Supra-ependymal serotoninergic nerves in mammalian brain: Morphological, pharmacological and functional studies

Brain Research

Bulletin,

Vol. 9, pp. 727-741, 1982. Printed

in the U.S.A.

Supra-Ependymal Serotoninergic Nerves in Mammalian Brain: Morphological, Pharmacological and Functional Studies H. P. LOREZ AND J. G. RICHARDS F. Hoffmann-La

Pharmaceutical Research Department Roche and Co., Ltd., CH-4002 Basel,

LORE& H. P. AND J. G. RICHARDS.

Supra-ependymal

serotoninergic

Switzerland

nerves in mammalian

brain:

Morphological,

BRAIN RES. BULL. 9(1-6) 727-741, 1982.-Supra-ependymal nerves in mammals (mainly rats) have been shown to contain serotonin (5-hydroxytryptamine, 5-HT) by combined Falck-Hillarp fluorescence histochemistry, ultrastructural monoamine cytochemistry and pharmacology as well as by immunohistochemistry and autoradiography. Supra-ependymal 5-HT cells do not occur. At least in rats, virtually aJl supra-ependymal nerves contain 5-HT and in our opinion the occasionally described non-5-HT supra-ependymal nerve cells and their processes contribute little to the supra-ependymal nerve plexus (with the possible exception of those cells above the median eminence). The cells of origin of the supra-ependymal 5-HT nerves are situated in raphe nuclei. The axons and terminals (varicosities) contain small and large dense core vesicles in both of which 5-HT is stored. A co-transmitter has not been found among the candidates investigated so far (leu- and met-enkephalin, substance P and y-aminobutyric acid (GABA)). The nerves possess uptake mechanisms specific for 5-HT and possibly GABA. Occasionally desmosome-like junctions are observed between 5-HT nerve terminals and ependymal cells but no true synapses. The function of these nerves is not known. They do not appear to regulate ciliary movement, but might influence the shape of ependymal cells.

pharmacological

and functional

studies.

Serotoninergic Supra-ependymal nerves Cerebrospinal fluid-contacting nerves

nerves

Ependyma

A KEY observation, which subsequently led to our interest in these nerves, was pure serendipity. In 1972, one of us (J. G. R.) was examining, ultrastructurally, the ventricular surface of rats injected intracerebroventricularly with Shydroxydopamine and found that virtually all profiles of supra-ependymal nerves were labelled, i.e., the large and were highly electron dense. SHysmall vesicles droxydopamine had earlier been proposed as a marker for monoaminergic nerve terminals [ 140,162]. Excited about the prospect of discovering a previously ignored aminergic nerve plexus, we decided to examine rat brain ventricle surfaces for the presence of a formaldehyde-induced amine-specific fluorescence. Indeed, tissues treated according to the Falck-Hillarp method [43,49] in the laboratory of H. P. L. clearly contained a dotted yellow fluorescence above most ependymal cells when examined with a fluorescence microscope (Figs. lA, 2A). These findings strongly indicated the presence of a supra-ependymal 5-HT nerve plexus which had remained undetected during the 10 years since the introduction of the FaIck-Hillarp method (probably because of its localization on the ependymal surface where a nonspecific fluorescence might have been suspected). We soon learned

Copyright

o 1982 ANKHO

International

Serotonin

Cerebral ventricles

that supra-ependymal nerves were long known in mammalian brain from light- [l, 15, 68, 1191 and unequivocally from electron microscopical studies [9, 17, 59, 70, 73, 78, 80, 81, 109, 111, 142, 143, 147, 1701. The present review is restricted to supra-ependymal5-HT nerves of mammals although other ependymal nervous elements are briefly mentioned. Non-mammalian vertebrates are not included since they appear to lack such 5-HT nerves, but possess monoamine-containing cerebrospinal fluidcontacting neurones which are not found in mammalian brain (for literature see [ 113, 154, 1671). Of the numerous scanning electron microscopical studies of ventricle surfaces which have appeared in the literature in the last few years, only a representative few will be discussed here. Unfortunately, although the morphology of the supra-ependymal 5-HT nerves has been extensively studied, their function has been rarely investigated. SUPRA-EPENDYMAL 5-HT NERVES VERSUS OTHER EPENDYMAL NON-5-HT NERVOUS ELEMENTS

Beside supra-ependymal5-HT nerves the following ependymal nervous elements devoid of 5-HT exist in mammals:

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As defined in 1969 [165] CSF-contacting neurones arc mostly bipolar cells located intra- or sub-ependymally. A short dendritic process, but not the axon, protrudes into the ventricular lumen. In non-mammalian vertebrates these neurones are more frequent than in mammals and are mainly found in two ventricular regions forming the hypothalamic and medullo-spinal CSF-contacting neuronal system [ 1671. Some CSF-contacting neurones in non-mammalian vertebrates contain 5-HT or a catecholamine (for literature see [113, 154, 1671). In contrast, in mammals intra- or sub-ependymal CSFcontacting neurones have been identified by conventional light and electron microscopy only in the caudal floor of the fourth ventricle [72, 75, 84, 1671 and in the central canal of the spinal cord [75,77, 159, 166. 167, 1711. Their transmitters have not yet been identified although monoamines have never been demonstrated. The floor of the third ventricle of rats and guinea pigs contains possible intraventricular endings of CSF-contacting neurones, which, however, are not located intra- or sub-ependymally [70, 96. 167, 1681, whereas in rabbits a peculiar bouquet-like structure has been described [74,76]. Recent immunohistochemical studies. moreover, have shown the possible existence of CSFcontacting neurones containing luliberin and somatostatin in rat third ventricle [18, 19,661. This and two further observaof catecholamine-containing subtions, the presence ependymal cells in rat diencephalon contacting the CSF via a ventricular process [ 1541 and CSF-contacting dendrites originating from noradrenergic A4 cells in rats 1471, await confirmation.

FIG. I. Fluorescence micrographs illustrating the formaldehydeinduced fluorescence of the left neostriatum (N) and on the free surface of the ependyma (E). Rats were sacrificed 6 hr after IP administration of the monoamine oxidase inhibitor nialamide (300 mg/kg) and 40 days after right lateral ventricle injections of (A) 10 ~1 0.02% ascorbic acid in Ringer, (B) 5 pg 5,6_dihydroxytryptamine, and (C) 51 pg 6-hydroxydopamine. In the vehicle-injected rat, the typical dotted yellow 5-HT fluorescence occurs above the ependyma and the diffuse blue-green dopamine fluorescence in the neostriatum. The monoamine neurotoxins, 5,6-dihydroxytryptamine and 6-hydroxydopamine, have selectively destroyed the 5-HT and catecholamine nerves, respectively. V, left lateral ventricle. x410.

Before reviewing the literature, we would like to stress two points. First, supra-ependymal 5-NT nerves clearly do not originate from supra-ependymal cells, since none of the fluorescence histochemical, immunohistochemical and autoradiographical studies published to date (see references below) have ever revealed a supra-ependymal 5-HT cell. Second, there remains some doubt about the nature of supra-ependymal cells (neuronal, glial or histocytic) observed in light and scanning electron microscopical (SEM) studies which are not combined with transmission electron microscopy (TEM) of the same preparations. Despite these limitations of the SEM technique several reports, based on TEM and/or SEM, on the occurrence of supra-ependymal nerve cells and their processes, mainly in the third ventricular floor, should be mentioned. Supraependymal nerve cells with processes have been observed in the third ventricle of: minks (floor and organum vasculosum laminae terminalis, SEM; [ 148]), rats (floor, TEM and SEM: [96. 115-l 17, 125, 1511, subfornical organ, TEM and SEM: [38], and habenular region, TEM and SEM; [34]), hamsters (floor, TEM and SEM; [22,23]), cats (floor, SEM; 1291). rabbits (hypothalamic region, TEM; [74,76]), hedgehogs (floor, TEM; [168]) and rhesus monkeys (floor and other regions, TEM and SEM; [149,150]). They have also been observed in the lateral ventricles of rats (SEM; [83]) and cats (SEM; [29,109]) as well as in the fourth ventricle of rabbits (apertura lateralis, TEM and SEM; [79]). From these studies it appears that. with the possible exception of localized areas such as the third ventricle floor.

supra-ependymal cells and their processes are rare. neuronal cell bodies being less frequent than histiocytes and glial cells.

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FIG. 2. Fluorescence micrographs illustrating the free surface of the ependyma (E) on the floor of the left (A) and right (B) interventricular foramen (IF). The rat was sacrificed 6 hr after the IP administration of nialamide (300 mg/kg) and 12 days after the electrolytical lesioning of the right medial forebrain bundle in the posterior hypothalamus. A supra-ependymal formaldehyde-induced yellow fluorescence typical of 5-HT is visible on the left side (A), but is virtually absent on the right side (B) due to the anterograde degeneration of 5-HT nerves passing through the medial forebrain bundle. CP, choroid plexus within the interventricular foramen. x457.

In our experience, extensive TEM and some SEM studies of normal rat and post-mortem human brain have rarely revealed supra-ependymal cells, but abundant supraependymal 5-HT nerves. PURELY ANATOMICAL STUDIES OF PRESUMPTIVE SUPRAEPENDYMAL 5-HT NERVES Unspecijic Light Microscopical Impregnation Methods)

Studies (Silver

It is quite possible that supra-ependymal 5-HT nerves were observed by light microscopy long ago in studies of adult mammalian brains treated with various silver impregnation methods. The most detailed reports date from 1930 [68] and 1940 [119] and describe dense supra-ependymal nerve plexuses in monkey, rat, cat and guinea pig brain, respectively, which correspond well with dense 5-HT nerve plexuses. Indeed, some supra-ependymal 5-HT (?) nerve endings, in the fourth ventricle floor of cats, seem to have been detected as early as 1922 [I]. Once the existence of supra-ependymal 5-HT nerves had been established, silver impregnation methods were again successfully employed to

study their morphology and distribution e.g., in whole mouse brain [39] and in the third and lateral ventricles of rat and mouse brain [5]. Electron Microscopical

Studies

Trunsmission electron microscopy

(TEM). The presence of supra-ependymal nerves was documented in numerous TEM studies [9, 17, 59, 70, 73, 74, 76, 78, 80, 81, 109-111, 142, 143, 147, 1701 long before 1973 when we showed that such nerves contain 5-HT [91,139]. From subsequent ultrastructural cytochemical[37,91,92, 139, 141, 1641, high resolution autoradiographical [5, 6, 16, 20, 21, 27, 37, 1321 and combined pharmacological-TEM studies [6, 16, 21, 27, 371, which enabled the specific ultrastructural characterization of supra-ependymal S-HT nerves, it appears, in retrospect, that also most of the former studies dealt with 5-HT nerves. 5-HT-specific and unspecific TEM studies of supraependymal 5-HT nerves have consistently shown the lack of marked regional or species differences. Thus, the main ultrastructural findings on supra-ependymal 5-HT nerves summarized below refer to studies of rats (lateral ventricle,

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[3,6, 8,58,91-93, 126, 139, 140, 1701; third ventricle, [3,6, 8, 16, 17, 21, 33, 34, 37, 38, 69, 70, 91-93, 104, 105, 129, 140, 1671; aqueduct, [8, 17, 27, 91-93, 1401 and fourth ventricle, 18, 9, 17, 27, 37, 65, 91-93, 140]), cats (lateral, [109-l II], third, [80] and fourth ventricles, [9]), rabbits (lateral, [56,1121, third, [78,801 and fourth ventricles, [73, 78-80]), monkeys (lateral 142,441, third, [30,149] and fourth ventricles [27] and aqueduct, [27]), mice (third, [6] and fourth ventricles, ]1431), guinea pigs [102,103], hamsters ([164], third ventricles), and man (several ventricle regions, [137]). For rats many details not referred to below are given in [6, 27,33, 34, 37, 65, 129, 139, 141 and 1701. It must also be stressed that we have not considered the TEM findings on the infundibulum e.g., of rats [96, 99, 117, 142, 147, 1511, monkeys [ 1491, hamsters [22], armadillos [67] and minks [ 1481, since at least the rat infundibulum does not appear to contain supraependymal 5-HT nerves, but nerves of another type. TEM of cerebral ventricles has revealed that the ependyma is covered by a nerve network consisting of crisscrossing thick (l-2.5 pm) and thin (0.1-0.5 pm, probably collateral) unmyelinated axons (for the rare myehnated axons see [73,79], with varicosities, occurring singly or frequently in groups of 2 to 7 elements (Fig. 3A). Large clusters of axons and varicosities were seen particularly at nodal points (crossing or branching sites). Axons were rarely observed between ependymal cells. The axons contain longitudinally arranged neurotubules (diameter 25 nm), neurofilaments and mitochondria, few electron translucent small (30-50 nm) and electron dense (dense core) large vesicles (80-120 nm). The varicosities (0.5-1.5 pm, for the rare giant terminals see [6,21]) contain some neurotubules, rare neurofilaments and mitochondria, but frequent round to oval small vesicles (30-50 nm) some with a dense core and occasional large dense core vesicles (80-120 nm, for details see [27, 33, 34, 37, 65, 79, 129, 139, 1701). As discussed below, the small and large vesicles store 5-HT. The varicosities may be surrounded by microvilli or ependymal cell protrusions. Synaptic junctions do not occur, but occasionally one can observe contact zones (desmosomes or zonulae adherentes) between nerves and between nerves and ependymal cells, with a minimum distance of 20 nm between the membranes. Stunning electron microscopy (SEM). Is SEM suitable for studying supra-ependymal5HT nerves? Of course, SEM does not visualize specifically these nerves. In order to unequivocally identify the neuronal (and, more specifically, the serotoninergic) nature of supra-ependymal Iibres, SEM has been correlated with TEM [22, 30, 33, 34, 38, 44, 56, 62, 69, 77,79, 84, 96,99, 101, 102, 117, 129, 136, 137, 148-151, 1671, but (more specifically) unfortunately not with immunocytochemistry, 5-HT autoradiography or manipulations inducing known effects on supra-ependymal 5-HT nerves e.g., their degeneration by intracerebroventricular 5,6_dihydroxytryptamine or lesions or raphe nuclei. How-

LOREZ AND RICHARDS ever, if one assumes that most beaded supra-ependymal fibres are nerves or that nerves can be distinguished with some certainty from non-neuronal filaments, and in view of the fact that (at least in the rat) virtually all nerves apart from those on the floor of the third ventricle contain 5-HT. SEM might be useful in the study of supra-ependymal 5-HT nerves. Since the floor of the third ventricle (infundibular recess) does not contain 5-HT nerves, at least in rats, the numerous SEM studies of this non-ciliated region showing various cells and beaded fibres (also in rats) will not be considered here [22,23,38,62,96,98-101, 115-l 17, 147-1521; certainly in the case of rats, these are examples of the unsuitability of SEM to identify 5-HT nerves. One must also remember that densely packed cilia may obscure underlying nerves, thus explaining many false negative SEM results. What has SEM contributed to our knowledge of supraependymal 5-HT nerves? It has elegantly demonstrated the networks of ramified fibres above various ependymal surfaces (Fig. 4). Fibres run either singly or twisting or parallel in fasciculated bundles. Nodal points (crossing or branching sites) were frequently described. Usually the fibres show spindle-shaped or bulb-like swellings (varicosities, 0.8-I .2 pm) and sometimes bulbous endings, but tapered processes were also seen. The fibres appear to give off collaterals. Occasionally, libres were seen extending into the ventricle from the ependymal surface. Generally the course of the fibres followed no particular direction. Correlative SEM and TEM studies have thus shown supra-ependymal(5-HT) nerves, e.g., in rat third [33, 34. 38. 69, 129, 1691 and fourth ventricles [136], in rabbit lateral [56] and fourth ventricles [79,84], in guinea pig third ventricle [102], in monkey lateral [44] and third ventricles [30] and in several ventricular regions in man [ 1371. There are also some purely SEM studies, e.g., of rat lateral [120] and third ventricles [121], cat fourth ventricle [82] and monkey lateral ventricle [57]. It must also be mentioned that SEM has confirmed the absence of intraventricular nerves on the choroid plexuses.

TRANSMITTER-RELATEDANATOMICAL STUDIES OF SUPRAEPENDYMAL 5-HT NERVES Identijication Combined trustructural

of 5-HT as the Neurotransmitto

Falck-Hillarp fluorescence monoamine

cytochemistry

histochemistry. ulund pharmucdogy.

Numerous fluorescence histochemical and ‘*specific” electron microscopical studies of rat brain ventricles have been reported [4,5, 16, 37,91-93, 139, 1411in addition to others of mouse [5] and human brain ventricles [ 1371. The following observations, allowing the identification of supra-ependymal nerves as monoaminergic, were typical of all supra-ependymal nerves:

FACING PAGE: FIG. 3. Transmission electron micrographs of supra-ependymal nerves of rats. (A) Ultrastructure after conventional fixation (glutaraldehyde and osmium tetroxide). Varicose regions of a nerve (*) contain several small electron translucent vesicles and a few large dense core vesicles (arrows). A, aqueduct; c, cilium; E, ependyma; m, mitochondrion; mv, microvillus; nt, neurotubule. (B) Ultrastructural monoamine cytochemistry. After the chromaffin reaction (fixation with chromate-buffered glutaraldehyde without osmium tetroxide) a varicose region of a nerve contains several smah and a few large electron dense cores specific for monoamines (arrows). E, ependyma; m, mitochondrion; V, lateral ventricle above the neostriatum. (C) Ultrastructural autoradiography. After intracerebroventricular injection of [3H]5-HT, silver grams can be observed exclusively over a varicose nerve. E, ependyma; V, lateral ventricle above the neostriatum. (D) Ultrastructural immunocytochemistry (PAP technique). After incubation of glutaraldehyde-fixed ventricle surfaces with 5-HT-specific antiserum, an immunoreactive (highly electron dense) nerve can be observed above the ependyma between cilia (c) and microvilli (mv). Large dense core vesicles (arrow) are markedly stained. A, aqueduct.

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5-HT NERVES IN MAMMALS

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FIG. 4. Scanning electron micrograph of varicose supra-ependymal5HT nerves (*) on the rat fourth ventricle floor. c. cilium; E, ependyma: mv, microvillus (kindly provided by Dr. R. Guggenheim. SEM Lab., Univ. Basel, Switzerland).

(1) The presence of a supra-ependymal varicose, formaldehyde-induced fluorescence (FIF, Figs. IA. 2A) which disappeared after treatment of the sections with sodium borohydride, but was partly regenerated by renewed formaldehyde treatment [3-5, 16, 31, 43, 49, 91, 92, 1391. (2) In conventionally fixed tissues, virtually all small and large vesicles of supra-ependymal nerves have a highly electron dense content after intracerebroventricular perfusion with 5-hydroxydopamine, a specific marker for monoamine storage vesicles [139. 140, 1621. (3) The appearance of electron dense dots in small synaptic vesicles and increased electron density of large dense core vesicles after treatment with nialamide or harmaline in conventionally fixed tissues [21,37]. (4) The presence of electron dense (chromaffin) small and large cores above the ependyma (Fig. 3B) of tissues treated for the specific cytochemical localization of biogenic monoamines at the ultrastructural level, namely the chromaffin reaction without post-osmification [37, 91, 92, 139, 141, 163, 1641. (5) The increase of both FIF and chromaffin dense cores in nialamide (and harmaline)-treated animals [37. 91. 92. 1391. (6) The absence of both FIF and chromaffin dense cores in reserpine-treated animals [37,91,92,94, 139, 1411, and the supra-ependymal localization of [“HI-1abelIed reserpine by light microscopical autoradiography [133-1351. Furthermore, the monoamine could be precisely identified as an indolealkylamine, very probably S-HT. by the following observations: (1) The yellow color of FIF rapidly fading upon irradiation [3-5. 11, 12, 16, 25, 32, 63, 91, 92, 1391; (Figs. lA, 2A). (2) The persistence of FIF in reserpine + nialamidetreated animals [24, 91, 92, 1391. (3) The diminution of FIF and the disappearance of

chromaffin dense cores after treatment with p-chlorophenylalanine, a potent inhibitor of tryptophan-5-hydroxylase 116, 67, 91, 92, 139, 1411, but their persistence after treatment with cu-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase [50. 91, 92, 139, 141. 161. (4) The appearance of electron dense dots in small synaptic vesicles and increased electron density of large core vesicles after intracerebroventricular injection of 5-HT with and without nialamide treatment [6, 16, 27, 371. (5) The presence of a high affinity uptake system in the axonal membrane for 5-HT (Fig. 3C). but not for noradrenaline and dopamine [4, 5. 8, 16, 20, 21, 27, 28, 37, 114, 1321; (these references include autoradiographical studies) and its inhibition by the specific 5-HT uptake inhibitors chlorimipramine and Ro 1l-2465 ([5-[3-(dimethylamino) propyl]-IO,11 ,-dihydro-5-H-dibenz[b,flazepine-3-carbonitrile [35,48], but not by the specific noradrenaline uptake inhibitor desmethylimipramine [ 131, 1321. (6) The high toxicity of 5,6_dihydroxytryptamine and other dihydroxytryptamines, injected intracerebroventricularly, to all supra-ependymal nerves as compared to the non-toxic 6-hydroxydopamine ([8, IO, 14. 27. 34. 93. 131. 1601; Fig. IB, IC). The reactions to treatment with nialamide, reserpine -t nialamide, p-chlorophenylalanine and 5,6_dihydroxytryptamine were characteristic of C-type neurones (5-HT containing) and not B-type neurones which probably contain a hitherto unknown indolealkylamine [ 12-141. However, since supra-ependymal nerves have never been studied microspectrofluorimetrically, ultimate confirmation for the (exclusive) presence of 5-HT was only recently obtained from immunohistochemical studies [ 138,157]. Another combined morphological-pharmacological approach was inconclusive in demonstrating the serotoninergic nature of the supra-ependymal S-HT nerves. Since haloge-

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FIG. 5. Fluorescence micrographs showing SHT- (A), substance P- (B), enkephalin- (C) and glutamic acid decarboxylase-like immunoreactivity (D) in the rat septum. Indirect immunofluorescence technique. Note the presence of an intense supra-ependymal (E, ependyma) immunofluorescence for SHT, but its notable absence for the other peptides despite the occurrence of specific immunofluorescence in the adjacent neuropil of the septum. The antisera to SHT, substance P and leucine enkephalin were obtained from Immuno Nuclear Corporation, Stillwater, MN, and the GAD antiserum from .I. Y. Wu, Department of Cell Biology, Baylor College, Houston, TX. The leucine enkephalin antiserum showed a 36% cross reactivity with methionine enkephalin when measured by radioimmunoassay. V, lateral ventricle. x316.

nated amphetamines creases of 5-HT

were

known

to induce

long-term

de-

content, uptake and turnover rate, 5-hydroxyindole-acetic content and tryptophan-5-hydroxylase activity in rat brain (for literature see [45,146]), suggesting a possible toxic effect on 5-HT neurones, we studied cerebral 5-HT systems including supra-ependymal nerves in rats after acute and chronic treatment with p-chloroamphetamine and p-chloro-N-methylamphetamine [87-901. FIF specific for 5-HT accumulated for a few days in swollen intracerebral 5-HT axons and was markedly decreased over a long period in some intracerebral5-HT terminal regions. However, a supra-ependymal 5-HT accumulation was rare and there was only a transient, i.e., reversible, decrease of 5-HT fluorescence intensity. The number of pontine 5-HT cell bodies was unchanged, in contrast to some reports [51-54, 971 but in line with others [124, 144, 1551. Electron microscopy of tissues from the same series of animals revealed no change, supra-ependymally, in the number of intact nerves and a lack of degenerating profiles in contrast to reports of nerve terminal degeneration in the neostriatum [55]. While the controversial findings on 5-HT cell bodies still cannot be explained, the observations on 5-HT nerves might indicate a selective toxic action of halogenated amphetamines on some intracerebral 5-HT systems leaving the supra-ependymal nerves virtually intact. Very recently, degenerating nerves were observed in the outer zone of the median eminence of rats treated with p-chloroamphetamine, but not in the third ventricle [144,145].

Interestingly, a seemingly similar difference between supra-ependymal and parenchymal 5-HT nerves has been observed in thiamine-deficient rats; while parenchymal5-HT systems were much less labelled after intracerebroventricular infusion of [“HI-labelled 5-HT in deficient as compared to control rats (as shown by autoradiography), the supraependymal nerves were equally labelled in both groups [28]. Immunohistochrmistry. Immunohistochemical studies using highly specific antibodies to 5-HT fully confirmed the existence and distribution (see below) of supra-ependymal 5-HT nerves in rats [138, 157, 1581; (Fig. 5A). These findings have also been supported by immunocytochemical studies although the general tissue preservation has not made it possible to unequivocally identify the small and large vesicles as storage sites of 5-HT (Fig. 3D). Unfortunately, there are no immunohistochemical reports on the presence (or even absence) of tryptophan-5hydroxylase in supra-ependymal nerves. Immunohistochemical studies have also provided evidence for the existence of luliberin- and somatostatincontaining nerve cells, possessing short CSF-contacting processes, beneath the ependyma of rat third ventricle [ 18, 19, 661. These neurones appear to resemble CSF-contacting neurones [ 165,167] rather than supra-ependymal 5-HT nerves. In addition to the visualization of 5-HT, we have used immunohistochemistry in a search for the possible coexistence of peptides in the supra-ependymal 5-HT nerves. The most likely peptide candidates were substance P and

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lateral ventricle

I \third

ventricle

FIG. 6. Relative density of supra-ependymal S-HT nerves in the rat third and lateral ventricles (hatched area) as shown in two frontal sections through the forebrain. Abbreviations: A, alveus hippocampi; Ac, amygdaloid cortex; ar. nucleus fn.) arcuatus; Ca, chiasma opticum; Cai, caps&a interna; Cc, corpus callosum; cp, neostriatum; Crc, crus cerebri; F, columna fornicis; Fh, fimbria hippocampi; Fmt, fasciculus mamillothalamicus; Fo. fornix; ha, n. anterior hypothalami; hpv, n. periventricularis hypothalami: Lm, lemniscus mediahs; mh, n. medialis habenulae; pvs, n. periventricularis stellatocellularis; Ri, recessus infundibularis; SC, n. suprachiasmaticus; Sgp, substantia grisea periventricuiaris; sl, n. septi lateralis; Sm, stria medutlaris thalami: St, stria terminalis: st, n. interstiti~is striae terminalis; T. tela chorioidea (choroid plexus).

leucine enkephalin which are known to occur in cerebral 5-HT neurones in rats (for literature see [60]) and cats [46], respectively. A detailed investigation of the whole cerebroventricular system of rats failed to reveal supraimmunore~t~vity for substance P and ependymal methionine and leucine enkephalin ([ 1381, Fig. 5B, 5C). Furthermore, we studied the immunohistochemical distribution of glutamic acid decarboxylase (GAD) in ventricular regions since the recent autoradiographical demonstration of a high affinity uptake (independent of the 5-HT uptake) of y-aminobutyric acid (GABA) by virtually all supraependymal 5-HT nerves has raised the possibility that they synthesize and store GABA [7,8]. However, in the whole ventricular system no supra-ependymal GAD immunoreactivity was observed ([138], Fig. SD); some figures in a more recent publication [118] also demonstrated the absence of GAD immunoreactivity in a few ventricular regions containing 5.HT nerve plexuses. Thus, in spite of the high affinity uptake of GABA [7,8] the supra-ependymal 5-HT nerves do not appear to synthesize GABA. Autoradiography. The autoradiographical studies reported in the literature are based on the high affinity (re)uptake and storage of 5-HT by central indolealkylamine

neurones pe~itting the specific identi~cation of supraependymaI5HT nerves by light [2, 4,5, 8, 16,20,21,27,28, 114, 1321 and electron microscopical autoradiography ([5, 6, 16, 20,21,27, 37, 1321; Fig. 3C) after intracerebroventricular injection (or infusion) of [3H]5-HT [2, 5, 6, 8, 16, 20, 21, 27. 28, 37, 114, 1321 or after the incubation of brain slices with the tracer [4,21]. However, it is not certain that all labelled nerves are serotoninergic since interspecific uptake into catecholaminergic nerves may occur. In some investigations the problem of specificity has been overcome by either the simultaneous use of excess cold, i.e., non-radioactive, catecholamines [6, 28, 1141 or parallel studies with low concentrations of [3H]-labelled catecholamines [4, 20, 21, 1321. Theoretically, auto~diographical labelhng with [3H]5-HT could visualize 5-I-IT nerves with an endogenous 5-HT content too low for its fluorescence histochemical or immunohistochemical demonstration. All the above mentioned studies showed that supraependym~ nerves possess marked (re)uptake and storage capacities for 5-HT, but not for noradrenaline and dopamine [4, 20, 27, 1321, thus confirming their serotoninergic nature. However, only two studies made use of the pharmacological characterization of the uptake system by selective 5-HT or

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5-HT NERVES

IN MAMMALS

catecholamine uptake inhibitors 148, 131, 1321. There are no reports of the absence of [3H]S-HT-labeUed nerves in ventricular regions known to contain supra-ependymal 5-HT nerves from Falck-Hillarp fluorescence histochemistry and 5-HT immunohistochemistry. Thus, light microscopical autoradiography has revealed the presence of supra-ependymal labelling in those regions of rat third ventricle [2, 4-6, 8, 16, 21, 27, 281, interventricular foramen [8, 27, 281, lateral ventricles 14, 5, 8, 27, 28, 114, 1321, aqueduct 12, 8,27, 281, floor of the fourth ventricle [4, 8, 27, 28, 1321 and foramen of Luschka [27,28]. In rhesus monkeys supra-ependymal labelling was also seen after int~cerebroventricular infusion of [3H]5-HT in regions probably rich in supra-ependymal S-HT nerves (inferred from the studies on rats) [27]. Similar observations were made on the third and lateral ventricles of mice [S] and on the lateral ventricles of cats [20]. The localization of the label over supra-ependymal axon and terminal profiles was contirmed by electron microscopical autoradiography in rats (third ventricle, [5,6, 16,21,37]; lateral ventricles, [5,132]; floor of the fourth ventricle, 127, 37, 132]), in rhesus monkeys (fourth ventricle, [27]), in mice (third and lateral ventricles, [5]) and in cats (lateral ventricles, 2201). In one study of rats, labelling was mainly located in regions exhibiting accumulations of small synaptic vesicles and large dense core vesicles indicating storage sites of 5-HT 1371. Poorly labelled ventricular surfaces corresponded well, apart from one report [28], with regions devoid of supraependymal 5-HT nerves as revealed by Falck-Hilly fluorescence histochemistry and 5-HT immunohistochemistry, e.g., rat median eminence 14, 21, 1321, subcommissural and subfornical organs [16,21] and roof of fourth ventricle [ 1321. However, in one detailed report 1281, light microscopical autoradiography of rat brain after intraventricular infusion of [3H]5-HT in the presence of an excess of cold (nonradioactive) noradrenaline revealed sups-ependymal labelling in all these regions, in fact on all ventricular surfaces including those of area postrema, central canal of medulla oblongata and spinal cord and even on the surface of the choroid plexuses which are devoid of intraventricular nerves. These results are not considered sufficiently relevant for inclusion in the following section on the distribution of supra-ependymal 5-HT nerves. Light microscopical autoradiography has also shown a supra-ependymal labelling in rats after intravenous injection of ~3H]-labe~ed reset-pine [133-1351. Since reserpine is known to bind specitically to intracellular amine-storing vesicles [36, 107, 1.531,this finding confirmed the monoaminergic nature of the supra-ependymal nerves and proved useful in their mapping. The autoradiographical demonstration of a high affinity GABA uptake by all supra-ependymal 5-HT nerves in rats [7,8], already discussed above, has also cont~buted to their mapping. ~istribation of ~apra-E~e~dymal 5-HT Nerves by Falck-Hillarp Fluorescence Histochemistry, tochemistry and Autoradiography

as Revealed Immunohis-

The distribution of sups-ependym~ 5-HT nerves in rats as revealed by the initial fluorescence histochemical and cytochemical studies (Figs. 6, 7; [91,92, 1391) has been fully confirmed by subsequent fluorescence histochemical[3,4,5, 161, immunohistochemical 1138, 157, 1581 and autoradiographical studies [4-6, 8, 16, 21, 27, 37, 114, 132, 1351; the

735

-

i

--

i

1

I

FIG. 7. Relative density of sups-ependym~ S-HT nerves in three frontal sections through the rat fourth ventricle (hatched area; a-c, cranial to caudal). Abbreviations: f, flocculus cerebelli; flm, fasciculus longitudinalis medialis; Ic, locus coeruleus; n, nodulus cerebehi; n XII, nucleus (n.) hypoglossus; ncv, n. cochlea& ventrahs; nd, n. dentatus; ndm, n. motorius dorsahs nervi vagi; ne, n. emboliformis; nf, n. fastigii; nnf, n. nervi facialis; nts, n. tractus solitarii; p, plexus chorioideus; pci, pedunculus cerebella& inferior; pcs, ~dunculus cerebella& superior; r&s, radix; tr. spinahs nervi trigemini; vma, velum medullare anterius.

exception is [28]. Briefly, supra-ependymal5-HT nerves are present with moderate to high density on all ventricular surfaces with the following main exceptions: they are sparse around the floor of the third ventricle, on the area praeoptica, the roof of the foramen interventriculare, the organum subfomicale and the fourth ventricle roof (velum medullare anterius and posterius, lateral parts). They are absent on the floor of the third ventricle (eminentia mediana), organum

LOKEZ AND RICHARDS sLlbcommissu~a~e. area postreme, roof of fourth ventricle (medial parts), central canal of medulla oblongata and spinal cord, and above all plexus chorioidei. In contrast, very high densities occur mainly on the floor of the interventricular foramen (Fig. 2A). nucleus medialis habenulae. recessus pinealis. most regions of the fourth ventricle floor and on all linings of the foramen of Luschka. The varicose axons form a network immediately above the ependymal cells which is best seen in oblique (tangential) thick sections of the ependyma ([S. 8, 27, 391, see also [68]). For further details on the distribution see the section on electron microscopical studies. Beside the supra-ependymal plexus. an interconnected sub-ependymal plexus of S-HT nerves exists in many ventricular regions ([4. 27. 91. 1391, see also [#,I IU]). Much less is known about the distribution of supraependymal S-HT nerves from specific studies in mammals other than rats, but they have been observed in some typical ventricular regions in mice ([S], unpublished observation), guinea pigs (unpublished observation}, cats ([20], unpublished observation), rhesus monkeys 1271and man [ 137.1381. We believe that, at least in rats, virtually all supraependymal nerves contain S-HT with the exception of those on the floor of the third ventricle. The area postrema is probably the only region with a few supra-ependymal noradrenaline-containing nerves as shown by fluorescence histochemistry 192) and dopamine-~-hydroxyjase immunohist~)chemistry (1611. ~~lthough the occurrence of supraependymal catecholamine-containing nervous elements has also been reported for rats from fluorescence histochemical studies of the fourth ventricle roof beneath the A4 cells [47]. the eminentia mediana [41] and the third ventricle [lS4]. We could not confirm the latter three findings. Although the relevance of I”H]GABA uptake by supraependymal nerves remains unclear. the pattern of supraependymal labelling by [:‘H]GABA [8], as well as: by [:‘H]reserpine [ 133-1351, were identical in their density and location to the supra-cpendymal S-HT network.

AIt findings relating to this problem have been obtained with rats except [39] (mice) and [ 1231 (cats). There is ample evidence that the cells of origin of all supra-ependymal S-HT nerves are situated in the raphe nuclei. (a) All nerves in the third and lateral ventricles and in the aqueduct degenerated after extensive electrolytic lesions of the mesencephalic raphe nuclei [3, 8. 341. (b) Electrical stimutati(~n of the mesencephatic and pontine raphe nuciei caused significant increases in the uptake of intracerebroventricularly injected [“HJS-HT by the supra-ependymal nerves of the aqueduct and the third and lateral ventricles [27]. (c) A few hours after injection of [““Iltetanus toxin into a lateral ventricle the labelling was located (by light and electron microscopicat autoradjography) in axon bundles and neuroncs in the ipsilaterat medial forebrain bundle and the dorsal and median raphe nuclei, respectively. This pattern of radiolabelling was prevented by pretreating rats with intracercbroventricular colchicine (blocking axonal transport). S,&dihydroxytryptamine (destroying S-HT nerve terminals) or with an ipsilateral medial forebrain bundle lesion (destroying S-HT axon bundles). It was concluded that [iZ51]tetanus toxin was accumulated by supra-ependymal nerve terminals and rapidly nuclei by

transported retrograde

to their cells of origin axonal flow 11331.

in the raphe

rd)

In cats.

radiolabelling

of supra-ependymal

was observed

after injection

surfaces in alI ventricles

of r”H]-labeiled

proline

into the

nucleus raphe dorsalis and centralis superior as a consequence of its anterograde axonal transport [133]. (e) Using silver impregnation techniques a pronounced exit zone of the supra-ependymal nerves in the fourth ventricle has been visualized over the raphe dotsalis region in mice 139f. (0 S-HT axons projecting from the nucleus raphe dorsaiis directly to the overlying aqueduct have very recently been visualized by S-HT immunohistochemistry [ 181. A few studies were mainly concerned with the intracerebral axonal pathways of the supra-ependymal S-HT nerves. (a) Experiments with small electrolytic lesions and local injections of S,6-dihydroxytryptamine have shown that all supra-ependymal nerves of the lateral ventricles and the cranial third ventricle originate uncrossed from S-HT axons passing through the media1 part of the medial forebrain bundle at the level of the posterior hypothalamus ([86], Fig. 2). (b) All supra-ependymal nerves reach the epithalamus through the fasciculus retroflexus [ 1311; more caudally these axons appeared to pass by the nucleus interpeduncularis. since extensive lesions of this nucleus destroyed all the epithalamic supra-ependymal nerves [130]. (c) In this context, it should be mentioned that the occasional penetration of S-HT nerves between ependymal cells into the ventricles has been described in some fluorescence histochemical and SEM studies but few TEM studies 13). To summarize, these studies showed that the cells of origin of all supra-ependymal 5-HT nerves are situaied in the raphe nuclei, and that some axon bundles pass intracerebrally along known 5-HT pathways to the ventriCkS. They presented additional evidence for the serotoninergic nature of all supra-ependymal nerves.

Speculations on the function(s) of the supra-ependymal S-HT nerves have centred mainly on three possibilities: (a) The nerves are receptors for the registration of the compusition of the cerebrospinal fluid. (b) The nerves act upon ependymal cells through the release of S-HT. influencing, e.g..

ependymal

secretion,

cell shape or ciliary activity.

nerves act upon a distant circumventricutar release of S-HT into the cerebrospinal

plexus chotioideus, neurohypophysis tures in the vicinity of the ventricles.

(cl The

site. through the

fluid. e.g.. on the or intracerebral struc-

So far, only hypothesis (h) has been experimentally tested: the absence of synapses between supra-ependymal 5;-HTnerves and ependymal cells does not exclude a priori this hypothesis, since in most brain regions S-HT nerves do not form synapses 120. 26. 40. 7 1. 121-j.It must also be stressed that there is only one example of ultrastructurally “classical” synapses on ependyma. namely the S-HT synapses on the base of specialized ependymal cells of the subcommissural organ 116. IOSl.

Since a regulatory role for 5-HT in ciliary movement in molluscs was well supported 11271, we investigated such a possible role for ependymal cilia in rat brain by measuring the velocity of particle (erythrocyte) transport by ciliary activity in vitro [85]. A suspension of erythrocytes (1 ~1) WC carefully introduced above the exposed fourth ventricle which was maintained at 37°C in Krebs-Ringer bicarb~)nate t-t glucose) saturated with 5% CO, in 0,; the time required for their extrusion out of the ventricle, controlled visually.

SUPRA-EPENDYMAL

5-HT NERVES

(average time in controls was 6 min 20 set k9.1 set). This time was not significantly changed by increasing or decreasing 5-HT receptor activity. Increased receptor activity was induced by adding 5-HT to the medium (1 mM S-HT + 1 mM pargyline) or by releasing 5-HT from supraependymal nerves (using 0.01 mM reset-pine or 0.1 mM D-amphetamine + 1 mM pargyline). Decreased receptor activation was induced by pretreatment with p-chloro-phenylalanine (to deplete 5-HT by inhibition of 5-HT synthesis, 3x 100 mg/kg IP 72, 48 and 24 hours before sacrifice), reserpine (to deplete 5-HT by interfering with the storage site of 5-HT, 10 mgikg IP, 20 hr), 5,6-dihydroxytryptamine (to destroy 5-HT nerves, 25 pg intracerebroventricularly, 4 days) or with the 5-HT antagonists methysergide (30 mg/kg IP, 0.5 hr), methiothepin (40 mg/kg IP, 1 hr) and cyproheptadine (20 mg/kg IP, 0.5 hr), or by adding methysergide (1 mM) to the medium. Nevertheless, with this experimental system, inhibition of ciliary activity could be readily demonstrated with low temperature (22°C and less), saturation of buffer with N&O,, potassium cyanide (0.1 mM?exclusion of glucose), 2,4dinitrophenol (0.1 mM + exclusion of glucose), iodoacetic acid (1 mM), sodium fluoride (1 mM) or g-strophantin (ouabain, 1 mM); exclusion of glucose from the medium was without effect. From these results it appeared that supra-ependymal 5-HT nerves, through release of 5-HT, do not exert a regulatory role on ciliary movement in the rat ependyma. However, stimulation of ciliary activity by 5-HT mechanisms cannot be completely excluded since adenine nucleotides (ATP or 3’,5’-cyclic AMP [l mM] + aminophylline (1 mM)), known to stimulate activity in various ciliary systems [95, 106, 1081, did not influence the transport of erythrocytes in our in vitro system. A second conclusion from these results was that the energy required for ciliary movement in ependyma can be generated by oxidation of either glucose or endogenous substrates; anaerobic glucolysis per se does not fully provide sufficient amounts of high-energy compounds. was measured

Possible

InJluence on Ependymal

737

IN MAMMALS

very narrow and the cuboidal ependymal cells were replaced by flattened cells after the disappearance of all supraependymal nerves. This was observed in the epithalamus, together with an enlargement of the ventricular cavity, six days after lesion of 5-HT axons reaching the epithalamus via the fasciculus retroflexus [ 1301; the ependymal cells showed a reduction in microvilli, a total disappearance of the cisternae of the smooth endoplasmic reticulum and an increase in lysosome-like bodies. A similar flattening and even degenerative signs of the ependyma were observed four to six days after electrolytical midbrain-raphe lesions in the habenular [34] and several other (unspecified) ventricle regions [8]. Similar changes were observed one day [34] and [8] after the intracerebroventricular or one week of a high dose of 5,7intracisternal injection dihydroxytryptamine. If confirmed it would be interesting to know if (specific) metabolic changes occurred. In this context one is reminded of the marked inhibition of protein synthesis in specialized ependymal cells (organum subcommissurale) by sub-ependymal 5-HT nerves; however, in this case typical synaptical contacts between 5-HT nerves and ependymal cells exist [ 1051. We have recently performed a purely morphological study on the possible involvement of supra-ependymal5-HT nerves in the development of a lethal congenital hydrocephalus in mice (hy-3/hy-3 mice, The Jackson Laboratory, Bar Harbor, ME 04609, [128]). As compared with controls the nerves were unchanged in the third and lateral ventricles when hy-3/hy-3 mice died approximately 20 days old, but the ependyma was very narrow and flattened. In the ten years since the observation of a yellow formaldehyde-induced supra-ependymal fluorescence in rat brain, the system of purely serotoninergic supra-ependymal nerves has been well defined by various morphological approaches in several mammalian species; in addition the usefulness of these nerves for morphological studies on drugs affecting serotoninergic mechanisms has been shown. However, the function of supra-ependymal 5-HT nerves remains to be elucidated.

Shape

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NOTE ADDED

IN PROOF

Two recent reports have shown dymal axons in the rat area postrema (Armstrong, D. M. et al., J. camp.

that some supraepenare catecholaminergic Neural. 206: 25!9-272,

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1982) and that CSF-contacting axons containing vasopressin occur in the lateral and third ventricles of mice and rats (Castel, M., First World Congress of IBRO, Lausanne, 1982).