Brain Specific CREB Loss in Mice

As mentioned in Section 3, the only viable genetically modified CREB mutant mouse model available for drug studies to date has been the CREBm hypomorph mouse. To study the brain-specific loss of CREB in adult mice, free of the complications inherent in classical knockout models, such as pleiotropic effects during embryonic development and postnatal physiology, we employed the Cre/loxP recombination system to conditionally eliminate CREB only in brain, leaving a normal intact CREB gene in all other tissues. To generate the nervous system-specific CREB mutant mice, we used homologous recombination in ES cells to generate a modified CREB allele in which CREB exon 10, encoding the first part of the bZIP domain, was flanked with loxP sites (Fig. 2). Mice harboring the CREBloxP allele were crossed with transgenic mice possessing a transgene for Cre recombinase under the control of the nestin promoter and enhancer (32) (Fig. 5A). The mice lacking CREB in brain are referred to as CREBNesCre mice.

CREBNesCre mice lacked CREB immunoreactivity in almost all neurons and glia, probing with either of three antibodies recognizing CREB epitopes from the N-termi-nal half to the C-terminal end, indicating that no CREB protein, including truncated forms, were present (Fig. 5B). Phenotypically, CREBNesCre mice are essentially normal except for a reduction in body size due to a deficiency in growth hormone (T. M., unpublished data).

Fig. 3. Behavioral signs measured during naloxone-precipitated morphine withdrawal syndrome in CREB hypomorph mice (white columns), and their wild-type controls (black columns). Opiate dependence was induced by repeated ip injections of morphine-HCl (increasing dose) every 8 h during 3 d. Withdrawal was precipitated once in each mouse by naloxone-HCl injection (1 mg/kg sc) 2 h after the last morphine injection. The mice were placed individually into test chambers 30 min before naloxone injection, and the behavioral signs of withdrawal were evaluated after injection for 30 min. Data were subjected to two-way analysis of variance between animals. The number of animals per group was 12-16. Black stars, comparison between morphine-treated mice (M) and saline-treated mice (S); white stars, comparisons between wild-type and mutant groups receiving the same treatment; one star, p <0.05; two stars, p <0.01; three stars, p <0.001.

Fig. 3. Behavioral signs measured during naloxone-precipitated morphine withdrawal syndrome in CREB hypomorph mice (white columns), and their wild-type controls (black columns). Opiate dependence was induced by repeated ip injections of morphine-HCl (increasing dose) every 8 h during 3 d. Withdrawal was precipitated once in each mouse by naloxone-HCl injection (1 mg/kg sc) 2 h after the last morphine injection. The mice were placed individually into test chambers 30 min before naloxone injection, and the behavioral signs of withdrawal were evaluated after injection for 30 min. Data were subjected to two-way analysis of variance between animals. The number of animals per group was 12-16. Black stars, comparison between morphine-treated mice (M) and saline-treated mice (S); white stars, comparisons between wild-type and mutant groups receiving the same treatment; one star, p <0.05; two stars, p <0.01; three stars, p <0.001.

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