Distribution of FMRFamide-related peptides and co-localization with glutamate in Cupiennius salei, an invertebrate model system
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FMRFamide-related proteins have been described in both vertebrate and invertebrate nervous systems and have been suggested to play important roles in a variety of physiological processes. One proposed function is the modulation of signal transduction in mechanosensory neurons and their associated behavioral pathways in the Central American wandering spider Cupiennius salei; however, little is known about the distribution and abundance of FMRFamide-related proteins (FaRPs) within this invertebrate system. We employ immunohistochemistry, Hoechst nuclear stain and confocal microscopy of serial sections to detect, characterize and quantify FMRFamide-like immunoreactive neurons throughout all ganglia of the spider brain and along leg muscle. Within the different ganglia, between 3.4 and 12.6% of neurons showed immunolabeling. Among the immunoreactive cells, weakly and strongly labeled neurons could be distinguished. Between 71.4 and 81.7% of labeled neurons showed weak labeling, with 18.3 to 28.6% displaying strong labeling intensity. Among the weakly labeled neurons were characteristic motor neurons that have previously been shown to express ɣ-aminobutyric acid or glutamate. Ultrastructural investigations of neuromuscular junctions revealed mixed presynaptic vesicle populations including large electron-dense vesicles characteristic of neuropeptides. Double labeling for glutamate and FaRPs indicated that a subpopulation of neurons may co-express both neuroactive compounds. Our findings suggest that FaRPs are expressed throughout all ganglia and that different neurons have different expression levels. We conclude that FaRPs are likely utilized as neuromodulators in roughly 8% of neurons in the spider nervous system and that the main transmitter in a subpopulation of these neurons is likely glutamate.
KeywordsSpider central nervous system Synaptic circuits Co-transmission Neuromuscular junctions Neuropeptides
Confocal imaging was performed at the University of Vermont Microscopy Imaging Center. The authors thank Dr. Douglas Taatjes, Nicole Bouffard and Nicole Bishop for continued support with confocal microscopy. We thank Duy Pham of Colchester High School/Vermont for the artwork in Fig. 1(a). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
The Zeiss 510 META laser scanning confocal microscope was supported by NIH Award Number 1S10RR019246 from the National Center for Research Resources. The Alden Foundation supported the purchase of essential equipment for the SMC Biology Department. KG and BF received generous research stipends from Mr. Michael J. Cunniff and Dr. Robert F. Tobin. This research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449 (RF-F, BF, ET, KC, CA, KG and AJ). Further support was received in the form of a research stipend from The John C Hartnett fund to NG.
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