Physiological changes induced by CRF are mediated through CR
Physiological changes induced by CRF are mediated through CRF1 and CRF2 receptors (Chang et al., 1993, Lovenberg et al., 1995). Previous findings with CRF1 receptor antagonists showed that activation of CRF1 receptor is involved in maternal separation-induced ultrasonic vocalizations (Kehne et al., 2000, Iijima and Chaki, 2005). However, the involvement of CRF2 receptor has not yet to be determined. In the present study, CRF1 receptor antagonist, but not CRF2 receptor antagonist, blocked the increased calls induced by exogenous CRF at 37 °C. A dose of 1–3 mg/kg of K41498 was sufficient to demonstrate blockade of CRF2 receptors, because 0.1 mg/kg of K41498 completely antagonized CRF-induced skin flush and hypothermia (P<0.0001, data not shown). These findings suggest that the CRF1 receptor is primarily responsible for vocalization responses elicited by CRF at 37 °C. In addition, CRF1 receptor antagonist decreased the baseline number of calls at both 24 °C and 37 °C, in a dose-dependent manner. These results suggest that the endogenous CRF–CRF1 receptor system is involved in the generation of calls under both high- and low-stress conditions. Since expression of CRF1 receptors is high in the SP2509 (Potter et al., 1992, Pett et al., 2000), exogenous CRF would activate central CRF1 receptors to cause generation of vocalization. However, some reports have indicated weak expression of CRF1 receptors in peripheral tissues (Baigent and Lowry, 2000, Chatzaki et al., 2004). The possibility that activation of peripheral CRF1 receptors was involved in the generation of vocalizations by CRF administration thus could not be completely excluded. A severe decrease in skin temperature caused by drugs would complicate interpretation of the increased number of ultrasonic vocalizations by rodent pups, as lowered body temperature could itself trigger calls (Blumberg et al., 2001). In the present study, pups exhibited skin flushing and hypothermia after peripheral administration of CRF (0.3, 3 and 10 mg/kg). However, decreases in body temperature themselves are less likely to have altered the generation of vocalizations, because: 1) low-dose CRF (0.3 mg/kg) decreased skin temperature, but did not affect vocalizations when pups were exposed to 37 °C; 2) NBI27914 (3 mg/kg) antagonized CRF-induced increases in number of vocalizations, but did not affect CRF-induced skin flush and hypothermia, while K41498 (1 and 3 mg/kg) antagonized skin flush and hypothermia, but not the increased number of vocalizations caused by CRF. The skin flushing and hypothermia observed following administration of CRF has been suggested to be due to vasodilatory effects, caused by activation of the CRF2 receptor (Iwakiri et al., 1997, Schilling et al., 1998, Jain et al., 1999). Unlike vocalizations, both physiological changes caused by CRF were completely antagonized by K41498, but not by NBI27914, confirming the involvement of CRF2 receptor on body temperature changes caused by CRF, but not on vocalizations. In conclusion, our results demonstrate that changes in ambient temperature alter the number of ultrasonic vocalizations emitted by isolated pup by activating the endogenous CRF–CRF1 receptor regulatory system. When a rat pup is isolated from its mother at low ambient temperature, the distress level of the pup enhances release of CRF, which activates CRFR1, in turn increasing the number of vocalizations to communicate a state of anxiety to the dam. These results support the idea that maternal separation-induced ultrasonic vocalizations represent distress signals from the pup and provide experimental evidence supporting the original hypothesis in previous reports (Insel and Harbaugh, 1989, Harvey and Hennessy, 1995, Dirks et al., 2002). Furthermore, this study indicates that maternal separation-induced ultrasonic vocalization at various temperatures may serve as one model not only for evaluation of anxiolytics and antidepressants-like effects (Olivier et al., 1998a, b), but also for investigating anti-stress effects with novel mechanisms influencing the CRF system.