862 to control the composition of the CSF and cerebral extra- cellular ﬂuid. Tight junction (TJ) proteins that link the cells forming these blood–brain interfaces form the anatomical basis for this control by preventing non-speciﬁc paracel- lular leakage between blood and the cerebral ﬂuids. Inﬂux and efﬂux transporters located at both barriers allow nutrient supply to the brain, while excluding a wide range of potentially deleterious compounds from this organ. The CPs, which are located in the different ventricles of the brain fulﬁll additional speciﬁc functions. They are a source of trophic factors during brain development, are responsi- ble for controlled secretion of CSF, and are major detoxi- fying organs within the brain (Davson and Segal 1996; Saunders et al. 2008; Strazielle and Ghersi-Egea 2000; Zappaterra and Lehtinen 2012). The brain is especially vulnerable during development, and perinatal brain injury can lead to altered myelination, reduced neurogenesis or inappropriate neuronal network organization with dramatic consequences throughout life (Dammann and Leviton 1999; Stolp et al. 2011). The blood–brain interfaces are often considered immature in the developing brain due to a high protein concentration in the CSF (e.g. Adinolﬁ 1985). However, electron micros- copy and tracer experiments in rodents and marsupials indicate that the paracellular pathway is already restricted in early developing brain (Ek et al. 2006; Johansson et al. 2006), and alternative explanations based on CSF space volume, CSF turnover, and transcellular transport of spe- ciﬁc proteins have been brought forward to explain the presence of proteins in the CSF of developing animals (Johansson et al. 2008b). Transfer of exogenous polar compounds and some endogenous proteins between the blood and the CSF has been demonstrated in the devel- oping rat brain, which partly occurs by diffusion (Habgood et al. 1992). A transcellular route has been described for both proteins and small lipid insoluble molecules during development (Balslev et al. 1997; Liddelow et al. 2009), but further studies are required to resolve whether there is also a paracellular component of this transfer before a deﬁnitive conclusion can be reached. Unraveling the complexity of TJs is important to appreciate the level of neuroprotection provided by the blood–brain interfaces at different stages of brain matura- tion, especially during the perinatal period, and to under- stand cerebral drug bioavailability in the context of pediatric treatments. The tightness of the cellular junctions results from the complex interplay of transmembrane and accessory proteins. The transmembrane proteins of the claudin (Cld) family play a crucial role in determining the efﬁciency and selectivity of TJs. In mammals Cld-5 is the main Cld identiﬁed in BBB tight junctions, which also comprise Cld-3 and -12 (Morita et al. 1999b; Nitta et al. 2003; Wolburg et al. 2003). The molecular identity of the 123 Histochem Cell Biol (2012) 138:861–879 TJ-forming Clds at the BCSFB remains more elusive. So far Cld-1/3,-2, and -11 have been described in the rodent or murine CP epithelium (Lippoldt et al. 2000; Wolburg et al. 2001). In human, Cld-5 was also shown to localize in intercellular junctions of the cerebral microvascular endo- thelium (Virgintino et al. 2004), but no data exist con- cerning the composition of TJ that link the choroidal epithelial cells forming the BCSFB. To decipher the molecular composition and understand the complexity of TJs at the BCSFB in the developing rat brain, we analyzed the pre- and post-natal developmental proﬁle of Cld expression in the CP, in comparison to the BBB-forming microvessels (MV), and determined the cellular localization of these proteins. On the basis of the current knowledge of TJ proteins in the brain, and preliminary transcriptomic analyses we performed on cho- roidal tissue, we selected 14 members of the Cld family. Occludin and the three zonula occludens (ZO) peripheral membrane proteins were also included. We report the presence of hitherto undescribed Cld members associated with junctional complexes of the BCSFB and BBB, and show differences in the developmental patterns of TJ pro- teins in the CP of the lateral and fourth ventricle. We ﬁnally demonstrate that knowledge of the early choroidal expres- sion of Clds can be extended to the human BCSFB. Materials and methods Tissue collection Animal care and procedures were conducted according to the guidelines approved by the French ethical committee (decree 87-848) and by the European Community (direc- tive 86-609-EEC). Sprague–Dawley rats, either adult males (250 g), pregnant time-dated females or females with litters were obtained from Janvier (Le Genest Saint Isle, France). All animals were kept under similar conditions in standard cages, with free access to food and tap water under a controlled environment (12 h day/light cycle). Animals were euthanized under isoﬂuorane anesthesia by decapita- tion and their brains were rapidly excised and placed at 4 C in Krebs–Ringer buffer (in mM: 135 NaCl, 4 KCl, 2.2 CaCl2, 1.2MgCl2, 6 NaHCO3,10 HEPES, 5 glucose, pH 7.4). The two lateral ventricle choroid plexuses (LCVP) and the fourth ventricle choroid plexus (4VCP) were dis- sected individually from postnatal day 2 (P2), postnatal day 9 (P9) and adult rats at 4 C under stereomicroscope as previously described and illustrated (Strazielle and Ghersi- Egea 1999, 2000). Nineteen day-old embryos (E19) were removed one by one from the mother, which was kept under isoﬂurane anesthesia on a heated pad and were used for brain sampling and further microdissection of the CPs