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  • The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

Cell and tissue research (2017-07-09)
Ruth Fabian-Fine, Carly M Anderson, Molly A Roush, Jessica A G Johnson, Hongxia Liu, Andrew S French, Päivi H Torkkeli
ABSTRACT

The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Horse serum, liquid
Sigma-Aldrich
SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-Free, for use in purification of Histidine-tagged proteins
Sigma-Aldrich
Goat serum
Sigma-Aldrich
Anti-Choline Acetyltransferase (ChAT) Antibody, serum, Chemicon®
Sigma-Aldrich
Anti-FMRFamide Antibody, serum, Chemicon®
Sigma-Aldrich
Agarose, BioReagent, for molecular biology, low EEO
Sigma-Aldrich
Bovine Serum Albumin, cold ethanol fraction, pH 5.2, ≥96%