ICV injection of UCN or
ICV injection of UCN2 or UCN 3 performed in the morning of the 8th day (12h after the last IP administration) seems to increase further the time spent in the open arms of mice exposed to saline or nicotine treatment. Interestingly, in mice exposed to saline treatment ICV injection of UCN 2 or UCN 3 tends to increase the swimming and the climbing activity, without influencing the time of immobility, in Cy5 carboxylic acid (non-sulfonated) with mice exposed to nicotine treatment, in which it tends to decrease them, without influencing the time of immobility. However, these results should be interpreted in the light of previous experiments, which suggest that the anxiolytic and the locomotor suppressive properties of nicotine are shared by urocortins too (Valdez et al., 2002, Valdez et al., 2003). Moreover, a single administration of UCN 2 or UCN 3 lowered the levels of the plasma corticosterone which were slightly elevated on the 8th day in both saline- and nicotine-treated animals, probably due the non-specific stress that is inevitable after testing, despite of the daily handling. Our experiments from the 9th day demonstrate that acute nicotine withdrawal after 24h (1day following 2mg/kg nicotine IP for 7 days, 4 times/day) produces signs of anxiety, since the number of entries into the open arms and the time spent in the open arms of the plus-maze decreased in the nicotine-treated group, compared to the saline-treated one. This result is in agreement with previous studies, which showed that acute withdrawal following chronic administration of nicotine (1 day of withdrawal following .1mg/kg/day IP treatment for 14 days or 12–24–48mg/kg/day treatment via minipump for 14 days) precipitates anxiety-like behavior in mice tested in light-dark box or elevated plus-maze. Our experiments from the 9th day also demonstrate that acute nicotine withdrawal after 24h induces signs of depression, since the time spent with swimming and climbing in the water increased in parallel with the time of immobilization in the nicotine-treated group, compared with the saline-treated one. This result coincides with that of a previous study using a similar treatment protocol (2mg/kg nicotine IP, 4 times/day), following which signs of depression were indicated during acute and chronic nicotine withdrawal in mice investigated in forced swim test (Mannucci et al., 2006). In concordance with these behavioral changes, significant elevation of the plasma corticosterone concentration, reflecting the hyperactivity of the HPA axis, was observed on the 9th day of our study. Indeed, hyperactivity of the HPA axis is associated frequently with nicotine withdrawal syndrome (Benwell and Balfour, 1979, Rasmussen, 1998) and generally with states of anxiety and depression (Binder and Nemeroff, 2010, Nemeroff, 1996b). ICV injection of UCN 2 or UCN 3 performed in the morning of the 9th day (at 24h after the last IP administration) increases the open-arm activity that was previously decreased by acute nicotine withdrawal. Concomitently, ICV injection of UCN 2 or UCN 3 reverses the swimming and the climbing activity and the immobility of mice, which were increased and decreased respectively by acute nicotine withdrawal. As a matter of fact, the anxiolytic and the antidepressant effects of the urocortins validated in the present study have been already suggested by previous studies using the same methods (Tanaka and Telegdy, 2008, Valdez et al., 2002, Valdez et al., 2003). Additionally, a single administration of UCN 2 or UCN 3 attenuated the levels of the plasma corticosterone which were remarkably and significantly augmented on the 9th day, at least in the nicotine-treated animals. This attenuation was achieved probably by activation of the CRF2 receptors that are expressed abundantly at the level of the hypothalamus and other subcortical regions (Bale and Vale, 2004, Van Pett et al., 2000). As regards the differences between the 12 and the 24h points of acute nicotine withdrawal these are in concert with the appearance of nicotine withdrawal syndrome that usually emerges after 12h, peaks around 24h and may even persist for 3 days (Kenny and Markou, 2001). Our results from the 12h paradigm suggest an altered mood, in form of concomitant signs of anxiolyis and depression, probably due to the non-specific stress that was induced by repeated nicotine treatment that occurred exactly 12h after the last IP administration from the previous day. Our results from the 24 paradigm, however, suggest that the state of anxiety and depression that is expected during acute nicotine withdrawal is already in full bloom following 1day. Putatively, the locomotor suppressive effects of nicotine that were exhibited after 12h of withdrawal may have been masked by the aversive signs expressed after 24h of withdrawal.