scholarly journals Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons and Isolated Nerve Terminals of the Rat

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 406-413 ◽  
Author(s):  
Yoichi Ueta ◽  
Hiroaki Fujihara ◽  
Ryota Serino ◽  
Govindan Dayanithi ◽  
Hitoshi Ozawa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Physiological Role ◽  
Transgenic Rats ◽  
Patch Clamp Technique ◽  
Axon Terminals ◽  
Green Fluorescent ◽  
Vasopressin Neurons

We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.

scholarly journals Robust Up-Regulation of Nuclear Red Fluorescent-Tagged Fos Marks Neuronal Activation in Green Fluorescent Vasopressin Neurons after Osmotic Stimulation in a Double-Transgenic Rat

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5633-5638 ◽  
Author(s):  
Hiroaki Fujihara ◽  
Yoichi Ueta ◽  
Hitoshi Suzuki ◽  
Akiko Katoh ◽  
Toyoaki Ohbuchi ◽  
...  
Keyword(s):  
Hypertonic Saline ◽  
Supraoptic Nucleus ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Regulatory Sequences ◽  
Neuronal Activation ◽  
Protein Fusion ◽  
Transgenic Rats ◽  
Green Fluorescent ◽  
Vasopressin Neurons

Abstract The up-regulation in the expression of mRNA or protein encoded by the c-fos gene is widely used as a marker of neuronal activation elicited by various stimuli. To facilitate the detection of activated neurons, we generated transgenic rats expressing a fusion gene consisting of c-fos coding sequences in frame with monomeric red fluorescent protein 1 (mRFP1) under the control of c-fos gene regulatory sequences (c-fos-mRFP1 rats). In c-fos-mRFP1 transgenic rats, 90 min after hypertonic saline ip administration, nuclear mRFP1 fluorescence was observed abundantly in brain regions known to be osmosensitive, namely the median preoptic nucleus, organum vasculosum lamina terminalis, supraoptic nucleus, paraventricular nucleus, and subfornical organ. Immunohistochemistry for Fos protein confirmed that the distribution of Fos-like immunoreactivity in nontransgenic rats was similar to those of mRFP1 fluorescence after ip administration of hypertonic saline in the transgenic rats. Several double-transgenic rats were obtained from matings between transgenic rats expressing an arginine vasopressin-enhanced green fluorescent protein fusion gene (AVP-eGFP rats) and c-fos-mRFP1 rats. In these double-transgenic rats, almost all eGFP neurons in the supraoptic nucleus and PVN expressed nuclear mRFP1 fluorescence 90 min after hypertonic saline administration. The c-fos-mRFP1 rats are a powerful tool that enables the facile identification of activated neurons in the nervous system. Furthermore, when combined with transgenes expressing another fluorophore under the control of cell-specific regulatory sequences, activation of specific neuronal cell types in response to physiological cues can be readily detected.

AVP-eGFP was significantly upregulated by hypovolemia in the parvocellular division of the paraventricular nucleus in the transgenic rats

2022 ◽  
Author(s):  
Kenya Sanada ◽  
Hiromichi Ueno ◽  
Tetsu Miyamoto ◽  
Kazuhiko Baba ◽  
Kentaro Tanaka ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Green Fluorescent Protein ◽  
Hpa Axis ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Posterior Pituitary ◽  
Transgenic Rats ◽  
Corticotrophin Releasing Factor ◽  
Green Fluorescent ◽  
Supraoptic Nuclei

Arginine vasopressin (AVP) is produced in the paraventricular (PVN) and supraoptic nuclei (SON). Peripheral AVP, which is secreted from the posterior pituitary, is produced in the magnocellular division of the PVN (mPVN) and SON. In addition, AVP is produced in the parvocellular division of the PVN (pPVN), where corticotrophin releasing factor (CRF) is synthesized. These peptides synergistically modulate the hypothalamic-pituitary-adrenal (HPA) axis. Previous studies have revealed that the HPA axis was activated by the hypovolemia. However, the detailed dynamics of AVP in the pPVN under hypovolemic state has not been elucidated. Here, we evaluated the effects of hypovolemia and hyperosmolality on the hypothalamus, using AVP-enhanced green fluorescent protein (eGFP) transgenic rats. Polyethylene glycol (PEG) or 3% hypertonic saline (HTN) was intraperitoneally administered in order to develop hypovolemia or hyperosmolality. AVP-eGFP intensity was robustly upregulated at 3 and 6 h after intraperitoneal (i.p.) administration of PEG or HTN in the mPVN. While in the pPVN, eGFP intensity was significantly increased at 6 h after i.p. administration of PEG with significant induction of Fos-immunoreactive (-ir) neurons. Consistently, eGFP mRNA, AVP hnRNA, and CRF mRNA in the pPVN and plasma AVP and corticosterone were significantly increased at 6 h after i.p. administration of PEG. The results suggest that AVP and CRF syntheses in the pPVN were activated by hypovolemia, resulting in the activation of the HPA axis.

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