Serotonin-containing projections to the mesencephalic trigeminal nucleus of the cat.код для вставкиСкачать
THE ANATOMICAL RECORD 241:136-142 (1995) Serotonin-Contai ning Projections to the M esenceph alic TrigeminaI Nucleus of the Cat NIKOLAI E. LAZAROV AND CHRIST0 N. CHOUCHKOV Department of Anatomy, Histology, and Embryology, Medical University, Stara Zagora, Bulgaria ABSTRACT Background: It is well known that the mesencephalic trigeminal nucleus (MTN) neurons transmit somatosensory information from proprioceptors in the oral-facial region. Several mechanisms of sensory transduction in these specialized receptors have been proposed, but the neurotransmitters which are responsible for mediating proprioceptive information are still unknown. The current study concentrates on the distribution of one putative neurotransmitter system, serotonin (SER),in the cat MTN. A second objective was to clarify the location and sources of serotoninergic projections on the MTN neurons. Methods: To determine whether SER was localized in the MTN, the peroxidase-antiperoxidase (PAP) immunocytochemical technique was applied at light and electron microscopic levels in colchicine-treated animals. The origin of SER-containing fibers in the MTN was studied using a doublelabeling method combining retrograde transport with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP)and SER immunocytochemistry. Results: There were no SER-containing neurons in the MTN. The cell bodies of immunonegative MTN neurons were closely surrounded by fine SER-positive fibers and terminals. The labeled fibers were in most cases very thin and sometimes varicose. Ultrastructurally, direct synaptic contacts between SER-containing terminals and perikarya of MTN neurons of all sizes could be seen. The majority of SER-labeled structures were synaptic terminals in which the immunoreactive material was located within the small round clear as well as the small granular vesicles (diameter 50-80 nm) and a few large dense-cored vesicles (up to 150 nm). Retrograde tracing demonstrated that most of cells in the nuclei raphe dorsalis, pontis and magnus were WGA-HRP-labeled. Conclusions: These results indicated that MTN neurons received serotoninergic projections from the raphe nuclei of the brainstem. In light of these morphological data, it is concluded that the MTN of the cat is under the influence of SER-containing axons and this serotoninergic input may modulate MTN neuronal activity at the first synaptic relay. 0 1995 Wiley-Liss, Inc. Key words: Mesencephalic trigeminal nucleus, Serotonin, WGA-HRP, Retrograde tracing, Immunocytochemistry, Cat The mesencephalic trigeminal nucleus (MTN) is a unique sector of the central nervous system. The cell bodies of primary sensory neurons composing this IIUcleus are situated within the rostra1 pons and all rostro-caudal levels of the entire midbrain. A characteristic peculiarity of the MTN in the cat is that about 40% of mesencephalic neurons are multipolar and only 60% of the population of first-order sensory neurons are (pseudo)unipolar (Walberg, 1984; Nomura et al., 1985). The peripheral processes of MTN neurons, running in the trigeminal nerve, reach the spindles of the masticatory muscles and periodontal mechanoreceptors (Shigenaga e t al., 1988a,b). c 1995 WIIXY-LISS, INC Therefore, morphologically and functionally MTN neurons are considered to be very similar or identical to the cranio-spinal ganglion cells. In contrast to sensory ganglia, however, where no synaptic contacts on somata and processes of primary sensory neurons have been detected, axosomatic and axodendritic synapses Received December 29, 1993; accepted July 8, 1994. Address reprint requests to Christo N. Chouchkov, Department of Anatomy, Histology, and Embryology, Medical University, 11 Armejska Street, 6003 Stara Zagora, Bulgaria. SEROTONIN IN MESENCEPHALIC TRIGEMINAL NUCLEUS 137 have been found in the MTN (Nomura et al., 1985). into the fourth ventricle. These animals were allowed Recently, Liem et al. (19921, examining the distribu- to survive for 24 h. A fifth cat was used a s control tion of synaptic boutons in the MTN of the rat, sug- (without colchicine). Under pentobarbital anesthesia gested that these synaptic boutons contained peptider- (30-40 mg/kg, i.p.1, the animals were fixed by perfugic, dopaminergic, or serotoninergic vesicles. The sion through the ascending aorta with 600 ml of hepexistence of a synaptic input to the primary sensory arinized phosphate buffered saline (PBS), immediately ganglion cells would suggest a n integrative function followed by 3 I of fixative-4% paraformaldehyde in 0.1 for the MTN to proprioceptive information carried by M phosphate buffer (PB) at pH 7.4 for light microscopy primary trigeminal afferents. (LM) and a fixative mixture containing 4% paraformalSeveral studies have dealt with the chemical content dehyde, 0.08% glutaraldehyde, and 0.15% picric acid in of MTN neurons of the rat, and some neurotransmit- 0.1 M PB, pH 7.4, for electron microscopy (EM). The ters and neuropeptides have recently been suggested brainstems were separated from the brains and placed (Copray et al., 1990a,b, 1991; Ginestal and Matute, for 4-24 h in the same fixative (without glutaralde1993). Information about the serotoninergic innerva- hyde for EM). They were then washed in a series of cold tion of MTN neurons is available for the rabbit a s well sucrose solutions overnight. Serial frontal sections (Kolta et al., 1993).Unfortunately, studies on the neu- (20-40 pm) were cut on a Vibratome (Lancer Series rotransmitter distribution in the MTN of the cat are 1000) and processed for LM and EM immunocytochemlacking, and the neurotransmitters which are respon- ical staining, respectively, using the conventional persible for mediating proprioceptive information are still oxidase-antiperoxidase (PAP) technique (Sternberger, unknown. There is only a light microscopic study 1986). (Tashiro et al., 1989) which has demonstrated that The sections were incubated with a 1:1,000 dilution perikarya of MTN neurons of the cat are often in direct of rabbit primary antiserum to serotonin, purchased contact with axonal varicosities showing enkephalin-, from INCSTAR (Stillwater, Mn; lot no. 8832021), for 24 substance P-, or serotonin-like immunoreactivity. h a t room temperature and 24 h at 4°C. As a second However, the source of these fibers and the physiolog- antibody a n anti-rabbit IgG (1:50) and in the third ical significance of these findings for the functioning of step of the staining a rabbit PAP (1:200) (both from MTN neurons has remained unclear. As the available Dakopatts, Glostrup, Denmark) were applied. The perdata on the distribution of neurotransmitter candi- oxidase activity was demonstrated by incubating the dates in the cat MTN are scanty, we have started to sections in 0.4 mg/ml 3,3'-diaminobenzidine tetrahyelucidate the potential neurotransmitters and the drochloride (DAB) and 0.01% H,O, in 0.05 M Tris-HC1 source of synaptic connections. buffer, pH 7.54, for 10 min. Between the separate steps, In the present study, we have described the origin of the sections were rinsed with cold PBS t 0.3% Triton the serotonin (SER)-containing axons in the MTN of X-100. Some of them were mounted on gelatin-coated the cat. For this purpose, we used a very sensitive ret- glass slides, dried, dehydrated, coverslipped with Enrograde tracing technique (that can be combined with tellan, and examined in a Zeiss research microscope. SER immunocytochemistry) with horseradish peroxiFor ultrastructural analysis, after the DAB incubadase a s a retrograde tracer in colchicine-treated ani- tion, some sections were postfixed for 30 min in 0.5% mals. A preliminary report has been published in ab- OsO, buffered with 0.1 M PB, dehydrated through a stract form (Lazarov and Chouchkov, 1993). graded ethanol series, and f lat-embedded in Durcupan ACM (Fluka). Ultrathin sections were cut on a MATERIALS AND METHODS Reichert-Jung ultratome. Lightly counterstained with Eight adult cats of either sex weighing 2-3 kg pro- Uranyl acetate and lead citrate, they were viewed with vided data. Five of them were treated for SER immu- an OPTON EM logelectron microscoPe. noreactivity only, and the other three were treated for Retrograde Labeling both immunoreactivity and retrograde tracing a t light Wheat germ agglutinin conjugated to horseradish and electron microscopic levels. peroxidase (WGA-HRP) (lot no. 0390; Sigma. St. Louis, MO) was used as a retrograde tracer to-determine the lrnrnunocytochernical Studies Four cats received 1,000 pl colchicine (Merck, Darm- origin of the serotoninergic axons in the MTN. Three cats were anesthetized with intraperitoneal sostadt, FRG) in 200 p1 saline injected stereotaxically adult dium pentobarbital (40 mg/kg b.wt.1, placed in a stereotaxic apparatus (Kopf Instruments Co., Tujunga, CA, USA), and given a single microinjection of the tracer (2 pl of 2.5% WGA-HRP, dissolved in 0.9% NaC1) into the MTN. The injection was made by iontophoresis A bbreuiations over a period of 30 min through a glass micropipette Aq cerebral aqueduct (tip diameter 10-20 pm) which was attached to a 2 pl CG central gray DPB dorsal parabrachial nucleus Hamilton microsyringe. Two days later, all the aniDR dorsal raphe nucleus mals were deeply re-anesthetized and perfused tranLC locus coeruleus scardially with 3 1 of a fixative composed of 2.5% gluMnR median raphe nucleus taraldehyde and 0.5% paraformaldehyde, containing MTN mesencephalic trigeminal nucleus PAG periaqueductal gray 4% sucrose in 0.05 M PB, pH 7.4. After the perfusion, RPn raphe pontis nucleus the brains were immediately removed, saturated with SCP superior cerebellar peduncle 30% sucrose in the same buffer a t 4"C, and then cut VPB ventral parabrachial nucleus serially into frontal sections of 40 pm thickness on a 4 v fourth ventricle 138 N.E. LAZAROV AND C.N. CHOUCHKOV freezing microtome. The sections were stained for the histochemical demonstration of WGA-HRP according to the tetramethylbenzidine (TMB) method of Mesulam (1978). To demonstrate the serotoninergic nature of WGA-HRP-labeled neurons in the brainstem raphe nuclei and the percentage frequency of immunoreactive cells giving rise to serotoninergic input to the cat MTN, the sections were treated for immunocytochemistry as described above, and those projecting to the MTN were identified by a retrograde tracer transport. Double-labeled neurons which contained SER immunoreactivity and WGA-HRP reactive granules were identified by the presence of both black and brown reaction product within their cytoplasm (cf. Fig. 8A-C). WGA-HRP was demonstrated, resulting in a black granular intracellular reaction product. On the other hand, SER immunoreactivity was visualized by a light brown cytoplasm staining. Control sections were processed in the same way, but the specific antibody was omitted from the incubation medium. As other controls, primary antiserum was replaced with normal (nonimmune) rabbit serum. In addition, the specificity of the immunostaining was checked by preabsorbing serotonin antiserum a t working dilution with a n excess (200 km/ml) of synthetic SER overnight at 4°C. RESULTS sented a small number, approximately 5 t 1%,of the total retrogradely labeled cell population. Incubation of control sections without primary or secondary antisera resulted in elimination of the immunocytochemical staining, a s in sections incubated with normal rabbit serum or antiserum preabsorbed with synthetic antiserum. DISCUSSION This paper has described for the first time in detail the serotoninergic innervation of primary sensory neurons in the cat MTN. In addition, we have demonstrated that serotoninergic terminals form synaptic contacts with cell bodies of MTN neurons. This observation is in agreement with the studies carried out on the distribution of SER-immunoreactive terminals within the rat mesencephalic trigeminal nucleus (Liem e t al., 1993). The serotoninergic input to the MTN appears to be part of a SER-positive fiber plexus covering the neighboring parabrachial nucleus and locus coeruleus. The present data have shown that serotoninergic fibers form a pericellular basket-like network around MTN neurons. In accordance with the previous reports on other species (Copray e t al., 1990b, 1991; Kolta et al., 1993), no perikaryal immunostaining has been seen in the MTN of the cat. Similarly, such encircled nonreactive neuronal somata have been identified in the cat trigeminal ganglion, as were described elsewhere (Chouchkov et al., 1988). Furthermore, our results have confirmed the observations in a n earlier re- Peroxidase immunoreactivity for SER was localized in fine and sometimes beaded nerve fibers of the MTN of the cat. None of the neurons within the MTN showed any specific immunoreactivity. The serotoninergic plexus extended over the adjacent locus coeruleus and nucleus parabrachialis (Fig. 1).The cell bodies of large 1. SER-immunostained frontal section through the caudal level immunonegative MTN neurons were closely sur- of Fig. the MTN. The MTN is located in between the LC and VPB. Note the rounded by SER-positive thin fibers and terminals high density of thin, varicose SER-positive fibers extending over the (Figs. 2, 3). On rare occasions, the axonal varicosities adjacent LC and VPB. x 100. covered the neuronal surface of small unstained MTN Fig. 2. Photomicrograph illustrating the distribution of SER-immucells. At higher magnification, some of these immuno- noreactive cell bodies and fibers in the brainstem of the cat at the reactive varicosities appeared to be in close proximity level of the rostra1 pons. Immunoreactive perikarya can be seen in the LC and VPB. A cluster of large unstained neurons in the MTN is to profiles of MTN neurons of all sizes (Fig. 3). At the ultrastructural level, fine labeled axonal pro- surrounded by thin serotoninergic fibers with intensively stained varfiles were in close apposition to the mesencephalic cell icosities and terminal boutons. x 160. bodies (Fig. 4),and direct axosomatic contacts could be Fig. 3. Higher magnification of the area inside the rectangle in Fig. seen (Fig. 5). Furthermore, some axodendritic (Fig. 6) 2 showing a plexus of varicose fibers (arrows) forming basket-like and synapses en passant (Fig. 7) were documented. The structures around five large immunonegative MTN neuron cell bodlabeled profiles were filled with small vesicles. In the ies. The SER-immunoreactive fibers pass to the LC and DPB. x 400. nerve terminals, electron-dense DAB reaction product Fig. 4. A perisomatic serotoninergic profile in the MTN. N o synaptic was located within numerous small clear round vesi- contact between the perikaryon and the labeled fiber is visible. The cles (diameter 50-80 nm) and a few small granular membranes are closely apposed and show some densification in some points (arrows). The reaction product is distributed in the axoplasm vesicles and was associated with the outer boundaries between the vesicles and inside some of the small vesicles. Mitochonof the mitochondria. A few immunolabeled large dense- drial profiles are also labeled. x 12,000. cored vesicles (up to 150 nm diameter) were also found Fig. 5. Electron micrograph of a synaptic contact between a SERin serotoninergic terminals. immunoreactive terminal and a MTN cell body (M).Note the presence The sites of WGA-HRP injection were restricted to of vesicles that are surrounded and only occasionally filled with DAB the MTN in the cats. Retrograde tracing with WGA- reaction product. X 50,000. HRP demonstrated that afferents of the MTN origiFig. 6. Demonstration of an axodendritic synapse between a SERnated from cells in serotoninergic areas (i.e., in the terminal and a small dendrite (D). The site of the contact brainstem raphe nuclei, mainly from the dorsal raphe containing is marked by an arrow. The axon terminal contains small clear and nucleus) (Fig. 8A-E). A few WGA-HRP-labeled cells granular (dense-cored) vesicles. Note the close membrane apposition were also found in the median and pontine raphe nu- (indicated by an open arrow) between the labeled terminal and a MTN cleus. After processing the sections for immunocyto- cell body (MI. x 50,000. chemistry, some of the SER-immunostained neurons in Fig. 7. Serotoninergic axon (Ax) making a synaptic contact with the the nucleus raphe dorsalis of the cat were also WGA- dendritic shaft en passant. Note the different synaptic vesicle conHRP-labeled. These double-labeled neurons repre- tents with small round and large dense-cored vesicles. x 50,000. SEROTONIN IN MESENCEPHALIC TRIGEMINAL NUCLEUS Figs. 1-7 139 140 N.E. LAZAROV AND C.N. CHOUCHKOV Fig. 8. A Coronal section of the brainstem a t the level of the rostra1 pons showing DR and RPn. Note a large number of SER-containing raphe nerve cells. Some of these raphe neurons are retrogradely labeled with a WGA-HRP applied to the MTN. Arrows point to the double-labeled cells and the arrowheads to SER-positive cells. x 100. B High magnification LM micrograph of the double-labeled (arrows) and single-labeled (arrowheads) neurons in the dorsal raphe nucleus as shown in A. Single-labeled neurons were easily identified by the presence of either a black granular reaction product (WGA-HRP- labeled neuron) or a brown reaction (SER-immunoreactive neuron) throughout the cytoplasm. Double-labeled neurons were demonstrated by the presence of both black and brown reaction product within their cytoplasm. x 630. C:Section of the DR at the level of the mesencephalon. Both magnifications x 100. D,E: Schematic drawing of the same sections as in A,C according to the stereotaxic atlas of Berman (1968).Injection sites of WGA-HRP and retrogradely labeled cells in the pons and mesencephalon as indicated in A and C, respectively. Each star represents one labeled neuronal cell body. port (Tashiro e t al., 1989) that SER-immunoreactive axonal varicosities are often seen in direct contact with profiles of MTN neurons. While the light microscopic analysis cannot demonstrate synaptic contacts, it is likely that the observed contacts represent sites of synaptic contacts. In the sensory ganglia, no synaptic contacts between a n axon terminal and a ganglion cell body or process have as yet been demonstrated (Lieberman, 1976). Only Yamamoto and Kondo (1989)have reported finding rare synaptic contacts on somata of primary afferent neurons in the rat trigeminal ganglion. Moreover, the axonal varicosities were immunoreactive to calcitonin gene-related peptide. SEROTONIN IN MESENCEPHALIC TRIGEMINAL NUCLEUS Our electron microscopical analysis has revealed that MTN neurons have synaptic contacts with SERcontaining terminals. However, the serotoninergic fibers engage only rarely in synaptic contacts. Serotonin-containing terminals form both axodendritic and axosomatic synapses. According to the classical observations of Hinrichsen and Larramendi (1968, 1970) and our own investigation of SER-labeled terminals, most of the synaptic profiles in the cat MTN contain numerous small round vesicles and several granular vesicles. In agreement to the data given by Maley et al. (1990), we could also find large granular vesicles in the SER-containing terminals of the cat MTN. The axosomatic contacts are rare and are formed by fine axons. It is likely that these correspond to the perisomatic serotoninergic fibers observed in this study. In addition, retrograde tracer studies combined with immunocytochemistry have demonstrated that MTN received serotoninergic afferents exclusively from the nuclei raphe dorsalis, pontis, and medianus. The first reported distribution of monoamine-containing neurons in the raphe system of the rat given by Dahlstrom and Fuxe (1964) has lately been confirmed in the cat (Poitras and Parent, 1975, 1978). More recently, SER has been localized a t the ultrastructural level in the nucleus raphe dorsalis of the cat (Chazal and Ralston, 1987), and direct projections from the brainstem raphe nuclei to the trigeminal sensory complex have been documented (Li et al., 1993b,c,d).The nuclei of origin of serotoninergic afferent fibers in the rat trigeminal motor, facial, and hypoglossal nuclei have earlier been described by Li et al. (1993a). This study has revealed that the SER-containing neurons project directly, except for the dorsal lateral geniculate nucleus (De Lima and Singer, 1987), to the MTN of the cat. Thus, the serotoninergic fibers revealed by immunocytochemistry in the MTN are most likely arising from the serotoninergic cells in the dorsal raphe nucleus. On the other hand, the double-labeled method has shown that in the cat, SER-containing raphe neurons contact with axonal varicosities exhibiting noradrenalin-, substance P-, and enkephalin-like immunoreactivity (Pretel and Ruda, 1988). The physiological significance of these findings for the functioning of the MTN neurons, however, is obscure and still remains to be determined. The activity of MTN neurons is modulated in different way (for review, see Cody et al., 1972, 1975; Inoue et al., 1981). Similar studies carried out on MTN in the r a t (Copray et al., 1991) and rabbit (Kolta et al., 1993) have suggested that SER can modulate the transmission of action potentials from muscle spindle receptors during mastication. Although until now the functional role of serotoninergic system has remained unknown, the results of a previous investigation (De Montigny and Lund, 1980) have shown that SER has a depressant effect on MTN spindle afferents in the cat. As direct projections from the brainstem raphe nuclei to the MTN of the cat have been observed in the present study and most of the double-labeled raphe neurons have shown SER immunoreactivity, it may be speculated that serotoninergic input modulates MTN neuronal activity. As in the rat, SER-containing raphe neurons may modulate the proprioceptive information in the cat MTN by directly sending axon collaterals to 141 this region. This is supported by our findings in earlier works on the neurochemical organization and the connections of the cat trigeminal sensory nuclear complex (Lazarov, 1991, 1994, Chouchkov and Lazarov, 1993, 1994). Besides the fact that the cell bodies of primary afferent neurons are SER-immunonegative, it is reasonable to assume that SER is not a transmitter candidate for MTN neurons of the cat. In conclusion, it seems t h a t in the MTN of the cat SER is likely to be involved not a s a classical neurotransmitter but as a pure neuromodulator. If i t is so, then other substances may act as transmitters, and only further investigations could clarify the neurotransmitter content of the cat MTN. LITERATURE CITED Berman, A.L. 1968 The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates. University of Wisconsin Press, Madison. Chazal, G., and H.J. Ralston 111 1987 Serotonin-containing structures in the nucleus raphe dorsalis of the cat: An ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals. J. Comp. Neurol., 259t317-329. Chouchkov, C., and N. Lazarov 1993 Comparative distribution of some neuropeptides and serotonin within the trigeminal principal and mesencephalic nuclei of the cat. Ann. Anatomy . Suppl., .. 175t7 (abstract). Chouchkov, C., and N. Lazarov 1994 Correlation of gamma-aminobutyric acid and tyrosine hydroxylase immunore&tivity in the cat mesencephalic trigeminal nucleus. Ann. Anatomy Suppl., 176r68 (abstract). Chouchkov, C., N. Lazarov, and M. Davidoff 1988 Serotonin-like immunoreactivity in the cat trigeminal ganglion. Histochemistry, 88:637-639. Cody, F.W.J., L.M. Harrison, and A. Taylor 1975 Analysis of activity of muscle spindles of the jaw-closing muscles during normal movements in the cat. J. Physiol., 253565-582, Cody, F.W.J., R.W.H. Lee, and A. Taylor 1972 A functional analysis of the components of the mesencephalic nucleus of the fifth nerve in the cat. J . Physiol., 226r249-261. Copray, J.C.V.M., R.S.B. Liem, G.J. Ter Horst, and J.D. van Willigen 1990a Dopaminergic afferents to the mesencephalic trigeminal nucleus of the rat: A light and electron microscope immunocytochemical study. Brain Res., 5I4r343-348. Copray, J.C.V.M.,R.S.B. Liem,G.J.TerHorst, J.D. van Willigen 1991 Origin, distribution and morphology of serotonergic afferents to the mesencephalic trigeminal nucleus of the rat. Neurosci. Lett., 12Ir97-101. Copray, J.C.V.M., G.J. Ter Horst, R.S.B. Liem, and J.D. van Willigen 1990b Neurotransmitters and neuropeptides within the mesencephalic trigeminal nucleus of the rat: An immunohistohemical analysis. Neuroscience, 37r399-411. Dahlstrom, A,, and K. Fuxe 1964 Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol. Scand. 62 (Suppl. 232):l-55. De Lima, A.D., and W. Singer 1987 The serotoninergic fibers in the dorsal lateral geniculate nucleus of the cat: Distribution and synaptic connections demonstrated with immunocytochemistry. J. Comp. Neurol., 258:339-351. De Montigny, C., and J.P. Lund 1980 A microiontophoretic study of the action of kainic acid and putative neurotransmitters in the rat mesencephalic trigeminal nucleus. Neuroscience, 5r16211628. Ginestal, E., and C. Matute 1993 Gamma-aminobutyric acid-immunoreactive neurons in the rat trigeminal nuclei. Histochemistry, 99:49-55. Hinrichsen, C.F.L., and L.M.H. Larramendi 1968 Synapses and cluster formation of the mouse mesencephalic fifth nucleus. Brain Res., 7,296-299. Hinrichsen, C.F.L., and L.M.H. Larramendi 1970 The trigeminal mesencephalic nucleus. 11. Electron microscopy. Am. J. Anat., I27r303-320. Inoue, H., T. Morimoto, and Y. Kawamura 1981 Response characteristics and classification of the muscle spindles of the masseter muscle in the cat. Exp. Neurol., 74r548-560. Kolta, A,, R. Dubuc, and J.P. Lund 1993 An immunocytochemical and 142 N.E. LAZAROV AND C.N. CHOUCHKOV autoradiographic investigation of the serotoninergic innervation of trigeminal mesencephalic and motor nuclei in the rabbit. Neuroscience, 53tlll3-1126. Lazarov, N. 1991 Histochemical and Immunocytochemical Peculiarities of Primary Trigeminal Afferent Neuron. Ph.D. thesis, Medical University of Stara Zagora. Lazarov, N. 1994 Primary trigeminal afferent neuron of the cat: 11. Neuropeptide- and serotonin-like immunoreactivity. J. Brain Res., 35r373-389. Lazarov. N., and C. Chouchkov 1993 Serotonineraic afferents to the mesencephalic trigeminal nucleus of the cat: light and electron microscopic immunocytochemical study. In: Proceedings of the Scientific Session 10 Years Medical University Stara Zagora, p. 1 (abstract). Li, Y.Q., M. Takada, and N. Mizuno 1993a The sites of origin of serotoninergic afferent fibers in the trigeminal motor, facial and hypoglossal nuclei in the rat. Neurosci. Res., 17r307-313. Li, Y.Q., M. Takada, and N. Mizuno 1993b Collateral projections of single neurons in the ueriaoueductal grav and dorsal rauhe nucleis to both the trigiminaf sensory Gmplex and spinal'cord in the rat. Neurosci. Lett., 153,153-156. Li, Y.Q., M. Takada, Y. Shinonaga, and N. Mizuno 1993c Direct projections from the midbrain periaqueductal gray and the dorsal raphe nucleus to the trigeminal sensory complex in the rat. Neuroscience, 54t431-443. Li, Y.Q., M. Takada, Y. Shinonaga, and N. Mizuno 1993d Collateral projections of single neurons in the nucleus raphe magnus to both the sensory trigeminal nuclei and spinal cord in the rat. Brain Res., 602.331-335. Lieberman, A.R. 1976 Sensory aanalia. In: The Periuheral Nerve. D.N. Landon, ed. Chapman andHall, London, pp.-188-278. Liem, R.S.B., J.C.V.M. Copray, and J.D. van Willigen 1992 Distribution of synaptic boutons in the mesencephalic trigeminal nucleus of the rat-a quantitative electron-microscopical study. Acta Anat., 143t74-78. Liem, R.S.B., J.C.V.M. Copray, and J.D. van Willigen 1993 Serotoninimmunoreactive terminals in the mesencepKalic trigeminal nucleus of the rat: An electron microscopic immunocytochemical study. Acta Anat., 148:34-41. Maky, B.E., M.G. Engle, S. Humphreys, D.A. Vascik, K.A. Howes, B.W. Newton, and R.P. Elde 1990 Monoamine synaptic structure and localization in the central nervous system. J. Electron Microsc. Tech., 15t20-33. Mesulam, M.-M. 1978 Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: A non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem., 26t106-117. Nomura, S., A. Konishi, K. Itoh, T. Sugimoto, Y. Yasui, A. Mitani, and N. Mizuno 1985 Multipolar neurons and axodendritic synapses in the mesencephalic trigeminal nucleus of the cat. Neurosci. Lett., 55t337-342. Poitras, D., and A. Parent 1975 Distribution of monoamine-containing neurons in the brain stem of the cat. Can. Fed. Biol. Sac. Proceed., 18:31. Pojtras, D., and A. Parent 1978 Atlas of the distribution of monoamine-containing nerve cell bodies in the brain stem of the cat. J. Camp. Neurol., 179r699-718. Pretel, S., and M.A. Ruda 1988 Immunocytochemical analysis of noradrenaline, substance P and enkephalin axonal contacts on serotonin neurons in the caudal raphe nuclei of the cat. Neurosci. Lett., 89r19-24. Shigenaga, Y., Y. Mitsuhiro, A. Yoshida, C. &in Cao, and H. Tsuru 1988a Morphology of single mesencephalic trigeminal neurons innervating masseter muscle of the cat. Brain Res., 445t392-399. Shigenaga, Y., A. Yoshida, Y. Mitsuhiro, K. Doe, and S. Suemune 198813 Morphology of single mesencephalic trigeminal neurons innervating periodontal ligament of the cat. Brain Res., 448~331338. Sternberger, L.A. 1986 The unlabeled antibody peroxidase-antiper.oxidase (PAP) method. In: Immunocytochemistry. L.A. Sternberger, ed. John Wiley & Sons, New York, pp. 90-209. Tashiro, T., T. Satoda, R. Matsushima, and N. Mizuno 1989 Enkephalin-, substance P- and serotonin-like immunoreactive axonal varicosities in close apposition to perikarya of mesencephalic trigeminal nucleus neurons in the cat. Brain Res., 494r162-167. Walberg, F. 1984 On the morphology of the mesencephalic trigeminal cells. New data based on tracer studies. Brain Res., 322t119-123. Yamamoto, M., and H. Kondo 1989 Calcitonin gene-related peptide (GGRP)-immunoreactive nerve varicosities in synaptic contact with sensory neurons in the trigeminal ganglion of rats. Neurosci. Lett., 104t253-257.