Selective tracking of FFAR3-expressing neurons supports receptor coupling to N-type calcium channels in mouse sympathetic neurons

Claudia Colina,Stephen R Ikeda,Henry L Puhl

doi:10.1038/s41598-018-35690-z

Abstract

Activation of short-chain free fatty acid receptors 3 (FFAR3) has been suggested to promote sympathetic outflow in postganglionic sympathetic neurons or hamper it by a negative coupling to N-type calcium (CaV2.2) channels. Heterogeneity of FFAR3 expression in sympathetic neurons, however, renders single neurons studies extremely time-consuming in wild-type mice. Previous studies demonstrated large variability of the degree of CaV2.2 channel inhibition by FFAR3 in a global population of rat sympathetic neurons. Therefore, we focused on a small subpopulation of mouse sympathetic neurons using an FFAR3 antibody and an Ffar3 reporter mouse to perform immunofluorescent and electrophysiological studies. Whole-cell patch-clamp recordings of identified FFAR3-expressing neurons from reporter mice revealed a 2.5-fold decrease in the CaV2.2-FFAR3 inhibitory coupling variability and 1.5-fold increase in the mean ICa2+ inhibition, when compared with unlabeled neurons from wild-type mice. Further, we found that the ablation of Ffar3 gene expression in two knockout mouse models led to a complete loss-of-function. Subpopulations of sympathetic neurons are associated with discrete functional pathways. However, little is known about the neural pathways of the FFAR3-expressing subpopulation. Our data indicate that FFAR3 is expressed primarily in neurons with a vasoconstrictor phenotype. Thus, fine-tuning of chemically-coded neurotransmitters may accomplish an adequate outcome.

Highlights

Free fatty acid receptors (FFAR1–3) were cloned in the search for novel receptors[1] and classified as orphan proteins in 1997
A screening of 24 different mouse tissues showed that superior cervical ganglia (SCG) and CSMG ganglia have the highest levels of Ffar[3] mRNA transcripts (Supplementary Fig. 1a)
Through the combined use of electrophysiological and immunofluorescent approaches, together with a fluorescent reporter mouse strain, we have examined free fatty acid receptors 3 (FFAR3)-expressing neurons in two mouse sympathetic ganglia

Summary

Introduction

Free fatty acid receptors (FFAR1–3) were cloned in the search for novel receptors[1] and classified as orphan proteins in 1997. Gα-GTP dissociates from Gβγ allowing each moiety to modulate discrete effector proteins. An example of such modulation is the association of dimeric Gβγ with the N-type calcium channels to negatively www.nature.com/scientificreports/. In this study, recording from identified neurons isolated from an established reporter mouse line resulted in 2.5-fold decrease in response variability and a 1.5-fold increase in the mean ICa2+ inhibition. The superior cervical ganglia (SCG) and the celiac-mesenteric ganglia (CSMG) are part of the sympathetic subdivision of the autonomic nervous system (ANS) Both ganglia contain discrete neuronal subpopulations with a distinctive “neurochemical code” that selectively projects to functionally heterogeneous peripheral targets. This provides a fine-tune control of different sympathetic pathways. Exceptions to this general rule are cholinergic sympathetic nerve endings innervating sweat glands[31], and non-cholinergic/non-noradrenergic vasodilator neurons supplying some skeletal muscle vessels[32,33], and skin blood vessels in rodents

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Results

Conclusion