109

Bruin Research Rel;iews, 17 (1992) 109-138

0 1992 Elsevier Science Publishers B.V. All rights reserved 0165-0173/92/$05.00

BRESR 90146

Neuroendocrine regulatory mechanisms in the choroid plexus-cerebrospinal fluid system Christer Nilsson, Maria Lindvall-Axelsson

and Christer Owman

Department of Medical Cell Research, Section of Neurobiology, Unirersity of Lund, Lund (Sweden)

(Accepted 26 May 1992)

Key words: Choroid plexus; Cerebrospinal

fluid; Neuroendocrine; Central nervous system; Vasoactive intestinal polypeptide; 5-Hydroxytryptamine; Atrial natriuretic peptide; Vasopressin; Insulin-like growth factor; Transthyretin

CONTENTS 1. Introduction

......................................................................................

110

2. Basic features of the choroid plexus ..................................................................... 2.1. Structure.. .................................................................................. 2.2. Development .................................................................................

110 110 Ill

3. Functional aspects of the choroid plexus and cerebrospinal fluid ................................................. 3.1. The blood-cerebrospinal fluid barrier ............................................................... 3.2. Secretion of cerebiospinal fluid .................................................................... 3.3. Synthesis and secretion of plasma proteins ............................................................ 3.4. Other functions of the choroid plexus ................................................................ 3.5. Functions of the cerebrospinal fluid .................................................................

112 112 113 113 113 113

4 Neurogenic regulatory mechanisms in the choroid plexus ...................................................... 4.1. Sympathetic innervation ......................................................................... 4.2. Cholinergic innervation .......................................................................... 4.3. Peptidergic innervation ..........................................................................

114 114 116 116

5 Endocrine regulatory mechanisms in the choroid plexus ....................................................... 5.1. 5-Hydroxytryptamine ... .. . ........ ........................... 5.2. Melatonin . . . . . . ... . . ........ ........................... 5.3. Histamine ..... .... ... ........ ........................... 5.4. Atrial and brain natriuretic peptide . ........ ........................... 5.5. Vasopressin .. . .. .... ........ ........................... 5.6. Angiotensin II . . . . ... ... ........ ........................... 5.7. Insulin and insulin-like growth factors ........ ........................... 5.8. Glucocorticoid hormones . . . . . ........ ........................... 5.9. Sex steroids and thyroid hormones . . . ........ ........................... _.a .. 5.10.1 urnermealarors.......................................................................,....,,.

118 118 119 120 120 121 122 122 123 123 124

6. The choroid plexus as source and pathway for endocrine signals targeted to the brain 6.1. Insulin-like growth factor-II . ... .... .. ... ... .. 6.2. Prolactin .. .. . ... ... ... . .. . . .

124 124 125

Correspondence (46) 10 79 27.

........................... ........................... ...........................

to: C. Nilsson, Institutionen for Medicinsk Cellforskning, Lunds Universitet, Biskopsgatan 5, S-223 62 Lund, Sweden. Fax: (46)

110 6.3. Otherpeptides..................................,.... 6.4. Transthyretin and the transport of thyroid hormones to the brain 7.Summary

~.........~..........~.~....~~...~~~.~.~.~~.~...~.~~~.....~.....~~~..~.........

Acknowledgements Abbreviations References

1.

........

. . . . . . . . . . . . . . . . . . . . . . . . . . .._..........~......I..I....I............................

... ....

175 115 .,...__.

128

I 2’)

. . . . . . . . . . ~....t............................r..................I.......................

I29

. . . . . . . . . . ..1.....__.........................t...................................~.......

129

INTRODUCX’ION

The cer~brospinal fluid (CSF) is produced by the choroid plexuses in the four ventricles of the brain, from where it flows to fill the subarachnoid spaces surrounding the brain and spinal cord. Although the CSF has an important role in the mechanical support and chemical homeostasis of the brain@, a more dynamic function as neuroendocrine pathway for communication and integration within the brain has also been sUggested2S”.284.3h~. The choroid plexus appears to constitute an important part of the CSF’s role in neuroend~rine signalling, beyond being the main site of CSF production. Modern methodology in receptor autoradiography, in situ hybridization and immunohistochemistry has revealed an autonomic innervation of the choroid plexus, but also high levels of receptors for, e.g., 5-HT, arginine vasopressin (AVP) and atria1 natriuretic peptide (ANP) in the choroid plexus epithelium. This suggests that centrally released transmitters released into the CSF can act on the choroid plexus29*‘40,2S0*3373357. While AVP and ANP appear to be involved in choroid plexus ion transport and CSF production48,~2~, central to their suggested role in brain volume reguiation”9X68~‘7~7X, the function of 5HT in the choroid plexus is not yet clear “(‘. The choroid plexus also appears to constitute a pathway for endocrine ~ommuni~tion between the periphery and the brain. It is the main site of synthesis of insulin-like growth factor-II (IGF-II) in the adult mammalian central nervous system (CNS)329. Receptors for IGF-II are present in many parts of the brain and IGF-II has been shown to have trophic and metabolic effects on both glial and neuronal cells9”~‘74~‘8’~223~235~2s’. Especially remarkable is the synthesis and secretion of the thyroid hormone transport protein transthyretin (‘ITR). The choroid plexus has the body’s highest levels of this plasma protein727301. This appears to be a phylo~eneti~a1ly old phenomenon, apppearing before TTR synthesis occurred in the liver13’, indicating that TTR has an important role in thyroid hormone transport and distribution in the

brain”“. Other hormones, such as prolactin, seem to be transported directly via specific carrier mechanisms in the choroid plexus’~~~7~zo6~ Thus, there is increasing evidence that the choroid plexus can act as a target, source and pathway for neuroendocrine signalling within the brain, as summarized in Fig. 1. The present review presents the data now available for such a role, much of which has accumulated during the last decade.

Fig. 1. Schematic drawing of neuroendocrine signaling pathways involving the choroid plexus-CSF system. Neurotransmitters released within the brain parenchyma can diffuse or flow between the cells of the ependyma (E) into the CSF and there be transported to apically located receptors on the choroid plexus epithelium (CP) (1). The choroid plexus might here participate as a part of a general functional response involving several parts of the brain. Two substances that might be released in such a fashion are AVP and ANP. In the case of 5-HT the releasing nente fibers have been identified. These are dense, supraependymal, serotonergic nerve fiber plexuses located in several parts of the ventricle walls. Several lines of evidence suggest that these nerve fibers release 5-HT into the CSF from where it reaches specific receptors in the choroid plexus epithelium (2). Apart from acting as a target for centralfy released transmitters (1.21, the choroid plexus also produces a growth factor (3) and transport proteins for peripheral hormones (41. These substances have been suggested to mediate endocrine signals to brain neurons and glia via CSF bulk flow and diffusion within the brain parenchyma 13,4X

111

2. BASIC FEATURES OF THE CHOROID PLEXUS

2.1. Structure The choroid plexus is present in all chordates except Amphioxus M, but shows large species variation in size, relative to brain size. In reptiles, birds and mammals, the choroid plexus is located in the lateral, third and

fourth ventricles of the brain, The surface area of the choroid plexus is increased by numerous villi, each villus consisting of a single, cuboidal epithelium overlying a highly vascularized connective tissue stroma. The capillaries of the choroid plexus are fenestrated while the epithelium is sealed by tight junctions between the epithelial cells, thus forming the blood-CSF barrier.

Fig. 2. Electron micrograph of a lateral ventricle choroid plexus from the rabbit. In A (X 1900), a one-layered cuboidal epithelium is seen overlying a connective tissue stroma consisting of a cross-sectioned capillary, a fibroblast and collagen fibres. The epithelial cells are characterized by numerous mitochondria and sheets of rough endoplasmic reticulum. Other prominent features are the apical microvilli forming a brush border, the large rounded nuclei, the basolateral interdigitations and occasional tufts of cilia. The large grey structures inside the epithelial cells are lipid droplets. In B, a high-magnification ( X 24000) electron micrograph of a part of a choroid plexus capillary is shown, demonstrating the numerous fenestrations of the endothelial cells (arrows). The endothelial cell is situated on a basal lamina.

112 The epithelial cells are characterized by large rounded nuclei, abundant mitochondria and rough endoplasmic reticulum, basolateral interdigitations and numerous microvilli protruding from the apical, luminal side (Fig. 2). 2.2. Development The choroid plexus appears early in embryonal development, between the 6th and 8th week of gestation in humans, arising from the neuroepithelium surrounding the neural tube and the underlying mesenchyme24*,242. The central role of the epithelium in the development of the villous choroid plexus has been demonstrated by transplantation of epithelial cells from the early choroid plexus anlage to the body wall, which induces the formation of a choroid plexus-like structure in this region36s. During the subsequent fetal development the choroid plexus of the lateral ventricles grow rapidly and fill one-third of the lateral ventricles between gestation weeks 9 and 17. The epithelial cells accumulate large amounts of glycogen in the cytoplasm, indicating a role for the choroid plexus in prenatal brain growth and development J6. The choroid plexus then gradually decreases in size relative to the whole brain as the growth of the latter accelerates towards the later part of the gestation. The functional development varies depending on the function studied. Morphometric and physiological studies indicate that CSF secretion is very low at birth and then increases rapidly during the first weeks postnatally2”J”‘.i72. Th is coincides with an increase in Na+/K+-ATPase activity and choline uptake capacity as well’99, which parallels the ingrowth of s~pathetic nerves1s7. These functions thus seem to be of greater importance in postnatal life. In contrast, the synthesis of many choroid plexus plasma proteins appear very early in plexus development 339*340,350. 3. FUNCTIONAL

ASPECTS OF THE CHOROID PLEXUS

AND CEREBROSPINAL

FLUID

Many functions are ascribed to the choroid plexus, which depend primarily on the epithelial cells of this tissue56. The main functions of the choroid plexus and the CSF are summarized below. 3.1. The blood-cerebrospinal fluid barrier It is essential for a brain function to keep its internal chemical environment as constant as possible”. To achieve this, the brain is separated from the blood by the blood-brain barrier (BBB). The BBB can be divided into two parts: (1) the endothelial cells of the

brain capillaries, which separate the blood from the extracellular fluid of the brain parenchyma and (2) the choroid plexus epithelium and arachnoid membrane which separate the blood from the CSF”s*hs,‘5”. For clarity only the former will be called the BBB in the present text, while the latter will be referred to as the blood-CSF barrier. While the brain capillaries are sealed by tight junctions between the endothelial ceils thus forming the actual BBB, the capillaries in the choroid plexus are fenestrated and permit the passage of macromolecules into the surrounding connective tissue (Fig. 2). instead, the epithelial cells of the choroid plexus are joined by continuous tight junctions and thereby forms the blood-CSF barrier. Lipophilic substances pass readily through the respective barriers, while smaller hydrophilic substances and macromolecules are excluded from the brain and CSF unless there is a specific carrier for them35. In addition, a very small ‘leak’ of the restricted substances can occur, probably through the junctions between the brain endothelial cell$‘. This ‘leak’ is somewhat larger for the choroid plexus epithelium than for the BBB3’. In addition to the choroid plexus, the arachnoid membrane has a large contact area with the CSF, impeding the passage of solutes from the relatively permeable capillaries of the dura mater to the CSF”. The CSF is in contact with the brain tissue in the ventricles which are lined by the ependyma and in the subarachnoid space where CSF and brain extracellular fluid are separated by the pia mater. There are, however, no barriers between the CSF and the brain extracellular fluid and substances can pass between the two compartments by simple diffusion or bulk flo~~‘@~‘~~+~s”. The different compartments and transport routes present in the brain are shown schematically in Fig. 3. Many substances that are necessary for proper brain function are transported across the BBB and bloodCSF barrier by either facilitated diffusion or active transport, e.g., glucose, amino acids, peptides, nucleotides and vitamins (cofactors)‘7.35.h5~‘58.2h”.‘22,374. The contribution of the CSF pathway appears to be too small to support the total demand of the brain and the role of the choroid plexus is probably to maintain a CSF concentration comparable to that of the brain extracellular fluid35,h5.This is due to the much greater surface area of the BBB compared to the blood-CSF barrier, traditionaily agreed to be approximately 5000 : 135 and the longer diffusion distances from CSF to brain compared to the distance between brain capillaries and glial cells/neurons. However, it should be mentioned that morphometric calculations by Keep

113

e

D

eECF

+ [C

Blood

T

CSF

Fig. 3. Schematic picture showing the four main fluid compartments of the brain: blood, cerebrospinal fluid (CSF), extracellular fluid (ECF) pnd the intracellular compartment (IC). Exchange of solutes occurs between all four compartments (arrows). Selective transport of substances occur in both directions between blood and ECF/CSF over the BBB and blood-CSF barrier, as well as between ECF and the IC. Direct transport between CSF and IC is also possible in neurons that are in contact with the CSF and although quantitatively small, this might in some cases be an important pathway for communication within the brain via the CSF. While the transport routes described above are over barriers (BBB, blood-CSF barrier and the plasma membranes of the cells; filled lines), there is free passage for polar substances and macromolecules in both directions between the ECF and CSF over the ependyma and pia mater (dashed line). and Jones’70~‘71 suggested that the ratio may be only 2 : 1, due to the well-developed microvillous brush border of the choroid plexus epithelium. There are a few substances (e.g., methyltetrahydrofolate, ascorbate and thymidine) that might enter the brain via the CSF, as there is a high-capacity active transport system in the choroid plexus, but no passage over the BBB322. In summary, it appears that substances that only require slow, long-term action, such as growth factors (IGF-II) and transport proteins (TT’R), or are required in very small amounts (micronutrients)322, could be transported from CSF to brain in a significant manner’59. In addition to being physical barriers, the blood-CSF and BBB are both enzymatic barriers in that they have the capacity for uptake and degradation of many substances originating either in the blood or the brain35*65*129. Also in this respect there are differences between the choroid plexus and the brain endothelial cells, for example in the concentrations of aminopeptidase M, puromycin-sensitive aminopeptidase and alkaline phosphatase, the latter two being more concentrated in the choroid plexus than in cortical microvessels316. Substances that are taken up and degraded in this manner are, among others, serotonin, noradrenaline and their metabolites, enkephalins and other peptides 32,33,104,196,229,341

3.2. Secretion of cerebrospinal fluid The composition of the CSF clearly shows that it cannot be a passively formed ultrafiltrate of plasma,

but rather a fluid modified by active secretion56*65.The secretory morphology of the choroid plexus, the localization of Na+/K+-ATPase to the apical part of the choroid plexus epithelium96*311 and the analysis of freshly secreted fluid from the choroid plexus*, strongly suggests that the choroid plexus secretes CSF. It is now generally agreeed that the choroid plexus is the major source of CSF production, while the proportion of CSF production contributed by the choroid plexus has not been firmly established, estimates varying between 60 and 90%59765,159,274. The main extrachoroidal source of CSF production is probably the endothelial cells of the brain capillaries59,65. The turnover rate of CSF is high in mammals, around 0.5% of the total volume per min or 4-5 times the total volume per day65. 3.3. Synthesis and secretion of plasma proteins During recent years the synthesis of a growing number of plasma proteins has been localized to the choroid plexus by Northern blot and in situ hybridization (for a recent review, see ref. 300). Among these proteins are transferrin, ceruloplasmin, cystatin C and /3,-microglobulin that appear to be synthesized both in the choroid plexus and the brain parenchyma3,4v46T’26, while others such as transthyretin (TTR; prealbumin), IGF-II and hitherto unidentified proteins seem to be synthesized exclusively in the choroid plexus epithelium and possibly the leptomeninges but not in other parts of the brainl26.298.300

Due to the nature of the BBB and blood-CSF barrier, the protein content of the CSF is very low. The reason for the high protein synthesis and secretion from the choroid plexus246 may be to supplement the CSF with certain important protein components. The actual importance and role of these proteins in brain function remain to be determined, however. TTR and IGF-II, which could be involved in endocrine signalling to the brain, will be further discussed below. 3.4. Other functions of the choroid plexus The choroid plexus might have a nutritive function in early embryological development and in those lower vertebrates with large choroid plexuses and thin brains, considering its large size and blood volume relative to whole brain in these species56,60,‘37.In adult mammals this seems unlikely due to the previously mentioned much greater surface area of the brain capillaries compared to the choroid plexus and long diffusion distance from CSF to much of the brain tissue, possibly with the exception of certain micronutrients such as ascorbate322. In the lumbar spinal cord, however, the CSF is an important nutritional pathway for spinal nerve roots291.

114 Nathanson and Chun24” have demonstrated that the choroid plexus can present antigen to and stimulate proliferation of, peripheral helper T lymphocytes and thus might play a role in immunological communication between the central nervous system (CNS) and the periphery. 3.5. Functions of the cerebrospinal fluid Several functions have been attributed to the CSF, including buoyancy and protection of the brain, excretion of metabolites, homeostasis of the brain’s chemical environment and as an endocrine pathway for intracerebral transport between different brain areas56,65,2*4,368. Naturally, the choroid plexus has a large influence on CSF function, e.g., in maintaining a constant CSF pH in the presence of large variations in plasma pH’69*237,or for keeping a stable concentration of ascorbate in the CSF322. As is thoroughly discussed in this review, there is also mounting evidence that the choroid plexus influences the composition of the CSF in respect to certain proteins and hormones. For an extensive review of CSF composition and function, see ref. 65, which is the major work of reference on this subject.

4. NEUROGENIC THE CHOROID

REGULATORY

MECHANISMS

IN

PLEXUS

The mammalian choroid plexus contains noradrenergic sympathetic, cholinergic and peptidergic nerves, probably of autonomic origin’“8,247. Nerve fibres from other sources than the autonomic ganglia have not been conclusively identified. Electron microscopy has demonstrated nerve terminals adjacent to epithelial and vascular elements in the choroid plexus”‘. Receptors for autonomic neurotransmitters can be found on choroid plexus epithelial ce11s’88~249~28x. These receptors and their intracellular effector mechanisms are shown in Table I and a summary of the known actions and interactions of the different neurotransmitters in the choroid plexus may be found in Fig. 4. 4.1. Sympathetic innervation The choroid plexus receives a relatively dense supply of sympathetic nerve fibres ‘86~188~192, deriving almost exclusively from the superior cervical ganglia”‘j. Ultrastructurally, the nerve fibres can be found in close relation to both epithelial cells and small arterioles, indicating a possibility for regulation of both these

TABLE I Putative receptors demonstrated in choroid plexus epithelium

Receptors or binding sites demonstrated in choroid plexus epithelium by either receptor binding (RB), immunohistochemistry of the receptor protein (IH), in situ hybridization of receptor mRNA (ISH), or by pharmacological characterization of the cellular, second messenger response (PC). The cellular response is defined as the primary effector mechanism after binding of the ligand to the receptor, e.g., stimulation (+I of cAMP production or tyrosine kinase activity. The other abbreviations are explained elsewhere in the text. Receptor

Endogenous ligand(s)

Cellular response

Method

Refs.

;: D, HZ 5-HT,,

noradrenaline noradrenaline dopamine histamine S-hydroxytryptamine

+ CAMP + CAMP + CAMP + CAMP PI-hydrolysis

Melatonin Muscarinic ANP-A

melatonin acetylcholine ANP, BNP

? ? + cGMP

Vi AVP,_, Ang-II IGF-I Insulin

AVP (and oxytocin) AVP4-9 angiotensin II IGF-I, IGF-II insulin, IGF-II

200 239 2,244 55 140, 230 266,370 355 288 29,216 295 114, 271, 344,357 36 112 63, 252 22,252

Man 6-P/IGF-II

IGF-II

Growth hormone VIP Endothelin Prolactin

Growth hormone VIP endothelin prolactin

PI-hydrolysis ? ? + tyrosine kinase + tyrosine kinase, transcytosis? uptake and degradation? ? + CAMP ? transcytosis

PC PC RB PC ISH RB RB RB RB IH RB RB RB RB RB

GABA A Benzodiazepine (peripheral type) T-2

GABA

3

RB IH RB RB RB RB IH RB

252 353 179 249 175 354 228 7

3 tryptamine

? ?

RB RB

364 268

115

structures by sympathetic nervesg7. Electrical stimulation of the superior cervical ganglia decreases CSF production in cat and rabbit136,192 (Fig. 51, without changing choroid plexus blood flow as measured in the cat5, while sympathetic denervation of the choroid plexus increases CSF production192, active transport of choline’97 and Na+/K+-ATPase activity198 in the rabbit.. Furthermore, noradrenaline has been shown to reduce CSF production both when administered intravenously in dog and rabbit and intraventricularly in cat and rabbit136*195,212. The effects of noradrenaline could be mediated by stimulation of cyclic AMP (CAMP) formation via activation of /3-receptors200,238. The nature of the P-receptors has only been characterized pharmacologically in functional studies and not by receptor binding. Thus, Nathanson found that adenylate cyclase was regulated by P,-receptors in the cat, while in vitro and in vivo studies in the rabbit indicated a /?l-receptor-mediated response195’200. Taken together, these findings have led to the hypothesis that the sympathetic nervous system has a tonic inhibitory influence on CSF production, probably by a CAMPmediated decrease of Na+/K+-ATPase activity in the choroid plexus epitheliumlg8.

EPITHELILH

Fig. 4. Actions and interactions of neurotransmitters in the choroid plexus. The presence of epithelial /3 and VIP receptors, which both mediate increases in CAMP formation in the epithelial cells, together with VIP’s stimulatory effect on noradrenaline release, suggests that a synergistic action exists for noradrenaline and VIP in this tissue. Both transmitters inhibit CSF production, although this has not been demonstrated in the same species, which lends further support to the hypothesis of synergism. There is also evidence for the presence of both p- and a-adrenoceptors, as well as VIP receptors, on the choroid plexus vasculature. For references, see text.

l

-

b

z

16

I”

Y-

c

c

5yx syx

C

Stim

Stimoff

Fig. 5. Demonstration of the regulation of CSF production in the rabbit by sympathetic nerves. a: denervation: 1 week following sympathetic denervation (SyX) of the rabbit choroid plexus there is a marked reduction in the noradrenaline concentration concomitant with a highly significant increase in the rate of CSF production compared with unoperated controls (0. Differences between mean values ( f S.E.M.) according to the Student’s r-test: P < 0.001 in both groups. b: stimulation: production rate of CSF before (C) and during (Stim) bilateral electrical stimulation of the superior cervical ganglia, which markedly reduces the rate of production (the difference, based on paired observations, was of highest significance: P < 0.001). After finishing stimulation (Stim off) there is a tendency to normalization of the production rate (Stim versus Stim off: P = 0.01). Bars indicate mean f S.E.M. Reproduced from ref. 192, with permission.

The hypothesis described above fits the results obtained in rabbit. Findings in other species are to a certain degree contradictory, however and yields a more complex picture. Thus, cholera toxin, which is a very potent stimulator of CAMP formation51, stimulates CSF production in dogs94. Saito and Wright296, in experiments on the isolated bull-frog choroid plexus, concluded that CAMP can increase HCO; secretion across the choroid plexus by increasing the apical HCO; conductance. Furthermore, sympathetic denervation of the choroid plexus in rats decreased the uptake of choline and Na+/K+-ATPase activity, opposite to the effects in rabbit197J98. Intravenous administration of the a-adrenergic agonist phenylephrine increased CSF production in cats, possibly via stimulation of a cholinergic pathway136, although it should be noted that the significant effects of phenylephrine was coupled to very large increases in blood pressure. Species differences and methodological considerations could be responsible for the different effects that were obtained. CSF production decreases in dogs during experimental communicating hydrocephalus’56. In rabbits, both CSF production and cerebral blood volume increase after sympathetic denervation, thereby increasing intracranial pressure259. Furthermore, induction of hydrocephalus in sympathetically denervated rabbits was not compatible with life. Electrical stimulation of the sympathetic nerves in rabbits after kaolin-induced acute obstructive hydrocephalus only reduced CSF production by 19%, compared to a reduction of 32%

116

and 38% in control animals and animals with chronic hydrocephalus, respectively ‘s9. These results were interpreted as follows: after induction of hydrocephalus the sympathetic nerves are activated, leading to a decrease in CSF production and of cerebral blood volume to reduce the intracranial pressure. Further stimulation of the sympathetic nerves in this situation can only reduce the CSF production slightly. In the chronic animals other compensatory mechanisms have reduced the intracranial pressure and the activity of the sympathetic nerves has diminished, making it possible to reduce the CSF production more substantially by electrical stimulation of the sympathetic nerves259. Sympathetic regulation of choroid plexus function could therefore have a role in the patho-physiological response to increases in intracranial pressure. 4.2. Cholinergic innervation In contrast to the numerous investigations of the sympathetic innervation few studies have been made of cholinergic nerves in the choroid plexus. This is largely due to the absence of sensitive and specific methods for detection of cholinergic nerve fibres in peripheral tissues. Histochemical demonstration of acetylcholinesterase has revealed presumably cholinergic nerve fibres varying in density depending on the species studied, the pig choroid plexus having the most well-developed supply of positive nerve fibres”‘. The origin of these nerve fibres is not known’**. In addition, dense labelling of muscarinic receptors was seen in rat choroid

plexus of the lateral ventricles, but not of the third or fourth ventricle plexuses, by receptor autoradiography 2x8. On the other hand, biochemical determinations of choline acetyltransferase (ChAT) activity in choroid plexus from different mammals demonstrated little or no neuronal ChAT activity and no release of [ “Hlacetylcholine could be detected following depolarization with either K+ or veratridine’27. Functional studies are equally scarce. Lindvall et a1.19” showed that the cholinergic agonist carbamylcholine inhibited CSF production in the rabbit by 20% at a concentration of lo-” M. It was also shown that the carbamylcholine-induced inhibition was mediated by muscarinic receptors and did not affect the inhibition of CSF production elicited by sympathetic nerve stimulation. In cat, opposite results were obtained, carbamylcholine increasing CSF production when given intravenously, in spite of a pronounced decrease in blood pressure. Also this effect was antagonized by atropine 13’. Again, species differences could be involved, but no real conclusions can be made about cholinergic function in the choroid plexus until further studies have been made. 4.3. Peptidergic innervation Vasoactive intestinal polypeptide (VIP) is a 28-amino acid neuropeptide with a widespread distribution in both the peripheral and central nervous system14’. VIP is a well-known vasodilator in peripheral and central arteries28,“32”352, but has numerous other effects as well,

Fig. 6. Fluorescence photomicrograph of VIP-immunoreactive nerve fibers in the lateral choroid plexus from pig. Immunoreactive fibers (arrowheads) can be seen both under the epithelium (A; X 600) and perivascularly (B; X 200). Reproduced from ref. 247, with permission.

117 e.g., stimulation of natural killer cell activity, regulation of cell growth and participation in ne~e-mediated stimulation of salivary gland secretion30~92~~z1~145~*s7. In the choroid plexus VIP-immunoreactive nerve fibres can be found in dense nerve plexuses around arteries, but also in the connective tissue stroma and (Fig. 6). VIP appears to be close to the epithelium 194,247 present in a population of nerve fibres that also store peptide histidine isoleucine (PHI), a peptide which belongs to the same peptide family and is a part of the same .precursor as VIP, and neuropeptide Y (further described below)247. The origin(s) of the VIP-immunoreactive nerve fibres in the choroid plexus are not known, but if the nerves enter the plexus tissue along the arteries they might originate in the same parasympathetic ganglia that supply the cerebral vessels with VIP fibres, namely the sphenopalatine, otic and internal carotid ganglia3%. A single VIP-binding protein of 5.5 kDa, with binding characteristics very similar to the VIP receptors previously described in other tissues208, has recently been demonstrated in isolated epithelial cells from pig choroid plexus 249(Fig. 7). This receptor might mediate the previously described VIP-induced stimulation of CAMP formation in cultured bovine epithelial cells and whole rabbit choroid plexus53,200. In vitro experiments have shown that both VIP and PHI enhance the release of [‘Hlnoradrenaline from pig lateral choroid plexus during electrical stimulation248 (Fig. 8) and that VIP dilates the anterior choroidal artery of cow ly4. To further study the function of VIP in the choroid plexus we have used an in vivo rat model, where CSF production and choroid plexus blood flow is measured simultaneously by the ventriculo-cisternal perfusion method developed by Pappenheimer 261 and laser-doppler flowmetry25’. In accordance with the in vitro studies, VIP increased the blood flow in the choroid plexus by 20% at concentrations of 10-9-10-7 M when given intraventricularly (Fig. 9) and, when infused in low doses, intravenous1~~~~.At higher intravenous doses no changes in blood flow were seen due to the VIP-induced systemic hypotension previously described in rat and man3’~176~31s. Ventriculo-cisternal perfusion with VIP (10-9-10-7 M) induces a decrease in CSF production up to 30% of control values2” (Fig. 9), indicating that choroid plexus blood flow and CSF production are not always directly coupled as has previously been suggested’“, although normaI CSF production might require a certain minimum blood supply. Similar results were obtained recently by Faraci et al.99, who found that the carbonic anhydrase inhibitor acetazolamide decreases CSF production by 55% while increasing the blood flow to the

MW(kDa)

205-

116-

92-

66-

Fig. 7. Autorad~ogram of the binding of [z251~VIPto choroid plexus epithelial cells, cross-linked to the receptor with disuccimidyl propionate and run overnight on a 7.5% SDS gel. The receptor has an apparent molecular weight of 55 kDa. The three lanes represent, from left to right, total binding of [‘2511VIP and binding in the presence of lo-” M unlabelled VIP and PHI, respectively. The position of the molecular-weight markers are indicated to the left. Reproduced from ref. 249, with permission.

choroid plexus twofold. That both noradrenaline and VIP stimulate CAMP production in choroid plexus epithelial cells and lower CSF production25’, in combination with the VIP-mediated presynaptic enhancement of noradrenaline re1ease248, is interesting in view of the synergistic actions of these two neurotransmitters demonstrated in the CNS103,213.It is possible that such synergism exists also in the choroid plexus. Information on other peptide neurotransmitters in the choroid plexus is more scarce. Neuropeptide Y (NPY) has been demonstrated in rat, guinea-pig, rabbit, cat, pig and human choroid plexus by immunohistochemistry and radioimmunoassay (RIA)X9,247. NPY

I

sY

70

% #m

60 t

ii

40

‘-

z c! 'II i!!

30

g

10

I l **

T

I

20

"Id =

0

.E

-10

ii ii

-20

;c

-30

Fig. 8. Maximum effects of VIP, PHI, NPY (all three at IO-’ M) and the a,-receptor antagonist yohimbine (3.10-s M) on the release of [ ‘Hlnoradrenaline (NA) from sympathetic nerves in the pig choroid plexus. All values are means + S.E.M. Statistically significant differences, as measured by the Student’s c-test for paired observations are shown as * P < 0.05, ** P < 0.01, ** * P < 0.001. The enhancing effects of VIP and PHI on [“H]noradrenaline release is in the same range as the effect of yohimbine which blocks the inhibitory effect of endogenous noradrenaline released from the sympathetic nerves.

levels measured by RIA were generally consistent between species, approximately 10 pmol/g in pooled plexus tissue from all ventricies247. In contrast, immunohistochemistry revealed large differences in the density of NPY-immunoreactive nerve fibres, with a moderate supply in pig and rabbit and a lower amount in rat and guinea-pig 247. Technical problems, e.g., fixation time, might explain the discrepancies, but extraneuronal sources of NPY cannot be excluded, e.g., by uptake or synthesis of NPY in the epithelium. NPY is well known to coexist with noradrenaline in sympathetic nervesy’~27h7but has also been demonstrated in non-sympathetic nerves, including nerves of

controi

VIP

Id3M

VIP

to.’M

Fig. 9. Relative changes in cerebrospinal fluid (CSF) production (open bars) and choroid plexus (CP) blood flow (filled bars; measured by laser-doppler flowmetry) during the second perfusion period of a ventriculocisternal perfusion experiment, compared to the initial perfusion period, with or without (control) intraventricular administration of VIP. The figures within parentheses are the number of experiments in each group. All values are means*S.E.M. Statistically significant differences, as measured by Student’s f-test for paired observations are shown as * P < 0.05, ** P < 0.01. Reproduced from ref. 251, with permission.

the cerebra1 circulation 'J1.105.l15.12~1.1t(2.22~.1.17..~.:i . Light microscopical immunohistochemist~ in the choroid plexus has shown two populations of NPY-immunoreactive nerve fibres: (1) a minor population harbouring NPY and the noradrenergic marker enzyme dopamine P-hydroxylase (DBH) and (21 a major population of nerve fibres where NPY, VIP and PHI coexist2”. Denervation studies demonstrated a non-significant reduction of 30% in NPY-content measured by RIA after sympathectomy in rabbit, while nerve fibres demonstrated by immunohist~hemist~ were completely abolished2~‘. The only functionaf data for NPY in the choroid plexus to date is a slight inhibition by 10% of [“HInoradrenaline release from sympathetic nerves in the pig choroid plexus evoked by electrical stimulation24”. This presynaptic inhibition of noradrenaline release by NPY has previously been demonstrated in several peripheral tissues8232y7,“0”. NPY inhibits CAMP formation induced by isoprenalin and VIP in what is believed to be a NPY receptor-mediated mechanism via activation of an inhibitory G-protein 24. It is possible that similar interactions between noradrenaline, VIP and NPY occur in the choroid plexus. Low levels of substance P immunoreactivity, shown by immunohistochemistry and RIA, have been reported in the choroid plexus’*. These results could not be confirmed in our laboratory247. 5. ENDOCRINE REGULATORY MECHANISMS IN THE CHOROID PLEXUS Hormones are traditionally described to be released from their site of origin and traveI with the blood to their target tissue, so called endocrine signalling. Also the CSF seems to be a signalling pathway for transport of ‘hormones’ from their site of synthesis to their target cells”“‘. Theoretically, a hormone can pass from blood to CSF (via the choroid plexus or brain regions lacking a BBB) and reach the whole or parts of the brain via bulk flow and diffusion to the brain extracellular fluid’sy~3”‘*3hX. Hormones can also be synthesized in the choroid plexus or meninges, secreted to the CSF and reach the brain as above252. Alternatively, substances synthesized in a brain region can diffuse to the CSF to reach another part of the brain by bulk flow 2513,368* Evidence exists that the choroid plexus is intimately involved in several of these processes, as will be discussed in this and the following sections. Advances in receptor autoradiography and immunohistochemistry together with traditional binding methods have revealed a great number of different neurohumorai receptors in the choroid plexus. In some cases, e.g., the 5-HT,, receptor, the receptor concentration is

119 the highest in the whole brain’40~370.Receptors for endocrine mediators in the blood or CSF, identified so far, are summarized in Table I. While methodological advances have made it possible to determine the presence, synthesis and characteristics of both the hormones and their receptors, functional studies of hormone action often suffer from the use of ‘non-physiological’ concentrations and routes of administration. This is particularly important as several peptides show a sharp peak in concentration for maximum effect90,153.

The choroid plexus contains large amounts of the receptor subtype143~266~370, with levels 10 times higher than other brain regions, as revealed by receptor autoradiography and in situ hybridization’40,‘66v230*267. The 5-HT,, receptor is a G-proteincoupled receptor I@‘,structurally and pha~acologically similar to the 5-HT, receptor’32,277 and acts on the cellular level by hydrolysis of phosphoinositides4’~‘~ without affecting adenylate cyclase activity260. The S-HT,, receptor appears to be localized to the apical membrane of the choroid plexus epithelial Chemical denervation of the 5-HT innervacells i17*131. tion of the brain by centrally injected 5,7-dihydroxytryptamine induced a marked receptor supersensitivity in the choroid plexus48, indicating that centrally released 5-HT influences the choroid plexus. Although raphe-lesions produce a pronounced decrease in choroid plexus 5-HT234, immunohistochemical and [3H]tryptamine uptake studies have been unable to demonstrate nerve fibres containing 5-HT in the choroid ple~s433%250,327. Instead there is a very rich plexus of 5-HT nerve fibres on the surface of the ventricular ependyma, without apparent contact with the ependymal celIs43,250*327. Thus the fall in choroid plexus 5-HT levels after lesion of the raphe nuclei234 probably reflects decreased reiease of 5-HT into the CSF followed by a parallel decrease in 5-HT uptake in the choroid plexus337,33X,34’. All things considered, we have proposed a model where 5-HT released from the supraependymal nerve fibres diffuse into the CSF337,338and is taken by bulk flow to the CSF side of the choroid plexus epithelium where it interacts with the apically located 5-HT,, receptors*” (Fig. 10). Comparison of cisternal 5-HT levels in the rat with the dissociation constant (Ku) of the 5-HT,, receptor is consistent with this hypothe5-i-IT,,

sis12,370

In cultured rat epithelial cells from the choroid plexus, 5-HT increases the gene expression and synthesis of the plasma protein transferrin349. This effect could be mimicked by a 5-HT,,-specific agonist but not

Fig. 10. Schematic picture of the rodent brain, showing a possible pathway for 5-HT influencing the choroid plexus (only the choroid plexus of a lateral ventricle is shown). Nerve fibers originating in serut~ergie neurons of the raphe nuclei project to the yenta of all four ventricles, te~inating in a dense supraependymal plexus in direct contact with CSF. Released 5-HT might diffuse into the CSF and reach the choroid plexus by bulk flow. Reproduced from ref. 250, with permission.

blocked by corresponding 5-HT,, receptor antagonists, indicating that this effect was not mediated by the 5-HT,, receptor or could be elicited in the presence of minimal receptor occupancy348. In monkeys and dogs, intravenous infusion of 5-HT increased choroid plexus blood flow by lo-200% without affecting cerebral blood flow9*. Ventriculo-cisternal perfusion in rabbits showed that intraventricular administration of 5-HT at relatively high concentrations (10-7-10-5 M) inhibits CSF production by 30 - 40%202,203.The effect could partially be blocked by the 5-HT, receptor antagonist ketanserin and the pi receptor antagonist practolol and was completely blocked by the specific 5-HT,, antagonist mesulergine202,203. Considering the relatively high concentrations used, an effect via the noradrenergic pi receptors cannot be excluded. However, in rats the partial 5-HT,, receptor agonist SCH-23390 has also been reported to decrease CSF production34. The physiological function of the 5-HT,, receptor might not be related to those described above. Banks and Kastin” proposed that the diurnal rhythms of opiate peptides and the tetrapeptide Tyr-MIF-1 codd partially be ascribed to diurnal variations in transport rate of these peptides over the blood-CSF barrier and BBB and later demonstrated that 5-HT could inhibit transport of Tyr-MIF-1 out of the brain” (Fig. 11). These findings correlate with the night-time peak levels of 5-HT observed in CSF”’ and it is possible that the 5-HT,, receptor mediates an inhibition of TyrMIF-1 transport from CSF at night to increase the levels in brain. If this is the case, then increased activity in a neuronal pathway could lead to overflow of a neurotransmitter, which will diffuse into the CSF and be transported to target areas along the CSF pathway as well as being absorbed into the blood as a part of the sink action of CSF65J50.

choroid plexus epithelial cells in a manner consistent with the presence of H, histamine receptorss”.‘5. Histamine enhances the passage of blood-borne “K into the CSFX”, while no effect was seen with histamine on rabbit CSF production measured by ventriculocisternal perfusion (Lindvall-Axelsson, unpublished observations).

Wlo

tnmol/mousc) ICV

Fig. 11. Effect of various doses of serotonin (black bars), atropine (hatched bars) and carbachol (grey bars) on the brain to blood transport of [ ‘251]Tyr-MIF-l. * P < 0.05. An average of ten mice were used per group. Reproduced from ref. 16, with permission.

5.2. Mela tonin Melatonin is secreted by the pineal gland, an endocrine gland located in the posterior part of the third ventricle and partially in contact with the CSF”‘“. The most prominent feature of melatonin is its diurnal rhythm in blood and CSF with high nighttime levels exceeding daytime concentrations severalfold’“,282, and appearing to be especially high in the lateral ventricle CSF as compared to cisternal CSF and jugular vein plasma”‘“. Central binding of melatonin occurs in a few discrete regions, notably the suprachiasmatic nucleus, median eminence and retina’77,‘s5, where it inhibits CAMP production via a pertussis toxin-sensitive G-protein’“. In the rat choroid plexus, receptor binding has been reported in the caudal region of the fourth ventricle choroid plexus”‘5, while chronic administration of melatonin in the hamster appeared to stimulate fluid secretion in the lateral ventricle choroid plexus (but not those of the third or fourth ventricle) as determined by electron microscopy and morphometry”6. These results are interesting in relation to the recently described circadian variation in human CSF production2.53

5.3. Histamine Histamine decarboxylase-immunoreactive and histidine decarboxylase (HDC)-immunoreactive cells with processes of varying number and length have been described in the rat choroid plexus, with morphology and distribution differing from that of mast cells24”, although these findings were not confirmed by in situ hybridization using a probe towards HDC mRNA42. The function of these cells is unknown, but histamine can stimulate CAMP production in cultured bovine

5.4. Atria1 and brain natriuretic peptide The atria of the heart synthesize and secrete atrial natriuretic peptide (ANP) which has an important role in the fluid balance of the body, with diuretic, natriuretic and vasodilatory actions and interactions with angiotensin II and aldosterone (for reviews, see refs. 113,151). ANP is also present in neurons of the brain where it appears to function as a central neuromediator”24,“67and can be found in the CSF at levels of 0.5-l PM. concentrations independent of the higher plasma concentrations2’“~22”~222.Binding sites for ANP are present in many brain regions including the brain capillaries and choroid plexus2Y~“h~2’h~27X, the apparent binding concentrations in the choroid plexus being among the highest in the brain 2y2. The ontogeny of these binding sites in rat brain show that while high levels of binding of ANP only occurs in the cerebral cortex in fetal and neonatal life and thereafter diminishes drastically, the binding sites in the choroid plexus and other circumventricular organs increase gradually after birth and reaches maximum levels postnatally’““. Interestingly, the increase in ANP binding parallels the gradual increase in CSF production seen after birth in rats”‘. The binding sites found in the choroid plexus I ave been identified as an ANP-binding particulate guanylate cyclase”4~4’~‘7X~2ys. ANP and the related brain natriuretic peptide, has been shown to stimulate cyclic GMP production in the choroid plexus”2,.14” and ANP reduces CSF production in rabbits”2h. Inhibition of CSF production might occur through inhibition of amiloride-sensitive Na+ transport, as demonstrated in cerebral capillaries”‘. Furthermore, ANP injected intraventricularly during hypoosmolar fluid loading led to sodium loss and water accumulation in the brain7x, indicating that ANP has a general role in brain fluid balance. In addition, ANP is rapidly degraded by the choroid plexus, as well as the pia mater, through the action of neutral endopeptidase 24.11 which is located at the apical brush border of the choroid plexus epithelial cells”2. Normal CSF levels of ANP (0.5 - 1 pM) are far below the association constant (K,,) reported for the ANP receptor binding and guanylate cyclase stimulation in the choroid plexus’2h,34”,and it seems likely that the ANP receptors in the choroid plexus are not acti-

121

pressure balance in the brain, with the choroid plexus and CSF production as one component.

Fig. 12. Autoradiographs showing [ ‘251]atrial natriuretic peptide (ANP) binding sites in the choroid plexus of 3-week-old rats with congenital hydrocephalus (LEW-HYR and HTX rats). In LEW-HYR rats, the extent of the dilatation of the lateral ventricle was divided into three subgroups: marked (I), medium (II) and slight (III). Arrows indicate the presence of [‘*‘INNP binding sites in the choroid plexus. Reproduced from ref. 232, with permission.

vated during physiological conditions. This might occur in several diseases, however. ANP binding sites are increased in kaolin-induced, but lowered in congenital, hydrocephalus 232,347(Fig. 12) and ANP levels in the CSF are higher in patients with raised intracranial pressure79. Brattleboro rats with hereditary diabetes insipidus (lack of vasopressin) showed no changes in ANP binding**‘, while the number of binding sites for ANP were lower in the choroid plexus and brain capillaries of spontaneously hypertensive rats, a rat strain that also shows dilated ventricles’47~232~258~292,293. These results indicate that atria1 and brain natriuretic peptides might be involved in volume, ion and

5.5. Vasopressin Vasopressin, often specified as arginine vasopressin (AVP), is a well-known nonapeptide neurotransmitter in neurons located in the hypothalamus projecting to the neurohypophysis where AVP is released into the blood stream. In addition, AVP has been demonstrated immunocytochemically outside the hypothalamo-neurohypophyseal system and binding sites exist in many areas of the brain, including the choroid plexus114~27’~356~357. The AVP receptor found in the choroid plexus appears to be of the V, subtype, which stimulates phospatidylinositol hydrolysis and appears to bind AVP, and the related peptides oxytocin, vasotocin and the AVP metabolite AVP,_,, with high affinity (1 nm