br Introduction The offspring of rodents emit vocalizations

Introduction The offspring of rodents emit vocalizations when isolated from their dam. The majority of vocalizations by rat pups are in the ultrasonic band frequency of 30–50 kHz (Insel et al., 1986). Maternal separation-induced ultrasonic vocalizations are believed to represent distress signals from the pup. This assumption is supported by the observation that the number of vocalizations can be attenuated by the administration of anxiolytics and antidepressants (Olivier et al., 1998a, Olivier et al., 1998b, Branchi et al., 2001). Previous studies have shown that the number of vocalizations from isolated pups is modulated by changes in environmental conditions such as ambient temperature, tactile stimuli and odors normally provided by the dam. Low ambient temperature (18 °C) increased callings by isolated rat pups, whereas at 37 °C, which was close to the temperature of the nest in which the dam and littermates lived, callings decreased (Olivier et al., 1998a, Olivier et al., 1998b). The increased callings of an isolated pup at room temperature were significantly reduced in the presence of the anesthetized dam (Shair et al., 1999). However, exactly how environmental factors affect the neuronal mechanisms controlling maternal separation-induced ultrasonic vocalizations is unclear. Corticotropin-releasing factor (CRF) is a hypothalamic-releasing peptide that activates the hypothalamic–pituitary–adrenal (HPA) axis and plays a pivotal role in anxiety- and distress-induced endocrine and behavioral responses (Vale et al., 1981, Heinrichs and Koob, 2004). Central administration of CRF to rats induces anxiogenic behaviors in an elevated plus-maze test (Dunn and Berridge, 1990, Heinrichs and Koob, 2004). In contrast, blockade of the CRF1 Isorhamnetin reduces anxiety and stress responses in an elevated plus-maze test and a social interaction test (Smith et al., 1998, Timpl et al., 1998, Chaki et al., 2004, Gehlert et al., 2005). A CRF1 receptor antagonist decreases the number of ultrasonic vocalizations induced by maternal separation under high-stress (low ambient temperature) condition (Kehne et al., 2000, Iijima and Chaki, 2005). These reports suggest that endogenous CRF modulates the emotional state by activating CRF1 receptor. The number of vocalizations elicited by maternal separation was thus expected to be exaggerated with administration of CRF (Insel and Harbaugh, 1989, Harvey and Hennessy, 1995). However, exogenous CRF did not increase the number of ultrasonic vocalizations induced by maternal separation in both high-stress (low ambient temperature) and low-stress (high ambient temperature) conditions (Insel and Harbaugh, 1989, Harvey and Hennessy, 1995, Dirks et al., 2002). Harvey and Hennessy (1995) thought that endogenous CRF was involved in the generation of a high number of vocalizations under high-stress conditions, but might be insufficient to generate ultrasonic vocalizations under low-stress conditions. How CRF influences the generation of maternal separation-induced ultrasonic vocalization under low-stress conditions thus remains unclear.
Results
Discussion Rat pups emitted a low number of vocalizations at 37 °C and maintained normal skin temperature. In contrast, when rat pups were isolated at 24 °C, a temperature significantly lower than skin temperature, calls were emitted approximately 11-times more frequently than pups at 37 °C. These results are consistent with previous findings (Allin and Banks, 1971, Blumberg et al., 1992, Olivier et al., 1998a, Olivier et al., 1998b). These results suggest that increases in the number of vocalizations at 24 °C compared with those at 37 °C are related to changes in emotional level in pups. In this study, administration of CRF dose-dependently increased the number of vocalizations at 37 °C, but not at 24 °C. The increased number of calls was blocked by CRF1 receptor antagonist, but not by CRF2 receptor antagonist. This is the first demonstration that administration of CRF increases the baseline number of vocalizations by activating CRF1 receptor. Harvey and Hennessy (1995) hypothesized that exogenously administered CRF could increase the number of vocalizations under the low-stress situations provided by high ambient temperature with bedded container, since exogenously administered CRF could mimic elevated stress situation (Dunn and Berridge, 1990, Heinrichs and Koob, 2004). However, the results did not support that assumption, with CRF showing little or no effect on the baseline number of vocalizations under a low-stress situation (33 °C) and causing a reduction in the number of vocalizations under a high-stress situation provided by room temperature and unbedded container. They thought that the effects of CRF showed an inverted U-shape, so other neuronal systems besides CRF systems might be responsible for inducing calls at a low stress level (33 °C), while exogenous CRF reduced the number of vocalizations under the already increased number of vocalizations by lowering ambient temperature (Room temperature), representing a high-stress situation. One explanation for the differing results between the present and previous studies may differences in the experimental protocol. We monitored vocalizations between 32 and 50 kHz, while Harvey and Hennessy (1995) only recorded a band of 42 kHz. We used systemic administration of CRF, whereas they used acute intracerebroventricular administration of CRF to conscious restrained animals. Since vocalizations were emitted over a broad range of frequency, their recording of vocalizations might have only recorded part of the vocalization. The effects of CRF thus could not be captured in their study. Regarding routes of administration of CRF, freehand injection into the cerebral ventricle might cause physical stress in addition to the stress caused by changes in environmental temperature, leading to activation of not only the CRF system, but also the noradrenergic or opioid system as they mentioned. Conversely, our study protocol might allow us to test effects of CRF without affecting other neuronal systems related to increased level of stress. The lack of additional increases in number of vocalizations by CRF at 24 °C indicates that effects of exogenous CRF were masked by high level of endogenous CRF that had been elevated by lower ambient temperature.