Elsevier

Brain Research

Volume 1429, 6 January 2012, Pages 18-28
Brain Research

Research Report
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

https://doi.org/10.1016/j.brainres.2011.10.033Get rights and content

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.

Highlights

► Adeno-associated virus containing A-CREB transduced neurons in the rat supraoptic nucleus in vivo. ► AAV A-CREB completely eliminated forskolin induced cfos expression in cell lines in vitro. ► AAV A-CREB injected in the SON in vivo reduced cfos mRNA by 20%. ► AAV A-CREB injected in the SON in vivo reduced NGFI-B mRNA by 30%. ► AAV A-CREB injected in the SON in vivo did not alter vasopressin gene expression.

Introduction

Intraperitoneal (i.p.) administration of hypertonic saline increases the expression of the immediate early genes (IEG), c-fos, ngfi-a, ngfi-b, along with the expression of the vasopressin (VP) gene in the supraoptic nucleus (SON) of rats (Kawasaki et al., 2005). The presence of a cyclic adenosine 3, 5-monophosphate (cAMP) response element (CRE) sequence in the promoter region of all of these genes (Boutillier et al., 1992, DeFranco et al., 1993, Inaoka et al., 2008, Yoshida et al., 2006) suggests that the increase in expression is mediated by the CRE binding protein (CREB).

CREB is a transcription factor that has been implicated in the transcriptional regulation of many genes (Sheng et al., 1990) and is involved in a myriad of cellular responses that are involved in nervous system development, learning and memory, addiction, and neuroprotection from disease among others (Lonze and Ginty, 2002). CREB is activated in response to a vast array of physiological stimuli together with the closely related members of the same bZIP superfamily of transcription factors such as cAMP response element modulator (CREM) and activating transcription factor 1 (ATF-1).

Efforts to study CREB function in vivo have been made by the use of traditional knockout models, but success has been limited since CREB −/− mice don't survive after birth largely because of respiratory distress (Lonze and Ginty, 2002, Mayr and Montminy, 2001). Although CREM −/− mice do survive to adulthood, the males are sterile, they exhibit disrupted circadian rhythms, and have altered behavioral patterns (Mayr and Montminy, 2001). Moreover, compensatory action by other CREB family members in CREM- or CREB-knockout mice prevents expression of a knockout phenotype (Mayr and Montminy, 2001). These drawbacks have led to the development of alternative transgenic (Carlezon et al., 1998, Herzig et al., 2001, Jancic et al., 2009, Lee et al., 2009) and viral vector (Barrot et al., 2005, Warburton et al., 2005, Yuan et al., 2003) strategies using mutant CREBs to perturb CREB function in vivo.

In the present study we stereotaxically injected A-CREB, a potent dominant negative inhibitor of CREB that disrupts the DNA binding of CREB and CREB family members (Ahn et al., 1998), into the rat SON using an adeno-associated virus (AAV) vector. A-CREB has an acidic amphipathic patch that is thought to mimic DNA in its binding to the N-terminal of the wild-type CREB leucine zipper. Experiments by Ahn et al. (1998) showed that A-CREB is able to completely and specifically inhibit CREB DNA binding. We injected an A-CREB bearing AAV vector in order to determine the role of CREB and CREB family members on the expression of c-fos, ngfi-a, ngfi-b, nr4a2, and vasopressin (VP) in this brain region. The effectiveness of using AAV to target molecules to the rat SON was recently demonstrated (Doherty et al., 2011). Since the acute, systemic administration of hypertonic saline causes rapid increases in expression of several of these genes, (Kawasaki et al., 2005) which are presumed to be primarily mediated by the transcription factor CREB, we expected that blocking CREB in vivo would significantly inhibit their increases in expression during hyperosmotic stimulation.

Section snippets

Localization of the AAV to the SON

A schematic diagram of the stereotaxic injection paradigm is illustrated in Fig. 2a, which shows a coronal section of a rat brain with a syringe lowered unilaterally to the SON. Figs. 2b and c show results from control experiments in which the SON was injected with the CMV-EGFP virus and subsequently double-labeled with IHC using antibodies against both EGFP and Neurophysin (an identifying marker for the magnocellular neurons). As shown in Fig. 2d, the virus (EGFP-ir) co-localized with nearly

Discussion

CREB target genes, which differ widely in their functional roles and mechanisms, all have in common the presence of a CREB binding site, the CRE sequence, in their promoter region. One of the most recognized of these target genes is the prototypic IEG, c-fos, whose transcription is rapidly activated by a variety of stimuli but peaks and returns to baseline within 1 h of the stimulus onset (Greenberg and Ziff, 1984, Sheng and Greenberg, 1990). C-fos was one of the first identified CREB target

Animals

Adult male Sprague–Dawley rats (270 g–370 g) obtained from Charles River Laboratories (Wilmington, MA) were maintained under normal laboratory conditions (temperature: 21–23 °C, 12 h light–dark cycles with light on at 6:00 AM) with access to unlimited food and drinking water. Following surgical procedures, rats were caged individually. All procedures were carried out in accordance with the National Institutes of Health (NIH) guidelines on the care and use of animals and according to an animal study

Acknowledgments

This research was supported by the Intramural Research Program of the NINDS, NIH. We would like to thank Dr. Kory Johnson (NINDS, NIH) for his advice about statistical tests, and Dr. C. Vinson (NCI, NIH) for his gift of the A-CREB plasmid.

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