DIURNAL PATTERN OF CLOCK GENE EXPRESSION IN THE HYPOTHALAMUS OF THE NEWBORN RABBIT I. CALDELAS, a * D. TEJADILLA, a B. GONZÁLEZ, a R. MONTÚFAR b AND R. HUDSON a a Instituto de Investigaciones Biomédicas, Universidad Nacional Au- tónoma de México, 70228, Ciudad Universitaria, 04510, Distrito Fed- eral, Mexico b Division de Ingenieria, Instituto Tecnológico de Monterrey, Campus Santa Fe, Distrito Federal, Mexico Abstract—In the European rabbit (Oryctolagus cuniculus) nurs- ing acts as a strong non-photic synchronizer of circadian rhyth- micity in the newborn young. Rabbits only nurse for a few minutes once every 24 h and previous studies have shown that the pups, blind at birth, display endogenous circadian rhythms in behavior and physiology entrained by this regular daily event. As a further step toward understanding the neural orga- nization of the rabbit’s early circadian system, we investigated the expression of clock genes in the suprachiasmatic nucleus of the hypothalamus (SCN; the principal circadian pacemaker in adult mammals) across the pups’ 24-h day. We used 43 pups from seven litters maintained in constant darkness and en- trained non-photically by nursing at the same time each day until P7. After nursing on day 7, pups were killed in the dark at 3-h intervals so as to obtain eight groups (n5– 6 pups/group) distributed evenly across the 24 h before the next scheduled nursing. Profiles in the expression of the clock genes Per1, Per2, Cry1 and Bmal1 were determined using in situ hybridiza- tion in brain sections through the hypothalamus at the level of the SCN. We report for the first time: 1) that Per1, Per2, Cry1 and Bmal1 are all expressed in the SCN of the newborn rabbit, 2) that the expression of Per1, Per2 and Bmal1 but not Cry1 shows diurnal rhythmicity similar to that in adult mammals, and 3) that the expression of Per1, Per2 and Bmal1 is consistent with the strong entraining effect of nursing found in previous studies. Unexpectedly, and contrasting somewhat to the pattern in the SCN, we also found diurnal rhythmicity in the expression of Cry1 and Bmal1 but not of Per1 in the anterior ventromedial hypothalamic nucleus. Overall, our findings suggest that the SCN is a functional part of the newborn rabbit’s circadian sys- tem and that it can be entrained by non-photic cues associated with the mother’s daily nursing visit. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: circadian rhythms, non-photic entrainment, su- prachiasmatic nucleus, anterior ventromedial hypothalamic nucleus, development, mammals. Circadian rhythmicity is a fundamental characteristic of organisms, which helps ensure that vital functions occur in an appropriate and precise temporal sequence and in accordance with cyclic environmental changes (Pitten- drigh, 1981). In mammals, exposure to a cyclic environ- ment already occurs in utero, where fetuses are subject to a variety of non-photic stimuli originating from the mother including fluctuations in hormones, nutrients and uterus motility (Morgan et al., 1992; Reppert, 1995; Ninomiya- Alarcón et al., 2004). The main circadian pacemaker, lo- cated in the hypothalamic suprachiasmatic nuclei (SCN), is already functional before birth (Reppert and Schwartz, 1984; Reppert and Uhl, 1987; Shibata and Moore, 1987; Davis et al., 1990) and can be entrained by such non- photic cues (Reppert and Schwartz, 1983, 1986; Shibata and Moore, 1988; Reppert, 1995). This has led to the suggestion that for altricial young, non-photic cues pro- vided by the mother are the main environmental signals synchronizing their circadian system until their eyes open, they have left the nest and are able to use the photic cues so important for adult circadian function (Reppert, 1995). A notable example of such maternal influence is pro- vided by the European rabbit (Oryctolagus cuniculus). Im- mediately after giving birth to the altricial young in a dark underground nest (or laboratory nest box), the mother leaves and only returns once approximately every 24 h to nurse for 3–5 min (Zarrow et al., 1965; Hudson and Distel, 1982, 1989; Hudson et al., 1999; Jilge and Hudson, 2001; Bautista et al., 2005). A variety of studies has shown that this visit is a strong synchronizing signal for the pups. In response to imposed nursing schedules pups display diur- nal rhythms in general arousal (Hudson and Distel, 1982, 1989; Jilge, 1993, 1995; Pongrácz and Altbäcker, 1999), body temperature (Jilge et al., 2000, 2001), plasma pro- lactin (Alvarez et al., 2005, 2006) and corticosterone levels (Rovirosa et al., 2005), serum and liver metabolites (Escobar et al., 2000), and c-Fos expression in thalamic and hypothalamic nuclei (Allingham et al., 1998). These rhythms appear adaptive, preparing pups for the competitive scramble for nipples accompanying nursing (Drummond et al., 2000; Bautista et al., 2005). Shortly before the mother’s arrival the pups become increasingly active, uncover from the nest material (Hudson and Distel, 1982, 1989; Jilge, 1993, 1995), and their body temperature and levels of plasma corticosterone rise (Jilge et al., 2000; Rovirosa et al., 2005). These changes persist even in pups fasted for up to 72 h (Hudson and Distel, 1982, 1989; Jilge, 1993; Jilge et al., 2000, 2001), a procedure which they readily survive. However, although newborn rabbits clearly possess a functional circadian system sensitive to and entrained by maternal non-photic cues, it is not known how these cues act on the pups’ circadian system, and more particularly on the SCN. *Corresponding author. Tel: 52-5622-3828; fax: 52-5555-0048. E-mail address: caldelas@biomedicas.unam.mx (I. Caldelas). Abbreviations: AVHN, anterior ventromedial hypothalamic nucleus; Cry1, cryptochrome gene 1; Cry2, cryptochrome gene 2; P, postnatal day; Per1, period gene 1; Per2, period gene 2; Per3, period gene 3; SCN, suprachiasmatic nucleus. Neuroscience 144 (2007) 395– 401 0306-4522/07$30.000.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.09.020 395