those recorded extracellularly by S.-Rozsa (1972) from the intestinal nerve in Helix pomatia in an isolated heart-nerve preparation. Burst-like firing patterns evoked by the stimulation of the mantle surface have also been reported by Bagust et al. (1979) in the intestinal nerve trunk of Helix aspersa. After stimulation of the heart and kidney, most of the nerve cells show similar change of activity and appear to receive a similar type of synaptic input from both organs. An extensive convergence of synaptic input on the sensory neurons have been described in the body wall of Lymnaea (Janse, 1974), and in motoneurons in the siphon nerve of Aplysia (Bailey et al., 1979). A convergence of several peripheral inputs of different origin has also been found on the identified sensory neurons of the CNS in Helix pomatia (S-Rbzsa, 1979; S.-Rozsa and Zhuravlev, 198 l), suggesting the overlapping of functionally different networks. For the operation of peripheral input of the neurons in the intestinal nerve the maintenance of the central connections proved, in many cases, to be essential. When the ganglionic ring is placed into a solution with high Mg2+ and low Ca2+ content uncoupling the chemical synaptic transmission, the response of the nerve cells is reduced or disappears even after the stimulation of the peripheral organs. In contrast, the inhibition of the responses is moderate and temporary when only the peripheral organs were bathed in the high Mg2+, low Ca2+-containing medium. As to the time relation of the spontaneously appearing patterns and the synaptically evoked responses, it is remarkable that the potentials appear in the central neuron prior to the activity of the peripheral neuronal counterpart. This observation suggests that the neurons of the central nervous system are in a presynaptic position to the nerve cells located in the intestinal nerve. They might mediate a general facilitation toward the peripheral neurons as described in Aplysia (Perlman, 1979). Nor can direct mono-
synaptic connections between the central and peripheral neurons be ruled out, similarly to the situation between peripheral and central neurons in Spisula (Prior, 1972) and Aplysia (Bailey et al., 1979). In Anisodoris, central neurons are also in a presynaptic position, compared to those located in the peripheral nerve plexus (Gorman and Mirolli, 1969). On the basis of our observations, it is suggested that the peripheral neurons located in the intestinal nerve of Helix pomatia may constitute the efferent part of a neuronal network regulating the function of the heart and kidney. An afferent motor function has been attributed to neurons in the peripheral nerves of Spisula (Prior, 1972), Aplysia (Bailey et al., 1979) and Helix aspersa (Bagust et al., 1979). The connections for integrative processes in the intestinal nerve of Helix pomatia have also been established by electron microscopy (Elekes et al., 1985). Both axo-axonic and axo-somatic synapses, some of them connected to the axon processes of identified central neurons, have been demonstrated. Our present results support the previous suggestion (Elekes et al., 1985) that in analysing the network regulating visceral functions one has to pay attention to the possible integrative role of neuronal elements situated between the central nervous system and peripheral target organs. REFERENCES
Bailey C. H., Castellucci V. F., Koester J. and Kandel E. R. (1979) Cellular studies of peripheral neurons in siphon skin of Aplysia californica. J. Neurophysiol. 42, 530-557. Bagust J., Fitzsimons J. T. R. and Kerkut G. A. (1975) Evidence for integrating activity in the isolated intestinal nerve of Helix aspersa. Comp. Biochem. Physiol. 62A, 397-408. Blackshaw S. E. (1976) Dye injection and electrophysiological mapping of giant neurons in the brain of krchido&. Proc.‘R. .?oc. iond. Ser. B. 142, 393-419. Bullock T. H. and Horridae G. A. (1965) Structure and Function in the Nervous System of bwertebrates. W. H. Freeman, San Francisco. Elekes K., S.-Rozsa K., Vehovszky A., Hernldi L. and Salanki J. (1985) Nerve cells and synaptic connections in the intestinal nerve of the snail, Helix pomotiu L. An ultrastructural and HRP study. Cell Tiss. Res. 239, 61 l-620. Gorman A. L. F. and Mirolli M. (1969) The input-output organization of a pair of giant neurons in the mollusc, Anisodoris nobilis (McFarland). J. exp. Biol. 51, 615-634. Janse C. (1974) A neurophysiological study of the peripheral tactile system of the pond snail Lymnaea stagnuhs (L.). Neth. J. 2001. 24, 93-161. Johansen J., Jensen L. H. and Holm Chr. (1982) Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganglia of Helix pomatia. Comp. Biochem. Physiol. 71A, 283-291. Kerkut G. A., Lambert J. D. C., Gayton R. J., Laker J. E. and Walker R. J. (1975) Mapping of nerve cells in the suboesophageal ganglia of Helix ospersa. Comp. Biochem. Physiol. 5OA, l-25. Lukowiak K. (1979) The development of central nervous system control of the gill withdrawal reflex evoked by siphon stimulation in Aplysia californica. Con. J. Physiol. Pharmacol. 57, 987-997. Lukowiak K. and Peretz B. (1977) The interaction between the central and peripheral nervous systems in the mediation of gill withdrawal reflex behavior in Aplysta. J. camp. Physiol. A, 157, 219-244. Perlman A. J. (1979) Central and peripheral control of
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S.-R&a K. (1976) Neuronal nerwork underlying the regulation of heart beat in Helix pomatia L. In Neurobiology of Invertebrates, Gastropoda Brain (Edited by Salanki J.), pp. 597613. Akademiai Kiado, Budapest. S.-R&a K. (1979) Analysis of the neural network regulating the cardio-renal system in the central nervous system of Helix pomatia L. Am. Zool. 19, 117-128. S.-R&a K. and Salanki J. (1973) Single neurone responses to tactile stimulation of the heart in the snail, Helix pomatia L. J. camp. Physiol. 84, 267-279. S.-R&a K. and Zhuravlev V. (1981) Central regulation and coordination of the activity of cardio-renal system and pneumostoma in the subodsophageal ganglion of Helix oomatia L. Comn. Biochem. Phvsiol. 69A. 85-98. Van Wilgenburg H. and Milligan- J. V. (1976) Neurons in the suboesophageal ganglion complex of the snail, Helix pomatia, having a functional relation to cardiorespiratory activity. In Neurobiology of Invertebrates, Gastropoda Brain (Edited by Sallnki J.), pp. 615-627. Akadbmiai Kiadb, Budapest.