ORIGINAL ARTICLE Circadian Nursing Induces PER1 Protein in Neuroendocrine Tyrosine Hydroxylase Neurones in the Rabbit Doe E. Meza*, S. M. Waliszewski and M. Caba* *Centro de Investigaciones Biome ´dicas, Universidad Veracruzana, Xalapa, Ver., Me ´xico.  Instituto de Medicina Forense, Universidad Veracruzana, Veracruz, Ver. Me ´xico. Lactation in the rabbit is circadian. Behavioural studies conducted in the wild and under laboratory conditions confirm that rabbits nurse their pups just one time every day for < 5 min, with circa- dian periodicity (1,2). At the molecular level, circadian rhythms are produced by a positive and negative transcriptional ⁄ translational autoregulatory feedback loop. The proteins CLOCK and BMAL pro- vide the positive transcriptional drive, whereas the proteins PERIOD (PER) 1, 2 and 3 and CRY1 and CRY2 provide the negative feedback (3). PER1 has a circadian expression in the suprachiasmatic nucleus (SCN), the master circadian clock, in several species such as mice (3), rats (4), hamsters (5) and rabbits (6). In the rabbit, maximum expression of PER1 is in the afternoon under light ⁄ dark conditions in intact nonpregnant, nonlactating females (6). Furthermore, sev- eral brain regions other than the SCN express circadian rhythms of clock genes. These include the olfactory bulb, hippocampus, cerebral cortex, pituitary gland, and arcuate, retrochiasmatic, supraoptic and paraventricular nuclei in the hypothalamus (7–10). However, both their neurochemical phenotype and functional significance remain mostly unexplored. The rabbit offers an excellent opportunity to explore the physio- logical significance of clock gene rhythms in neuroendocrine cells, considering the particular circadian lactation of this species (11). Accordingly, we previously reported that brief daily nursing entrains oxytocinergic (OT) neurones in the hypothalamus, as indicated by the expression of the PER1 protein, a product of the Per1 clock gene. PER1 in the SCN does not change with the physiological con- dition of the doe but, in contrast, brief daily nursing shifts PER1 protein rhythm in OT neurones, which peaks 4–8 h after the timing Journal of Neuroendocrinology Correspondence to: M. Caba, Centro de Investigaciones Biome ´dicas, Universidad Veracruzana, A.P. # 114, Xalapa, Ver., Me ´xico (e-mail: mcaba@uv.mx). Rabbit does nurse their pups once a day with circadian periodicity and pups ingest up to 35% of their body weight in milk in < 5 min. In the doe, there is a massive release of prolactin. We hypothesised that periodic suckling synchronises dopaminergic populations that control prolactin secretion. We explored this by immunohistochemical colocalisation of PER1 protein, the product of the clock gene Per1 on tyrosine hydroxylase (TH) cells in three dopaminergic populations: tu- beroinfundibular dopaminergic (TIDA), periventricular hypophyseal dopaminergic (PHDA) and in- certohypothalamic dopaminergic (IHDA) cells. PER1 ⁄ TH colocalisation was explored every 4 h through a complete 24-h cycle at postpartum day 7 in does that nursed their pups either at 10.00 h (ZT03) or at 02.00 h (ZT19; ZT0 = 07.00 h, time of lights on). Nonpregnant, nonlactating females were used as controls. In control females, there was a rhythm of PER1 that peaks at ZT15. By contrast, in nursed does, the PER1 peak shifted in parallel to scheduled nursing in TIDA and PHDA cells but not in IHDA cells, which are not related to the control of prolactin. Next, we determined that the absence of suckling for 48 h significantly decreases the number of PER1 ⁄ TH colocalised cells in PHDA but not TIDA cells. Locomotor behaviour in control subjects was maxi- mal at around the time of lights on but, in nursed females, shifted at around the time of sched- uled nursing. Finally, in the suprachiasmatic nucleus, there is a maximal expression of PER1 at ZT11 in the three groups. However, this maximal expression was significantly lower in the nursed groups in relation to the control group and in the groups deprived of nursing for 48 h. We conclude that suckling synchronises dopaminergic cells related to the control of prolactin and appears to be a nonphotic stimulus for the suprachiasmatic nucleus. Key words: prolactin, clock genes, circadian lactation, dopamine, maternal behaviour. doi: 10.1111/j.1365-2826.2011.02138.x Journal of Neuroendocrinology 23, 472–480 ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd Journal of Neuroendocrinology From Molecular to Translational Neurobiology