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

Abstract

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.

In vivo processing and release into the circulation of GFP fusion protein in arginine vasopressin enhanced GFP transgenic rats: response to osmotic stimulation

“…AVP preproprotein (A) and AVP-GFP fusion preproprotein (B) consist of the signal peptide (SP), AVP hormone, AVP-associated neurophysin (NP) II and the glycoprotein copeptin (CP), where the glycan is represented by a diamond. The translation as a CP [1][2][3][4][5][6][7][8][9][10][11][12][13][14] is expected in this transgene, and the sixth asparagine of CP is expected to be a glycosylation site. The numbers denote the amino acids present in each region.…”

“…3B). Subsequently, we performed a western analysis using anti-CP [7][8][9][10][11][12][13][14] antiserum, because we expected the translation product in the Tg rats to be CP 1-14 (see Fig. 1).…”

“…After stripping CP [7][8][9][10][11][12][13][14] antibodies, the blots were re-incubated with a GFP mAb. Although some higher molecular weight bands were visible at about 32-36 kDa for CP [7][8][9][10][11][12][13][14] (Fig. 4A), an intense and single GFP-ir band at 28 kDa was observed in each blot (Fig.…”

“…After GFP-IP, the CP 7-14 positive band was only detected at the same level of 28 kDa (B). The second incubation with GFP antibodies demonstrated that the CP 7-14 positive proteins appeared to exist as CP [1][2][3][4][5][6][7][8][9][10][11][12][13][14] and GFP fusion proteins (B). Western analyses were repeated independently three times by using different samples and gave similar results.…”

“…A transgenic (Tg) rat line which expresses the AVPeGFP fusion gene was generated about 10 years ago [8][9][10][11][12] (see Fig. 1B).…”