Recent evidence showing that basal forebrain cholinergic neurons with projections to the frontal cortex and hippocampus are activated by behaviorally salient stimuli suggests that these neurons are involved in arousal and/or attentional processes. We sought in the present experiments to test this hypothesis by examining whether unconditioned stimuli (a tone and flashing light) that normally increase cortical nad hippocampal acetylcholine (ACh) release would fail to do so after habituation (i.e., repeated presentation with no programmed consequences). In addition, the extent to which presentation of these stimuli would continue to increase ACh release when they had previously been paired with an aversive stimulus was investigated. Three experimental groups were used: habituation, novel stimuli, and conditioned fear. Subjects in each of these groups were placed in a training apparatus for twelve 200 min sessions. While the habituation group received extensive exposure to the tone and light during the training sessions, subjects in the novel stimuli group were placed in the apparatus but were never exposed to the tone or light during these sessions. The conditioned fear group was treated identically to the habituation group, with the addition that the tone and light were paired with footshock. On completion of these training schedules, all animals were implanted with microdialysis probes in the frontal cortex and hippocampus. Two days later, they were placed in the apparatus and the tone and light were presented to all subjects during microdialysis. In the novel stimuli group, the tone and light (unconditioned stimuli) produced significant increases in frontal cortical and hippocampal ACh release. Similarly, in the conditioned fear group, presentation of the tone and light (conditioned stimuli) also significantly increased ACh release in frontal cortex and hippocampus. In contrast, in the habituation group the tone and light failed to significantly enhance ACh release in either structure. During the test session, the tone and light elicited a variety of arousal- and fear-related behaviors in the novel stimuli and conditioned fear groups. In contrast, subjects in the habituation group generally failed to respond to these stimuli. These data indicate that cortically and hippocampally projecting basal forebrain cholinergic neurons are activated by conditioned and unconditioned stimuli that produce arousal in rats (novelty or conditioned fear). In contrast, presentation of these stimuli to habituated animals fails to enhance ACh release. These findings are consistent with a growing body of information indicating that ACh release in the cortex and hippocampus is reliably activated by behaviorally relevant stimuli. They also provide strong support for the hypothesis that cholinergic neurons in the basal forebrain are involved in arousal and/or attentional processes.

In vivo brain microdialysis was used to monitor changes in dopamine (DA) release in the nucleus accumbens (NAc) during anticipatory and consummatory components of feeding behavior. During 10 daily training sessions, rats were first confined to one compartment of a testing chamber for 10 minutes. During this period (anticipatory phase) they were prevented from gaining access to a highly palatable liquid meal by a wire mesh screen. The screen was then removed and the animals were permitted to consume the meal for 20 min (consummatory phase). On removal of the screen, the latency to begin drinking decreased and the amount consumed increased as a function of days of training, both measures reaching asymptotic levels by day 7. Trained animals were implanted with dialysis probes in the NAc on day 10, and on day 12 DA release was monitored during the feeding session. Compared to controls, trained animals failed to show significantly greater increases in accumbal DA release during the anticipatory phase, all groups showing small (approximately 10%) increases on being placed in the test chamber. In contrast, compared to controls, DA release increased significantly in the NAc during consumption of the palatable meal. The magnitude of this increase was significantly enhanced (30% vs 71% peak increase) in animals that were 20 hr food deprived at the time of testing. The latter animals also showed a statistically significant increase (24%) in DA release during the anticipatory phase. A subsequent experiment in which consumption of the palatable liquid was limited to 5 ml in deprived and nondeprived animals indicated that only part of the deprivation-induced potentiation of accumbal DA release could be attributed to the larger volume consumed by the deprived animals. That is, the same volume and rate of consumption of a small amount of the liquid diet produced a significantly greater increase in accumbal DA release in deprived than in nondeprived animals (42% vs 23% peak increase). Feeding-induced increases in accumbal DA release were not due to postingestional factors as direct injections of the liquid diet into the stomach by gavage failed to produce this effect. The results of these experiments indicate (1) that consummatory rather than anticipatory aspects of feeding are robustly associated with increases in DA release in the NAc, and (2) that motivational state can influence the magnitude of the neurochemical events that are associated with goal- directed behaviors.

Caspase-2 is an initiator caspase, which has been implicated to function in apoptotic and non-apoptotic signalling pathways, including cell-cycle regulation, DNA-damage signalling and tumour suppression. We previously demonstrated that caspase-2 deficiency enhances E1A/Ras oncogene-induced cell transformation and augments lymphomagenesis in the ElMyc mouse model. Caspase-2 À / À mouse embryonic fibroblasts (casp2 À / À MEFs) show aberrant cell-cycle checkpoint regulation and a defective apoptotic response following DNA damage. Disruption of cell-cycle checkpoints often leads to genomic instability (GIN), which is a common phenotype of cancer cells and can contribute to cellular transformation. Here we show that caspase-2 deficiency results in increased DNA damage and GIN in proliferating cells. Casp2 À / À MEFs readily escape senescence in culture and exhibit increased micronuclei formation and sustained DNA damage during cell culture and following c-irradiation. Metaphase analyses demonstrated that a lack of caspase-2 is associated with increased aneuploidy in both MEFs and in ElMyc lymphoma cells. In addition, casp2 À / À MEFs and lymphoma cells exhibit significantly decreased telomere length. We also noted that loss of caspase-2 leads to defective p53-mediated signalling and decreased trans-activation of p53 target genes upon DNA damage. Our findings suggest that loss of caspase-2 serves as a key function in maintaining genomic integrity, during cell proliferation and following DNA damage.

Caspase-2 has been implicated in apoptosis and in non-apoptotic processes such as cell cycle regulation, tumor suppression and ageing. Using caspase-2 knockout (casp2 À/À ) mice, we show here that the putative anti-ageing role of this caspase is due in part to its involvement in the stress response pathway. The old casp2 À/À mice show increased cellular levels of oxidized proteins, lipid peroxides and DNA damage, suggesting enhanced oxidative stress. Furthermore, murine embryonic fibroblasts from casp2 À/À mice showed increased reactive oxygen species generation when challenged with pro-oxidants. Reduced activities of antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were observed in the old casp2 À/À mice. Interestingly, in the old casp2 À/À animals expression of FoxO1 and FoxO3a was significantly reduced, whereas p21 levels and the number of senescent hepatocytes were elevated. In contrast to young wild-type mice, the casp2 À/À animals fed an on ethanol-based diet failed to show enhanced GSH-Px and SOD activities. Thus, caspase-2, most likely via FoxO transcription factors, regulates the oxidative stress response in vivo.