- Email: [email protected]
Interneuronal monosynaptic peptidergic contact responsible for the bursting activity generation in the rpal neuron of the snail Helix pomatia L. is axo-axonal
Camp. Biochem. Physiol. Vol. 99A, No. 3, pp. 371-373, 1991
0300-9629/91 53.00 + 0.00 0 1991 Pergamon Press plc
Printed in Great Britain
INTERNEURONAL MONOSYNAPTIC PEPTIDERGIC CONTACT RESPONSIBLE FOR THE BURSTING ACTIVITY GENERATION IN THE RPal NEURON OF THE SNAIL HELIX POMATIA L. IS AXO-AXONAL OLEG N. OSIPENKO* and GY~RGY KEMENES Balaton Limnological Research Institute of the Hungarian Academy of Science, H-8237 Tihany, Hungary. (Received 21 September 1990)
Abstract-I. The connection between an identified intemeuron and RPal bursting neuron from Helix L. brain was studied electrophysiologically and morphologically. 2. Electrical stimulation of intemeuron induced an increase or evoked bursting activity in the RPal neuron. 3. The intemeuron and RPal neurons were stained by CoCl, and NiCl, intracellular injections. 4. The axon from intemeuron had two large branches, the first branch was traced in visceral nerve and the second branch was traced in the direction of RPal axon, which in turn went into visceral nerve. 5. According to these and the literature data, it is proposed that contact between intemeuron and RPal neuron is axo-axonal.
porn&a
INTRODUCI’ION It has been found that connection between an identified interneuron in the visceral ganglion and the Real bursting neuron from the right parietal ganglion (Pin and Gola, 1983; Kononenko and Osipenko, 1986) is monosynaptic and peptidergic (Kononenko and Osipenko, 1987a,b). The morphological and electro-
The present data will describe the axoaxonal nature of the contact between an interneuron and RPal neuron. MATERIALS AND METHODS The experiments were performed on the neurons on the dorsal surface of the sub-oesophageal ganglia of the snail
physiological characteristics of RPal neuron (RPal) neuron in the nomenclature of Sakharov and Salanki (1969), Fl or Br neuron in the nomenclatures of Kerkut et al. (1975) and Pin and Gola (1983) were studied widely. In particular it is known that RPal neuron is a unipolar cell which sends axon into the intestinal nerve (Kerkut et al., 1975; Vehovsky and Sallnki, 1981; Johansen et al., 1982; Elekes et al., 1985). Also earlier Koval et al. (1984) found axosomatic contacts on the RPal neuron soma which are formed by peptidergic terminals of the nonidentified neuron or neurons. These contacts were characterized a gap of up to 100 nM between the terminals and neuronal soma. These results were also suggested by results of electrophysiological experiments (Kononenko and Osipenko, 1986,1987a,b; Osipenko, 1984,1986; Pin and Gola, 1983,1987). They found the peptidergic nature of identified interneuron initiated bursting activity in RPal neuron (new neuropeptide with molecular weight (MW) 2100Da), with a prolonged latency between stimulation of the interneuron and RPal neuronal response (near 2 set). However the location of contact of the identified interneuron responsible for the bursting activity generation in RPal neuron remained unknown. *All correspondence should be addressed to: Oleg N. Osipenko, Department of General Physiology of Nervous System, A. A. Bogomoletz, Institute of Physiology of the Ukrainian Academy of Sciences, Bogomoletz str. 4, Kiev-24, GSP, 252601, U.S.S.R.
Fig. 1. Effect of the interneuron stimulation on the activity of RPal neuron. (A) RPal neuron demonstrates bursting activity. (B) RPal generates bursting activity. Top recordsactivity of the interneuron, lower records-activity of the RPal neuron. A and B are different preparations. 371
312
OLEG N. OSIPENKO and GY~RGY KEMENFS
Helix pomatia L. collected
locally in Kiev and Tihany Peninsula. Physiological saline with the following composition was used (mM): NaCl 80. KC1 4. CaCl, 10. MaCl, 5. alucose 10. Tris-HCl (pH 7.4) 10. - - For electrophysiological experiments conventional methods were used. Glass microelectrodes were filled with 2.5 M KC1 and had resistances of 3-5 MR. Potentials were measured relative to a grounded Ag+-AgCl electrode and were amplified by standard amplifier (Vera, 1974). Neuronal activity was displayed on a Tetronix R5103 N storage oscilloscope (U.S.A.) and recorded on a Gould Brush 2400 (France) pen recorder. For intracellular staining, the neurons were penetrated with a standard microelectrode filled with Co’+ or
Ni2+-lysine complex solution. Metal concentration were diluted to 250 mM. Metal-lysine complexes were prepared according to Fredman (1987). Metals were injected from the recording electrode using conventional iontophoresis (positive current with amplitude of 100 nA and duration 100 ms, frequency 1 Hz during 1 hr). Three to four hours after injection the metals were precipitated by adding 2-3 ml of rubeanic acid (dithiooximide) to the Petri dish containing the brain. A solution of rubeanic acid was also prepared according to Fredman (1987). After allowing l-2 hr for all the cobalt and nickel to react, the cells became brown- or blu+black (accordingly). After precipitating the metals, the brain was fixed in 10% formalin (in physiological saline). After formalin fixation, ethylene glycol monomethyl ether (methyl cellosolve) was used to bleach the neurons. Fixation
Fig. 2. Co2+-staining of both interneuron and RPal neuron. (A) and (B) the same preparation with different extension. V.n.-visceral nerve. Triangles indicate the axon branch of the interneuron which could be in contact with axon of the RPal neuron. Arrows indicate the axon branch of the intemeuron which has been traced in the visceral nerve.
Axo-axonal peptidergic contact was followed by dehydration in an alcohol series and cleared in methyl salicylate. Brains were photographed in EPON.
and stored
RESULTS
Figure 1 presents the results of electrophysiological identification of the connection studied. Stimulation of the inteme~on (top line on Fig. 1A and B) leads to generation of action potentials in the identified interneuron, followed by initiation (Fig. 1A) or an increase (Fig. 1B) in “spontaneous” bursting electrical activity in the RPal neuron (lower line on Fig. lA,B). After identification of the connection between interneuron and RPal neuron, intracellular injections of metal was applied. Figure 2 presents an example of typical Co2+ stained preparation (view from ventral surface of the sub-oesophageal ganglia). The unipolar axon of the RPal neuron goes in the visceral (intestinal) nerve (V.n.). The interneuron axon demonstrates two main branches. First axonal branch also goes in the visceral (intestinal) nerve (arrows) and the second branch goes in the RPal axon direction (triangles). The second branch probably contacts with the axon of the RPal neuron and forms axo-axonal connection, which could be a cause of the “spontaneous” bursting electrical activity generation in the RPal neuron. DISCUSSION
It has been proposed according to the data presented above (Fig. 2) that identified interneuronal contact with RPal neuron (Fig. 1) could be axoaxonal. Our unpublish~ results dealing with Lucifer Yellow and fluore~eine staining of the identified intemeuron suggest this view. We found, in part, that after fluorescent dyes staining, an identified interneuron also shows two main branches of the axon similar to that presented in Fig. 2 for Co*+ staining preparation. In some cases however we also observed low intensity fluorescence in RPal neuron. It is known that fluorescent dyes in some cases could leak across the synaptic contact (Masinovsky and Lloyd, 1985). Axo-axonic contact was also observed between RPal neuron and another V21 neuron which similar to the identified interneuron also goes in the visceral ganglion (Elekes et al., 1985). These contacts were studied by means of electronic microscope, but this very interesting contact between RPal and V21 neurons has not been characterized electrophysiologically. In spite of that these contacts, as proposed, implies that the function of Helix central neurons participating in central integrative processes and in the regulation of peripheral (visceral) organs may be influenced by a synaptic input at the peripheral level (Elekes et al., 1985).
373 REFERENCES
Elekes K., S.-Rosa K., Vehovszky A., Hemadi L. and Salinli J. (1985) Nerve cells and svnautic connections in the intestinal nerve of the snail k&x porn&a L. Cell Tissue Res. 239, 61 l-620. Fredman S. M. (1987) Intracellular staining of neurons with nickel-lysine. J. Neurosci. Meth. 20, 181-194. Johansen J., Jensen L. H. & Holm C. (1982) Morphological and el~trophysiolo~~l mapping of giant neurons in the sub-esophageal ganglia of Helix pom~tia. Camp. Biothem. Physiol. 71A, 283-291. Kerkut G. A., Lambert J. D. C., Gayton R. J., Loker J. E. and Walker R. J. (1975) Mapping of nerve cells in the suboesophageal ganglia of Helix aspersa. Camp. Biochein. Phystol. SOA, l-25. Kononenko N. I. and Osipenko 0. N. (1986) A study of the connection between the intemeuron initiating pacemaker activity in a bursting neuron and the bursting neuron of the snail Helix pomatia. Cell Mol. Neurobiol. 6, 177-l 90. Kononenko N. I. and Gsipenko 0. N. (1987a) Monosynaptic pathway responsible for the bursting activity generation in RPal neuron of the snail H&x pomatia. Neurophysiology (Kiev} 19, 20-28.
Kononenko N. I, and Osinenko 0. N. (1987b) Postsvnaotic mechanisms of initiation of the bursting activity in‘the RPal neuron of snail Helix pomoriu by influences from an intemeuron. Neurophys~oiogy (Kim) 19, 28-36. Koval L. M., Kononenko N. 1. and Skibo G. G. (1984) The axosomatic contacts on the bursting neuron of the snail Helix pomutiu-1. Ultrastructural features of the axosomatic contacts. Ceil Mol. Neurobiol. 4, 3141. Masinovsky B. and Lloyd P. E. (1985) Morphology of two pairs of identified peptidergic neurons -in the buccal ganglia of the mollusc Tritonia diomedea. J. Neurobiol. 16. z7-i9. Osipenko 0. N. (1984) Isolation of a peptide initiating bursting activity in an identified neuron of the snail Heltx pomatia. Neurophysiology (Kiev) 16, 488492.
Osipenko 0, N. (1986) Effect of endogenous peptide initiating bursting activity on isolated “bursting” neuron of snail. N~rophysioiogy (Kiev) 18, 552-554. Pin T., Darbon H., Jacquet G. and Gola M. (1986) Peptidergic modulation of burst firing of neurosecretory Helix nerve cells. Abstracts of the Symposium on Molluscan Neurobiology, pp. 78. 18-22 August, 1986, Amsterdam, Netherlands. Pin T. and Gola M. (1983) Two identified interneurons modulate the firing pattern of pacemaker bursting cells in Helix. Neurosci. Lett. 37, 117-122. Pin T. and Gola M. (1987) Decreased resting membrane conductance as the proximate cause of burst modulation by an identified interneuron in Helix. Bruin Res. 412, 165-168. Sakharov D. A. and Salanki J. (1969) Physiological and pha~acolo~cal identification of neurones in the CNS of Helix pomatia L. Acta Physiol. Acod. Sci. Hung. 35, 19-30.
Vehovszky A. and Salanki J. (1981) Properties of postsynaptic potentials in the bimodal pacemaker neuron of Helix pomatiu L. Neurobiology of invertebrates. Adv. Physiol. Sci., Vol. 23, pp. 55-66. Pergamon Press, Oxford and Akademiai Kiado, Budapest. V&6 M. (1974) Voltage-clamp measurement set-up for investigation of membrane parameters. Ann. Viol. Tihany 41, 111-117.
0300-9629/91 53.00 + 0.00 0 1991 Pergamon Press plc
Printed in Great Britain
INTERNEURONAL MONOSYNAPTIC PEPTIDERGIC CONTACT RESPONSIBLE FOR THE BURSTING ACTIVITY GENERATION IN THE RPal NEURON OF THE SNAIL HELIX POMATIA L. IS AXO-AXONAL OLEG N. OSIPENKO* and GY~RGY KEMENES Balaton Limnological Research Institute of the Hungarian Academy of Science, H-8237 Tihany, Hungary. (Received 21 September 1990)
Abstract-I. The connection between an identified intemeuron and RPal bursting neuron from Helix L. brain was studied electrophysiologically and morphologically. 2. Electrical stimulation of intemeuron induced an increase or evoked bursting activity in the RPal neuron. 3. The intemeuron and RPal neurons were stained by CoCl, and NiCl, intracellular injections. 4. The axon from intemeuron had two large branches, the first branch was traced in visceral nerve and the second branch was traced in the direction of RPal axon, which in turn went into visceral nerve. 5. According to these and the literature data, it is proposed that contact between intemeuron and RPal neuron is axo-axonal.
porn&a
INTRODUCI’ION It has been found that connection between an identified interneuron in the visceral ganglion and the Real bursting neuron from the right parietal ganglion (Pin and Gola, 1983; Kononenko and Osipenko, 1986) is monosynaptic and peptidergic (Kononenko and Osipenko, 1987a,b). The morphological and electro-
The present data will describe the axoaxonal nature of the contact between an interneuron and RPal neuron. MATERIALS AND METHODS The experiments were performed on the neurons on the dorsal surface of the sub-oesophageal ganglia of the snail
physiological characteristics of RPal neuron (RPal) neuron in the nomenclature of Sakharov and Salanki (1969), Fl or Br neuron in the nomenclatures of Kerkut et al. (1975) and Pin and Gola (1983) were studied widely. In particular it is known that RPal neuron is a unipolar cell which sends axon into the intestinal nerve (Kerkut et al., 1975; Vehovsky and Sallnki, 1981; Johansen et al., 1982; Elekes et al., 1985). Also earlier Koval et al. (1984) found axosomatic contacts on the RPal neuron soma which are formed by peptidergic terminals of the nonidentified neuron or neurons. These contacts were characterized a gap of up to 100 nM between the terminals and neuronal soma. These results were also suggested by results of electrophysiological experiments (Kononenko and Osipenko, 1986,1987a,b; Osipenko, 1984,1986; Pin and Gola, 1983,1987). They found the peptidergic nature of identified interneuron initiated bursting activity in RPal neuron (new neuropeptide with molecular weight (MW) 2100Da), with a prolonged latency between stimulation of the interneuron and RPal neuronal response (near 2 set). However the location of contact of the identified interneuron responsible for the bursting activity generation in RPal neuron remained unknown. *All correspondence should be addressed to: Oleg N. Osipenko, Department of General Physiology of Nervous System, A. A. Bogomoletz, Institute of Physiology of the Ukrainian Academy of Sciences, Bogomoletz str. 4, Kiev-24, GSP, 252601, U.S.S.R.
Fig. 1. Effect of the interneuron stimulation on the activity of RPal neuron. (A) RPal neuron demonstrates bursting activity. (B) RPal generates bursting activity. Top recordsactivity of the interneuron, lower records-activity of the RPal neuron. A and B are different preparations. 371
312
OLEG N. OSIPENKO and GY~RGY KEMENFS
Helix pomatia L. collected
locally in Kiev and Tihany Peninsula. Physiological saline with the following composition was used (mM): NaCl 80. KC1 4. CaCl, 10. MaCl, 5. alucose 10. Tris-HCl (pH 7.4) 10. - - For electrophysiological experiments conventional methods were used. Glass microelectrodes were filled with 2.5 M KC1 and had resistances of 3-5 MR. Potentials were measured relative to a grounded Ag+-AgCl electrode and were amplified by standard amplifier (Vera, 1974). Neuronal activity was displayed on a Tetronix R5103 N storage oscilloscope (U.S.A.) and recorded on a Gould Brush 2400 (France) pen recorder. For intracellular staining, the neurons were penetrated with a standard microelectrode filled with Co’+ or
Ni2+-lysine complex solution. Metal concentration were diluted to 250 mM. Metal-lysine complexes were prepared according to Fredman (1987). Metals were injected from the recording electrode using conventional iontophoresis (positive current with amplitude of 100 nA and duration 100 ms, frequency 1 Hz during 1 hr). Three to four hours after injection the metals were precipitated by adding 2-3 ml of rubeanic acid (dithiooximide) to the Petri dish containing the brain. A solution of rubeanic acid was also prepared according to Fredman (1987). After allowing l-2 hr for all the cobalt and nickel to react, the cells became brown- or blu+black (accordingly). After precipitating the metals, the brain was fixed in 10% formalin (in physiological saline). After formalin fixation, ethylene glycol monomethyl ether (methyl cellosolve) was used to bleach the neurons. Fixation
Fig. 2. Co2+-staining of both interneuron and RPal neuron. (A) and (B) the same preparation with different extension. V.n.-visceral nerve. Triangles indicate the axon branch of the interneuron which could be in contact with axon of the RPal neuron. Arrows indicate the axon branch of the intemeuron which has been traced in the visceral nerve.
Axo-axonal peptidergic contact was followed by dehydration in an alcohol series and cleared in methyl salicylate. Brains were photographed in EPON.
and stored
RESULTS
Figure 1 presents the results of electrophysiological identification of the connection studied. Stimulation of the inteme~on (top line on Fig. 1A and B) leads to generation of action potentials in the identified interneuron, followed by initiation (Fig. 1A) or an increase (Fig. 1B) in “spontaneous” bursting electrical activity in the RPal neuron (lower line on Fig. lA,B). After identification of the connection between interneuron and RPal neuron, intracellular injections of metal was applied. Figure 2 presents an example of typical Co2+ stained preparation (view from ventral surface of the sub-oesophageal ganglia). The unipolar axon of the RPal neuron goes in the visceral (intestinal) nerve (V.n.). The interneuron axon demonstrates two main branches. First axonal branch also goes in the visceral (intestinal) nerve (arrows) and the second branch goes in the RPal axon direction (triangles). The second branch probably contacts with the axon of the RPal neuron and forms axo-axonal connection, which could be a cause of the “spontaneous” bursting electrical activity generation in the RPal neuron. DISCUSSION
It has been proposed according to the data presented above (Fig. 2) that identified interneuronal contact with RPal neuron (Fig. 1) could be axoaxonal. Our unpublish~ results dealing with Lucifer Yellow and fluore~eine staining of the identified intemeuron suggest this view. We found, in part, that after fluorescent dyes staining, an identified interneuron also shows two main branches of the axon similar to that presented in Fig. 2 for Co*+ staining preparation. In some cases however we also observed low intensity fluorescence in RPal neuron. It is known that fluorescent dyes in some cases could leak across the synaptic contact (Masinovsky and Lloyd, 1985). Axo-axonic contact was also observed between RPal neuron and another V21 neuron which similar to the identified interneuron also goes in the visceral ganglion (Elekes et al., 1985). These contacts were studied by means of electronic microscope, but this very interesting contact between RPal and V21 neurons has not been characterized electrophysiologically. In spite of that these contacts, as proposed, implies that the function of Helix central neurons participating in central integrative processes and in the regulation of peripheral (visceral) organs may be influenced by a synaptic input at the peripheral level (Elekes et al., 1985).
373 REFERENCES
Elekes K., S.-Rosa K., Vehovszky A., Hemadi L. and Salinli J. (1985) Nerve cells and svnautic connections in the intestinal nerve of the snail k&x porn&a L. Cell Tissue Res. 239, 61 l-620. Fredman S. M. (1987) Intracellular staining of neurons with nickel-lysine. J. Neurosci. Meth. 20, 181-194. Johansen J., Jensen L. H. & Holm C. (1982) Morphological and el~trophysiolo~~l mapping of giant neurons in the sub-esophageal ganglia of Helix pom~tia. Camp. Biothem. Physiol. 71A, 283-291. Kerkut G. A., Lambert J. D. C., Gayton R. J., Loker J. E. and Walker R. J. (1975) Mapping of nerve cells in the suboesophageal ganglia of Helix aspersa. Camp. Biochein. Phystol. SOA, l-25. Kononenko N. I. and Osipenko 0. N. (1986) A study of the connection between the intemeuron initiating pacemaker activity in a bursting neuron and the bursting neuron of the snail Helix pomatia. Cell Mol. Neurobiol. 6, 177-l 90. Kononenko N. I. and Gsipenko 0. N. (1987a) Monosynaptic pathway responsible for the bursting activity generation in RPal neuron of the snail H&x pomatia. Neurophysiology (Kiev} 19, 20-28.
Kononenko N. I, and Osinenko 0. N. (1987b) Postsvnaotic mechanisms of initiation of the bursting activity in‘the RPal neuron of snail Helix pomoriu by influences from an intemeuron. Neurophys~oiogy (Kim) 19, 28-36. Koval L. M., Kononenko N. 1. and Skibo G. G. (1984) The axosomatic contacts on the bursting neuron of the snail Helix pomutiu-1. Ultrastructural features of the axosomatic contacts. Ceil Mol. Neurobiol. 4, 3141. Masinovsky B. and Lloyd P. E. (1985) Morphology of two pairs of identified peptidergic neurons -in the buccal ganglia of the mollusc Tritonia diomedea. J. Neurobiol. 16. z7-i9. Osipenko 0. N. (1984) Isolation of a peptide initiating bursting activity in an identified neuron of the snail Heltx pomatia. Neurophysiology (Kiev) 16, 488492.
Osipenko 0, N. (1986) Effect of endogenous peptide initiating bursting activity on isolated “bursting” neuron of snail. N~rophysioiogy (Kiev) 18, 552-554. Pin T., Darbon H., Jacquet G. and Gola M. (1986) Peptidergic modulation of burst firing of neurosecretory Helix nerve cells. Abstracts of the Symposium on Molluscan Neurobiology, pp. 78. 18-22 August, 1986, Amsterdam, Netherlands. Pin T. and Gola M. (1983) Two identified interneurons modulate the firing pattern of pacemaker bursting cells in Helix. Neurosci. Lett. 37, 117-122. Pin T. and Gola M. (1987) Decreased resting membrane conductance as the proximate cause of burst modulation by an identified interneuron in Helix. Bruin Res. 412, 165-168. Sakharov D. A. and Salanki J. (1969) Physiological and pha~acolo~cal identification of neurones in the CNS of Helix pomatia L. Acta Physiol. Acod. Sci. Hung. 35, 19-30.
Vehovszky A. and Salanki J. (1981) Properties of postsynaptic potentials in the bimodal pacemaker neuron of Helix pomatiu L. Neurobiology of invertebrates. Adv. Physiol. Sci., Vol. 23, pp. 55-66. Pergamon Press, Oxford and Akademiai Kiado, Budapest. V&6 M. (1974) Voltage-clamp measurement set-up for investigation of membrane parameters. Ann. Viol. Tihany 41, 111-117.