RFamidergic neurons in the olfactory centers of the terrestrial slug Limax

Abstract

BackgroundThe terrestrial slug Limax has long been used as a model for the study of olfactory information processing and odor learning. Olfactory inputs from the olfactory epithelium are processed in the tentacular ganglion and then in the procerebrum. Glutamate and acetylcholine are the major neurotransmitters used in the procerebrum. Phe-Met-Arg-Phe-NH2 (FMRFamide) has been shown to be involved in the regulation of the network activity of the procerebrum. Although there are thought to be various RFamide family peptides other than FMRFamide that are potentially recognized by anti-FMRFamide antibody in the central nervous system of mollusks, identifying the entire repertoire of RFamide peptides in Limax has yet to be achieved.MethodsIn the present study, we made a comprehensive search for RFamide peptide-encoding genes from the transcriptome data of Limax, and identified 12 genes. The expression maps of these RFamide genes were constructed by in situ hybridization in the cerebral ganglia including the procerebrum, and in the superior/inferior tentacles.ResultsTen of 12 genes were expressed in the procerebrum, and nine of 12 genes were expressed in the tentacular ganglia. Gly-Ser-Leu-Phe-Arg-Phe-NH2 (GSLFRFamide), which is encoded by two different genes, LFRFamide1 (Leu-Phe-Arg-Phe-NH2–1) and LFRFamide2 (Leu-Phe-Arg-Phe-NH2–2), decreased the oscillatory frequency of the local field potential oscillation in the procerebrum when exogenously applied in vitro. We also found by immunohistochemistry that the neurons expressing pedal peptide send efferent projections from the procerebrum back to the tentacular ganglion.ConclusionOur findings suggest the involvement of a far wider variety of RFamide family peptides in the olfactory information processing in Limax than previously thought.Electronic supplementary materialThe online version of this article (10.1186/s40851-018-0108-9) contains supplementary material, which is available to authorized users.

Regional expression of neuropeptides in the retina of the terrestrial slug Limax valentianus (Gastropoda, Stylommatophora, Limacidae)

“…The cDNA regions were from 2157 to 2654 bases of vGluT2, and 428 to 1008 bases of WWa, 1119 to 1659 bases of MIP1, 1231 to 1752 bases of PP2, 1098 to 1636 bases of PH4, 100 to 683 bases of clionin, 1240 to 1791 bases of PH4var, 2521 to 2984 bases of MIP2, and 143 to 615 bases of Myo1. Those for PP, Enterin, FxRIa1, and FxRIa2 have been reported previously (Matsuo et al., 2020; Matsuo et al., 2018; Yamanaka et al., 2021). Left superior tentacles were isolated from the slugs deeply anesthetized with an intrabody injection of ice‐cold Mg 2+ buffer (57.6 m M MgCl 2 , 5.0 mM glucose, and 5.0 mM HEPES, pH 7.0) and rapidly frozen in Tissue‐Tek optimal cutting temperature (O.C.T.)…”

“…We first investigated whether Glu is used as a neurotransmitter in the retina of the eye of Limax , like in B. gouldiana (Michel et al., 2000) and L. stagnalis (Zhukov, 2007). Because we failed to detect the expression of vGluT (Matsuo et al., 2009) in the eye by in situ hybridization (data not shown), we searched for other variants of vGluT in the transcriptome data of Limax CNS (Matsuo et al., 2018), and found partial sequences of putative vGluT cDNA. As the two sequence data in the transcriptome data were expected to be the 5′ and 3′ ends of a novel vGluT cDNA, we obtained the intermediate part by RT‐PCR.…”

“…The concentrations of the primary antibodies were 0.2 μg/mL (WWa), 0.1 μg/mL (PP2), 0.2 μg/mL (MIP1/2), 0.2 μg/mL (Enterin), 0.1 μg/mL (FxRIamide), and 0.1 μg/mL (Myo1). As for PP, antiserum diluted in blocking buffer (1:5000) was used as the primary antibody (Matsuo et al., 2018). Following three washes in PBS, the sections were incubated with the secondary antibody (Alexa488‐labeled anti‐rabbit IgG, Thermo Fisher Scientific) diluted in blocking buffer (1:500) for 1 h at room temperature.…”

“…Antisera against the neuropeptides were obtained in rabbits by immu- against PP, Enterin, and FxRIa were described in our previous articles Matsuo et al, 2018;Yamanaka et al, 2021).…”

Co‐expression of opsins in the eye photoreceptor cells of the terrestrial slug Limax valentianus

“…We first searched for transcripts that were predicted to encode opsin‐like proteins in the transcriptome data of the central nervous system of L. valentianus (Matsuo, Yamanaka, & Matsuo, ) by Blast search, and found five such genes. Molecular phylogenetic analysis revealed the evolutionary relationships of these opsin‐like proteins with the opsin genes of other species (Figure , and the list of the dataset is shown in Table S1).…”