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Distribution of serotonin cells projecting to the pontomedullary reticular formation in the cat
NEUROSCIENCE RESEARCH ELSEVIER
Neuroscience Research 20 (1994) 43-55
Distribution of serotonin cells projecting to the pontomedullary reticular formation in the cat Yoshifumi Kobayashi *a, Kiyoji Matsuyama b, Shigemi Mori b aDepartment of Physiology, Asahikawa Medical College, Nishikagura, Asahikawa, 078, Japan bNational Institute for Physiological Sciences, Myodaiji, Okazaki, 444, Japan (Received 3 February 1994; revision received 21 March 1994; accepted 4 April 1994)
Abstract
Using immunohistochemistry and retrograde transport techniques, this study demonstrates that serotonin (5-HT) cells in the dorsal tegmental gray of the pons at rostrocaudal from P1 to P6 levels are sources of bilateral serotonergic projections to the gigantocellular tegmental field of the medial pontine and medullary reticular formation in the cat. The cells observed were a relatively homogeneous population of small (30/zm) to medium-sized (40/~m) cells, oval, fusiform or spindle-shaped. Rostrally and caudally located 5-HT cells in the dorsal tegmental gray tended to project to the pontine and medullary reticular formation respectively. The dendrites of these cells spread in a plane perpendicular to the long axis of the brainstem. Thin 5-HT fibers with a series of fine varicosities (0.5-1.0/zm in diameter) surrounded reticular neurons, and some of these varicosities closely apposed the proximal dendrites and somata of the reticular neurons. Both large (50/zm) and small (20-30 #m) reticular neurons were uniformly innervated by 5-HT fibers. As revealed in the present study, the serotonergic system, together with the monoaminergic and cholinergic systems, seems to be involved in behavioral state regulation.
Keywords." Serotonin (5-HT); Cholera-toxin B subunit (CTb); Dorsal tegmental gray; Pontomedullary reticular formation; Cat; Double labeling; Pontomedullary reticular neurons
1. Introduction
The distribution of monoamine-containing neurons was originally described by Dahlstr6m and Fuxe (1964, 1965) in the rat brainstem, and subsequently in the cat brainstem by Pin et al. (1968) and Poitras and Parnet (1978). The distribution, morphology and number of serotonin (5-HT) cells within the brainstem of the cat were first extensively studied by Wiklund et al. (1981) with the Falck-Hillarp fluorescence histochemical technique. Using an antibody which is highly specific and sensitive to the 5-HT molecule, Jacobs et al. (1984) also studied the localization and relative number of 5-HT cells in the brainstem of the cat. These studies led to a schematic atlas of 5-HT immunoreactive cells. In agree* Corresponding author, Tel.: +81 166 65 2111; Fax: +81 166 65 7563.
ment with previous studies in the rat (Steinbusch et al., 1978; Steinbusch, 1981; Lidov et al., 1982), a great majority of 5-HT cells were located in the raphe nuclei. A significant number of 5-HT cells were located outside the raphe nuclei at all brainstem levels. Distribution of 5-HT in the central nervous system of the rat was also studied with the use of a specific and sensitive enzyme-isotopic technique (Palkovits et al., 1974; Saavedra et al., 1974). With regard to the brainstem, the nucleus raphe dorsalis had the highest concentration of 5-HT. The ventral periaqueductal gray and dorsal tegmental gray of the mesencephalon and pons, as well as the nuclei of the pontomedullary reticular formation, contained a moderate amount of 5-HT (Palkovits et al., 1974). With the indirect immunofluorescence technique using a highly specific and wellcharacterized antibody to 5-HT, Steinbusch (1981) also studied the distribution of 5-HT cell bodies and ter-
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Neur.s~'i. Re~. 20 ,' 1994) 43-55
minals in the rat and found 5-HT-positive nerve terminals to be widespread throughout the central nervous system. In the reticular formation, dense populations of 5-HT-immunoreactive nerve terminals were present in the dorsolateral part of the nucleus reticularis pontis oralis. The magnocellular pontine reticular formation, which is composed of the nuclei reticularis pontis oralis (NRPo) and caudalis (NRPc) (Brodal, 1957), has been implicated by a number of physiological and behavioral studies to be involved in several functional domains (Shammah-Lagnado et al., 1987), with some of their functions presumed to be under the serotonergic control of reticular neurons (Jouvet, 1969; Steriade and McCarley, 1990). A precise determination of the sources of serotonergic input to the pontomedullary reticular formation is a prerequisite for elucidation of the anatomical basis for understanding the complex biological phenomena. In this study, therefore, an attempt was made to identify the localization of the cells of origin, which contained 5-HT and projected to the mesencephalic, pontine and medullary reticular formations, by utilizing 5-HT immunohistochemistry (Sano et al., 1982; Takeuchi et al., 1982) combined with the retrograde tracing method (Luppi et al., 1990). A preliminary account of the present study has been reported in abstract form (Kobayashi et al., 1991a). 2. Materials and methods
Experiments were performed on 2 groups of cats. In the first group, 5-HT immunohistochemistry was employed in combination with retrograde tracing techniques, and in the second group, only 5-HT immunohistochemistry was employed.
2.1. 5-HT immunohistochemistry and retrograde tracing of 5-HT cells Experiments were performed on 5 adult (2 male and 3 female) cats weighing 2.0-3.1 kg. Under halothane nitrous oxide gas anesthesia, the head of the animal was fixed in a stereotaxic apparatus and a small hole was made in the calvarium. Two different micropipettes (tip diameter 20/zm) were prepared for the stereotaxic injections and filled with a 1.0% solution of horseradish peroxidase (HRP)-conjugated cholera-toxin B subunit (CTb-HRP) (List Biological Laboratories, USA) or a 0.05% solution of CTb-conjugated fluorescein isothiocyanate (CTb-FITC) (List Biological Laboratories, USA) dissolved in 0.1 M sodium phosphate buffered saline (pH 7.4). These micropipettes were inserted into the brainstem through the cerebellum at an angle of 30° with respect to the horizontal plane. Before injecting CTb-HRP and CTb-FITC solutions into the brainstem, stereotaxic coordinates for these two
pipettes were carefully calibrated so that the tips of the pipettes were located at the same position. Each injection of 0.5-1.0 ~1 was performed over a period of 15-20 min by means of a micrometer-driven oil pressure pump attached to the injecting micropipette (Matsuyama et al., 1988, 1993; Ohta et al., 1988). After termination of" the injection, the pipette was left in place for an additional 15 rain. CTb-FITC was injected into the sites at which CTb-HRP had previously been injected. In each animal, CTb-HRP and CTb-FITC were injected into one of the following regions on the left side: the medial portion of the caudal mesencephalic and rostral pontine (Horsley-Clarke coordinates, P1 to P3, LI.5, H-3 to H-5), caudal pontine (P2 to P4, L1.5, H-3 to H-5) and rostrat medullary reticular formation (P8 to P10, L I.5, H-5 to H-8). Forty-eight hours after injection, the cats were deeply anesthetized with pentobarbital (40 mg/kg, i.p.) and perfused transcardially with 0.01 M phosphate buffered saline (pH 7.4) followed by 4% paraformaldehyde, 0.2% picrate and 0.35% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C, The brain was removed and postfixed for 2 days at 4°C in the same fixative without glutaraldehyde. It was then immersed in 0.1 M phosphate buffer containing 15% sucrose for at least 2 days. Serial frontal sections of the brainstem were cut with a cryotome at a thickness of 25 or 50/~m. In each set of 6 consecutive sections, the 1st, 2nd, 5th and 6th sections were cut at a thickness of 25 tzm while the 3rd and 4th sections were cut at a thickness of 50 #m. These sections with different thicknesses were used for different purposes as will be shown below. The 50 #m-thick 3rd section of each set was used for the analysis of the distribution of 5-HT-containing perikarya. These sections, in a free floating state, were incubated for 2 days with rabbit 5-HT antiserum (diluted 1:20 000) followed by incubations with biotinylated goat anti-rabbit lgG (2 h) and avidinbiotin-peroxidase complex (1 h). Finally, the sections were reacted with 0.02% 3,3'-diaminobenzidine (DAB), 0.3% nickel ammonium sulfate and 0.005% H202 in 0.05 M Tris-HC1 buffer (Kimura et al., 1981; Takeuchi et al., 1982). The 50 ~tm-thick 4th section of each set was used for CTb-HRP retrograde tracing. These sections were reacted with 3,3',5,5'-tetramethylbenzidine (TMB) (Mesulam, 1982). The 25 p.m-thick sections were used for identification of both CTb-F1TC for the retrograde labeling and tetramethylrhodamine isothiocyanate (TRITC) for immunohistochemistry of the 5HT-positive cells. They were incubated overnight with rabbit 5-HT antiserum (diluted 1:200) followed by goat anti-rabbit |gG coupled to TRITC (List Biological Laboratories, USA). The 50/~m-thick sections were mounted on gelatincoated glass slides, lightly counterstained with neutral red, dehydrated and cover-slipped. Sections were exam-
Y. Kobayashi et al./ Neurosci. Res. 20 (1994) 43-55
ined under a light microscope equipped with a brightfield condenser (BHB, Olympus Co., Japan; Microphoto-FX, Nikon Co., Japan). The 25 ~m-thick sections were mounted in 0.2% p-phenylenediamine and glycerol. After cover-slipping, they were examined under a fluorescence microscope equipped with an incident illuminator (Microphoto-FX, Nikon, Japan). 2.2. 5-HT immunohistochemistry of nerve terminals Experiments were performed on 5 2 female) cats weighing 2.8-3.7 kg. anesthetized with pentobarbital (40 perfused with the same fixative
adult (3 male and They were deeply mg/kg, i.p.), and described in the
45
preceding section. Serial frontal sections (50 t~m) of the brainstem were cut with a cryotome. They were reacted for 5-HT immunohistochemistry as described above. These sections were used to examine the 5-HT nerve terminals which contact neurons in the caudal mesencephalic, pontine and rostral medullary reticular formations. Camera lucida drawings were made to demonstrate such relationships. The distribution pattern of 5-HT cells was analyzed and compared with that of the CTb-HRP retrogradely labeled cells, and the CTb-FITC and TRITC doublelabeled cells. The terminology and delineation of the brainstem structures used followed those of Berman (1968) and Snider (1961).
Pl
P2
P3
P4
P5
Fig. 1. Distribution and localization of 5-HT cells identified by 5-HT immunohistochemistry and retrograde labeling. (A) 5-HT cells, (BI CTb-HRP labeled cells, (C) CTb-FITC and TRITC double-labeled cells in a single cat. Plotting of double-labeled cells was superimposed from 4 sections. In each frontal plane of the brainstem, the locations of individual cells are plotted by closed circles. Injection sites of the CTb-HRP and CTb-FITC are indicated by hatched areas in (B) and (C), respectively. Both injection sites are located in the area corresponding to the NRPo. IC, inferior colliculus; PAG, periaqueductal gray; RD, nucleus raphe dorsalis; CS, nucleus central superior; BC, brachium conjunctivum: CNF, cuneiform nucleus; DTG, dorsal tegrnental gray; MLF, medial longitudinal fasciculus; TB, trapezoid body; 5M, motor trigeminal nucleus; VS, superior vestibular nucleus; RPo, nucleus raphe pontis; SO, superior olive; P, pyramidal tract.
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~. Kobavashi et al. / Neurosci, Res, 20 f19942 4 3 - 5 5
3. Results In the brainstem, 5-HT-positive perikarya were distributed from the A 4 level rostrally, corresponding to the caudal portion of the mesencephalic reticular formation, to the PI7 level caudally, corresponding to the decussation of the pyramidal tract. On the other hand, CTbFITC and TRITC double-labeled cells were found exclusively in the dorsal tegrnental gray from P1 to P6 levels.
3.1. Location and distribution of 5-HT-positive perikarya In Figs. 1, 2 and 3, the distributions of 5-HT cells (A), CTb-HRP-labeled cells (B) and CTb-FITC and TRITC double-labeled cells (C) are shown. These results were obtained from independent experiments. Throughout the brainstem, the 5-HT cells were rather homogeneously distributed, small to medium-sized (30-40 #m in diameter), and oval, fusiform or spindle-shaped (Fig. 4).
a
P
At the caudal mesencephalic (Fig. IA) and pontme levels (Fig. 2A) from P1 to P6, 5-HT cells were concentrated in the following areas: dorsal tegmental gray, the nucleus raphe dorsalis, the nucleus centralis superior and the midline region between the medial longitudinal fasciculi (MLF) on both sides. The nucleus raphe dorsalis, which appeared most distinctly at P I and P2 levels as a dense paramedian group, contained many 5-HT cells from the ventral aspect of the MLF to the floor of the fourth ventricle. In addition to many 5-HT cells in the nucleus centralis superior, which is composed of two thin, closely apposed paramedian columns, a small number of 5-HT cells extended laterally from the ventral portion of this nucleus. There was also a moderate number of 5-HT cells throughout the ventral periaqueductal gray. Characteristic clusters of 5-HT cells were found within the dorsal tegmental gray forming two lateral wings beneath the ependymal lining of the tourth ventricle. More caudally, 5-HT cells were arranged along the midline in the nucleus raphe pontis. A few 5-HT cells
r'
P2
P3
P4
P5
P6
Fig. 2. Distribution and localization of 5-HT cells, CTb-HRP labeled cells and double-labeledcells. The results were obtained from a different animal, and the injectionsites of the CTb-HRPand CTb-FITCare locatedcaudallyto those in the cat in Fig. 1.6, abducens nucleus: 7N, facial nerve.
Y. Kobayashi et al./ Neurosci. Res. 20 (1994) 43-55
were also present adjacent the dorsal border to the trapezoid body. In the rostral medulla (Fig. 3A) from P4 to P9 levels, the 5-HT cells were located along the median raphe in the nucleus raphe obscurus, in the small ventral collection of cells in the nucleus raphe pallidus and in and around the more dorsal and rostral nucleus raphe magnus. 5-HT cells were also distributed in the medullary reticular formation dorsolateral to the inferior olivary complex in an arc extending to the ventrolateral margin of the medulla. In the nucleus reticularis lateralis, lateral and ventral to the medullary raphe nuclear complex, many polygonal and fusiform neurons were found to be 5-HT positive. At the posterior level of the inferior olivary complex, these cells were concentrated in an area close to the ventrolateral surface of the medulla.
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3.2. Location of CTb-HRP retrogradely labeled cells in the brainstem CTb-HRP microinjections into the caudal mesencephalic, pontine and rostral medullary reticular formations resulted in retrograde labeling of cells throughout the brainstem. Injection areas of CTb-HRP included the NRPo, the NRPc (Figs. 1B and 2B) and the nucleus reticularis gigantocellularis (NRGc) (Fig. 3B). In all cases, the largest axes of the injection areas in the mediolateral, dorsoventral and rostrocaudal directions were 3.0, 2.0 and 2.5 mm, respectively. Following injections of CTb-HRP into the left NRPo or NRPc at P1 to P4 levels, many retrogradely labeled cells were found in the ventral periaqueductal gray and the dorsal tegrnental gray. A moderate number of cells were present in the caudal mesencephalic and pontine
E P4
P5
P6
P8
P9
PIO
Fig. 3. Distribution and localization of 5-HT cells, CTb-HRP labeled cells and double-labeled cells. Injection sites of CTb-HRP and the CTb-FITC are located in the medullary reticular formation. VLD, lateral vestibular nucleus; 5ST, spinal trigeminal tract; 7, facial nucleus; RM, nucleus raphe magnus; VIN, inferior vestibular nucleus; RO, nucleus raphe obscurus; NRGc, nucleus reticularis gigantocelluralis; PH, nucleus prepositus hypoglossi; VM, medial vestibular nucleus; RP, nucleus raphe pallidus; IO, inferior olive; LR, lateral reticular nucleus.
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~ Kohayashi eta/. i NeuroscL Re,s. 20 ~1994) 43-55
[
A
Fig. 4. Microphotographs on the left column are the CTb-FITC labeled cells, while those on the right column are the same cells with TRITC-posit~ve reaction. All the double-labeled cells were located in the dorsal tegmental gray. Scale bar = 50 #m. See text for details.
Y. Kobayashi et aL /Neurosci. Res. 20 (1994) 43-55
reticular formations bilaterally. Other pontine structures containing a fewer number of labeled cells were regions dorsal and ventral to the brachium conjunctivum, ventral to the nucleus trigeminus, the nucleus raphe dorsalis and the nucleus raphe pontis. Following injections of CTb-HRP into the left NRGc at P8 to P10 levels, a large number of retrogradely labeled cells were present in the dorsal tegmental gray and the vestibular nuclear complex bilaterally. A moderate number of cells were located in the pontine reticular formation (PRF) bilaterally. Other medullary structures, such as the nucleus prepositus hypoglossi and the area ventral to the brachium conjunctivum, contained only a few labeled cells. 3.3. Location of CTb-FITC and T R I T C double-labeled cells
The present study of dual retrograde transport and immunofluorescence labeling revealed 5-HT cells which
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varied in distribution, and provided evidence for substantial 5-HT input to the pontomedullary reticular formation. Injection areas of CTb-FITC included the NRPo (Fig. IC), the NRPc (Fig. 2C) and the NRGc (Fig. 3C). In all cases, the largest axes of the injection areas in mediolateral, dorsoventral and rostrocaudal directions were 1.5, 2.0 and 2.5 mm, respectively. Following injection of CTb-FITC into the left NRPo at PI to P3 levels and into the left NRPc at P2 to P4 levels, CTb-FITC and TRITC (for 5-HT labeling) doublelabeled cells were found exclusively in the dorsal tegmental gray bilaterally, just beneath the floor of the fourth ventricle from P1 to P5 levels (Fig. 1C), and from P2 to P6 levels (Fig. 2C), respectively. The total number of double-labeled cells in the dorsal tegmental gray was larger on the side of CTb-FITC injection. When the injection sites of CTb-FITC were in the left NRGc at P8 to P10 levels (Fig. 3C), double-labeled cells were again found exclusively in the dorsal tegmental gray from P4 to P6 levels bilaterally, with almost equal numbers of
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.
.
Fig. 5.5-HT terminal fibers and large-sized reticular neurons located in the rostral (A) and caudal (B) mesencephalic reticular formation. Left column: microphotographs of mesencephalic reticular neurons and 5-HT-immunoreactive fibers. Right column: camera lucida drawings of them. Scale bar = 50 t~m.
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Y. Kobayasht et a l . / Neurosci. Res. 20 ( 1 9 9 4 ) 4 3 - 5 5
cells on both sides. The dendrites of most of the doublelabeled cells were spread in a plane perpendicular to the long axis of the brainstem. Representative cells double labeled with CTb-FITC and TRITC are shown in Fig. 4 (arrows). With optimal filter settings, differences in CTb-FITC and TRITC labeling could be distinguished. Labeled cells were located in the dorsal tegrnental gray at levels of the caudal mesencephalon (A), pons (B) and rostral medulla (C). CTb-FITC revealed clear labeling of cell soma (left column), while TRITC revealed labeling of both somata and proximal dendrites (right column). In addition, 5HT-positive terminal fibers and their varicosities were observed with TRITC (right column). Double-labeled cells were distributed among other cells labeled with TRITC or CTb-FITC alone. The results clearly indicated that cells in the mesencephalic, pontine and medullary reticular formations are innervated selectively
by only those 5-HT cells which are located in the dorsal tegrnental gray. Some topological organization was also found; e.g., rostrally and caudally located 5-HT cells in the dorsal tegmental gray innervate the pontine and medullary reticular formations, respectively.
3.4. lnnervation of mesencephalic, pontine and medullary reticular neurons with 5-HT-positive terminals In the mesencephalic and pontomedullary reticular formations, 5-HT fibers usually did not form compact and distinct fiber bundles as observed in the pyramidal tract and the MLF (Sano et al., 1982). Instead, 5-HT fibers and their terminals formed mesh-like plexuses. DAB-reacted sections revealed a number of fine black elements representing 5-HT-immunoreactive varicosities in the reticular formation. Intervaricose segments were thin, resulting in the varicosities appearing as separate
Fig. 6. 5-HT terminal fibers and large-sized reticular neurons located in the rostral (A) and caudal {B) PRF. Scale bar = 50 #m.
Y. Kobayashi et al./Neurosci. Res. 20 (1994) 43-55
dots. Some of them closely apposed the perikarya and proximal dendrites of variously sized reticular neurons. Three sets of microphotographs and camera lucida drawings in Figs. 5, 6 and 7 illustrate representative reticular neurons in the mesencephalic, pontine and medullary reticular formations on the left side, respectively. Each camera lucida drawing was made from the corresponding micrograph of a single section. These reticular neurons were large (40-50 #m in diameter) with multipolar somata, typically with thick proximal dendrites. 5-HT fibers which surrounded reticular neurons were mostly very thin. Along each of these fibers, a series of fine varicosities (0.5-1.0 #m in diameter) was present. As can be seen from the drawings, some of the 5-HT fibers appeared to extend from every direction to the soma of neuron in (A) and form contacts with a cluster of round terminal boutons (arrows). One of the prox-
51
imal dendrites and the soma of neuron in (B) (arrows) were in contact with a number of fine varicosities which were arranged along a single terminal fiber running from the right side. On neuron in (A) in the rostral PRF (Fig. 6), a single terminal fiber with a series of fine varicosities was closely apposed to the left side of the soma (arrows). Two terminal fibers with a number of fine varicosities, running from the right side, appeared to be in contact with the two different proximal dendrites of pontine reticular neuron in (B) (arrows). A cluster of round terminal boutons was also present at the left dorsal side of the soma (short arrows). A small reticular neuron surrounded by fine varicosities was also observed (thick arrows). On the medullary reticular neurons (Fig. 7), a similar distribution pattern of 5-HT fibers on the somata and the proximal dendrites was observed. A single thin fiber, which extended towards the soma of neuron in (A) from
Fig. 7 . 5 - H T terminal fibers and large-sized reticular neurons located in the rostral (At and caudal (B) medullary reticular formation. Scale bar = 50 #m.
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Y Koha.vashi eta/. ,, Neurosci. Res. 20 (1994) 43-55
the right side of the drawing, bifurcated at a site close to the soma, ascending and descending along the right side of the soma (arrows), accompanied by a number of fine varicosities. Near neuron in (B), a single terminal fiber extended from the distal portion of the thick proximal dendrite to the soma (arrows). A series of fine varicosities was present both at the thick proximal dendrite and at the soma. The terminal fiber with fine varicosities appeared to encircle the proximal dendrite and even a part of the soma. A small reticular cell was also observed with a few fine varicosities on the soma (thick arrows). 4. Discussion
A number of neurotransmitters have been shown to affect the activity of pontomedullary reticular neurons, including acetylcholine (Ach), 5-HT, noradrenaline (NA) and histamine, all implicated in behavioral state regulation (Steriade and McCarley, 1990). Recently, sources of cholinergic projections to the gigantocellular tegmental field of the pons in the cat were identified (Mitani et al., 1988). Cells of the laterodorsal tegmental nucleus and pedunculopontine nucleus (PPN) were double labeled utilizing choline acetyltransferase (CHAT) immunohistochemistry combined with retrograde transport of WGA-HRP. In the mammalian central nervous system, the presence of 5-HT terminals and NA throughout the PRF had been demonstrated using the histochemical fluorescence method (Dahlstr6m and Fuxe, 1964). In the pons and medulla, a very high density of 5-HT fibers was observed also in the lateral portion of the reticular formation, such as the dorsolateral part of the NRPo, corresponding partly to the medial PRF (Steinbusch, 1981).
4.1. Sources of serotonergic afferents to the pontomedullary reticular formation The immunohistochemical staining of 5-HT in the brainstem of the cat showed an extensive distribution of 5-HT cell bodies, fibers and terminals. In the rat, serotonergic afferents to the medial PRF were considered to originate from the raphe nuclei at all levels, with heavier projections from the pontine rather than the medullary raphe nuclei (Semba et al., 1990). However, corresponding results have not been obtained in the cat (Poitras and Parnet, 1978; Maeda et al., 1989). The connectivities of 5-HT fibers with the pontomedullary reticular neurons have not been studied in detail (Semba, 1993). For our purposes, it was first necessary to study the distribution and location of 5-HT cells in the brainstem. Our results were similar to those of 5-HT cells previously identified in the cat brainstem (Jacobs et al., 1984). Therefore, the distribution and location of 5-HT cells
were used as a reference to study the sources ol serotonergic afferents projecting to the pontomedullary reticular formation in each animal. With our method of double labeling, cells in the dorsal tegmental gray were identified as such sources. These cells were located mainly at P1 to P6 levels, just beneath the floor of the fourth ventricle. It was also found that rostrally and caudally located 5-HT cells in the dorsal tegmental gray projected to the pontine and medullary reticular tbrmations, respectively. It is interesting that Ach cells, which projected to the cat PRF, were located in the dorsal tegmental gray laterally to the dorsal tegmental nucleus at its pericentral division and ventromedially to the mesencephalic trigeminal nucleus, partly overlapping with the location of the nucleus locus coeruleus (LC). Mitani et al. (1988) referred to this region as the laterodorsal tegmental nucleus (LDT) in the cat. In our preliminary study (Kobayashi et al., 1991b), we have also found that ChAT-reactive cells were present in the lateral part of the dorsal tegmental gray, corresponding to the LDT in addition to the PPN. Some of these ChAT-reactive cells were found to project to the medial PRF, to which medially located 5-HT cells in the dorsal tegmental gray also projected. These results indicated that the pontomedullary reticular neurons are under the control of serotonergic-cholinergic interactions (Luebke et al., 1992). There is also anatomical evidence that 5-HT cells in the dorsal and median raphe nuclei project to the LDT (Cornwall et al., 1990).
4.2. Innervation patterns of 5-HT fibers in the pontomedullary reticular formation In the brainstem, widespread networks of 5-HT fibers are present. As in the previous studies by Takeuchi et al. (1983) and Maeda et al. (1989), 5-HT fibers repeatedly ramified and anastomosed, forming mesh-like plexuses. By light microscopic examination, 5-HT fibers with a number of fine varicosities were found to form ring-like structures of various shapes and sizes. Such ring-like structures constituted an axonal reticulum that is present in almost all areas of the central nervous system (Takeuchi and Sano, 1983). It is characteristic that most of the dendrites of reticular neurons are spread out in a plane perpendicular to the long axis of the brainstem (Brodal, 1981). The reticular formation is considered a series of neuropil segments of the reticular neurons. Our results demonstrated that most of the projections of 5HT cells are also spread out in a plane perpendicular to the long axis of the reticular formation. The majority of 5-HT fibers were unmyelinated, though myelinated 5-HT fibers have been detected in some areas, i.e., in the medial forebrain bundle of the rat and monkey (Azmitia and Gannon, 1983) and in the nucleus raphe dorsalis of the monkey (Kapadia et al.~
Kobayashi et al./Neurosci. Res. 20 (1994) 43-55
1985). Of particular interest were the dense plexiform 5HT fibers present in the pontomedullary reticular formation. 5,HT varicosities frequently surrounded the somata of pontomedullary reticular neurons, interlaced with the fine 5-HT fibers. A number of fine 5-HT varicosities often closely apposed the proximal dendrites of reticular neurons. When a part of the soma or dendrite and an apposed varicosity were in the same focal plane and no space could be detected between them, observed contacts were considered to represent the sites of synaptic interaction (Van Dongen et al., 1985). Such contacts between 5-HT fibers and pontomedullary neurons were both axo-somatic and axo-dendritic. 4.3. Physiological role o f 5 - H T
The dorsal tegmental gray of the pons contains several neurotransmitter-specific nuclei located adjacent to each other, including the noradrenergic LC, the cholinergic LDT and the serotonergic dorsal raphe nuclei. Based on the fine morphology and the connectivities of the cells in the dorsal tegmental gray with the pontomedullary reticular neurons, the dorsal tegmental gray can be thought of as an integral part of the reticular formation as suggested by Sutin and Jacobowitz (1991). Due to the wide array of chemicals within cells in the dorsal tegmental gray, a suggestion has already been made that this area is a highly active region, playing some role in a variety of biological functions and behaviors such as nociception, cardiovascular function, thermoregulation and the general level of arousal (Wilkinson et al., 1991). The primary role of 5-HT cells in physiology and behavior may be to coordinate the activity of target structures with the level of behavioral arousal (Fornal and Jacobs, 1988). Intracellular recordings of rat P R F neurons in slice preparations have already demonstrated that Ach, carbachol, 5-HT and NA elicit a variety of changes in the membrane properties (Greene et al,, 1989). Stevens et al. (1989) demonstrated two major 5-HT effects on medial P R F neurons. One population of neurons responded with membrane hyperpolarization and another showed membrane depolarization. Greene and Carpenter (1985) found a population of neurons in the para-abducens reticular formation in which some neurons exhibited excitatory 5-HT and inhibitory Ach responses, while others revealed inhibitory 5-HT and NA, and excitatory Ach responses. Luebke et al. (1992) demonstrated that 5-HT induced hyperpolarization of bursting cholinergic LDT neurons in in vitro preparations. We have recently found that intrapontine microinjection of 5-HT and NA in the acutely decerebrate cat resulted in not only augmentation of postural muscle tone but also in restoration of carbachol-induced postural atonia (Mori et al., 1990; Takakusaki et al., 1993a,b). These results suggest that monoaminergic, serotonergic and cholinergic interactions take place at the
53
cellular levels of both the dorsal tegmental gray and the pontomedullary reticular formation with serotonergiccholinergic interactions providing probably one of the cellular bases of mammalian behavioral state control (Steriade and McCarley, 1990c; Luebke et al., 1992). It has been well demonstrated that 5-HT cells exhibit marked state-related changes in spontaneous activity, firing most often during the alert state, firing less often during slow-wave sleep, and not firing during rapid eye movement (REM) sleep (McGinty and Harper, 1976). These results indicate that central 5-HT cells are involved in state-related changes in physiological regulation, supporting the notion that the 5-HT system, together with the monoaminergic and cholinergic systems, plays an important role in coordinating the activity of several heterogeneous physiological and behavioral systems (Fornal and Jacobs, 1988).
Acknowledgements We express our sincere thanks to Professor H. Kimura, Shiga Medical College, for his guidance in the immunohistochemistry technique and the generous supply of 5-HT antibody. The advice and support of Dr. K. Takakusaki is greatly appreciated. Y.K. is indebted to Prof. T. Unno, Dept. of Otolaryngology, Asahikawa Medical College, for his continued encouragement during this investigation. This study was submitted by Y. Kobayashi in partial fulfillment of the requirements for a Ph.D. degree at Asahikawa Medical College. This study was supported by a grant from the Uehara Memorial Foundation to S. Mori and by a Grant-in-Aid for Encouragement of Young Scientists (A) 05780611 from the Ministry of Education, Science and Culture of Japan to Y. Kobayashi.
6. References Azmitia, E. and Gannon, P. (1983) The ultrastructural localization of serotonin immunoreactivityin myelinated and unmyelinatedaxons within the medial forebrain bundle of rat and monkey. J. Neurosci., 3: 2083-2090. Berman, A.L. (1968) The Brain Stem of the Cat: A Cytoarchitectonic Atlas with StereotaxicCoordinates, Universityof Wisconsin Press, Madison. Brodal, A. (1957) The reticular formation of the brain stem. Anatomical aspects and functional correlations. The Henderson Trust Lecture, Oliver and Boy& Edingburgh, pp. 1-87. Brodal, A. (1981) The reticular formation and some related nuclei. In: A. Brodal (Ed.), Neurological Anatomy in Relation to Clinical Medicine (Third Edn.), Oxford University Press, New York/Oxford, pp. 394-447. Cornwall, J., Cooper, J.D. and Phillipson, O.T. (1990) Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat. Brain Res. Bull., 25: 271-284. Dahlstr6m, A. and Fuxe, K. (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: 1-49.
54
Y Kobayashi eta/./Neurosci Res. 20 (1994) 43-55
Dahlstr6m, A. and Fuxe, K. (1965) Evidence lor the existence of monoamine-containing neurons in the central nervous system. 11. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron system. Acta Physiol. Scand., 64: 1-36. Fornal C.A. and Jacobs, B.L. (1988) Physiological and behavioral correlates of serotonergic single-unit activity. In: N.H. Osborne and M. Hamon (Eds.), Neuronal Serotonin, Wiley, New York, pp. 305-345. Greene, R.W. and Carpenter, D.O. (1985)Actions of neurotransmitters on pontine medial reticular formation neurons of the cat. J. Neurophysiol., 54: 520-531. Greene, R.W., Gerber, U. and McCarley, R.W. (1989) Cholinergic activation of medial pontine reticular formation neurons in vitro. Brain Res., 476:154-159. Jacobs, B.L., Gannon, P.J. and Azmitia, E.C. (1984) Atlas of serotonergic cell bodies in the cat brainstem: an immunocytochemical analysis. Brain Res. Bull., 13: 1-31. Jouvet, M. (1969) Biogenic amines and the states of sleep. Science, 163: 32-41. Kapadia, S.E., De Lanerolle, N.C. and LaMotte, C.C. (1985) lmmunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey. Neuroscience, 15: 729-746. Kimura, H., McGeer, P.L., Peng, J.H. and McGeer, E.G. (1981) The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat. J. Comp. Neurol., 200: 151-201. Kobayashi, Y., Matsuyama K. and Mori, S. (1991a) Origins of serotonergic and cholinergic neurons projecting to the nucleus reticularis pontis oralis in cats. Neurosci. Res. Suppl., 16: S105. Kobayashi, Y., Tanaka, H. and Mori, S. (1991b) Distribution for 5HT containing neurons projecting to the nucleus reticularis pontis oralis in cats. Jpn. J. Physiol. Suppl., 41: $242. Lidov, H.G.W. and Molliver, M.E. (1982) lmmunohistochemical study of the development of serotonergic neurons in the rat CNS. Brain Res. Bull., 9: 559-604. Luebke, J.l., Greene, R.W., Semba, K., Kamondi, A., McCarley, R.W. and Reiner, P.B. (1992) Serotonin hyperpolarizes cholinergic low-threshold burst neurons in the rat laterodorsal tegmental nucleus in vitro. Proc. Natl. Acad. Sci. USA, 89: 743-747. Luppi, P.H., Fort, P. and Jouvet, M. (1990) lontophoretic application of unconjugated choleratoxin B subunit (CTb) combined with immunohistochemistry of neuroehemical substances: a method for transmitter identification of retrogradely labeled neurons. Brain Res., 534: 209-224. Maeda, T., Fujimiya, M., Kitahama, K., lmai, H. and Kimura, H. (1989) Serotonin neurons and their physiological roles. Arch. Histol. Cytol., 52: 113-120. Matsuyama, K., Ohta, Y. and Mori, S. (1988) Ascending and descending projections of the nucleus reticularis gigantocellularis in the cat demonstrated by the anterograde neural tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res., 460: 124-141. Matsuyama, K., Kobayashi, Y., Takakusaki, K., Mori, S. and Kimura, H. (1993) Termination mode and branching patterns of reticuloreticular and reticulospinal fibers of the nucleus reticularis pontis oralis in the cat: an anterograde PHA-L tracing study. Neurosci. Res., 17: 9-21. McGinty, D.J. and Harper, R.M. (1976) Dorsal raphe neurons: depression of firing during sleep in cats. Brain Res., 101: 569-575. Mesulam, M.M. (1982) Principles of horseradish peroxidase neurohistochemistry and their application for tracing neural pathways-axonal transport, enzyme histochemistry and light microscopic analysis. In: M.M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase, Wiley-lnterscience, New York, pp. 1-151. Mitani, A., Ito, K., Hallanger, A.E., Wainer, B.H., Kataoka, K. and MeCarley, R.W. (1988) Cholinergic projections from the
laterodorsal and pedunculopontine tegmental nuclei to the pontinc gigantocellular tegrnental field in the cat. Brain Res, 45 ! : 397-402 Mori, S., Shimoda, N., Tanaka, H., Oka, T. and Takakusaki, K (1990) Contribution of pontine reticular formation to the full execution of controlled locomotion in decerebrate cats. Somatosens. Motor Res., 7: 246-247. Ohta, Y., Mori, S. and Kimura, H. (1988) Neuronal structures of the brainstem participating in postural suppression in cats. Neurosci. Res., 5: 181-202. Palkovits, M., Brownstein, M. and Saavedra, J.M. (1974) Serotonin content of the brain stem nuclei in the rat. Brain Res., 80: 237-249. Pin, C., Jones, B. and Jouvet, M. (1968) Topographic des neurones monoaminergiques du tronc cdrdbral du chat: &ude par histofluorescence. Comptes Rendus des Sdances de la Soci&6 de Biologie et de ses Filiales, 18: 2136-2141. Poitras, D. and Parnet, A. (1978) Atlas of the distribution of monoamine-containing nerve cell bodies in the brain stem of the cat. J. Comp. Neurol., 179: 699-718. Saavedra, J.M., Brownstein, M. and Palkovits, M. (1974) Serotonin distribution in the limbic system of the rat. Brain Res.. 79: 437-441. Sano, Y., Takeuchi, Y., Kimura, H., Goto, M., Kawata, M., Kojima, M., Matsuura, T., Ueda, S. and Yamada, H. (1982) lmmunohistochemical studies on the processes of serotonin neurons and their ramification in the central nervous system - - with regard to the possibility of the existence of Golgi's rete nervosa diffusa. Arch. Histol. Jpn., 45 305-316. Semba, K. (1993) Aminergic and cholinergic afferents to REM sleep induction regions of the pontine reticular formation in the rat. J. Comp. Neurol., 330: 543-556. Semba, K., Reiner, P.B. and Fibiger, H.C. (1990) Single cholinergic mesopontine tegmental neurons project to both the pontine reticular formation and the thalamus in the rat. Neurosci., 38: 643-654. Shammah-Lagnado, S.J., Negr~io, N., Silva, B.A. and Ricardo, J.A. (1987) Afferent connections of the nuclei reticularis pontis oralis and caudalis: a horseradish peroxidase study in the rat. Neurosci., 20: 961-989. Snider, R.S. and Niemer, W.T. (1961) A Stereotaxic Atlas of the Cat Brain, University of Chicago Press, Chicago. Steinbusch, H.W.M. (1981) Distribution of serotoninimmunoreactivity in the central nervous system of the rat cell bodies and terminals. Neurosci., 6: 557-618. Steinbusch, H.W.M., Verhofstad, A.A.J. and Joosten, H.W.J. (1978) Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications. Neurosci., 3:811-819. Steriade, M. and McCarley, R.W. (1990) Brainstem Control of Wakefulness and Sleep. Plenum, New York. Stevens, D.R., Greene, R.W. and McCarley, R.W. (1989)Excitatory and inhibitory actions of serotonin on medial pontine reticular formation neurons mediated by opposing actions on potassium conductance(s). Sleep Res., 18: 23. Sutin, E.L. and Jacobowitz, D.M. (1991) Neurochemicals in the dorsal pontine tegmentum. Prog. Brain Res., 88: 3-14. Takakusaki, K., Kohyama, J., Matsuyama, K. and Mori, S. (1993a) Synaptic mechanisms acting on lumbar motoneurons during postural augmentation induced by serotonin injection into the rostral pontine reticular formation in decerebrate cats. Exp. Brain Res., 93: 471-482. Takakusaki, K., Matsuyama, K., Kobayashi, Y., Kohyama, J. and M ori, S. (1993b) Pontine microinjection of carbachol and critical zone for inducing postural atonia in reflexively standing decerebrate cats. Neurosci. Lett., 153: 185-188. Takeuchi, Y. and Sano, Y. (1983) Immunohistochemical demonstration of serotonin-containing nerve fibers in the inferior olivary
Y. Kobayashi et.al./ Neurosci. Res. 20 (1994) 43-55 complex of the rat, cat and monkey. Cell Tissue Res., 231: 17-28. Takeuchi, Y., Kimura, H. and Sano, Y. (1982) lmmunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat. Cell Tissue Res., 224: 247-267. Takeuchi, Y., Kimura, H., Matsuura, T., Yonezawa, T. and Sano, Y. 0983) Distribution of serotonergic neurons in the central nervous system: a peroxidase-antiperoxidase study with anti-serotonin antibodies. J. Histochem. Cytochem., 31: 181-185. Van Dongen, P.A.M., H6kfelt, T., Grillner, S., Verhofstad, A. A.J. and Steinbusch, H.W.M. (1985) Possible target neurons of
55
5-hydroxytryptamine fibers in the lamprey spinal cord: immunohistochemistry combined with intracellular staining with Lucifer yellow. J. Comp. Neurol., 234: 523-535. Wiklund, L., lager, L. and Persson, M. (1981) Monoamine cell distribution in the cat brain stem. A fluorescence histochemical study with quantification of indolaminergic and locus coeruleus cell groups. J. Comp. Neurol., 203: 613-647. Wilkinson, L.O., Auerbach, S.B. and Jacobs, B.L. (1991) Extracellular serotonin levels change with behavioral state but not with pyrogeninduced hyperthermia. J. Neurosci., l I: 2732-2741.
Neuroscience Research 20 (1994) 43-55
Distribution of serotonin cells projecting to the pontomedullary reticular formation in the cat Yoshifumi Kobayashi *a, Kiyoji Matsuyama b, Shigemi Mori b aDepartment of Physiology, Asahikawa Medical College, Nishikagura, Asahikawa, 078, Japan bNational Institute for Physiological Sciences, Myodaiji, Okazaki, 444, Japan (Received 3 February 1994; revision received 21 March 1994; accepted 4 April 1994)
Abstract
Using immunohistochemistry and retrograde transport techniques, this study demonstrates that serotonin (5-HT) cells in the dorsal tegmental gray of the pons at rostrocaudal from P1 to P6 levels are sources of bilateral serotonergic projections to the gigantocellular tegmental field of the medial pontine and medullary reticular formation in the cat. The cells observed were a relatively homogeneous population of small (30/zm) to medium-sized (40/~m) cells, oval, fusiform or spindle-shaped. Rostrally and caudally located 5-HT cells in the dorsal tegmental gray tended to project to the pontine and medullary reticular formation respectively. The dendrites of these cells spread in a plane perpendicular to the long axis of the brainstem. Thin 5-HT fibers with a series of fine varicosities (0.5-1.0/zm in diameter) surrounded reticular neurons, and some of these varicosities closely apposed the proximal dendrites and somata of the reticular neurons. Both large (50/zm) and small (20-30 #m) reticular neurons were uniformly innervated by 5-HT fibers. As revealed in the present study, the serotonergic system, together with the monoaminergic and cholinergic systems, seems to be involved in behavioral state regulation.
Keywords." Serotonin (5-HT); Cholera-toxin B subunit (CTb); Dorsal tegmental gray; Pontomedullary reticular formation; Cat; Double labeling; Pontomedullary reticular neurons
1. Introduction
The distribution of monoamine-containing neurons was originally described by Dahlstr6m and Fuxe (1964, 1965) in the rat brainstem, and subsequently in the cat brainstem by Pin et al. (1968) and Poitras and Parnet (1978). The distribution, morphology and number of serotonin (5-HT) cells within the brainstem of the cat were first extensively studied by Wiklund et al. (1981) with the Falck-Hillarp fluorescence histochemical technique. Using an antibody which is highly specific and sensitive to the 5-HT molecule, Jacobs et al. (1984) also studied the localization and relative number of 5-HT cells in the brainstem of the cat. These studies led to a schematic atlas of 5-HT immunoreactive cells. In agree* Corresponding author, Tel.: +81 166 65 2111; Fax: +81 166 65 7563.
ment with previous studies in the rat (Steinbusch et al., 1978; Steinbusch, 1981; Lidov et al., 1982), a great majority of 5-HT cells were located in the raphe nuclei. A significant number of 5-HT cells were located outside the raphe nuclei at all brainstem levels. Distribution of 5-HT in the central nervous system of the rat was also studied with the use of a specific and sensitive enzyme-isotopic technique (Palkovits et al., 1974; Saavedra et al., 1974). With regard to the brainstem, the nucleus raphe dorsalis had the highest concentration of 5-HT. The ventral periaqueductal gray and dorsal tegmental gray of the mesencephalon and pons, as well as the nuclei of the pontomedullary reticular formation, contained a moderate amount of 5-HT (Palkovits et al., 1974). With the indirect immunofluorescence technique using a highly specific and wellcharacterized antibody to 5-HT, Steinbusch (1981) also studied the distribution of 5-HT cell bodies and ter-
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Neur.s~'i. Re~. 20 ,' 1994) 43-55
minals in the rat and found 5-HT-positive nerve terminals to be widespread throughout the central nervous system. In the reticular formation, dense populations of 5-HT-immunoreactive nerve terminals were present in the dorsolateral part of the nucleus reticularis pontis oralis. The magnocellular pontine reticular formation, which is composed of the nuclei reticularis pontis oralis (NRPo) and caudalis (NRPc) (Brodal, 1957), has been implicated by a number of physiological and behavioral studies to be involved in several functional domains (Shammah-Lagnado et al., 1987), with some of their functions presumed to be under the serotonergic control of reticular neurons (Jouvet, 1969; Steriade and McCarley, 1990). A precise determination of the sources of serotonergic input to the pontomedullary reticular formation is a prerequisite for elucidation of the anatomical basis for understanding the complex biological phenomena. In this study, therefore, an attempt was made to identify the localization of the cells of origin, which contained 5-HT and projected to the mesencephalic, pontine and medullary reticular formations, by utilizing 5-HT immunohistochemistry (Sano et al., 1982; Takeuchi et al., 1982) combined with the retrograde tracing method (Luppi et al., 1990). A preliminary account of the present study has been reported in abstract form (Kobayashi et al., 1991a). 2. Materials and methods
Experiments were performed on 2 groups of cats. In the first group, 5-HT immunohistochemistry was employed in combination with retrograde tracing techniques, and in the second group, only 5-HT immunohistochemistry was employed.
2.1. 5-HT immunohistochemistry and retrograde tracing of 5-HT cells Experiments were performed on 5 adult (2 male and 3 female) cats weighing 2.0-3.1 kg. Under halothane nitrous oxide gas anesthesia, the head of the animal was fixed in a stereotaxic apparatus and a small hole was made in the calvarium. Two different micropipettes (tip diameter 20/zm) were prepared for the stereotaxic injections and filled with a 1.0% solution of horseradish peroxidase (HRP)-conjugated cholera-toxin B subunit (CTb-HRP) (List Biological Laboratories, USA) or a 0.05% solution of CTb-conjugated fluorescein isothiocyanate (CTb-FITC) (List Biological Laboratories, USA) dissolved in 0.1 M sodium phosphate buffered saline (pH 7.4). These micropipettes were inserted into the brainstem through the cerebellum at an angle of 30° with respect to the horizontal plane. Before injecting CTb-HRP and CTb-FITC solutions into the brainstem, stereotaxic coordinates for these two
pipettes were carefully calibrated so that the tips of the pipettes were located at the same position. Each injection of 0.5-1.0 ~1 was performed over a period of 15-20 min by means of a micrometer-driven oil pressure pump attached to the injecting micropipette (Matsuyama et al., 1988, 1993; Ohta et al., 1988). After termination of" the injection, the pipette was left in place for an additional 15 rain. CTb-FITC was injected into the sites at which CTb-HRP had previously been injected. In each animal, CTb-HRP and CTb-FITC were injected into one of the following regions on the left side: the medial portion of the caudal mesencephalic and rostral pontine (Horsley-Clarke coordinates, P1 to P3, LI.5, H-3 to H-5), caudal pontine (P2 to P4, L1.5, H-3 to H-5) and rostrat medullary reticular formation (P8 to P10, L I.5, H-5 to H-8). Forty-eight hours after injection, the cats were deeply anesthetized with pentobarbital (40 mg/kg, i.p.) and perfused transcardially with 0.01 M phosphate buffered saline (pH 7.4) followed by 4% paraformaldehyde, 0.2% picrate and 0.35% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C, The brain was removed and postfixed for 2 days at 4°C in the same fixative without glutaraldehyde. It was then immersed in 0.1 M phosphate buffer containing 15% sucrose for at least 2 days. Serial frontal sections of the brainstem were cut with a cryotome at a thickness of 25 or 50/~m. In each set of 6 consecutive sections, the 1st, 2nd, 5th and 6th sections were cut at a thickness of 25 tzm while the 3rd and 4th sections were cut at a thickness of 50 #m. These sections with different thicknesses were used for different purposes as will be shown below. The 50 #m-thick 3rd section of each set was used for the analysis of the distribution of 5-HT-containing perikarya. These sections, in a free floating state, were incubated for 2 days with rabbit 5-HT antiserum (diluted 1:20 000) followed by incubations with biotinylated goat anti-rabbit lgG (2 h) and avidinbiotin-peroxidase complex (1 h). Finally, the sections were reacted with 0.02% 3,3'-diaminobenzidine (DAB), 0.3% nickel ammonium sulfate and 0.005% H202 in 0.05 M Tris-HC1 buffer (Kimura et al., 1981; Takeuchi et al., 1982). The 50 ~tm-thick 4th section of each set was used for CTb-HRP retrograde tracing. These sections were reacted with 3,3',5,5'-tetramethylbenzidine (TMB) (Mesulam, 1982). The 25 p.m-thick sections were used for identification of both CTb-F1TC for the retrograde labeling and tetramethylrhodamine isothiocyanate (TRITC) for immunohistochemistry of the 5HT-positive cells. They were incubated overnight with rabbit 5-HT antiserum (diluted 1:200) followed by goat anti-rabbit |gG coupled to TRITC (List Biological Laboratories, USA). The 50/~m-thick sections were mounted on gelatincoated glass slides, lightly counterstained with neutral red, dehydrated and cover-slipped. Sections were exam-
Y. Kobayashi et al./ Neurosci. Res. 20 (1994) 43-55
ined under a light microscope equipped with a brightfield condenser (BHB, Olympus Co., Japan; Microphoto-FX, Nikon Co., Japan). The 25 ~m-thick sections were mounted in 0.2% p-phenylenediamine and glycerol. After cover-slipping, they were examined under a fluorescence microscope equipped with an incident illuminator (Microphoto-FX, Nikon, Japan). 2.2. 5-HT immunohistochemistry of nerve terminals Experiments were performed on 5 2 female) cats weighing 2.8-3.7 kg. anesthetized with pentobarbital (40 perfused with the same fixative
adult (3 male and They were deeply mg/kg, i.p.), and described in the
45
preceding section. Serial frontal sections (50 t~m) of the brainstem were cut with a cryotome. They were reacted for 5-HT immunohistochemistry as described above. These sections were used to examine the 5-HT nerve terminals which contact neurons in the caudal mesencephalic, pontine and rostral medullary reticular formations. Camera lucida drawings were made to demonstrate such relationships. The distribution pattern of 5-HT cells was analyzed and compared with that of the CTb-HRP retrogradely labeled cells, and the CTb-FITC and TRITC doublelabeled cells. The terminology and delineation of the brainstem structures used followed those of Berman (1968) and Snider (1961).
Pl
P2
P3
P4
P5
Fig. 1. Distribution and localization of 5-HT cells identified by 5-HT immunohistochemistry and retrograde labeling. (A) 5-HT cells, (BI CTb-HRP labeled cells, (C) CTb-FITC and TRITC double-labeled cells in a single cat. Plotting of double-labeled cells was superimposed from 4 sections. In each frontal plane of the brainstem, the locations of individual cells are plotted by closed circles. Injection sites of the CTb-HRP and CTb-FITC are indicated by hatched areas in (B) and (C), respectively. Both injection sites are located in the area corresponding to the NRPo. IC, inferior colliculus; PAG, periaqueductal gray; RD, nucleus raphe dorsalis; CS, nucleus central superior; BC, brachium conjunctivum: CNF, cuneiform nucleus; DTG, dorsal tegrnental gray; MLF, medial longitudinal fasciculus; TB, trapezoid body; 5M, motor trigeminal nucleus; VS, superior vestibular nucleus; RPo, nucleus raphe pontis; SO, superior olive; P, pyramidal tract.
46
~. Kobavashi et al. / Neurosci, Res, 20 f19942 4 3 - 5 5
3. Results In the brainstem, 5-HT-positive perikarya were distributed from the A 4 level rostrally, corresponding to the caudal portion of the mesencephalic reticular formation, to the PI7 level caudally, corresponding to the decussation of the pyramidal tract. On the other hand, CTbFITC and TRITC double-labeled cells were found exclusively in the dorsal tegrnental gray from P1 to P6 levels.
3.1. Location and distribution of 5-HT-positive perikarya In Figs. 1, 2 and 3, the distributions of 5-HT cells (A), CTb-HRP-labeled cells (B) and CTb-FITC and TRITC double-labeled cells (C) are shown. These results were obtained from independent experiments. Throughout the brainstem, the 5-HT cells were rather homogeneously distributed, small to medium-sized (30-40 #m in diameter), and oval, fusiform or spindle-shaped (Fig. 4).
a
P
At the caudal mesencephalic (Fig. IA) and pontme levels (Fig. 2A) from P1 to P6, 5-HT cells were concentrated in the following areas: dorsal tegmental gray, the nucleus raphe dorsalis, the nucleus centralis superior and the midline region between the medial longitudinal fasciculi (MLF) on both sides. The nucleus raphe dorsalis, which appeared most distinctly at P I and P2 levels as a dense paramedian group, contained many 5-HT cells from the ventral aspect of the MLF to the floor of the fourth ventricle. In addition to many 5-HT cells in the nucleus centralis superior, which is composed of two thin, closely apposed paramedian columns, a small number of 5-HT cells extended laterally from the ventral portion of this nucleus. There was also a moderate number of 5-HT cells throughout the ventral periaqueductal gray. Characteristic clusters of 5-HT cells were found within the dorsal tegmental gray forming two lateral wings beneath the ependymal lining of the tourth ventricle. More caudally, 5-HT cells were arranged along the midline in the nucleus raphe pontis. A few 5-HT cells
r'
P2
P3
P4
P5
P6
Fig. 2. Distribution and localization of 5-HT cells, CTb-HRP labeled cells and double-labeledcells. The results were obtained from a different animal, and the injectionsites of the CTb-HRPand CTb-FITCare locatedcaudallyto those in the cat in Fig. 1.6, abducens nucleus: 7N, facial nerve.
Y. Kobayashi et al./ Neurosci. Res. 20 (1994) 43-55
were also present adjacent the dorsal border to the trapezoid body. In the rostral medulla (Fig. 3A) from P4 to P9 levels, the 5-HT cells were located along the median raphe in the nucleus raphe obscurus, in the small ventral collection of cells in the nucleus raphe pallidus and in and around the more dorsal and rostral nucleus raphe magnus. 5-HT cells were also distributed in the medullary reticular formation dorsolateral to the inferior olivary complex in an arc extending to the ventrolateral margin of the medulla. In the nucleus reticularis lateralis, lateral and ventral to the medullary raphe nuclear complex, many polygonal and fusiform neurons were found to be 5-HT positive. At the posterior level of the inferior olivary complex, these cells were concentrated in an area close to the ventrolateral surface of the medulla.
47
3.2. Location of CTb-HRP retrogradely labeled cells in the brainstem CTb-HRP microinjections into the caudal mesencephalic, pontine and rostral medullary reticular formations resulted in retrograde labeling of cells throughout the brainstem. Injection areas of CTb-HRP included the NRPo, the NRPc (Figs. 1B and 2B) and the nucleus reticularis gigantocellularis (NRGc) (Fig. 3B). In all cases, the largest axes of the injection areas in the mediolateral, dorsoventral and rostrocaudal directions were 3.0, 2.0 and 2.5 mm, respectively. Following injections of CTb-HRP into the left NRPo or NRPc at P1 to P4 levels, many retrogradely labeled cells were found in the ventral periaqueductal gray and the dorsal tegrnental gray. A moderate number of cells were present in the caudal mesencephalic and pontine
E P4
P5
P6
P8
P9
PIO
Fig. 3. Distribution and localization of 5-HT cells, CTb-HRP labeled cells and double-labeled cells. Injection sites of CTb-HRP and the CTb-FITC are located in the medullary reticular formation. VLD, lateral vestibular nucleus; 5ST, spinal trigeminal tract; 7, facial nucleus; RM, nucleus raphe magnus; VIN, inferior vestibular nucleus; RO, nucleus raphe obscurus; NRGc, nucleus reticularis gigantocelluralis; PH, nucleus prepositus hypoglossi; VM, medial vestibular nucleus; RP, nucleus raphe pallidus; IO, inferior olive; LR, lateral reticular nucleus.
48
~ Kohayashi eta/. i NeuroscL Re,s. 20 ~1994) 43-55
[
A
Fig. 4. Microphotographs on the left column are the CTb-FITC labeled cells, while those on the right column are the same cells with TRITC-posit~ve reaction. All the double-labeled cells were located in the dorsal tegmental gray. Scale bar = 50 #m. See text for details.
Y. Kobayashi et aL /Neurosci. Res. 20 (1994) 43-55
reticular formations bilaterally. Other pontine structures containing a fewer number of labeled cells were regions dorsal and ventral to the brachium conjunctivum, ventral to the nucleus trigeminus, the nucleus raphe dorsalis and the nucleus raphe pontis. Following injections of CTb-HRP into the left NRGc at P8 to P10 levels, a large number of retrogradely labeled cells were present in the dorsal tegmental gray and the vestibular nuclear complex bilaterally. A moderate number of cells were located in the pontine reticular formation (PRF) bilaterally. Other medullary structures, such as the nucleus prepositus hypoglossi and the area ventral to the brachium conjunctivum, contained only a few labeled cells. 3.3. Location of CTb-FITC and T R I T C double-labeled cells
The present study of dual retrograde transport and immunofluorescence labeling revealed 5-HT cells which
49
varied in distribution, and provided evidence for substantial 5-HT input to the pontomedullary reticular formation. Injection areas of CTb-FITC included the NRPo (Fig. IC), the NRPc (Fig. 2C) and the NRGc (Fig. 3C). In all cases, the largest axes of the injection areas in mediolateral, dorsoventral and rostrocaudal directions were 1.5, 2.0 and 2.5 mm, respectively. Following injection of CTb-FITC into the left NRPo at PI to P3 levels and into the left NRPc at P2 to P4 levels, CTb-FITC and TRITC (for 5-HT labeling) doublelabeled cells were found exclusively in the dorsal tegmental gray bilaterally, just beneath the floor of the fourth ventricle from P1 to P5 levels (Fig. 1C), and from P2 to P6 levels (Fig. 2C), respectively. The total number of double-labeled cells in the dorsal tegmental gray was larger on the side of CTb-FITC injection. When the injection sites of CTb-FITC were in the left NRGc at P8 to P10 levels (Fig. 3C), double-labeled cells were again found exclusively in the dorsal tegmental gray from P4 to P6 levels bilaterally, with almost equal numbers of
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.
.
Fig. 5.5-HT terminal fibers and large-sized reticular neurons located in the rostral (A) and caudal (B) mesencephalic reticular formation. Left column: microphotographs of mesencephalic reticular neurons and 5-HT-immunoreactive fibers. Right column: camera lucida drawings of them. Scale bar = 50 t~m.
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Y. Kobayasht et a l . / Neurosci. Res. 20 ( 1 9 9 4 ) 4 3 - 5 5
cells on both sides. The dendrites of most of the doublelabeled cells were spread in a plane perpendicular to the long axis of the brainstem. Representative cells double labeled with CTb-FITC and TRITC are shown in Fig. 4 (arrows). With optimal filter settings, differences in CTb-FITC and TRITC labeling could be distinguished. Labeled cells were located in the dorsal tegrnental gray at levels of the caudal mesencephalon (A), pons (B) and rostral medulla (C). CTb-FITC revealed clear labeling of cell soma (left column), while TRITC revealed labeling of both somata and proximal dendrites (right column). In addition, 5HT-positive terminal fibers and their varicosities were observed with TRITC (right column). Double-labeled cells were distributed among other cells labeled with TRITC or CTb-FITC alone. The results clearly indicated that cells in the mesencephalic, pontine and medullary reticular formations are innervated selectively
by only those 5-HT cells which are located in the dorsal tegrnental gray. Some topological organization was also found; e.g., rostrally and caudally located 5-HT cells in the dorsal tegmental gray innervate the pontine and medullary reticular formations, respectively.
3.4. lnnervation of mesencephalic, pontine and medullary reticular neurons with 5-HT-positive terminals In the mesencephalic and pontomedullary reticular formations, 5-HT fibers usually did not form compact and distinct fiber bundles as observed in the pyramidal tract and the MLF (Sano et al., 1982). Instead, 5-HT fibers and their terminals formed mesh-like plexuses. DAB-reacted sections revealed a number of fine black elements representing 5-HT-immunoreactive varicosities in the reticular formation. Intervaricose segments were thin, resulting in the varicosities appearing as separate
Fig. 6. 5-HT terminal fibers and large-sized reticular neurons located in the rostral (A) and caudal {B) PRF. Scale bar = 50 #m.
Y. Kobayashi et al./Neurosci. Res. 20 (1994) 43-55
dots. Some of them closely apposed the perikarya and proximal dendrites of variously sized reticular neurons. Three sets of microphotographs and camera lucida drawings in Figs. 5, 6 and 7 illustrate representative reticular neurons in the mesencephalic, pontine and medullary reticular formations on the left side, respectively. Each camera lucida drawing was made from the corresponding micrograph of a single section. These reticular neurons were large (40-50 #m in diameter) with multipolar somata, typically with thick proximal dendrites. 5-HT fibers which surrounded reticular neurons were mostly very thin. Along each of these fibers, a series of fine varicosities (0.5-1.0 #m in diameter) was present. As can be seen from the drawings, some of the 5-HT fibers appeared to extend from every direction to the soma of neuron in (A) and form contacts with a cluster of round terminal boutons (arrows). One of the prox-
51
imal dendrites and the soma of neuron in (B) (arrows) were in contact with a number of fine varicosities which were arranged along a single terminal fiber running from the right side. On neuron in (A) in the rostral PRF (Fig. 6), a single terminal fiber with a series of fine varicosities was closely apposed to the left side of the soma (arrows). Two terminal fibers with a number of fine varicosities, running from the right side, appeared to be in contact with the two different proximal dendrites of pontine reticular neuron in (B) (arrows). A cluster of round terminal boutons was also present at the left dorsal side of the soma (short arrows). A small reticular neuron surrounded by fine varicosities was also observed (thick arrows). On the medullary reticular neurons (Fig. 7), a similar distribution pattern of 5-HT fibers on the somata and the proximal dendrites was observed. A single thin fiber, which extended towards the soma of neuron in (A) from
Fig. 7 . 5 - H T terminal fibers and large-sized reticular neurons located in the rostral (At and caudal (B) medullary reticular formation. Scale bar = 50 #m.
52
Y Koha.vashi eta/. ,, Neurosci. Res. 20 (1994) 43-55
the right side of the drawing, bifurcated at a site close to the soma, ascending and descending along the right side of the soma (arrows), accompanied by a number of fine varicosities. Near neuron in (B), a single terminal fiber extended from the distal portion of the thick proximal dendrite to the soma (arrows). A series of fine varicosities was present both at the thick proximal dendrite and at the soma. The terminal fiber with fine varicosities appeared to encircle the proximal dendrite and even a part of the soma. A small reticular cell was also observed with a few fine varicosities on the soma (thick arrows). 4. Discussion
A number of neurotransmitters have been shown to affect the activity of pontomedullary reticular neurons, including acetylcholine (Ach), 5-HT, noradrenaline (NA) and histamine, all implicated in behavioral state regulation (Steriade and McCarley, 1990). Recently, sources of cholinergic projections to the gigantocellular tegmental field of the pons in the cat were identified (Mitani et al., 1988). Cells of the laterodorsal tegmental nucleus and pedunculopontine nucleus (PPN) were double labeled utilizing choline acetyltransferase (CHAT) immunohistochemistry combined with retrograde transport of WGA-HRP. In the mammalian central nervous system, the presence of 5-HT terminals and NA throughout the PRF had been demonstrated using the histochemical fluorescence method (Dahlstr6m and Fuxe, 1964). In the pons and medulla, a very high density of 5-HT fibers was observed also in the lateral portion of the reticular formation, such as the dorsolateral part of the NRPo, corresponding partly to the medial PRF (Steinbusch, 1981).
4.1. Sources of serotonergic afferents to the pontomedullary reticular formation The immunohistochemical staining of 5-HT in the brainstem of the cat showed an extensive distribution of 5-HT cell bodies, fibers and terminals. In the rat, serotonergic afferents to the medial PRF were considered to originate from the raphe nuclei at all levels, with heavier projections from the pontine rather than the medullary raphe nuclei (Semba et al., 1990). However, corresponding results have not been obtained in the cat (Poitras and Parnet, 1978; Maeda et al., 1989). The connectivities of 5-HT fibers with the pontomedullary reticular neurons have not been studied in detail (Semba, 1993). For our purposes, it was first necessary to study the distribution and location of 5-HT cells in the brainstem. Our results were similar to those of 5-HT cells previously identified in the cat brainstem (Jacobs et al., 1984). Therefore, the distribution and location of 5-HT cells
were used as a reference to study the sources ol serotonergic afferents projecting to the pontomedullary reticular formation in each animal. With our method of double labeling, cells in the dorsal tegmental gray were identified as such sources. These cells were located mainly at P1 to P6 levels, just beneath the floor of the fourth ventricle. It was also found that rostrally and caudally located 5-HT cells in the dorsal tegmental gray projected to the pontine and medullary reticular tbrmations, respectively. It is interesting that Ach cells, which projected to the cat PRF, were located in the dorsal tegmental gray laterally to the dorsal tegmental nucleus at its pericentral division and ventromedially to the mesencephalic trigeminal nucleus, partly overlapping with the location of the nucleus locus coeruleus (LC). Mitani et al. (1988) referred to this region as the laterodorsal tegmental nucleus (LDT) in the cat. In our preliminary study (Kobayashi et al., 1991b), we have also found that ChAT-reactive cells were present in the lateral part of the dorsal tegmental gray, corresponding to the LDT in addition to the PPN. Some of these ChAT-reactive cells were found to project to the medial PRF, to which medially located 5-HT cells in the dorsal tegmental gray also projected. These results indicated that the pontomedullary reticular neurons are under the control of serotonergic-cholinergic interactions (Luebke et al., 1992). There is also anatomical evidence that 5-HT cells in the dorsal and median raphe nuclei project to the LDT (Cornwall et al., 1990).
4.2. Innervation patterns of 5-HT fibers in the pontomedullary reticular formation In the brainstem, widespread networks of 5-HT fibers are present. As in the previous studies by Takeuchi et al. (1983) and Maeda et al. (1989), 5-HT fibers repeatedly ramified and anastomosed, forming mesh-like plexuses. By light microscopic examination, 5-HT fibers with a number of fine varicosities were found to form ring-like structures of various shapes and sizes. Such ring-like structures constituted an axonal reticulum that is present in almost all areas of the central nervous system (Takeuchi and Sano, 1983). It is characteristic that most of the dendrites of reticular neurons are spread out in a plane perpendicular to the long axis of the brainstem (Brodal, 1981). The reticular formation is considered a series of neuropil segments of the reticular neurons. Our results demonstrated that most of the projections of 5HT cells are also spread out in a plane perpendicular to the long axis of the reticular formation. The majority of 5-HT fibers were unmyelinated, though myelinated 5-HT fibers have been detected in some areas, i.e., in the medial forebrain bundle of the rat and monkey (Azmitia and Gannon, 1983) and in the nucleus raphe dorsalis of the monkey (Kapadia et al.~
Kobayashi et al./Neurosci. Res. 20 (1994) 43-55
1985). Of particular interest were the dense plexiform 5HT fibers present in the pontomedullary reticular formation. 5,HT varicosities frequently surrounded the somata of pontomedullary reticular neurons, interlaced with the fine 5-HT fibers. A number of fine 5-HT varicosities often closely apposed the proximal dendrites of reticular neurons. When a part of the soma or dendrite and an apposed varicosity were in the same focal plane and no space could be detected between them, observed contacts were considered to represent the sites of synaptic interaction (Van Dongen et al., 1985). Such contacts between 5-HT fibers and pontomedullary neurons were both axo-somatic and axo-dendritic. 4.3. Physiological role o f 5 - H T
The dorsal tegmental gray of the pons contains several neurotransmitter-specific nuclei located adjacent to each other, including the noradrenergic LC, the cholinergic LDT and the serotonergic dorsal raphe nuclei. Based on the fine morphology and the connectivities of the cells in the dorsal tegmental gray with the pontomedullary reticular neurons, the dorsal tegmental gray can be thought of as an integral part of the reticular formation as suggested by Sutin and Jacobowitz (1991). Due to the wide array of chemicals within cells in the dorsal tegmental gray, a suggestion has already been made that this area is a highly active region, playing some role in a variety of biological functions and behaviors such as nociception, cardiovascular function, thermoregulation and the general level of arousal (Wilkinson et al., 1991). The primary role of 5-HT cells in physiology and behavior may be to coordinate the activity of target structures with the level of behavioral arousal (Fornal and Jacobs, 1988). Intracellular recordings of rat P R F neurons in slice preparations have already demonstrated that Ach, carbachol, 5-HT and NA elicit a variety of changes in the membrane properties (Greene et al,, 1989). Stevens et al. (1989) demonstrated two major 5-HT effects on medial P R F neurons. One population of neurons responded with membrane hyperpolarization and another showed membrane depolarization. Greene and Carpenter (1985) found a population of neurons in the para-abducens reticular formation in which some neurons exhibited excitatory 5-HT and inhibitory Ach responses, while others revealed inhibitory 5-HT and NA, and excitatory Ach responses. Luebke et al. (1992) demonstrated that 5-HT induced hyperpolarization of bursting cholinergic LDT neurons in in vitro preparations. We have recently found that intrapontine microinjection of 5-HT and NA in the acutely decerebrate cat resulted in not only augmentation of postural muscle tone but also in restoration of carbachol-induced postural atonia (Mori et al., 1990; Takakusaki et al., 1993a,b). These results suggest that monoaminergic, serotonergic and cholinergic interactions take place at the
53
cellular levels of both the dorsal tegmental gray and the pontomedullary reticular formation with serotonergiccholinergic interactions providing probably one of the cellular bases of mammalian behavioral state control (Steriade and McCarley, 1990c; Luebke et al., 1992). It has been well demonstrated that 5-HT cells exhibit marked state-related changes in spontaneous activity, firing most often during the alert state, firing less often during slow-wave sleep, and not firing during rapid eye movement (REM) sleep (McGinty and Harper, 1976). These results indicate that central 5-HT cells are involved in state-related changes in physiological regulation, supporting the notion that the 5-HT system, together with the monoaminergic and cholinergic systems, plays an important role in coordinating the activity of several heterogeneous physiological and behavioral systems (Fornal and Jacobs, 1988).
Acknowledgements We express our sincere thanks to Professor H. Kimura, Shiga Medical College, for his guidance in the immunohistochemistry technique and the generous supply of 5-HT antibody. The advice and support of Dr. K. Takakusaki is greatly appreciated. Y.K. is indebted to Prof. T. Unno, Dept. of Otolaryngology, Asahikawa Medical College, for his continued encouragement during this investigation. This study was submitted by Y. Kobayashi in partial fulfillment of the requirements for a Ph.D. degree at Asahikawa Medical College. This study was supported by a grant from the Uehara Memorial Foundation to S. Mori and by a Grant-in-Aid for Encouragement of Young Scientists (A) 05780611 from the Ministry of Education, Science and Culture of Japan to Y. Kobayashi.
6. References Azmitia, E. and Gannon, P. (1983) The ultrastructural localization of serotonin immunoreactivityin myelinated and unmyelinatedaxons within the medial forebrain bundle of rat and monkey. J. Neurosci., 3: 2083-2090. Berman, A.L. (1968) The Brain Stem of the Cat: A Cytoarchitectonic Atlas with StereotaxicCoordinates, Universityof Wisconsin Press, Madison. Brodal, A. (1957) The reticular formation of the brain stem. Anatomical aspects and functional correlations. The Henderson Trust Lecture, Oliver and Boy& Edingburgh, pp. 1-87. Brodal, A. (1981) The reticular formation and some related nuclei. In: A. Brodal (Ed.), Neurological Anatomy in Relation to Clinical Medicine (Third Edn.), Oxford University Press, New York/Oxford, pp. 394-447. Cornwall, J., Cooper, J.D. and Phillipson, O.T. (1990) Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat. Brain Res. Bull., 25: 271-284. Dahlstr6m, A. and Fuxe, K. (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: 1-49.
54
Y Kobayashi eta/./Neurosci Res. 20 (1994) 43-55
Dahlstr6m, A. and Fuxe, K. (1965) Evidence lor the existence of monoamine-containing neurons in the central nervous system. 11. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron system. Acta Physiol. Scand., 64: 1-36. Fornal C.A. and Jacobs, B.L. (1988) Physiological and behavioral correlates of serotonergic single-unit activity. In: N.H. Osborne and M. Hamon (Eds.), Neuronal Serotonin, Wiley, New York, pp. 305-345. Greene, R.W. and Carpenter, D.O. (1985)Actions of neurotransmitters on pontine medial reticular formation neurons of the cat. J. Neurophysiol., 54: 520-531. Greene, R.W., Gerber, U. and McCarley, R.W. (1989) Cholinergic activation of medial pontine reticular formation neurons in vitro. Brain Res., 476:154-159. Jacobs, B.L., Gannon, P.J. and Azmitia, E.C. (1984) Atlas of serotonergic cell bodies in the cat brainstem: an immunocytochemical analysis. Brain Res. Bull., 13: 1-31. Jouvet, M. (1969) Biogenic amines and the states of sleep. Science, 163: 32-41. Kapadia, S.E., De Lanerolle, N.C. and LaMotte, C.C. (1985) lmmunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey. Neuroscience, 15: 729-746. Kimura, H., McGeer, P.L., Peng, J.H. and McGeer, E.G. (1981) The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat. J. Comp. Neurol., 200: 151-201. Kobayashi, Y., Matsuyama K. and Mori, S. (1991a) Origins of serotonergic and cholinergic neurons projecting to the nucleus reticularis pontis oralis in cats. Neurosci. Res. Suppl., 16: S105. Kobayashi, Y., Tanaka, H. and Mori, S. (1991b) Distribution for 5HT containing neurons projecting to the nucleus reticularis pontis oralis in cats. Jpn. J. Physiol. Suppl., 41: $242. Lidov, H.G.W. and Molliver, M.E. (1982) lmmunohistochemical study of the development of serotonergic neurons in the rat CNS. Brain Res. Bull., 9: 559-604. Luebke, J.l., Greene, R.W., Semba, K., Kamondi, A., McCarley, R.W. and Reiner, P.B. (1992) Serotonin hyperpolarizes cholinergic low-threshold burst neurons in the rat laterodorsal tegmental nucleus in vitro. Proc. Natl. Acad. Sci. USA, 89: 743-747. Luppi, P.H., Fort, P. and Jouvet, M. (1990) lontophoretic application of unconjugated choleratoxin B subunit (CTb) combined with immunohistochemistry of neuroehemical substances: a method for transmitter identification of retrogradely labeled neurons. Brain Res., 534: 209-224. Maeda, T., Fujimiya, M., Kitahama, K., lmai, H. and Kimura, H. (1989) Serotonin neurons and their physiological roles. Arch. Histol. Cytol., 52: 113-120. Matsuyama, K., Ohta, Y. and Mori, S. (1988) Ascending and descending projections of the nucleus reticularis gigantocellularis in the cat demonstrated by the anterograde neural tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res., 460: 124-141. Matsuyama, K., Kobayashi, Y., Takakusaki, K., Mori, S. and Kimura, H. (1993) Termination mode and branching patterns of reticuloreticular and reticulospinal fibers of the nucleus reticularis pontis oralis in the cat: an anterograde PHA-L tracing study. Neurosci. Res., 17: 9-21. McGinty, D.J. and Harper, R.M. (1976) Dorsal raphe neurons: depression of firing during sleep in cats. Brain Res., 101: 569-575. Mesulam, M.M. (1982) Principles of horseradish peroxidase neurohistochemistry and their application for tracing neural pathways-axonal transport, enzyme histochemistry and light microscopic analysis. In: M.M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase, Wiley-lnterscience, New York, pp. 1-151. Mitani, A., Ito, K., Hallanger, A.E., Wainer, B.H., Kataoka, K. and MeCarley, R.W. (1988) Cholinergic projections from the
laterodorsal and pedunculopontine tegmental nuclei to the pontinc gigantocellular tegrnental field in the cat. Brain Res, 45 ! : 397-402 Mori, S., Shimoda, N., Tanaka, H., Oka, T. and Takakusaki, K (1990) Contribution of pontine reticular formation to the full execution of controlled locomotion in decerebrate cats. Somatosens. Motor Res., 7: 246-247. Ohta, Y., Mori, S. and Kimura, H. (1988) Neuronal structures of the brainstem participating in postural suppression in cats. Neurosci. Res., 5: 181-202. Palkovits, M., Brownstein, M. and Saavedra, J.M. (1974) Serotonin content of the brain stem nuclei in the rat. Brain Res., 80: 237-249. Pin, C., Jones, B. and Jouvet, M. (1968) Topographic des neurones monoaminergiques du tronc cdrdbral du chat: &ude par histofluorescence. Comptes Rendus des Sdances de la Soci&6 de Biologie et de ses Filiales, 18: 2136-2141. Poitras, D. and Parnet, A. (1978) Atlas of the distribution of monoamine-containing nerve cell bodies in the brain stem of the cat. J. Comp. Neurol., 179: 699-718. Saavedra, J.M., Brownstein, M. and Palkovits, M. (1974) Serotonin distribution in the limbic system of the rat. Brain Res.. 79: 437-441. Sano, Y., Takeuchi, Y., Kimura, H., Goto, M., Kawata, M., Kojima, M., Matsuura, T., Ueda, S. and Yamada, H. (1982) lmmunohistochemical studies on the processes of serotonin neurons and their ramification in the central nervous system - - with regard to the possibility of the existence of Golgi's rete nervosa diffusa. Arch. Histol. Jpn., 45 305-316. Semba, K. (1993) Aminergic and cholinergic afferents to REM sleep induction regions of the pontine reticular formation in the rat. J. Comp. Neurol., 330: 543-556. Semba, K., Reiner, P.B. and Fibiger, H.C. (1990) Single cholinergic mesopontine tegmental neurons project to both the pontine reticular formation and the thalamus in the rat. Neurosci., 38: 643-654. Shammah-Lagnado, S.J., Negr~io, N., Silva, B.A. and Ricardo, J.A. (1987) Afferent connections of the nuclei reticularis pontis oralis and caudalis: a horseradish peroxidase study in the rat. Neurosci., 20: 961-989. Snider, R.S. and Niemer, W.T. (1961) A Stereotaxic Atlas of the Cat Brain, University of Chicago Press, Chicago. Steinbusch, H.W.M. (1981) Distribution of serotoninimmunoreactivity in the central nervous system of the rat cell bodies and terminals. Neurosci., 6: 557-618. Steinbusch, H.W.M., Verhofstad, A.A.J. and Joosten, H.W.J. (1978) Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications. Neurosci., 3:811-819. Steriade, M. and McCarley, R.W. (1990) Brainstem Control of Wakefulness and Sleep. Plenum, New York. Stevens, D.R., Greene, R.W. and McCarley, R.W. (1989)Excitatory and inhibitory actions of serotonin on medial pontine reticular formation neurons mediated by opposing actions on potassium conductance(s). Sleep Res., 18: 23. Sutin, E.L. and Jacobowitz, D.M. (1991) Neurochemicals in the dorsal pontine tegmentum. Prog. Brain Res., 88: 3-14. Takakusaki, K., Kohyama, J., Matsuyama, K. and Mori, S. (1993a) Synaptic mechanisms acting on lumbar motoneurons during postural augmentation induced by serotonin injection into the rostral pontine reticular formation in decerebrate cats. Exp. Brain Res., 93: 471-482. Takakusaki, K., Matsuyama, K., Kobayashi, Y., Kohyama, J. and M ori, S. (1993b) Pontine microinjection of carbachol and critical zone for inducing postural atonia in reflexively standing decerebrate cats. Neurosci. Lett., 153: 185-188. Takeuchi, Y. and Sano, Y. (1983) Immunohistochemical demonstration of serotonin-containing nerve fibers in the inferior olivary
Y. Kobayashi et.al./ Neurosci. Res. 20 (1994) 43-55 complex of the rat, cat and monkey. Cell Tissue Res., 231: 17-28. Takeuchi, Y., Kimura, H. and Sano, Y. (1982) lmmunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat. Cell Tissue Res., 224: 247-267. Takeuchi, Y., Kimura, H., Matsuura, T., Yonezawa, T. and Sano, Y. 0983) Distribution of serotonergic neurons in the central nervous system: a peroxidase-antiperoxidase study with anti-serotonin antibodies. J. Histochem. Cytochem., 31: 181-185. Van Dongen, P.A.M., H6kfelt, T., Grillner, S., Verhofstad, A. A.J. and Steinbusch, H.W.M. (1985) Possible target neurons of
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5-hydroxytryptamine fibers in the lamprey spinal cord: immunohistochemistry combined with intracellular staining with Lucifer yellow. J. Comp. Neurol., 234: 523-535. Wiklund, L., lager, L. and Persson, M. (1981) Monoamine cell distribution in the cat brain stem. A fluorescence histochemical study with quantification of indolaminergic and locus coeruleus cell groups. J. Comp. Neurol., 203: 613-647. Wilkinson, L.O., Auerbach, S.B. and Jacobs, B.L. (1991) Extracellular serotonin levels change with behavioral state but not with pyrogeninduced hyperthermia. J. Neurosci., l I: 2732-2741.