Fluorescent Visualisation of the Hypothalamic Oxytocin Neurones Activated by Cholecystokinin‐8 in Rats Expressing c‐fos‐Enhanced Green Fluorescent Protein and Oxytocin‐Monomeric Red Fluorescent Protein 1 Fusion Transgenes

Abstract

The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

Activation of Nesfatin‐1‐Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin‐1 in Rats

“…Co‐administration of low‐dose GLP‐1 (33 μg/kg) and leptin (300 μg/kg), neither of which decrease food intake when administered alone, dramatically decreases food intake in rats . The dosage of CCK‐8 was referenced from our previous studies .…”

Up‐regulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin‐enhanced green fluorescent protein

“…We previously generated transgenic rats expressing AVP‐enhanced green fluorescent protein (eGFP), which is a quantitative indicator of AVP . Expression of this transgene is characterised by robust fluorescence in the SON, PVN and PP in close parallel with the biosynthesis, transport and storage of endogenous AVP gene products …”

Fluorescent visualization of oxytocin in the hypothalamo-neurohypophysial system

“…It is postulated that these noradrenergic inputs activate OXT-secreting neurons in the SON and the PVN and cause the secretion of OXT into the systemic circulation in rats (Hamamura et al, 1991). Recently, we have developed a novel transgenic rat that enables the trivial visualization of c- fos expression using an eGFP tag (Katoh et al, 2014). These rats express a transgene consisting of c- fos gene regulatory sequences that drive the expression of a c- fos -eGFP fusion protein.…”

“…Three-dimensional reconstruction imaging enables the visualization of nuclear eGFP in the cytoplasm of OXT neurons illuminated and identified by virtue of their expression of mRFP1. In these neurons, abundant OXT granules in the cytoplasm are clearly visible by a plane image obtained from a higher magnification by confocal laser microscopy (Katoh et al, 2014). …”