L., 2002). Offered the reduced anxiety in Rcan1 KO mice, we tested these mice for

L., 2002). Offered the reduced anxiety in Rcan1 KO mice, we tested these mice for abnormal PPI. We discovered no difference in PPI between Rcan1 KO mice and WT littermates across a range of acoustic prepulse intensities (Fig. 4D; percentage D1 Receptor Antagonist medchemexpress inhibition of startle response: 74 dB, t(26) 0.123, p 0.9; 78 dB, t(26) 0.601, p 0.five; 82 dB, t(26) 1.232, p 0.two; 86 dB, t(26) 1.222, p 0.2; 90 dB, t(26) 1.753, p 0.091; startle test: t(26) 0.113, p 0.9; null period: t(26) 0.109, p 0.9). This demonstrates that the anxiety phenotype in Rcan1 KO mice is just not the result of abnormal sensorimotor gating. Considering that RCAN1 removal reduced the show of anxiety in Rcan1 KO mice, we subsequent tested whether RCAN1 overexpression could improve anxiety behaviors. We took advantage of two conditional flox-ON RCAN1 transgenic mouse lines (Tg1 and Tg1a) that overexpress human RCAN1 protein at high or low levels, respectively, within the presence of Cre recombinase (Oh et al., 2005). We utilized two Cre-driver lines to activate RCAN1 overexpression at distinct developmental time points, Nse-Cre for the duration of improvement (onset at about embryonic day 16.five; Forss-Petter et al., 1990) and T29-CamkII -Cre postdevelopmentally (onset at about postnatal day 14; Hoeffer et al., 2008). Overexpression of RCAN1 was confirmed by Western blots using antibodies against RCAN1 (Vega et al., 2003; Hoeffer et al., 2007) and also the FLAG epitope tagged towards the RCAN1 transgenic construct (Oh et al., 2005; Fig. 4E). RCAN1 overexpression employing either Cre driver had no detectable impact in the OFA assay (Table 1). In the EPM assay, even so, RCAN1 overexpression early in improvement under Nse-Cre in RCAN1Tg1a mice was shown to reduce open-arm time compared with handle WT (no Cre) littermates (Mann?Whitney U(83) two.010, p 0.044; Fig. 4F ). This impact was not as a result of group differences in locomotor activity (distance moved t(18) 1.683, p 0.110) or sensorimotor gating (Table 2), which supports the idea that the decreased open-arm time in NseRCAN1Tg1a mice represents greater anxiety. Nevertheless, overexpression from the other RCAN1 construct (RCAN1Tg1) below the same Nse-Cre driver did not impact EPM open-arm time, (Mann?Whitney U(18) 0.140, p 0.9; Fig. 4F ). Also, postdevelopmental RCAN1 overexpression under CamkII -Cre did not affect EPM open-arm time (CamkII -RCAN1Tg1a open-arm time, Mann hitney U(70) 0.018, p 0.9; CamkII RCAN1Tg1 open-arm time, Mann hitney U(28) 0.873, p 0.four; Fig. 4F ). Combined with the behavioral benefits in16936 ?J. Neurosci., October 23, 2013 ?33(43):16930 ?Hoeffer, Wong et al. ?RCAN1 Modulates Anxiousness and Responses to SSRIsADBECFFigure 4. Rcan1 KO mice show decreased measures of anxiety within the EPM. A, Rcan1 KO mice invest significantly a lot more time exploring the open arms with the EPM compared with their WT littermates. N 10 KO, 12 WT. B, Rcan1 KO mice enter the open arms early within the EPM test (minute 1) whereas their WT littermates increased open-arm exploration starting in the third minute of testing compared with minute 1. N ten KO, 9 WT. C, Total distance moved and speed of Rcan1 KO mice are indistinguishable from WT mice in the EPM. N ten KO, 12 WT. D, Rcan1 KO mice show equivalent PPI of acoustic startle responses compared with their WT littermates. E, Western blot analysis of RCAN1 expression within the PFC of RCAN1 transgenic (Tg) mice utilised for this study. Upper blot is stained with an RCAN1 antibody that recognizes endogenously expressed RCAN1.1L ( 38 kDa) and RCAN1.4 ( 28 kDa) protein Brd Inhibitor Formulation isoforms and transgenicall.