876 observed in both CPs an increase of Cld-9, -22, and mostly Cld-2 gene expression, conﬁrmed by Western blot for Cld- 9 and -2, between the embryonic stage E19 and the later developmental stages. Conversely, the expression of Cld-6 and Cld-3 was signiﬁcantly down-regulated in adult rats compared to perinatal animals, concomitantly with a decrease in Cld-3 protein level. Cld-3 is considered a sealing component of the TJ towards mono- and divalent ions as well as non-charged small solutes (Milatz et al. 2010), while Cld-2 was described as a monovalent cation- selective pore-forming junctional protein displaying a high selectivity for Na? and K? (Furuse et al. 2001; Amasheh et al. 2002; Yu 2009). Its overexpression in MDCK cells increases the transfer capacity of tight junctions for small (up to 250 Da) organic compounds (Van Itallie et al. 2008). It was also described as a paracellular water channel (Rosenthal et al. 2010). Thus, the mirror image of Cld-2 and Cld-3 developmental expression proﬁle in the choroi- dal epithelial monolayer suggests that the BCSFB is actu- ally less permeable to selected ions or small molecular weight compounds in developing brain than in adult. This CP speciﬁc developmental regulation of Clds could be related to the CSF secretory function of this tissue. The choroidal CSF secretion rate increases prominently after birth in mammals (Catala 1997). In adult CSF secretion is driven by the apically located Na?–K?–ATPase and car- bonic anhydrase II, which are both up-regulated during perinatal brain development in rat (Johansson et al. 2008a). CSF secretion results from a complex coordinated transport of various inorganic anions and cations across the choroi- dal epithelial monolayer, leading to transcellular ﬂuxes of Na?, Cl- and HCO3- from the blood to the CSF. Our current understanding of CSF secretion implies that only the transfer of K?, necessary to provide adequate K? levels in the CSF, occurs via the paracellular route (Damkier et al. 2010). Thus, the pore-forming Cld-2, whose upregulation in CP parallels the increased rate of CSF secretion, may be involved in K? paracellular movement. Water movement, which is driven by ion exchanges across the choroidal epithelium, was thought to occur transcellularly through aquaporin1 channels. Recently this view was challenged by the phenotypic analysis of aquaporin1 knockout animals. This and other osmolarity data point to mechanisms other than aquaporin-related pathways in CSF water secretion, and revives the paracellular pathway as a route for water transfer from blood to CSF (reviewed in Damkier et al. 2010). In this context a role for Cld-2 in paracellular water movement across the BCSFB, as observed in MDCK cells (Rosenthal et al. 2010) is another appealing hypothesis that could explain the developmental regulation of this Cld. The selective expression of Cld-19 in choroidal epithe- lial cells is another common feature that this barrier shares with the kidney epithelium along with a number of 123 Histochem Cell Biol (2012) 138:861–879 transport systems. In the peripheral organ, Cld-19 associ- ates with Cld-16 in selected areas of the thick ascending limb of the loop of Henle to form pores for cations including Na? and the divalent cations Ca2? and Mg2? (Angelow et al. 2007; Hou et al. 2008, 2009). In contrast, in TJ-like structures of peripheral myelinating Schwann cells, Cld-19 acts as a barrier to cations, and thus achieves the electrical sealing of myelin sheaths within the internodal region, which is necessary to maintain saltatory conduction (Miyamoto et al. 2005). In the brain, we found that Cld-16 is localized at the BBB-forming MVs rather than at CPs. Choroidal Cld-19 is therefore more likely to act as a barrier towards Na? and divalent cations rather than as a pore- forming Cld. This hypothesis is in accordance with the existence of an unidirectional, energy-dependent blood to CSF Ca2? and Mn2? transport across the CP epithelium (Schmitt et al. 2011), whose efﬁciency depends on a closed paracellular pathway to these cations. The sealing function of Cld-19 towards selected cations at the choroidal tight junctions yet remains to be experimentally demonstrated, and the capacity of Cld-19 to homo- or heterodimerize with Clds other than Cld-16 needs further investigations. Like Cld-19 in Schwann cells, Cld-11 fulﬁlls electrical sealing functions in myelinating oligodendrocytes (Morita et al. 1999a). Cl-9 acts as a paracellular permeability bar- rier towards Na? and K? in the cochlea (Nakano et al. 2009). Cld-9 and -11 may have similar functions in the choroidal epithelium and, in conjunction with Cld-19, may confer upon the BCSFB junctions a sealed phenotype towards non-speciﬁc cation leakage. The reason for the reverse developmental regulation of Cld-9 and Cld-11 remains however to be clariﬁed. Another unexpected ﬁnding was the reverse ratios in LVCP to 4VCP expression levels for Cld-2 and -11 during the perinatal period. Very few differences between the various CPs have been documented, partly because LVCPs are preferentially studied. Our results point to such dif- ferences, whose functional signiﬁcance remains to be determined. Evaluation of the respective contribution of LVCPs and 4VCPs to CSF secretion around birth could support the potential involvement of Cld-2 in CSF secretion. Overall, the developmental differences in tight junction gene expression that we observed at the BCSFB appear to be necessary for physiological adaptation and/or microen- vironmental changes during brain development, rather than reﬂecting an immaturity of the barrier. The three most highly expressed Clds (Cld-1, -2 and -3) in rat CPs were also examined by immunohistochemistry in CPs of human fetuses of different gestational stages. Human data paralleled the ﬁndings observed in rat. Immunoreactivity of all three Clds was localized at the epithelial tight junctions in both LVCP and 4VCP at all