Ongoing Analyses of CREB Hypomorph and Conditional Mutant Mice

The previous work on CREBm mice has recently been extended, in parallel with novel studies on CREBNesCre mice. CREBm mice used in this work were backcrossed for seven generations into a C57/BL6 strain, to determine the contribution of genetic background on the withdrawal behavior. The backcrossed CREBm mice exhibited almost identical withdrawal responses to those described previously, showing that the attenuated withdrawal syndrome in these mice is a robust phenotype apparently independent of genetic background.

Fig. 4. Development of tolerance to the analgesic effects of morphine. Prior to chronic morphine treatment, mice were examined in the hot-plate test. Fifteen minutes after acute morphine administration (3 or 9 mg/kg ip) the percentage of analgesia was calculated as (test latency minus control latency) divided by (cutoff time minus control latency) 100. Test latency is the time it takes for the animal to jump off the hot plate after saline injection. Cutoff time is 120 s. Mice were treated with morphine (5 mg/kg ip) for 4 d and reexamined on the hot-plate test 9 h after the last morphine injection. Circles, CREBm mutant mice; triangles, wild-type mice; open symbols, percentage analgesia before chronic morphine; filled symbols, percentage analgesia after chronic morphine.

Fig. 4. Development of tolerance to the analgesic effects of morphine. Prior to chronic morphine treatment, mice were examined in the hot-plate test. Fifteen minutes after acute morphine administration (3 or 9 mg/kg ip) the percentage of analgesia was calculated as (test latency minus control latency) divided by (cutoff time minus control latency) 100. Test latency is the time it takes for the animal to jump off the hot plate after saline injection. Cutoff time is 120 s. Mice were treated with morphine (5 mg/kg ip) for 4 d and reexamined on the hot-plate test 9 h after the last morphine injection. Circles, CREBm mutant mice; triangles, wild-type mice; open symbols, percentage analgesia before chronic morphine; filled symbols, percentage analgesia after chronic morphine.

Preliminary results indicate that CREBNesCre mice exhibit significantly attenuated withdrawal responses, similar to CREBm mice, supporting the notion that the pheno-type observed in CREBm mice is primarily a consequence of CREB loss in the nervous system. Furthermore, the CREB □ and □ are probably the major CREB isoforms involved in the expression of morphine withdrawal syndrome. A more detailed analysis of the conditional CREB mutants will allow for the distinction between CNS and peripheral CREB-dependent mechanisms.

An elevated activity of LC neurons has been postulated to contribute to the expression of opiate withdrawal in morphine-dependent rats. Controversial data have been previously reported on the role played by the LC in the expression of morphine abstinence. The firing rate of LC neurons was strongly increased during spontaneous and antagonist-precipitated morphine withdrawal, which seems to contribute to the behavioral expression of the somatic signs of abstinence. Moreover, the LC was the most sensitive brain structure to precipitate the somatic signs of morphine withdrawal by microinjection of opioid antagonists, and its electrolytic lesion strongly inhibited opioid abstinence. Other studies, however, found that morphine treated rats failed to exhibit opiate withdrawal hyperactivity in the LC or that lesions of the noradrenergic brain pathways emanating from the LC failed to attenuate the somatic signs of opioid withdrawal. To examine whether CREB plays a role in this withdrawal-induced hyperactiv-

Fig. 5. Disruption of the CREB gene in brain by Cre/loxP-mediated recombination. (A) Once mice homozygous for the CREBloxP allele are crossed with mice expressing Cre recombinase specifically in brain, the result is CREB loss restricted to the nervous system. Use of various Cre transgenic lines would result in distinct anatomical and temporal patterns of CREB gene ablation. (B) Cre-recombinase expression under the control of the nestin promoter and enhancer results in almost complete CREB loss in brain. CREBloxP brains show normal widespread nuclear protein expression revealed by using anti-CREB antibodies, while CREBNesCre mutant mice exhibit almost complete loss of CREB protein. The anatomical specificity of CREB loss is highlighted by the failure of CREB recombination in the pituitary cells of CREBNesCre mice.

Fig. 5. Disruption of the CREB gene in brain by Cre/loxP-mediated recombination. (A) Once mice homozygous for the CREBloxP allele are crossed with mice expressing Cre recombinase specifically in brain, the result is CREB loss restricted to the nervous system. Use of various Cre transgenic lines would result in distinct anatomical and temporal patterns of CREB gene ablation. (B) Cre-recombinase expression under the control of the nestin promoter and enhancer results in almost complete CREB loss in brain. CREBloxP brains show normal widespread nuclear protein expression revealed by using anti-CREB antibodies, while CREBNesCre mutant mice exhibit almost complete loss of CREB protein. The anatomical specificity of CREB loss is highlighted by the failure of CREB recombination in the pituitary cells of CREBNesCre mice.

ity, single-unit extracellular recordings of LC neurons in brain slices from wild-type, CREBNesCre, and CREBm-deficient mice will be performed following chronic morphine treatment.

Interesting studies focussing on the role of CREB in rewarding behavior have recently been reported. Using rats in the conditioned place preference paradigm, where a herpes simplex virus vector expressing dominant-negative CREB was injected into the NAc of rat brain, a significant enhancement in cocaine rewarding effects was seen, while overexpression of wild-type CREB had an aversive effect (27). More recently, studies using CREBm mice suggest that there may be differences in the way CREB modulates downstream target genes, depending on whether morphine or cocaine is used to induce reward. In this study, CREBm-deficient mice do not respond to the reinforcing properties of morphine but do show an enhanced response to cocaine (9). We are currently using both our hypomorph and conditional knockout CREB mutant models to investigate these reward responses. In contrast to this last study, our preliminary data suggest that both CREBm mice and CREBNesCre mice show a reward response to morphine.

The conditional CREB mutant mice will prove to be useful in further studies as more Cre transgenic mice become available, allowing for more precise anatomical and temporal control over CREB ablation. For example, we now have Cre transgenic mice that will allow for the selective postnatal loss of CREB in either all neurons or dopamine D1 receptor-positive neurons, further refining the neuroanatomical and developmental molecular dissection of CREB function in mouse behavioral studies. The conditional disruption of CREB in either the peripheral or central nervous system will also allow us to distinguish between effects dependent on either or both the central or peripheral nervous system.

Was this article helpful?

0 0

Post a comment