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

Abstract

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.

Fig. 1

A–C, Structure of the oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) transgene (A) and representative autoradiographs of brain sections hybridised to a ³⁵S-labeled oligodeoxynucleotide probe for eCFP mRNA in a transgenic rat (B and C). D, Enlargement from the boxed area in B. E, Enlargement from the boxed area in C. White is the most intense signal, and black is the least intense signal. F–K, Endogenous fluorescence of eCFP in the paraventricular nucleus (PVN; F and I). G, OXT antibodies were visualised as red fluorescence, using an Alexa 546-conjugated secondary antibody. J, arginine vasopressin (AVP) antibodies were visualised as red fluorescence, using an Alexa 546-conjugated secondary antibody. H, merged view of fluorescence of eCFP and specific OXT is seen as white. K, merged view of fluorescence of eCFP and specific AVP is seen as white. Scale bars, 1 mm (B and C), 0.5 mm (D and E) and 50 Nm (F–K). O, oxytocin; V, vasopressin; OT, optic tract.

Fig. 2

Endogenous florescence of enhanced cyan fluorescent protein (eCFP) in the SON (A), the PVN (B), the ME (C), and the PP (D). Effects of salt loading for 5 days on the eCFP fluorescence of the SON (E), the PVN (F), the ME (G), and the PP (H). Visually identified neurones with high-intensity endogenous fluorescence of eCFP in a freshly isolated supraoptic nucleus neurone (I, J) and axon terminals (K, L). Under light (I, K) and fluorescent microscopy (J, L). Scale bars, 50 Nm. OT, optic tract; 3V, third ventricle; AP, anterior pituitary.

Fig. 3

A–H, Representative autoradiographs of brain sections hybridised to ³⁵S-labeled oligodeoxynucleotide probe for enhanced cyan fluorescent protein (eCFP) mRNA in transgenic rats. The expression of the eCFP gene was detected in the SON (A–D) and the PVN (E–H). Sections (A, E) were obtained from non-salt-loaded male rats. Sections (B, F) were obtained from salt-loaded male rats. Sections (C, G) were obtained from non-salt-loaded female rats. Sections (D, H) were obtained salt-loaded female rats. The changes in eCFP mRNA levels in the SON (I) and PVN (J) after salt loading for 5 days in OXT-eCFP transgenic rats. The numbers each of non-salt-loaded (Control) or salt-loaded male and non-salt-loaded (Control) or salt-loaded female rats was 6, respectively. White is the most intense signal, and black is the least intense signal. Scale bars, 1 mm (A–D) and 0.5 mm (E–H).

Fig. 4

A–H, Representative autoradiographs of brain sections hybridised to ³⁵S-labeled oligodeoxynucleotide probe for oxytocin (OXT) mRNA in OXT-enhanced cyan fluorescent protein (eCFP) transgenic rats. The expression of the OXT gene was detected in the SON (A–D) and the PVN (E–H). Sections (A, E) were obtained from non-salt-loaded male transgenic rats. Sections (B, F) were obtained from salt-loaded male transgenic rats. Sections (C, G) were obtained from non-salt-loaded female transgenic rats. Sections (D, H) were obtained salt-loaded female transgenic rats. Scale bars, 1 mm (A–D) and 0.5 mm (E–H). Changes in OXT mRNA levels in the SON (I, J) and the PVN (K, L) after salt loading for 5 days in non-transgenic rats and OXT-eCFP transgenic rats. The numbers each of non-transgenic [eCFP(−)] non-salt or salt-loaded male, non-transgenic [eCFP(−)] non-salt or salt-loaded female, [eCFP(+)] non-salt or salt-loaded male, and [eCFP(+)] non-salt or salt-loaded female rats was 6, respectively. *P< 0.05 and **P< 0.01 versus control of each group (non-transgenic and transgenic).

Fig. 5

A–H, Representative autoradiographs of brain sections hybridised to ³⁵S-labeled oligodeoxynucleotide probe for arginine vasopressin (AVP) mRNA in oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) transgenic rats. The expression of the AVP gene was detected in the SON (A–D) and the PVN (E–H). Sections (A, E) were obtained from non-salt-loaded male transgenic rats. Sections (B, F) were obtained from salt-loaded male transgenic rats. Sections (C, G) were obtained from non-salt-loaded female transgenic rats. Sections (D, H) were obtained salt-loaded female transgenic rats. White is the most intense signal, and black is the least intense signal. Scale bars, 1 mm (A–D) and 0.5 mm (E–H). Changes in AVP mRNA levels in the SON (I, J) and the PVN (K, L) after salt loading for 5 days in non-transgenic rats and OXT-eCFP transgenic rats. For each experimental condition, 6 rats were used. *P< 0.05 and **P< 0.01 versus control of each group (non-transgenic and transgenic).

Fig. 6

Plasma osmolality (A, B), Na⁺ (C, D), oxytocin (OXT; E, F) and arginine vasopressin (AVP; G, H) in 5 days salt-loaded non-transgenic and OXT-enhanced cyan fluorescent protein (eCFP) transgenic rats. The number each of eCFP(−), eCFP(+) non-salt or salt-loaded, male or female rats was 6. (*P<0.05 and **P<0.01).