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Crustacean cardioactive peptide (CCAP)-related molluscan peptides (M-CCAPs) are potential extrinsic modulators of the buccal feeding network in the pond snail Lymnaea stagnalis
Neuroscience Letters 373 (2005) 200–205
Crustacean cardioactive peptide (CCAP)-related molluscan peptides (M-CCAPs) are potential extrinsic modulators of the buccal feeding network in the pond snail Lymnaea stagnalis ´ Agnes Vehovszkya,∗ , Hans-J¨urgen Agricolab , Christopher J.H. Elliottc , Masahiro Ohtanid , Levente K´arp´atie , L´aszl´o Hern´adia a
b
Department of Experimental Zoology, Balaton Limnological Research Institute, Hungarian Academy of Sciences, P.O. Box 35, 3 Klebelsberg, Tihany H-8237, Hungary Institute of General Zoology, Department of Animal Physiology, Friedrich Schiller University, D-07743 Jena, Germany c Department of Biology, University of York, P.O. Box 373, York YO15YW, UK d Aimoto Laboratory, Institute for Protein Research, Osaka University, Suita, Osaka, Japan e Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, P.O. Box 40, Debrecen H-4012, Hungary Received 31 March 2004; received in revised form 1 October 2004; accepted 4 October 2004
Abstract We combine electrophysiological and immunocytochemical analyses in the snail Lymnaea stagnalis of M-CCAP1 and M-CCAP2, two molluscan peptides with structure similar to crustacean cardioactive peptide CCAP, originally isolated from the snail Helix pomatia. Both M-CCAP peptides (M-CCAP1 and M-CCAP2, 1 M) had an excitatory effect, depolarizing all the identified neurons of the buccal feeding network (including motoneurons: B1, B2, B4 and modulatory interneurons SO, OC: 62 neurons in 33 preparations). Additionally, in 67% of preparations, rhythmic activity (fictive feeding) was recorded with a mean rate of 7 cycles/min. No significant difference in the proportion of preparations showing fictive feeding or mean feeding rate was found between M-CCAP1 and M-CCAP2. The extrinsic feeding modulator, the serotonergic CGC neuron, responds by increase of the spontaneous activity after M-CCAP application (9 of 18 preparations). Crustacean CCAP (1 M) evokes a slight membrane depolarization in 3 out of 8 preparations but never evokes fictive feeding. Immunostaining revealed no cell bodies in the buccal ganglia, but a dense network of CCAP immunopositive fibers arborizing in the buccal neuropil. Many of these fibers originate from a symmetrical pair of CCAP-immunoreactive cerebro-buccal interneurons, which are the most likely candidates for extrinsic modulatory interneurons in the buccal feeding network. Our data are the first results suggesting that M-CCAP-peptides exist as effective modulators in mollusc. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Central pattern generator; CPG; Feeding; Neuromodulation; Mollusc; Electrophysiology; Immunocytochemistry
1. Introduction Crustacean cardioactive peptide (CCAP) is a potent cardioexcitator, originally identified in the pericardial organs [21] then in the nervous system of the crab, Carcinus maenas [4,5,22]. This peptide is a modulator in many arthropods, affecting the pyloric rhythm in crabs [24], oviduct contrac∗
Corresponding author. Tel.: +36 87 448 244; fax: +36 87 448 006. ´ Vehovszky). E-mail address: [email protected] (A.
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.10.020
tion in Locusta [6], ecdysis [9,20] and the circadian clock in Drosophila [16] (for review see [4]). A peptide family related to CCAP, called molluscan CCAPs (M-CCAPs) was isolated from the central nervous system of the gastropod Helix pomatia [17,19]. The crustacean CCAP and the M-CCAP peptides show a high sequence homology, with a disulphide bridge as a common structural feature (Table 1). Additionally, applying the antibody raised against the crustacean CCAP showed specific CCAP-immunoreactivity in the CNS of two gastropods, H. pomatia and Lymnaea stagnalis [11].
´ Vehovszky et al. / Neuroscience Letters 373 (2005) 200–205 A. Table 1 Structure of the crustacean cardioactive peptide (CCAP) isolated from Carcinus [21] and the two fractions of molluscan CCAP-like peptides (M-CCAP1 and M-CCAP2) [17] found to be effective on Lymnaea neurons studied
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The physiological role of CCAP and M-CCAP peptides was assessed using electrophysiological recordings from identified motoneurons and interneurons in the isolated CNS. Double labeling (Lucifer Yellow backfilling of the cerebrobuccal connective and CCAP immunostaining) on the buccal and cerebral ganglia identified the neurons likely to release M-CCAP.
2. Materials and methods
The neuronal effects of CCAP have been tested on two CPG networks in crustaceans, one which drives the crustacean swimmeret systems [18] and one which drives the pyloric rhythm [24]. In both cases CCAP affected both the premotor circuit and the motor neurons of the CPG network. No data are available, however, on the physiological significance of the CCAP peptide family in the molluscan (snail) nervous system. In L. stagnalis CCAP immunoreactive cell bodies can be seen throughout the whole CNS, except the paired buccal ganglia. The buccal neuropil contains a network of dense CCAP-immunoreactive fibres as do the buccal musculature and the esophagus (Hern´adi et al., in preparation). The paired buccal ganglia contain the main elements of the central feeding network including the CPG system, while the buccal muscles and oesophagus are the main structures executing cyclic feeding movements [3,7]. This suggests that CCAP-related peptides play a role in generation and modulation of rhythmic feeding movements. To obtain information on the possible physiological significance of CCAP and M-CCAP peptides in feeding, we studied the neuronal effects of these peptides on identified members of the central feeding network of the pond snail L. stagnalis. In Lymnaea, the buccal ganglia contain the central pattern generating (CPG) interneurons for feeding and modulatory interneurons, as well as motor neurons which innervate the feeding muscles [7]. The paired cerebral ganglia contain the extrinsic feeding modulator, the giant serotonergic interneuron (CGC). In the isolated CNS preparation members of the feeding network (both interneurons and motoneurons) display a characteristic pattern of rhythmic synaptic inputs and action potentials called fictive feeding, which can be correlated to particular phases of the cyclic motor activity of the intact feeding animal: radula protraction (N1), rasp (N2) and swallowing (N3) [3,23,26].
Adult specimens of the pond snail L. stagnalis were sampled in spring, summer and autumn. The central nervous system was isolated and neurons identified and impaled as described previously [23]. The CCAP-peptide, M-CCAP1 and M-CCAP2 peptides were synthesized by the authors; CCAP by L. K´arp´ati and the M-CCAP peptides by M. Ohtani using the published structure and amino acid sequences [17,21]. Peptides were diluted before use into standard saline [23] to provide final concentrations from 0.1 M to 0.1 mM, and pumped through the perfusion system. For immunocytochemistry the polyclonal antibody (raised against CCAP in rabbit) was produced by Agricola et al. [1]. Immunocytochemistry was carried out on both whole-mount CNS preparations and cryostat sections [10], and visualized with the avidin/biotin peroxidase immunostaining method [11]. Samples were dehydrated and embedded into Canada balsam. The specificity of the antibody has been tested in two ways. 1. The antibody was omitted and replaced with bovine serum albumin during the immunostaining procedure; no immunostaining was observed. 2. The CCAP antibody diluted 1:10,000 was preabsorbed with M-CCAP forms (M-CCAP1 and M-CCAP2) at 1.5 mg/ml (approximately 1 mM) concentration at 4 o C overnight and then used for immunostaining. We failed to obtain immunoreactivity on sections applying the preabsorbed antibody with either of the two forms of M-CCAP. This shows that the CCAP antibody crossreacted with the applied M-CCAP forms indicating that the antibody specifically stains those neurons which contain forms of M-CCAP. To visualize cerebro-buccal interneurons (cerebral neurons which send axons to the buccal ganglia via the cerebrobuccal connectives, cbc), Lucifer Yellow dye (LY) was used as retrograde tracer [10,13]. To determine the CCAP containing neurons among the cerebro-buccal interneurons double immunostaining was used. The LY-backfilled preparations were used for CCAP immunostaining as whole mount and cryostat sections. The application of TRIC-labeled anti rabbit secondary antibody (diluted 1:50) to visualize bound CCAP antibody gave only weak CCAP staining with high background either in wholemount preparations or sections. This enabled us to determine the double stained neurons but was
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Fig. 1. Responses of buccal feeding motoneurons after M-CCAP application. (A) M-CCAP2 (0.1 M) evokes strong excitatory effect on B2 motoneuron leading to action potential firing (lower trace) while the B4 motoneuron is slightly depolarized followed by short bursts of excitatory postsynaptic potentials (upper trace). (Bi) M-CCAP2 (1 M) evokes rhythmic intracellular activity (fictive feeding) on B5 (upper trace) and B4 (lower trace) motoneurons. (Bii) Part of the record, indicated by a box, taken during fictive feeding, showing the bursts and synaptic inputs which correspond to feeding cycles in detail: slow inhibition on B5 and B4 motoneuron (N1, radula protraction), fast inhibition on both cells (N2, rasp), firing in burst (N3, swallowing).
not suitable for photographic documentation. Therefore, the LY-backfilled preparations were sectioned and the cryostat sections were used for CCAP immunostaining [11] applying the very sensitive avidin/biotin peroxidase method [12] to visualize immunoreactivity. LY stained and CCAP-stained neurons were photographed applying double exposition in bright field and FITC filters.
3. Results 3.1. Physiological effects of CCAP-related peptides on identified feeding neurons Preliminary experiments showed that two peptides, MCCAP1 and M-CCAP2 (Table 1) produced responses on Lymnaea neurons. We therefore tested these two M-CCAP peptides and compared them with crustacean CCAP peptide. The threshold is 0.1 M, with consistent responses at 1 M. Both M-CCAP1 and M-CCAP2 elicit a slow depolarization on all the 62 buccal neurons tested in 33 preparations (Figs. 1–3, representative data from both peptides shown). In 67% of these preparations, this depolarization is followed by rhythmic synaptic inputs and a series of bursts (Fig. 1Bi, Fig. 2 and Fig. 3A and B). Analysis of the pattern of synaptic inputs (Fig. 1Bii, Fig. 2B and Fig. 3Aii and Bii) shows that the full feeding sequence (N1 protraction, N2 rasp and N3 swallow [3]) is occurring, showing that the central pattern generator is fully active. The mean fictive feeding rate with 1 M M-CCAP1 and M-CCAP2 was the same, 6.9 ± 0.7 and 7.0 ± 0.9 cycles/min, respectively, though these preparations showed no initial feeding activity. There is no significant difference between M-CCAP1 and M-CCAP2 in the proportion
Fig. 2. Both feeding motoneurons and buccal modulatory interneurons respond by fictive feeding in 1 M M-CCAP. (A) The modulatory SO (upper trace) and the B1 motoneuron (lower trace) are depolarized leading to bursting activity pattern. After M-CCAP1 application the B1 motoneuron starts bursting while SO receives N2 phase inhibitory inputs. (Bi) Expanded from the first box in A shows the N2 inputs. (Bii) expanded from the second box shows full rhythmic bursting of fictive feeding (N1, N2 and N3 marking individual feeding cycles). (C) Recording from B3 and B4 motoneurons (upper and lower traces, respectively) shows rhythmic bursts during fictive feeding in response to M-CCAP2 while the octopaminergic modulator interneuron OC (middle trace) receives pattern inhibitory and excitatory synaptic inputs without firing activity.
2 = 3.03, of preparations responding with fictive feeding (χ1df P > 0.05). Two buccal modulatory interneurons, SO (N = 3) and OC (N = 2) also respond to M-CCAP peptides (Fig. 2). Rhythmic inputs of fictive feeding occur before the slow depolarization of SO, caused by direct membrane effect of M-CCAP1, is enough to fire action potentials (Fig. 2A, Bi), and these occur in the rhythmic bursts typical of the feeding pattern (Fig. 2Bii). The other buccal modulatory neuron, OC was also depolarized by M-CCAP2, but not always enough to fire action potentials during fictive feeding (Fig. 2C). The cerebral serotonergic neuron, the CGC, responds to 1 M M-CCAP1 application (Fig. 3A and B) by an increased firing rate in 9 out of 18 preparations. On other 9 preparations there is no increase in CGC firing despite the depolarization and rhythmic inputs in the buccal motoneuron (Fig. 3A). To compare the effect of M-CCAP1 and M-CCAP2 with crustacean CCAP, we tested the peptides on the same eight preparations. The molluscan peptides evoked just depolar-
´ Vehovszky et al. / Neuroscience Letters 373 (2005) 200–205 A.
Fig. 3. Intracellular responses of the cerebral modulatory CGC neuron and identified buccal feeding neurons after application of CCAP-like peptide. (Ai) After application of 1 M M-CCAP1 the firing pattern of the serotonergic cerebral giant cell (CGC) does not change, while the buccal B1 motoneuron receives regular (protraction phase) depolarizing inputs from the feeding network. (Aii) Expansion of (Ai) showing series of compound excitatory synaptic potentials on B1 motoneuron. (Bi) M-CCAP2 (1 M) evokes higher firing frequency on the CGC interneuron and rhythmic pattern of synaptic inputs on B4 motoneuron. (Bii) Detail of record (Bi) showing CGC spikes (upper trace) which did not correlate with the regular pattern of inhibitory and excitatory inputs on B4 motoneuron. C. Application the crustacean CCAP (1 M) on the same preparation as shown in B, has no effect on either the CGC (upper trace) or B4 motoneuron (lower trace). (Cii) Detail of record (Ci).
ization in two preparations, while the other six showed the series of synaptic inputs typical of fictive feeding (Fig. 3B). The crustacean peptide, CCAP, however, produced no visible response in five preparations, and a much smaller depolarization in three preparations (Fig. 3C) and never evoked fictive 2 = 9.6 P < 0.01. feeding. This difference is significant: χ1df 3.2. Origin of the CCAP immunoreactive neuronal elements in the buccal ganglia Immunolabelling shows no CCAP-positive cell bodies in the buccal ganglia, but densely stained thick fibers in the cerebro-buccal connective (cbc), which enter the buccal ganglia and run to the contralateral ganglion and richly arborise in their neuropil (Fig. 4Ai and Aii). The richly arborized fine varicose CCAP-ir fibers are confined to the neuropil area and only rarely run to the cell body layer (Fig. 4Aii). Backfilling the cbc with Lucifer Yellow labels several small groups of neurons in the cerebral ganglia on both the dorsal (Fig. 4Ci) and ventral surface (Fig. 4Bi). The CCAP immunostaining of LY-backfilled cerebral ganglia visualized only two double (CCAP and LY) labeled neurons, located on the dorsomarginal area of the cerebral ganglion (Fig. 4Cii and Ciii). Double staining also revealed that CCAP-ir fibers surround
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Fig. 4. CCAP immunopositive elements in the Lymnaea central nervous system. (Ai) In the buccal ganglion no CCAP-immunopositive cell bodies only immunopositive fibers are seen (Ai) which enter from the cerebral ganglion (long arrows) through the cerebro-buccal connective (cbc). (Aii) Thick axonal branches (short arrows) run to the contralateral ganglia through the buccal commissure (bc) and arborize in the neuropil area in both ganglia. Whole mount preparations. lbn: laterobuccal nerve. B Whole mount of the cerebral ganglion after LY-backfilling the cbc. The majority of LY-labeled cerebro-buccal interneurons are located on the ventral surface of the cerebral ganglion (Bi). The labeled neurons send processes to the contralateral side through the cerebral commissure (cc) and to the lip nerves (mln, uln). The cerebral giant cell (CGC) shows pale LY fluorescence (Bi). CCAP stained fibers surround the axonal process of the CGC) (long arrows) suggesting direct connections (LY fluorescence was faded in the cell body) (Bii). Asterisk shows small LY-labeled cerebro-buccal interneurons. uln: upper lip nerve; mln: medial lip nerve. (C) Higher magnification of labelled neurons at the dorso-marginal area of the cerebral ganglion. LY-backfilling of the cbc labels three cerebro-buccal interneurons (1,2,3, Ci). Among them two neurons, the neuron 2 (Cii and Ciii) and neuron 3 (Ciii) show LY and CCAP immunostaining after the application of double staining on consecutive sections. Calibration bars: 100 m.
the axonal process of the giant cerebro-buccal interneuron the CGC (Fig. 4Bii)
4. Discussion 4.1. Molluscan CCAP-related peptides evoke fictive feeding in buccal feeding neurons Electrophysiological recordings from identified buccal feeding neurons in Lymnaea show the molluscan CCAP-like peptides (M-CCAP1 and M-CCAP2) modulate the spontaneous activity of all feeding neurons tested, and in 67% of the preparations evoked the pattern of fictive feeding on iden-
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tified buccal feeding neurons. The effectiveness of both peptides is not significantly different, and so we use M-CCAP to refer to them jointly. Physiologically, they are different from the crustacean CCAP peptide, which evoked a slight membrane depolarization in 31% of the preparations, but never fictive feeding. Thus, while the M-CCAP and CCAP peptides were originally isolated from Helix and crustacea, respectively, they are recognized by Lymnaea neurons, with significantly higher affinity to M-CCAP peptides. This, together with the M-CCAP pre-absorption tests and the fact that CCAP-immunoreactive neurons show similar numbers and distribution in Helix and Lymnaea (Hern´adi et al., in preparation), suggests the presence of M-CCAP-like molecules in both species, although the exact structure of the CCAP-like peptides in Lymnaea is yet unknown. M-CCAP1 and M-CCAP2 peptides evoke fictive feeding in 67% of preparations by stimulating the buccal feeding CPG system. The feeding rate induced by 1 M M-CCAP, 7 cycles/ min is lower than the feeding rate of the intact snail 16–18 cycles/min [14] or from the isolated CNS after stimulating modulatory interneurons (12.9 ± 1.9 cycles/min for SO stimulation and 11.8 ± 1.3 cycles/min after OC stimulation [23]). In isolated CNS preparations, fictive feeding may be evoked by intracellular current injection into modulatory neurons, e.g. SO, N1L, OC interneurons [3,26]. However, recording from SO during M-CCAP application revealed that fictive feeding is not caused by increased SO activity as M-CCAP activates the central pattern generator before the SO interneuron. Similarly, OC activity is not necessary for M-CCAP to drive fictive feeding. We conclude that the buccal CPG network is the most likely direct target for the M-CCAP, and the modulatory SO and OC interneurons are excited by their connections from the central pattern generator [3,23]. Double staining revealed CCAP immunoreactive fibers surrounding the initial axonal segment of another cerebral modulatory interneuron, the CGC, which contributes to the responsiveness of the feeding system [7,25]. Although some preparations showed that the CGC fired faster in M-CCAP we concluded that the M-CCAP effect on the buccal network did not depend on activation of the CGC, because other preparations showed activation of the central pattern generator without any change in CGC activity. Our observations suggest that the main effect of the MCCAP neurons is on the central pattern generator and motoneurons: this is different from the effect of FMRFamide, released by a pleural interneuron onto the feeding system, where the targets include all of the modulatory interneurons in addition to the central pattern generator interneurons and motoneurons [2]. Backfilling the cerebro-buccal connective reveals several neurons [18–22] in the cerebral ganglia, including three cells on the dorso-lateral surface. Only two of these show CCAP immunostaining; the transmitter used by the third cell is unknown. Another cerebro-buccal interneuron, located ven-
trally in the cerebral ganglia, containing APGWamide, is previously identified in Lymnaea to have a similar modulatory effect on the buccal feeding system [15]. The CCAP has closely similar effects in the crab stomatogastric ganglion [24] and crayfish swimmeret system [18]. In both cases the CCAP peptide activates the premotor circuit (the CPG network), and also directly modulates the motoneurons. In Lymnaea the pericardial organ and the connective sheath of the CNS including the buccal ganglia are also densely innervated by CCAP-stained fibers (Hern´adi et al., in preparation) therefore, we suggest that in vivo, the M-CCAP peptides reach the buccal neurons not only as neuromodulators liberated in the buccal neuropil close to the axons of the buccal neurons but also as neurohormone through the circulating haemolymph which is a very important rout of CCAP in arthropods [4,8]. Although we have shown consistent responses, which differ between the peptides applied, we still need to take the physiological relevance of these findings with caution, as the precise actions of the Lymnaea CCAP orthologous sequences have not been identified or functionally tested.
Acknowledgements The authors wish to thank for the technical assistance of Ms. Henriette Szab´o during the electrophysiological experiments and Mr. Boldizs´ar Bal´azs for the digital images. This work was supported by the Hungarian Academy of Sciences OTKA grant (T037389), DAAD (Germany), and the Wellcome Trust CRIG Programme.
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Crustacean cardioactive peptide (CCAP)-related molluscan peptides (M-CCAPs) are potential extrinsic modulators of the buccal feeding network in the pond snail Lymnaea stagnalis ´ Agnes Vehovszkya,∗ , Hans-J¨urgen Agricolab , Christopher J.H. Elliottc , Masahiro Ohtanid , Levente K´arp´atie , L´aszl´o Hern´adia a
b
Department of Experimental Zoology, Balaton Limnological Research Institute, Hungarian Academy of Sciences, P.O. Box 35, 3 Klebelsberg, Tihany H-8237, Hungary Institute of General Zoology, Department of Animal Physiology, Friedrich Schiller University, D-07743 Jena, Germany c Department of Biology, University of York, P.O. Box 373, York YO15YW, UK d Aimoto Laboratory, Institute for Protein Research, Osaka University, Suita, Osaka, Japan e Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, P.O. Box 40, Debrecen H-4012, Hungary Received 31 March 2004; received in revised form 1 October 2004; accepted 4 October 2004
Abstract We combine electrophysiological and immunocytochemical analyses in the snail Lymnaea stagnalis of M-CCAP1 and M-CCAP2, two molluscan peptides with structure similar to crustacean cardioactive peptide CCAP, originally isolated from the snail Helix pomatia. Both M-CCAP peptides (M-CCAP1 and M-CCAP2, 1 M) had an excitatory effect, depolarizing all the identified neurons of the buccal feeding network (including motoneurons: B1, B2, B4 and modulatory interneurons SO, OC: 62 neurons in 33 preparations). Additionally, in 67% of preparations, rhythmic activity (fictive feeding) was recorded with a mean rate of 7 cycles/min. No significant difference in the proportion of preparations showing fictive feeding or mean feeding rate was found between M-CCAP1 and M-CCAP2. The extrinsic feeding modulator, the serotonergic CGC neuron, responds by increase of the spontaneous activity after M-CCAP application (9 of 18 preparations). Crustacean CCAP (1 M) evokes a slight membrane depolarization in 3 out of 8 preparations but never evokes fictive feeding. Immunostaining revealed no cell bodies in the buccal ganglia, but a dense network of CCAP immunopositive fibers arborizing in the buccal neuropil. Many of these fibers originate from a symmetrical pair of CCAP-immunoreactive cerebro-buccal interneurons, which are the most likely candidates for extrinsic modulatory interneurons in the buccal feeding network. Our data are the first results suggesting that M-CCAP-peptides exist as effective modulators in mollusc. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Central pattern generator; CPG; Feeding; Neuromodulation; Mollusc; Electrophysiology; Immunocytochemistry
1. Introduction Crustacean cardioactive peptide (CCAP) is a potent cardioexcitator, originally identified in the pericardial organs [21] then in the nervous system of the crab, Carcinus maenas [4,5,22]. This peptide is a modulator in many arthropods, affecting the pyloric rhythm in crabs [24], oviduct contrac∗
Corresponding author. Tel.: +36 87 448 244; fax: +36 87 448 006. ´ Vehovszky). E-mail address: [email protected] (A.
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.10.020
tion in Locusta [6], ecdysis [9,20] and the circadian clock in Drosophila [16] (for review see [4]). A peptide family related to CCAP, called molluscan CCAPs (M-CCAPs) was isolated from the central nervous system of the gastropod Helix pomatia [17,19]. The crustacean CCAP and the M-CCAP peptides show a high sequence homology, with a disulphide bridge as a common structural feature (Table 1). Additionally, applying the antibody raised against the crustacean CCAP showed specific CCAP-immunoreactivity in the CNS of two gastropods, H. pomatia and Lymnaea stagnalis [11].
´ Vehovszky et al. / Neuroscience Letters 373 (2005) 200–205 A. Table 1 Structure of the crustacean cardioactive peptide (CCAP) isolated from Carcinus [21] and the two fractions of molluscan CCAP-like peptides (M-CCAP1 and M-CCAP2) [17] found to be effective on Lymnaea neurons studied
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The physiological role of CCAP and M-CCAP peptides was assessed using electrophysiological recordings from identified motoneurons and interneurons in the isolated CNS. Double labeling (Lucifer Yellow backfilling of the cerebrobuccal connective and CCAP immunostaining) on the buccal and cerebral ganglia identified the neurons likely to release M-CCAP.
2. Materials and methods
The neuronal effects of CCAP have been tested on two CPG networks in crustaceans, one which drives the crustacean swimmeret systems [18] and one which drives the pyloric rhythm [24]. In both cases CCAP affected both the premotor circuit and the motor neurons of the CPG network. No data are available, however, on the physiological significance of the CCAP peptide family in the molluscan (snail) nervous system. In L. stagnalis CCAP immunoreactive cell bodies can be seen throughout the whole CNS, except the paired buccal ganglia. The buccal neuropil contains a network of dense CCAP-immunoreactive fibres as do the buccal musculature and the esophagus (Hern´adi et al., in preparation). The paired buccal ganglia contain the main elements of the central feeding network including the CPG system, while the buccal muscles and oesophagus are the main structures executing cyclic feeding movements [3,7]. This suggests that CCAP-related peptides play a role in generation and modulation of rhythmic feeding movements. To obtain information on the possible physiological significance of CCAP and M-CCAP peptides in feeding, we studied the neuronal effects of these peptides on identified members of the central feeding network of the pond snail L. stagnalis. In Lymnaea, the buccal ganglia contain the central pattern generating (CPG) interneurons for feeding and modulatory interneurons, as well as motor neurons which innervate the feeding muscles [7]. The paired cerebral ganglia contain the extrinsic feeding modulator, the giant serotonergic interneuron (CGC). In the isolated CNS preparation members of the feeding network (both interneurons and motoneurons) display a characteristic pattern of rhythmic synaptic inputs and action potentials called fictive feeding, which can be correlated to particular phases of the cyclic motor activity of the intact feeding animal: radula protraction (N1), rasp (N2) and swallowing (N3) [3,23,26].
Adult specimens of the pond snail L. stagnalis were sampled in spring, summer and autumn. The central nervous system was isolated and neurons identified and impaled as described previously [23]. The CCAP-peptide, M-CCAP1 and M-CCAP2 peptides were synthesized by the authors; CCAP by L. K´arp´ati and the M-CCAP peptides by M. Ohtani using the published structure and amino acid sequences [17,21]. Peptides were diluted before use into standard saline [23] to provide final concentrations from 0.1 M to 0.1 mM, and pumped through the perfusion system. For immunocytochemistry the polyclonal antibody (raised against CCAP in rabbit) was produced by Agricola et al. [1]. Immunocytochemistry was carried out on both whole-mount CNS preparations and cryostat sections [10], and visualized with the avidin/biotin peroxidase immunostaining method [11]. Samples were dehydrated and embedded into Canada balsam. The specificity of the antibody has been tested in two ways. 1. The antibody was omitted and replaced with bovine serum albumin during the immunostaining procedure; no immunostaining was observed. 2. The CCAP antibody diluted 1:10,000 was preabsorbed with M-CCAP forms (M-CCAP1 and M-CCAP2) at 1.5 mg/ml (approximately 1 mM) concentration at 4 o C overnight and then used for immunostaining. We failed to obtain immunoreactivity on sections applying the preabsorbed antibody with either of the two forms of M-CCAP. This shows that the CCAP antibody crossreacted with the applied M-CCAP forms indicating that the antibody specifically stains those neurons which contain forms of M-CCAP. To visualize cerebro-buccal interneurons (cerebral neurons which send axons to the buccal ganglia via the cerebrobuccal connectives, cbc), Lucifer Yellow dye (LY) was used as retrograde tracer [10,13]. To determine the CCAP containing neurons among the cerebro-buccal interneurons double immunostaining was used. The LY-backfilled preparations were used for CCAP immunostaining as whole mount and cryostat sections. The application of TRIC-labeled anti rabbit secondary antibody (diluted 1:50) to visualize bound CCAP antibody gave only weak CCAP staining with high background either in wholemount preparations or sections. This enabled us to determine the double stained neurons but was
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Fig. 1. Responses of buccal feeding motoneurons after M-CCAP application. (A) M-CCAP2 (0.1 M) evokes strong excitatory effect on B2 motoneuron leading to action potential firing (lower trace) while the B4 motoneuron is slightly depolarized followed by short bursts of excitatory postsynaptic potentials (upper trace). (Bi) M-CCAP2 (1 M) evokes rhythmic intracellular activity (fictive feeding) on B5 (upper trace) and B4 (lower trace) motoneurons. (Bii) Part of the record, indicated by a box, taken during fictive feeding, showing the bursts and synaptic inputs which correspond to feeding cycles in detail: slow inhibition on B5 and B4 motoneuron (N1, radula protraction), fast inhibition on both cells (N2, rasp), firing in burst (N3, swallowing).
not suitable for photographic documentation. Therefore, the LY-backfilled preparations were sectioned and the cryostat sections were used for CCAP immunostaining [11] applying the very sensitive avidin/biotin peroxidase method [12] to visualize immunoreactivity. LY stained and CCAP-stained neurons were photographed applying double exposition in bright field and FITC filters.
3. Results 3.1. Physiological effects of CCAP-related peptides on identified feeding neurons Preliminary experiments showed that two peptides, MCCAP1 and M-CCAP2 (Table 1) produced responses on Lymnaea neurons. We therefore tested these two M-CCAP peptides and compared them with crustacean CCAP peptide. The threshold is 0.1 M, with consistent responses at 1 M. Both M-CCAP1 and M-CCAP2 elicit a slow depolarization on all the 62 buccal neurons tested in 33 preparations (Figs. 1–3, representative data from both peptides shown). In 67% of these preparations, this depolarization is followed by rhythmic synaptic inputs and a series of bursts (Fig. 1Bi, Fig. 2 and Fig. 3A and B). Analysis of the pattern of synaptic inputs (Fig. 1Bii, Fig. 2B and Fig. 3Aii and Bii) shows that the full feeding sequence (N1 protraction, N2 rasp and N3 swallow [3]) is occurring, showing that the central pattern generator is fully active. The mean fictive feeding rate with 1 M M-CCAP1 and M-CCAP2 was the same, 6.9 ± 0.7 and 7.0 ± 0.9 cycles/min, respectively, though these preparations showed no initial feeding activity. There is no significant difference between M-CCAP1 and M-CCAP2 in the proportion
Fig. 2. Both feeding motoneurons and buccal modulatory interneurons respond by fictive feeding in 1 M M-CCAP. (A) The modulatory SO (upper trace) and the B1 motoneuron (lower trace) are depolarized leading to bursting activity pattern. After M-CCAP1 application the B1 motoneuron starts bursting while SO receives N2 phase inhibitory inputs. (Bi) Expanded from the first box in A shows the N2 inputs. (Bii) expanded from the second box shows full rhythmic bursting of fictive feeding (N1, N2 and N3 marking individual feeding cycles). (C) Recording from B3 and B4 motoneurons (upper and lower traces, respectively) shows rhythmic bursts during fictive feeding in response to M-CCAP2 while the octopaminergic modulator interneuron OC (middle trace) receives pattern inhibitory and excitatory synaptic inputs without firing activity.
2 = 3.03, of preparations responding with fictive feeding (χ1df P > 0.05). Two buccal modulatory interneurons, SO (N = 3) and OC (N = 2) also respond to M-CCAP peptides (Fig. 2). Rhythmic inputs of fictive feeding occur before the slow depolarization of SO, caused by direct membrane effect of M-CCAP1, is enough to fire action potentials (Fig. 2A, Bi), and these occur in the rhythmic bursts typical of the feeding pattern (Fig. 2Bii). The other buccal modulatory neuron, OC was also depolarized by M-CCAP2, but not always enough to fire action potentials during fictive feeding (Fig. 2C). The cerebral serotonergic neuron, the CGC, responds to 1 M M-CCAP1 application (Fig. 3A and B) by an increased firing rate in 9 out of 18 preparations. On other 9 preparations there is no increase in CGC firing despite the depolarization and rhythmic inputs in the buccal motoneuron (Fig. 3A). To compare the effect of M-CCAP1 and M-CCAP2 with crustacean CCAP, we tested the peptides on the same eight preparations. The molluscan peptides evoked just depolar-
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Fig. 3. Intracellular responses of the cerebral modulatory CGC neuron and identified buccal feeding neurons after application of CCAP-like peptide. (Ai) After application of 1 M M-CCAP1 the firing pattern of the serotonergic cerebral giant cell (CGC) does not change, while the buccal B1 motoneuron receives regular (protraction phase) depolarizing inputs from the feeding network. (Aii) Expansion of (Ai) showing series of compound excitatory synaptic potentials on B1 motoneuron. (Bi) M-CCAP2 (1 M) evokes higher firing frequency on the CGC interneuron and rhythmic pattern of synaptic inputs on B4 motoneuron. (Bii) Detail of record (Bi) showing CGC spikes (upper trace) which did not correlate with the regular pattern of inhibitory and excitatory inputs on B4 motoneuron. C. Application the crustacean CCAP (1 M) on the same preparation as shown in B, has no effect on either the CGC (upper trace) or B4 motoneuron (lower trace). (Cii) Detail of record (Ci).
ization in two preparations, while the other six showed the series of synaptic inputs typical of fictive feeding (Fig. 3B). The crustacean peptide, CCAP, however, produced no visible response in five preparations, and a much smaller depolarization in three preparations (Fig. 3C) and never evoked fictive 2 = 9.6 P < 0.01. feeding. This difference is significant: χ1df 3.2. Origin of the CCAP immunoreactive neuronal elements in the buccal ganglia Immunolabelling shows no CCAP-positive cell bodies in the buccal ganglia, but densely stained thick fibers in the cerebro-buccal connective (cbc), which enter the buccal ganglia and run to the contralateral ganglion and richly arborise in their neuropil (Fig. 4Ai and Aii). The richly arborized fine varicose CCAP-ir fibers are confined to the neuropil area and only rarely run to the cell body layer (Fig. 4Aii). Backfilling the cbc with Lucifer Yellow labels several small groups of neurons in the cerebral ganglia on both the dorsal (Fig. 4Ci) and ventral surface (Fig. 4Bi). The CCAP immunostaining of LY-backfilled cerebral ganglia visualized only two double (CCAP and LY) labeled neurons, located on the dorsomarginal area of the cerebral ganglion (Fig. 4Cii and Ciii). Double staining also revealed that CCAP-ir fibers surround
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Fig. 4. CCAP immunopositive elements in the Lymnaea central nervous system. (Ai) In the buccal ganglion no CCAP-immunopositive cell bodies only immunopositive fibers are seen (Ai) which enter from the cerebral ganglion (long arrows) through the cerebro-buccal connective (cbc). (Aii) Thick axonal branches (short arrows) run to the contralateral ganglia through the buccal commissure (bc) and arborize in the neuropil area in both ganglia. Whole mount preparations. lbn: laterobuccal nerve. B Whole mount of the cerebral ganglion after LY-backfilling the cbc. The majority of LY-labeled cerebro-buccal interneurons are located on the ventral surface of the cerebral ganglion (Bi). The labeled neurons send processes to the contralateral side through the cerebral commissure (cc) and to the lip nerves (mln, uln). The cerebral giant cell (CGC) shows pale LY fluorescence (Bi). CCAP stained fibers surround the axonal process of the CGC) (long arrows) suggesting direct connections (LY fluorescence was faded in the cell body) (Bii). Asterisk shows small LY-labeled cerebro-buccal interneurons. uln: upper lip nerve; mln: medial lip nerve. (C) Higher magnification of labelled neurons at the dorso-marginal area of the cerebral ganglion. LY-backfilling of the cbc labels three cerebro-buccal interneurons (1,2,3, Ci). Among them two neurons, the neuron 2 (Cii and Ciii) and neuron 3 (Ciii) show LY and CCAP immunostaining after the application of double staining on consecutive sections. Calibration bars: 100 m.
the axonal process of the giant cerebro-buccal interneuron the CGC (Fig. 4Bii)
4. Discussion 4.1. Molluscan CCAP-related peptides evoke fictive feeding in buccal feeding neurons Electrophysiological recordings from identified buccal feeding neurons in Lymnaea show the molluscan CCAP-like peptides (M-CCAP1 and M-CCAP2) modulate the spontaneous activity of all feeding neurons tested, and in 67% of the preparations evoked the pattern of fictive feeding on iden-
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tified buccal feeding neurons. The effectiveness of both peptides is not significantly different, and so we use M-CCAP to refer to them jointly. Physiologically, they are different from the crustacean CCAP peptide, which evoked a slight membrane depolarization in 31% of the preparations, but never fictive feeding. Thus, while the M-CCAP and CCAP peptides were originally isolated from Helix and crustacea, respectively, they are recognized by Lymnaea neurons, with significantly higher affinity to M-CCAP peptides. This, together with the M-CCAP pre-absorption tests and the fact that CCAP-immunoreactive neurons show similar numbers and distribution in Helix and Lymnaea (Hern´adi et al., in preparation), suggests the presence of M-CCAP-like molecules in both species, although the exact structure of the CCAP-like peptides in Lymnaea is yet unknown. M-CCAP1 and M-CCAP2 peptides evoke fictive feeding in 67% of preparations by stimulating the buccal feeding CPG system. The feeding rate induced by 1 M M-CCAP, 7 cycles/ min is lower than the feeding rate of the intact snail 16–18 cycles/min [14] or from the isolated CNS after stimulating modulatory interneurons (12.9 ± 1.9 cycles/min for SO stimulation and 11.8 ± 1.3 cycles/min after OC stimulation [23]). In isolated CNS preparations, fictive feeding may be evoked by intracellular current injection into modulatory neurons, e.g. SO, N1L, OC interneurons [3,26]. However, recording from SO during M-CCAP application revealed that fictive feeding is not caused by increased SO activity as M-CCAP activates the central pattern generator before the SO interneuron. Similarly, OC activity is not necessary for M-CCAP to drive fictive feeding. We conclude that the buccal CPG network is the most likely direct target for the M-CCAP, and the modulatory SO and OC interneurons are excited by their connections from the central pattern generator [3,23]. Double staining revealed CCAP immunoreactive fibers surrounding the initial axonal segment of another cerebral modulatory interneuron, the CGC, which contributes to the responsiveness of the feeding system [7,25]. Although some preparations showed that the CGC fired faster in M-CCAP we concluded that the M-CCAP effect on the buccal network did not depend on activation of the CGC, because other preparations showed activation of the central pattern generator without any change in CGC activity. Our observations suggest that the main effect of the MCCAP neurons is on the central pattern generator and motoneurons: this is different from the effect of FMRFamide, released by a pleural interneuron onto the feeding system, where the targets include all of the modulatory interneurons in addition to the central pattern generator interneurons and motoneurons [2]. Backfilling the cerebro-buccal connective reveals several neurons [18–22] in the cerebral ganglia, including three cells on the dorso-lateral surface. Only two of these show CCAP immunostaining; the transmitter used by the third cell is unknown. Another cerebro-buccal interneuron, located ven-
trally in the cerebral ganglia, containing APGWamide, is previously identified in Lymnaea to have a similar modulatory effect on the buccal feeding system [15]. The CCAP has closely similar effects in the crab stomatogastric ganglion [24] and crayfish swimmeret system [18]. In both cases the CCAP peptide activates the premotor circuit (the CPG network), and also directly modulates the motoneurons. In Lymnaea the pericardial organ and the connective sheath of the CNS including the buccal ganglia are also densely innervated by CCAP-stained fibers (Hern´adi et al., in preparation) therefore, we suggest that in vivo, the M-CCAP peptides reach the buccal neurons not only as neuromodulators liberated in the buccal neuropil close to the axons of the buccal neurons but also as neurohormone through the circulating haemolymph which is a very important rout of CCAP in arthropods [4,8]. Although we have shown consistent responses, which differ between the peptides applied, we still need to take the physiological relevance of these findings with caution, as the precise actions of the Lymnaea CCAP orthologous sequences have not been identified or functionally tested.
Acknowledgements The authors wish to thank for the technical assistance of Ms. Henriette Szab´o during the electrophysiological experiments and Mr. Boldizs´ar Bal´azs for the digital images. This work was supported by the Hungarian Academy of Sciences OTKA grant (T037389), DAAD (Germany), and the Wellcome Trust CRIG Programme.
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