5-HT1 receptor binding sites in the guinea pig superior colliculus are predominantly of the 5-HT1D class and are presynaptically located on primary retinal afferents

Brain Researah, 528 (1990) 207-211

207

Elsevier BRES 15871

5-HTI receptor binding sites in the guinea pig superior colliculus are predominantly of the 5-HT m class and are presynaptically located on primary retinal afferents Christian Waeber and Jos6 M. Palacios Preclinical Research, Sandoz Pharma Ltd., Basel (Switzerland)

(Accepted 20 March 1990) Key words: 5-HT m receptor; Guinea pig brain; Superior colliculus; Visual system; Autoradiography

The superficial layers of the guinea pig superior colliculus are characterized by high densities of [3H]5-HT binding sites. We have chosen receptor autoradiography to establish the drug binding profile and the localization of these sites. The binding of [3H]5-HT to guinea pig superior colliculus was nearly completely blocked by drugs such as 5-carboxamido-tryptamine and yohimbine, but only slightly sensitive to the 5-HT1 receptor agonist 8-hydroxy-dipropylamino-tetralin. 5-HTlc antagonists such as mesulergine or the fl-adrenergic receptor blocking compound SDZ 21-009 did not show any effect. The profile of the majority of these sites corresponds to that of 5-HT m sites. Unilateral enucleation resulted in a nearly complete depletion of [3H]5-HT binding in the contralateral superior colliculus, whereas [~I]Bolton-Hunter8-methoxy-N-propylaminotetralin binding sites, corresponding to 5-HT~A receptors, were preserved. These results indicate that 5-HTlo (and not 5-HT~A) receptors might be presynaptically localized on non-serotoninergic neuronal pathways. The guinea pig visual system may be a useful model for the study of the properties of these presynaptic 5-HT heteroreceptors. INTRODUCTION The physiological effects of serotonin (5-hydroxytryptamine, 5-HT) are mediated by the interaction of this amine with several subtypes of receptor which can be distinguished on the basis of their diverse drug binding profiles (for review, see ref. 24). The different receptor subtypes are coupled to distinct signal transduction mechanisms: some elicit activation or inhibition of adenylate cyclases, others stimulate the hydrolysis of phosphatidyl inositols (for review, see ref. 1l). In addition, the subtypes of the 5-HT receptors have different anatomical and cellular localizations (for review, see ref. 19). In particular, receptors with the properties of the 5-HTIB or 5-HT m sites are abundant in the basal ganglia, the substantia nigra and the neocortex of rodents and other mammals 32'35. The 5-HT receptor modulating the release of 5-HT from the terminals of the serotoninergic neurons (5-HT autoreceptor) has in the rat and mouse brain the characteristics of a 5-HT m receptor whereas in other species the 5-HT autoreceptor has the properties of a 5-HTID receptor (for review, see ref. 12). Using an anatomical technique such as receptor autoradiography, it has been difficult to demonstrate the presence of significant densities of presynaptic 5-HT receptors on serotoninergic neurons a9,31'37. This is prob-

ably due to the fact that these sites are present in brain only at very low densities. We have shown that the guinea pig is a convenient laboratory animal for the study of 5-HTID receptors 34'35. The investigations presented in this paper are focused on 5-HT binding sites present in the superior colliculus of this animal, a component of the visual pathway which is characterized by a high density of 5-HT binding sites 2°. Furthermore, the afferences and efferences of this brain region are well characterized and could offer a potential area where the properties of 5-HT receptors could be examined at the biochemical and functional levels. One of the features of this area is the fact that it receives crossed monosynaptic afferents from the ganglion cells of the retina 26. Experiments in the rat suggest that this area could contain presynaptic 5-HT receptors 27. Here we show that in the guinea pig the primary retinal afferents to the superior colliculus are enriched in 5-HT receptors which are probably presynaptically located. MATERIALS AND METHODS Adult, male guinea pigs (ca. 300 g b. wt.) were used in all experiments. For the pharmacological characterization of 5-HT binding sites in the superior colliculus, animals were sacrificed by decapitation and the brains rapidly removed, dissected into l cm segments and frozen until used. Other animals underwent unilateral

Correspondence: J.M. Palacios, Preclinical Research, Sandoz Ltd., CH-4002 Basel, Switzerland.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

208

100" -~ 80°~ 60. ,~ ,~

~

T

~ total ~iiiiiiii~8-0H-DPAT ~ +mosulorgine [ ~ +SDZ21009 ~ +y0himbine a m + 5"CT

~ ~

40. iiiiiiii!i ..... i!!!!!!!!!!

20' ~'

Guinea Pig

Rat

Fig. 1. Bar graph illustrating the effects of various drugs on the density of specific [3H]5-HT binding in the superficial grey layer of the superior colliculus, measured on the autoradiograms by computer-aided microdensitometry. Results are expressed as percent of [3H]5-HT binding in the absence of blocking drug. 5-HTIA sites account for about one third of all [3H]5-HT binding sites, as shown by the effect of 100 nM 8-OH-DPAT, whereas further addition of 100 nM mesulergine does not result in any displacement, ruling out the presence of 5-HT1¢ receptors in this region. In the guinea pig (in the presence of 100 nM 8-OH-DPAT), the 5-HT m agent SDZ 21009 (30 nM) has no effect, contrarily to 1/~M yohimbine, which displaces most of the remaining binding to the level obtained using 10 nM 5-carboxamido-tryptamine (5-CT). The sites labelled in the rat differ from those in the guinea pig by their higher sensitivity to 30 nM SDZ 21009 and the smaller displacement obtained with yohimbine. These profiles are consistent with that of 5-HT~D receptors in the guinea pig and 5-HT m receptors in the rat brain.

enucleation. These animals were anesthetized according to the following protocol: s.c. injection of 0.1% atropine (0.05 mg/kg; Dr. E. Graub SA, Bern, Switzerland) 10 min before i.m. injection of a mixture of Ketalar (150 mg/kg; Parke-Davis, Ziirich, Switzerland) and Rompun (5 mg/kg, Bayer, Leverkusen, ER.G.). The right eye was enucleated and the animals were allowed to survive for 2 weeks. The brains of these lesioned animals were dissected after decapitation and tissue blocks prepared and kept frozen until use. Thick tissue sections of 10 gm thick were obtained using a cryostatmicrotome and mounted on gelatine-coated microscope slides. 5-HT receptors in these tissue sections were labelled with [3H]5-HT as previously described32. In addition, 5-HT1A receptors were selectively labelled with [z25I]Bolton-Hunter-8-MeO-N-PAT (final concentration: 0.5 nM), using the same procedure as for [3H]5-HT. Consecutive sections from the control animals were used to examine drug binding characteristics of [3H]5-HT to the superior colliculus, using a series of compounds presenting differential selectivity for the subtypes of 5-HT 1 receptors. Autoradiograms were generated by apposing the labelled sections to 3H-Hyperfilms (Amersham, Sweden) along with tritiated plastic standard (Amersham, Sweden). Quantification was performed using a computerized image analysis system (MCID, Imaging Research, St Catherines, Ont., Canada) as previously described33. RESULTS Fig. 1 illustrates the drug sensitivity of [3H]5-HT binding to the guinea pig and rat superior colliculus. W h i l e the 5-HT1A selective agonist 8 - O H - D P A T 9'17 blocks a b o u t one third of [3H]5-HT binding sites in the guinea pig brain, n e i t h e r the 5 - H T l c antagonist mesulergine 21 nor the fl-adrenergic r e c e p t o r blocking and 5 - H T m selective c o m p o u n d S D Z 2100922 significantly

Fig. 2. Coronal sections of guinea pig brain sacrificed 14 days after ablation of the left eye (organized in a rostro-caudal progression). The decrease of [3H]5-HT binding sites (in the presence of 100 nM 8-OH-DPAT and mesulergine) is readily observable in the contralateral superficial grey layer of the superior colliculus (SGL), where retinal neurons are known to project. The level of labelling is not affected in the dentate gyrus (DG) or in other areas of the mesencephalon, such as the central grey (CG) and the substantia nigra (SN). Bar = 5 mm.

block [3H]5-HT binding to the s u p e r i o r colliculus in this species. In contrast, in the presence o f 100 n M 8O H - D P A T and 100 n M mesulergine, [3H]5-HT binding is almost c o m p l e t e l y displaced by S D Z 21-009 in the rat brain. A n o t h e r difference b e t w e e n the two species is the higher efficacy of y o h i m b i n e at c o m p e t i n g for [3H]5-HT binding sites in the guinea pig. T h e p o t e n t 5 - H T 1 r e c e p t o r antagonist 5 - c a r b o x a m i d o - t r y p t a m i n e c o m p l e t e l y blocks [3H]5-HT binding at n a n o m o l a r concentration. Fig. 2 shows a r e p r e s e n t a t i v e e x a m p l e of the effects of unilateral enucleation on [3H]5-HT binding in the super-

209 TABLE I Effects of unilateral enucleation on total and 5-HTID [3H]5-HT specific binding in several regions of the guinea pig brain

Results are expressed as the percentage of the density on the contralateral sides over the ipsilateral side. Data are mean of at least 5 different animals + S.E.M. Region

Total

5-HTID

P

Superior colliculus Substantia nigra Hippocampus

50.6 + 2.2 99.9 + 3.4 95.5 + 3.4

28.57 + 3.28 93.42 + 3.11 109.77 + 8.10

0.001 0.091 0.453

ficial grey layer of the superior colliculus of the guinea pig. Whereas no effect can be observed in the hippocampal formation or in the substantia nigra, a marked decrease in [3H]5-HT binding results from the lesion of the primary retinal afferents to this area. Table I presents the results expressed as the percentage of the receptor density on the contralateral side compared to the ipsilateral side. As the majority of the retinal fibers are

Fig. 3. Sections of the same animal as in Fig. 2, incubated with

[125I]Bolton-Hunter-8-MeO-N-PAT to label specifically 5-HTIA sites. A shows specific binding whereas B illustrates non-specific binding as obtained by adding 10 /~M 5-HT to the incubation medium. The dorsal raph6 nucleus, which contains high concentrations of 5-HTIA receptors, is densely labelled. Note that the level of labelling in the superior grey layer of the superior colliculus (SGL) is fully comparable on both sides. Bar = 5 mm.

crossed, at least in the rat 14, the use of the ipsilateral side as a reference is justified. The analysis of the pharmacological characteristics of the remaining receptors indicates that they belong to the 5-HT1A class. The selective labelling of these sites with [12SI]Bolton-Hunter-8-MeON-PAT (Fig. 3) clearly demonstrate that they are present at equal concentrations in both superior colliculi. DISCUSSION The main findings of these investigations are that the 5-HT receptors in the guinea pig superior colliculus belong to the 5-HT1D class and that they appear to be presynaptically located on the terminals of the primary retinal afferents. These results taken together with data obtained in human pathological brains 33 and experimentally lesioned guinea pig brains 36 as well as with evidence from functional investigations 12 indicate that 5-HTID receptors possess at least 3 different cellular localizations in the mammalian brain. They are present on the terminals of serotoninergic neurons where they modulate the release of 5-HT (for review, see ref. 12). They are also presynaptically localized on the terminals of nonserotoninergic neurons, i.e. as presynaptic heteroreceptors 3a'36. In addition, 5-HT~D receptors are also postsynaptically located on the dendrites or cell bodies of neurons in other brain areas, as for instance the caudateputamen or the globus pallidus 33'36. Evidence for a presynaptic localization of 5-HTlo receptors on non-serotoninergic neurons have been found in functional systems other than the visual pathway. In the striatonigral loop, 5-HTID receptors appear to be presynapticaUy located on the terminals of t h e pathway descending from the striatum to the substantia nigra pars reticulata 33"36. These presynaptic receptors in the substantia nigra have been shown to be linked negatively to an adenylate cyclase 25. It will be interesting to establish whether the 5-HT~D receptors located presynaptically in the superior colliculus are also negatively linked to adenylate cyclase. After unilateral enucleation, the binding sites for the plant diterpene derivative [3H]forskolin, a potent stimulator of adenylate cyclase, have the same fate as 5-HT m and 5-HT1D receptors under similar conditions ~°. This would be compatible with a similar cellular localization for these two molecules and with the linkage of presynaptic 5-HT1D receptors to this second messenger system 25"3a. It is noteworthy that a 5-HT receptor stimulating an adenylate cyclase has been described in cell cultures from mouse superior colliculus. This site, characterized by its high affinity for benzamide derivatives, has been named 5-HT4, owing to its completely new pharmacological profile 4a. However the precise regional localisation of these receptors in adult

210 brain has not been determined. The signal transducing systems used by 5-HT receptors in this region thus remain to be studied in detail. The present results indicate a similarity already described by us and others between 5 - H T l o and 5-HT m receptors. Although pharmacologically different, these subtypes of 5-HT receptors, present in different species, appear to be very similar in their linkage to a second messenger system as well as in their regional distribution in the mammalian brain 35. Both are negatively linked to an adenylate cyclase activity in the substantia nigra 2'25 and both appear to be located presynaptically in this brain area 33'36. O u r results in the guinea pig superior colliculus correlate well with similar studies in the rat brain 27, where 5-HT m receptors (Palacios and collaborators, unpublished results), decrease after unilateral enucleation. Several studies have been carried out to investigate the presence of serotoninergic terminals in the superior colliculus. From the early fluorescence studies of Fuxe 6, the superior colliculus of the rat has been found to contain a remarkable density of serotoninergic processes 29'30. These processes appear to be particularly enriched in the most superficial layers of the rat superior coUiculus. Tracing experiments in the rat and in the cat have shown that most of these terminals arise from the nucleus raph6 dorsalis 1'18. Biochemical, physiological and behavioral studies support a role for 5-HT on the functions of the superior colliculus. 5-HT turnover in the

superior colliculus can be increased after visual stimulus 5. Radiolabelled 5-HT can be released from superior colliculus slices after stimulation of the optic tract 15. Electrophysiological experiments have shown that iontophoretically applied 5-HT can inhibit some collicular cells 7. The pharmacological profile of the 5-HT receptors involved in this inhibition is, however, poorly characterized 8'16. The superior colliculus plays a role in the coordination of eye m o v e m e n t and in the orientation of head and eyes to external stimuli 28. It has been shown that 5-HT neuron activity may correlate with eye movement during R E M sleep 13. All these data taken together suggest that serotoninergic mechanisms can be involved in many different functions of this brain area and that the activity of 5-HT could be mediated, at least partly, via 5-HTID receptors located on primary retinal afferents. The transmitter present in these afferences in the mammalian brain is not completely characterized. It has been proposed that it could be an excitatory amino acid, substance P or both 3'4. Our finding of a predominant 5-HT1D receptor concentration in these terminals suggest that biochemical or physiological tests could be developed for further characterization of 5-HT1D receptors within this functionally well characterized pathway in the mammalian brain.

REFERENCES

Ther., 188 (1974) 688-699. 8 Haigler, H.J. and Aghajanian, G.K., Peripheral serotonin antagonists: failure to antagonize serotonin in brain areas receiving a prominent serotonergic input, J. Neural Transm., 35 (1974) 257-273. 9 Gozlan, H., El Mestikawy, S., Pichat, L., Glowinski, J. and Hamon, M., Identification of presynaptic serotonin autoreceptors using a new ligand: [3H]PAT, Nature, 305 (1983) 140-142. 10 Horsburgh, K., Chalmers, D. and McCulloch, J., [3H]forskolin binding in the visual system of the rat after unilateral orbital enucleation, Br. J. Pharmacol., 95 (1988) 9022 11 Hoyer, D., 5-Hydroxytryptamine receptors and effector coupling mechanisms in peripheral tissues. In J.R. Fozard (Ed.), The Peripheral Actions of 5-HT, Oxford University Press, Oxford, 1989, pp. 72-99. 12 Hoyer, D. and Middlemiss, D.N., The pharmacology of the terminal 5-HT autoreceptors in mammalian brain: evidence for species differences, Trends Pharmacol. Sci., 10 (1989) 130-132. 13 Jakobs, B.L., Heym, J. and Trulson, M.E., Behavioral and physiological correlates of brain serotoninergic unit activity, J. Physiol. (Paris), 77 (1981) 431-436. 14 Jeffery, G., Retinal ganglion cell death and terminal field retraction in the developing rodent visual system, Dev. Brain Res., 13 (1984) 81-96. 15 Kawai, N., Release of 5-hydroxytryptamine from slices of superior colliculus by optic tract stimulation, Neuropharmacology, 9 (1970) 395-397. 16 Kawai, N. and Yamamoto, C., Effect of 5-hydroxytryptamine, LSD, and related compounds on electrical activities evoked in vitro in thin sections from the superior colliculus, Int. J.

1 Beitz, A.J., Clements, J.R., Mullet, M.A. and Ecklund, L.J., Differential origin of brainstem serotoninergic projections to the midbrain periaqueductal gray and superior colliculus of the rat, J. Comp. Neurol., 250 (1986) 1498-1509. 2 Bouhelal, R., Smounya, L. and Bockaert, J., 5-HTla receptors are negatively coupled with adenylate cyclase in rat substantia nigra, Eur. J. Pharmacol., 151 (1988) 189-196. 3 Brecha, N., Johnson, D., Bolz, J., Sharma, S., Parnevalas, J.G. and Lieberman, A.R., Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit, Nature, 327 (1987) 155-158. 4 Canzek, V., Wolfenberger, M., Amsler, U. and Curnod, M., In vivo release of glutamate and aspartate following optic nerve stimulation, Nature, 293 (1981) 572-574. 4a Dumuis, A., Bouhelal, R., Sebben, M., Cory, R. and Bockaert, J., A nonclassical 5-hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system, Mol. Pharmacol., 34 (1989) 880-887. 5 Fukui, R. and Vogt, M., The effect of visual stimuli on the turnover of 5-hydroxytryptamine (5-HT) in the superior colliculus, J. Physiol., 248 (1975) 39P-402 6 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of the monoamine nerve terminals in the central nervous system, Acta Physiol. Scand., 64, Suppl. 247 (1965) 38-85. 7 Haigler, H.J. and Aghajanian, G.K., Lysergic acid diethylamide and serotonin: a comparison of effects on serotonergic neurons and neurons receiving a serotonergic input, J. Pharmacol. Exp.

Acknowledgements. We would like to thank Dr. H. Gozlan and Dr. M. Hamon for the generous gift of 8-MeO-N-PAT and Dr. J.R. Fozard for critically reading the manuscript.

211

Neuropharmacol., 8 (1969) 437-449. 17 Middlemiss, D.N. and Fozard, J.R., 8-Hydroxy-2-(di-n-propylamino)-tetralin discriminates between subtypes of the 5-HTt recognition sites, Eur. J. Pharmacol., 90 (1983) 151-153. 18 Mize, R.R. and Homer, L.H., Origin, distribution and morphology of serotonergic afferents to the cat superior colliculus: a light and electron microscope immunocytochemistry study, Exp. Brain Res., 75 (1989) 83-98. 19 Palacios, J.M. and Dietl, M.M., Autoradiographic studies on 5-HT receptors. In E. Sanders-Bush (Ed.), The Serotonin Receptors, Humana, Clifton, NJ, 1988, pp. 89-138. 20 Pazos, A. and Palacios, J.M., Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors, Brain Research, 346 (1985) 205-230. 21 Pazos, A., Hoyer, D. and Palacios, J.M., The binding of serotoninergic ligands to porcine choroid plexus: characterization of a new type of serotonin recognition site, Eur. J. Pharmacol., 106 (1985) 539-546. 22 Pazos, A., Engel, G. and Palacios, J.M., fl-Adrenoreceptor blocking agents recognize a subpopulation of serotonin receptors in brain, Brain Research, 343 (1985) 403-408. 23 Pazos, A., Hoyer, D., Dietl, M.M. and Palacios, J.M., Autoradiography of serotonin receptors. In N.N. Osborne and M. Hamon (Eds.), Neuronal Serotonin, Wiley, Chichester, 1988, pp. 507-544. 24 Peroutka, S.J., 5-Hydroxytryptamine receptor subtypes: molecular, biochemical and physiological characterization, Trends Neurosci., 11 (1988) 496-500. 25 Schoeffter, P., Waeber, C., Palacios, J.M. and Hoyer, D., The 5-hydroxytryptamine 5-HT1D receptor subtype is negatively coupled to adenylate cyclase in calf substantia nigra, NaunynSchmiedeberg's Arch. Pharmacol, 337 (1988) 602-608. 26 Sefton, A.J. and Dreher, B., Visual system. In G. Paxinos (ed.), The Rat Nervous System, Vol. 1, Forebrain and Midbrain, Academic Press, Sydney, 1984, pp. 169-221. 27 Segu, L., Abdelkefi, J., Dusticier, G. and Lanoir, J., Highaffinity serotonin binding sites: autoradiographic evidence for their location on retinal afferents in the rat superior colliculus, Brain Research, 384 (1986) 205-217.

28 Sprague, J.M. and Meikle, T.H., The role of the superior colliculus in visually guided behavior, Exp. Neurol., 11 (1965) 115-146. 29 Steinbusch, H.V.M., Distribution of serotonin immunoreactivity in the central nervous system of the rat: cell bodies and terminals, Neuroscience, 6 (1981) 557-618. 30 Steinbusch, H.W.M., Serotonin-immunoreactive neurons and their projections in the CNS. In A. Bj6rklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy: Classical Transmitters and TransmitterReceptors in the CNS, Part II, Elsevier, Amsterdam, 1984, pp. 68-125. 31 Verg6, D., Daval, G., Marcinkiewicz, M., Patey, A., El Mestikawy, S., Gozlan, H. and Hamon, M., Quantitative autoradiography of multiple 5-HT 1 receptor subtypes in the brain of control or 5,7-dihydroxy-tryptamine-treated rats, J. Neurosci., 6 (1986) 3474-3482. 32 Waeber, C., Dietl, M.M., Hoyer, D., Probst, A. and Palacios, J.M., Visualization of a novel serotonin recognition site (5HT~D) in the human brain by autoradiography, Neurosci. Lett., 88 (1988) 11-16. 33 Waeber, C. and Palacios, J.M., Serotonin-1 receptor binding sites in the human basal ganglia are decreased in Huntington's chorea but not in Parkinson's disease: a quantitative in vitro autoradiography study, Neuroscience, 340 (1989) 486-494. 34 Waeber, C., Schoeffter, P., Palacios, J.M. and Hoyer, D., 5-HTID receptors in the guinea pig and pigeon brain: radioligand binding and biochemical studies, Naunyn-Schmiedeberg's Arch. Pharmacol., 340 (1989) 479-485. 35 Waeber, C., Dietl, M.M., Hoyer, D. and Palacios, J.M., 5-HT 1 receptors in the vertebrate brain: regional distribution examined by autoradiography, Naunyn-Schmiedeberg's Arch. Pharmacol., 32 (1989) 337-347. 36 Waeber, C., Zhang, L. and Palacios, J.M., 5-HTID receptors in the guinea pig brain: pre- and postsynaptic localizations in the striato-nigral pathway, 528 (1990) 197-206. 37 Weissmann, D., Mach, E., Oberlander, C., Demassey, Y. and Pujol, J.F., Evidence for byperdensity of 5-HT m binding sites in the substantia nigra of the rat after 5,7-dihydroxytryptamine intraventricular injection, Neurochem. Int., 9 (1986) 191-200.