Sympathetic influence on sodium-potassium activated adenosine triphosphatase activity of rabbit and rat choroid plexus

Ouabain-sensitive Na+-K+-ATPase was measured spectrophotometrically in the lateral choroid plexuses of rabbit and rat. In the rabbit, a significant increase in the enzyme activity was seen at one week after unilateral sympathectomy (removal of the superior cervical ganglion), but not at three days or two weeks postoperatively, as compared with the intact, contralateral plexus. Unilateral sympathetic denervation of the rat's choroid plexus induced a nearly 40% decrease in Na+-K+-ATPase activity at 6 days after the operation, while no effect was seen after 12 days. The results agree with a local sympathetic inhibition of CSF production in rabbit (corresponding studies on rat have not been performed), and favor the assumption that the adrenergic nerves in the choroid plexus mediate direct effects on transport functions in the plexus epithelium.     

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.     

The neurohistology of quiescent mammary tissue in Lepus albus

The neurohistology of quiescent mammary tissue from adult female albino rabbits was investigated by cholinesterase histochemistry. Dense plexuses of nerve fibers enter the nipple in the dermis surrounding the lactiferous ducts, and unite at the summit of the nipple to form an apical plexus. From the plexuses at the base of the nipple nerves pass between deep epidermal invaginations, to form a basal nipple sub-epidermal plexus, A sub-epidermal plexus of fibers of small diameter arises from the apical dermal plexus. At the base of the nipple extensive nerve plexuses occur around smooth muscle. The only endings are intraepidermal nerve terminals from the apical sub-epidermal plexus. In mammary skin outside the nipple area the dermal and sub-epidermal plexuses are more attenuated. Longitudinally or circularly arranged nerves occur around hair follicles. Peri-arterial plexuses are present throughout the mammary tissue. The myo-epithelial cells and alveoli are not innervated. The neurohistological pattern suggests a spatio-temporal sensory innervation concerned in neurohormonal reflexes of milk secretion and ejection, and neural reflexes of nipple erection. Autonomic motor innervation regulates blood flow and promotes nipple erection. The absence of myo-epithelial cell and alveolar innervation indicates a solely endocrine effector regulation of milk ejection and secretion.     

Small pial vessels, but not choroid plexus, exhibit specific biochemical correlates of functional cholinergic innervation

In an attempt to provide the biochemical foundations for a putative cholinergic innervation of small pial vessels and choroid plexus, we have assessed their ability to specifically accumulate choline, synthesize and release acetylcholine (ACh) in response to depolarization. Our results show that both small pial vessels and choroid plexus avidly accumulate choline via a sodium-dependent mechanism which could be inhibited by hemicholinium-3 (IC50 in pial vessels = 47.8 microM). Light microscopic examination of radioautographs from vessels incubated with [3H]choline revealed two distinct sites of accumulation in the vessel wall. One site probably corresponded to nerve terminals and the other was closely associated with the endothelial cells. In small pial vessels, a major proportion (60%-70%) of the choline acetyltransferase (ChAT) activity could be inhibited by 4-naphthylvinylpyridine (4-NVP), a potent inhibitor of neuronal ChAT; and, following either K+ or veratridine depolarization, a Ca2(+)-dependent release of authentic [3H]ACh could be measured. In contrast, the choroid plexus exhibited a rather low ChAT activity which was not inhibited by 4-NVP and no release of ACh could be detected in this tissue following depolarization. Altogether, the results of the present study show that (1) small pial vessels exhibit all the most selective biochemical markers that are characteristic of cholinergic nerves; (2) [3H]choline in pial vessels can be accumulated in non-neuronal elements which probably correspond to the endothelial cells; and (3) the choroid plexus failed to exhibit convincing biochemical markers that would attest in favor of a functional cholinergic innervation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early postnatal development of transport functions in the rabbit choroid plexus

The development of transport functions in the rabbit choroid plexus was followed postnatally up to 2 months after birth. The activity of ouabain-sensitive Na+, K+-ATPase in newborn rabbit choroid plexus composes about one-fourth (lateral and third ventricle) to one-half (fourth ventricle) of the activity in the adult animal, and it increases markedly within the first 3 weeks of early life. A similar profile of postnatal changes is observed for the capacity to take up and accumulate the organic base choline, which is about three to five times higher for the adult rabbit than for the newborn animal. This coincides with the maturation of the epithelial cells as well as with the development of the sympathetic nerve supply in the choroid plexus. The results suggest that energy-dependent translocation systems influenced by local sympathetic nerves in the choroid plexus, at the interface between blood and CSF, have a functional role shortly after birth.     

Sympathetic influence on transport functions in the choroid plexus of rabbit and rat

In vitro uptake of an organic base (choline) and an organic acid (p-aminohippuric acid; PAH), both radiolabelled, was measured in isolated choroid plexus of rat and rabbit, and expressed as tissue/medium ratios. A significant in the tissue accumulation of choline was seen in denervated rabbit choroid plexus 1 week following unilateral cervical sympathectomy. The accumulation of PAH was not affected. The uptake of both test substances was significantly reduced after sympathetic denervation of the rat choroid plexus. The results agree with the local sympathetic inhibition of cerebrospinal fluid production in rabbit (corresponding studies on rat have not been performed), and favour the assumption that the adrenergic nerves in the choroid plexus mediate direct effects on the plexus epithelium.     

QUANTITATIVE DEVELOPMENT of CHOROID PLEXUSES IN CHICK EMBRYO CEREBRAL VENTRICLES

Quantitative development of choroid plexuses in cerebral ventricles of chick embryos was investigated by means of the planary projection of the choroid plexuses from the time the plexuses reached a consistent flattened structure. Choroid plexuses in the lateral cerbral ventricles were studied from day 6, the plexus in the third cerebral ventricle from day 8, and the plexus in the fourth cerebral ventricle from day 11 of incubation. Regardless of the microscopic origin of these choroid plexuses, their development reached a growth maximum on day 15 of incubation, after which there was a slight regression. The regression was gradual in the plexus of the third cerebral ventricle but a transient enlargement of plexuses in the laternal and in the fourth cerebral ventricle was observed between days 18 and 19. The enlargement of choroid plexuses in the lateral cerebral ventricles was caused by a flattening of the villi, whereas that of the plexus in the fourth cerebral ventricle was caused by thinning and yawning of the villi. The area of choroid plexuses in the lateral cerebral ventricles was six or seven times larger than the sum of the areas of the remaining choroid plexuses.     

The lateral,third, and fourth ventricle choroid plexus of the dog: A structural and ultrastructural study

The morphology of the choroid plexuses of the lateral, third, and fourth ventricles in dogs was studied by histological and ultrastructural techniques after in situ fixation. Most portions of the plexus in the lateral ventricle showed a parallel arrangement of capillaries, producing a fine corrugation of the overlying layer of epithelial cells. Relatively large amounts of connective tissue separated the capillaries from the epithelium. The fourth ventricle choroid plexus showed short capillary loops projecting into the ventricle and more intimately covered by epithelium. Smaller amounts of connective tissue separated the capillaries from the epithelium. The choroid plexus of the third ventricle showed characteristics seen in the plexuses of both the lateral and fourth ventricles. The total surface of the choroid plexuses in the dog averaged 10.7 cm², of which 55% was fourth ventricle choroid plexus.     

The effect of cholera toxin on choroid plexus carbonic anhydrase activity in vitro

The effects of the adenylate cyclase agonists cholera toxin and prostaglandin E2 on carbonic anhydrase activity in vitro was measured in choroid plexuses isolated from Sprague-Dawley rats. Choroid plexuses were incubated in buffer at 38 degrees C (pH 7.4) with either cholera toxin or prostaglandin E2 (PGE2) at a concentration and for a time period that had been shown in earlier studies to result in maximal stimulation of cyclic AMP production. Cholera toxin (10 micrograms/ml) caused a twofold increase (p < .001) in choroid plexus carbonic anhydrase activity when cholera toxin treated plexuses [20.92 +/- .46 mol CO2/(min)(mg protein X 10(-8)] were compared with plexuses exposed to heat inactivated cholera toxin (10.92 +/- .43). When choroid plexuses were homogenized and separated into a 10,000 g pellet and a supernatant fraction, the supernatant carbonic anhydrase was unresponsive to cholera toxin stimulation. In the pellet fraction, which contained all the cellular adenylate cyclase, challenge with cholera toxin produced a significant increase in carbonic anhydrase activity (p < .01). Control activity was 10.9 +/- 1.2 mol CO2/(min)(mg protein X 10(-8), while carbonic anhydrase activity in fractions exposed to cholera toxin was 28.4 +/- 0.8. PGE2 had no effect, however, upon choroid plexus carbonic anhydrase activity. Since both PGE2 and cholera toxin stimulate cyclic AMP production in vitro, a compartmental model of secretory control is proposed.     

The stability of the hippocampal slice preparation: An electrophysiological and ultrastructural analysis

To determine if canine and rat choroid plexus Na+,K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified canine kidney medulla Na+,K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified canine kidney Na+,K+-ATPase and canine kidney microsomes were treated with antiserum followed by [125I]protein A and autoradiography, the canine microsomes and purified Na+,K+-ATPase showed a prominent radioactive band coincident with the alpha-, beta- and gamma-subunits of the purified canine kidney enzyme. When the rabbit immunoglobulin that was purified from the Na+,K+-ATPase antiserum through DEAE-cellulose ion exchange chromatography was used for immunoperoxidase staining of the choroid plexus fixed with Bouin's fixative, intense immunoreactive staining was present on the epithelial cells of both choroid plexuses but was not found in the tissue around the vessel. The staining was especially confined to apical surfaces of the epithelial cells. The same procedure was performed in the canine kidney, and immunostaining was obtained in the tubules where Baskin and Stahl described the enzyme localization. No staining was seen with pre-immune serum of the normal rabbit. We concluded that both the canine and rat choroid plexus are rich in Na+,K+-ATPase, which plays an important role in cerebrospinal fluid (CSF) secretion.     

Intrinsic nerves in the mammalian colon: confirmation of a plexus at the circular muscle-submucosal interface

Slow waves in the small intestine seem to arise in plexuses of neurites with interstitial cells of Cajal. In the colon, slow waves appear to arise at the circular muscle - submucosal interface. We therefore sought a plexus at this surface in the colon in the cat, dog, ferret, opossum, rabbit, rat, guinea-pig and man. Segments from all levels of the colon were stained by the Champy-Maillet osmic acid-zinc iodide method and cut into serial 25 micron sections in the plane of the muscle layers. A dense network of neurites with abundant interstitial cells of Cajal was found at the circular muscle - submucosal interface in all species except rabbit. Neurites in this plexus appeared to arise from the deep plexus of the submucosa (Schabadasch's or Henle's plexus). It was not found in the small intestine and stomach. A similar plexus was found in the interstices of the myenteric plexus in the colon. Interstitial cells of Cajal in both plexuses were positive for the NADH-diaphorase stain, but not for silver impregnation. The possible roles of the plexuses of neurites and interstitial cells of Cajal at the circular muscle - submucosal interface and at the plane of the myenteric plexus in the generation of rhythmic activity in the colon are discussed.     

Actions of sex steroids and corticosteroids on rabbit choroid plexus as shown by changes in transport capacity and rate of cerebrospinal fluid formation

The effect of betamethasone on choroid plexus transport and CSF formation in rabbits was studied. Following 5 days of daily treatment with betamethasone the CSF production rate was reduced by 43% as measured by ventriculo-cisternal perfusion with radioactive inulin. Accordingly, the Na(+)-K(+)-ATPase activity and the transport capacity in the choroid plexus, measured in terms of choline (10(-5) M) uptake and accumulation in vitro, decreased (in the lateral ventricles by 31% in both cases). Isolated choroid plexuses from rabbits were also used to determine uptake and accumulation of choline and the activities of various types of ATPases following pretreatment of the animals with 17-beta-oestradiol, alone or in combination with progesterone. The combined treatment reduced the choline uptake by 35% and also lowered the activity of Na(+)-K(+)-ATPase by 31% without influencing tissue wet weight. Thus, the demonstrated influences of glucocorticoids and sex steroids on the transport capacity in the choroid plexus seem to be important components in their postulated effects on intracranial hypertension.     

Demonstration of a coupled metabolism-efflux process at the choroid plexus as a mechanism of brain protection toward xenobiotics.

Brain homeostasis depends on the composition of both brain interstitial fluid and CSF. Whereas the former is largely controlled by the blood-brain barrier, the latter is regulated by a highly specialized blood-CSF interface, the choroid plexus epithelium, which acts either by controlling the influx of blood-borne compounds, or by clearing deleterious molecules and metabolites from CSF. To investigate mechanisms of brain protection at the choroid plexus, the blood-CSF barrier was reconstituted in vitro by culturing epithelial cells isolated from newborn rat choroid plexuses of either the fourth or the lateral ventricle. The cells grown in primary culture on semipermeable membranes established a pure polarized monolayer displaying structural and functional barrier features, (tight junctions, high electric resistance, low permeability to paracellular markers) and maintaining tissue-specific markers (transthyretin) and specific transporters for micronutriments (amino acids, nucleosides). In particular, the high enzymatic drug metabolism capacity of choroid plexus was preserved in the in vitro blood-CSF interface. Using this model, we demonstrated that choroid plexuses can act as an absolute blood-CSF barrier toward 1-naphthol, a cytotoxic, lipophilic model compound, by a coupled metabolism-efflux mechanism. This compound was metabolized in situ via uridine diphosphate glururonosyltransferase-catalyzed conjugation, and the cellular efflux of the glucurono-conjugate was mediated by a transporter predominantly located at the basolateral, i.e., blood-facing membrane. The transport process was temperature-dependent, probenecid-sensitive, and recognized other glucuronides. Efflux of 1-naphthol metabolite was inhibited by intracellular glutathione S-conjugates. This metabolism-polarized efflux process adds a new facet to the understanding of the protective functions of choroid plexuses.     

Innervation of the guinea pig trachea: a quantitative morphological study of intrinsic neurons and extrinsic nerves

The innervation of the guinea pig trachea was studied in wholemount preparations stained for acetylcholinesterase, catecholamines, and substance P immunoreactivity and by electron microscopy. The majority of parasympathetic and afferent nerve fibres arrive from the vagus via branches of the recurrent laryngeal nerves. The recurrent laryngeal nerves are composed of several fascicles comprising 600-700 small myelinated fibres (2-5 microns diameter) and about 1,000-2,000 unmyelinated fibres; both components exit from the nerve and project in fine branches to the trachea. A separate component of 200-250 large myelinated fibres (more than 5 microns diameter) runs the full length of the nerve and innervates the striated muscles of the larynx. The recurrent laryngeal nerves are slightly asymmetric in their origin, length, number, and composition of fibres, with the right nerve being shorter but with more numerous and thinner myelinated fibres. At the distal end of the recurrent nerve, a fine branch called the ramus anastomoticus connects it to the superior laryngeal nerve. In the tracheal plexus, there are on average 222 ganglion cells (range 166-327), distributed mostly in small ganglia of 12 or fewer neurons. The ganglionated plexus is situated entirely outside the tracheal wall, overlying the smooth muscle. Ligation experiments show that sympathetic nerve fibres reach the trachea with the recurrent nerves via anastomoses between the sympathetic chain and vagus nerves, or occasionally with recurrent nerves directly, the largest being at the level of the ansa subclavia. There are also perivascular sympathetic nerve plexuses. Substance P immunoreactive fibres enter the trachea from the vagus nerves and by pathways similar to those of sympathetic nerves. There are also paraganglion cells within the recurrent laryngeal nerve that contain catecholamines and are surrounded by substance P immunoreactive fibres. After cervical vagotomy, all the large myelinated fibres of the ipsilateral recurrent laryngeal nerve degenerate and so do all but 10 or 20 small myelinated fibres and all but a few unmyelinated fibres. Degenerating fibres are found within the entire tracheal plexus, indicating bilateral innervation. The small myelinated fibres that survive cervical vagotomy probably represent sympathetic or afferent nerves with their cell bodies located in sympathetic or dorsal root ganglia.     

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.     

A morphometric study on the development of the lateral ventricle choroid plexus, choroid plexus capillaries and ventricular ependyma in the rat

Morphometric changes in the rat lateral ventricle choroid plexus epithelium and endothelium and in the ventricular ependyma were studied between 16 days gestation and 30 days after birth, using stereological techniques. The epithelial apical surface density increased from 0.6 to 3.3 microns 2/microns 3 and the mitochondrial volume fraction from 3.2 to 7.6% during this period. The endothelial fenestrations increased from 0.05 to 0.39 micron-1. These changes may be related to postnatal increases in choroid plexus function. Morphological changes in basolateral surface density, cell height and nucleus and glycogen volume fraction have also been measured. The development of the lateral ventricle choroid plexus was qualitatively similar to the fourth ventricle plexus reported previously, but small quantitative differences occurred. The ventricular ependyma also showed a significant increase in mitochondrial volume fraction after birth, though to a lesser extent than the plexus epithelium. The total apical surface area of the choroid plexuses was estimated at 75 cm2 for 30-day-old rats. This figure, which takes into account the apical microvilli, is much greater than previous estimates and is similar to the surface area of the cerebral capillaries (155 cm2), and suggests that the choroid plexuses may play a more important role in the regulation of the brain microenvironment than previously thought.     

Choroidal bodies: a new structure in the fourth ventricular choroid plexus of the rat and mouse

Histologic study of the caudal end of the fourth ventricular choroid plexus of the rat and mouse revealed 1-4 small, discrete collections of cells that differed from the surrounding choroidal epithelial cells in appearance. They did not occur in other parts of the choroid plexuses. These choroidal bodies were not affected by a chemical toxin that caused hydropic degeneration of all the epithelial cells in the choroid plexuses. The function, if any, of the choroidal bodies is unknown. They were present in all rats that were studied by serial sections.     

The Effect of Cholera Toxin on Choroid Plexus Carbonic Anhydrase Activity In Vitro

The effects of the adenylate cyclase agonists cholera toxin and prostaglandin E₂ on carbonic anhydrase activity in vitro was measured in choroid plexuses isolated from Sprague-Dawley rats. Choroid plexuses were incubated in buffer at 38° C (pH 7.4) with either cholera toxin or prostaglandin E₂ (PGE₂) at a concentration and for a time period that had been shown in earlier studies to result in maximal stimulation of cyclic AMP production. Cholera toxin (10 μg/ml) caused a twofold increase (p < .001) in choroid plexus carbonic anhydrase activity when cholera toxin treated plexuses [20.92 ± .46 mol CO₂/(min)(mgprotein X 10^-8)] were compared with plexuses exposed to heat inactivated cholera toxin (10.92 ± .43).

When choroid plexuses were homogenized and separated into a 10,000 g pellet and a supernatant fraction, the supernatant carbonic anhydrase was unresponsive to cholera toxin stimulation. In the pellet fraction, which contained all the cellular adenylate cyclase, challenge with cholera toxin produced a significant increase in carbonic anhydrase activity (p <.01). Control activity was 10.9 ± 1.2 mol CO₂/(min)(mg protein × 10^-8), while carbonic anhydrase activity in fractions exposed to cholera toxin was 28.4±0.8. PGE₂ had no effect, however, upon choroid plexus carbonic anhydrase activity. Since both PGE₂ and cholera toxin stimulate cyclic AMP production in vitro, a compartmental model of secretory control is proposed.     

A quantitative analysis of the afferent and extrinsic efferent innervation of specific regions of the bladder and urethra in the cat

Using the fluorescent tracer dyes bisbenzimide, nuclear yellow and fast blue, the possibility of differential innervation of various regions of the bladder and urethra was tested in cats. The dyes were injected into the lateral detrusor, bladder base, and urethra. Fluorescent cell bodies were counted in serial 48 micron sections of dorsal root, inferior mesenteric, sympathetic chain and pelvic plexus ganglia. Several dorsal root ganglia, primarily S2, were the principal source of afferent innervation to all locations injected. The bladder and urethra received significant efferent innervation from both the inferior mesenteric ganglia and sympathetic chain ganglia (particularly L7 to S2) however, pelvic plexus ganglia made only small contribution to the innervation of these areas. The sympathetic chain and inferior mesenteric ganglia contributed equally to the innervation of the detrusor and bladder base but the sympathetic chain made double the contribution of the inferior mesenteric ganglia to the innervation of the urethra. There was a very low incidence (less than 1%) of neurons which projected to more than one injection site.