Somatostatin-IRES-Cre Mice: Between Knockout and Wild-Type?

Abstract

The neuropeptide somatostatin (SOM) is widely expressed in rodent brain and somatostatin-IRES-Cre (SOM-cre) mouse strains are increasingly used to unravel the physiology of SOM-containing neurons. However, while knock-in targeting strategy greatly improves Cre-Lox system accuracy, recent reports have shown that genomic insertion of Cre construct per se can markedly affect physiological function. We show that Cre transgene insertion into the 3'UTR of the somatostatin gene leads to the selective and massive depletion of endogenous SOM in all tested brain regions. It also strongly impacts SOM-related neuroendocrine responses in a similar manner to what has been reported for SST KO mice: increased corticosterone levels after 30-min restraint stress, decreased amplitude and regularity of ultradian growth hormone secretory patterns accompanied by changes in sexually dimorphic liver gene expression (serpina1, Cyp2b9, Cyp2a4, Cyp2d9, and Cyp7b1). In addition to demonstrating the need for examination of the consequences of Cre transgenesis, these results also reveal how this SOM-cre strain may be a useful tool in studying the functional consequences of moderate to low SOM levels as reported in neurological and psychiatric disorders.

Keywords: corticosterone; cre mice; feminization; growth hormone secretory patterns; hepatic expression.

Figure 1

Effect of genotype on peptide and somatostatin receptor subtype levels in mouse brain. (A) Somatostatin (SST), cortistatin (CST), and neuropeptide Y (NPY) expression levels in wild-type (WT), heterozygous (0/Tg), or homozygous (Tg/Tg) SOM-Cre male (black bars) and female (open bars) cortex (n = 3–4 mice per group). Error bars indicate SEM. ***P < 0.001 vs WT, ^°P < 0.01 vs 0/Tg. (B) SSTR1, SSTR2, SSTR3, SSTR4 expression levels in WT, heterozygous (0/Tg) or homozygous (Tg/Tg) SOM-Cre male (black bars) and female (filled bars) cortex (n = 3–4 mice per group). Error bars indicate SEM. (C) SOM and NPY peptide levels in male WT, heterozygous (0/Tg) or homozygous (Tg/Tg) SOM-Cre cortex (n = 6–7 mice per group). Error bars indicate SEM. **P < 0.01 vs WT. (D) SOM peptide levels in the hypothalamus, cortex, cerebellum, and olfactory bulb of male WT, homozygous SOM-Cre (Tg/Tg) or SST KO (SST^−/−) mice (n = 2–5 mice per group). Error bars indicate SEM. *P < 0.05, **P < 0.01 vs WT.

Figure 2

Representative pictures of SOM immunoreactivity in cortex (A), hippocampus (B), hypothalamus (C), olfactory bulb (D), and amygdala (E) of WT (1, left) or homozygous Tg/Tg SOM-Cre (2, right) mice. Scale bars: 100 µm.

Figure 3

Increased stress responses in homozygous SOM-Cre and SST KO mice. (A) Plasma corticosterone before (black bars) or after a 30-min restraint stress (gray bars) (n = 5–9 per group). Error bars indicate SEM. *P < 0.05, **P < 0.01 vs WT, ANOVA; ^°P < 0.05, ^°°P < 0.01 vs Basal, Wilcoxon test. (B) Serpina6 expression levels in wild-type mice (WT), homozygous SOM-Cre (Tg/Tg), or SST KO (SST^−/−) male (black bars) and female (white bars) mice (n = 3–9 mice per group). Error bars indicate SEM. *P < 0.05 vs WT, ^°°P < 0.001 vs male, ANOVA. (C–E) Behavioral responses of WT (white bars) and SOM-Cre Tg/Tg (gray bars) male mice. (C) Percent of total entries and time spent in the open arms in the elevated-plus maze. (D) Global activity in the open field test (20 min) (E) Percent of time spent in immobility, or swimming or climbing activity during the four first minutes of the forced-swim test (n = 10 mice per group). Error bars indicate SEM.

Figure 4

Feminized molecular and hormonal profiles of homozygous SOM-Cre and SST KO male mice. (A) Expression levels of female-predominant gene Cyp2b9 and Cyp2a4 and male-predominant Cyp2d9 and Cyp7d1 genes in wild-type mice (WT), homozygous SOM-Cre (Tg/Tg), or SST KO (SST^−/−) male (black bars) and female (filled bars) mice (n = 3–4 mice per group). Error bars indicate SEM. **P < 0.01, ***P < 0.001 vs WT, ^°P < 0.05, ^°°P < 0.001 vs male, ANOVA. (B) Mean growth hormone (GH) profiles over 6 h sampling. (C) GH cumulative distribution in WT (open symbols) and SOM-Cre Tg/Tg (black symbols) males (circles) and females (triangles) (n = 2–4 mice per group). Error bars indicate SEM. *P < 0.05, **P < 0.01 vs WT male, Kolmogorov–Smirnov test.