Mammalian Choroid Plexus Research Articles

Augurin production in the mammalian choroid plexus: Implications for CSF and hydrocephalus

Whereas augurin, a protein encoded by the ecrg4 gene, is highly conserved across species, bioinformatic algorithms predict the existence of several other potential hormonelike peptide products transcribed from the same gene. With gene expression highest in the mammalian choroid plexus (CP) compared to all other tissue types however, we deemed it critical to know which peptide(s) is produced by the CP so as to determine its potential release into, and activity in, cerebrospinal fluid (CSF). Our previous data has shown that gene knockdown in developing zebrafish causes severe and dose-dependent hindbrain edema/hydrocephalus. Accordingly, we suggested a novel function for ecrg4 gene products in CP physiology and implicated them as new hormone(s) regulating CSF and CP function. Immunohistochemical staining showed protein in CP epithelium in vitro and in vivo, and ligandtargeting shows internalization into ependymal cells. Materials and methods Ecrg4 gene products were detected by immunoblotting and immunofluorescence with chicken and rabbit polyclonal or mouse monoclonal antibodies that we raised. DNA sequences for fragments (31-148), (31-70) and (71-148) were cloned into pET15b vector, expressed in BL21DE3pLysS and purified. The human gene was cloned into the pLVx-IRES-ZsGreen vector and virus generated using the LentiX kit (Clontech) while siRNA lentivirus was obtained from Santa Cruz Biotechnology.

Molecular mechanisms of cerebrospinal fluid production

The epithelial cells of the choroid plexuses secrete cerebrospinal fluid (CSF), by a process which involves the transport of Na +, Cl − and HCO 3 − from the blood to the ventricles of the brain. The unidirectional transport of ions is achieved due to the polarity of the epithelium, i.e. the ion transport proteins in the blood-facing (basolateral) membrane are different to those in the ventricular (apical) membrane. The movement of ions creates an osmotic gradient which drives the secretion of H 2O. A variety of methods (e.g. isotope flux studies, electrophysiological, RT-PCR, in situ hybridization and immunocytochemistry) have been used to determine the expression of ion transporters and channels in the choroid plexus epithelium. Most of these transporters have now been localized to specific membranes. For example, Na +-K +ATPase, K + channels and Na +-2Cl −-K + cotransporters are expressed in the apical membrane. By contrast the basolateral membrane contains Cl −- HCO 3 exchangers, a variety of Na + coupled HCO 3 − transporters and K +-Cl − cotransporters. Aquaporin 1 mediates water transport at the apical membrane, but the route across the basolateral membrane is unknown. A model of CSF secretion by the mammalian choroid plexus is proposed which accommodates these proteins. The model also explains the mechanisms by which K + is transported from the CSF to the blood.

Structural characteristics and barrier properties of the choroid plexuses in developing brain of the opossum (Monodelphis Domestica).

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.

Mechanisms of CSF secretion by the choroid plexus.

The epithelial cells of the choroid plexus secrete cerebrospinal fluid (CSF), by a process that involves the movement of Na(+), Cl(-) and HCO(3)(-) from the blood to the ventricles of the brain. This creates the osmotic gradient, which drives the secretion of H(2)O. The unidirectional movement of the ions is achieved due to the polarity of the epithelium, i.e., the ion transport proteins in the blood-facing (basolateral) are different to those in the ventricular (apical) membranes. Saito and Wright (1983) proposed a model for secretion by the amphibian choroid plexus, in which secretion was dependent on activity of HCO(3)(-) channels in the apical membrane. The patch clamp method has now been used to study the ion channels expressed in rat choroid plexus. Two potassium channels have been observed that have a role in maintaining the membrane potential of the epithelial cell, and in regulating the transport of K(+) across the epithelium. An inward-rectifying anion channel has also been identified, which is closely related to ClC-2 channels, and has a significant HCO(3)(-) permeability. This channel is expressed in the apical membrane of the epithelium where it may play an important role in CSF secretion. A model of CSF secretion by the mammalian choroid plexus is proposed that accommodates these channels and other data on the expression of transport proteins in the choroid plexus.

Choroid plexus recovery after transient forebrain ischemia: role of growth factors and other repair mechanisms.

1. Transient forebrain ischemia in adult rats, induced by 10 min of bilateral carotid occlusion and an arterial hypotension of 40 mmHg, caused substantial damage not only to CA-1 neurons in hippocampus but also to epithelial cells in lateral ventricle choroid plexus. 2. When transient forebrain ischemia was followed by reperfusion (recovery) intervals of 0 to 12 hr, there was moderate to severe damage to many frond regions of the choroidal epithelium. In some areas, epithelial debris was sloughed into cerebrospinal fluid (CSF). Although some epithelial cells were disrupted and necrotic, their neighbors exhibited normal morphology. This patchy response to ischemia was probably due to regional differences in reperfusion or cellular metabolism. 3. Between 12 and 24 hr postischemia, there was marked restoration of the Na+, K+, water content, and ultrastructure of the choroid plexus epithelium. Since there was no microscopical evidence for mitosis, we postulate that healthy epithelial cells either were compressed together on the villus or migrated from the choroid plexus stalk to more distal regions, in order to "fill in gaps" along the basal lamina caused by necrotic epithelial cell disintegration. 4. Epithelial cells of mammalian choroid plexus synthesize and secrete many growth factors and other peptides that are of trophic benefit following injury to regions of the cerebroventricular system. For example, several growth factors are upregulated in choroid plexus after ischemic and traumatic insults to the central nervous system. 5. The presence of numerous types of growth factor receptors in choroid plexus allows growth factor mediation of recovery processes by autocrine and paracrine mechanisms. 6. The capability of choroid plexus after acute ischemia to recover its barrier and CSF formation functions is an important factor in stabilizing brain fluid balance. 7. Moreover, growth factors secreted by choroid plexus into CSF are distributed by diffusion and convection into brain tissue near the ventricular system, e.g., hippocampus. By this endocrine-like mechanism, growth factors are conveyed throughout the choroid plexus-CSF-brain nexus and can consequently promote repair of ischemia-damaged tissue in the ventricular wall and underlying brain.

Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells.

The function of the apical Na+-K+-2Cl- cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl-/HCO-3 (anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 microM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+ removal or by addition of 100 microM bumetanide. Exposure of CP cells to 5 mM BaCl2 induced rapid swelling that was inhibited by 100 microM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+ channels to reabsorb K+ from cerebrospinal fluid to blood.

Microdialysis analysis of effects of loop diuretics and acetazolamide on chloride transport from blood to CSF

With the hypothesis that the NaCl cotransporter in mammalian choroid plexus (CP) has a role in CSF formation, we postulated that loop diuretic agents would curtail transport of Cl from blood to CSF. Microdialysis in the cisterna magna of Sprague-Dawley rats was used to assess the ability of furosemide and ethacrynic acid (i.e. loop agents that interfere directly with cotransport) to inhibit 36Cl transport from blood to CSF over a 3-h period. Cl uptake by CSF was quantified as % volume of distribution ( V d) of 36Cl, i.e. 100 ×cpm/g CSF÷cpm/ml plasma. Uptake curves of V d vs. time were constructed for the various treatments; then, to compare drug effects, the curves were analyzed for: (i) the early slope of uptake ( K d), (ii) the steady-state value for V d, and (iii) the area-under-curve (AUC). Assessment of the curve parameters collectively revealed that at 5 mg/kg, both furosemide (FUR) and ethacrynic acid (EA) reduced Cl penetration into CSF by one quarter; at 50 mg/kg, these loop agents decreased Cl uptake by about a third. On the other hand, 50 mg/kg of the carbonic anhydrase inhibitor, acetazolamide, reduced Cl uptake into CSF by 55–60%. Thus, NaCl cotransport inhibitors maximally reduced Cl transport in the rat by about 35%; this inhibition was less extensive than that brought about by acetazolamide, which interferes with CSF secretion by a different mechanism.

Expression of retinol-binding protein mRNA in mammalian choroid plexus

1. 1. The expression of the gene for retinol-binding protein in choroid plexus from rats, mice, sheep and cattle is demonstrated by Northern analysis and reverse transcription coupled with polymerase chain reaction. 2. 2. The content of retinol-binding protein mRNA in total RNA from choroid plexus of ruminants was higher than that for RNA from any other extrahepatic tissues of adult animals reported so far. 3. 3. The ratios of retinol-binding protein mRNA levels in choroid plexus RNA to those in liver RNA varied greatly among rodents, ruminants and carnivores.

Cotransport of sodium and chloride by the adult mammalian choroid plexus

Cerebrospinal fluid formation stems primarily from the transport of Na and Cl in choroid plexus (CP). To characterize properties and modulation of choroidal transporters, we tested diuretics and other agents for ability to alter ion transport in vitro. Adult Sprague-Dawley rats were the source of CPs preincubated with drug for 20 min and then transferred to cerebrospinal fluid (CSF) medium containing 22Na or 36Cl with [3H]mannitol (extracellular correction). Complete base-line curves were established for cellular uptake of Na and Cl at 37 degrees C. The half-maximal uptake occurred at 12 s, so it was used to assess drug effects on rate of transport (nmol Na or Cl/mg CP). Bumetanide (10(-5) and 10(-4) M) decreased uptake of Na and Cl with maximal inhibition (up to 45%) at 10(-5) M. Another cotransport inhibitor, furosemide (10(-4) M), reduced transport of Na by 25% and Cl by 33%. However, acetazolamide (10(-4) M) and atriopeptin III (10(-7) M) significantly lowered uptake of Na (but not Cl), suggesting effect(s) other than on cotransport. The disulfonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 10(-4) M), known to inhibit Cl-HCO3 exchange, substantially reduced the transport of 36Cl. Bumetanide plus DIDS (both 10(-4) M) caused additive inhibition of 90% of Cl uptake, which provides strong evidence for the existence of both cotransport and antiport Cl carriers. Overall, this in vitro analysis, uncomplicated by variables of blood flow and neural tone, indicates the presence in rat CP of the cotransport of Na and Cl in addition to the established Na-H and Cl-HCO3 exchangers.

The Mammalian Choroid Plexus

T he brain is choosy about what it allows to enter its tissues. Because it can function only in a chemically stable environment, the brain shuts out most water-soluble substances in the blood-even some potentially helpful medications. Three structures stand as gatekeepers to the brain's interior: the network of cere­ bral capillaries, the arachnoid mem­ brane that covers the brain's surface and the diffuse, highly vascularized tissue called the choroid plexus. The choroid plexus and the arach­ noid membrane act together as barri­ ers between the blood and the cere­ brospinal fluid (CSF), a watery broth of nutrients, ions and other essential molecules. The CSF bathes the exterior of the central nervous system and fills the ventricles, the four large cavities inside the brain. Because the CSF ex­ changes substances freely with the interstitial fluid that surrounds the brain's neurons and supportive glial cells, the blood-CSF barrier is vital for keeping dangerous compounds out of the brain. The arachnoid membrane is generally impermeable to water-solu­ ble substances, and its role in forming a blood-CSF barrier is largely passive.

Cerebrospinal fluid calcium homeostasis: evidence for a plasma membrane Ca 2+-pump in mammalian choroid plexus

A major unanswered question in central nervous system physiology concerns the mechanism by which cerebrospinal fluid (CSF) Ca 2+ homeostasis is maintained in the face of hypo- or hypercalcemia. To address this question, we sought and found a protein of M r ∼ 140,000 in choroid plexus plasma membranes that forms a phosphorylated intermediate with characteristics of a plasma membrane Ca 2+-pump. A choroid plexus plasma membrane protein of this molecular weight also bound to a monoclonal antibody prepared against the human erythrocyte plasma membrane Ca 2+-Mg 2+ ATPase Ca 2+-pump. When this monoclonal antibody was used for immunohistochemical localization, the plasma membrane Ca 2+-pump was found primarily in the CSF-facing membranes of choroid plexus cells from rats, cats, and man. The localization of a plasma membrane Ca 2+-pump in the CSF-facing membranes of the choroid plexus suggests that the choroid plexus, by mechanisms including this pump, may regulate CSF Ca 2+ concentrations.

125I]LSD labels 5-HT 1C recognition sites in pig choroid plexus membranes. Comparison with [ 3H]mesulergine and [ 3H]5-HT binding

The mammalian choroid plexus is enriched in a newly described serotonin recognition site, the 5-HT 1C site. In order to further characterize these sites, the binding characteristics of [ 125I]LSD, [ 3H]mesulergine and [ 3H]serotonin to pig choroid plexus membranes were compared. These ligands labelled with high affinity a similar number of sites. The binding profiles of the sites labelled with these radioligands are indistinguishable as illustrated by highly significant correlation parameters. These sites are very similar to those labelled by N 1-methyl-2-[ 125I]LSD and [ 125I]LSD in pig and rat choroid plexus membranes. The data demonstrate that these ligands label 5-HT 1C recognition sites in pig and rat choroid plexus membranes.

Cl-HCO3 exchange in choroid plexus: analysis by the DMO method for cell pH

[14C]DMO distribution was used to measure steady-state intracellular pH (pHi) and [HCO3]i in adult rat choroid plexus (CP) incubated in synthetic cerebrospinal fluid (CSF) for 30 min. In controls at 37 degrees C, mean pHi (6.95 at PCO2 = 30 mmHg) was close to corresponding in vivo values; and [HCO3]i/[HCO3]csf, i.e., rHCO3, was 0.37. At normal [HCO3]csf = 18 mM, cell HCO3 was accumulated threefold above electrochemical equilibrium (as estimated from Em = -50 mV). [HCO3]i decreased proportionally with [HCO3]csf, as the latter was altered from 47 to 9 mM; in severe extracellular acidosis [( HCO3]csf = 3.7 mM), [HCO3]i was not reduced further and so rHCO3 rose to 0.66. Except in low [HCO3]csf, acetazolamide and ouabain (10(-4) M) caused small depletion of cell HCO3. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid lowered [HCO3]i by 60%, thus decreasing rHCO3 (0.16) and rCl (0.25) to values close to estimated equilibrium distribution (0.15). Substitution of CSF Cl with isethionate resulted in marked alkalinization of pHi when [Cl]csf was depleted to 12 mM. Augmented PCO2 associated with temperature reduction to 15 degrees C elevated [HCO3]i, thereby increasing rHCO3 (to 0.66) as well as rCl. Anion distribution ratios indicate heteroanion exchange in mammalian CP.

Autonomic nerves in the mammalian choroid plexus and their influence on the formation of cerebrospinal fluid.

The choroid plexuses of all ventricles receive a well-developed adrenergic and cholinergic innervation reaching both the secretory epithelium and the vascular smooth muscle cells. Also peptidergic nerves, containing vasoactive intestinal polypeptide, are present but primarily associated only with the vascular bed. A sympathetic inhibitory effect on the plexus epithelium has been indicated in determinations of carbonic anhydrase activity and by studies of various aspects of active transport in isolated plexus tissue. Pharmacological analysis in vitro has shown the choroidal arteries to possess both vasoconstrictory alpha-adrenergic and vasodilatory beta-adrenergic receptors. Electrical stimulation of the sympathetic nerves, which originate in the superior cervical ganglia, induces as much as 30% reduction in the net rate of cerebrospinal fluid (CSF) production, while sympathectomy results in a pronounced increase, about 30% above control, in the CSF formation. There is strong reason to believe that the choroid plexus is under the influence of a considerable sympathetic inhibitory tone under steady-state conditions. From pharmacological and biochemical experiments it is suggested that the sympathomimetic reduction in the rate of CSF formation is the result of a combined beta-receptor-mediated inhibition of the secretion from the plexus epithelium and a reduced blood flow in the choroid plexus tissue resulting from stimulation of the vascular alpha-receptors. The choroid plexus probably also represents an important inactivation site and gate mechanism for sympathomimetic amines, as evidenced by considerable local activity of catechol-O-methyl transferase and monoamine oxidase, primarily type B. The CSF production rate is also reduced by cholinomimetic agents, suggesting the presence of muscarinic-type cholinergic receptors in the choroid plexus.

Peptidergic (VIP) nerves in the mammalian choroid plexus

The mammalian choroid plexus was examined by immunohistochemistry and radioimmunoassay for the presence of nerves containing vasoactive intestinal polypeptide (VIP). A moderate number of VIP-immunoreactive nerve fibres were found in choroid plexuses from pig. The corresponding concentration of chemically measured VIP was approx. 10 pmol/g. VIP nerves were few in plexuses from cow, cat and rabbit and rare in plexuses from mouse and rat. Pure porcine VIP produced relaxation of isolated bovine anterior choroidal artery in vitro.

Histochemical study on regional differences in the cholinergic nerve supply of the choroid plexus from various laboratory animals

The cholinergic nerve supply of the mammalian choroid plexus was investigated by histochemical demonstration of acetylcholinesterase following inhibition of pseudocholinesterase. Material from mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, cow, and baboon was included in the study. Positive staining was found in all animals except the hamster. The plexuses of all ventricles received parasympathetic cholinergic nerves, which were associated not only with the blood vessels but also with the plexus epithelium. Usually, the plexus of the third ventricle contained somewhat more nerves than those of the lateral ventricles, which were better supplied than the plexus of the fourth ventricle. The most well-developed cholinergic innervation was found in the plexus tissue from pig and cat, whereas mouse and dog plexuses received comparatively few nerve fibers. The choroid plexus of the other animals contained a cholinergic nerve plexus of intermediary density.

Adrenergic innervation of the mammalian choroid plexus

AbstractChoroid plexuses from the four cerebral ventricles of mice, rats, guinea‐pigs, rabbits, cats, cows, and monkeys were either sectioned after freeze‐drying or stretched on microscope slides for subsequent exposure to formaldehyde gas to demonstrate fluorescent adrenergic nerves. All plexuses received a substantial amount of noradrenaline‐containing axons which originated in the superior cervical sympathetic ganglia. The nerve terminals enclosed both arterial and venous vessels. Some of the terminals in the tufts of the choroid plexus ran between the base of the epithelial cells and the underlying vascular wall. Thus, there are structural possibilities for a sympathetic innervation of the plexus epithelium, the plexus blood vessels, or both.

Lipolytic Peptides in Mammalian Stomach and Choroid Plexus

Acetone powders of porcine stomach stimulated lipolysis in the rabbit’s adipose tissue at a concentration of 1000 μg/ml. By extraction with glacial acetic acid, fractional precipitation with organic solvents, gel filtration and ion exchange chromatography, the lipolytic activity was concentrated 1000 times. This activity was destroyed by trypsin and by chymotrypsin. The porcine stomach lipolytic factor, apparently a peptide with molecular weight 1000–3500 (as indicated by volume of elution from Sephadex G-25), stimulated lipolysis in adipose tissues of rabbit, rat, hamster, guinea pig, cat and opossum, but not in that of the chicken. Lipolytic activity with similar properties was found in omassum and abomassum of bovine stomach, and in leporine, cavian, canine and simian stomach. Acetone powder of bovine choroid plexus was also active at 1000 μg/ral on adipose tissue of the rabbit. The lipolytic activity was concentrated 100-fold by extraction with glacial acetic acid, fractional precipitation with organi...

Dark and light epithelial cells in the choroid plexus of mammals

Dark and light cells were observed in the epithelium of mammalian choroid plexus fixed with glutaraldehyde and/or osmium using immersion or perfusion techniques. Previous studies interpreted dark and light cells in various tissues as artifacts of poor fixative penetration when tissue was fixed by immersion. Since choroidal villi consisted of a single layer of epithelial cells directly overlying a capillary core, the appearance of dark and light choroidal epithelial cells was probably not due to degree or rate of fixative penetration. It was concluded that the dark and light cells of the choroidal epithelium, which appeared fine structurally similar to one another, might represent varying states of cellular hydration at the time of fixation.