Search

Studies of the choroid plexus lag behind those of the more widely known blood-brain barrier, despite a much longer history. This review has two overall aims. The first is to outline long-standing areas of research where there are unanswered questions, such as control of cerebrospinal fluid (CSF) secretion and blood flow. The second aim is to review research over the past 10 years where the focus has shifted to the idea that there are choroid plexuses located in each of the brain's ventricles that make specific contributions to brain development and function through molecules they generate for delivery via the CSF. These factors appear to be particularly important for aspects of normal brain growth. Most research carried out during the twentieth century dealt with the choroid plexus, a brain barrier interface making critical contributions to the composition and stability of the brain's internal environment throughout life. More recent research in the twenty-first century has shown the importance of choroid plexus-generated CSF in neurogenesis, influence of sex and other hormones on choroid plexus function, and choroid plexus involvement in circadian rhythms and sleep. The advancement of technologies to facilitate delivery of brain-specific therapies via the CSF to treat neurological disorders is a rapidly growing area of research. Conversely, understanding the basic mechanisms and implications of how maternal drug exposure during pregnancy impacts the developing brain represents another key area of research.

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

The protective barriers of the brain provide a complex series of physical and chemical obstacles to movement of macromolecules from the periphery into the central nervous system. Studies on these barriers have been focused on two main research areas: (i) anatomical and physiological descriptions of their properties, including during development where functioning barriers are likely to be important for normal neuronal growth; and (ii), investigations of these barriers during disease and attempts at overcoming their defenses in order to deliver drugs to the central nervous system. Both fields are now advanced by the application of molecular gene expression studies of cerebral endothelia (blood vasculature, site of the blood-brain barrier) and choroid plexus epithelia (site of the blood-cerebrospinal fluid barrier) from developing and adult brains, particularly with respect to solute-linked carriers and other transporters. These new techniques provide a wealth of information on the changing nature of transporters at barrier interfaces during normal development and following disease. This review outlines published findings from transcriptome and qPCR studies of expression of genes coding for transporters in these barriers, with a focus on developing brain. The findings clearly support earlier published physiological data describing specific transport mechanisms across barrier interfaces both in the adult and in particular in the developing brain., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Specialized populations of choroid plexus epithelial cells have previously been shown to be responsible for the transfer of individual plasma proteins from blood to the cerebrospinal fluid (CSF), contributing to their characteristically high concentrations in CSF of the developing brain. The mechanism of this protein transfer remains elusive. Using a marsupial, Monodelphis domestica, we demonstrate that the albumin-binding protein SPARC (osteonectin/BM-40/culture-shock protein) is present in a subset of choroid plexus epithelial cells from its first appearance, throughout development, and into adulthood. The synthesis of SPARC by the lateral ventricular plexus was confirmed with real-time PCR. The expression level of SPARC was higher in plexuses of younger than older animals. Western blot analysis of the gene product confirmed the quantitative PCR results. The co-localization of SPARC and albumin shown by immunocytochemistry and its cellular location indicate that this glycoprotein may act as a recognition site for albumin. In addition, the numbers of SPARC-immunopositive cells and its expression were responsive to experimental changes of albumin concentration in the blood. It is suggested that SPARC may be one of the molecules that govern the uptake and delivery of proteins from blood to the CSF. The results also confirm that protein transfer across the blood-CSF barrier is developmentally and physiologically regulated., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Choroid plexus epithelial cells secrete cerebrospinal fluid (CSF) and transfer molecules from blood into CSF. Tight junctions between choroidal epithelial cells are functionally effective from early in development: the route of transfer is suggested to be transcellular. Routes of transfer for endogenous and exogenous plasma proteins and dextrans were studied in Monodelphis domestica (opossum). Pups at postnatal (P) days 1-65 and young adults were injected with biotinylated dextrans (3-70 kDa) and/or foetal protein fetuin. CSF, plasma and brain samples were collected from terminally anaesthetized animals. Choroid plexus cells containing plasma proteins were detected immunocytochemically. Numbers of plasma protein-positive epithelial cells increased to adult levels by P28, but their percentage of plexus cells declined. Numbers of cells positive for biotinylated probes increased with age, while their percentage remained constant. Colocalization studies showed specificity for individual proteins in some epithelial cells. Biotinylated probes and endogenous proteins colocalized in about 10% of cells in younger animals, increasing towards 100% by adulthood. Injections of markers into the ventricles demonstrated that protein is transferred only from blood into CSF, whereas dextrans pass in both directions. These results indicate that protein and lipid-insoluble markers are transferred by separate mechanisms present in choroid plexuses from the earliest stage of brain development, and transfer of proteins from plasma across choroid plexus epithelial cells contributes to the high protein concentration in CSF in the immature brain.

The choroid plexuses secrete cerebrospinal fluid (CSF) and regulate the brain's internal environment via the blood-CSF barrier. The permeability properties of the blood-CSF interface have been studied previously in adult and immature brains, however, little is known about the development of CSF secretion and its modulation. ATP influences secretion in other epithelia via ionotropic P2X or metabotropic P2Y receptors. P2 receptors have frequently been found to be down-regulated in the postnatal period, suggesting a developmental role for purinergic and pyrimidine signalling. The present study investigated the expression of P2 receptors in lateral ventricular choroid plexus in relation to recent studies of aquaporin-1 expression and rapid expansion of the lateral ventricles in rat embryos. In the present study mRNAs for all known mammalian nucleotide receptor subtypes, except P2X(7), were identified from as early as E15. P2X(7) mRNA was detected from E18. Indications of differential expression patterns were observed for the different subtypes during development: an apparent increase in expression for P2Y(2) and P2X(7), a decline in P2X(1-2,4), no detectable difference in expression levels for P2X(6) and P2Y(12-13) and transient expression peaks for P2X(3,5) and P2Y(1,4,6,14). P2X(4,5,7) and P2Y(1,4) receptor proteins were detected immunohistochemically in the choroidal epithelium from early in development (E15 or E18). Their differing developmental profiles suggest specific roles in the development of CSF secretion that may have particular relevance for the rapid expansion of the ventricles that occurs in the embryo. P2X(5) and P2Y(6) were also detected in the developing neuropendyma from P0 and P9, respectively.

The normal brain develops within a well-controlled stable internal "milieu" protected by specialised mechanisms referred to collectively as blood-brain barriers. A fundamental feature of this environment is the control of water flow in and out of the developing brain. Because of limited vascularisation of the immature brain, choroid plexuses, via the cerebrospinal fluid, have been proposed as the main route of fluid exchange between the blood and brain interfaces. We describe the temporal expression and appearance of aquaporin-1 (AQP1) which is important for water transfer across adult choroid plexuses. AQP1 expression was studied in rat embryos using real time reverse transcription/polymerase chain reaction. mRNA for AQP1 was present in rat brain at embryonic day 12 (E12) one day before the protein was detectable in the fourth ventricular choroid plexus (the first plexus to appear); its relative levels increased at E13-E14 when more AQP1-immunoreactive cells appeared in all plexuses. The presence of AQP1 was determined immunocytochemically in five different mammalian species (rat, mouse, human, sheep and opossum) in all four choroid plexuses from their earliest appearance. In all five species studied, the appearance of AQP1 immunoreactivity followed the same developmental sequence: the fourth, lateral and, finally, third ventricular choroid plexus. The stage of choroid plexus development when AQP1 was first detected in all five species and in all four choroid plexuses corresponded to the transition between Stages I and II. The cellular localisation of AQP1 in all choroid plexuses, as soon as it was detectable, had the characteristic apical membrane distribution previously described in the adult; a basolateral membrane localisation was also observed.

The structural and functional development of the choroid plexuses, the site of the blood-cerebrospinal fluid (CSF) barrier, in an opossum (Monodelphis domestica) was studied. Marsupial species are extremely immature at birth compared with more conventional eutherian species. Choroid plexus tissue of each brain ventricle, from early stages of development, was collected for light and electron microscopy. During development, the choroidal epithelium changes from a pseudostratified to a cuboidal layer. Individual epithelial cells appear to go through a similar maturation process even though the timing is different between and within each plexus. The ultrastructural changes during development in the choroidal epithelial cells consist of an increase in the number of mitochondria and microvilli, and changes in structure of endoplasmic reticulum. There are also changes in the core of plexuses with age. In contrast, the structure of the tight junctions between epithelial cells does not appear to change with maturation. In addition, the route of penetration for lipid insoluble molecules from blood to CSF across the choroid plexuses was examined using a small biotin-dextran. This showed that the tight junctions already form a functional barrier in early development by preventing the paracellular movement of the tracer. Intracellular staining shows that there may be a transcellular route for these molecules through the epithelial cells from blood to CSF. Apart from lacking a glycogen-rich stage, cellular changes in the developing opossum plexus seem to be similar to those in other species, demonstrating that this is a good model for studies of mammalian choroid plexus development., (Copyright 2003 Wiley-Liss, Inc.)

Mammalian choroid plexuses develop at four sites in the roof of the neural tube shortly after its closure, in the order IVth, lateral, and IIIrd ventricles. Bone morphogenetic proteins and tropomyosin are involved in early specification of these sites and in early plexus growth. Four stages of lateral ventricular plexus development have been defined, based on human and sheep fetuses; these depend mainly on the appearance of epithelial cells and presence or absence of glycogen. Other plexuses and other species are probably similar, although marsupials may lack glycogen. Choroid plexuses form one of the blood-brain barrier interfaces that control the brain's internal environment. The mechanisms involved combine a structural diffusion restraint (tight junctions between the plexus epithelial cells) and specific exchange mechanisms. In this review, it is argued that barrier mechanisms in the developing brain are different in important respects from those in the adult brain, but these differences do not necessarily reflect immaturity of the system. Absence of a barrier mechanism or presence of one not found in the adult may be a specialisation that is appropriate for that stage of brain development. Emphasis is placed on determining which mechanisms are present in the immature brain and relating them to brain development. One mechanism unique to the developing brain transfers specific proteins from blood to cerebrospinal fluid (CSF), via tubulocisternal endoplasmic reticulum in plexus epithelial cells. This results in a high concentration of proteins in early CSF. These proteins do not penetrate into brain extracellular space because of "strap" junctions between adjacent neuroependymal cells, which disappear later in development, when the protein concentration in CSF is much lower. Functions of the proteins in early CSF are discussed in terms of generation of a "colloid" osmotic pressure that expands the ventricular system as the brain grows; the proteins may also act as specific carriers and growth factors in their own right. The pathway for low molecular weight compounds, which is much more permeable in the developing choroid plexuses, appears also to be a transcellular one, rather than paracellular via tight junctions. There is thus good evidence to support a novel view of the state of development and functional significance of barrier mechanisms in the immature brain. It grows in an environment that is different from that of the rest of the fetus/neonate and that is also different in some respects from that of the adult. But these differences reflect developmental specialisation rather than immaturity., (Copyright 2001 Wiley-Liss, Inc.)

The nature of the barriers that keep proteins out of the developing brain has been studied in tissues obtained from fetal sheep in experiments conducted under controlled physiological conditions. In anaesthetised pregnant ewes, 60 day gestation fetuses (term is 150 days) were exposed to human albumin injected intravenously for periods up to 6 h. The immunocytochemical distribution of exogenous human albumin was compared with that of endogenous sheep albumin at both the light and electron-microscopical level. Immunogold labelling of ultracryosections suggests that a tubulocisternal endoplasmic reticulum system in immature choroid-plexus epithelial cells is the route by which albumin crosses from blood to cerebrospinal fluid (CSF) in the developing brain. The integrity of the blood-brain barrier, the blood-cerebrospinal fluid barrier and the cerebrospinal fluid-brain barrier to protein, was confirmed. In addition, at the outer surface of the developing brain there also appears to be a restriction on the passage of albumin from CSF into the brain. These observations support earlier proposals that the immature brain develops within an internal environment from which proteins in plasma and CSF are largely excluded.

1. Blood-cerebrospinal fluid (CSF) transfer of various exogenous albumins has been investigated in developing Monodelphis domestica (South American grey short-tailed opossum) and compared with the steady-state CSF: plasma ratios for endogenous (Monodelphis) albumin. Ratios for Monodelphis albumin and human albumin were similar and were the highest at postnatal day 5 (P5) (48.2 +/- 4.4 and 40.6 +/- 4.5%, respectively). The ratio for bovine albumin was similar to the steady-state ratio for Monodelphis albumin at P7-8 but became consistently lower than the Monodelphis albumin ratio at all other ages until P32-36 when all albumins tested attained a similar low ratio. The CSF:plasma ratio of chemically modified (succinylated) bovine albumin was always significantly lower than that of other albumins, except at the oldest age examined (P32-36). 2. Immunocytochemistry showed that within the brain, albumin was confined to the lumen and endothelial cells of blood vessels. In the choroid plexus only a small proportion (0.2-1.7% of the total cell number) of epithelial cells was positive for albumin, both endogenous and exogenous, at all ages studied (except the 3rd ventricle where cells were only positive from P8). The CSF was strongly positive for all albumins. The peak proportion of positive cells and of albumin concentrations in CSF occurred at P8. These findings suggest that the primary route for penetration of albumin into CSF is directly across the choroid plexus rather than via the brain. 3. Double-labelling immunocytochemistry revealed that the same epithelial cells contained both endogenous (Monodelphis) and exogenous (human) albumin. In contrast, for succinylated albumin, at P7 only about 35% (lateral ventricle) and 50% (4th ventricle) of Monodelphis albumin-positive cells were also positive for succinylated albumin, but by P30 this proportion increased to 90% at both sites. 4. Thus the developing choroid plexus distinguishes between different albumins. Chemical modification of albumin (succinylation) disrupts this mechanism. It is proposed that in older animals (P32-36) all of the albumin in the CSF is derived from plasma by diffusion (as in adult animals). At earlier stages of development, a proportion of the albumin in CSF also appears to be transferred from the plasma by diffusion with an additional component transferred by a mechanism that can distinguish between different species of albumin. The main route of entry of albumin to CSF seems likely to be via the choroid plexus epithelial cells.

The distributions of transthyretin and albumin in the choroid plexus during brain development have been compared. The South American opossum was chosen because the young are born around the time of choroid plexus formation. Previous work showed that in the adult opossum, transthyretin is expressed in the choroid plexus cells. However, systematic studies of transthyretin in the choroid plexus during development have not been carried out before. Transthyretin was present in 90-95% of the choroidal cells form birth to adulthood. In most cells, transthyretin immunoreactivity was concentrated in the apical region of the cytoplasm. Double labelling of choroid plexus sections with antibodies to albumin and transthyretin showed that 1-2% of cells were positive for both proteins. These findings suggest that from the very earliest stage of choroid plexus formation most epithelial cells both synthesize and contain transthyretin, and a few of these transthyretin-synthesizing cells also contain albumin that is probably being transferred form blood to the cerebrospinal fluid.