Effects of A-CREB, a dominant negative inhibitor of CREB, on the expression of c-fos and other immediate early genes in the rat SON during hyperosmotic stimulation in vivo

Abstract

Intraperitoneal administration of hypertonic saline to the rat supraoptic nucleus (SON) increases the expression of several immediate early genes (IEG) and the vasopressin gene. These increases have usually been attributed to action of the cyclic-AMP Response Element Binding Protein (CREB). In this paper, we study the role of CREB in these events in vivo by delivering a potent dominant-negative form of CREB, known as A-CREB, to the rat SON through the use of an adeno-associated viral (AAV) vector. Preliminary experiments on HEK 293 cells in vitro showed that the A-CREB vector that we used completely eliminated CREB-induced c-fos expression. We stereotaxically injected this AAV-A-CREB into one SON and a control AAV into the contralateral SON of the same rat. Two weeks following these injections we injected hypertonic saline intraperitoneally into the rat. Using this paradigm, we could measure the relative effects of inhibiting CREB on the induced expression of c-fos, ngfi-a, ngfi-b, and vasopressin genes in the A-CREB AAV injected SON versus the control AAV injected SON in the same rat. We found only a small (20%) decrease of c-fos expression and a 30% decrease of ngfi-b expression in the presence of the A-CREB. There were no significant changes in expression found in the other IEGs nor in vasopressin that were produced by the A-CREB. This suggests that CREB may play only a minor role in the expression of IEGs and vasopressin in the osmotically activated SON in vivo.

Fig. 1

(a) Illustration of the plasmid used to construct the A-CREB AAV. In addition to the A-CREB, the construct contains an EGFP reporter separated by a T2A peptide, and therefore equimolar expression of the A-CREB and EGFP would be expected in each transduced cell. The expression of these genes in the AAV vector is driven by a CMV promoter. (b) Determination of virus titer was done by serially diluting the purified AAVs and subsequently measuring the corresponding amount of the EGFP moiety by quantitative real time PCR. The cycle threshold (Ct) values for the AAVs were compared to standard curves such as the standard dilution curve shown here (see Experimental procedure for details). In general, we injected AAVs with titers around 5×10¹² vg/ml.

Fig. 2

(a) Schematic drawing of cannula placements used to inject the AAVs into the rat SON. The A-CREB AAV was injected over one SON, and the control AAV was injected over the contralateral SON. Hence, each rat contained its own control SON. Abbreviations: SON = supraoptic nucleus; SCN = suprachiasmatic nucleus; PVN = paraventricular nucleus. The images in panels b–d are from the same SON that had been injected with an AAV that expressed EGFP driven by a CMV promoter. (b) Illustrates the expression of EGFP as green immunofluorescence in SON cells that were successfully transduced by the CMV-EGFP AAV. OC = optic chiasm (c) Illustrates red immunofluorescent labeling of neurophysin in both oxytocin and vasopressin magnocellular neurons in the same SON. (d) Shows the merged overlay of green (b) and red (c) fluorescence demonstrating that the AAV successfully transduced most of the magnocellular neurons in the SON and robustly expressed EGFP in virtually all of them.

Fig. 3

Validation of the quantitative RT-PCR assays of the immediate early genes: c-fos (a), ngfi-a (b), ngfi-b (c), nr4a-2 (d) after forskolin stimulation. The figure depicts fluorescent growth curves and cycle threshold values for each of these immediate-early genes in cultured rat neuroblastoma (B35) cells that were stimulated with forskolin (solid line) versus those that were not (dotted line). The Cfos, NGFIA, NGFIB, and NR4A2 primers are shown in Table 1. In each case, there was an increase (lower Ct value) in the IEG mRNA in the forskolin-stimulated cultures as compared with the mRNA in non-stimulated cultures.

Fig. 4

Efficacy of the A-CREB-T2A-EGFP construct to inhibit induced c-fos expression in vitro. The graph shows fluorescent growth curves and cycle threshold values for c-fos mRNA in HEK-293 t cells under control conditions and after stimulation with forskolin. Cells were either transduced with A-CREB-T2A-EGFP AAV, EGFP-AAV, or not transduced with any virus (control). Human c-fos primer was used in the qPCR assay (see Table 1). Note that there was a large reduction in c-fos mRNA (higher Ct value) in the cells that were transduced with A-CREB AAV relative to those that were transduced with the EGFP-only control AAV. The non-transduced control cells and those that were transduced with the EGFP-only AAV exhibited similar fluorescent growth curves.

Fig. 5

Immunohistochemical staining for c-Fos protein in the SONs of rats in which the left SON was injected with the AAV containing A-CREB and the right SON with a control AAV. (a) Fos immunoreactivity. Both left and right SONs (arrows) are demonstrated here at low power (4× magnification). Note that there is more c-Fos immunofluorescence in the control SON after an acute salt loading stimulus. The 3rd ventricle is labeled 3 V, the optic chiasm, OC. (b and c) Illustrate higher magnification views (10×) of Fos immunofluorescence in A-CREB AAV treated and control AAV SONs in a different rat. The SONs are outlined by white lines. (b) Shows Fos immunoreactivity in the A-CREB treated SON versus the contralateral control SON (c) after an acute salt loading stimulus. This image demonstrates, in an additional rat, that c-Fos immunoreactivity is reduced in the A-CREB treated SON relative to the contralateral, control SON. See Table 2 for related changes in mRNA for all the intermediate genes under these experimental conditions.