Distribution and morphology of putative catecholaminergic and serotonergicneurons in the medulla oblongata of a sub-adult giraffe,

Giraffa camelopardalis

N. Ludo Badlangana a, Adhil Bhagwandin a, Kjell Fuxe b, Paul R. Manger a,*a School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193,

Johannesburg, Republic of South AfricabDepartment of Neuroscience, Karolinska Institutet, Retzius vag 8, S-171 77 Stockholm, Sweden

Received 19 March 2007; received in revised form 2 May 2007; accepted 2 May 2007

Available online 22 May 2007

Abstract

The current study details the nuclear parcellation and appearance of putative catecholaminergic and serotonergic neurons within the medullaoblongata of a sub-adult giraffe, using immunohistochemistry for tyrosine hydroxylase and serotonin.We hypothesized that the unusual phenotype ofthe giraffe, this being the long neck and potential axonal lengthening of these neurons,may pose specific problems in terms of the efficient functioningof these systems, as several groups of catecholaminergic and serotonergic neurons, especially of themedulla, are known to project to the entire spinalcord. This specific challenge may lead to observable differences in the nuclear parcellation and morphology of these systems in the giraffe. Ourpersonal observations in the giraffe reveal that, as with other Artiodactyls, the spinal cord extends to the caudal end of the sacral vertebrae.Within thegiraffemedullawe found evidence for five putative catecholaminergic (neurons containing tyrosine hydroxylase) andfive serotonergic nuclei. In termsof bothmorphological appearance of the neurons and nuclear parcellationwe did not find any evidence for features thatmay be considered affected bythe phenotypeof thegiraffe.The nuclear parcellation andappearance of both theputative catecholaminergic and serotonergic systems in themedulla ofthe giraffe studied are strikingly similar to that seen in previous studies of otherArtiodactyls.We interpret these findings in terms of a growing literaturedetailing order specific phylogenetic constraints in the evolution of these neuromodulatory systems.# 2007 Elsevier B.V. All rights reserved.

Keywords: Tyrosine hydroxylase; Serotonin; Evolution; Artiodactyl; Neural systems

1. Introduction

The phenotypically unusual long neck of the giraffe (Giraffacamelopardalis), which can be well over 1 m long, presents arange of potential problems for a variety of neural systems. Thespinal cord terminates at the sacral level and in itself can be over1.5 m in length in sub-adult giraffe (personal observations).Furthermore, there are nerves that project to the viscera such asthe vagus and phrenic nerves that presumably have significantlylonger distances to cover than would be the case in otherArtiodactyla and mammals. In this study, the catecholaminergicand serotonergic systems of the medulla of the giraffe wereexamined using immunohistochemistry for tyrosine hydro-xylase to reveal putative catecholaminergic neurons andserotonin to reveal serotonergic neurons.

The specific nuclei forming the catecholaminergic systemsnormally observed in the medulla of mammals are the rostral

www.elsevier.com/locate/jchemneuJournal of Chemical Neuroanatomy 34 (2007) 69–79

Abbreviations: 10, dorsal motor vagus nucleus; 12, hypoglossal nucleus;

12n, hypoglossal nerve; A1, caudal ventrolateral medullary tegmental group;A2, caudal dorsomedial medullary group; AP, area postrema; C1, rostral

ventrolateral medullary tegmental group; C2, rostral dorsomedial medullary

group; Cu, cuneate nucleus; CVL, caudal ventrolateral medullary tegmental

serotonergic cell column; DC, dorsal cochlear nucleus; DMS, dorsomedialspinal trigeminal nucleus; ECu, external cuneate nucleus; flm, medial long-

itudinal fasciculus; Ge5, gelatinous layer of the caudal spinal trigeminal

nucleus; Gr, nucleus gracilis; icp, inferior cerebellar peduncle; io, inferiorolive; LRt, lateral reticular nucleus; MdD, medullary reticular nucleus dorsal

part; MdV, medullary reticular nucleus ventral part; N.Amb, nucleus ambiguus;

oc, olivocerebellar tract; py, pyramidal tract; pyx, pyramidal decussation; RMg,

raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidusnucleus; RVL, rostral ventrolateral medullary tegmental serotonergic cell

column; Sp5, spinal trigeminal nucleus; Sp5c, spinal trigeminal nucleus caudal

part; SpVe, spinal vestibular nucleus; vc, ventral cochlear nucleus; VII, facial

nerve nucleus* Corresponding author. Tel.: +27 11 717 2497; fax: +27 11 717 2422.

E-mail address: mangerpr@anatomy.wits.ac.za (P.R. Manger).

0891-0618/$ – see front matter # 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jchemneu.2007.05.003

ventrolateral tegmental group (C1), the caudal ventrolateraltegmental group (A1), the rostral dorsomedial group (C2), thecaudal dorsomedial group (A2), and the area postrema(Dahlstrom and Fuxe, 1964; Hokfelt et al., 1974; Kaliaet al., 1985a,b; Smeets and Gonzalez, 2000). Of these nuclei,the C1 neurons project to the intermediolateral cell column andthe intermediate grey matter of the spinal cord (Smeets andGonzalez, 2000), and the neurons of area postrema are interalia involved in the regulation of heart rhythm and innervatesmooth muscle (Koizumi and Brooks, 1974). Given the spinalprojection of the C1 neurons, and the involvement of the areapostrema in vascular control, the phenotype of the giraffe maypose a unique challenge that is reflected in the anatomy of thesecatecholaminergic nuclei in comparison to other mammals.

While the majority of these catecholaminergic nuclei havebeen observed in the medulla of cow, pig and sheep (Tillet andThibault, 1989; Ruggiero et al., 1992; Tillet and Kitahama,1998), it has been reported that sheep lack the C1 group (Tillet,1988). The reported absence of C1 in sheep is based on a lack ofphenylethanolamine-N-methyltransferase (PNMT) immunopo-sitive neurons in the region where one would expect these to befound. Despite this, Tillet and Thibault (1989), usingdopamine-b-hydroxylase (DBH) and tyrosine hydroxlase(TH) immunohistochemistry revealed neurons that are foundin a location of the medulla that might be considered C1. Whilein many mammals the neurons of C1 are immunoreactive forPNMT (Smeets and Gonzalez, 2000), this may not be the casefor the C1 neurons of the sheep, as a conformational change inthe 3D structure of the protein reacting with the PNMTantibody may have occurred, leading to a potential falsenegative in this case. But if there is really a lack of a C1 in thesheep, the identity of the neurons that are immunoreactive toboth TH and DBH in the region where C1 neurons would beexpected to be located in sheep needs to be established (Tilletand Thibault, 1989). They may in fact represent the C1 systembut without expression of the PNMT protein thus operating vianoradrenaline transmission.

All serotonergic neurons of the medulla, the caudalserotonergic cluster, project to the spinal cord (Dahlstromand Fuxe, 1965; Tork, 1990). There are normally fiveserotonergic nuclei found in the medulla which are: the raphemagnus nucleus (RMg), raphe pallidus nucleus (RPa), rapheobscurus nucleus (ROb) and the rostral and caudal ventrolateralmedullary tegmental (RVL and CVL) groups (Dahlstrom andFuxe, 1964; Tork, 1990; Bjarkam et al., 1997), all of which arefound in sheep (Tillet, 1987). The neurons of RMg project tolaminas I and II of the dorsal horn of the spinal cord, RPa andROb neurons project to layers VIII and IX of the ventral horn ofthe spinal cord, and the axons of the RVL/CVL neurons projectto the intermediolateral column of the spinal cord (Tork, 1990).This intermediolateral projection is of interest specifically inthe giraffe in terms of its involvement in cardiovascular control(Howe et al., 1983). Again, the giraffe phenotype may pose achallenge to the caudal serotonergic neurons in that they mustproject over a large distance in order to maintain their specificfunctions in the spinal cord and this may cause changes in themorphology and organization of these neurons.

While other mammalian species, such as camels and thelarger cetaceans will also have long spinal cords, the giraffe isunique in that it must pump blood against the force of gravity toenable sufficient blood flow to the brain—this is not anappreciable factor in these other long-necked or large animals.This anti-gravity circulation of the giraffe may provide aspecific challenge to the neural control of circulation andsmooth muscle that involves the catecholaminergic andserotonergic systems.

2. Materials and methods

One sub-adult giraffe (Giraffa camelopardalis) was used in the present

study. The animal was male, approximately 400 kg and 2 years old. The animal

was treated in accordance to the University of the Witwatersrand Animal EthicsCommittee guidelines for the care and use of animals in scientific experiments.

Appropriate permissions for use of the animal were also obtained from the local

provincial governmental bodies responsible for wildlife management in SouthAfrica. Upon death, the giraffe was immediately perfused with 0.9% normal

saline solution (200 l, or approximately 0.5 l/kg) to flush out the blood. This

process took approximately 20 min. This was immediately followed with a

perfusion of 200 l of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) forfixation, a process which again took about 20 min. The saline rinse and fixative

were manually pumped through tubes inserted into the carotid arteries. The

rinse and fixative were directed rostrally within the arteries, and thus caudally

these arteries were severed to allow the escape of blood, excess rinse andfixative. Following fixation the brain as well as the spinal cord was removed.

The whole brain and the entire length of the spinal cord were clear of blood and

rubbery to the touch indicating a successful whole body perfusion. Theperfusion was carried out in the field, with excess fixative being collected

upon a plastic tarpaulin that was placed under the animal and stored in drums for

later appropriate waste disposal. After weighing the brain (509 g), it was then

immersed overnight in the buffered paraformaldehyde for further fixation. Itwas then allowed to equilibrate in 30% sucrose solution in 0.1 M PB, and

subsequently stored in antifreeze solution at!20 8C. Themedullawas dissected

from the remainder of the brain (Fig. 1) and allowed to re-equilibrate in sucrose

solution and then frozen in crushed dry ice.Serial 50 mm sections of the medulla were made in a coronal plane using a

freezing sledge microtome. A one in four series of sections was stained for Nissl

(cresyl violet), myelin (modified from Gallyas, 1979), serotonin, and tyrosinehydroxylase (TH) (e.g. Manger et al., 2003, 2004; Da Silva et al., 2006). For

immunocytochemical staining, the sections were treated first with a peroxidase

inhibitor (49.2% methanol:49.2% 0.1 M PB:1.6% of 30% H2O2) for 30 min to

inhibit endogenous peroxidase, followed by three 10 min rinses in 0.1 M PB.This was followed by a 2 h preincubation (on a shaker), at room temperature, in

a solution containing 3% normal goat serum (NGS) (Chemicon Int.), 2% bovine

serum albumin (Sigma), and 0.25% Triton X100 (Merck) 0.1 M PB. After

Fig. 1. Photograph of the left side of the giraffe brain (lateral view) showing the

region of the brainstem investigated in the present study.

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–7970

preincubation, the sections were placed in primary antibody solution containingthe appropriately diluted antibody in 0.25% Triton X-100 in 0.1 M PB, 3%

NGS, and 2% bovine serum albumin, for 48 h at 4 8C on a shaker.

To reveal catecholaminergic neurons we used a polyclonal anti-tyrosine

hydroxylase primary antibody (TH) (AB151, Chemicon, raised in rabbit) ata dilution of 1:6500. To reveal serotoninergic neurons we used a polyclonal

anti-serotonin primary antibody (AB938, Chemicon, raised in rabbit) at a

dilution of 1:10,000. After the 48 h incubation in primary antibody solution,

the sections underwent three 10 min rinses in 0.1 M PB, and were thenplaced in a secondary antibody solution for 2 h at room temperature. The

secondary antibody solution contained a 1:500 dilution of biotinylated anti-

rabbit IgG (BA 1000, Vector Labs), with 3% normal goat serum, and 2%bovine serum albumin, in 0.1 M PB. After a further three 10 min rinses in

0.1 M PB, the sections were incubated in AB solution (Vector Labs) for 1 h

and rinsed in three 10 min rinses of 0.1 M PB. The sections were then

treated in a 0.05% solution of 3.30 diamino-benzidine (DAB) in 0.1 M PB for5 min. This was followed by adding 3 ml of 30% H2O2 per 0.5 ml solution.

The development process was followed visually and checked under a low

power stereomicroscope. The staining was allowed to continue until the

background staining was at a level that would enable us to match archi-tectonic features of the sections with the adjacent Nissl and myelin stained

sections without obscuring the immunopositive neurons. The development

was stopped by placing the sections in 0.1 M PB and then rinsing them twice

in 0.1 M PB.The sections were mounted on 0.5% gelatine coated glass slides and left to

dry overnight. They were then dehydrated in a graded series of alcohols, cleared

in xylene and coverslipped with Depex mounting medium. Two controls wereemployed during the immunostaining. The first control consisted of omitting the

primary antibody and the second control omitted the secondary antibody, in

selected sections.

The sections were examined under a low power stereomicroscope and thearchitectonic borders were traced according to the nissl and myelin sections

using a camera lucida. After completing the traces, the immunoreacted sections

were matched to the drawings and the immunopositive neurons marked.

Immunopositive neurons were those that clearly showed stained soma and

dendrites. The drawings were scanned and redrawn using the Canvas 8 drawingprogram.

High power photomicrographs were taken using a digital camera mounted

on a Zeiss Axioskop. No pixilation adjustments or manipulations of the

captured images were made, except for brightness and contrast adjustmentsusing Adobe Photoshop 7. In this study we used the nomenclature suggested

by Dahlstrom and Fuxe (1964) and Hokfelt et al. (1974, 1984) in conjunction

with that provided by Smeets and Gonzalez (2000) and Tillet and Thibault

(1989) for the catecholaminergic system, and that suggested by Tork (1990)for the serotonergic system (as opposed to that provided by Dahlstrom and

Fuxe, 1964; Tillet, 1987; Bjarkam et al., 1997) (see Table 1 for comparative

nomenclatures). While we use the nomenclature for the catecholaminergicsystem in this paper, we realise that the neuronal groups we revealed with

tyrosine hydroxylase immunohistochemistry may not correspond directly

with these nuclei as has been described in previous studies by Meister

et al. (1988), Kitahama et al. (1990, 1996), and Ruggiero et al. (1992).However, given the striking similarity of the results of the tyrosine hydro-

xylase immunohistochemistry to that seen in the sheep and other mammals we

feel this terminology is appropriate. Clearly further studies in more giraffe

with a wider range of antibodies, such as those to PNMT, DBH and aromatic L-amino acid decarboxylase (AADC) would be required to fully determine the

implied homologies ascribed in this study and the generality of the results to

the giraffe as a species.

3. Results

The giraffe brain is typically mammalian (Fig. 1). Thepresent study found immunocytochemically positive putativecatecholaminergic (tyrosine hydroxylase immunoreactive,TH+) and serotonergic (serotonin immunoreactive, 5-HT+)neurons throughout the medulla of the giraffe. The TH+neurons were for the most part similar to those seen in manyother mammals previously studied (Dahlstrom and Fuxe,

Table 1

Terminology of the catecholaminergic and serotonergic cell groups of the medulla oblongata of mammals and its link to their neuroanatomical location as described

by several groups

Name of nucleus in

present study

Smeets and

Gonzalez (2000)

Tillet and

Thibault

(1989)

Hokfelt

et al.

(1974)

Dahlstrom and

Fuxe (1964)

Tork (1990) Tillet

(1987)

Bjarkam

et al. (1997)

Catecholaminergic nuclei

C1 Rostral ventrolateraltegmental group

Group C1 C1

C2 Rostral dorsomedial

group

Group C2 C2

A1 Caudal ventrolateraltegmental group

Group A1 A1 A1

A2 Caudal dorsomedial

group

Group A2 A2 A2

Area postrema Area postrema Area

postrema

Area

postrema

Area postrema

Serotonergic nucleiRaphe magnus

nucleus, RMg

Nuc. Raphe Magnus,

RM, part of B3

Raphe magnus

nucleus, RMg

Group

B1/B3

Raphe magnus

nucleus, NRM

Raphe pallidus

nucleus, RPa

Nuc. Raphe pallidus,

RP, part of B1

Raphe pallidus

nucleus, RPa

Group

B1/B3

Raphe pallidus

nucleus, NRPRaphe obscurus

nucleus, ROb

Nuc. Raphe

obscurus, RO, B2

Raphe obscurus

nucleus, ROb

Group B2 Raphe obscurus

nucleus, NRO

Rostral ventrolateral

medulla, RVL

Rostral ventrolateral

medulla; part of B3/B1

Rostral

ventrolateralmedulla, RVL

Group S2 Rostral ventrolateral

medulla, NMVR

Caudal ventrolateral

medulla, CVL

Caudal ventrolateral

medulla; part of B3/B1

Caudal ventrolateral

medulla, CVL

Group S1 Caudal ventrolateral

medulla, NMVC

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–79 71

1964; Tillet and Thibault, 1989; Smeets and Gonzalez,2000). There was however, one major difference in theputative catecholaminergic nuclear complement, this beingan apparent lack of the C3 subdivision typically found inrodents (Smeets and Gonzalez, 2000). The 5-HT+ neuronswere very similar to those seen in other mammals (Dahlstromand Fuxe, 1964; Tork, 1990; Tillet, 1987; Bjarkam et al.,1997). The TH+ and 5-HT+ neurons that were observed are

described from the rostral to the caudal aspect of themedulla.

3.1. Putative catecholaminergic neurons

The neurons revealed with TH immunocytochemistry werereadily divided into five nuclei based on their anatomicallocation and neuronal morphology. We found evidence

Fig. 2. Diagrammatic reconstructions of the left half of the giraffe medulla. The open squares represent neurons immunoreactive to tyrosine hydroxylase and the filledcircles represent neurons immunoreactive to serotonin. Each circle and square indicates the location of an individual neuron. Section A is the most rostral, M the most

caudal, and each section is approximately 1000 mm apart.

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–7972

indicating the presence of the putative catecholaminergic nucleiC1, A1, C2, A2 and area postrema.

3.1.1. C1, rostral ventrolateral tegmental groupThe putative C1 neurons are found in the rostral ventro-

lateral portion of the medullary tegmentum extending from thefloor of the medulla to approximately the mid dorso-ventrallevel of the medullary tegmentum. At their most rostral extentthey are found surrounding the caudal pole of the facial nucleus(Fig. 2B–D). Caudal to this they are seen ventral to and

surrounding nucleus ambiguus, and the caudal pole of thenucleus ambiguus is the most posterior level at which neuronsthat form part of this nucleus could be identified (Fig. 2E–G).The most medially located neurons of this nucleus are found ina position just lateral to the inferior olive and they extend to justlateral of the lateral edges of facial nucleus and nucleusambiguus (Fig. 2B–H). The neurons are for the most part ovoidin shape and range between bipolar and multipolar forms(Fig. 3A) and are medium to large in size (30–45 mm diameter).No specific orientation to the dendrites could be observed.

Fig. 2. (Continued ).

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–79 73

These TH+ neurons exhibit a low to moderate densitythroughout the extent of this nuclear grouping.

3.1.2. A1, caudal ventrolateral tegmental groupThe TH+ neurons assigned putatively to the A1 nucleus are

found as a caudal continuation of those that form the putative C1group (Fig. 2H). They are found from the level of the posteriorpole of nucleus ambiguus to the anterior level of the cervicalspinal cord within the medullary tegmentum (Fig. 2H–L). Theyoccur in a position that can be described as significantly morelateral within the medullary tegmentum as compared with theneurons of the C1 nucleus and extend from the ventrolateral edgeof themedulla in a dorsomedial direction approximately halfwayacross the medullary tegmentum (Fig. 2K–H). They are found inthe region of the lateral reticular nucleus (LRt) of the medullarytegmentum (Fig. 2I–L). These medium size (around 20 mmdiameter) TH+ neurons are ovoid in shape, range betweenbipolar and multipolar forms (Fig. 3B), and exhibit no specificdendritic orientation. They have a very low density throughoutthe range in which they are found.

3.1.3. C2, rostral dorsomedial groupThe putative rostral dorsomedial group is represented by a

relatively substantial number of TH+ neurons located in thedorsal aspect of the medulla in a position just lateral to the areapostrema (see below) and adjacent to the floor of the fourthventricle. This nuclear cluster is found dorsal and lateral to theanterior pole of the dorsal motor vagus nucleus (Fig. 2G) wherethe majority of neurons are found; however, there is a smallnumber of neurons anterior to this main cluster in a similarlocation. The antero-posterior, medio-lateral, and dorso-ventralextents of the caudally located major cluster of this nucleus areless than 1 mm. Within this region the TH+ neurons show amoderate to high density caudally, with a lower numberanteriorly. All neurons observed were ovoid in shape and bipolarand small tomedium in size (10–20 mmdiameter) (Fig. 3C). Thedendrites were orientated parallel to the floor of the 4th ventricle.

3.1.4. A2, caudal dorsomedial groupThe number of TH+ neurons that could be reliably identified

as belonging to the putative A2 nucleus were very few and only

Fig. 3. Photomicrographs of the giraffemedulla showing neurons immunoreactive to tyrosine hydroxylase in four locations. (A) C1, rostral ventrolateral group,medium

sized neurons; (B) A1, caudal ventrolateral group, medium sized neurons; (C) dorsal strip of C2, rostral dorsomedial group belonging to the nucleus tractus solitarius,

showing a high density of small neurons, and (D) area postrema, showing a high density of small neurons. Scale bar = 200 mm, dorsal is up and medial is to the left.

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–7974

found over a very restricted antero-posterior extent. Thoseneurons assigned to this nucleus were found lying just dorsal tothe dorsal motor vagus nucleus and between the dorsal motorvagus nucleus and hypoglossal nucleus close to the centralcanal (Fig. 2K), close to the level of the spinal cord at the levelof the pyramidal decussation, and slightly lateral to thislocation within the dorsal medullary tegmentum. Theseneurons were ovoid and bipolar, medium in size (20 mmdiameter) and exhibited a very low density and had no specificdendritic orientation.

3.1.5. Area postremaThe area postrema was found at the same antero-posterior

level as the major part of the putative C2 nucleus, but waslocated at the midline dorsal to the anterior most part of thedorsal motor vagus nucleus and forming part of the floor of thefourth ventricle (Fig. 2G and H). The TH+ neurons thatdemarcate the area postrema form two distinct clusters on eitherside of the midline, but there are several neurons located

between these two main clusters, across the midline, that mergethe left and right halves of this distinct structure. There is amoderate to high density of small (10 mm diameter) ovoid tocircular shaped TH+ soma that show a mixture of bipolar andmultipolar forms (Fig. 3D). The majority of the neurons arebipolar and the dendrites are orientated in a medio-lateralaspect parallel to the floor of the fourth ventricle.

3.2. Serotonergic neurons

The serotonergic immunopositive (5-HT+) neurons werereadily divided into five nuclei based on their anatomicallocation and neuronal morphology. These were the: raphemagnus, raphe pallidus, rostral and caudal ventrolateral, andraphe obscurus nuclei.

3.2.1. RMg, raphe magnus nucleusThe neurons of raphe magnus were found close to the

midline forming two columns either side of the midline

Fig. 4. Photomicrographs of the giraffe medulla showing large neurons immunoreactive to serotonin in four locations. (A) RMg, raphe magnus nucleus; (B) RVL,

rostral ventrolateral group, medial portion; (C) RVL, rostral ventrolateral group, middle portion, and (D) RVL, rostral ventrolateral group, extreme lateral portion—

located lateral to facial nucleus. Note the varying orientation of the dendrites in the different regions. Scale bar = 200 mm, dorsal is up and medial to the right.

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–79 75

(Fig. 2A and B). The 5-HT+ neurons forming this nucleus werefirst observed at the level of the trigeminal motor nucleus, andcontinued caudally to the anterior most level of the nucleusambiguus (Fig. 2A–E). The neurons comprising this nucleuswere all large (25–35 mm diameter), piriform in shape andbipolar (Fig. 4A). For those neurons located immediatelyadjacent to the midline, the dendrites were orientated dorso-ventrally. The dendrites of those neurons located within500 mm of the midline were orientated medio-laterally. Theneurons had a low density throughout the extent of the nucleus,but appeared to fuse with the more extensive RVL (see below).

3.2.2. RPa, raphe pallidus nucleusThe 5-HT+ neurons that form the raphe pallidus nucleus

were all found in and around the pyramidal tracts (Fig. 2A–M).These neurons were found in the midline between the twopyramidal tracts, as well as ventral and lateral to thesepathways. The lateral most neurons were found ventral to theRVL (see below), but the difference in size allowed them to beassigned to the RPa. This nucleus exhibited an antero-posteriorextent that ranged from the level of the caudal pole of CNVII tothe cervical spinal cord (Fig. 2A–M). The neurons of thisnucleus were never seen beyond the inferior olive, eitherlaterally or dorsally. These neurons were small to medium insize (15–20 mm diameter) and multipolar (Fig. 5A). No distinctorientation of the dendrites could be readily observed.

3.2.3. RVL and CVL, rostral and caudal ventrolateralmedullary tegmental serotonergic cell columns

These two nuclear groups are described together as theyappear to be continuous in their distribution and the distinctionbetween them is more or less arbitrary based on anatomicallocation more so than connections or function (Fig. 2A–L). Therostrocaudal boundary given for these two nuclei is thetrapezoid body in the rabbit (Bjarkam et al., 1997), but otherdescriptions in mammals do not provide a clearly delimitedboundary (e.g. Tork, 1990). The 5-HT+ neurons assigned tothese nuclei are found from the level of the facial nucleusthrough to the cervical spinal cord (Fig. 2A–L). The neurons arefor the most part found in the ventral lateral portions of themedullary tegmentum, extending approximately 4 mm lateralto the inferior olive. Here they form a continuous antero-posterior column along their extent in this locality.

Anterior to the inferior olive, the neurons of the putativeRVL sweep across the midline above the pyramidal tracts,fusing the anterior portions of the RVL with the most ventralportions of the RMg. The RVL is then split into left and righthalves by the paired inferior olives and the appearance of theraphe pallidus nucleus (Fig. 2A–H). The RVL continuescaudally in the ventrolateral medullary tegmentum to the levelof the caudal pole of the nucleus ambiguus, where, somewhatarbitrarily, we can demarcate the beginning of the putative CVL(Fig. 2D–H). The CVL neurons continue this ventrolateralFig. 5. Photomicrographs of the giraffe medulla showing neurons immunor-

eactive to serotonin in three further locations. (A) RPa, raphe pallidus nucleus,

smaller neurons; (B) CVL, caudal ventrolateral group; (C) ROb, raphe

obscurus. Note the differing shape of the soma and dendritic orientationbetween the neurons closest to the midline (fusiform and dorsoventral) as

opposed to those away from the midline (triangular and irregular) in the ROb.Scale bar = 200 mm, dorsal is up and medial is to the right.

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–7976

medullary column of 5-HT+ neurons to the level of the cervicalspinal cord (Fig. 2H–L).

The neurons forming these two nuclei are most numerousanteriorly and steadily decline in number caudally. The neuronsof these nuclei are medium to large in size (20–40 mmdiameter) and are multipolar (Figs. 4B–D and 5B), giving thesoma a stellate appearance. The larger size of these neuronsallowed us to distinguish those neurons of the RVL from thosethat form the most lateral part of the RPa. The dendrites exhibita rough medio-lateral orientation. At the most anterior extent ofthe RVL there is a small cluster of 5-HT+ neurons in a positionjust lateral to CNNVII, and while the neuronal morphology issimilar to that of the other neurons of these nuclei, the dendritesare not oriented in any specific direction.

3.2.4. ROb, raphe obscurus nucleusThe 5-HT+ neurons of ROb are seen to form two columns

either side of the midline between the hypoglossal nucleus andthe inferior olive (Fig. 2I–K). They are found from the level ofthe exiting hypoglossal nerve to the anterior most portion of thecervical spinal cord (Fig. 2I–K). Within this nucleus there areneurons located immediately adjacent to the midline and thosethat are a short distance away from the midline. These mediumsized neurons (20 mm diameter) are found in a low to moderatedensity throughout their distribution and all are multipolar.Those neurons located immediately adjacent to the midline arefusiform in shape (Fig. 5C) and the dendrites are orientateddorsoventrally. The neurons located a short distance from themidline are triangular in shape and there is no specificorientation to the dendrites.

4. Discussion

Overall our results show that the putative catecholaminergicand serotonergic neurons in the medulla of the sub-adult giraffestudied are very similar to those seen in other Artiodactyls(Tillet, 1987, 1988; Tillet and Thibault, 1989; Ruggiero et al.,1992; Tillet and Kitahama, 1998). The catecholaminergicnuclei, as demarcated with tyrosine hydroxylase immunohis-tochemistry, found in this giraffe medulla include C1, A1, C2,A2 and area postrema. We identified the putative C1 nucleus inthe giraffe medulla with tyrosine hydroxylase (TH) immuno-histochemistry. This nucleus has been identified with TH andPNMT immunohistochemistry in pig and cow (Ruggiero et al.,1992; Tillet and Kitahama, 1998), but in sheep this nucleus isnot visualized with PNMT immunohistochemistry (Tillet,1988), despite neurons in this region being immunoreactive forboth TH and DBH (Tillet, 1988; Tillet and Thibault, 1989). Theserotonergic nuclei found in the giraffe medulla include RMg,RPa, RVL/CVL and ROb, all of which were seen in sheep(Tillet, 1988) and indeed most other mammals (Dahlstrom andFuxe, 1964; Tork, 1990).

4.1. Putative catecholaminergic nuclei in the giraffe

The catecholaminergic nuclei found in the giraffe medullastudied are similar to those seen in most other mammals, the

one exception being the lack of a C3 nucleus seen in thelaboratory rat, hamsters, neonatal swine and human (Ruggieroet al., 1992; Tillet and Kitahama, 1998; Smeets and Gonzalez,2000); however, this nucleus has not been reported inmonotremes (Manger et al., 2002a), sheep (Tillet and Thibault,1989), cow (Tillet and Kitahama, 1998), rabbit (Blessing et al.,1981), cat (Poitras and Parent, 1978; Blessing et al., 1980), dog(Barnes et al., 1988; Dormer et al., 1993), tree shrew (Murrayet al., 1982), or any non-human primates that have been studied(Hubbard and Di Carlo, 1974; Garver and Sladek, 1975;Jacobwitz and MacLean, 1978; Schofield and Everitt, 1981;Pearson et al., 1983). The A2 division was comprised of only afew TH+ neurons, but these were found in the expected location(above the dorsal motor vagus nucleus) as well as in a position(between the dorsal motor vagus nucleus and the hypoglossalnucleus), that is similar to what is seen in sheep and cow (Tilletand Thibault, 1989; Tillet and Kitahama, 1998) but differs tothat seen in the rat (Kalia et al., 1985a). We did not see thevariety in cellular morphology described in the sheep for theneurons of this group (Tillet and Thibault, 1989). The neuronsforming the A1 nucleus appeared similar to that of othermammals studied, in being multipolar (Tillet and Thibault,1989), but there were more neurons in the dorsal aspect of thisnucleus compared to the ventral aspect where the opposite is thecase in the sheep and the rat (Tillet and Thibault, 1989; Kaliaet al., 1985a). The C2 nucleus of the giraffe exhibited only a fewneurons in the region that contains the most neurons in the rat(Kalia et al., 1985b), but exhibited many neurons in the regionof the rat that can be described as the dorsal strip of the C2nucleus, being part of the nucleus tractus solitarius, animportant autonomic center in the CNS (Kalia et al., 1985b).This dorsal strip of C2 in the giraffe appeared to be far moredensely populated with neurons compared to other mammalsand in particular the sheep and cow (Tillet and Thibault, 1989;Tillet and Kitahama, 1998). The appearance and location ofarea postrema was similar to that seen in all other mammals butagain was more densely packed with TH immunoreactiveneurons compared with what is seen in other mammals (Tilletand Kitahama, 1998). Given the overall involvement of the C2nucleus and the area postrema in cardiovascular functions(Koizumi and Brooks, 1974), this may be expected in the long-necked giraffe.

The C1 nucleus in this giraffe medulla has been identified inthe present study with tyrosine hydroxylase immunohisto-chemistry. Using TH and DBH immunohistochemistry, Tilletand Thibault (1989) also found immunoreactive neurons in thesame location in the sheep as described here for the giraffe; butin the sheep, there were no neurons immunoreactive for PNMT(Tillet, 1988); however, Tillet and Kitahama (1998) andRuggiero et al. (1992) described a C1 division on the basis ofPNMT immunohistochemistry in cow and pig respectively. Itmay be considered that the gene/s related to the expression ofPNMT in the sheep has/have become inactivated. Furtherstudies in the giraffe must be undertaken using severalimmunochemical markers (TH, DBH, PNMT, AADC) todetermine if C1 is indeed present or absent in this species.Overall, the phenotype of the giraffe does not appear to have

N.L. Badlangana et al. / Journal of Chemical Neuroanatomy 34 (2007) 69–79 77

had a dramatic effect on the nuclear subdivisions or neuronalmorphology of the putative medullary catecholaminergicneurons in comparison to most other mammals, and the manysimilarities to that seen in other members of the Artiodactyla,strengthens this conclusion.

4.2. Serotonergic nuclei

All the 5-HT+ subdivisions (RMg, RPa, RVL/CVL 5HTcolumns and ROb) in the giraffe medulla studied were found tohave direct homologs in the sheep and indeed other eutherianmammals. The CVL 5HT column appears to be absent in themonotremes (Manger et al., 2002b), and the Virginia oppossum(Crutcher and Humbertson, 1978), but is present in the wallaby(Ferguson et al., 1999). The one difference in the giraffe studiedis that the RVL 5HT column appeared to be more extensiveanteriorly than that seen in other mammals. The projection ofthe RVL 5HT column to the intermediate lateral horn of thespinal cord in other mammals (Tork, 1990), where it regulatesblood pressure, may explain the relative expansion of thisnucleus in the giraffe compared to the sheep, for example(Tillet, 1987). Despite projecting down a long spinal cord, wefound no evidence for abnormally large 5-HT+ neurons in thegiraffe medulla examined. The neuronal morphology in termsof number of poles and shape is for the most part quite similar tothat seen in the sheep for the different subdivisions of thissystem (Tillet, 1987).

4.3. Order specific patterns in the nuclear complement ofneuromodulatory systems

Manger (2005) and Da Silva et al. (2006) have suggestedthat animals from the same mammalian order will exhibit thesame complement of nuclei in the neuromodulatory systems ofthe brain regardless of phenotype, life history or brain size. Thegiraffe has a significantly different phenotype to the sheep interms of the length of the limbs and neck, has a far larger brain(around 500 g in the giraffe studied compared to around 150 gin the sheep), and a very different life history (e.g. a browser oftrees compared with a grazer of grass). Despite thesedifferences, there is a striking resemblance in the morphologyand subdivisions of the putative catecholaminergic andserotonergic systems of the medulla of the giraffe studiedand those of the sheep, pig and cow (Tillet, 1987, 1988; Tilletand Thibault, 1989; Ruggiero et al., 1992; Tillet and Kitahama,1998). Some variations were evident, especially in the relativedensity of the neurons within various nuclei (e.g. dorsal strip ofC2, area postrema, dorsal A1, and the anterior portion of theRVL 5HT column), and these may be reflective of certainfunctional properties of the specific nuclei, but these differencesare found within a nucleus and do not relate to the nuclearparcellation of these systems. Thus, regardless of its brain size,phenotype and life history, it appears that the sub-adult giraffebrain studied was constrained in its ability to construct changesat the systems level (in terms of increased nuclear parcellation)that may be adaptive to its phenotype within a given Artiodactylframework. Galis (1999) has suggested that changing the

expression of one gene that may lead to changes in themorphophysiology of different parts of the body brings a highrisk. It may lead to lethal mutations as there are several othergenes that are intricately tied to that one gene and they toowould be affected and cause phenotypic change (pleiotropy),that may not be compatible with overall organismal success.Given that the neuronal systems examined in the present studyappear very early in development, it may be that suchconsequences of pleiotropy during the evolutionary changesleading to altered morphology may be the major factorrestricting change in the nuclear parcellation of theseneuromodulatory systems within a mammalian order.

Acknowledgments

The work reported herein was funded by a University of theWitwatersrand Faculty of Health Sciences Research Grant(NLB) and by the South African National Research Foundation(PRM).

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