Transmitter identification in neurons involved in male copulation behavior in Lymnaea stagnalis

Abstract

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.

Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite

“…We confirm the presence of this peptide in the HP neurons only. This earlier immunocytochemical study also showed that anti-DEILSR and anti-LIP ( Lymnaea inhibitory peptide) are mutually exclusive in the backfilled neurons of the right parietal ganglion, suggesting that at least some of the silent, DEILSR-negative parietal cells with projections into only the penis nerve are LIP-containing cells (de Lange et al 1998a ). However, our MALDI-TOF-MS data do not support this hypothesis since they did not confirm the presence of LIP in the nonHP neurons.…”

“…Male mating in the pond snail, Lymnaea stagnalis , was the focus of many multidisciplinary studies aimed at getting a deep insight into the regulation of this behavior. It has been studied in great detail and at different levels: behavioral (van Duivenboden 1984 ; de Boer et al 1996 ; Koene and Ter Maat 2005 , 2007 ), electrophysiological (de Boer et al 1997 ), biochemical (Li et al 1992 ; 1995 ), immunohistochemical (de Lange et al 1997 , 1998a , b ) and functional (van Golen et al 1995a , b ). Male mating is a complex behavior consisting of a series of stereotypic stages, movements and decisions that reflect an accurate communication between two partners that both have the ability to be male or female (van Duivenboden 1984 ; Koene and Ter Maat 2005 ).…”

“…The clustered cells have been intensively studied, particularly the anterior lobe of the right cerebral ganglion (de Lange et al 1997 ; Koene et al 2000 ). However, the dispersed neurons, not being grouped and therefore not so easily visually defined, are less amenable for single-cell analysis, and their peptide contents remain largely unknown (Koene 2010 ; de Lange et al 1998a ). It is known that even neighboring neurons in the same ganglion may have different neuropeptide contents and hence different biological functions.…”

“…Thus, it is important to apply a single-cell approach to reveal the chemical identity of dispersed cells that form part of the network controlling male mating. Therefore, our aim here is to provide a comprehensive description of these neurons in terms of peptide content and to relate that to their electrophysiological characteristics previously studied (de Lange et al 1998a ), in order to understand their involvement in the neural network underlying this behavior.…”

“…Earlier studies on the right parietal ganglion using electrophysiological and immunocytochemical techniques revealed two groups of neurons: (i) spontaneously active neurons, containing heptapeptides, and (ii) silent neurons containing Lymnaea inhibitory neuropeptide (LIP) and no heptapeptides (de Lange et al 1998a ). However, the global peptidergic identity of these neurons remains largely unknown.…”