Cell Tissue Res (1992) 270:149-156
Cell&Tissue
Research
9 Springer-Verlag 1992
Evidence for projections from medullary nuclei onto serotonergic and dopaminergic neurons in the midbrain dorsal raphe nucleus of the rat Horst Herbert Tierphysiologie, Universit/it Tfibingen, Auf der Morgenstelle 28, W-7400 Tiibingen, Federal Republic of Germany Received February 26, 1992 / Accepted June 22, 1992
Summary. The anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into the medial nucleus of the solitary tract and into the rostral dorsomedial medulla. A sequential two-color immunoperoxidase staining was accomplished in order to demonstrate the co-distribution o f presumed terminal axons with chemically distinct neurons in the dorsal raphe nucleus of the midbrain central gray, i.e,, B7 serotonergic and A10dc dopaminergic neurons. Black-stained efferent fibers from the medial nucleus of the solitary tract and the rostral dorsomedial medulla intermingled with brown-stained serotonergic (5-hydroxytryptamine-immunoreactive) or dopaminergic (tyrosine hydroxylase-immunoreactive) neurons. Light microscopy revealed that the black-stained efferent axons exhibited numerous en passant and terminal varicosities that were often found in close apposition to brown-stained serotonergic and dopaminergic somata, and to proximal and distal dendrites and dendritic processes. The close association of immunoreactive elements suggests the presence of axo-somatic and axodendritic synaptic contacts of medullary fibers with serotonergic and dopaminergic neurons in the dorsal raphe nucleus. These projections could be involved in the modulation of dorsal raphe neurons, depending on the autonomic status of an animal. Key words: Nucleus tractus solitarii - Dorsomedial medulla - Midbrain central gray - Autonomic control 5-Hydroxytryptamine - Tyrosine hydroxylase - Central gray, mesencephalic, periaqueductal - Rat (SpragueDawley)
CG, including motor, limbic, sensory, and autonomic functions (see Beitz 1990a, for review). Recently, we have further analyzed this pathway demonstrating that a large number of adrenergic and noradrenergic neurons belonging to the AI/C1 group in the ventrolateral medulla, the A2/C2 group in the medial nucleus of the solitary tract (NTS), and the C3 group in the rostral dorsomedial medulla ( D M M ) participate in this projection (Herbert and Saper 1992). Despite our considerable knowledge of the afferents to the CG, little is known about the nature of individual C G neurons, which are the postsynaptic targets of fibers from the autonomic cell groups. This information, however, is a key to the better understanding of the possible functional implications of these afferents. Two neuroanatomical approaches are conceivable for identifying and further characterizing potential target neurons in the CG. First, these neurons could be distinguished by retrograde tracing experiments as neurons projecting to a specific brain region. Secondly, the neurons could be characterized by immunohistochemical staining in order to demonstrate their chemical identity. The aim of the present study was to identify those neurons in the C G that are potential targets of medullary efferents from the NTS and the D M M . I have concentrated on neurochemically characterized neurons in the mesencephalic dorsal raphe nucleus (DR), i.e., serotonergic neurons of the B7 cell group (Dahlstr6m and Fuxe 1964; Steinbusch 1981) and dopaminergic neurons of the A10dc cell group (Dahlstr6m and Fuxe 1964; H6kfelt et al. 1984).
Materials and methods The midbrain central gray (CG) receives a prominent innervation from autonomic cell groups in the medulla (Bandler and T6rk 1987; Kwiat and Basbaum 1990; Woulfe et al. 1990; Herbert and Saper 1992). These cell groups have a distinct termination pattern in certain subdivisions of the CG, thus suggesting a differential influence on the processing of the diverse functions of the
The data presented here are derived from 7 female Sprague-Dawley rats weighing 300-350 g. They were anesthetized with 7% chloral hydrate (400 mg/kg) and received iontophoretic injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the medial NTS (n = 4) or the rostral DMM just ventral to the nucleus prepositus and lateral to the medial longitudinal fasciculus (n = 3). Large injections were deliberately made in order to label many efferent fibers pro-
150 jecting into the CG (for the location of the injection sites, see Herbert and Saper 1992). Briefly, 2.5% PHA-L in 10 mM phosphate buffer, pH 8.0, was injected using glass micropipettes with 25- to 40-gm tip diameters by means of a pulsed positive current of 5-6 gA with a 50% duty cycle; this current was applied for 15 to 20 rain. After 8- to 12-day survival, the animals were reanesthetized and perfused through the aorta with 0.9% saline, followed by a two-step pH-change procedure (Berod et al. 1981). This procedure consisted of perfusing rats with 180 ml ice-cold 4% paraformaldehyde in 0.1 M sodium acetate buffer, pH 6.5, followed by 300 ml ice-cold 4% paraformaldehyde in 0.1 M borate buffer, pH 11.0. Thereafter, the brains were removed and postfixed overnight at 4~ C in the borate buffer fixative containing 5% sucrose. Tissue blocks were then cut and soaked for 2 days at 4~ in 30% sucrose in 0.1 M phosphate buffer, pH 7.4. Coronal sections were cut at 40 gm on a freezing microtome, and alternate series were then processed by different immunocytochemical procedures.
Two-color immunoperoxidase staining In order to demonstrate the co-distribution of PHA-L labeled fibers with serotonergic or dopaminergic cell groups in the CG, sequential peroxidase-antiperoxidase (PAP)-staining was carried out on freefloating sections. Three series of sections through the midbrain were rinsed in several changes of TRIS-buffered saline (TBS: 50 mM, pH 7.6) and incubated in a blocking solution containing 10% normal swine serum, 2% bovine serum albumin (BSA), and 0.3% Triton X-100 in TBS. After 1 h, the sections were transferred into the primary antibody solution (rabbit anti-PHA-L; Dako, Hamburg, FRG), diluted 1:3000 in a carrier solution containing 1% normal swine serum, 1% BSA, and 0.3% Triton X-100 in TBS, and incubated for 2 days at 4~ C. Sections were then rinsed in several changes of TBS, transferred into swine anti-rabbit IgG antiserum (Dako) diluted 1:50, and incubated for 1.5 h at room temperature. After several rinses in TBS, sections were incubated for 1.5 h in a rabbit-PAP complex (Dako) diluted 1:200. The final visualization of the anterogradely transported PHA-L was accomplished by processing the sections with nickel-enhanced diaminobenzidine (Ni-DAB) (Hancock 1986; Wouterlood 1988). Sections were rinsed thoroughly in TBS and reacted for 10~15 min in 50 mM TBS, pH 8.0, containing 0.6% nickel-ammonium sulfate, 0.02% DAB, and 0.01% hydrogen peroxide (HaOa). The PHA-L-immunoreactive (-ir) fibers visualized with Ni-DAB appeared black. In the second step, sections were incubated in an antiserum for the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) or in an antiserum for serotonin (5-hydroxytryptamine, 5HT). Two series of sections were rinsed in TBS overnight at 4~ C. One series was incubated in a 1:600 dilution of rabbit anti-TH (Eugene Tech, Allendale, N J, USA) and the other series in a 1 : 1500 dilution of rabbit anti-5-HT (Eugene Tech) in order to visualize the dopaminergic or the serotonergic cells, respectively, in the DR. All subsequent incubations followed the protocol outlined above. The final visualization of TH-ir or 5-HT-ir cells was performed by incubating the sections, for 8-10 min, in TBS containing 0.05% DAB and 0.01% H2Oz; this resulted in a brown reaction product. Sections were mounted on gelatine-coated slides, air-dried, dehydrated in graded alcohols, cleared in xylene, and c0verslipped with Entellan.
Controls As all primary antisera used were raised in rabbits, the specificity of the immunohistochemical staining was assured by controls. Omission of either one of the primary antisera in the two-color immunoperoxidase protocol always resulted in the specific labeling of one antigen only. Moreover, sections that were stained for PHAL alone always exhibited the same fiber pattern as adjacent sections that were taken from the same brain and that were processed fol-
lowing the double-stainingprotocol. In addition, the Ni-DAB reaction was always carried out first, thereby avoiding blackening of formerly brown reaction product. Furthermore, the morphological differences between axons and dendritic processes were clearly distinguishable, so that false-positive labeling caused by cross-reactivity could have been easily detected by light microscopy. Finally, it was assumed that the DAB-polymerization product masked the immunocomplex and thus prevented the antisera applied in the second incubation step binding to the first immunocomplex (Joseph and Piekut 1986).
Data analysis Sections were analyzed by light microscopy using a 100 x oil-immersion objective. Photomicrographs were taken of PHA-L labeled varicosities in contact with TH-ir or 5-HT-ir somata or processes when both structures were in the same plane of focus, when beaded fibers tracked the shape of somatic or dendritic profiles, or when axons exhibited varicosities exactly at the point where they crossed TH-ir or 5-HT-ir somata or processes. These points of close association were regarded as presumed synaptic contacts between medullary fibers and TH-ir or 5-HT-ir neurons in the DR.
Specificity of TH-staining TH is the key enzyme involved in the catecholamine biosynthetic pathway converting L-tyrosine into L-DOPA, the precursor of dopamine. Dopamine may be consecutively converted into noradrenaline and adrenaline by the enzymes dopamine/%hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), respectively. Thus, TH-ir neurons need not necessarily represent dopaminergic neurons but could also be neurons that contain noradrenalin or adrenalin as a transmitter. However, sections through the midbrain CG that were immunohistochemically stained for DBH or PNMT never exhibited DBH-ir or PNMT-ir somata in the DR proper (Herbert and Saper 1992). I therefore conclude that the TH-ir neurons in the midbrain CG as demonstrated in the present study indeed represent dopaminergic cells of the A10dc group.
Results Injections o f the a n t e r o g r a d e tracer P H A - L into the medial N T S resulted in a x o n a l labeling i n the v e n t r o l a t e r a l C G , i n c l u d i n g the D R . S u b s e q u e n t i m m u n o h i s t o c h e m i cal s t a i n i n g for 5 - H T showed a large degree o f overlap of N T S a x o n s with the rostral m e d i a n p o r t i o n a n d with the " l a t e r a l w i n g s " o f the B7 serotonergic cell g r o u p , whereas the c a u d a l m e d i a n aspect o f the D R was a l m o s t devoid o f fibers. T h e b l a c k - s t a i n e d fiber plexus was m o s t dense w i t h i n the b r o w n - s t a i n e d 5 - H T - i r s o m a t a o f the " l a t e r a l w i n g s " (Fig. 1 A) a n d also j u s t dorsal to this cluster o f s o m a t a , where n u m e r o u s lightly stained 5 - H T ir d e n d r i t i c processes were p r e s e n t (Fig. 1 B). Light microscopy revealed t h a t the b l a c k - s t a i n e d varicose fibers were often f o u n d j u x t a p o s e d to b r o w n - s t a i n e d 5 - H T - i r structures (Fig, 3 A D ) . Both en passant varicosities (Fig. 3 A , B) a n d p r e s u m e d t e r m i n a l varicosities (Fig. 3 C ) w e r e f o u n d in close a p p o s i t i o n to 5 - H T - i r som a t a (Fig. 3 A, B), p r i m a r y dendrites (Fig. 3 C, D), a n d 5 - H T - i r processes, the latter possibly r e p r e s e n t i n g fragm e n t s o f higher o r d e r dendrites (not illustrated). I m m u n o h i s t o c h e m i c a t s t a i n i n g o f a d j a c e n t sections for T H la-
151
Fig. 1A, B. Two-color immunoperoxidase-stained coronal section through the DR in the midbrain CG illustrating the concomitant distribution of 5-HT-ir neurons 0ightly stained) and anterogradely labeled fibers (black-stained) after an injection of PHA-L into the medial NTS. The box in A indicates the area enlarged in B. Note
that a dense fiber plexus is situated within, and dorsal to, the "lateral wing" of the DR, whereas the caudal median portion is only weakly innervated. Arrowheads in B indicate an individual PHA-L labeled fiber. Aq aqueduct; m0~mediallongitudinal fasciculus. Bar in A: 250 [xm,in B: 100 gm
beled dopaminergic neurons o f the A l 0 d c cell group. These were primarily located rostrally to the D R proper just ventromedially to the aqueduct (Fig. 2A) and, to a lesser extent, further caudally also in the lateral portion of the D R (Fig. 2 B). Light-microscopic analysis revealed several black-stained varicose fibers in the vicinity o f brown-stained TH-ir somata and processes (Fig. 3 E-I). Many fibers with en passant varicosities were seen approaching TH-ir neurons, tracking the shape of the somata, and apparently moving on to another target (Fig. 3 E, F). Other fibers appeared to end on somata (Fig. 3 G, H) or dendritic processes (Fig. 3 I) by forming a large terminal bouton. Following P H A - L injections into the rostral D M M , anterogradely labeled fibers were found widespread in the ventral CG. Within the DR, the D M M fibers were evenly distributed in all aspects, i.e., in the "lateral wings" and in the rostral and caudal median DR. The density of fibers from the D M M was lower compared with those from the NTS (see, Fig. 1 B versus Fig. 2A, B). Nevertheless, several contiguities between blackstained PHA-L-labeled fibers and brown-stained 5-HTir (Fig. 4 A - C ) and TH-ir neurons (Fig. 4 D - G ) were found. Varicose fibers were closely apposed to somata (Fig. 4B-E), proximal dendrites, (Fig. 4A) and higher
order dendritic processes (Fig. 4F, G). In general, the morphology of terminal fibers from the D M M and the types o f presumed synaptic contacts observed in the D R were similar to those reported for axonal projections originating from the NTS. To summarize, both NTS and D M M fibers appear to contact serotonergic and dopaminergic neurons in the DR. With respect to the A10dc dopaminergic neurons, the number of presumed contacts with ascending fibers from either the NTS or the D M M was similar. In contrast, a gradient in the number of presumed synaptic contacts with NTS fibers could be seen on B7 serotonergic neurons: neurons in the "lateral wings" exhibited large numbers, those in the rostral median D R moderate numbers, and those in the caudal median D R only low numbers of presumed contacts. Finally, D M M fibers appear to contact serotonergic neurons in all parts of the B7 group, although less frequently than NTS fibers.
Discussion
The present data provide light-microscopic evidence that numerous varicose fibers from the NTS and the rostral D M M form putative synaptic contacts with serotonergic
152 Fig. 2A, B. Two-color immunoperoxidasestained coronal sections through the D R in the midbrain CG illustrating the concomitant distribution of TH-ir neurons (lightly stained) and anterogradely labeled fibers (black-stained) after an injection of PHA-L into the rostral DMM. Rostral in the CG, the dopaminergic neurons of the A10dc group are primarily located ventromedially to the aqueduct (A), whereas caudal in the CG, dopaminergic neurons are also found in lateral aspects (B). Arrowheads in A indicate a beaded, PHA-L labeled fiber. Aq aqueduct. Bars in A and B: 100 Izm
153
Fig. 3A-L Dual-immunostainedsections through the DR after an injection of PHA-L into the medialNTS. A-D Black-stainedPHAL-Jr fibers and varicosities closelyapposed to 5-HT-ir somata and dendritic processes. Arrows indicate presumed synaptic contacts between black-stainedvaricositiesand lighter stained postsynaptic
targets. E-I Black-stainedPHA-L-ir fibers and varicosities closely apposed to TH-ir somata and processes. Note the smaller soma size of dopaminergic (E-H) versus serotonergic neurons (A-D). Bar: 10 ~m
and dopaminergic neurons in the midbrain DR. This is further indicated by ultrastructural investigations demonstrating that varicosities of PHA-L labeled axons represent presynaptic boutons (Wouterlood and Groenewegen 1985). However, close appositions between two stained elements, as seen under the light microscope, need not necessarily represent synaptic contacts. This has been convincingly demonstrated in a combined lightand electron-microscopic (EM) study by Meredith and Wouterlood (1990). Thus, my statement concerning apparent axo-somatic and axo-dendritic contacts between medullary fibers and serotonergic and dopaminergic neurons should be interpreted cautiously; it requires verification at the EM level in order to provide a clearcut estimation of the number of synaptic contacts. In the double-stained material, I have also observed PHA-L labeled varicose axons that are not apposed to 5-HT-ir or TH-ir elements. Thus, it is likely that, in addition to monoaminergic neurons, other neurochemically distinct cells in the midbrain CG receive input from NTS and DMM fibers. Among these potential targets are peptidergic neurons containing enkephalin, neurotensin, cholecystokinin, substance P, or somatostatin (Beitz et al. 1983; Moss et al. 1983; Andrezik and Beitz 1985; Shipley et al. 1987; Li et al. 1990), and neurons that contain amino-acid transmitters, such as 7-aminobutyric acid, glutamate, or aspartate (Beitz 1990b; Reichling and Basbaum 1990). What is the nature of the information transmitted from the NTS and the DMM to the TH-ir and 5-HT-ir
neurons in the DR? The medial NTS is the major recipient of visceral sensory afferents carried in the glossopharyngeal and the vagus nerve. These afferents include baroreceptive and chemoreceptive fibers of the carotid sinus and aortic branch, and chemoreceptive and mechanoreceptive subdiaphragmatic vagal fibers from the intestine, stomach, and the hepatic portal vein (Leslie et al. 1982; Rogers and Hermann 1983; Higgins et al, 1984; Shapiro and Miselis 1985; Housley et al. 1987). Sumal and coworkers (1983) have provided ultrastructural evidence that the vagal fibers in the medial NTS synapse on catecholaminergic cells of the A2/C2 group and non-catecholaminergic neurons. These cells in the medial NTS are therefore in a position to transmit visceral sensory information of various modalities to the DR and thus, may provide information about the actual autonomic status, Little information is available about the functions of the rostral DMM, including the C3 adrenergic cell group. Electrical and chemical stimulation in this area result in a pressor response (Ross et al. 1984; Chai et al. 1988; Lin et al. 1989), thereby indicating a role of the DMM in autonomic regulation. This is supported by anatomical findings showing that adrenergic and nonadrenergic neurons of the DMM project to the paraventricular nucleus of the hypothalamus (Cunningham et al. 1990) and to spinal preganglionic neurons in the intermediolateral cell column (Minson et al. 1990) where they may directly modulate sympathetic outflow. It is interesting to note that adrenergic and non-adrenergic neu-
154
Fig. 4A-G. Dual-immunostainedsections through the DR after an injection of PHA-L into the rostral DMM. A-C Black-stained PHA-L-ir fibers and varicositiescloselyapposed to 5-HT-ir somata and dendrites. Arrows indicatepresumed synapticcontacts between black-stained varicosities and lighter-stained postsynaptic targets.
D-G Black-stained PHA-L-ir fibers and varicosities ciosely apposed to TH-ir somata and dendritic processes. Note the smaller soma size of dopaminergic (D, E) versus serotonergicneurons (AC). Bar: 10 gm
rons of the DMM are also major sources of projections to the locus coeruleus (Aston-Jones et al. 1986; Pieribone etal. 1988; Pieribone and Aston-Jones 1991), which resembles the DR with respect to its widespread projections in the brain and its involvement in global brain functions, such as behavioral arousal, vigilance, emotion, or sleep (Aston-Jones and Bloom 1981 ; Foote et al. 1983; Aston-Jones et al. 1986). It is tempting to speculate that neurons in the DMM co-regulate the sympathetic outflow and the activity of major transmitter systems, including the noradrenergic locus coeruleus group and the dopaminergic and serotonergic DR group. A large number of studies have documented the anatomical connections of the DR, including the B7 serotonergic cell group, which comprises approximately one third of the total DR cell population (Descarries et al. 1982). A widespread distribution of ascending projections to various diencephalic and telencephalic regions has been reported (for reviews, see Steinbusch 1984; T6rk 1985; Vertes 1991). The diverse forebrain projections are in agreement with the diverse functions influenced by this cell group, for example, locomotor activity, thermoregulation, slow-wave sleep, and sexual behavior (see, e.g., Vertes 1990; Hillegaart 1991). The DR neurons have a highly collateralized axon
system by which multiple targets can be modulated simultaneously (see, e.g., de Olmos and Heimer 1980; Vil, lar et al. 1988). However, there is also a certain degree of topographic organization with respect to the rostral versus caudal DR efferents (see, e.g., Imai et al, 1986; Vertes 1991) and with respect to the "lateral wings" of the B7 group (O'Hearn and Molliver 1984; Villar et al. 1987, 1988; Klepper and Herbert 1991). It has previously been shown that the DMM projection is evenly distributed in the rostrocaudal dimension of the DR and in the "lateral wings" (Herbert and Saper 1992). Thus, the DMM may equally influence all aspects of the serotonergic and non-serotonergic forebrain projections that arise in the DR. In contrast, projections from the NTS terminate predominantly in the "lateral wings" of the DR and in the rostral midline portion, whereas the caudal median DR is almost devoid of NTS fibers (Herbert and Saper 1992). Consequently, the NTS may modulate the serotonergic input to visual and auditory nuclei, an input that arises exclusively in the "lateral wings" of the B7 group (Villar et al. 1987, 1988; Klepper and Herbert 1991); it may also modulate the serotonergic input to all regions of the cortex, caudate-putamen, substantia nigra, amygdala, lateral hypothalamus, preoptic area, and the substantia innominata, an input that arises predominantly in the rostral DR and in the "later-
155 al wings" (see, e.g., Imai etal. 1986; Hallanger and Wainer 1988; Vertes 1991). Serotonergic and non-serotonergic neurons in the caudal DR, i.e., neurons that project predominantly to the frontal cortex, lateral septum, bed nucleus of the stria terminalis, hippocampus, and the locus coeruleus, and that are thus closely related to the limbic system (Imai et al. 1986; Vertes 1991), are apparently not markedly influenced by NTS fibers. This topography indicates a role of the NTS in modulating the activity of serotonergic projections to nuclei concerned with the processing of sensory, autonomic, and m o t o r functions, whereas the D M M also appears to influence the serotonergic input to the limbic system. In a recent study, we have shown that a subpopulation of the NTS and D M M projection neurons is catecholaminergic (Herbert and Saper 1992). It is therefore conceivable that some of the varicosities, which are found in close apposition to serotonergic or dopaminergic neurons in the DR, represent adrenergic or noradrenergic synapses. This is suggested by an EM study in the D R demonstrating that a majority of noradrenergic terminals, of hitherto undefined origin, form synaptic contacts with serotonergic neurons (Baraban and Aghajanian 1981). Furthermore, physiological experiments show that noradrenalin excites serotonergic neurons in the D R , whereas a blockade o f e-adrenoceptors results in a cessation of their spontaneous activity (Baraban and Aghajanian 1980). The tonic noradrenergic input to the D R is presumably responsible for the unique pacemaker activity of the serotonergic neurons (Vandermaelen and Aghajanian 1983). Physiological data on the effect of noradrenalin or e-adrenergic antagonists on A10dc dopaminergic neurons in the D R are presently not available. The A10dc dopaminergic group consists of approximately 1000 neurons and is considered a small subpopulation of the A10 mesolimbic/mesolimbocortical dopaminergic system of the ventral tegmental area (Descarries et al. 1986; Stratford and Wirtshafter 1990). As yet, only a few studies have demonstrated projections of the A10dc cells to the prefrontal cortex, nucleus accumbens, lateral septum, hippocampus, and the neostriatum (Pohle et al. 1984; Descarries et al. 1986; Yoshida et al. 1989; Stratford and Wirtshafter 1990), indicating a role in m o t o r function and also in the regulation of emotional behavior (Fuxe et al. 1985). The pathways reported in the present study could participate in controlling the expression of these behaviors. In conclusion, this study provides evidence that the medial NTS and the rostral D M M innervate serotonergic and dopaminergic neurons in the DR, and could thus modulate the activity of these cell groups, depending on the autonomic status of an animal.
Acknowledgements. I wish to thank H. Zillus for excellent technical assistance, Drs. E. Friauf, A. Klepper, and M. Koch for valuable comments on the manuscript, and Gwynn Goldring for correcting the English. This work was supported by the Deutsche Forschungsgemeinschaft (He 1842/3-1).
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tal gray and nucleus raphe magnus. J Comp Neurol 302:370377 Rogers RC, Hermann GH (1983) Central connections of the hepatic branch of the vagus nerve: a horseradish peroxidase histochemical study. J Auton Nerv Syst 7:165-174 Ross CA, Ruggiero DA, Park DH, Joh TH, Sved AF, FernandezPardal J, Saavedra JM, Reis DJ (1984) Tonic vasomotor control by the rostral ventrolateral medulla: effects of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J Neurosci 4:474M94 Shapiro RE, Miselis RR (1985) The central organization of the vagus nerve innervating the stomach of the rat. J Comp Neurol 238 :473-488 Shipley MT, McLean JH, Behbehani MM (1987) Heterogeneous distribution of neurotensin-like immunoreactive neurons and fibers in the midbrain periaqueductal gray of the rat. J Neurosci 7 :2025-2034 Steinbusch HWM (1981) Distribution of serotonin-immunoreactivity in the central nervous system of the rat. Cell bodies and terminals. Neuroscience 6: 557-618 Steinbusch HWM (1984) Serotonin-immunoreactive neurons and their projections in the CNS. In: Bj6rklund A, H6kfelt T, Kuhar MJ (eds) Handbook of chemical neuroanatomy, vol 3. Classical transmitters and transmitter receptors in the CNS, part II. Elsevier, Amsterdam, pp 68-125 Stratford TR, Wirtshafter D (1990) Ascending dopaminergic projections from the dorsal raphe nucleus in the rat. Brain Res 511:173-176 Sumal KK, Blessing WW, Joh TH, Reis DJ, Pickel VM (1983) Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius. Brain Res 277:3140 T6rk I (1985) Raphe nuclei and serotonin containing systems. In: Paxinos G (ed) The rat nervous system, vol 2. Hindbrain and spinal cord. Academic Press, Sydney, pp 43-78 Vandermaelen CP, Aghajanian GK (1983) Electrophysiological and pharmacological caracterization of serotonergic dorsal raphe neurons recorded extracellularly and intracellularly in rat brain slices. Brain Res 289:109-119 Vertes RP (1990) Fundamentals of brainstem anatomy: a behavioral perspective. In: Klemm WR, Vertes RP (eds) Brainstem mechanisms of behavior. Wiley, New York, pp 33-103 Vertes RP (1991) A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat. J Comp Neurol 313:643-668 Villar M J, Vitale ML, Parisi MN (1987) Dorsal raphe serotonergic projection to the retina. A combined peroxidase tracing-neurochemical/high performance liquid chromatography study in the rat. Neuroscience 22:681-686 Villar MJ, Vitale ML, H6kfelt T, Verhofstad AAJ (1988) Dorsal raphe serotonergic branching neurons projecting both to the lateral geniculate body and superior colliculus: a combined retrograde tracing-immunohistochemicalstudy in the rat. J Comp Neurol 277:126-140 Woulfe JM, Flumerfelt BA, Hrycyshyn AW (1990) Efferent connections of the A1 noradrenergic cell group: a DBH immunohistochemicat and PHA-L anterograde tracing study. Exp Neurol 109:308-322 Wouterlood FG (1988) Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid, choline acetyltransferase or serotonin. Histochemistry 89:421-428 Wouterlood FG, Groenewegen HJ (1985) Neuroanatomical tracing by use of Phaseolus vulgaris-leucoagglutiniu (PHA-L) : electron microscopy of PHA-L filled neuronal somata, dendrites, axons and axon terminals. Brain Res 326:188-191 Yoshida M, Shirouzu M, Tanaka M, Semba K, Fibinger HC (1989) Dopaminergic neurons in the nucleus raphe dorsalis innervate the prefrontal cortex in the rat: a combined retrograde tracing and immunohistochemical study using anti-dopamine serum. Brain Res 496: 373-376
Cell&Tissue
Research
9 Springer-Verlag 1992
Evidence for projections from medullary nuclei onto serotonergic and dopaminergic neurons in the midbrain dorsal raphe nucleus of the rat Horst Herbert Tierphysiologie, Universit/it Tfibingen, Auf der Morgenstelle 28, W-7400 Tiibingen, Federal Republic of Germany Received February 26, 1992 / Accepted June 22, 1992
Summary. The anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into the medial nucleus of the solitary tract and into the rostral dorsomedial medulla. A sequential two-color immunoperoxidase staining was accomplished in order to demonstrate the co-distribution o f presumed terminal axons with chemically distinct neurons in the dorsal raphe nucleus of the midbrain central gray, i.e,, B7 serotonergic and A10dc dopaminergic neurons. Black-stained efferent fibers from the medial nucleus of the solitary tract and the rostral dorsomedial medulla intermingled with brown-stained serotonergic (5-hydroxytryptamine-immunoreactive) or dopaminergic (tyrosine hydroxylase-immunoreactive) neurons. Light microscopy revealed that the black-stained efferent axons exhibited numerous en passant and terminal varicosities that were often found in close apposition to brown-stained serotonergic and dopaminergic somata, and to proximal and distal dendrites and dendritic processes. The close association of immunoreactive elements suggests the presence of axo-somatic and axodendritic synaptic contacts of medullary fibers with serotonergic and dopaminergic neurons in the dorsal raphe nucleus. These projections could be involved in the modulation of dorsal raphe neurons, depending on the autonomic status of an animal. Key words: Nucleus tractus solitarii - Dorsomedial medulla - Midbrain central gray - Autonomic control 5-Hydroxytryptamine - Tyrosine hydroxylase - Central gray, mesencephalic, periaqueductal - Rat (SpragueDawley)
CG, including motor, limbic, sensory, and autonomic functions (see Beitz 1990a, for review). Recently, we have further analyzed this pathway demonstrating that a large number of adrenergic and noradrenergic neurons belonging to the AI/C1 group in the ventrolateral medulla, the A2/C2 group in the medial nucleus of the solitary tract (NTS), and the C3 group in the rostral dorsomedial medulla ( D M M ) participate in this projection (Herbert and Saper 1992). Despite our considerable knowledge of the afferents to the CG, little is known about the nature of individual C G neurons, which are the postsynaptic targets of fibers from the autonomic cell groups. This information, however, is a key to the better understanding of the possible functional implications of these afferents. Two neuroanatomical approaches are conceivable for identifying and further characterizing potential target neurons in the CG. First, these neurons could be distinguished by retrograde tracing experiments as neurons projecting to a specific brain region. Secondly, the neurons could be characterized by immunohistochemical staining in order to demonstrate their chemical identity. The aim of the present study was to identify those neurons in the C G that are potential targets of medullary efferents from the NTS and the D M M . I have concentrated on neurochemically characterized neurons in the mesencephalic dorsal raphe nucleus (DR), i.e., serotonergic neurons of the B7 cell group (Dahlstr6m and Fuxe 1964; Steinbusch 1981) and dopaminergic neurons of the A10dc cell group (Dahlstr6m and Fuxe 1964; H6kfelt et al. 1984).
Materials and methods The midbrain central gray (CG) receives a prominent innervation from autonomic cell groups in the medulla (Bandler and T6rk 1987; Kwiat and Basbaum 1990; Woulfe et al. 1990; Herbert and Saper 1992). These cell groups have a distinct termination pattern in certain subdivisions of the CG, thus suggesting a differential influence on the processing of the diverse functions of the
The data presented here are derived from 7 female Sprague-Dawley rats weighing 300-350 g. They were anesthetized with 7% chloral hydrate (400 mg/kg) and received iontophoretic injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the medial NTS (n = 4) or the rostral DMM just ventral to the nucleus prepositus and lateral to the medial longitudinal fasciculus (n = 3). Large injections were deliberately made in order to label many efferent fibers pro-
150 jecting into the CG (for the location of the injection sites, see Herbert and Saper 1992). Briefly, 2.5% PHA-L in 10 mM phosphate buffer, pH 8.0, was injected using glass micropipettes with 25- to 40-gm tip diameters by means of a pulsed positive current of 5-6 gA with a 50% duty cycle; this current was applied for 15 to 20 rain. After 8- to 12-day survival, the animals were reanesthetized and perfused through the aorta with 0.9% saline, followed by a two-step pH-change procedure (Berod et al. 1981). This procedure consisted of perfusing rats with 180 ml ice-cold 4% paraformaldehyde in 0.1 M sodium acetate buffer, pH 6.5, followed by 300 ml ice-cold 4% paraformaldehyde in 0.1 M borate buffer, pH 11.0. Thereafter, the brains were removed and postfixed overnight at 4~ C in the borate buffer fixative containing 5% sucrose. Tissue blocks were then cut and soaked for 2 days at 4~ in 30% sucrose in 0.1 M phosphate buffer, pH 7.4. Coronal sections were cut at 40 gm on a freezing microtome, and alternate series were then processed by different immunocytochemical procedures.
Two-color immunoperoxidase staining In order to demonstrate the co-distribution of PHA-L labeled fibers with serotonergic or dopaminergic cell groups in the CG, sequential peroxidase-antiperoxidase (PAP)-staining was carried out on freefloating sections. Three series of sections through the midbrain were rinsed in several changes of TRIS-buffered saline (TBS: 50 mM, pH 7.6) and incubated in a blocking solution containing 10% normal swine serum, 2% bovine serum albumin (BSA), and 0.3% Triton X-100 in TBS. After 1 h, the sections were transferred into the primary antibody solution (rabbit anti-PHA-L; Dako, Hamburg, FRG), diluted 1:3000 in a carrier solution containing 1% normal swine serum, 1% BSA, and 0.3% Triton X-100 in TBS, and incubated for 2 days at 4~ C. Sections were then rinsed in several changes of TBS, transferred into swine anti-rabbit IgG antiserum (Dako) diluted 1:50, and incubated for 1.5 h at room temperature. After several rinses in TBS, sections were incubated for 1.5 h in a rabbit-PAP complex (Dako) diluted 1:200. The final visualization of the anterogradely transported PHA-L was accomplished by processing the sections with nickel-enhanced diaminobenzidine (Ni-DAB) (Hancock 1986; Wouterlood 1988). Sections were rinsed thoroughly in TBS and reacted for 10~15 min in 50 mM TBS, pH 8.0, containing 0.6% nickel-ammonium sulfate, 0.02% DAB, and 0.01% hydrogen peroxide (HaOa). The PHA-L-immunoreactive (-ir) fibers visualized with Ni-DAB appeared black. In the second step, sections were incubated in an antiserum for the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) or in an antiserum for serotonin (5-hydroxytryptamine, 5HT). Two series of sections were rinsed in TBS overnight at 4~ C. One series was incubated in a 1:600 dilution of rabbit anti-TH (Eugene Tech, Allendale, N J, USA) and the other series in a 1 : 1500 dilution of rabbit anti-5-HT (Eugene Tech) in order to visualize the dopaminergic or the serotonergic cells, respectively, in the DR. All subsequent incubations followed the protocol outlined above. The final visualization of TH-ir or 5-HT-ir cells was performed by incubating the sections, for 8-10 min, in TBS containing 0.05% DAB and 0.01% H2Oz; this resulted in a brown reaction product. Sections were mounted on gelatine-coated slides, air-dried, dehydrated in graded alcohols, cleared in xylene, and c0verslipped with Entellan.
Controls As all primary antisera used were raised in rabbits, the specificity of the immunohistochemical staining was assured by controls. Omission of either one of the primary antisera in the two-color immunoperoxidase protocol always resulted in the specific labeling of one antigen only. Moreover, sections that were stained for PHAL alone always exhibited the same fiber pattern as adjacent sections that were taken from the same brain and that were processed fol-
lowing the double-stainingprotocol. In addition, the Ni-DAB reaction was always carried out first, thereby avoiding blackening of formerly brown reaction product. Furthermore, the morphological differences between axons and dendritic processes were clearly distinguishable, so that false-positive labeling caused by cross-reactivity could have been easily detected by light microscopy. Finally, it was assumed that the DAB-polymerization product masked the immunocomplex and thus prevented the antisera applied in the second incubation step binding to the first immunocomplex (Joseph and Piekut 1986).
Data analysis Sections were analyzed by light microscopy using a 100 x oil-immersion objective. Photomicrographs were taken of PHA-L labeled varicosities in contact with TH-ir or 5-HT-ir somata or processes when both structures were in the same plane of focus, when beaded fibers tracked the shape of somatic or dendritic profiles, or when axons exhibited varicosities exactly at the point where they crossed TH-ir or 5-HT-ir somata or processes. These points of close association were regarded as presumed synaptic contacts between medullary fibers and TH-ir or 5-HT-ir neurons in the DR.
Specificity of TH-staining TH is the key enzyme involved in the catecholamine biosynthetic pathway converting L-tyrosine into L-DOPA, the precursor of dopamine. Dopamine may be consecutively converted into noradrenaline and adrenaline by the enzymes dopamine/%hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), respectively. Thus, TH-ir neurons need not necessarily represent dopaminergic neurons but could also be neurons that contain noradrenalin or adrenalin as a transmitter. However, sections through the midbrain CG that were immunohistochemically stained for DBH or PNMT never exhibited DBH-ir or PNMT-ir somata in the DR proper (Herbert and Saper 1992). I therefore conclude that the TH-ir neurons in the midbrain CG as demonstrated in the present study indeed represent dopaminergic cells of the A10dc group.
Results Injections o f the a n t e r o g r a d e tracer P H A - L into the medial N T S resulted in a x o n a l labeling i n the v e n t r o l a t e r a l C G , i n c l u d i n g the D R . S u b s e q u e n t i m m u n o h i s t o c h e m i cal s t a i n i n g for 5 - H T showed a large degree o f overlap of N T S a x o n s with the rostral m e d i a n p o r t i o n a n d with the " l a t e r a l w i n g s " o f the B7 serotonergic cell g r o u p , whereas the c a u d a l m e d i a n aspect o f the D R was a l m o s t devoid o f fibers. T h e b l a c k - s t a i n e d fiber plexus was m o s t dense w i t h i n the b r o w n - s t a i n e d 5 - H T - i r s o m a t a o f the " l a t e r a l w i n g s " (Fig. 1 A) a n d also j u s t dorsal to this cluster o f s o m a t a , where n u m e r o u s lightly stained 5 - H T ir d e n d r i t i c processes were p r e s e n t (Fig. 1 B). Light microscopy revealed t h a t the b l a c k - s t a i n e d varicose fibers were often f o u n d j u x t a p o s e d to b r o w n - s t a i n e d 5 - H T - i r structures (Fig, 3 A D ) . Both en passant varicosities (Fig. 3 A , B) a n d p r e s u m e d t e r m i n a l varicosities (Fig. 3 C ) w e r e f o u n d in close a p p o s i t i o n to 5 - H T - i r som a t a (Fig. 3 A, B), p r i m a r y dendrites (Fig. 3 C, D), a n d 5 - H T - i r processes, the latter possibly r e p r e s e n t i n g fragm e n t s o f higher o r d e r dendrites (not illustrated). I m m u n o h i s t o c h e m i c a t s t a i n i n g o f a d j a c e n t sections for T H la-
151
Fig. 1A, B. Two-color immunoperoxidase-stained coronal section through the DR in the midbrain CG illustrating the concomitant distribution of 5-HT-ir neurons 0ightly stained) and anterogradely labeled fibers (black-stained) after an injection of PHA-L into the medial NTS. The box in A indicates the area enlarged in B. Note
that a dense fiber plexus is situated within, and dorsal to, the "lateral wing" of the DR, whereas the caudal median portion is only weakly innervated. Arrowheads in B indicate an individual PHA-L labeled fiber. Aq aqueduct; m0~mediallongitudinal fasciculus. Bar in A: 250 [xm,in B: 100 gm
beled dopaminergic neurons o f the A l 0 d c cell group. These were primarily located rostrally to the D R proper just ventromedially to the aqueduct (Fig. 2A) and, to a lesser extent, further caudally also in the lateral portion of the D R (Fig. 2 B). Light-microscopic analysis revealed several black-stained varicose fibers in the vicinity o f brown-stained TH-ir somata and processes (Fig. 3 E-I). Many fibers with en passant varicosities were seen approaching TH-ir neurons, tracking the shape of the somata, and apparently moving on to another target (Fig. 3 E, F). Other fibers appeared to end on somata (Fig. 3 G, H) or dendritic processes (Fig. 3 I) by forming a large terminal bouton. Following P H A - L injections into the rostral D M M , anterogradely labeled fibers were found widespread in the ventral CG. Within the DR, the D M M fibers were evenly distributed in all aspects, i.e., in the "lateral wings" and in the rostral and caudal median DR. The density of fibers from the D M M was lower compared with those from the NTS (see, Fig. 1 B versus Fig. 2A, B). Nevertheless, several contiguities between blackstained PHA-L-labeled fibers and brown-stained 5-HTir (Fig. 4 A - C ) and TH-ir neurons (Fig. 4 D - G ) were found. Varicose fibers were closely apposed to somata (Fig. 4B-E), proximal dendrites, (Fig. 4A) and higher
order dendritic processes (Fig. 4F, G). In general, the morphology of terminal fibers from the D M M and the types o f presumed synaptic contacts observed in the D R were similar to those reported for axonal projections originating from the NTS. To summarize, both NTS and D M M fibers appear to contact serotonergic and dopaminergic neurons in the DR. With respect to the A10dc dopaminergic neurons, the number of presumed contacts with ascending fibers from either the NTS or the D M M was similar. In contrast, a gradient in the number of presumed synaptic contacts with NTS fibers could be seen on B7 serotonergic neurons: neurons in the "lateral wings" exhibited large numbers, those in the rostral median D R moderate numbers, and those in the caudal median D R only low numbers of presumed contacts. Finally, D M M fibers appear to contact serotonergic neurons in all parts of the B7 group, although less frequently than NTS fibers.
Discussion
The present data provide light-microscopic evidence that numerous varicose fibers from the NTS and the rostral D M M form putative synaptic contacts with serotonergic
152 Fig. 2A, B. Two-color immunoperoxidasestained coronal sections through the D R in the midbrain CG illustrating the concomitant distribution of TH-ir neurons (lightly stained) and anterogradely labeled fibers (black-stained) after an injection of PHA-L into the rostral DMM. Rostral in the CG, the dopaminergic neurons of the A10dc group are primarily located ventromedially to the aqueduct (A), whereas caudal in the CG, dopaminergic neurons are also found in lateral aspects (B). Arrowheads in A indicate a beaded, PHA-L labeled fiber. Aq aqueduct. Bars in A and B: 100 Izm
153
Fig. 3A-L Dual-immunostainedsections through the DR after an injection of PHA-L into the medialNTS. A-D Black-stainedPHAL-Jr fibers and varicosities closelyapposed to 5-HT-ir somata and dendritic processes. Arrows indicate presumed synaptic contacts between black-stainedvaricositiesand lighter stained postsynaptic
targets. E-I Black-stainedPHA-L-ir fibers and varicosities closely apposed to TH-ir somata and processes. Note the smaller soma size of dopaminergic (E-H) versus serotonergic neurons (A-D). Bar: 10 ~m
and dopaminergic neurons in the midbrain DR. This is further indicated by ultrastructural investigations demonstrating that varicosities of PHA-L labeled axons represent presynaptic boutons (Wouterlood and Groenewegen 1985). However, close appositions between two stained elements, as seen under the light microscope, need not necessarily represent synaptic contacts. This has been convincingly demonstrated in a combined lightand electron-microscopic (EM) study by Meredith and Wouterlood (1990). Thus, my statement concerning apparent axo-somatic and axo-dendritic contacts between medullary fibers and serotonergic and dopaminergic neurons should be interpreted cautiously; it requires verification at the EM level in order to provide a clearcut estimation of the number of synaptic contacts. In the double-stained material, I have also observed PHA-L labeled varicose axons that are not apposed to 5-HT-ir or TH-ir elements. Thus, it is likely that, in addition to monoaminergic neurons, other neurochemically distinct cells in the midbrain CG receive input from NTS and DMM fibers. Among these potential targets are peptidergic neurons containing enkephalin, neurotensin, cholecystokinin, substance P, or somatostatin (Beitz et al. 1983; Moss et al. 1983; Andrezik and Beitz 1985; Shipley et al. 1987; Li et al. 1990), and neurons that contain amino-acid transmitters, such as 7-aminobutyric acid, glutamate, or aspartate (Beitz 1990b; Reichling and Basbaum 1990). What is the nature of the information transmitted from the NTS and the DMM to the TH-ir and 5-HT-ir
neurons in the DR? The medial NTS is the major recipient of visceral sensory afferents carried in the glossopharyngeal and the vagus nerve. These afferents include baroreceptive and chemoreceptive fibers of the carotid sinus and aortic branch, and chemoreceptive and mechanoreceptive subdiaphragmatic vagal fibers from the intestine, stomach, and the hepatic portal vein (Leslie et al. 1982; Rogers and Hermann 1983; Higgins et al, 1984; Shapiro and Miselis 1985; Housley et al. 1987). Sumal and coworkers (1983) have provided ultrastructural evidence that the vagal fibers in the medial NTS synapse on catecholaminergic cells of the A2/C2 group and non-catecholaminergic neurons. These cells in the medial NTS are therefore in a position to transmit visceral sensory information of various modalities to the DR and thus, may provide information about the actual autonomic status, Little information is available about the functions of the rostral DMM, including the C3 adrenergic cell group. Electrical and chemical stimulation in this area result in a pressor response (Ross et al. 1984; Chai et al. 1988; Lin et al. 1989), thereby indicating a role of the DMM in autonomic regulation. This is supported by anatomical findings showing that adrenergic and nonadrenergic neurons of the DMM project to the paraventricular nucleus of the hypothalamus (Cunningham et al. 1990) and to spinal preganglionic neurons in the intermediolateral cell column (Minson et al. 1990) where they may directly modulate sympathetic outflow. It is interesting to note that adrenergic and non-adrenergic neu-
154
Fig. 4A-G. Dual-immunostainedsections through the DR after an injection of PHA-L into the rostral DMM. A-C Black-stained PHA-L-ir fibers and varicositiescloselyapposed to 5-HT-ir somata and dendrites. Arrows indicatepresumed synapticcontacts between black-stained varicosities and lighter-stained postsynaptic targets.
D-G Black-stained PHA-L-ir fibers and varicosities ciosely apposed to TH-ir somata and dendritic processes. Note the smaller soma size of dopaminergic (D, E) versus serotonergicneurons (AC). Bar: 10 gm
rons of the DMM are also major sources of projections to the locus coeruleus (Aston-Jones et al. 1986; Pieribone etal. 1988; Pieribone and Aston-Jones 1991), which resembles the DR with respect to its widespread projections in the brain and its involvement in global brain functions, such as behavioral arousal, vigilance, emotion, or sleep (Aston-Jones and Bloom 1981 ; Foote et al. 1983; Aston-Jones et al. 1986). It is tempting to speculate that neurons in the DMM co-regulate the sympathetic outflow and the activity of major transmitter systems, including the noradrenergic locus coeruleus group and the dopaminergic and serotonergic DR group. A large number of studies have documented the anatomical connections of the DR, including the B7 serotonergic cell group, which comprises approximately one third of the total DR cell population (Descarries et al. 1982). A widespread distribution of ascending projections to various diencephalic and telencephalic regions has been reported (for reviews, see Steinbusch 1984; T6rk 1985; Vertes 1991). The diverse forebrain projections are in agreement with the diverse functions influenced by this cell group, for example, locomotor activity, thermoregulation, slow-wave sleep, and sexual behavior (see, e.g., Vertes 1990; Hillegaart 1991). The DR neurons have a highly collateralized axon
system by which multiple targets can be modulated simultaneously (see, e.g., de Olmos and Heimer 1980; Vil, lar et al. 1988). However, there is also a certain degree of topographic organization with respect to the rostral versus caudal DR efferents (see, e.g., Imai et al, 1986; Vertes 1991) and with respect to the "lateral wings" of the B7 group (O'Hearn and Molliver 1984; Villar et al. 1987, 1988; Klepper and Herbert 1991). It has previously been shown that the DMM projection is evenly distributed in the rostrocaudal dimension of the DR and in the "lateral wings" (Herbert and Saper 1992). Thus, the DMM may equally influence all aspects of the serotonergic and non-serotonergic forebrain projections that arise in the DR. In contrast, projections from the NTS terminate predominantly in the "lateral wings" of the DR and in the rostral midline portion, whereas the caudal median DR is almost devoid of NTS fibers (Herbert and Saper 1992). Consequently, the NTS may modulate the serotonergic input to visual and auditory nuclei, an input that arises exclusively in the "lateral wings" of the B7 group (Villar et al. 1987, 1988; Klepper and Herbert 1991); it may also modulate the serotonergic input to all regions of the cortex, caudate-putamen, substantia nigra, amygdala, lateral hypothalamus, preoptic area, and the substantia innominata, an input that arises predominantly in the rostral DR and in the "later-
155 al wings" (see, e.g., Imai etal. 1986; Hallanger and Wainer 1988; Vertes 1991). Serotonergic and non-serotonergic neurons in the caudal DR, i.e., neurons that project predominantly to the frontal cortex, lateral septum, bed nucleus of the stria terminalis, hippocampus, and the locus coeruleus, and that are thus closely related to the limbic system (Imai et al. 1986; Vertes 1991), are apparently not markedly influenced by NTS fibers. This topography indicates a role of the NTS in modulating the activity of serotonergic projections to nuclei concerned with the processing of sensory, autonomic, and m o t o r functions, whereas the D M M also appears to influence the serotonergic input to the limbic system. In a recent study, we have shown that a subpopulation of the NTS and D M M projection neurons is catecholaminergic (Herbert and Saper 1992). It is therefore conceivable that some of the varicosities, which are found in close apposition to serotonergic or dopaminergic neurons in the DR, represent adrenergic or noradrenergic synapses. This is suggested by an EM study in the D R demonstrating that a majority of noradrenergic terminals, of hitherto undefined origin, form synaptic contacts with serotonergic neurons (Baraban and Aghajanian 1981). Furthermore, physiological experiments show that noradrenalin excites serotonergic neurons in the D R , whereas a blockade o f e-adrenoceptors results in a cessation of their spontaneous activity (Baraban and Aghajanian 1980). The tonic noradrenergic input to the D R is presumably responsible for the unique pacemaker activity of the serotonergic neurons (Vandermaelen and Aghajanian 1983). Physiological data on the effect of noradrenalin or e-adrenergic antagonists on A10dc dopaminergic neurons in the D R are presently not available. The A10dc dopaminergic group consists of approximately 1000 neurons and is considered a small subpopulation of the A10 mesolimbic/mesolimbocortical dopaminergic system of the ventral tegmental area (Descarries et al. 1986; Stratford and Wirtshafter 1990). As yet, only a few studies have demonstrated projections of the A10dc cells to the prefrontal cortex, nucleus accumbens, lateral septum, hippocampus, and the neostriatum (Pohle et al. 1984; Descarries et al. 1986; Yoshida et al. 1989; Stratford and Wirtshafter 1990), indicating a role in m o t o r function and also in the regulation of emotional behavior (Fuxe et al. 1985). The pathways reported in the present study could participate in controlling the expression of these behaviors. In conclusion, this study provides evidence that the medial NTS and the rostral D M M innervate serotonergic and dopaminergic neurons in the DR, and could thus modulate the activity of these cell groups, depending on the autonomic status of an animal.
Acknowledgements. I wish to thank H. Zillus for excellent technical assistance, Drs. E. Friauf, A. Klepper, and M. Koch for valuable comments on the manuscript, and Gwynn Goldring for correcting the English. This work was supported by the Deutsche Forschungsgemeinschaft (He 1842/3-1).
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