serotonergic neurons in the brainstem of the wallaby,macropus eugenii

Serotonergic Neurons in the Brainstemof the Wallaby, Macropus eugenii

I.A. FERGUSON,1 C.D. HARDMAN,1 L.R. MAROTTE,2 A. SALARDINI,1

P. HALASZ,1 D. VU,1 AND P.M.E. WAITE1*1School of Anatomy, University of New South Wales, Sydney NSW 2052, Australia

2Developmental Neurobiology Group, RSBS, Australian National University,Canberra ACT 2601, Australia

ABSTRACTThe organisation and cytoarchitecture of the serotonergic neurons in a diprotodont

marsupial were examined by using serial sections of the brainstem processed for serotoninimmunohistochemistry and routine histology. The topographic distribution of serotonergicneurons in the brainstem of the adult wallaby (Macropus eugenii) was similar to that ofeutherian mammals. Serotonergic neurons were divided into rostral and caudal groups,separated by an oblique boundary through the pontomedullary junction. Approximately 52%of the serotonergic neurons in the wallaby brainstem were located in the rostral midline nuclei(caudal linear nucleus, dorsal, median, and pontine raphe nuclei and the interpeduncularnucleus), whereas 21% were found in the caudal midline region (nuclei raphe magnus,obscurus, and pallidus). The remaining serotonergic neurons (27%) were located in morelateral regions such as the pedunculopontine tegmental nuclei, the supralemniscal nuclei (B9group), and the ventrolateral medulla. The largest serotonergic group, the dorsal raphe,contained one-third of the brainstem serotonergic neurons and showed five subdivisions,similar to that described in other species. In contrast, the median raphe did not show clearsubdivisions. The internal complexity of the raphe nuclei and the degree of lateralisation ofserotonergic neurons suggest that the wallaby serotonergic system is similar in organisationto that described for the cat and rabbit. This study supports the suggestion that theserotonergic system is evolutionally well conserved and provides baseline data for aquantitative study of serotonergic innervation of the developing cortex in the wallaby. J.Comp. Neurol. 411:535–549, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: marsupials; immunohistochemistry; raphe; evolution; cortex

The serotonergic system is a phylogenetically ancientneurotransmitter system which is thought to subservefundamental brain functions in all vertebrates (reviews:Parent, 1981; Jacobs and Azmitia, 1992; Fuller, 1995).Besides its widespread functions in adults, there hasrecently been considerable interest in the role the systemmay play in cortical development (Killackey et al., 1995).Forebrain development is well advanced by the time ofbirth in commonly used experimental animals such as therat, cat, and primate; thus, experimental studies that aimto test the involvement of the serotonergic system in thenormal developmental process have been limited by thedifficulties of performing experiments in utero. It has beensuggested that the wallaby may provide a useful model forovercoming such experimental difficulties, because muchof the brain development takes place after birth (Mark andMarotte, 1992).

Eutherians and marsupials diverged from a commonancestor about 135 million years ago (see Richardson,

1988; Clemens et al., 1989). The serotonergic system isessentially similar in all vertebrate groups (Parent, 1981)and is likely to have been present in this common ancestor.The organisation, cytoarchitecture, and projections of thesystem have been extensively investigated in eutherianmammals (reviews: Jacobs and Azmitia, 1992; Tork, 1992;Halliday et al., 1995). Serotonergic neurons are foundpredominantly in midline regions of the brainstem and canbe divided into a rostral group which provides ascendinginnervation to the forebrain, and a caudal group which

Grant sponsor: National Health and Medical Research Council ofAustralia.

*Correspondence to: Dr. P.M.E. Waite, School of Anatomy, University ofNew South Wales, Sydney NSW 2052, Australia.E-mail: [email protected]

Received 21 September 1995; Revised 1 February 1999; Accepted 2 April1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 411:535–549 (1999)

r 1999 WILEY-LISS, INC.

provides descending projections (Jacobs and Azmitia, 1992;Tork, 1992). Although the overall organisation is similar inall mammalian species studied to date, increased laterali-sation has been noted as a feature of more complex brains,such as the primates (Parent, 1981; Hornung and Fritschy,1988; Jacobs and Azmitia, 1992).

The serotonergic system has only been studied in onemarsupial species, the polyprotodontid North Americanopossum (Didelphis virginiana; Martin et al., 1985, 1988).Martin et al. (1985) found that the topographic distribu-tion of serotonin-immunoreactive neurons in the brain-stem of the opossum was similar to that in eutherians.However, their plots of cell distribution, although notquantified, indicated considerable numbers of laterallyplaced serotonergic cells, suggesting a degree of lateralisa-tion more like that seen in primates.

The purpose of the present study was to assess therelevance of the wallaby as a suitable model for studiesrelating to the involvement of the serotonergic system inbrain development. To achieve this aim, we undertook adetailed immunohistochemical study of the adult wallabybrainstem, with the objective of comparing the overallorganisation and degree of lateralisation with thatpreviously published for more commonly used experi-mental animals. We report here the first descriptionof the organisation and cytoarchitecture of the raphesystem in a diprotodont marsupial. Quantitative informa-tion on the relative density and distribution of serotonergicneurons in the midline raphe nuclei and in lateral regionshas been calculated for comparison with other speciesand with the aim of providing baseline information neededfor a study of the development of the serotonergic systemin the wallaby.

MATERIALS AND METHODS

Immunohistochemistry

The animals used for immunohistochemistry comprisedthree adult wallabies (Macropus eugenii) weighing be-tween 5.0 and 6.0 kg, obtained from a breeding colony.Approval for the experiments was obtained from theAnimal Ethics Experimentation Committee and con-formed to NIH guidelines. Anaesthesia was induced withintramuscular ketamine (20 mg/kg body weight) and xyla-zine (8 mg/ml) followed by intravenous 5% sodium thiopen-tane. Once deeply anaesthetised, each animal was per-fused through the aorta with 0.9% NaCl for 30 seconds,followed by 4% paraformaldehyde in 0.1 M phosphatebuffer (pH 7.4) containing 15% vol/vol saturated picric acidat 4°C for 45 minutes. The head was immersed in fixativefor an additional 1 hour. The brain was removed andtransferred to 30% sucrose in phosphate buffer (pH 7.4) at4°C for cryoprotection. The brainstem (Fig. 1) was frozenin dry ice powder and stored at 280°C until sectioned.Fifty-micron serial coronal sections through the entirebrainstem were collected.

Every second serial section throughout the entire rostro-caudal length of the brainstem was processed for immuno-histochemistry, by using an adaptation of previously de-scribed procedures (Vu and Tork, 1992). Adjacent sectionswere Nissl-stained to facilitate subsequent anatomicallocalisation and cytoarchitectural analysis. Briefly, theimmunohistochemical procedures were performed on free-floating sections with antibodies to serotonin raised inrabbits (Incstar, Stillwater, MN, used at 1:10,000 dilution).Antibodies were diluted in phosphate-buffered saline (pH7.4) containing 0.1% Triton X-100 and incubated withcontinuous agitation at 4°C for 24–48 hours. The rest ofthe reaction was performed at room temperature. The

Abbreviations

3 oculomotor nucleus3n oculomotor root or nerve4x trochlear decussation5HT 5-hydroxytryptamine7 facial nucleus7n facial nerve8n vestibulocochlear nerve10 dorsal motor nucleus of vagus12 hypoglossal nucleusAmb ambiguus nucleusAq aqueductB9 B9 (supralemniscal) groupCC central canalCLi caudal linear nucleusCG central grey or periaqueductal greyCu cuneate nucleuscu cuneate fasiculuscp cerebral peduncleDR dorsal raphe nucleusDRc dorsal raphe, caudal subnucleusDRd dorsal raphe, dorsal subnucleusDRif dorsal raphe, interfascicular subnucleusDRv dorsal raphe, ventral subnucleusDRvl dorsal raphe, ventrolateral subnucleusdtg dorsal tegmental fasciculusECu external cuneate nucleusGi gigantocellular retitular nucleusGr gracile nucleusg7 genu of facial nerveIO inferior oliveIP interpeduncular nucleusLC locus coeruleus

LL nucleus of lateral lemniscusmcp middle cerebellar peduncleml medial lemniscusmlf medial longitudinal fasciculusMnR median raphe nucleusMo5 motor trigeminal nucleusPR pontine reticular nucleiPn pontine nucleiPPTg pedunculopontine tegmental nucleusPr5 principal trigeminal nucleuspy pyramidal tractpyx pyramidal decussationRamb retro-ambiguus nucleusRMg raphe magnusROb raphe obscurusRPa raphe pallidusRPn raphe pontisSC superior colliculusscp superior cerebellar peduncleSO superior oliveSol nucleus of solitary tractsp5 spinal trigeminal tractSp5C spinal trigeminal nucleus caudalisSp5I spinal trigeminal nucleus interpolarisSp5O spinal trigeminal nucleus oralistfp transverse fibres of ponstz trapezoid bodyTz nucleus of trapezoid bodyVe vestibular nucleiVTg ventral tegmental nucleusxscp superior cerebellar peduncle, decussation

536 I.A. FERGUSON ET AL.

location of the antigen was demonstrated by the avidin-biotin technique of Hsu et al. (1981). The secondaryantibody was a biotinylated anti-rabbit antibody (Amer-sham, Little Chalfont, U.K.) used at a dilution of 1:100, for1 hour, and the ABC reagent (Vector Labs, Burlingame,CA) was applied at a concentration of 1:80, also for 1 hour.The peroxidase label was demonstrated by an incubationwith 3,38-4,48-diaminobenzidine (Sigma, St. Louis, MO) ata concentration of 0.05% in the presence of 0.003% H2O2for 5 minutes. This peroxidase reaction was enhanced byusing 0.04% nickel ammonium sulphate in the sectionsfrom two animals. The finished sections were mountedonto gelatin-coated slides and coverslipped in DePeX. Thespecificity of the immunohistochemistry for serotonin wastested by substituting nonimmune rabbit antibodies forthe affinity-purified rabbit antibodies to serotonin or byomitting the primary and/or secondary antibodies.

Determination of cell position,number, and size

The cellular location of immunoreactive neurons wasdetermined with reference to both adjacent Nissl-stainedsections, the rat brain atlas of Paxinos and Watson (1986),and an opossum atlas (Oswaldo-Cruz and Rocha-Miranda,1968) as well as previous studies of the distribution ofserotonergic neurons in the rat (Tork, 1985), cat (Wiklundet al., 1981; Jacobs et al., 1984), and primate (Hornung andFritschy, 1988; Baker et al., 1990, 1991a,b).

A specially designed computer program (Magellan Ver-sion 3.1, Halasz and Martin, 1985; Halasz and Tork, 1989)was used for quantitative analysis and the three-dimen-sional reconstructions. The outline of each fourth sectionand the position and outline of every immunohistochemi-cally stained neuron was drawn. A conventional drawingtube was used to project an optically matched image of thecomputer screen onto the image of the section viewedthrough the microscope. By focussing down through thesection, the outline of each immunohistochemically stainedcell was drawn as it came into focus by using a magnifica-tion of 200 times or higher. A hand-operated computermouse linked to the computer was used to draw around thetissue sections and labelled cells. The x, y, and z coordi-nates, traced using the mouse, were stored in computerfiles in a form that was subsequently used to plot theanatomical features, prepare the three-dimensional recon-structions of the brainstems, and to generate quantitativedata on the number and position of cells and on celldiameter and form factor. The computer calculated the celldiameter as the diameter of a circle having the equivalentarea of the cell profile. The number of immunoreactiveneurons in each region of the brainstem was counted inone representative animal, using the optical dissectormethod. The relative proportion of serotonergic neurons inthe rostral and caudal midline nuclei and in the nonmid-line regions was then quantified. The total number ofserotonergic neurons was calculated by multiplying thenumber of immunoreactive neurons in the whole brain-stem by four.

RESULTS

General anatomy

Following immunohistochemical staining of serial sec-tions cut from the entire length of the wallaby brainstems(Fig. 1), specific staining for serotonin was observed in

all sections extending from the rostral end of the midbrainto the rostral end of the spinal cord. The reaction productwas dispersed throughout the cytoplasm of serotonergicneurons with both the somata and the larger dendritesof the immunoreactive neurons densely labelled (Fig.2A–C). Axons and dendrites could sometimes be followedover considerable distances. The specificity of the immuno-histochemistry for serotonin was confirmed by absence ofstaining of the neurons described below when nonimmunerabbit antibodies were substituted for the affinity-purifiedrabbit antibodies to serotonin or when the primary orsecondary antibodies were omitted (results not shown).

The distribution of immunohistochemically stained sero-tonergic neurons in the wallaby brainstem is shown inphotographs of immunostained coronal sections takenfrom different animals (Figs. 3–5), in line drawings ofserial coronal sections throughout the whole length of thebrainstem (Figs. 6, 7, 9) and in a three-dimensionalreconstruction of the brainstem (Fig. 8) prepared by usingthe data from one animal. Serotonergic neurons in thewallaby brainstem were assigned to nuclei within rostralgroups or caudal groups on the basis of their topographicdistribution and morphology, which was similar to thatreported for the rat, rabbit, cat, and primate (Jacobs et al.,1984; Tork, 1985; Azmitia and Gannon, 1986; Hornungand Fritschy, 1988; Tork and Hornung, 1990; Jacobs andAzmitia, 1992; Bjarkam et al., 1997). The serotonergicneurons in the rostral group (Figs. 3, 4, 6) comprisedneurons in the four midline raphe nuclei: the caudal linearnucleus (CLi), the median raphe (MnR), dorsal raphe (DR),and raphe pontis (RPn), as well as the interpeduncularnucleus (IP); besides these nuclei, serotonergic neuronswere present more laterally in the supralemniscal region(B9 group) and in the pedunculopontine tegmental nucleiand deep mesencephalic nucleus. The caudal group (Figs.5, 7) comprised neurons in three raphe nuclei: raphemagnus (RMg), raphe obscurus (ROb), and raphe pallidus(RPa), as well as a lateral group in the ventrolateralmedulla, dorsal and medial to the facial nucleus. Largebipolar immunoreactive neurons which extended longdendrites in various directions were also observed sur-rounding the central canal of the cervical spinal cord (Fig.7H). The overall distribution of all serotonergic cells isindicated by the reconstructions in Figures 8 and 9. Thisshows the separation of the rostral and caudal groups (Fig.8A) and the extent of lateral spread (Figs. 8B, 9). Histo-grams of the diameter of serotonergic neurons (Fig. 2)indicate the wide range of cell sizes found throughout thebrainstem (range for all cells 3–38 µm, mean diameter 14.46 4.5 µm, Fig. 2a). Serotonergic neurons with the smallestaverage diameter were located in the RPa (mean diameter12.6 6 3.1 µm, Fig. 2b), whereas the serotonergic neuronswith the largest average diameter were located in thegroup lateral to the DR (mean 19.2 6 5.1 µm, Fig. 2c).

The proportion of serotonin-immunoreactive neurons inthe rostral midline, caudal midline, and the nonmidlinegroups of the representative wallaby brainstem is shownin Table 1. The total number of serotonergic neurons wascalculated to be approximately 26,000. Approximately 52%of the serotonin-immunoreactive neurons were located inthe rostral midline nuclei; 21% in the caudal midlinenuclei, and 27% in nonmidline regions. The DR containedthe largest number of serotonergic neurons, with approxi-mately one-third (32.5%) of all immunoreactive cells lo-

WALLABY SEROTONERGIC NEURONS 537

cated in this nucleus. A more detailed description of theserotonergic neurons in the three major groups follows.

Rostral midline nuclei

Caudal linear nucleus (CLi). This relatively smallnucleus is located in the midbrain and is the most rostralof the serotonergic midline nuclei (Figs. 3A–C,E, 6A,B, 8,yellow). The CLi extends rostrally to the level of theinterfascicular nucleus where it is bounded laterally by the

roots of the oculomotor nucleus. At this level, scatteredimmunoreactive cells can be observed dorsal to the interpe-duncular nucleus within the rostral decussating fibres ofthe ventral tegmentum (Fig. 3A). Further caudally (Fig.3B,C,E), the nucleus is displaced dorsally, so that theboundary between it and the dorsal raphe intrafascicularsubnucleus (DRif) becomes obscure and then disappears.Serotonergic neurons in the CLi are small to medium-sizedand round or oval.

Fig. 1. Dissected wallaby brainstem prior to sectioning to show major surface landmarks. A: Lateralview. B: Dorsal view. SC, superior colliculus; mcp, middle cerebral peduncle; py, pyramids; ECu, externalcuneate. Scale bar 5 2 mm.


Median raphe nucleus (MnR). The MnR is located inthe caudal midbrain and rostral pons (Figs. 3B–D,E, 5A,6A–D). For most of the nucleus, the neurons lie in twoparallel lines, on each side of the midline (Fig. 3C,E). Thepaired lines of blood vessels (best seen in Fig. 3B–D) in themidbrain define the lateral limit of spread of serotonergicneurons in the MnR. At its rostral extent, serotonergicneurons of the MnR are found scattered within the decus-

sating fibres of the tegmentum (Fig. 3B) and just dorsal tothe interpeduncular nucleus (Fig 3C). Further caudally,the MnR lengthens dorsally as the CLi tapers (Fig. 3D). Atits caudal pole, the MnR is ventral to the DRif. Theserotonergic neurons throughout the MnR are small tomedium-sized and round or oval. (Fig. 3E).

Dorsal raphe nucleus (DR). This is the largest of theraphe nuclei in the wallaby and contains about one-third of

Fig. 2. A–C: Examples of different morphologies of serotonergicneurons: fusiform cells from raphe magnus (RMg; A); small roundneurons (arrows) from raphe pallidus (RPa; B); large multipolar cellsfrom the dorsal nonmidline serotonergic region, lateral to the dorsalraphe, dorsal subnucleus (DRd; C). a–c: Histograms showing the

range of cell diameters of serotonergic neurons: in the whole brain-stem (a); in RPa (b); lateral to the DRd (c). The mean diameter 6 S.D.for a–c are: 14.4 6 4.5 µm, n 5 2,624; 12.6 6 3.1 µm, n 5 214; and19.2 6 5.1 µm, n 5 68, respectively. Scale bar 5 50 µm.


Figure 3

the immunoreactive cells in the brainstem (Table 1). Themajority of these neurons are located in the midbrain(Figs. 6A–C, 8, red) but the nucleus extends caudally intothe rostral pons (Fig. 6D–F). The cells are located in theventral periaqueductal grey and around the medial longi-tudinal fasciculus (Figs. 3B–D, 5A). The ventral edge of themedial longitudinal fasciculi on each side marks theventralmost extent of the DR (Fig. 3E). At the most rostralextent of the DR, a few scattered immunoreactive cells arefound around the midline (Fig. 6A). The density of immuno-reactive cells then increases markedly (Fig. 6B,C). Thecaudal pole of DR is indistinct with the density of imunore-active neurons decreasing progressively in a caudal direc-tion (Figs. 6E,F, 7A). In the human, the DR can be dividedinto distinct subnuclei (Baker et al., 1990, 1991b) andthese subnuclei can also be discerned in the wallaby.

Intrafascicular subnucleus (DRif). This subnucleus islocated on the midline between and dorsal to the mediallongitudinal fasciculus (Fig. 3E). It extends from its rostralpole at the transition between the oculomotor nucleus andthe trochlear nucleus to its caudal pole level with therostral pole of the sphenoid nucleus. Immunoreactiveneurons in the DRif are small to medium-sized andfusiform in shape with their long axis oriented along themidline.

Ventral subnucleus (DRv). This subnucleus is locatedon or just lateral to the midline and dorsal to the DRif (Fig.3F). It extends from the rostral end of the dorsal raphenucleus to the isthmus. Neurons in the DRv are small tomedium-sized and round or ovoid.

Dorsal subnucleus (DRd). Serotonergic neurons in theDRd are found ventral to the cerebral aqueduct anddorsolateral to the DRv (Fig. 3F). Scattered serotonergicneurons in this region extend laterally to merge withserotonergic neurons in the nonmidline regions (see later).As with the DRv, this subnucleus extends from the rostralpole of the DR to the decussation of the isthmus. Neuronsin the DRd are medium to large in diameter, round orovoid, and extend dendrites considerable distances.

Ventrolateral subnucleus (DRvl). Serotonergic neuronsin the DRvl (Fig. 3F) lie ventral to the DRd and lateral tothe DRv and DRif. Scattered neurons extending into themedial longitudinal fasciculus can be occasionally ob-served. The immunoreactive neurons in the DRvl aremedium-sized to large, and round or multipolar.

Caudal subnucleus (DRc). The DRc is the most caudalof the DR subnuclei and is equivalent to the B6 division ofDahlstrom and Fuxe (1964). It extends from its caudal endat the decussation of the trochlear nerve to the raphepontis nucleus (RPn; Figs. 6E,F, 7A). The density ofserotonergic neurons decreases progressively towards thecaudal pole. The immunoreactive neurons are small tomedium-sized and oval or fusiform.

Raphe pontis nucleus (RPn). This small group ofimmunoreactive neurons is found ventral to the mediallongitudinal fasciculus, scattered around the midline andextending caudally from the MnR to just anterior to themotor nucleus of the trigeminal nerve (Fig. 6F). Rostrally,these cells are medium-sized to large and multipolar inmorphology. Further caudally, the serotonergic neuronsform into two parallel lines on either side of the midline.These neurons are small to medium-sized and round oroval, and abut onto the rostral pole of the RMg.

Interpeduncular nucleus (IP). Although not one ofthe midline raphe nuclei, the IP is included here in therostral midline group, as its cells are located around themidline as well as being more laterally dispersed (Figs.3C,D,G, 6B,C). At the rostral pole of the IP, two diagonallysloping groups of immunoreactive cells are present on thelateral borders of IP (Fig. 3C,G). As in the rat (Singhani-yom et al., 1982), the serotonergic neurons in the IP of thewallaby are continuous dorsally with the MnR. However,unlike the rat, the neurons in the IP of the wallaby extendfurther laterally to merge with the serotonergic neurons inthe B9 group (Figs. 3C, 6B, and see below) with thetransition between the IP and B9 groups occurring at theparamedian columns of blood vessels (Fig. 3C,D). Towardsthe caudal pole of IP, the density of scattered serotonergicneurons in between the paramedian clusters increasesconsiderably (Figs. 3D, 6C). These neurons are scattered inthe decussating fibres and send projections horizontally.Serotonergic neurons in the IP are small to medium-sizedand round, oval, or fusiform.

Caudal serotonergic nuclei

Raphe magnus nucleus (RMg). The RMg (Figs. 5B,7A–E, 8, orange) is found in the ventral pons and medullaand extends from the level of the pontine nucleus, at therostral pole, to the inferior olive. At the most rostral level,the serotonergic neurons in the RMg are scattered aroundthe midline, dorsal to the pyramids. They are frequentlyfound among the decussating fibres of the medial lemnis-cus where they extend horizontally projecting dendrites(Figs. 2A, 5B). In more caudal sections (Fig. 7D,E), thescattered cells extend more laterally and appear continu-ous with the lateral groups (see later). The neurons of theRMg are typically medium-sized to large, round or ovalnear to the midline, and fusiform with a horizontal orienta-tion further laterally (Fig. 2A).

Raphe pallidus nucleus (RPa). This nucleus consistsof parallel lines of cells on both sides of the midline, withinor ventral to the RMg (Figs. 5A,D, 7C–F, 8, dark pink). Thenucleus extends from the level of the rostral pole of the

Fig. 3. Photomicrographs of coronal sections from the rostralbrainstem processed for serotonin immunohistochemistry. A: Rostralmidbrain at the level of the emerging roots of the oculomotor nerve.Immunoreactive neurons of the caudal linear nucleus (CLi) are withinthe rostral decussating fibres of the ventral tegmentum and dorsal tothe interpeduncular nucleus. B: Midbrain at the level of oculomotornucleus. Immunoreactive neurons are mostly located in the medianraphe nucleus (MnR), within the decussating fibres of the ventraltegmentum, in the CLi, dorsal to the decussating fibres of the superiorcerebellar peduncle (xscp), and in the dorsal raphe nucleus (DR),within the periaqueductal grey and between the medial longitudinalfasciculus (mlf) dorsal to CLi. The boxed region can be seen at higherpower in F. C: Midbrain just rostral to the ventral tegmental nucleus.The boxed regions can be seen at higher power in E, G, and H.Immunoreactive neurons can be seen in the DR, CLi, MnR, and theinterpeduncular nucleus (IP) as well as the more lateral B9 group.D: Midbrain just rostral to the isthmus at the level of the ventraltegmental nucleus (VTg). Immunoreactive neurons are mostly locatedin the DR, MnR, IP, and B9 group. E: Higher magnification montage ofthe box in C showing the boundary between immunostained neuronsin the CLi and the dorsal raphe interfascicular subnucleus (DRif;dashed lines). F: Higher magnification of box in B, showing the dorsal(DRd), ventral (DRv), and ventrolateral (DRvl) subnuclei (dashedlines) in the DR. G: Higher magnification of box in C, showingimmunoreactive neurons in the dorsal and lateral aspects of the IP.H: Higher magnification of box in C, showing neurons in the B9 group.Scale bars 5 1 mm in A–D; 200 µm in E–H.


facial nucleus (Fig. 7C) to the decussation of the pyramidaltract. The RPa cells are found within a plexus of denselystaining serotonergic processes (Fig. 5D). The serotonergicneurons in RPa are uniformly small and round or oval (Fig.2B,b).

Raphe obscurus nucleus (ROb). The ROb extendsfrom the level of the caudal pole of the facial nucleus to thedecussation of the pyramidal tract (Figs. 5D, 7D–G, 8,mauve). Rostrally, ROb neurons are found along the

ventral half of the midline, abutting ventrally onto theRPa. Further caudally, the ROb lengthens dorsally anddivides into a paired group of serotonergic neurons on bothsides of the midline (Fig. 4D). The region occupied by theROb also contains numerous serotonin- positive fibresaligned parallel to the midline. The serotonergic neuronsin the ROb are medium-sized, round or fusiform, andgenerally extend dendrites in a vertical direction, parallelwith the midline.

Fig. 4. Photomicrographs of coronal section from the caudal brain-stem processed for serotonin immunohistochemistry. A: Low-powermagnification showing immunoreactive neurons in the medulla at thelevel of rostral pole of the ambiguus nucleus. The boxed regions can beseen at higher power in B–D. B: Higher magnification of box in A,showing raphe magnus (RMg) neurons dorsal to the pyramidal tract.

C: Higher magnification of most lateral box in A, showing serotonergicneurons (arrows) in the ventrolateral medulla, lateral to the RMg.D: Higher magnification of elongated midline box in A, showingserotonergic neurons in the rostral raphe obscurus (ROb) and raphepallidus (RPa). Scale bars 5 1 mm in A; 200 µm in B–D.


Nonmidline regions

Rostral groups. A significant number of the immuno-reactive neurons in the midbrain are found lateral to themidline raphe nuclei (Figs. 5, 6, 8 [blue], 9). As most clearlyseen in Figure 9, the majority of lateral serotonergicneurons in the midbrain and pons are located in twodistinct clusters, termed here the central subgroup and thedorsal subgroup. The first subgroup is centered around thepedunculopontine tegmental nucleus, lateral to the supe-rior cerebellar peduncle (Figs. 5D, 6A–C, 9); the dorsalsubgroup is scattered in the periaqueductal grey and deepmesencephalic nucleus, lateral to the dorsal raphe (Figs.5B,C, 6B–D, 9). A third group, the supralemniscal group orB9, is located lateral to the interpeduncular nucleus anddorsal to the medial lemniscus (Figs. 3C,D,H, 5A, 9).Serotonergic neurons were also found between these three

groups, although these were relatively sparsely distrib-uted (Figs. 9).

Central subgroup. Neurons in this region extendedfrom the level of the caudal pole of the oculomotor nucleusto the caudal pole of the locus coeruleus (Figs. 6A–E, 9). Atits rostral extent, the serotonergic neurons are scattered inthe region of the pedunculopontine tegmental nucleus andsurrounding reticular formation. Further caudally, thelocation of serotonergic neurons shifts somewhat dorsome-dially towards the medial longitudinal fasciculus. Theneurons in the central subgroup are heterogeneous inmorphology, being small, medium or large, and round,irregular, or multipolar. (Fig. 4D).

Dorsal subgroup. The most rostral extent of the seroton-ergic neurons in the dorsal subgroup is found in theperiaqueductal grey lateral to the dorsal raphe (Fig. 6A).

Fig. 5. Photomicrographs of serotonin-immunoreactive neuronslateral to the raphe nuclei. A: Low-power magnification of the coronalsection at the level of the rostral pole of the ventral tegmental nucleusshowing serotonergic neurons in the dorsal raphe nucleus (DR),median raphe nucleus (MnR), and B9 group. Boxed regions are shownat higher power in B–D. B: Higher magnification of most dorsal box inA, showing scattered neurons (arrows) lateral to the DR in the

periaqueductal grey. C: Higher magnification of central box in A,showing serotonergic neurons (arrows) within the deep mesencephalicnucleus, adjacent to the dorsal tegmental fasciculus (dtg). D: Highermagnification of most ventral box in A, showing scattered serotonergicneurons (arrows) within the pedunculopontine tegmental nucleus atthe lateral extent of the superior cerebellar peduncle. Scale bars 5 1mm in A; 200 µm in B–D.


Fig. 6. Computer plots of coronal sections showing the distribution of serotonin-immunoreactiveneurons (dots) at 1-mm intervals (A–F) throughout the midbrain and pons. For abbreviations, see list.Scale bar 5 2 mm.


Slightly more caudally, these serotonergic neurons in-crease in number and extent so that they are found dorsaland lateral to the oculomotor nucleus as well as lateral tothe DRd (Figs. 4B, 6B). This subgroup can be distin-guished from the neurons in the DRd by their larger size,more prominent dendrites, and multipolar shape (Figs.2C, 4B). These cells are amongst the largest serotonergicneurons seen in the brainstem (Fig. 2c). Further caudally,cells in this dorsal subgroup are found scattered around,and occasionally in, the locus coeruleus (Fig. 6E,F).

B9 group. The serotonergic neurons in the B9, orsupralemniscal, group (Figs. 3C,D,H, 6B,C, 8 [light pink],9) appear in coronal section as a continuous crescent-shaped band of cells, extending laterally from the interpe-duncular nucleus. The rootlets of the oculomotor nucleusmark the most rostral boundary of the B9 group. At thisrostral limit, immunoreactive cells are found at the dorsaland lateral edge of the interpeduncular nucleus and dorsalto, and associated with, the medial lemniscus. Furthercaudally, the density of serotonergic cells in these regionsincreases considerably and serotonergic neurons appear inthe ventral tegmental area and just dorsal to the trans-verse fibres of the pons (Fig. 6B,C). The caudal extent ofthe B9 group is located at the level of the rostral pole of thenucleus of the trapezoid body. The serotonergic neurons inthe B9 group are medium–sized to large, round, multipo-lar, or fusiform, and can extend immunopositive dendritesfor considerable distances (Fig. 3H).

Caudal groups. A population of laterally located sero-tonergic neurons are observed in the ventrolateral me-dulla, lateral to the RMg (Figs. 4A,C, 7C, 8, green). Thesescattered cells lie at the ventral edge of the lateralparagigantocellular nucleus, the gigantocellular reticularnucleus, and intermediate reticular nucleus, medial anddorsal to the facial nucleus (Fig. 7–E). These serotonergicneurons are medum-sized to large, and multipolar inshape.

DISCUSSION

This study provides a detailed description of the seroton-ergic neurons in the brainstem of a diprotodont marsupial,the wallaby, Macropus eugenii. On the basis of the distribu-tion and morphology of serotonin-immunoreactive neu-rons, distinct nuclei corresponding to those in eutherianmammals were identified. We have established that, ingeneral, the organisation and morphology of serotonergicneurons in the brainstem of the wallaby is similar to thatof eutherian mammals. The present results from thewallaby are discussed below in terms of the major similari-ties found between species, as well as the differences whichoccur, for instance, in the degree of lateralisation.

Relative distribution of serotonergic neuron:Comparison with other species

The distribution of serotonergic neurons in the brain-stem of marsupials has only been studied in one othermarsupial, a polyprotodont marsupial, the North Ameri-can opossum, Didelphis virginiana (Martin et al., 1985).The data reported in both that study and this current one

Fig. 7. Computer plots of coronal sections showing the distributionof serotonin-immunoreactive neurons (dots) at 2-mm intervals (A–H)throughout the medulla. For abbreviations, see list. Scale bar 5 2 mm.


Fig. 8. Computer-generated reconstructions showing the distribution of serotonergicneurons in the wallaby brainstem as seen in a lateral view (A) and dorsal view (B). Theoblique separation of serotonergic cells into rostral and caudal groups at the level of thepontomedullary junction can be seen the lateral view. Rostral group: (for abbrviations, see

list) red, DR; yellow, CLi, MnR, IP, and RPn; blue, central and dorsal lateral groups; lightpink, B9 group. Caudal group: orange, RMg; dark pink, RPa; mauve, ROb; green, ventrolat-eral medulla. The lateral spread of the rostral (blue, light pink) and caudal (green) groups isseen in the dorsal view. Scale bar 5 2 mm.

provide support for the notion that the distribution ofserotonergic neurons is remarkably stable across phylog-eny (Parent, 1981; Jacobs and Azmitia, 1992) with themajority of the serotonergic neurons in placental andeutherian mammals as well as in nonmammalian speciesbeing located in the midline raphe nuclei.

The number of serotonergic neurons in the wallabybrainstem (approximately 26,000) is more than that pre-sent in the rat (20,500 neurons; Jacobs and Azmitia, 1992,Table 1) and less than that in the cat (60,000 neurons;Wiklund et al., 1981). As in eutherian species, the seroton-

ergic neurons in the wallaby brainstem could be assignedto specific nuclei by using cell location and morphologicalcriteria previously established (Tork, 1992; Jacobs andAzmitia, 1992). The nuclei identified in the wallaby are ingeneral agreement with those described in the rat (Tork,1985), cat (Wiklund et al., 1981), and primate, includinghuman (Hornung and Fritschy, 1988; Tork and Hornung,1990). In all species, the brainstem contains rostral andcaudal midline groups, as well as more laterally lyingserotonergic cells. These rostral and caudal subdivisionshave been shown to differ not only in their position in thebrainstem, but also in their development and projections(Azmitia and Gannon, 1986; Jacobs and Azmitia, 1992;Halliday et al., 1995). Comparison of the computer recon-struction of the serotonergic neurons in the wallaby withthat for the rat (Halliday et al., 1995) and the marmoset,Callithrix jacchus (Hornung and Fritschy, 1988), illus-trates that the overall organisation of the serotonergicneurons in all three species is largely similar.

Although the general organisation indicates similaritiesacross species, differences in the relative size and densityof individual serotonergic nuclei are present. Most studiesin eutherians describe four rostral raphe nuclei (CLi, MnR,DR, RPn) and three caudal midline nuclei (RMg, ROb,RPa) and the same was found in the wallaby. In all cases,DR contains the largest number of serotonergic cells,although this nucleus appears to be relatively smaller inthe wallaby (32% of all serotonergic neurons) compared tothe rat (56%, Jacobs and Azmitia, 1992) or cat (40%,Wiklund et al., 1981). We have identified the small raphepontis (the B5 group of Dahlstrom and Fuxe, 1964) as aseparate nucleus in the wallaby, as described for the rat(Steinbusch and Nieuwenhuys, 1983), cat (Wilkund et al.,1981; Jacobs et al., 1984), and marmoset (Hornung andFritschy, 1988), rather than including it within MnR, as inthe human (Tork and Hornung, 1990). Serotonergic neu-rons were prominent in the lateral and dorsal aspects ofthe IP of the wallaby. Although significant numbers ofserotonergic neurons have been reported in the IP of therat (Singhaniyom et al., 1982) and monkey (Takeuchi etal., 1982), serotonergic neurons in the IP of humans werenot considered as a separate group (Baker et al., 1991a).

Internal subdivisions of raphe nuclei

Although the MnR of the wallaby has a similar organisa-tion of paramedian columns seen in other species (e.g., cat,Jacobs and Azmitia, 1992) no internal divisions intosubnuclei could be discerned in the wallaby. Nonhumanprimates have dorsal and ventral subdivisions within thisnucleus (Hornung and Fritschy, 1988) whereas three dis-tinct subregions can be delineated in the human (Baker etal., 1991a). This suggests that the MnR of the wallaby maynot be as complex as that of primates.

In cross-sections of the midbrain, the DR has beenappropriately described as having ‘‘the outline of a foun-tain’’ (Tork, 1992) with a central column of cells spreadingdorsally into lateral wings. Most studies have consideredthis nucleus to have several subdivisions, with intrafascicu-lar, medial, and lateral components. Distinct subdivisionshave been described for DR in the rat (Steinbusch et al.,1981): a dorsomedian, ventromedian and two lateral groupsin the midbrain, and a caudal group in the pons. Similarsubdivisions are present in the cat and rabbit with medial,lateral, and intrafascicular groups (Jacobs et al., 1984;Bjarkam et al., 1997). However, in primates the lateral

Fig. 9. Computer reconstruction of 12 midbrain sections rostral tothe isthmus, showing a composite plot of all serotonergic neuronsexcept those in dorsal raphe nucleus (DR). The nonmidline serotoner-gic cells can be seen to cluster into three groups: the dorsal, central,and B9 groups. Serotonergic cells in median raphe nucleus (MnR) andinterpeduncular nucleus (IP) are also shown. Scale bar 5 2 mm.

TABLE 1. Proportion of Serotonergic Neurons in the Rostral, Caudal, andNonmidline Groups in the Adult Wallaby Brainstem1

Brainstem region Cell number % total

Rostral midline groupCLi 325 1.2MnR 3,304 12.7DR 8,468 32.5RPn 167 0.6IP 1,320 5.1Total 13,584 52

Caudal midline groupRMg 2,921 11.2RPa 1,730 6.6ROb 833 3.2Total 5,484 21

Nonmidline groupsRostral central/dorsal 4,180 16.1B9 group 2,350 9.0Ventrolateral medulla 444 1.7Total 6,974 27

Total 26,042 100

1For abbreviations, see list.


wings are particularly prominent (Jacobs and Azmitia,1992), and in humans, can be divided into dorsal andventral subdivisions (Baker et al., 1990). Although subdivi-sions were also present in the wallaby, the lateral wingswere relatively homogeneous. Thus, the wallaby DR ap-pears to have an internal structure that is at least ascomplex as the rat, rabbit, and cat, but possibly not ascomplex as that of primates.

Cell morphology

Cells in the raphe nuclei in most species show consider-able variability in cell size and morphology, and a similarvariety was noted for the wallaby. In general, there wasgood correspondence between the typical cell morphologyreported for the individual raphe nuclei and that reportedin the literature for other mammals (see reviews: Tork,1985, 1992; Jacobs and Azmitia, 1992). For example, in theDR of the wallaby, as is seen in the rat (Tork, 1985) and thehuman (Baker et al., 1991b), the DRif contained medium-sized, fusiform serotonergic neurons; the ventromedialregion or DRv contained primarily small to medium-sizedround or oval neurons, and large multipolar neurons werelocalised to the lateral regions of the DR. An interestingtrend noted in previous studies in the rabbit (Bjarkam etal., 1997) and marmoset (Hornung and Fritschy, 1988) andsupported here, is for more laterally located neurons to belarger and generally multipolar in shape. Thus, in thepresent study, the serotonergic groups located lateral tothe DR (dorsal subdivision) and those in the ventrolateralmedulla contained mainly multipolar cells which wereamongst the largest immunopositive cells present in thebrainstem.

Nonmidline serotonergic neurons

The evolution of more complex brains has been relatedto increased lateralisation of serotonergic neurons (Par-ent, 1981). Only birds and mammals have been reported tohave significant spread of serotonergic neurons into thelateral tegmentum throughout the brainstem (Dube andParent, 1981). This theme of evolution of more complexnervous systems being associated with increased laterali-sation of serotonergic neurons is also seen within theeutherians and is particularly well developed in primates(Jacobs and Azmitia, 1992). This increased lateralisationrefers particularly to neurons in the rostral brainstem andis presumably associated with differences in ascendingprojections (Jacobs and Azmitia, 1992). Comparison of theserotonergic cell distribution in the wallaby with that ofthe cat and rat (Wiklund et al., 1981; Tork, 1985), suggeststhat the degree of lateralisation within the wallaby (27%)is greater than that in the rat (12.5%, Jacobs and Azmitia,1992) and similar to that in the cat (22.5%, Wiklund et al.,1981). As in other species, laterally placed cells in thewallaby were found in the pedunculopontine tegmentalnucleus, the deep mesencephalic nucleus, as well as moreventrally in B9. In the rat, B9 was the second largestserotonergic group in the midbrain and pons, being largerthan MnR and having nearly one-third of the neuronnumber of DR (Vertes and Crane, 1997). Although stillsizable, B9 in the wallaby was relatively smaller than therat, with less cells than in DR, MnR, and the more dorsallateral groups. An interesting recent study has reportedmarked lateralisation in the New Zealand white rabbit(Bjarkam et al., 1997). Although not quantified, the distri-bution of the cells they describe was similar to that found

here and, as in other species, these cells were mainly largeand multipolar.

Lateralisation in the caudal brainstem is similar acrossa wide range of mammalian species and may reflectsimilar spinal projections (Bjarkam et al., 1997). Thus, inthe wallaby medulla, as in the rat (Tork, 1985), cat(Wiklund et al., 1981), and several primates (Azmitia andGannon, 1986; Takeuchi et al., 1982; Hornung and Fritschy,1988), serotonergic neurons appear to extend ventrolater-ally into the reticular formation from RMg. In the rabbitthese neurons of the ventroloateral medulla were dividedinto rostral and caudal groups. However, in the wallaby noobvious subdivision was apparent and this group did notextend caudally into the closed medulla.

CONCLUSIONS

In the present study, we have demonstrated that theorganisation of serotonergic neurons in the marsupialbrainstem, the internal complexity of the brainstem sero-tonergic nuclei, and the morphology of serotonergic neu-rons are similar to that of eutherians. This providessupport for the suggestion that the serotonergic system isa phylogenetically ancient neurotransmitter system whichis evolutionally well conserved (Parent, 1981). Withinmammals, the evolution of the primate brain has beenassociated with a trend both towards increased lateralisa-tion of serotonergic neurons and towards a more restrictedterminal innervation of the cortex (Jacobs and Azmitia,1992). Features of the wallaby serotonergic system, suchas the degree of lateralisation and the extent of subdivi-sions within the raphe nuclei, suggest that the complexityof the wallaby brain is similar to that in the cat and rabbit.Unlike eutherian mammals, most of the development ofthe cortex of the wallaby occurs after birth (Renfree et al.,1982) and marsupials such as the wallaby can provide auseful model for overcoming the experimental difficultiesassociated with in utero studies of cortical development(Mark and Marotte, 1992). Thus, the present paper pro-vides necessary baseline data for future investigationsusing the wallaby as a model to address the involvement ofthe serotonergic system in normal cortical development.

ACKNOWLEDGMENTS

We thank Dr. G.M. Halliday and Dr. K. Ashwell for theircomments on the manuscript; Ms. A. Devlin and Mr. K.Williams for animal care; and Dr. R. Meischke for veteri-nary advice.

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serotonergic neurons in the brainstem of the wallaby,macropus eugenii

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