Elsevier

Peptides

Volume 31, Issue 6, June 2010, Pages 1007-1018
Peptides

Peptidergic modulation of serotonin and nerve elicited responses of the salivary duct muscle in the snail, Helix pomatia

https://doi.org/10.1016/j.peptides.2010.03.004Get rights and content

Abstract

In the present study, the ability of a range of endogenous neuropeptides to modulate neuromuscular transmission was examined in the salivary duct neuromuscular preparation of the terrestrial snail, Helix pomatia. Immunohistochemical and physiological techniques were used to localize the neuropeptides (GSPYFVamide, CARP, FMRFamide and APGWamide) and to investigate whether contractions elicited by the stimulation of the salivary nerve or by exogenously applied 5-HT are subject to peptidergic modulation. All of the neuropeptides studied decreased the tonus by a direct action on the muscle fibers in a concentration dependent manner in a range of 10−9 to 10−6 M. Neuropeptides distinctly affected the 5-HT evoked contraction or relaxation and GSPYFVa and APGWa decreased also the amplitude of contractions elicited by the stimulation of the salivary nerve. All four neuropeptides facilitated the relaxation phase providing further evidence for the postsynaptic action of neuropeptides. Low Ca2+/high Mg2+ saline abolished the nerve-elicited contractions, however the denervated muscle retained the ability to contract due to the mobilization of the Ca2+ from intracellular stores. It was concluded, that peptides belonging to different peptide families exerted their effects through pre- and postsynaptic mechanisms. The modulatory effect of neuropeptides can be assigned to the partial co-localization of 5-HT and neuropeptides in the nerves innervating muscles of the salivary duct, as it was demonstrated by double-labeling immunohistochemistry. A double origin of the 5-HTergic innervation was demonstrated, including efferents originating from both the cerebral and visceral ganglia.

Introduction

Modulation of neuromuscular transmission is an effective tool by which the nervous system controls synaptic efficacy. Neuromodulation is mediated by different signal molecules, such as transmitters and neuropeptides via cascades of different intracellular second messengers. An important observation on neuropeptides was the recognition that they usually coexist with one or more classical transmitters [6], [23], [26]. Thus, neuropeptides are complementary to classic transmitters, modulating their actions by enhancement or depression. The existence of a combination of neuropeptides and neurotransmitters at the same synapse enables synaptic actions to take place in both fast (2–5 ms) and slow (100–500 ms) time window [29]. Because neuropeptidergic transmission lacks a high-affinity uptake system, the termination of their action as well as their degradation is slow, resulting in a long lasting effect. Consequently, they can also activate receptors located at larger distances from their release sites [3], [17]. Modulatory effects of neuropeptides at neuromuscular contacts have been extensively studied in the regulation of feeding system and in the reproductive behavior of different mollusks [5], [10], [34], [35], [36]. Feeding behavior of gastropods consists of concerted, rhythmic movements of different muscles regulated by network(s) of interneurons called the central pattern generator (CPG) [2], [15], [27]. As to the signal molecules, feeding is regulated by serotonin (5-HT) and dopamine (DA) and in addition, by neuropeptides mainly present in the motor neurons of feeding muscles [5], [8], [9], [16], [36]. An important part of the feeding system is the salivary glands (SG) contributing to the process of ingestion by lubrication, agglutination and early enzymatic digestion [25], [30]. Previously we have shown that the DA and 5-HT play an important role as transmitters and/or modulators in the regulation of the salivary duct (SD) musculature [24]. SD was shown to respond to the external application of DA by sustained contraction, acting at D1-like receptors. On the other hand, 5-HT evoked either relaxation or contraction of the SD realized through 5-HT2A- or 5-HT3-like receptors, depending on the agonist concentration. Cellular localization of transmitters and several neuropeptides that control the SG complex have been partly demonstrated earlier. For example, 5-HTergic (MGC, metacerebral giant cell) and cholinergic (B2) neurons innervating the SG were found to be located in the cerebral and buccal ganglia [1], [7]. Mytilus inhibitory peptide (GSPYFVa), catch-relaxing peptide (CARP) and FMRFa immunoreactive elements were also detected in the buccal ganglia neurons other than B2 [13], [21]. Using correlative light- and electron microscopy, it was demonstrated that muscle cells of the SG and SD are directly innervated by deeply embedded FMRFa-ir and MIP-ir varicosities of non-identified neurons, contacting the muscle and gland cells along unspecialized membrane segments [12], [14]. Co-localization of 5-HT immunoreactivity and FMRFa or CARP immunoreactivity was observed in some cerebral neurons, but not in the MGC [18].

Based on the above findings, the aim of the present study was: (i) to examine the modulatory effects of endogenous neuropeptides on contractions of the SD muscle elicited by 5-HT or by stimulation of the salivary nerve; (ii) to examine the anatomical relationship and possible co-localization of 5-HT and different neuropeptides in the SD, applying double-labeling immunocytochemistry. In addition, the origin of 5-HT-ergic fibers innervating the SD musculature was localized using neurobiotin tracing and immunostaining.

Section snippets

Bioassay

Experiments were performed on adult specimens of the snail, Helix pomatia L. collected in the surroundings. The SDs with the tightly attached blood vessel were dissected and ligated at the both ends with thin threads. One end of the SD was attached to a stable hook located on the bottom of a 14 ml tissue chamber, while the other end was attached to a force transducer (WPI, FORT 1000). Contractions were recorded in a standard snail physiological solution containing 80 mM NaCl, 4 mM KCl, 10 mM CaCl2,

Effect of neuropeptides on the SD muscle

In order to determine the effects of individual endogenous neuropeptides on the muscle APGWa, CARP, FMRFa and GS were added to the bath at increasing concentrations (10−9 to 10−5 M) and responses were recorded isometrically. APGWa, FMRFa and CARP relaxed denervated SD muscle cells in a concentration dependent way between 10−9 and 10−6 M. However, the peptides at concentrations >10−6 M elicited a transient effect first relaxing the muscle which was followed by phasic contractions. Representative

Peptidergic modulation is both pre- and postsynaptic in the SDM

The neuropeptides, APGWa, GS, FMRFa or CARP applied at 10−9 to 10−6 M evoked a concentration dependent decrease of basal tension of the denervated muscle. At higher concentrations, however FMRFa (10−5 M) and CARP (10−6 M) had an opposite effect: they increased the tension of the muscle with superimposed phasic contractions. From these data we concluded that peptides exerted their effect directly interacting with receptors at the postsynaptic site.

Following the external application of 5-HT, which

Acknowledgments

This work was supported by grants from OTKA to T.K. (No. T43216) and to K.E. (Nos. T049090 and K78224). Authors thank are due to Prof. Miklós Palkovits and Dr. Árpád Dobolyi (Department of Anatomy, Semmelweis Medical University, Budapest) providing the possibility to work with the confocal microscope.

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