Our study for the first time established an ontogenetic postnatal profile of Ucn 1 and CART in the perioculomotor region of the midbrain. The developmental profile of these peptides was investigated simultaneously to compare the timing of their appearance. We observed the absence of Ucn 1 –ir in pIIIu in B6 mice at birth and the progressive development of Ucn 1 peptide and mRNA in these mice through infancy until adulthood. We also did not observe Ucn 1 –ir in any other brain region in the slices stained laterally from the midline at the ages examined during the first postnatal week. Theoretically, the absence of immunoreactivity for a peptide during development can be interpreted as lack of expression of this peptide or as absence of neurons expressing this peptide. However, since Ucn 1 co-localizes with CART in pIII (Kozicz, 2003
; Lazar et al., 2004
; Lima et al., 2008
; Xu et al., 2009
), and CART –ir is already present at PND 1, our studies clearly indicate that pIIIu neurons are already present in this brain area at this developmental stage, but Ucn 1 is not expressed in them. Our findings suggest that it is unlikely that Ucn 1 plays a role in pIII or other brain areas during the first postnatal week. Our finding is in agreement with an increase of Ucn 1 – immunoreactive fibers observed in rat cerebellum at PND 8 and PND 12 (Swinny et al., 2004a
). As described previously for adult mice (Weitemier et al., 2005
), we also did not observe Ucn 1 –ir in the cerebellum, suggesting that postnatal appearance of Ucn 1 in this brain region is a species-specific effect. The fact that Ucn 1 –immunoreactive cells were detected in the inferior olive at PND 3 by Swinny et al (2004a)
, but not by us in this study suggests that developmental expression of Ucn 1 in the inferior olive is also species-specific. The existence of species-specific expression in the Ucn 1 neurocircuit has been documented earlier (Lim et al., 2006
; Weitemier et al., 2005
It can be theorized that although we did not detect Ucn 1 by immunohistochemistry at PND 1 and detected Ucn 1 –ir only in one out of five animals at PND 4, the peptide is still present and active but not detected at these early postnatal ages (for example, due to active release). This idea is contradicted by observations that when Ucn 1 is reported in terminal fibers of adult animals, the Ucn 1 –ir in positive cells appears more intense, suggesting that more peptide is produced than released (Bachtell et al., 2003
; Bittencourt et al., 1999
; Weitemier et al., 2005
). It seems unlikely that in neonatal animals the release is more efficient than in adults. However, a more definitive confirmation could be obtained by future studies simultaneously examining the presence of different CRF-like peptides, as well as receptors and CRF-binding protein in cells and terminals during development.
The patterns of Ucn 1 –ir and Ucn 1 mRNA levels largely showed a parallel development, although it appeared that Ucn 1 mRNA levels continue to increase further into adulthood. This however, could be due to technical issues because on one hand qRT-PCR is more quantitative than immunohistochemical detection, but on the other hand, qRT-PCR relies on correct dissection of the pIIIu, which is difficult at early postnatal ages. Importantly both methods show that Ucn 1 levels in pIII increase during postnatal development by more than an order of magnitude, while levels of CART stay mostly unchanged.
It is worth noting that the very low levels of Ucn 1 during early postnatal development coincide with the hyporesponsive period of the hypothalamic-pituitary-adrenal axis (Levine, 1994
; Sapolsky and Meaney, 1986
). Although pIIIu neurons are activated by stress, it has been shown that this activation is independent of glucocorticoids (Gaszner and Kozicz, 2003
; Kozicz et al., 2004
; Weninger et al., 2000
). However, the postnatal development of Ucn 1 expression and stress sensitivity is in agreement with the contribution of Ucn 1 to regulation of stress-related functions (Kozicz, 2007
The role of Ucn 1 in pIIIu in sensitivity to ethanol and addictive drugs, hypothermic responses, and food and ethanol intake in adult B6 mice has been extensively documented (reviewed in (Ryabinin and Weitemier, 2006
)). For example, Ucn 1 immunoreactivity in pIIIu is higher in two replicate lines of mice selectively bred for high alcohol preference versus two replicate control lines (Bachtell et al., 2003
), in two replicate lines of mice selectively bred for high alcohol-induced hypothermia versus two replicate control lines (Bachtell et al., 2002b
), in mice selectively bred for high alcohol conditioned place preference versus its control line (Kiianmaa et al., 2003
), and in four lines of rats selectively bred to prefer alcohol versus corresponding low alcohol preferring control lines (Fonareva et al., 2009
; Turek et al., 2005
). In agreement with these genetic studies, lesions of pIIIu significantly decrease alcohol preference and food consumption in mice (Bachtell et al., 2004
; Weitemier and Ryabinin, 2005
). However, little is known regarding the potential role of this neuropeptide during development. Since we observe that Ucn 1 –ir in pIIIu develops at later postnatal stages, it is unlikely that Ucn 1 plays a role in these behaviors prior to PND 8. Nevertheless, it is remarkable that this time course parallels the development of several relevant behaviors. Specifically, previous findings showed that sensitivity to the hypothermic effect of ethanol in mice, a response regulated by Ucn 1 (Bachtell et al., 2002b
; Turek and Ryabinin, 2005
), develops around PNDs 12–15 (French et al., 1995
; Wood et al., 1999
). On the other hand, sensitivity to the locomotor-stimulating effects of ethanol in mice, a behavior that is likely not influenced by Ucn 1 (Bachtell et al., 2003
), develops around PND 30 (Wood et al., 1999
), an age when Ucn 1 –ir in pIIIu in the present study reached levels close to those observed in adults. While alcohol intake has not been studied in mice at early postnatal ages, it has been shown that rats consume high amounts of ethanol before PND 8 (Sanders and Spear, 2007
) and that intake of ethanol is higher during PND 9–14 than in adults (Hall, 1979
; McKinzie et al., 1999
), a finding in agreement with inhibitory effects of centrally-administered Ucn 1 on ethanol intake (Ryabinin et al., 2008
). Finally, it should be noted that the development of Ucn 1 expression clearly coincides with the development of independent feeding in pups (Hall and Browde, 1986
). The parallel development of Ucn 1 –ir with developmental changes in these behaviors is in agreement with the importance of this peptide for regulation of food and alcohol self-administration.
It has also been shown that administration of exogenous Ucn 1 can have neuroprotective and neurotrophic effects (Abuirmeileh et al., 2007
; Brar et al., 2000
; Calle et al., 2005
; Choi et al., 2006
; Facci et al., 2003
; Gounko et al., 2005
; Swinny et al., 2004b
). Therefore, it was theoretically possible that Ucn 1, as a peptide released from pIII, could play a trophic or guiding role for other neurons or their processes. Since Ucn 1 appears to be almost absent during the first postnatal days, our results argue against such a role for Ucn 1 during early postnatal development.
Additionally, our results indicate that there is certain degree of variability when Ucn 1 –ir starts to appear in pIII. Thus, while six of seven mice examined prior to PND 8 using brightfield and fluorescent immunohistochemistry did not show Ucn 1 –ir, one PND 4 mouse did. This occurred despite the fact that these mice are inbred, and are at an age when social hierarchy in a cage is unlikely to play a role. It is also unlikely that this observation is due to sex differences because it appears much earlier than gonadal maturation. While the reason for a different start of Ucn 1 expression in pIIIu deserves future investigations, our findings indicate that in a majority of animals Ucn 1 –ir is absent prior to PND 8.
In contrast to Ucn 1, CART –ir has been shown to migrate to the perioculomotor region prenatally and occupy the location of pIII already at embryonic day 16 in rats. In fact, CART may be one of the earliest neuropeptides with a neuromodulatory role that is expressed in the brain (Brischoux et al., 2002
; Risold et al., 2006
). Therefore, while we argue that a developmental role of Ucn 1 is unlikely, it is quite possible that CART from pIIIu neurons can have such a role. Moreover, finding that expression of CART and Ucn 1 appears in this brain region at such different ages, indicates differential regulation of genes expressed in pIII neurons during development. The pIIIu shows preferential expression of several additional genes involved in regulation of food consumption, stress and energy homeostasis, such as cholecystokinin, nesfatin and the growth hormone secretagogue receptor (also known as ghrelin receptor) (Brailoiu et al., 2007
; Innis and Aghajanian, 1986
; Maciewicz et al., 1984
; Xu et al., 2009
). It would be important to comparatively investigate developmental profiles of different behaviorally-important genes in this brain region.
Taken together, our studies for the first time show a differential expression of genes (Ucn 1 versus CART) within a specific subregion of pIII during postnatal development. This population of pIII neurons has been shown previously to play a role in regulation of stress-related behaviors, alcohol sensitivity, alcohol drinking, and food consumption. Future studies should pay attention to age of animals when elucidating roles of genes expressed in pIII in these behaviors. We suggest that Ucn 1’s role in these behaviors is age-dependent.