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