We have generated rats bearing an oxytocin (OXT)-monomeric red fluorescent protein 1 (mRFP1) fusion transgene. The mRFP1 fluorescence was highly visible in ventral part of the supraoptic nucleus (SON) and the posterior pituitary in a whole mount. mRFP1 fluorescence in hypothalamic sections was also observed in the SON, the paraventricular nucleus (PVN), and the internal layer of the median eminence. Salt loading for 5 d caused a marked increase in mRFP1 fluorescence in the SON, the PVN, the median eminence, and the posterior pituitary. In situ hybridization histochemistry revealed that the expression of the mRNA encoding the OXT-mRFP1 fusion gene was observed in the SON and the PVN of euhydrated rats and increased dramatically after chronic salt loading. The expression of the endogenous OXT and the arginine vasopressin (AVP) genes were significantly increased in the SON and the PVN after chronic salt loading in both nontransgenic and transgenic rats. These responses were not different between male and female rats. Compared with nontransgenic rats, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration, OXT, and AVP levels. Finally, we succeeded in generating a double-transgenic rat that expresses both the OXT-mRFP1 fusion gene and the AVP-enhanced green fluorescent protein fusion gene. Our new transgenic rats are valuable new tools to study the physiology of the hypothalamo-neurohypophysial system.
Nociceptive stimulation elicits neuroendocrine responses such as arginine vasopressin (AVP) release as well as activation of the hypothalamo-pituitary-adrenal axis. We have generated novel transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene, and we examined the effects of nociceptive stimulation on transgene expression in the hypothalamus after subcutaneous injection of saline or formalin into the bilateral hindpaws in these rats. We have assessed (1) AVP levels in plasma and the changes of eGFP mRNA and AVP heteronuclear RNA (hnRNA) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) using in situ hybridization histochemistry, (2) gene expression changes in distinct magnocellular and parvocellular divisions of the PVN, (3) eGFP fluorescence in the SON, the PVN, the median eminence (ME), and the posterior pituitary gland (PP). Plasma AVP levels were significantly increased 15 min after formalin injection. In the same time period, the AVP hnRNA levels in the PVN were increased, especially in the parvocellular division of the PVN in formalin-injected rats. In the same region, eGFP mRNA levels after formalin injection were also significantly increased to a much greater extent than those of AVP hnRNA. The eGFP fluorescence in the SON, the PVN, the ME, and the PP was markedly increased in formalin-injected rats and especially increased in the parvocellular divisions of the PVN. Together, our results demonstrate robust and rapid changes in the expression of the AVP-eGFP transgene in the rat hypothalamus after acute nociceptive stimulation.
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.
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