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Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro

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Abstract

Relaxin-3/RXFP3 signalling is proposed to be involved in the neuromodulatory control of arousal- and stress-related neural circuits. Furthermore, previous studies in rats have led to the proposal that relaxin-3/RXFP3 signalling is associated with activation of the hypothalamic-pituitary-adrenal axis, but direct evidence for RXFP3-related actions on the activity of hypothalamic corticotropin-releasing hormone (CRH) neurons is lacking. In this study, we investigated characteristics of the relaxin-3/RXFP3 system in mouse hypothalamus. Administration of an RXFP3 agonist (RXFP3-A2) intra-cerebroventricularly or directly into the paraventricular nucleus of hypothalamus (PVN) of C57BL/6J mice did not alter corticosterone levels. Similarly, there were no differences between serum corticosterone levels in Rxfp3 knockout (C57BL/6JRXFP3TM1) and wild-type mice at baseline and after stress, despite detection of the predicted stress-induced increases in serum corticosterone. We examined the nature of the relaxin-3 innervation of PVN in wild-type mice and in Crh-IRES-Cre;Ai14 mice that co-express the tdTomato fluorophore in CRH neurons, identifying abundant relaxin-3 fibres in the peri-PVN region, but only sparse fibres associated with densely packed CRH neurons. In whole-cell voltage-clamp recordings of tdTomato-positive CRH neurons in these mice, we observed a reduction in sEPSC frequency following local application of RXFP3-A2, consistent with an activation of RXFP3 on presynaptic glutamatergic afferents in the PVN region. These studies clarify the relationship between relaxin-3/RXFP3 inputs and CRH neurons in mouse PVN, with implications for the interpretation of current and previous in vivo studies and future investigations of this stress-related signalling network in normal and transgenic mice, under normal and pathological conditions.

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Acknowledgements

The authors thank Berenice Chua and Mouna Haidar for technical assistance, Dr Mohammad Akhter Hossain (The Florey Institute of Neuroscience and Mental Health) for providing RXFP3-A2 peptide, and Prof Paul Sawchenko and Dr Jean Rivier (Salk Institute, San Diego, USA) for supplying the rabbit CRH antiserum originally prepared in the Wylie Vale Laboratory, which was used in these studies.

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  1. The Florey Institute of Neuroscience and Mental Health, 30 Royal Parade, Parkville, VIC, 3052, Australia

    C. Zhang, C. M. Smith & Andrew L. Gundlach

  2. Florey Department of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, 3010, Australia

    C. Zhang, C. M. Smith & Andrew L. Gundlach

  3. Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, AB, T2N 4N1, Canada

    D. V. Baimoukhametova & J. S. Bains

Authors
  1. C. Zhang
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  2. D. V. Baimoukhametova
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  3. C. M. Smith
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  4. J. S. Bains
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  5. Andrew L. Gundlach
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Corresponding author

Correspondence to Andrew L. Gundlach.

Ethics declarations

Experiments were conducted with approval from The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee, in compliance with guidelines of the National Health and Medical Research Council of Australia, and from the University of Calgary Animal Care and Use Committee in compliance with guidelines established by the Canadian Council on Animal Care.

Financial disclosure

This research was supported by a National Health and Medical Research Council of Australia Research Fellowship and Project Grant (1106330, 1024885, ALG), a Brain & Behavior Research Foundation (USA) NARSAD Independent Investigator Award (ALG); by the Canadian Institutes of Health Research and Brain Canada (JSB); and by the Victorian Government Operational Infrastructure Support Programme. CZ was the recipient of a Bethlehem Griffiths Research Foundation Postgraduate Scholarship and a Rebecca Hotchkiss International Scholar Exchange Travelling Fellowship.

Additional information

C. M. Smith, J. S. Bains and A. L. Gundlach jointly supervised this research.

Electronic supplementary material

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Fig. S1

Comparative distribution of RXFP3 mRNA with that of oxytocin and arginine vasopressin mRNAs in mouse PVN determined by in situ hybridisation histochemistry (adapted from the Allen Brain Institute Atlas of Brain Gene Expression). (A) Schematic image of the anterior PVN (coronal level ~Bregma -0.70 mm). (B) RXFP3 mRNA is present in neurons located in a region of PVN that is similar to that occupied by neurons expressing oxytocin mRNA (C) and arginine vasopressin mRNA (D), suggesting a relationship between RXFP3 signalling and modulation of these neurohormone neurons. Abbreviations 3V, third ventricle; f, fornix; OT, oxytocin; AVP, arginine vasopressin. Scale bar, 300 μm. (PDF 259 kb)

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Fig. S2

Comparative distribution of RXFP3 mRNA and oxytocin and arginine vasopressin mRNAs in mouse supraoptic nucleus (SON), determined by in situ hybridisation histochemistry (adapted from the Allen Brain Institute Atlas of Brain Gene Expression). (A) Schematic image of the SON (coronal level ~Bregma -0.82 mm). (B) RXFP3 mRNA is present in the magnocellular neurons within the supraoptic nucleus (SON), which express oxytocin mRNA (C) and arginine vasopressin mRNA (D), consistent with the presumed expression of RXFP3 mRNA by magnocellular neurons in the PVN (see Fig. S1), and further suggesting a relationship between RXFP3 signalling and these neurohormone neurons. In the supraoptic nucleus (SON), RXFP3 is expressed (B) in close proximity to oxytocin- (C) and vasopressin- (D) expressing neurons, further suggesting a relationship between RXFP3 signalling and these neuromodulatory peptides. Abbreviations: oc, optic chiasm. Scale bar, 100 μm. For further details of the distribution of RXFP1, RXFP3, CRH, OT and AVP mRNAs in the Allen Brain Institute Atlas of Brain Gene Expression, see <www.brain-map.org>. (PDF 196 kb)

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Zhang, C., Baimoukhametova, D.V., Smith, C.M. et al. Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro. Psychopharmacology 234, 1725–1739 (2017). https://doi.org/10.1007/s00213-017-4575-z

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