Toyoaki Ohbuchi

Highly Visible Expression of an Oxytocin-Monomeric Red Fluorescent Protein 1 Fusion Gene in the Hypothalamus and Posterior Pituitary of Transgenic Rats

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.

Exaggerated Response of a Vasopressin–Enhanced Green Fluorescent Protein Transgene to Nociceptive Stimulation in the Rat

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.

Centrally administered relaxin-3 induces Fos expression in the osmosensitive areas in rat brain and facilitates water intake.

The expression of the relaxin-3 gene, detected as a new member of the insulin superfamily using human genomic databases, is abundantly present in the brain and testis. Intracerebroventricularly (icv) administered relaxin-3 stimulates food intake. Icv administered relaxin (identical to relaxin-2 in humans) affects the secretion of vasopressin and drinking behavior. Relaxin-3 partly binds relaxin family peptide receptor 1, which is a specific receptor to relaxin. Thus, we hypothesized that relaxin-3 would have physiological effects in the body fluid balance. However, the effects of relaxin-3 in the body fluid balance remain unknown. In the present study, we revealed that icv administered relaxin-3 induced dense Fos-like immunoreactivity (Fos-LI) in the rat hypothalamus and circumventricular organs including the organum vasculosum of the lamina terminalis, the median preoptic nucleus, supraoptic nucleus (SON), the subfornical organ (SFO) and the paraventricular nucleus (PVN), that are related to the central regulation of body fluid balance. Icv administered relaxin-3 (54, 180 and 540 pmol/rat) also induced a significant increase in c-fos gene expression in a dose-dependent manner in the SON, SFO and PVN. Further, icv administered relaxin-3 (180 pmol/rat) significantly increased water intake, and the effect was as strong as that of relaxin-2 (180 pmol/rat). These results suggest that icv administered relaxin-3 activates osmosensitive areas in the brain and plays an important role in the regulation of body fluid balance.

Acid-sensing ion channels in rat hypothalamic vasopressin neurons of the supraoptic nucleus

Body fluid balance requires the release of arginine vasopressin (AVP) from the neurohypophysis. The hypothalamic supraoptic nucleus (SON) is a major site of AVP synthesis, and AVP release is controlled somatodendritically or at the level of nerve terminals by electrical activities of magnocellular neurosecretory cells (MNCs). Acid‐sensing ion channels (ASICs) are neuronal voltage‐insensitive cationic channels that are activated by extracellular acidification. Although ASICs are widely expressed in the central nervous system, functional ASICs have not been assessed in AVP neurons. ASICs are modulated by lactate (La−), which reduces the extracellular calcium ion concentration. We hypothesize that ASICs modify neuronal function through La− that is generated during local hypoxia resulting from osmotic stimulation in the SON. In the present study, we used the whole‐cell patch‐clamp technique to show that acid‐induced ASIC current is enhanced by La− in isolated rat SON MNCs that express an AVP‐enhanced green fluorescent protein (eGFP) transgene. Immunohistochemistry and multi‐cell reverse transcriptase‐polymerase chain reaction experiments revealed that these neurons express the ASIC1a and ASIC2a subunits. In addition, increased La− production was specifically observed in the SON after osmotic stress. These results suggest that interaction between ASICs and La− in the SON plays an important role in the regulatory mechanism of body fluid homeostasis.

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

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.

Brain-derived neurotrophic factor inhibits spontaneous inhibitory postsynaptic currents in the rat supraoptic nucleus.

Body fluid balance requires the release of arginine vasopressin (AVP) from the neurohypophysis. The hypothalamic supraoptic nucleus (SON) is one of the major sites for the synthesis of AVP, and secretion of AVP is controlled by the electrical activities of magnocellular neurosecretory cells (MNCs), which in turn are regulated by neuronal excitatory glutamatergic and inhibitory GABAergic inputs and humoral factors such as plasma osmolality. Previous studies have shown that brain-derived neurotrophic factor (BDNF) mRNA was increased by osmotic stress in the rat SON. In the present study, the effects of BDNF on excitatory and inhibitory synaptic inputs were examined in the MNCs of rat SON, using the whole-cell patch-clamp technique in in vitro brain slice preparations. BDNF application caused a significant reduction in the frequency and amplitude of the spontaneous inhibitory postsynaptic currents of the MNCs without affecting the spontaneous excitatory postsynaptic currents. Next, whole-cell patch-clamp recordings from dissociated SON MNCs expressing AVP-enhanced green fluorescent protein (eGFP) transgene revealed that the amplitude of GABA-induced currents were significantly smaller after BDNF treatment. Moreover, multi-cell reverse transcriptase-polymerase chain reaction (RT-PCR) experiments revealed the expression of TrkB mRNA in AVP-eGFP neurons. These results suggest that BDNF in the rat SON may decrease the postsynaptic GABAergic activity and may be involved in the regulatory mechanisms of body fluid homeostasis.

Efficacy of intratympanic steroid administration on idiopathic sudden sensorineural hearing loss in comparison with hyperbaric oxygen therapy

The efficacy of intratympanic steroid administration was examined in comparison with hyperbaric oxygen (HBO) therapy in patients with idiopathic sudden sensorineural hearing loss (ISSNHL).

Centrally administered ghrelin potently inhibits water intake induced by angiotensin II and hypovolemia in rats

Ghrelin is a potent, centrally acting orexigenic hormone. Recently, we showed that centrally administered ghrelin is a potent antidipsogenic hormone in 24-h water deprived rats. In this study, we examined the effect of intracerebroventricular (icv) injection of ghrelin on angiotensin II (AII)-induced water intake in rats. We also examined the effects of icv injection of ghrelin on drinking induced by intraperitoneal injection of an isotonic polyethylene glycol (PEG) solution that causes isotonic hypovolemia. Water intake induced by the icv injection of AII or ip injection of PEG was significantly reduced after icv injection of ghrelin, although food intake was stimulated by the hormone. The drinking induced by AII was also inhibited by the icv administration of 4α-phorbol 12, 13-didecanoate, an agonist of the osmosensitive TRPV4 channel. This study showed that ghrelin is a potent antidipsogenic peptide by antagonizing general dipsogenic mechanisms including those activated by AII and hypovolemia in rats.

Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary

We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the Oxt-eCfp fusion gene was expressed in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT immunofluorescence, but not with AVP immunofluorescence in the SON and the PVN. Although the expression levels of the Oxt-eCfp fusion gene in the SON and the PVN showed a wide range of variations in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the Oxt gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared with wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration and OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rats are a valuable new tool to identify OXT-producing neurones and their terminals.

Diurnal changes of arginine vasopressin-enhanced green fluorescent protein fusion transgene expression in the rat suprachiasmatic nucleus

We have recently developed a new transgenic rat line expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The AVP-eGFP transgene is expressed in the paraventricular (PVN) and supraoptic (SON) nuclei and the suprachiasmatic nucleus (SCN) of the hypothalamus. Transgene expression in the PVN and SON showed an exaggerated response to salt loading and nociceptive stimulation. However, the expression of the AVP-eGFP transgene in the SCN did not change under these stressful conditions. Here, we examined daily profiles of the expression of the AVP-eGFP transgene in the SCN in comparison with the endogenous AVP and Period (Per1 and Per2) genes. While all of these genes elicited diurnal patterns of expression in the SCN, the rate of rhythmic change of transgene expression was significantly greater than that of the endogenous AVP gene. We also examined the effect of a light stimulus on the expression of the AVP-eGFP, AVP, Per1 and Per2 genes in the SCN of transgenic rats. Ninety minutes after a light stimulus, AVP-eGFP mRNA and AVP hnRNA levels in the SCN were significantly decreased, while Per2 mRNA levels were significantly increased. In addition, we observed the eGFP fluorescence in the SCN and recorded the electrophysiological properties of a dissociated SCN eGFP-positive neuron. The AVP-eGFP transgenic rat is a useful animal model to study the diurnal change and dynamics of the AVP system, and enables the facile identification of SCN AVP neurons both in vivo and in vitro.