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Autoradiographic evidence for the selective accumulation of [3H]5-HT by supra-ependymal nerve terminals
Brain Research, 134 (1977) 151-157 © Elsevier/North-HollandBiomedical Press
151
Autoradiographic evidence for the selective accumulation of [3H]5-HT by supra-ependymal nerve terminals*
J. G.
RICHARDS
Pharmaceutical Research Department, F. HoJfmann-La Roche, CHo4002 Basle (Switzerland)
(Accepted June 2rid, 1977)
A network of supra-ependymal serotonergic (5-HT) nerve fibres has been shown to exist in most ventricular regions of rat brain 16,17,22,23. In the forebrain these nerve fibres originate from the raphe nuclei of the rues- and/or metencephalon 3,15. It is well known that aminergic nerves selectively accumulate monoamines and that this uptake mechanism shows its highest affinity for the endogenous amine 13. A synaptic mechanism for the reuptake of 5-HT (into synaptosomes) has been demonstrated for serotonin neurons ~4. Indeed, supra-ependymal nerve terminals have been shown to accumulate 5-hydroxydopamine22, so that it was reasonable to assume 5-HT would be preferentially taken up by these nerves and when radiolabelled should be visualized by the technique of autoradiographya0, 24. Several periventricular regions of rat brain were therefore investigated for their ability to accumulate [3H]5-HT and, for comparison, [3H]dopamine (DA) and [3H]noradrenaline (NA). This study also provided a unique opportunity to investigate the possible effects of known antidepressants (5-HT or NA-uptake inhibitors) on the accumulation of [3H]5-HT in a pure serotonergic nerve terminal network. Attempts were also made to localize specific sites of accumulation of [3H]lysergic acid diethylamide (LSD) on periventricular regions after its intraventricular injection, and thereby identify 5-HT receptors4, H. The results indicate a highly selective accumulation of [3H]5-HT by supra-ependymal nerve terminals and a significant but random localization of [aH]LSD. Male albino outbred rats of Wistar origin weighing 180-200 g were used. One half of the animals received a single injection of pargyline (100 mg/kg i.p., 18 h before sacrifice); this drug inhibits the enzyme, monoamine oxidase, metabolizing intraneuronal monoamines. For intraventricular injections the freehand technique of Noble et al. 19 was used. [3H]5-HT (spec. act. 12.6 Ci/mmole; radioact, conc. 1.0 mCi/ml) was injected into a lateral ventricle (0.25 /~g in 10/~1; 5 #l/rain); [3H]DA (spec. act. 10.0 Ci/mmole; 0.6/~g in l0 #1), [3H]NA (spec. act. 25.0 Ci/mmole; 0.2/~g in 10/~1) and [3H]LSD (2.35 Ci/mmole; 1.4 #g in 10 #1) were similarly administered. * Recently published in part (see refs. 20 and 21).
152 The effects of the following drugs on the accumulation of radiolabelled amines were investigated: reserpine (Serpasil, 10 mg/kg i.p., 18 h before injection of radiolabel); chlorimipramine (hydrochloride salt, 50 mg/kg i.p., 2 h) and desmethylimipramine (hydrochloride salt, 30 mg/kg i.p., 3 h). One hour after injection of the radiolabel, the animals (groups of 4) were fixed by vascular perfusion (20 rain, 19 °C) for light and electron microscopic autoradiography. The perfusate, delivered under an initial ether anesthesia, consisted of l o//oglutaraldehyde -F 1 ',~'iiformaldehyde in 0.0~ M sodium cacodylate buffer, pH 7.2. Various periventricular brain regions were then isolated and fixed for a further 60 min in the same aldehyde solution with some agitation; they included nucleus caudatus and corpus callosum of the injected lateral ventricle (cranial and caudal to the site of injection), hypothalamus ventralis anterior and eminentia mediana of the third ventricle and the floor and roof (velum medullare) of the fourth ventricle. After storage for 18 h in 0.2 M sodium cacodylate, pH 7.2 containing 7";i sucrose, the tissues were postfixed in 2 °Jo osmium tetroxide in the same buffer tbr I h, Semi-thin (1 #m) and ultrathin (70 nm) Epon sections were prepared for light and electron microscopic autoradiography. For light microscopy, semi-thin sections were mounted on gelatin-coated glass slides and dipped in Ilford K5 nuclear research emulsion; for electron microscopy, ultrathin sections were mounted on silver grids (200 mesh) and coated with llford L4 nuclear research emulsion using a loop technique7. Both emulsions were handled under a darkroom safelight (Kodak OA). Exposures for light and electron microscopy, at 4 °C, were 12 and 18 days respectively (with the exception of [aH]LSD, 42 days and 2 months respectively). Unstained semithin sections were viewed by phase-contrast microscopy while ultrathin sections were stained with lead citrate before examination in an electron microscope. (3H]5-HT uptake. Light microscopic examination of periventricular regions of rat brain (with or without pargyline treatment) revealed contrasting aspects in the different regions: while the parenchyma of most regions was labelled to varying degrees, the ependymal surface was not uniformly labelled. For example, there was a marked accumulation of silver grains in the lateral ventricle, e.g. above the nucleus caudatus and corpus callosum (Figs. 1 and 2) and on the floor of the fourth ventricle, whereas the ventricular surface of the hypothalamus ventralis anterior and eminentia mediana of the third ventricle (Figs. 3 and 4) and the roof of the fourth ventricle was free of label. Electron microscopy of the former regions revealed that supraependymal labelling was selectively localized to nerve fibres (varicosities and preterminal axons) (Fig. 5); clusters of silver grains were regularly found on the ependymal surface where varicosities or axons occurred but were totally absent elsewhere on the ventricle surface. The parenchymal labelling in the corpus callosum, nucleus caudatus and floor of the fourth ventricle, as well as in all other regions examined, was restricted to nerve terminals and myelinated and unmyelinated axons. Effects of drugs. Reserpine prevented the supra-ependymal accumulation of radiolabel in the absence, but not the presence, of the monoamine oxidase inhibitor. While chlorimipramine prevented uptake of [aH]5-HT in both cases (Fig. 6), desmethylimipramine on the other hand did not significantly affect this uptake (Fig. 7). [aH]DA and [ZH]NA uptake. In contrast to the accumulation of [3H]5-HT, the
153
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Figs. 1-4• Phase-contrast micrographs of various periventricular brain regions from a pargylinetreated rat one hour after injection of [3H]5-HT into a lateral ventricle• Note the supra-ependymal localization of silver grains in the nucleus caudatus and corpus callosum (Figs. 1 and 2 respectively) but not in the hypothalamus ventralis anterior and eminentia mediana (Figs. 3 and 4 respectively). The radiolabel in the parenchyma of all regions is not uniformly distributed and clusters of silver grains are observed. E, ependyma; P, parenchyma; V, ventricle. Magnifications: × 530; bar = 20/tin. Fig. 5. Electron micrograph of the ventricle surface above the nucleus caudatus from a pargylinetreated rat one hour after injection of [3H]5-HT into a lateral ventricle. Note the cluster of silver grains (radiolabel) over a profile o f a supra-ependymal nerve terminal in which small (--,-)and large (-~) densecored vesicles are observed. Magnification: :x: 47,000; bar -- 0.5/~m.
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Figs. 6 and 7. Phase-contrast micrographs of the corpus callosum from pargyline-treatcd i'ais, l:ig. 6: chlorimipramine prior to injection of [:~H]5-HT and Fig. 7: desmethylimipramine prior to [:~H]5-HT. Note the absence of supra-ependymal labelling and fewer silver grains in the parenchyma in the f\~rmer and presence of supra-ependymal and parenchymal labelling in the latter. Magnifications: 530: bar 20/~m. Figs. 8 and 9. Phase-contrast micrographs of the corpus callosum from pargyline-t reared rats one hotir after injection of ['~H]DA (Fig. 8) and ['~H]NA (Fig. 9) into a lateral ventricle. Note the absence of supra-ependymal labelling in both cases but the presence of silver grains partly in clusters in the parenchyma. Magnifications: .: 530; bar : 20t~m. Fig. 10. Phase-contrast micrograph of the nucleus caudatus from a rat one hour after injection of [:~H]LSD into a lateral ventricle. Note the significant but random distribution of silver grains. Magnification: ~ 1650; bar 10/~m.
155 two catecholamines rarely labelled the ventricular surface; radiolabelled supra-ependymal nerve terminals were not observed (.Figs. 8 and 9). Parenchymal labelling was confined to nerve terminals and myelinated and unmyelinated axons. Localization of[3H]LSD. The exposures required for [3H]LSD localization were much longer than for the amines. Nevertheless, a significant localization of radiolabel was observed which appeared to be highest in brain regions adjacent to the ventricle lumen, i.e. the ependyma and subependyma (Fig. 10). The distribution of silver grains, unlike that for [3H]5-HT or the other amines, was random and a selective association with supra-ependymal or other nerve terminals, axons or cell surfaces was not apparent. These findings provide evidence for the existence of a specific uptake mechanism for 5-HT in supra-ependymal nerve fibres and thus for their serotonergic nature. Previous fluorescence histochemical and cytochemical investigations of rat cerebral ventricles 16,17,22,2a have shown that a yellow formaldehyde-induced fluorescence and electron-dense (chromaffin) cores specific for 5-HT occur above the ependyma in several brain regions, e.g. nucleus caudatus, corpus callosum and floor of the fourth ventricle but rarely, if ever, in the hypothalamus ventralis anterior, eminentia mediana and roof (velum medullare) of the fourth ventricle. Earlier autoradiographic studies on the localization of [aH]5-HT taken up from the cerebral ventricles 12 had shown, by light and electron microscopy, that the amine penetrates into the brain parenchyma where it is predominantly localized in nerve terminals and unmyelinated axons; no evidence for its localization on the ependymal surface was mentioned presumably because the authors were aware of the possibility that the radiolabel might be merely 'absorbed upon a non-neuronal element such as the ependymal lining'. The present findings provide strong evidence for the neuronal localization of the [3H]5-HT accumulated on the ependymal surface. Alonso et al. ~ have also described an intense autoradiographic reaction on the surface of the fourth ventricle of the rat brain after incubation of halfbrain with [aH]5-HT; recent investigations have shown that supra-ependymal 5-HT nerve fibres also occur in the fourth ventricle (mainly the floor) and foramina of Luschka iv. The failure of [aH]DA and [3H]NA to accumulate in supra-ependymal nerve terminals suggests that the small quantities of these catecholamines injected prevented interspecific labelling and favoured affinity uptake of the endogenous amine. The selective uptake and neurotoxicity of 5,6-dihydroxytryptamine, compared to 6-hydroxydopamine, in supra-ependymal nerves TM would seem to support these findings. Very recent studies by Chan-Palay s on [3H]5-HT labelling of supra-ependymal and sub-ependymal plexuses of rats and rhesus monkeys have shown that this amine, but not [3H]DA or [3H]NA, is preferentially taken up by these nerves and that the uptake is enhanced by stimulation in the dorsal and median raphe neurons: it was concluded that these are the cells of origin of many of these nerve fibres. These findings therefore confirm observations of Aghajanian and Gallager a and Lorez et al. 15 who described the disappearance of supra-ependymal nerve fibres in several ventricular regions after either chemical or electrolytic lesions of dorsal and median raphe nuclei and median forebrain bundle. The inhibition of [aH]5-HT uptake by chlorimipramine
156 but not by desmethylimipramine agrees with previous findings which have shown that tertiary amines are more potent inhibitors of 5-HT uptake than secondary :tmines, the converse being true for inhibition of NA uptakel:L The physiological significance of the specific uptake mechanism in supra-ependymal nerve terminals is likely to be the removal of 5-HT from 'synaptic areas' in order to terminate the possible neurotransmitter action of 5-HT. However, at present the effector organ (target cell) is not known, although several speculations have been put forwardS,2L The presence of 5-HT in the cerebrospinal fluid 12, probably secreted in part by supra-ependymal nerve terminals, suggests that there may be specific receptors and a physiological role for this amine in periventricular brain regions. This may also be true for the human brain since supra-ependymal nerve terminals have recently been observed in the lateral and fourth ventricles of human postmortem brain tissue by electron microscopy (unpublished observations); it might be expected that these nerves also store and accumulate 5-HT although this is yet to be demonstrated. Since the hallucinogenic drug D-LSD is believed to have a high affinity lk)r 5-HT receptors ~ and in addition for DA receptors :~ acting as an agonist, the localization of specific sites of [3H]LSD accumulation in periventricular brain regions, e.g. ependymal cells, choroid plexus epithelia by autoradiography, would identify the target cell (,assuming the absence of DA receptors on such cells). The failure to demonstrate such a specific localization in the present study does not necessarily preclude the presence of 5-HT receptors on periventricular regions of the brain since the random distribution of radiolabelling, possibly masking the presence of specifically bound label, would first have to be tested for its displacement by D-LSD or 2-bromo-LSD, but not by I,-LSD: these experiments are in progress. In conclusion, supra-ependymal nerve terminals possess a specific uptake mechanism for 5-HT which is inhibited by chlorimipramine but not by desmethylimipramine. Clinically, potent 5-HT uptake inhibitors have predominantly moodelevating properties and/or sedative, anti-anxiety effects. Due to the complexity of the brain and the lack of techniques for the preparation of relatively pure fractions of aminergic neurons or nerve terminals, uptake studies have had to rely on the use of freshly prepared brain slices or crude synaptosomes as test preparations or even the use of blood platelets which also have a specific transport system for 5-tiT. These preparations have the disadvantage of being a mixed population of nerve terminals or synaptosomes and, in the case of platelets, may not react to drugs in the same manner as central 5-HT neurons. In these respects an approach using a pure population of 5H T nerve terminals would be a valuable complement and could be employed in semiquantitative autoradiographic studies on the relative potencies of potential antidepressants in vitro or in vivo. I thank Dr. H. Kuhn for help in setting up the autoradiography and Mrs. Z. Bleuel and Mr. M. Konte for excellent technical assistance.
157 1 Aghajanian, G. K. and Bloom, F. E., Localization of tritiated serotonin in rat brain by electron microscopic autoradiography, J. Pharmacol. exp. Ther., 156 (1967) 23-30. 2 Aghajanian, G. K., Bloom, F. E. Lovell, R. A., Sheard, M. H. and Freedman, D. X., The uptake of 5-hydroxytryptamine[aH] from the cerebral ventricles: autoradiographic localization, Biochem. Pharmacol., 15 (1966) 1401-1403. 3 Aghajanian, G. K. and Gallager, D. W., Raphe origin of serotonergic nerves terminating in the cerebral ventricles, Brain Research, 88 (1975) 221-231. 4 Aghajanian, G. K., Haigler, H. J. and Bennett, J. L., Amine receptors in CNS. 111. 5-Hydroxytryptamine in brain. In L. L. Iversen, S. D. lversen and S. H. Snyder (Eds.), Handbook of P,wchopharmacology, Vol. 6, Plenum Press, New York, 1975, pp. 63 96. 5 A/onso, G., Pons, F. et Cadilhac, J., Mise en evidence par radioautographie de terminaisons indolaminergiques dans les parois ventriculaires c6r6brales chez le rat, C.R. Soc. Biol. (Paris), 168 (1974) 1021 1024. 6 Bennett, J. P. and Snyder, S. H., Stereospecific binding of D-lysergic acid diethylamide (LSD) to brain membranes: relationship to serotonin receptors, Brain Research, 94 (1975) 523-544. 7 Caro, L. and Van Tubergen, R. P., High resolution autoradiography. I. Methods, J. Cell Biol., 15 (1962) 173 188, 8 Chan-Palay, V., Serotonin axons in the supra- and subependymal plexuses and in the leptomeninges ; their roles in local alterations of cerebrospinal fluid and vasomotor activity, Brain Research, 102 (1976) 103-130. 9 Creese, I., Burt, D. and Snyder, S. H., The dopamine receptor: differential binding of o-LSD and related agents of agonist and antagonist states, Life Sci., 17 (1976) 1715 1720. 10 Descarries, L. and Droz, B,, lntraneuronal distribution of exogenous norepinephrine in the central nervous system of the rat, J. Cell BioL, 44 (1970) 385-399. 11 Diab, 1. M., Freedman, D. X. and Roth, L. J., [aH]lysergic acid diethylamide: cellular autoradiographic localization in rat brain, Science, 173 (1971) 1022-1024. 12 Holman, R. B. and Vogt, M., Release of 5-hydroxytryptamine from caudate nucleus and septtlm, J. Physiol. (Lond.), 223 (1972) 243-254. 13 Iversen, L. L., Uptake processes for biogenic amines. In L. L. Iversen, S. D. lversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 3, Plenum Press, New York, 1975, pp. 381442. 14 Kuhar, M. J., Aghajanian, G. K. and Roth, R. H., Serotonin neurons: a synaptic mechanism for the reuptake of serotonin, Advanc. Biochem. Psychopharmacol., 10 (1974) 287 295. 15 Lorez, H. P., Pieri, L. and Richards, J. G., Disappearance of supra-ependymal 5-HT axons in the rat forebrain after electrolytic and 5,6-DHT-induced lesions of the median forebrain bundle, Brain Research, 100 (1975) 1-12. 16 Lorez, H. P. and Richards, J. G., Distribution of indolealkylamine nerve terminals in the ventricles of the rat brain, Z. ZellJorsch., 144 (1973) 511-522. 17 Lorez, H. P. and Richards, J. G., 5-HT nerve terminals in the fourth ventricle of the rat brain: their identification and distribution studied by fluorescence histochemistry and electron microscopy, Cell Tiss. Res., 165 (1975) 37~[8. 18 Lorez, H. P. and Richards, J. G., Effects of intracerebroventricular injection of 5,6-dihydroxytryptamine and 6-hydroxydopamine on supra-ependymal nerves, Brain Research, 116 (1976) 165 171. 19 Noble, E. P., Wurtman, R. J. and Axelrod, J., A simple and rapid method for injecting H3-norepinephrine into the lateral ventricle of the rat brain, Life Sci., 6 (1967) 281-29l. 20 Richards, J. G., Selective uptake of 3H-5HT by supra-ependymal nerve terminals: localization by autoradiography, Experientia (Basel), 32 (1976) 808. 21 Richards, J. G., Autoradiographic evidence for the effects of specific uptake-inhibitors on the selective accumulation of [aH]5-HT by supraependymal nerve terminals and for the localization of binding sites for [3H]BLSD, Brit. J. Pharmacol., 58 (1976) 424P-425P. 22 Richards, J, G., Lorez, H. P. and Tranzer, J. P., Indolealkylamine nerve terminals in cerebral ventricles : identification by electron microscopy and fluorescence histochemistry, Brain Research, 57 (1973) 277-288. 23 Richards, J. G. and Tranzer, J. P., Ultrastructural evidence for the localization of an indolealkylamine in supra-ependymal nerves from combined cytochemistry and pharmacology, Experientia (Basel), 30 (1974) 287-289. 24 Wolfe, D. E., Potter, L. T., Richardson, K. C. and Axelrod, J., Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography, Science, 138 (1962) 440-442.
151
Autoradiographic evidence for the selective accumulation of [3H]5-HT by supra-ependymal nerve terminals*
J. G.
RICHARDS
Pharmaceutical Research Department, F. HoJfmann-La Roche, CHo4002 Basle (Switzerland)
(Accepted June 2rid, 1977)
A network of supra-ependymal serotonergic (5-HT) nerve fibres has been shown to exist in most ventricular regions of rat brain 16,17,22,23. In the forebrain these nerve fibres originate from the raphe nuclei of the rues- and/or metencephalon 3,15. It is well known that aminergic nerves selectively accumulate monoamines and that this uptake mechanism shows its highest affinity for the endogenous amine 13. A synaptic mechanism for the reuptake of 5-HT (into synaptosomes) has been demonstrated for serotonin neurons ~4. Indeed, supra-ependymal nerve terminals have been shown to accumulate 5-hydroxydopamine22, so that it was reasonable to assume 5-HT would be preferentially taken up by these nerves and when radiolabelled should be visualized by the technique of autoradiographya0, 24. Several periventricular regions of rat brain were therefore investigated for their ability to accumulate [3H]5-HT and, for comparison, [3H]dopamine (DA) and [3H]noradrenaline (NA). This study also provided a unique opportunity to investigate the possible effects of known antidepressants (5-HT or NA-uptake inhibitors) on the accumulation of [3H]5-HT in a pure serotonergic nerve terminal network. Attempts were also made to localize specific sites of accumulation of [3H]lysergic acid diethylamide (LSD) on periventricular regions after its intraventricular injection, and thereby identify 5-HT receptors4, H. The results indicate a highly selective accumulation of [3H]5-HT by supra-ependymal nerve terminals and a significant but random localization of [aH]LSD. Male albino outbred rats of Wistar origin weighing 180-200 g were used. One half of the animals received a single injection of pargyline (100 mg/kg i.p., 18 h before sacrifice); this drug inhibits the enzyme, monoamine oxidase, metabolizing intraneuronal monoamines. For intraventricular injections the freehand technique of Noble et al. 19 was used. [3H]5-HT (spec. act. 12.6 Ci/mmole; radioact, conc. 1.0 mCi/ml) was injected into a lateral ventricle (0.25 /~g in 10/~1; 5 #l/rain); [3H]DA (spec. act. 10.0 Ci/mmole; 0.6/~g in l0 #1), [3H]NA (spec. act. 25.0 Ci/mmole; 0.2/~g in 10/~1) and [3H]LSD (2.35 Ci/mmole; 1.4 #g in 10 #1) were similarly administered. * Recently published in part (see refs. 20 and 21).
152 The effects of the following drugs on the accumulation of radiolabelled amines were investigated: reserpine (Serpasil, 10 mg/kg i.p., 18 h before injection of radiolabel); chlorimipramine (hydrochloride salt, 50 mg/kg i.p., 2 h) and desmethylimipramine (hydrochloride salt, 30 mg/kg i.p., 3 h). One hour after injection of the radiolabel, the animals (groups of 4) were fixed by vascular perfusion (20 rain, 19 °C) for light and electron microscopic autoradiography. The perfusate, delivered under an initial ether anesthesia, consisted of l o//oglutaraldehyde -F 1 ',~'iiformaldehyde in 0.0~ M sodium cacodylate buffer, pH 7.2. Various periventricular brain regions were then isolated and fixed for a further 60 min in the same aldehyde solution with some agitation; they included nucleus caudatus and corpus callosum of the injected lateral ventricle (cranial and caudal to the site of injection), hypothalamus ventralis anterior and eminentia mediana of the third ventricle and the floor and roof (velum medullare) of the fourth ventricle. After storage for 18 h in 0.2 M sodium cacodylate, pH 7.2 containing 7";i sucrose, the tissues were postfixed in 2 °Jo osmium tetroxide in the same buffer tbr I h, Semi-thin (1 #m) and ultrathin (70 nm) Epon sections were prepared for light and electron microscopic autoradiography. For light microscopy, semi-thin sections were mounted on gelatin-coated glass slides and dipped in Ilford K5 nuclear research emulsion; for electron microscopy, ultrathin sections were mounted on silver grids (200 mesh) and coated with llford L4 nuclear research emulsion using a loop technique7. Both emulsions were handled under a darkroom safelight (Kodak OA). Exposures for light and electron microscopy, at 4 °C, were 12 and 18 days respectively (with the exception of [aH]LSD, 42 days and 2 months respectively). Unstained semithin sections were viewed by phase-contrast microscopy while ultrathin sections were stained with lead citrate before examination in an electron microscope. (3H]5-HT uptake. Light microscopic examination of periventricular regions of rat brain (with or without pargyline treatment) revealed contrasting aspects in the different regions: while the parenchyma of most regions was labelled to varying degrees, the ependymal surface was not uniformly labelled. For example, there was a marked accumulation of silver grains in the lateral ventricle, e.g. above the nucleus caudatus and corpus callosum (Figs. 1 and 2) and on the floor of the fourth ventricle, whereas the ventricular surface of the hypothalamus ventralis anterior and eminentia mediana of the third ventricle (Figs. 3 and 4) and the roof of the fourth ventricle was free of label. Electron microscopy of the former regions revealed that supraependymal labelling was selectively localized to nerve fibres (varicosities and preterminal axons) (Fig. 5); clusters of silver grains were regularly found on the ependymal surface where varicosities or axons occurred but were totally absent elsewhere on the ventricle surface. The parenchymal labelling in the corpus callosum, nucleus caudatus and floor of the fourth ventricle, as well as in all other regions examined, was restricted to nerve terminals and myelinated and unmyelinated axons. Effects of drugs. Reserpine prevented the supra-ependymal accumulation of radiolabel in the absence, but not the presence, of the monoamine oxidase inhibitor. While chlorimipramine prevented uptake of [aH]5-HT in both cases (Fig. 6), desmethylimipramine on the other hand did not significantly affect this uptake (Fig. 7). [aH]DA and [ZH]NA uptake. In contrast to the accumulation of [3H]5-HT, the
153
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Figs. 1-4• Phase-contrast micrographs of various periventricular brain regions from a pargylinetreated rat one hour after injection of [3H]5-HT into a lateral ventricle• Note the supra-ependymal localization of silver grains in the nucleus caudatus and corpus callosum (Figs. 1 and 2 respectively) but not in the hypothalamus ventralis anterior and eminentia mediana (Figs. 3 and 4 respectively). The radiolabel in the parenchyma of all regions is not uniformly distributed and clusters of silver grains are observed. E, ependyma; P, parenchyma; V, ventricle. Magnifications: × 530; bar = 20/tin. Fig. 5. Electron micrograph of the ventricle surface above the nucleus caudatus from a pargylinetreated rat one hour after injection of [3H]5-HT into a lateral ventricle. Note the cluster of silver grains (radiolabel) over a profile o f a supra-ependymal nerve terminal in which small (--,-)and large (-~) densecored vesicles are observed. Magnification: :x: 47,000; bar -- 0.5/~m.
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Figs. 6 and 7. Phase-contrast micrographs of the corpus callosum from pargyline-treatcd i'ais, l:ig. 6: chlorimipramine prior to injection of [:~H]5-HT and Fig. 7: desmethylimipramine prior to [:~H]5-HT. Note the absence of supra-ependymal labelling and fewer silver grains in the parenchyma in the f\~rmer and presence of supra-ependymal and parenchymal labelling in the latter. Magnifications: 530: bar 20/~m. Figs. 8 and 9. Phase-contrast micrographs of the corpus callosum from pargyline-t reared rats one hotir after injection of ['~H]DA (Fig. 8) and ['~H]NA (Fig. 9) into a lateral ventricle. Note the absence of supra-ependymal labelling in both cases but the presence of silver grains partly in clusters in the parenchyma. Magnifications: .: 530; bar : 20t~m. Fig. 10. Phase-contrast micrograph of the nucleus caudatus from a rat one hour after injection of [:~H]LSD into a lateral ventricle. Note the significant but random distribution of silver grains. Magnification: ~ 1650; bar 10/~m.
155 two catecholamines rarely labelled the ventricular surface; radiolabelled supra-ependymal nerve terminals were not observed (.Figs. 8 and 9). Parenchymal labelling was confined to nerve terminals and myelinated and unmyelinated axons. Localization of[3H]LSD. The exposures required for [3H]LSD localization were much longer than for the amines. Nevertheless, a significant localization of radiolabel was observed which appeared to be highest in brain regions adjacent to the ventricle lumen, i.e. the ependyma and subependyma (Fig. 10). The distribution of silver grains, unlike that for [3H]5-HT or the other amines, was random and a selective association with supra-ependymal or other nerve terminals, axons or cell surfaces was not apparent. These findings provide evidence for the existence of a specific uptake mechanism for 5-HT in supra-ependymal nerve fibres and thus for their serotonergic nature. Previous fluorescence histochemical and cytochemical investigations of rat cerebral ventricles 16,17,22,2a have shown that a yellow formaldehyde-induced fluorescence and electron-dense (chromaffin) cores specific for 5-HT occur above the ependyma in several brain regions, e.g. nucleus caudatus, corpus callosum and floor of the fourth ventricle but rarely, if ever, in the hypothalamus ventralis anterior, eminentia mediana and roof (velum medullare) of the fourth ventricle. Earlier autoradiographic studies on the localization of [aH]5-HT taken up from the cerebral ventricles 12 had shown, by light and electron microscopy, that the amine penetrates into the brain parenchyma where it is predominantly localized in nerve terminals and unmyelinated axons; no evidence for its localization on the ependymal surface was mentioned presumably because the authors were aware of the possibility that the radiolabel might be merely 'absorbed upon a non-neuronal element such as the ependymal lining'. The present findings provide strong evidence for the neuronal localization of the [3H]5-HT accumulated on the ependymal surface. Alonso et al. ~ have also described an intense autoradiographic reaction on the surface of the fourth ventricle of the rat brain after incubation of halfbrain with [aH]5-HT; recent investigations have shown that supra-ependymal 5-HT nerve fibres also occur in the fourth ventricle (mainly the floor) and foramina of Luschka iv. The failure of [aH]DA and [3H]NA to accumulate in supra-ependymal nerve terminals suggests that the small quantities of these catecholamines injected prevented interspecific labelling and favoured affinity uptake of the endogenous amine. The selective uptake and neurotoxicity of 5,6-dihydroxytryptamine, compared to 6-hydroxydopamine, in supra-ependymal nerves TM would seem to support these findings. Very recent studies by Chan-Palay s on [3H]5-HT labelling of supra-ependymal and sub-ependymal plexuses of rats and rhesus monkeys have shown that this amine, but not [3H]DA or [3H]NA, is preferentially taken up by these nerves and that the uptake is enhanced by stimulation in the dorsal and median raphe neurons: it was concluded that these are the cells of origin of many of these nerve fibres. These findings therefore confirm observations of Aghajanian and Gallager a and Lorez et al. 15 who described the disappearance of supra-ependymal nerve fibres in several ventricular regions after either chemical or electrolytic lesions of dorsal and median raphe nuclei and median forebrain bundle. The inhibition of [aH]5-HT uptake by chlorimipramine
156 but not by desmethylimipramine agrees with previous findings which have shown that tertiary amines are more potent inhibitors of 5-HT uptake than secondary :tmines, the converse being true for inhibition of NA uptakel:L The physiological significance of the specific uptake mechanism in supra-ependymal nerve terminals is likely to be the removal of 5-HT from 'synaptic areas' in order to terminate the possible neurotransmitter action of 5-HT. However, at present the effector organ (target cell) is not known, although several speculations have been put forwardS,2L The presence of 5-HT in the cerebrospinal fluid 12, probably secreted in part by supra-ependymal nerve terminals, suggests that there may be specific receptors and a physiological role for this amine in periventricular brain regions. This may also be true for the human brain since supra-ependymal nerve terminals have recently been observed in the lateral and fourth ventricles of human postmortem brain tissue by electron microscopy (unpublished observations); it might be expected that these nerves also store and accumulate 5-HT although this is yet to be demonstrated. Since the hallucinogenic drug D-LSD is believed to have a high affinity lk)r 5-HT receptors ~ and in addition for DA receptors :~ acting as an agonist, the localization of specific sites of [3H]LSD accumulation in periventricular brain regions, e.g. ependymal cells, choroid plexus epithelia by autoradiography, would identify the target cell (,assuming the absence of DA receptors on such cells). The failure to demonstrate such a specific localization in the present study does not necessarily preclude the presence of 5-HT receptors on periventricular regions of the brain since the random distribution of radiolabelling, possibly masking the presence of specifically bound label, would first have to be tested for its displacement by D-LSD or 2-bromo-LSD, but not by I,-LSD: these experiments are in progress. In conclusion, supra-ependymal nerve terminals possess a specific uptake mechanism for 5-HT which is inhibited by chlorimipramine but not by desmethylimipramine. Clinically, potent 5-HT uptake inhibitors have predominantly moodelevating properties and/or sedative, anti-anxiety effects. Due to the complexity of the brain and the lack of techniques for the preparation of relatively pure fractions of aminergic neurons or nerve terminals, uptake studies have had to rely on the use of freshly prepared brain slices or crude synaptosomes as test preparations or even the use of blood platelets which also have a specific transport system for 5-tiT. These preparations have the disadvantage of being a mixed population of nerve terminals or synaptosomes and, in the case of platelets, may not react to drugs in the same manner as central 5-HT neurons. In these respects an approach using a pure population of 5H T nerve terminals would be a valuable complement and could be employed in semiquantitative autoradiographic studies on the relative potencies of potential antidepressants in vitro or in vivo. I thank Dr. H. Kuhn for help in setting up the autoradiography and Mrs. Z. Bleuel and Mr. M. Konte for excellent technical assistance.
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