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0270.6474/85/0512-3178$02.00/O The Journal of Neuroscrence Copyrrght 0 Society for Neuroscrence Vol. 5, No. 12, pp. 3178-3183 Printed I” U S A December 1985

‘*% Biethylamide Binds to a Novel Site on Rat Choroid Plexus Epithelial Cells’

KEITH A. YAGALOFF AND PAUL R. HARTIG’

Department of Biology, Johns Hopkins University, Baltimore, Maryland 2 1218

Abstract

‘251-Lysergic acid diethylamide (‘251-LSD) binds with high Materials and Methods affinity to serotonergic sites on rat choroid plexus. These Autoradrography. Coronal sections (6 to 8 pm) of frozen adult rat brains sites were localized to choroid plexus epithelial cells by use were thaw-mounted onto subbed mrcroscope slides, air-drred for 20 min, of a novel high resolution stripping film technique for light and stored at -20°C In dessrcated microscope boxes. The mounted trssue microscopic autoradiography. In membrane preparations sections were brought to room temperature and then labeled with ‘?LSD from rat choroid plexus, the serotonergic site density was bv a technique srmilar to the method of Nakada et al. (1984). Sections were 3100 fmol/mg of , which is lo-fold higher than the incubated for 60 min at room temperature In 50 mM Tris-HCI buffer (pH 7.6) density of any other serotonergic site in brain homogenates. containina either 1.5 nM ‘?LSD (2000 Ci/mmol) or 1.5 nM “51-LSD and 1 UM The choroid plexus site exhibits a novel that ketansen;, to determine nonspecific binbrng. ‘?LSD was synthesized ‘by does not match the properties of 5hydroxytryptamine-la (5 the method of Morettr-Rojas et al. (1983) and purified by the method of Hartig et al. (1985a). The labeled sections were washed four trmes for 20 min each HT,,), 5-HTlb, or 5-HT2 serotonergic sites. ‘251-LSD binding to in ice-cold 50 mM Tris-HCI (pH 7.6) dipped briefly in ice-cold distilled water, the choroid plexus site is potently inhibited by , and blown dry with cold air. Labeled slides were exposed either against , and (+)-LSD. Other serotonergic, , Kodak AR.10 strrpping film-coated microscope slides or against LKB Ultrofilm and and antagonists exhibit moderate 3H. Ultrofilm was exposed for 24 hr in an x-ray cassette at 4°C developed to weak affinities for this site. The rat choroid plexus ‘251-LSD in a Kodak GBX developer for 2 min at 2O”C, processed, and dried. Stripping binding site appears to represent a new type of serotonergic film slides were prepared by floatrng preces of stripprng film (emulsion side site which is located on non-neuronal cells in this tissue. up) onto drstrlled water, picking them up from below with subbed microscope slides, wrapping the film around the slide, and drying thoroughly. Slides with labeled sections were clamped tightly against stripping film slides (using ‘“51-Lysergic acid diethylamide (“51-LSD) binds to serotonin 5- binder clips) and placed in a Irght-tight, dessicated box for 4 days at 4°C. hydroxytryptamine-2 (5HT2) receptors in the mammalian brain (Engel The film was developed for 2 min In Kodak D19 developer at 2O”C, washed 30 set In water, fixed 12 min in Kodak fixer, rinsed, and air dried. Specific et al., 1984; Kadan et al., 1984). In a recent autoradiographic study, binding was typically 80% of the total “~51-LSD bound. Labeled sections were Nakada et al. (1984) noted a high level of “51-LSD binding in the starned with Harris’ hematoxylin and counterstained with eosin Y after the lateral ventricles, which was displaced by , a potent autoradiographtc exposure. serotonin 5HT, . Other investigators have described the bind- Homogenate-binding assays. The choroid plexus was dissected from the ing of 3H-LSD and 3H-serotonin to a particular ventricular structure, lateral and third ventricles of 40 adult rat brains (Pel Freeze). From each rat the choroid plexus (Diab et al., 1971; Meibach et al., 1980; Palacios brarn we obtained approximately 1.3 mg (wet weight) of choroid plexus et al., 1983), but these studies did not describe the pharmacological &sue. The combined tissue was homogenrzed in 2 ml of ice-cold 0.32 M properties of the choroid plexus binding sites. We decided to sucrose (eight strokes in a Teflon pestle tissue homogenizer rotating at 500 investigate ventricular ‘*?LSD-binding sites to determine their cellular rpm) and centrifuged for 15 min at 750 X g (4°C). The supernatant was centrrfuged again at 750 x g for 15 min. The resulting supernatant was location and binding properties. Our results show that binding sites carefully removed and centrifuged 20 min at 35,000 x g. The pellet was for lz51-LSD are located on epithelial cells of the choroid plexus. resuspended in 2 ml of 50 mM Tris-HCI (pH 7.6) with six strokes of the These binding sites exhibit a unique serotonergic pharmacology homogenizer and incubated 15 min at 37°C. The homogenate was cooled which does not match the properties of either 5HT, or 5-HT2 sites and centrifuged for 20 min at 35,000 x g (4’C). The resulttng pellet was in the brain, These sites are present at a density IO-fold higher than resuspended by homogenization rn 2 ml of 50 mM Tris-HCI (pH 7.6) and was the density of any other serotonergic site in brain membrane ho- stored under lrquid nitrogen until use. Membrane preparations of choroid mogenates The choroid plexus ‘251-LSD-binding site appears to plexus from adult mouse, pig, rabbit, and cow brains were prepared as represent a new type of mammalian brain serotonergic site. A described for rat brain using choroid plexuses dissected from 2 to 25 brains preliminary report of this work has been published (Hartig and and homogenized in an appropriate volume of 0.32 M sucrose. Protein concentrations were determined by the method of Lowry et al. (1951). Yagaloff, 1985). A buffer of 50 mM Tris-HCI, pH 7.6 (at 23°C) and a tissue concentration of 0.18 mg of protein/ml were used for the determination of the ‘Z51-LSD Received January 7, 1985; Revised March 25, 1985; drssociation constant (Scatchard analysis). The ‘“51-LSD concentration was separately measured for each dilution used in the Scatchard analysis. This Accepted Aprrl 3, 1985 avoids errors which can arise from adsorption of the radiolrgand during dilutions and transfers (Hartig et al., 1985a). Nonspecrfic binding was defined ’ This work was supported by Natronal Science Foundation Grant BNS- by 1 PM ketanserin in the Scatchard experiments and all other bindrng 8407432. K. A. Y. was supported by Natronal lnstrtutes of Health Trarning studies. Sample incubatron, filtration, and counting conditions were the same Grant 5T32GM07231. We thank Dr. J. Palacios for providing a preprint of his as previously described for ‘251-LSD binding to rat frontal cortex membranes work (Pazos and Palacios, 1985) prior to publicatron. (Kadan et al., 1984). * To whom correspondence should be addressed. In the competition (I&) experiments, binding equilrbrium was reached 3178 The Journal of Neuroscience ‘251-LSD Binding in Rat Choroid Plexus 3179 during the 15min incubation at 37°C at which time less than 10% of the the cellular location of binding sites within the choroid plexus. In added radioligand was bound. Specific binding was consistently 85 to 90% order to obtain hrgher resolution autoradiographs, we used Kodak of total binding at the tissue concentration used (0.18 mg of protein/ml), with AR.10 stripping film. The grain size of this stripping film is approxi- the radioligand concentration near 3.5 nM. The ‘“I-LSD concentration was mately 6 times smaller than that of Ultrofilm 3H, which results in between 3 and 4 nM for all competition studies. A buffer containing 50 mM greater resolutron with a proportionate decrease in sensitivity. In Tris, 10 PM , 5 mM EDTA, 1 mM sodium ascorbate, pH 7.6, was used for all assays, Only small changes in total and specific binding were addition, the film IS of consrstently uniform thickness, is easily seen with this buffer as compared to 50 mM Tris-HCI alone. Total binding manipulated, and has a longer shelf life than nuclear emulsions was typically 40,000 dpm in each 30.@I sample used for these experiments. (NTB2, NTB3) which are commonly used for high resolution autora- Thirty microliters represents the lower limit of final sample volume that can diographs. The stripping film autoradiograph reveals (Fig. 2) that ‘251- be accurately handled in these binding experiments. Lower tissue concen- LSD-binding sites are located on eprthelial cells distributed through- trations can be used, but the ratio of specific to nonspecific binding de- out the choroid plexus. These epithelial cells form a monolayer of creases due to an increased contribution from radioligand binding to the cuboidal cells in direct contact with the cerebrospinal fluid (Tennyson glass fiber filters. (+)-LSD and (-)-LSD for these studies were obtained from and Pappas, 1968). Within the ventricular region, ‘251-LSD labeling is the National Institute on Abuse. found exclusively on the choroid plexus. Ependymal cells which line Results the walls of the ventricles are not labeled by ‘251-LSD. Homogenate binding assays. We dissected the choroid plexus Autoradiography. The binding of “51-LSD to a coronal section from adult rat brains, prepared a membrane homogenate, and from adult rat brain is shown in Figure 1. In agreement with previous examined the pharmacological properties of the ‘251-LSD-binding studies (Engel et al., 1984; Nakada et al., 1984) this Ultrofilm site. The binding of ‘251-LSD to the choroid plexus homogenate (Fig. autoradiograph shows a high density of ‘251-LSD binding in layer IV 3) yields a linear Scatchard plot (Hill slope = 0.98) and an average of the cortex, in the claustrum, and in the , which is reduced dissociation constant (Kd) of 3.4 + 0.1 nM (mean + SEM). In contrast, to background levels by inclusion of 1 PM ketanserin during the “51-LSD has been shown to bind to 5HT2 receptors with a Kd of 1 labeling. Especially high levels of lz51-LSD binding are seen (Fig. 1) to 1.5 nM (Engel et al., 1984; Kadan et al., 1984; Hartig et al., 198513) in the choroid plexus of the lateral and third ventricles. The density to 02 sites with a /Cdof 9.1 nM (Hartig et al., 1985b), to 5 of ‘251-LSD labeling in choroid plexus exceeds the labeling density HT,,sites with an apparent K, of 30 nM (Engel et al., 1984) and very observed in any other region of the rat brain. Although it was possible weakly, rf at all, to 5.HTlb, Hl, or adrenergic sites (Engel to localize lz51-LSD binding to the choroid plexus using Ultrofilm, the et al., 1984). The site density (B,,) for “51-LSD binding to the relatively low resolution of this film prevented us from determining choroid plexus homogenate is approximately 3100 fmol/mg of pro-

Figure 1. Autoradiograph of ‘z51-LSD binding to a coronal section from adult rat brain. The image is reversed such that areas with a high density of labeling appear as white grains on a dark background. The section was labeled with 1.5 nM ‘?LSD and exposed against Ultrofilm for 24 hr. High levels of labeling are seen in the choroid plexus of the lateral and third ventricles (arrows), in layer IV of the cortex, in the claustrum, and in the striatum. 3180 Yagaloff and Hartig Vol. 5, No. 12, Dec. 1985

Figure 2. High resolution autoradiograph of ‘*%LSD labeling of rat lateral ventricle. A, Photomicrograph of a rat lateral ventricle stained with hematoxylin and eosin Y after autoradiography. B, Autoradiograph of ‘z51-LSD binding to this same section. Regions of high density labeling appear as white grains on a black background. The section was labeled with 1.5 nM ‘251-LSD and exposed against AR.10 stripping film for 4 days. The bar represents 200 pm. CC, corpus callosum; CN, caudate nucleus; CP, choroid plexus; LS, lateral septal nucleus; V, lateral ventricle.

tein, which is more than 10 times greater than any other serotonergic site density reported in rat brain homogenates (Pedigo et al., 1981; Mallat and Hamon, 1982; Engel et al., 1984). Table I summarizes the results of competition binding studies (C, assays) using various ligands for serotonergic, dopaminergic, and adrenergic sites. Serotonin and the serotonergic antago- nists mianserin, (+)-LSD, and ketanserin are all potent inhibitors of ‘?-LSD binding. Serotonrn is over 200-fold more potent than the E other agonists we tested. (-)-LSD, with a K, of >5 - 1500 PM, is at least 1000 times less potent than (+)-LSD in displacing “?- 53 m LSD binding, demonstrating the stereospecificity of binding at this i 1000 site. Dopamine and the are both 8+ t 1 weak displacers of ‘251-LSD binding. The (Y- and P-adrenergic antag- onists and both show weak displacement of ‘251-LSD as does the adrenergic , epinephrine. Chlorphen- 4 500-o/4.Amy, iramine, a histamine Hl antagonist, is a very weak inhibitor of ‘251- 0 I 2 3 4 5 6 7 8 9 LSD binding, as is the selective 5HT,, agonist, 8-OH-DPAT (8- “‘I-LSD CONCENTRATION (nM) hydroxy-2-(di-n-propylamino) tetralin). Addition of 1 mM sodium ascorbate, 5 mM EDTA, and 10 PM Figure 3. Binding of ?-LSD to rat choroid plexus membrane preparations, Total (Cl) and nonspecific (m) binding were determined in the absence or pargyline to the incubation mixture had little effect on the total or presence of 1 PM ketanserin, respectively. Data are shown as a saturation specific binding of ‘251-LSD. However, the addition of ascorbate in plot and the corresponding Scatchard plot (inset) from a single representative the absence of EDTA caused a large decrease (50%) in specific experiment. Data points represent mean f SD values from triplicate assays 12’l-LSD binding. This may be due to ascorbate-catalyzed lipid at each concentration. The K,, value is 3.45 nM and the B,, is 3117 fmol/mg peroxidation, as was shown to occur in several receptor systems of protein in this single experiment. (Heikkila et al., 1982; Heikkila, 1983; Muakkassah-Kelly et al., 1983). The Journal of Neuroscience ‘251-LSD Binding in Rat Choroid Plexus 3181

TABLE I LSD-binding site was also detected in homogenates from mouse, Apparent K, values (K, = /C&l + [L]/Kd)) for ‘%LSD binding to rat pig, rabbit, and cow choroid plexus, but the apparent site density choroid plexus homogenates and ratio of specific to nonspecific binding was much higher in the Assay conditions are described under “Materials and Methods. The data rat brain. represent mean values + SEM for three to six experiments, each assayed in The serotonergic choroid plexus site does not resemble any of triplicate. The data from a previous study of ‘?LSD binding to 5-HT2 sites the three serotonergic sites previously described in the mammalian In rat frontal cortex (Kadan et al., 1984) are also provided for comparison. brain: 5-HT,,, 5.HT,,,, or 5-HT,. GTP and divalent cations modulate agonist affinity for the 5-HT’ (Mallat and Hamon, 1982; Hamon et Displacer Apparent K, in Apparent K, in Chorold Plexus (no) Frontal Cortex (nu) al., 1983; Sills et al., 1984a) and 5HT2 sites (Battaglia et al., 1984) Miansenn 1.9 f 0.2 8.1 in a manner characteristic of adenylate cyclase-linked receptors. We (+)-LSD 4.4 f 0.4 2.9 did not detect significant GTP modulation of agonist binding to rat Ketanserin 28 rfr. 1 3.8 choroid plexus sites under conditions that cause large effects on Clnanserln 74 f 2 10 serotonin 5-HT, sites. Propranolol 736k 17 2,200 In comparison to the 5-HT2 site labeled by “?LSD in rat frontal Phentolamine 1,250k 100 870 cortex (Kadan et al., 1984), the choroid plexus site exhibits a 37. Haloperidol 2,050f 120 153 fold higher affinity for serotonin and a 7-fold lower affinity for the Chlorphenlramine >5,000 antagonists ketanserin and . One serotonergic antagonist, (-)-LSD >5,000 >5,000 mianserin, is much more potent at the choroid plexus site than at the 5-HT2 site. In addition, the affinity of ‘251-LSD for the choroid Serotonln 30.4 -c 3.6 1,120 plexus site is 2- to 3-fold lower than its affinity for serotonin 5-HT2 8-OH-DPAT 7,700+ 460 sites (Engel, et al., 1984; Kadan et al., 1984; Hartig et al., 1985b). Dopamlne 43,100 + 4,100 162,000 The choroid plexus site also differs significantly from the 5-HT,, Eplnephnne 330,000 + 12,300 760,000 site. Cinanserin and ketanserin are weak inhibitors (K, = 1 and 4 PM, respectively) of 5-HT’, binding in the (Gozlan et al., TABLE II 1983), whereas these same compounds are relatively potent inhibi- Species survey of “‘I-LSD binding to choroid plexus tors of ?LSD binding to the choroid plexus site (,Y = 74 and 28 Membrane preparations of choroid plexus from each species were pre- nM, respectively). Furthermore, the selective 5-HTl, agonist 8-OH- pared and assayed as described under “Materials and Methods.” lZ51-LSD DPAT (Middlemiss and Fozard, 1983) has a weak affinity for the binding was measured using 3.5 nM ‘*‘l-LSD in 50 mM Tris buffer (pH 7.6), choroid plexus site (K, = 7.7 PM), and the ligand affinity profile for and membranes were adjusted to 0.2 mg of protein/ml in the assay. Values 3H-serotonin binding to the 5-HT’, site (Pedigo et al., 1981; Sills et are averages from two to three separate membrane preparations, each al., 1984b) differs significantly from that of the choroid plexus site. assayed in triplicate. The level of specific binding in the rat was defined as The choroid plexus site also differs from the 5-HTlb site. The 5-HTlb 100%. site exhibits a weak affinity for most serotonergic antagonists (Pedigo Speclflc Blnding Speclflc/Total Binding et al., 1981; Fardin et al., 1984; Sills et al., 1984b), whereas the (%) (%) choroid plexus site exhibits a strong to moderate affinity for all serotonergic antagonists we tested. Rat 100 85 The rat choroid plexus “51-LSD-binding site was localized to the Mouse 18 58 epithelial cell layer by high resolution light microscopic autoradiog- Pig 16 57 raphy using a stripping film technique. The sites are distributed Rabbit 13 39 throughout the choroid plexus epithelial cell layer but are absent cow 6 35 from ependymal cells which line the ventricles. Various serotonergic sites have also been found on other non-neuronal cells. 3H-Serotonin Addition of 5 mM calcium chloride caused a 25% decrease in the binding sites have been described on crude glial cell membrane specific binding of 3.5 nM ‘251-LSD to choroid plexus membranes in fractions (Fillion et al., 1980), on C6 glioma cells (Whitaker-Azmitia a buffer of 1 mM sodium ascorbate, 10 FM pargyline, 50 mM Tris, and Azmitia, 1984), and on astrocytes in primary cultures (Hertz et pH 7.6. Addition of 0.1 mM GTP or GppNHp (5’-guanylylimidodi- al., 1979; Whitaker-Azmitia and Azmitia, 1984). In our own laboratory phosphate) did not noticeably alter the affinity of serotonin (I&,, we have found serotonergic 3H-LSD-binding sites on astrocyte- value) for the lz51-LSD-binding site when tested in 1 mM sodium enriched fractions isolated from adult rat brains (Gal and Hartig, ascorbate, 1 mM Mg&, 10 PM pargyline, 50 mM Tris, pH 7.6. 1982). These studies demonstrate that serotonergic sites are found ?LSD binding was also measured in choroid plexus membrane on a variety of non-neuronal cells and emphasize the uncertainty preparations from mouse, pig, rabbit, and cow brain. Table II shows that currently exists regarding the cellular location of serotonergic the binding of 3.5 nM lz51-LSD to these membrane preparations. Both sites in the mammalian brain. the apparent site densities and the ratio of specific to nonspecific Following completion of this work, we learned of a paper by binding were much lower than the values obtained from rat choroid Palacios and co-workers (Pazos et al., 1984) which describes the plexus homogenates. The next highest site density was found in the properties of a new serotonergic site (termed 5-HT’,) labeled by 3H- mouse, which exhibits 18% of the binding sites seen in rat and 58% and 3H-serotonin in pig choroid plexus. The serotonergic specific binding compared to 85 to 90% specific binding in rat site they characterized in pig choroid plexus is present at a much choroid plexus preparations. lower density than is the rat choroid plexus binding site. Although there is a clear similarity between the 5-HT’, site defined by 3H- Discussion serotonin and the lz51-LSD-binding site in choroid plexus, there are Rat choroid plexus possesses a novel serotonergic ‘251-LSD- also differences which may be important. First, the number of choroid binding site. This site exhibits a high affinity for serotonin and for plexus binding sites (B,,) detected by 3H-LSD, 3H-serotonin, 3H- several serotonergic antagonists but a weak affinity for adrenergic mesulergine, or ‘251-LSD differs by as much as a factor of 2 (Pazos and dopaminergic ligands. The ligand affinity profile of the choroid et al., 1984; Pazos and Palacios, 1985; this paper). Second, the plexus site is unique and its site density (B,, value) of 3100 fmol/ dissociation constant (&) for 3H-serotonin binding to the choroid mg of protein is more than IO-fold higher than the density of any plexus 5-HT,,site is 6.5 nM (Pazos et al., 1984), whereas the K,value other serotonergic site in brain homogenates. A high affinity 1251- for serotonin competition at 3H-mesulergine, and ‘?LSD sites is 20 3182 Yagaloff and Hartig Vol. 5, No. 12, Dec. 1985 to 30 nM (Pazos et al., 1984; this paper). Finally, various compounds latory or hormone-like action) have been observed in other systems exhibit similar but not identical K, values in displacing ‘251-LSD, 3H- (for reviews see Kupfermann, 1979; Aghajanian, 1981; Bloom, 1981; mesulergine, or 3H-serotonin from choroid plexus binding sites. For Gelperin, 1981; Shain and Carpenter, 1981). example, cinanserin is a much weaker inhibitor of both 3H-serotonin The current study extends our understanding of the choroid plexus and 3H-mesulergine binding (K, = 1585 and 200 nM, respectively serotonergic site in several new directions. We have localized this (Pazos et al., 1984)) than of ‘?LSD binding to choroid plexus (K, = site to the epithelial cell layer of choroid plexus. The utility of “%LSD 74 nM; Table I). These differences do not appear to be species as a label for choroid plexus sites has been demonstrated and the effects since the binding properties in rat (Pazos and Palacios, 1985) high sensitivity of this ligand has made it possible to study the and in pig (Pazos et al., 1984) are quite similar. pharmacological properties of this site in membrane homogenates The physiological function of the choroid plexus serotonergic site from rat choroid plexus, where the amount of available tissue is has not been identified; thus, it cannot yet be classified as a receptor extremely limited. The choroid plexus should provide an excellent site. The choroid plexus is a richly vascularized structure which acts source for future studies on the cell biology of this serotonergic site as a major, but not the only, site of cerebrospinal fluid (CSF) because it is a relatively simple tissue which contains only a few production. The epithelial cell serotonergic site in this tissue is well different cell types and it can be maintained in culture (Crook et al., situated to modulate some aspect of CSF production or composition. 1981). Since the choroid plexus serotonergic site is present at very In a recent ventriculocisternal perfusion study, serotonin was shown high densities, this tissue should also be useful for biochemical to be the most effective monoamine modulator of CSF, causing a studies. With the localization of this novel serotonergic site to choroid 40% reduction in the rate of CSF production when the serotonin plexus epithelial cells, we can focus on these cells in an effort to precusor 5hydroxytryptophan was injected intravenously (Maeda, understand this intriguing ventricular serotonergic system. 1983). The choroid plexus serotonergic site may act as a receptor site for serotonergic modulation of CSF production. It is also possible References that the choroid plexus serotonergic site serves as a transport site Aghatanran, G. K. (1981) The modulatory role of serotonrn at multrple for serotonin or a closely related monoamine. Two studies (Tochino receptors in the brarn. In Serotonin Neurotransmission and Behavior, B. L. and Schanker, 1965; Lindvall et al., 1980) have described a choroid Jacobs and A. Gelperin, eds., pp. 156-185, MIT Press, Cambridge, MA. plexus active transport system for serotonin, whereas another study Aghajanian, G. K., and D. W. Gallager (1975) Raphe origin of serotonergic (Chan-Palay, 1976) failed to find serotonin uptake by this structure. nerves terminatrng in the cerebral ventricles. Brain Res. 88: 221-231. If the choroid plexus site is a serotonin transport carrier, its binding Battaglia, G., M. Shannon, and M. Titeler (1984) Guanyl nucleotide and divalent cation regulation of cortical S2 serotonin receptors. J. Neurochem. properties differ markedly from those of other serotonin uptake 43: 1213-1219. systems (Whipple et al., 1983; Wong et al., 1983). Bloom, F. E. (1981) A comparrson of serotonergic and noradrenergic neu- It is not clear whether the choroid plexus serotonergic site is rotransmission in the mammalian CNS. In Serotonm Neurotransmission activated by serotonin released locally within the choroid plexus or and Behavior, B. L. Jacobs and A. Gelperin, eds., pp. 403-424, MIT Press, by serotonin released from a remote site. Extensive immunohisto- Cambridge, MA. chemical studies with serotonin antibodies (Lidov et al., 1980; Stein- ChanPalay, V. (1976) Serotonin axons in the supra- and subependymal busch, 1981) and histochemical fluorescence studies (Aghajanian plexuses and in the leptomeninges; their roles in local alterations of and Gallager, 1975) failed to note any serotonergic innervation of cerebrosprnal fluid and vasomotor activity. Brain Res. 702: 103-130. the choroid plexus. Recently, however, indoleaminergic innervation Crook, R. B., H. Kasagami, and S. B. Prusrner (1981) Culture and character- rzation of epithelial cells from bovine choroid plexus. J. Neurochem. 37: of rat choroid plexus was detected when rats were pretreated with 845-854. and 5-hydroxytryptophan (Napoleone et al., 1982) and Diab, I. M., D. X. Freedman, and L. J. Roth (1971) [3H]Lysergic acid low levels of serotonin were detected in the choroid plexus by a drethylamrde: Cellular autoradiographic localization in rat brain. Science sensitive enzymatic assay (Moskowitz et al., 1979). The indoleami- 7 73: 1022-l 024. nergic innervation of choroid plexus occurs mostly on the walls of Engel, G., E. Mdller-Schweinitzer, and J. M. Palacios (1984) 2-[‘*‘lodo]LSD, blood vessels in the choroid plexus with one report suggesting that a new ligand for the characterization and localisation of 5-HTZ receptors. a few indoleaminergic fibers contact the epithelial cells (Napoleone Naunyn-Schmiedebergs Arch. Pharmacol. 325: 328-336. et al., 1982) whereas another study (Nakamura and Moriyasu, 1978) Fardin, V., D. L. Nelson, and H. I. Yamamura (1984) Pharmacological found no nerve endings in direct contact with epithelial cells. Another characterizatron of serotonrn-1 binding sites in the rat spinal chord: Com- possibility is that serotonin reaches the choroid plexus following parison with cortex. Sot. Neurosci. Abstr. 70: 221. FrIllon, G., D. Beaudorn, J. C. Rousselle, and J. Jacob (1980) [3H]5-HT release from rich presynaptic stores in the ventricles. A dense binding sites and 5HT sensitive adenylate cyclase in glial cell membrane network of serotonergic fibers is present on the surface of ependy- fraction. Brain Res. 198: 361-374. mal cells which line the walls of the ventricles (Aghajanian and Gal, S., and P. R. Hartrg (1982) Cellular drstribution of 3H-LSD binding sites Gallager, 1975). These supra-ependymal fibers contain numerous in rat brain. J. Cell 8101. 95: 139a. varicosities with small and large vesicles in which serotonin is stored Gelpenn, A. (1981) Synaptic modulation by identified serotonin . In (Richards and Guggenheim, 1982) as well as an active serotonin Serotonin Neurotransmission and Behavior, B. L. Jacobs and A. Gelperin, uptake system which is stimulated by electrical excitation (Chan- eds., pp. 288-306, MIT Press, Cambridge, MA. Palay, 1976) of the corresponding cell bodies in the raphe nuclei Gozlan, H., S. El Mestikawy, L. Pichat, J. Glowinski, and M. Hamon (1983) Identification of presynaptic serotonin autoreceptors using a new ligand: (Aghajanian and Gallager, 1975). Interestingly, the supra-ependymal 3H-PAT. Nature 305: 140-l 42. serotonergic fibers do not form recognizable with the Hamon, M., C. Goetz, and H. Gozlan (1983) Recrprocal modulations of ependymal cells (Richards and Guggenheim, 1982) but they are central 5-HT receptors by GTP and cations. Adv. Biochem. Psychophar- situated in contact with CSF fluid which may serve to receive and macol. 37: 349-359. distribute serotonin released by these fibers, The target sites do not Hartig, P., and K. Yagaloff (1985) “‘I-LSD binds to a novel serotonergic site appear to be ependymal cells since these cells are devoid of in rat chorord plexus. Fed. Proc. 44: 1244. serotonergic binding sites (Meibach et al., 1980; see Fig. 2). Sus- Hartig, P. R., A. M. Krohn, and S. A. Hirschman (1985a) Microchemical pended in the CSF, however, is the choroid plexus which is richly synthesis of the serotonin receptor ligand, ‘“%LSD. Anal. Biochem. 144: endowed with serotonergic binding sites. It is tempting to speculate 441-446. that serotonin serves a hormonal or neuromodulatory role in this Hanig, P. R., M. J. Evans, A. M. Krohn, S. A. Leder, P. C. Sze, and D. A. Stoffers (1985b) ‘“%LSD binding to serotonin and dopamine receptors in system in which it is released into the CSF under control of the bovine caudate membranes. Neurochem. Int. 7: 699-707. raphe nuclei, diffuses through the CSF, and activates receptor sites Heikkila, R. E. (1983) Ascorbateinduced lipid peroxidation and the binding on the choroid plexus which control some aspect of CSF production. of [3H]. Eur. J. Pharmacol. 93: 79-85. Examples of similar action-at-a-distance by serotonin (neuromodu- Herkkila, R. E., F. S. Cabbat, and L. Manzino (1982) Inhibitory effects of The Journal of Neuroscience lz51-LSD Binding in Rat Choroid Plexus 3183

ascorbrc acrd on the binding of [3H]dopamine antagonists to neostriatal Nakamura, S., and N. Moriyasu (1978) Nerve fibers and nerve ending in the membrane preparations: Relatronshtp to lipid peroxrdation. J. Neurochem. choroid plexus: Electron mrcroscoprc study. Brain Nerve 30: 259-266. 38: 1000-1006. Napoleone, P., G. Sancesario, and F. Amenta (1982) lndoleaminergic inner- Hertz, L., F. Baldwin, and A. Schousboe (1979) Serotonin receptors on vation of rat choroid plexus: A fluorescence histochemical study. Neurosci. astrocytes in primary cultures: Effects of and fluoxettne. Lett. 34: 143-147. Can. J. Physrol. Pharmacol. 57: 223-226. Palacios, J. M., A. Probst, and R. Cortes (1983) The drstributron of serotonrn Kadan, M. J., A. M. Krohn, M. J. Evans, R. L. Waltz, and P. R. Hartig (1984) receptors in the human brain: Hugh density of [3H]LSD binding sites in the Characterizatron of “51-LSD binding to serotonin receptors in rat frontal raphe nuclei of the brainstem. Brain Res. 274: 150-I 55. cortex. J. Neurochem. 43; 601-606. Pazos, A., and J. M. Palacios (1985) Quantrtative autoradiographic mapping Kupfermann, I. (1979) Modulatory actions of . Annu. Rev. of serotonin receptors In the rat brain. I. Serotonin-1 receptors. Brain Res., Neurosci. 2: 447-465. in press. Lidov, H. G. W., R. Grzanna. and M. E. Molliver (1980) The serotonin Pazos, A., D. Hoyer, and J. M. Palacros (1984) The binding of serotonergic innervatron of the cerebral cortex in the rat-An immunohistochemical ligands to the porcine choroid plexus: Characterization of a new type of analysis. Neuroscience 5: 207-227. serotonin recognition site. Eur. J. Pharmacol. 706: 539-546. Lindvall, M.. J. E. Hardebo. and C. Owman (1980) Barrier mechanisms for Pedigo, N. W., H. I. Yamamura, and D. L. Nelson (1981) Discrimination of monoamines in the choroib plexus. Acta Physiol. Stand. multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic 108: 215-221. in rat brain. J. Neurochem. 36: 220-226. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951) Protein Richards, J. G., and R. Guggenheim (1982) Serotonergic axons in the brain: measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. A bird’s eye view. Trends Neuroscr. 5: 4-5. Maeda, K. (1983) Monoaminergic effect on cerebrospinal fluid production. Shain, W., and D. 0. Carpenter (1981) Mechanisms of synaptic modulation. Nihon Univ. J. Med. 25: 155-174. Int. Rev. Neurobiol. 22: 205-245. Mallat, M., and M. Hamon (1982) Ca++-guanrne nucleotide rnteractrons in Sills, M. A., B. B. Wolfe, and A. Frazer (1984a) Multiple states of the 5- brarn membranes. I. Modulation of central 5-hydroxytryptamine receptors hydroxytryptamtne, receptor as indicated by the effects of GTP on [3H]5- In the rat. J. Neurochem. 38: 151-161. hydroxytryptamrne binding in rat frontal cortex. Mol. Pharmacol. 26: lo- Meibach, R. C., S. Maayani, and J. P. Green (1980) Characterization and 18. radroautography of [%]LSD binding by rat brain slices in vitro: The effect Sills, M. A., B. B. Wolfe, and A. Frazer (198413) Determination of selective of 5-hvdroxvtrvptamine. Eur. J. Pharmacol. 267: 371-382. and nonselective compounds for the 5-HT,, and 5.HTlb receptor subtypes Middlem&s, d. N., and J. R. Fozard (1983) 8-Hydroxy-2(di-n-propylamino) in rat frontal cortex. J. Pharmacol. Exp. Ther. 237: 480-487. tetralrn drscriminates between subtypes of the 5HT, recognition site. Eur. Steinbusch, H. W. M. (1981) Distribution of serotonin-immunoreactivrty In the J. Pharmacol. 90: 151-l 53. central nervous system of the rat--Cell bodies and terminals. Neurosci- Moretti-Rojas, I., E. G. Ezrailson, L. Birnbaumer, M. L. Entman, and A. J. ence 6: 557-618. Garber (1983) Serotonergic and adrenergic regulation of skeletal muscle Tennyson, V. M., and G. D. Pappas (1968) The fine structure of the choroid metabolrsm in the rat. II. The use of [“51]rodolysergic acrd diethylamide and plexus: Adult and developmental stages. Prog. Brain Res. 29: 63-85. [‘Z51]iodoprndolol as probes of sarcolemmal receptor function and specific- Tochino, Y., and L. S. Schanker (1965) Transport of serotonin and norepi- ity. J. Biol. Chem. 258: 12499-12508. nephrine by the rabbit choroid plexus in vitro. Biochem. 74: 1557-1566. Moskowitz, M. A., J. E. Lrebmann, J. F. Reinhard, Jr., and A. Schlosberg Whipple, M. R., M. G. Reinecke, and F. H. Gage (1983) Inhibitron of (1979) Raphe origin of serotonin-containing neurons within choroid plexus synaptosomal neurotransmitter uptake by . J. Neurochem. of the rat. Brain Res. 769: 590-594. 40: 1185-l 188. Muakkassah-Kelly, S. F., J. W. Andresen, J. C. Shih, and P. Hochstein (1983) Whitaker-Azmitia, P. M., and E. C. Azmitia (1984) High affinity [3H]serotonrn Dual effects of ascorbate on serotonin and spiperone binding in rat cortical binding sites on intact brain astroglial cells. Sot. Neurosct. Abstr. 70: 222. membranes. J. Neurochem. 47: 1429-1439. Wong, D. T., F. P. Bymaster, L. R. Rerd, and P. G. Threlkeld (1983) Nakada, M. T., C. M. Wieczorek, and T. C. Rainbow (1984) Localization and and two other serotonin uptake inhibitors without affinity for neuronal characterization by quantitative autoradiography of [‘?]LSD binding sites receptors. Biochem. Pharmacol. 32: 1287-l 293. in rat brain. Neurosci. Lett. 49: 13-18.