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Radioautographic study of serotoninergic axon terminals in the rat trigeminal motor nucleus
Neuroscience Letters, 44 (1984) 31-36 Elsevier Scientific Publishers Ireland Ltd.
31
NSL 02541
RADIOAUTOGRAPIIlC STUDY OF SEROTONINERGIC AXON TERMINALS IN TIlE RAT TRIGEMINAL MOTOR NUCLEUS
N. S C H A F F A R 1, A. J E A N l'* and A. C A L A S 2
~Ddpartement de Physiologie et Neurophysiologie, Facult~ des Sciences et Techniques St JOr6me, 13397 Marseille Cedex 13 and 2Laboratoire de Physiologie des Interactions cellulaires, UniversitO de Bordeaux I, 33405 Talence Cedex (France) (Received June 30th, 1983; Revised version received and accepted November 14th, 1983)
Key words: trigeminal motor nucleus - serotoninergic terminals - radioautography - light and electron microscopy - rat
Serotonin (5-HT) axon terminals were demonstrated in the rat trigeminal motor nucleus (VmN) using radioautography following local injection of tritiated 5-HT. The light microscope radioautographs of the V m N showed silver grain aggregates typical of axonal varicosities having taken up the exogenous 5-HT. In electron microscope radioautographs, labeled 5-HT axon terminals took the form of unmyelinated enlargements, 0.3-2 t~m in diameter (average 0.9/zm) containing small clear and large granular vesicles. Out of 182 labeled 5-HT profiles, only 4.9°70 showed a clear synaptic junction with dendritic branches, indicating the existence of non-synaptic as well as synaptic mechanisms for the 5-HT terminals within the VmN. An estimate of the density of 5-HT varicosities within the VmN was performed on histological radioautographs: the concentration of labeled terminals was evaluated at (1.8_+ 0.38) × 106 per m m 3.
The trigeminal motor nucleus (VmN), which is involved in several oral functions such as mastication and swallowing [13, 16], contains serotoninergic (5-HT) axon terminals which were first demonstrated by fluorescence histochemistry [11]. Biochemical measurements of endogenous 5-HT content [17] and a study using the immunofluorescence technique [20] have confirmed these initial results. However, a study of 5-HT terminals in the VmN at the electron microscope level is lacking, thus the anatomical organization and the morphofunctional features of these terminals remain unknown. Radioautography based on the uptake of radiolabeled monoamines is a suitable tool to enable the study of the fine structure and cellular interrelationships of monoaminergic terminals [4, 6, 9]. The aim of the present study was to achieve specific identification of 5-HT terminals in the VmN, to examine their ultrastructural features and intercellular relationships, and to estimate the density of the 5-HT innervation within the VmN. Parts of the results have been communicated earlier in abstract form [19]. *Author for correspondence.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
32 Experiments were performed on 5 Wistar rats (200-300 g). One hour before the injection o f the tracer (tritiated 5-HT, [3HI5-HT, spec. act. 10.8-17.5 C i / m m o [ , A m e r s h a m , U.K,) the animals were pretreated with a m o n o a m i n e oxidase inhibitor (pargyline, 75 m g / k g i.p.). The animals were then anesthetized (Nembutal, 50 m g / k g i.p.) and attached to a stereotaxic device. A solution (2-2.5 ~1) containing the tracer, [3H]5-HT, at 6 . 1 0 ~ M with non-radioactive noradrenaline (NA, 6 . 10- 4 M) and ascorbic acid (0.5 m g / m l ) was injected stereotaxically in the VmN, over 20-30 min, through either a metallic cannula (o.d. 300 /xm) ot- a glass micropipette (o.d. 80 p.m) connected to a H a m i l t o n microsyringe. Half an hour after the injection the brains were then fixed by aortic perfusion of 3.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The r h o m b e n c e p h a l o n o f one rat was processed for paraffin embedding. For the others, 1 mm-thick frontal slices were removed at the VmN level, postfixed for 2 h in 2% osmium tetroxide, dehydrated in ethanol and embedded in E p o n 812. Paraffin (7 #m), E p o n semi-thin (1 #m) and ultra-thin (70-80 nm) frontal sections were appropriately r a d i o a u t o g r a p h e d according to k a r r a and Droz [14] by dipping in llford K5 or L4 nuclear emulsions. R a d i o a u t o g r a p h s were developed with D19 B Kodak (paraffin, semi-thin sections) after 21 days o f exposure or with Microdol X Kodak or paraphenylene diamine
Fig. 1. Light microscope radioautographs of histological sections in the VmN after local [3HI5-HT injection. A: paraffin section (7 am thickness) stained with cresyl violet after a 30-day exposure. B: Epon semi-thin section (1 ~m thickness) after a 21-day exposure. Note the silver grain aggregates typical of axonal varicosities. Scale bars- 50 ~m.
33 ( u l t r a - t h i n sections) after 30-60 d a y s o f exposure, a n d e x a m i n e d u n d e r light or electron microscope. Light microscope radioautography. T w o types o f r a d i o a u t o g r a p h i c reactions c o u l d be distinguished within the V m N p a r a f f i n or E p o n semi-thin section r a d i o a u t o g r a p h s (Fig. 1A, B): a diffuse r e a c t i o n consisting o f scattered silver grains, r a n d o m l y d i s t r i b u t e d over the w h o l e tissue; and an intense r e a c t i o n which t o o k the f o r m o f silver grain aggregates, d i s t r i b u t e d u p o n the whole V m N a n d significantly p r o m i n e n t on the nucleus as c o m p a r e d to the s u r r o u n d i n g structures. Electron microscope radioautography. At the electron m i c r o s c o p e level the diffuse r e a c t i o n t o o k the f o r m o f scattered silver grains d i s t r i b u t e d over all the cellular c o m p o n e n t s o f the tissue. The intense reactions resulted in dense a c c u m u l a t i o n s o f
Fig. 2. Electron microscope radioautographs from ultrathin sections after local [3HI5-HT injection within the VmN. A, B and C: labeled 5-HT varicosities containing both small clear and large granular vesicles. In A, the 5-HT-labeled varicosities can be seen in close contact with a dendritic profile, while in C the labeled bouton displays a well differentiated axo-dendritic synapse. Scale bars = 1 /zm.
34 silver grains which were superimposed on unmyelinated enlargements corresponding to axonal varicosities having taken up and retained the tracer (Fig. 2). The profiles of labeled axonal varicosities measured 0.3-2/~m, averaging 0.9/zm in diameter. The reactive boutons contained some common features consisting of mitochondria and a heterogeneous population of vesicles: mainly small agranular synaptic vesicles (30-40 nm in diameter) with a round or flattened shape and large granular vesicles (50-120 nm in diameter), round or oval in shape. A small number of labeled axonal profiles showed a clear synaptic specialization of their surface membrane. Indeed, on a sample of 182 labeled profiles, only 9 showed a clear synaptic junction (4.9o70), whereas on 351 unlabeled axonal profiles 127 clear synaptic junctions (36.2O7o) could be distinguished. The labeled synaptic nerve endings observed were exclusively in contact with dendritic branches (Fig. 2C) and most seemed to be of the asymmetrical variety. It may be noted that several labeled varicosities were located very close to the cell bodies of trigeminal neurons, without exhibiting a clear synaptic junction as far as could be observed. Quantitative analysis. To estimate the density of 5-HT varicosities in the VmN a quantitative analysis was performed on the radioautographs of 8 non-serial semithin sections f r o m the same experiment, using an O p t o m a x (Analytical Instruments) [12]. This digitalized video system, according to a procedure previously described by Segu and Calas [18], allows measurement of the density of varicosities in a given structure. In our experimental conditions, the density of varicosities per unit area (Na) in the VmN was evaluated at: N a = 3121 +_437 per m m 2. It is thus possible to estimate the concentration of labeled terminals in a given volume of V m N by using the equation (Na)/(D + t - 2 h) [10] (where D is the mean diameter of varicosities 0.9 gm; t, the thickness of the sections 1 ~m; h, the lost caps height evaluated as 0.1 ~m). Thus, the estimated concentration of 5-HT terminals in the VmN was (1.8 +- 0.38) × 106 varicosities per m m 3. The specificity of the radioautographic method under our experimental conditions has by now been well established [1, 4, 6, 9]. The pretreatment of the animals with an inhibitor of monoamine oxidase and the injection of the tracer, [3H]5-HT, at a concentration of 6- 10- 5 M to which is added a 10-fold higher concentration of non-radioactive noradrenaline, are consistent with a selective identification of 5-HT axon terminals [8, 9, 15]. Thus, our results confirm the existence of 5-HT terminals in the VmN as previously demonstrated by fluorescence histochemistry and immunofluorescence [11, 20]. Concerning the density of the labeled varicosities in the VmN, the evaluated number might depend on the functional state of the terminals, and also on the concentration of the tracer and the duration of radioautographic exposure [21. Under our experimental conditions, it can be assumed that most of the 5-HT terminals in the VmN were labeled. However, in view of our time of exposure (21 days), our evaluation may be slightly underestimated; thus the density of 5-HT varicosities in the VmN should be said to be at least 1.8 × 10 6 per m m 3 [2]. Our results indicate
35 a rather high density of 5-HT terminals within the VmN. They are in good agreement with the histochemical results reported by Fuxe [11] and Steinbusch [20] and also with the biochemical measurement o f endogenous 5-HT in the VmN [17]. In the latter study, the 5-HT concentration in the VmN was evaluated at 9.5 n g / m g protein. Thus, according to our measurement (1.8 × 10 6 varicosities per mm3), and taking into account the estimated value of 0.1 mg protein/ram 3, the mean 5-HT content per varicosity can be estimated at 0.53 • 10-3 pg. It can be noted that values of the same order of magnitude were evaluated in the rat frontoparietal cortex (0.35.10 -3 pg) [2] and in the cat spinal cord ( 1 . 8 . 1 0 -3 pg) [18]. Considering a varicosity as a sphere with an average diameter o f 0.9 #m, the mean 5-HT concentration within a varicosity could be evaluated at 0 . 7 5 . 1 0 - 2 M. Our results, moreover, indicate that only a low proportion of labeled profiles within the VmN exhibited clear morphologically defined synaptic junctions, as also demonstrated in various regions of the central nervous system [4, 9]. It is clear that the proportion evaluated (4.9°-/0) from observation of the labeled profiles does not indicate the true percentage of 5-HT synaptic junctions in the VmN, a tridimensional reconstruction from serial thin sections would be required [7, 9]. However, a formula developed by Beaudet and Sotelo [5] allows us to estimate the probability of seeing a synaptic junction. In our experimental conditions this probability can be estimated at 37°7o ( P = ( d / D ) - ( 2 / T r ) + c o / D ; where D is the varicosities mean diameter 0.9 #m; d is the active zone mean diameter 0.4 #m; and c0 is the section thickness 0.08 #m); thus the percentage of 5-HT synaptic junctions within the VmN would be 13-14°70. Nevertheless, our results suggest that non-synaptic as well as synaptic mechanisms may be involved in the 5-HT terminals within the VmN. At this level serotonin might be considered to be a true neurotransmitter as well as a neuromodulator, as has been postulated for other regions of the central nervous sytem [3, 4]. We thank L. Segu for his help with the quantitative analysis, and Mrs. E. Dussauze, A.M. Lajard and Y. Minguella for technical and secretarial help. Supported in part by CNRS LA 205 (N.S., A.J.), INSERM (C.R.E. 836016, N.S., A.J.), FRMI (A.C.) and MRI (A.C., Grant 82 J. 1430).
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 Beaudet, A. and Descarries, L., Quantitative data on serotonin nerve terminals in adult rat neocortex, Brain Res., 111 (1976) 301-309. 3 Beaudet, A. and Descarries, L., The monoamine innervation of the rat cerebral cortex: synaptic and nonsynaptic relationship, Neuroscience, 3 (1978) 851-860. 4 Beaudet, A. and Descarries, L., The fine structure of central serotonin neurons, J. Physiol. (Paris), 77 (1981) 193-203. 5 Beaudet, A. and Sotelo, C., Synaptic remodeling of serotonin axon terminals in rat agranular cerebellum, Brain Res., 206 (1981) 305-329.
36 6 Bosler, O. and Calas, A., Radioautographic investigation of monoaminergic neurons: an evaluation, Brain Res. Bull., 9 (1982) 151-169. 7 Chan-Palay, V., The paratrigeminal nucleus. II. Identification and inter-relations of catecholamine axons, indoleamine axons and substance P immunoreactive cells in the neuropil, J. Neurocytol., 7 (1978) 419-442. 8 Descarries, L. and Droz, B., Interneural distribution of exogenous norepinephrine in the central nervous system of the rat, J. Cell. Biol., 44 (1970) 385-399. 9 Descarries, L., Beaudet, A. and Watkins, K.C., Serotonin nerve terminals in adult rat neocortex, Brain Res., 100 (1975) 563-588. 10 Elias, H., Hennig, A. and Schwartz, D.E., Stereology: applications to biomedical research, Physiol. Rev., 51 (1971) 158-200. 11 Fuxe, K., Evidence for the existence of m o n o a m i n e neurons in the central nervous system, ll. Distribution of m o n o a m i n e nerve terminals in the central nervous system, Acta physiol, scand., 64, Suppl. 247 (1965), 37-85. 12 Hopkins, B.M., A quantitative image analysis system, Opt. Engin., 15 (1976) 236-240. 13 Jean, A., Amri, M. and Calas, A., Connections between the ventral medullary swallowing area and the trigeminal motor nucleus of the sheep studied by tracing techniques, J. Auton. Nerv. Syst., 7 (1983) 87-96. 14 Larra, F. and Droz, B., Techniques radioautographiques et leur application a l'6tude du renouvellement des constituants cellulaires, J. Microsc., 9 (1970) 845-880. 15 Leger, L., Mouren-Mathieu, A.M. and Descarries, L., Identification radioautographique de neurones monoaminergiques centraux par micro-instillation locale de s6rotonine ou de noradrenaline triti6e chez le chat, C.R. Acad. Sci. (Paris), 286 (1978) 1523-1526. 16 Miller, A.J., Deglutition, Physiol. Rev., 62 (1982) 129-184. 17 Palkovits, M., Brownstein, M. and Saavedra, J.M., Serotonin content of the brain stem nuclei in the rat, Brain Res., 80 (1974) 237-249. 18 Segu, L. and Calas, A., The topographical distribution of serotoninergic terminals in the spinal cord of the cat: quantitative radioautographic studies, Brain Res., 153 (1978) 449-464. 19 Schaffar, N., Jean, A. and Calas, A., Identification of serotoninergic axon terminals in the rat trigeminal motor nucleus. A radioautographic study, Neurosci. Lett., Suppl. I0 (1982) $433. 20 Steinbusch, H . W . M . , Distribution of serotonin immunoreactivity in the central nervous system of the rat - cell bodies and terminals, Neuroscience, 6 (1981) 557-618.
31
NSL 02541
RADIOAUTOGRAPIIlC STUDY OF SEROTONINERGIC AXON TERMINALS IN TIlE RAT TRIGEMINAL MOTOR NUCLEUS
N. S C H A F F A R 1, A. J E A N l'* and A. C A L A S 2
~Ddpartement de Physiologie et Neurophysiologie, Facult~ des Sciences et Techniques St JOr6me, 13397 Marseille Cedex 13 and 2Laboratoire de Physiologie des Interactions cellulaires, UniversitO de Bordeaux I, 33405 Talence Cedex (France) (Received June 30th, 1983; Revised version received and accepted November 14th, 1983)
Key words: trigeminal motor nucleus - serotoninergic terminals - radioautography - light and electron microscopy - rat
Serotonin (5-HT) axon terminals were demonstrated in the rat trigeminal motor nucleus (VmN) using radioautography following local injection of tritiated 5-HT. The light microscope radioautographs of the V m N showed silver grain aggregates typical of axonal varicosities having taken up the exogenous 5-HT. In electron microscope radioautographs, labeled 5-HT axon terminals took the form of unmyelinated enlargements, 0.3-2 t~m in diameter (average 0.9/zm) containing small clear and large granular vesicles. Out of 182 labeled 5-HT profiles, only 4.9°70 showed a clear synaptic junction with dendritic branches, indicating the existence of non-synaptic as well as synaptic mechanisms for the 5-HT terminals within the VmN. An estimate of the density of 5-HT varicosities within the VmN was performed on histological radioautographs: the concentration of labeled terminals was evaluated at (1.8_+ 0.38) × 106 per m m 3.
The trigeminal motor nucleus (VmN), which is involved in several oral functions such as mastication and swallowing [13, 16], contains serotoninergic (5-HT) axon terminals which were first demonstrated by fluorescence histochemistry [11]. Biochemical measurements of endogenous 5-HT content [17] and a study using the immunofluorescence technique [20] have confirmed these initial results. However, a study of 5-HT terminals in the VmN at the electron microscope level is lacking, thus the anatomical organization and the morphofunctional features of these terminals remain unknown. Radioautography based on the uptake of radiolabeled monoamines is a suitable tool to enable the study of the fine structure and cellular interrelationships of monoaminergic terminals [4, 6, 9]. The aim of the present study was to achieve specific identification of 5-HT terminals in the VmN, to examine their ultrastructural features and intercellular relationships, and to estimate the density of the 5-HT innervation within the VmN. Parts of the results have been communicated earlier in abstract form [19]. *Author for correspondence.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
32 Experiments were performed on 5 Wistar rats (200-300 g). One hour before the injection o f the tracer (tritiated 5-HT, [3HI5-HT, spec. act. 10.8-17.5 C i / m m o [ , A m e r s h a m , U.K,) the animals were pretreated with a m o n o a m i n e oxidase inhibitor (pargyline, 75 m g / k g i.p.). The animals were then anesthetized (Nembutal, 50 m g / k g i.p.) and attached to a stereotaxic device. A solution (2-2.5 ~1) containing the tracer, [3H]5-HT, at 6 . 1 0 ~ M with non-radioactive noradrenaline (NA, 6 . 10- 4 M) and ascorbic acid (0.5 m g / m l ) was injected stereotaxically in the VmN, over 20-30 min, through either a metallic cannula (o.d. 300 /xm) ot- a glass micropipette (o.d. 80 p.m) connected to a H a m i l t o n microsyringe. Half an hour after the injection the brains were then fixed by aortic perfusion of 3.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The r h o m b e n c e p h a l o n o f one rat was processed for paraffin embedding. For the others, 1 mm-thick frontal slices were removed at the VmN level, postfixed for 2 h in 2% osmium tetroxide, dehydrated in ethanol and embedded in E p o n 812. Paraffin (7 #m), E p o n semi-thin (1 #m) and ultra-thin (70-80 nm) frontal sections were appropriately r a d i o a u t o g r a p h e d according to k a r r a and Droz [14] by dipping in llford K5 or L4 nuclear emulsions. R a d i o a u t o g r a p h s were developed with D19 B Kodak (paraffin, semi-thin sections) after 21 days o f exposure or with Microdol X Kodak or paraphenylene diamine
Fig. 1. Light microscope radioautographs of histological sections in the VmN after local [3HI5-HT injection. A: paraffin section (7 am thickness) stained with cresyl violet after a 30-day exposure. B: Epon semi-thin section (1 ~m thickness) after a 21-day exposure. Note the silver grain aggregates typical of axonal varicosities. Scale bars- 50 ~m.
33 ( u l t r a - t h i n sections) after 30-60 d a y s o f exposure, a n d e x a m i n e d u n d e r light or electron microscope. Light microscope radioautography. T w o types o f r a d i o a u t o g r a p h i c reactions c o u l d be distinguished within the V m N p a r a f f i n or E p o n semi-thin section r a d i o a u t o g r a p h s (Fig. 1A, B): a diffuse r e a c t i o n consisting o f scattered silver grains, r a n d o m l y d i s t r i b u t e d over the w h o l e tissue; and an intense r e a c t i o n which t o o k the f o r m o f silver grain aggregates, d i s t r i b u t e d u p o n the whole V m N a n d significantly p r o m i n e n t on the nucleus as c o m p a r e d to the s u r r o u n d i n g structures. Electron microscope radioautography. At the electron m i c r o s c o p e level the diffuse r e a c t i o n t o o k the f o r m o f scattered silver grains d i s t r i b u t e d over all the cellular c o m p o n e n t s o f the tissue. The intense reactions resulted in dense a c c u m u l a t i o n s o f
Fig. 2. Electron microscope radioautographs from ultrathin sections after local [3HI5-HT injection within the VmN. A, B and C: labeled 5-HT varicosities containing both small clear and large granular vesicles. In A, the 5-HT-labeled varicosities can be seen in close contact with a dendritic profile, while in C the labeled bouton displays a well differentiated axo-dendritic synapse. Scale bars = 1 /zm.
34 silver grains which were superimposed on unmyelinated enlargements corresponding to axonal varicosities having taken up and retained the tracer (Fig. 2). The profiles of labeled axonal varicosities measured 0.3-2/~m, averaging 0.9/zm in diameter. The reactive boutons contained some common features consisting of mitochondria and a heterogeneous population of vesicles: mainly small agranular synaptic vesicles (30-40 nm in diameter) with a round or flattened shape and large granular vesicles (50-120 nm in diameter), round or oval in shape. A small number of labeled axonal profiles showed a clear synaptic specialization of their surface membrane. Indeed, on a sample of 182 labeled profiles, only 9 showed a clear synaptic junction (4.9o70), whereas on 351 unlabeled axonal profiles 127 clear synaptic junctions (36.2O7o) could be distinguished. The labeled synaptic nerve endings observed were exclusively in contact with dendritic branches (Fig. 2C) and most seemed to be of the asymmetrical variety. It may be noted that several labeled varicosities were located very close to the cell bodies of trigeminal neurons, without exhibiting a clear synaptic junction as far as could be observed. Quantitative analysis. To estimate the density of 5-HT varicosities in the VmN a quantitative analysis was performed on the radioautographs of 8 non-serial semithin sections f r o m the same experiment, using an O p t o m a x (Analytical Instruments) [12]. This digitalized video system, according to a procedure previously described by Segu and Calas [18], allows measurement of the density of varicosities in a given structure. In our experimental conditions, the density of varicosities per unit area (Na) in the VmN was evaluated at: N a = 3121 +_437 per m m 2. It is thus possible to estimate the concentration of labeled terminals in a given volume of V m N by using the equation (Na)/(D + t - 2 h) [10] (where D is the mean diameter of varicosities 0.9 gm; t, the thickness of the sections 1 ~m; h, the lost caps height evaluated as 0.1 ~m). Thus, the estimated concentration of 5-HT terminals in the VmN was (1.8 +- 0.38) × 106 varicosities per m m 3. The specificity of the radioautographic method under our experimental conditions has by now been well established [1, 4, 6, 9]. The pretreatment of the animals with an inhibitor of monoamine oxidase and the injection of the tracer, [3H]5-HT, at a concentration of 6- 10- 5 M to which is added a 10-fold higher concentration of non-radioactive noradrenaline, are consistent with a selective identification of 5-HT axon terminals [8, 9, 15]. Thus, our results confirm the existence of 5-HT terminals in the VmN as previously demonstrated by fluorescence histochemistry and immunofluorescence [11, 20]. Concerning the density of the labeled varicosities in the VmN, the evaluated number might depend on the functional state of the terminals, and also on the concentration of the tracer and the duration of radioautographic exposure [21. Under our experimental conditions, it can be assumed that most of the 5-HT terminals in the VmN were labeled. However, in view of our time of exposure (21 days), our evaluation may be slightly underestimated; thus the density of 5-HT varicosities in the VmN should be said to be at least 1.8 × 10 6 per m m 3 [2]. Our results indicate
35 a rather high density of 5-HT terminals within the VmN. They are in good agreement with the histochemical results reported by Fuxe [11] and Steinbusch [20] and also with the biochemical measurement o f endogenous 5-HT in the VmN [17]. In the latter study, the 5-HT concentration in the VmN was evaluated at 9.5 n g / m g protein. Thus, according to our measurement (1.8 × 10 6 varicosities per mm3), and taking into account the estimated value of 0.1 mg protein/ram 3, the mean 5-HT content per varicosity can be estimated at 0.53 • 10-3 pg. It can be noted that values of the same order of magnitude were evaluated in the rat frontoparietal cortex (0.35.10 -3 pg) [2] and in the cat spinal cord ( 1 . 8 . 1 0 -3 pg) [18]. Considering a varicosity as a sphere with an average diameter o f 0.9 #m, the mean 5-HT concentration within a varicosity could be evaluated at 0 . 7 5 . 1 0 - 2 M. Our results, moreover, indicate that only a low proportion of labeled profiles within the VmN exhibited clear morphologically defined synaptic junctions, as also demonstrated in various regions of the central nervous system [4, 9]. It is clear that the proportion evaluated (4.9°-/0) from observation of the labeled profiles does not indicate the true percentage of 5-HT synaptic junctions in the VmN, a tridimensional reconstruction from serial thin sections would be required [7, 9]. However, a formula developed by Beaudet and Sotelo [5] allows us to estimate the probability of seeing a synaptic junction. In our experimental conditions this probability can be estimated at 37°7o ( P = ( d / D ) - ( 2 / T r ) + c o / D ; where D is the varicosities mean diameter 0.9 #m; d is the active zone mean diameter 0.4 #m; and c0 is the section thickness 0.08 #m); thus the percentage of 5-HT synaptic junctions within the VmN would be 13-14°70. Nevertheless, our results suggest that non-synaptic as well as synaptic mechanisms may be involved in the 5-HT terminals within the VmN. At this level serotonin might be considered to be a true neurotransmitter as well as a neuromodulator, as has been postulated for other regions of the central nervous sytem [3, 4]. We thank L. Segu for his help with the quantitative analysis, and Mrs. E. Dussauze, A.M. Lajard and Y. Minguella for technical and secretarial help. Supported in part by CNRS LA 205 (N.S., A.J.), INSERM (C.R.E. 836016, N.S., A.J.), FRMI (A.C.) and MRI (A.C., Grant 82 J. 1430).
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 Beaudet, A. and Descarries, L., Quantitative data on serotonin nerve terminals in adult rat neocortex, Brain Res., 111 (1976) 301-309. 3 Beaudet, A. and Descarries, L., The monoamine innervation of the rat cerebral cortex: synaptic and nonsynaptic relationship, Neuroscience, 3 (1978) 851-860. 4 Beaudet, A. and Descarries, L., The fine structure of central serotonin neurons, J. Physiol. (Paris), 77 (1981) 193-203. 5 Beaudet, A. and Sotelo, C., Synaptic remodeling of serotonin axon terminals in rat agranular cerebellum, Brain Res., 206 (1981) 305-329.
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