Chemical Architecture of Arthropod Neuromuscular Junctions as Phylogenetic Characters
Final Report Abstract
In contrast to the vertebrates and many other phylogenetic groups which use acetylcholine as chemical messenger at the neuromuscular junction, in some well-studied crustacean and insect preparations glutamate has been shown as the excitatory transmitter. However, it is currently unknown whether glutamate acts as neuromuscular transmitter in other arthropod lineages. In this cytochemical/immunofluorescence investigation, we studied potential neurotransmitters in ventral ganglia and neuromuscular junctions of selected species of Myriapoda, Chelicerata, basal Hexapoda. and pterygote insects. We could co-localize glutamate with synapsin labelling in synaptic boutons on skeletal muscles in all studied arthropod specimens. Acetylcholine esterase (AChE) activity as a marker for cholinergic synapses was found abundantly in the central nervous systems, but not at neuromuscular junctions. Our data indicate that glutamate, and to a lesser extent, GABA are most likely neurotransmitters at arthropod neuromuscular junctions, whereas acetylcholine is very unlikely to play a role in neuromuscular transmission. As outgroup we localized AChE at the neuromuscular junctions of the Annelida Platynereis, Lumbricus and Eisenia. Together with our previous findings of mixed cholinergic/glutamatergic neuromuscular innervations in the onychophoran sister group, the new data support our hypothesis of the skeletal neuromuscular transmitter glutamate as a phylogenetic character of Arthropoda. During the evolution of rapid arthropod locomotion, GABAergic inhibitory neuromuscular transmission may have been an advantageous character, but is missing in several taxa. We found neuromuscular GABA labelling in all non-holometabolous hexapods. In chelicerates we found GABA in spider and scorpion, but not pseudoscorpion NMJs. In Myriapods, we found GABA in walking leg NMJs, but body wall NMJs displayed GABA-immunoreactivity only in Chilopoda. In addition to revealing glutamate and GABA as the most likely neurotransmitters at the neuromuscular junction, we made the surprising discovery of GABA and its biosynthetic enzyme glutamate decarboxylase (GAD) in sensory neurons of a centipede. Some arthropod mechanosensory neurons may not only use acetylcholine as a common neurotransmitter, but GABA is also a candidate. This is in line with published data from other labs on neurotransmitter diversity in the arthropod sensory system, showing histaminergic mechanosensory neurons in Drosophila and a spider. To address neurochemical characters the central nervous system, we investigated the morphology of identifiable serotonin-containing neurons in the wingless hexapod taxa Archeognata and Zygentoma. Comparisons with the patterns of serotonin-containing neurons in major tetraconate taxa suggested a close phylogenetic relationship of Remipedia, Cephalocarida, and Hexapoda. A recently published phylogenomic analysis considered Remipedia as the sole crustacean sister group of Hexapoda. Using a lipophilic dye, we traced the remipedian olfactory projection neuron pathway up to the hemiellipsoid bodies. All parts of the hemiellipsoid body expressed the catalytic subunit of the cAMP-dependent protein kinase A, an enzyme particularly enriched in in insect mushroom bodies. Moreover, immunofluorescence of the GAD enzyme revealed a cluster of GABAergic interneurons in the hemiellipsoid body, reminiscent of the characteristic feedback neurons of the mushroom body. These findings support the hypothesis of homology between remipedian hemiellipsoid bodies and hexapod mushroom bodies, despite differences in the neuroanatomical architecture and the malacostracan ground pattern of the olfactory pathway.
Publications
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(2018) Neuromuscular transmitter candidates of a centipede (Lithobius forficatus, Chilopoda). Frontiers in zoology 15 28
Langeloh, Hendrik; Wasser, Hannah; Richter, Nicole; Bicker, Gerd; Stern, Michael
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(2014) Olfactory projection neuron pathways in two species of marine Isopoda (Peracarida, Malacostraca, Crustacea). Tissue Cell 46:260-263
Stemme T, Eickhoff R, and G Bicker
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(2016) Olfactory pathways in Xibalbanus tulumensis: Remipedian hemiellipsoid body as counterpart of hexapod mushroom body. Cell Tissue Res 363: 635-648
Stemme T, Iliffe TM, and G Bicker G
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(2017) Serotonin containing neurons in basal insects: in search of ground patterns among Tetraconata. J. Comp. Neurol. 525: 79 – 115
Stemme T, Stern M, and G Bicker
