Distribution and morphology of catecholaminergic and serotonergicneurons in the brain of the highveld gerbil, Tatera brantsii

Don-Joon Moon a, Busisiwe C. Maseko a, Amadi O. Ihunwo 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 Africa

bDepartment of Neuroscience, Karolinska Institutet, Retzius vag 8, S-171 77 Stockholm, Sweden

Received 23 March 2007; received in revised form 29 May 2007; accepted 3 June 2007Available online 8 June 2007

Abstract

The distribution, morphology and nuclear subdivisions of the putative catecholaminergic and serotonergic systems within the brain of thehighveld gerbil were identified following immunohistochemistry for tyrosine hydroxylase and serotonin. The aim of the present study was toinvestigate possible differences in the complement of nuclear subdivisions of these systems when comparing those of the highveld gerbil with thoseof the laboratory rat. The highveld gerbil was chosen as it is relatively closely related to the laboratory rat, but theGerbillinae andMurinae lineagesdiverged over 20 million years ago. Moreover, even though brain sizes are similar, the life history and phenotypes between these two species aresubstantially different. The gerbils used in the present study were caught from the wild, which is again another contrast to the laboratory rat. Whilethese differences may lead to the prediction of significant differences in the nuclear complement of these systems, we found that all nuclei identifiedin both systems in the laboratory rat in several earlier studies had direct homologs in the brain of the highveld gerbil. Moreover, there were noadditional nuclei in the brain of the highveld gerbil that are not found in the laboratory rat. The only discernable difference between the two specieswas a greater density and number of catecholaminergic neurons in the olfactory bulb of the highveld gerbil. Thus, the evolution of nuclearparcellation in these systems appears to demonstrate a form of phylogenetic constraint related to the order Rodentia.# 2007 Elsevier B.V. All rights reserved.

Keywords: Tyrosine hydroxylase; Serotonin; Evolution; Mammal; Rodent; Neural systems

www.elsevier.com/locate/jchemneuJournal of Chemical Neuroanatomy 34 (2007) 134–144

Abbreviations: 3v, third ventricle; A1, caudal ventrolateral medullary tegmental group; A2, caudal dorsomedial medullary group; A4, dorsomedial division of

locus coeruleus; A5, fifth arcuate nucleus; A6d, diffuse portion of locus coeruleus; A7d, diffuse portion of subcoeruleus; A7sc, compact portion of subcoeruleus; A8,retrorubral region; A9l, lateral division substantia nigra; A9m, medial division substantia nigra; A9pc, substantia nigra pars compacta; A9v, substantia nigra pars

reticulata (ventral); A10, ventral tegmental area; A10c, central division ventral tegmental area; A10d, dorsal division ventral tegmental area; A10dc, dorsal caudal

division ventral tegmental area; A11, caudal diencephalic group; A12, tuberal cell group; A13, zona incerta; A14, rostral periventricular nucleus; A15d, anterior

hypothalamic group, dorsal division; A15v, anterior hypothalamic group, ventral division; ac, anterior commissure; Amyg, amygdala; AP, area postrema; B9,supralemniscal serotonergic cluster; BP, basilar pontine nucleus; ca, cerebral aqueduct; C1, rostral ventrolateral medullary tegmental group; C2, rostral dorsomedial

medullary group; C3, rostral dorsal midline medullary group; C/P, caudate and putamen nuclei; Cb, cerebellum; cc, corpus callosum; Cn, deep cerebellar nuclei; CLi,

caudal linear nucleus; CVL, caudal ventrolateral medullary tegmental serotonergic group; DRc, caudal division of dorsal raphe; DRd, dorsal division of dorsal raphe;DRif, interfascicular division of dorsal raphe; DRl, lateral division of dorsal raphe; DRp, peripheral division of dorsal raphe; DRv, ventral division of dorsal raphe;

DT, dorsal thalamus; f, fornix; GC, central grey/periaqueductal grey matter; GP, globus pallidus; Hip, hippocampus; IC, inferior colliculus; io, inferior olivary nuclei;

IP, interpeduncular nucleus; LGd, dorsal lateral geniculate nucleus; lot, lateral olfactory tract; lv, lateral ventricle; mcp, middle cerebellar peduncle; MnR, median

raphe nucleus; NEO, cerebral neocortex; oc, optic chiasm; ot, optic tract; PC, cerebral peduncle; PIR, piriform cortex; PoA, preoptic area; py, pyramidal tract; R,thalamic reticular nucleus; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; RVL, rostral ventrolateral medullary tegmental

serotonergic group; S, septal nuclear complex; SC, superior colliculus; scp, superior cerebellar peduncle; Vgang, trigeminal sensory ganglion; VIIv, ventral division

of facial nerve nucleus; Vm, trigeminal motor nucleus; Vs, trigeminal sensory nucleus; Vsp, trigeminal spinal nucleus; X, dorsal motor vagus nerve nucleus; XII,

hypoglossal nerve nucleus; zi, zona incerta* 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.2007.06.001

1. Introduction

Within the order Rodentia there are over 400 genera and2300 species, representing nearly half of all knownmammalian species, and of these the family Muridae is themost diverse with over 1300 recognized species (Jansa andWeksler, 2004). Two species from the family Muridae,subfamily Murinae, Rattus norvegicus and Mus musculus,are two of the most commonly used species in modernneuroscience. These two species are often used in clinical andapplied studies with the results extrapolated to expand ourunderstanding of the human brain. Despite this, very little isknown about the evolution of brains among rodents and inparticular what systems of the brain may show variance withchanges in phenotype, lifestyle and evolutionary history.Knowledge of rodent brain evolution is important in terms ofunderstanding what extrapolations can be made from thelaboratory rat and mouse to humans.

A recent study of cholinergic neurons within the cerebralcortex of various rodent species demonstrated that corticalcholinergic interneurons were present in the members of thesubfamily Murinae studied, but not in the cortex of anothermember of the Muridae family of the subfamily Gerbillinae,Tatera brantsii, demonstrating variance amongst the Muridaesubfamilies in the occurrence of this characteristic (Bhag-wandin et al., 2006). On the other hand, another recent studyin a rodent distantly related to the Muridae, the commonmolerat Cryptomys hottentotus, demonstrated that thecholinergic, catecholaminergic and serotonergic systems ofthe midbrain and pontine region retained the same nuclearparcellation as described for the Muridae (Da Silva et al.,2006).

These two aforementioned studies were designed to test anearlier proposal that the nuclear parcellation of the neuromo-dulatory systems were conserved or constrained within amammalian order such that irrespective of brain size, lifestyleor phenotype, the same complement of nuclei of these systemswill be found in all species of a single mammalian order(Manger, 2005). Thus, given the observation of variance in thecortical cholinergic system of T. brantsii compared to theMurinae, but the observations of invariance in the midbrain andpontine nuclei in C. hottentotus compared with the Murinae, itis clear further comparison is required to aid our understandingof this variance and invariance in the evolution of rodent brains.To this end, we decided to examine the full complement ofputative catecholaminergic and serotonergic nuclei in the brainof T. brantsii to compare to the previously published reports ofthese systems in the Murinae (Dahlstrom and Fuxe, 1964; Fuxeet al., 1969; Lindvall and Bjorklund, 1974; Bjorklund andLindvall, 1984; Steinbusch, 1981; Hokfelt et al., 1977, 1984;Tork, 1990; Smeets and Gonzalez, 2000).

2. Materials and methods

In the present study, six adult males of the nocturnal, omnivorous highveldgerbil (T. brantsii, average body weight of 125.1 g, average brain weight of

1.61 g, Fig. 1), were caught from the wild in late summer and used. These

gerbils breed throughout the year, with a lull in breeding during the drier

months of winter. The animals were treated and used according to the guide-

lines of the University of the Witwatersrand Animal Ethics Committee, whichparallel those set down by the NIH for use of animals in scientific experiments.

While under deep barbiturate anaesthesia (Euthanaze, 40 mg/kg, i.p.) the

animals were perfused intracardially following cessation of respiration. Theanimals were initially perfused with a cold (4 8C) rinse of 0.9% saline,

followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (approx.

1l kg!1 of each). The brains were removed from the skull and post-fixed

overnight in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), and thenallowed to equilibrate in 30% sucrose in PB. The brains were then frozen in dry

ice and sectioned in either a coronal or sagittal plane (50 mm section thickness,

collected in PB) on a slidingmicrotome.A one in four series of stainswasmade

for Nissl (with cresyl violet), myelin (a modification of Gallyas, 1979),tyrosine hydroxylase (TH, AB151, Chemicon, at a dilution of 1:6500, raised

in rabbit) and serotonin (AB938, Chemicon, at a dilution of 1:10,000, raised in

rabbit).

For immunohistochemical staining the sections were first treated for30 min with an endogenous peroxidase inhibitor (49.2% methanol:49.2%

0.1 M PB:1.6% of 30% H2O2) followed by three 10-min rinses in 0.1 M PB.

This was followed by a 2 h pre-incubation, at room temperature, in a solutioncontaining 3% normal goat serum (Chemicon Int.), 2% bovine serum albumin

Fig. 1. Photographs of the dorsal, ventral and lateral aspects of the brain of the

highveld gerbil. Note that despite showing the typical rodent gross brain

morphology the olfactory bulbs and optic nerve are large for a rodent. Scalebar = 5 mm.

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144 135

(Sigma) and 0.25% Triton X-100 (Merck) in 0.1 M PB. Following three 10-min rinses in 0.1 M PB, the sections were placed in the primary antibody

solution that contained the appropriately diluted antibody in 0.25% Triton X-

100 in 0.1 M PB, 3% normal goat serum and 2% bovine serum albumin,

for 48 h at 4 8C. Three 10-min rinses in 0.1 M PB followed this step, andthe sections were then placed in a secondary antibody solution for a 2-h

incubation. The secondary antibody solution contained a 1:500 dilution of

biotinylated anti-rabbit IgG (BA 1000, Vector Labs), with 3% normal goat

serum, 2% bovine serum albumin, in 0.1 M PB. After three more rinses in0.1 M PB, the sections were incubated for 1 h in AB solution (Vector Labs),

and again rinsed three times. Following this, the sections were treated in a

solution of 0.05% diaminobenzidine (DAB) in 0.1 M PB for 5 min. This wasfollowed by adding 3 ml of 30%H2O2 per 0.5 ml of solution. The development

process was followed visually, and checked under a low power stereomicro-

scope. This was allowed to continue until the background staining was at such

a level as to not obscure the immunopositive neurons. The development wasarrested by placing the sections in 0.1 M PB after which they were rinsed two

more times in the same solution. The sections were mounted on 0.5% gel

coated glass slides and left to dry overnight. The next day they were

dehydrated in a graded series of alcohols and cleared in xylene before beingcoverslipped with Depex. Two controls were employed during the immunos-

taining. The first control consisted of omitting the primary antibody, while the

second omitted the secondary antibody. No staining was evident in the control

sections.The sections were examined under a low power stereomicroscope and the

architectonic borders were traced according to the Nissl and myelin sections

using a camera lucida. After completing the traces, the immunoreacted sectionswere 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 drawing

program. Digital photomicrographs were captured using a Zeiss Axioskop andthe Axiovision software. No pixilation adjustments, or manipulation of the

captured images were undertaken, except for the adjustment of contrast,

brightness and levels using Adobe Photoshop 7. The present study used

previously published reports for nomenclature (Dahlstrom and Fuxe, 1964;Fuxe et al., 1969; Lindvall and Bjorklund, 1974; Steinbusch, 1981; Bjorklund

and Lindvall, 1984; Hokfelt et al., 1977, 1984; Tork, 1990; Paxinos et al., 1999;

Smeets and Gonzalez, 2000). While we use the standard nomenclature for thecatecholaminergic system 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 hydroxylase immu-

nohistochemistry to that seen in other rodents we feel this terminology is

appropriate. Clearly further studies in the highveld gerbil with a wider range of

antibodies, such as those to PNMT, DBH and aromatic L-amino acid decarbox-ylase (AADC) would be required to fully determine the implied homologies

ascribed in this study.

3. Results

In the current study using immunohistochemical stains fortyrosine hydroxylase and serotonin, the distribution, nuclearparcellation and morphology of neurons belonging to theputative catecholaminergic and serotonergic systems werereadily identified. Although the neuronal somatawere themostheavily stained portion of the neurons, the primary dendriticbranches were also often readily visible. The putativecatecholaminergic system in the highveld gerbil followedthe pattern typically seen in rodents, with nuclear clustersbeing found in the olfactory bulb, diencephalon, midbrain,pons and medulla (Fig. 2A). The serotonergic system wasreadily divisible into the rostral and caudal brainstem clustersas described in other rodent species and other mammals(Fig. 2B).

3.1. Catecholaminergic nuclei

3.1.1. A16—Olfactory bulbA high density of strongly tyrosine hydroxylase immunor-

eactive (TH+) periglomerular neurons was present in theolfactory bulb, mostly within the glomerular layer, but theoccasional neuron was visible in the outer plexiform layer(Fig. 2A). The neurons had triangular shaped somata and weremultipolar, exhibiting a dense dendritic network surroundingthe glomeruli.

3.1.2. A15–A11—Diencephalic nucleiWithin the diencephalon the TH+ neurons located within the

hypothalamus could be readily subdivided in the A15–A11nuclei previously described for rodents (Fig. 2A). A dorsaldivision of the A15 group was found lateral to the dorsal aspectof the anterior part of the third ventricle adjacent to the inferioraspect of the anterior commissure (Fig. 3A). The neurons of thisgroup were ovoid in shape and bipolar in nature. Within theventral lateral aspect of the hypothalamus a cluster of bipolarTH+ neurons were located and assigned to the A15 ventralgroup (Fig. 3C). These neurons exhibited a mixture of ovoidand fusiform shapes with dendrites oriented parallel to the floorof the diencephalon.

A group of TH+ neurons forming a column on either side ofthe anterior aspect of the wall of the third ventricle wasrecognizable as the A14 nucleus (rostral periventricular cellgroup) (Fig. 3B). These neurons were bipolar and ovoid shaped,but no specific dendritic orientation was observed (Fig. 4A). Acluster of TH+ neurons located caudal to the A15d group andadjacent to the zona incerta, but within the hypothalamic greymatter, was designated as the A13 (zona incerta) nucleus(Fig. 3B and C). These neurons were small, bipolar andfusiform in shape with dendrites oriented in a mediolateraldirection (Fig. 4B). Surrounding the ventral aspect of the thirdventricle, in the vicinity of the tuber cinereum was anothergroup of TH+ neurons that collectively could be assigned to theA12 nucleus (tuberal cell group) (Fig. 3B and C). These ovoidshaped, bipolar neurons evinced dendrites that were eitheroriented parallel to the wall of the third ventricle or the base ofthe hypothalamus (Fig. 4C). The last cluster of TH+ neuronsrecognizable as a distinct nucleus within the hypothalamus wasthe A11 nucleus, or caudal diencephalic group. The bipolar,ovoid shaped neurons of this nucleus were found looselyscattered around the diminishing posterior aspect of the thirdventricle close to the midline (Fig. 3D). There were not a greatnumber of neurons in this nucleus, and the dendrites wereoriented roughly parallel to the wall of the third ventricle.

3.1.3. A10—Ventral tegmental area nuclear complexWithin the ventral medial aspect of the midbrain

tegmentum a distinct TH+ nuclear complex divisible intoseveral nuclei could be located (Figs. 2A and 3E–G). Thisnuclear complex represents the ventral tegmental area of thehighveld gerbil. Within this region we could identify the A10,A10c (central), A10d (dorsal) and A10dc (dorsocaudal)nuclei. The bipolar and round neurons of the A10 nucleus were

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144136

located between the interpeduncular nucleus and theoculomotor nerve (corresponding to the paranigral nucleusin the rat). Immediately dorsal to the interpeduncular nucleusand medial to the A10 nucleus, the bipolar, fusiform shapedneurons of the A10c nucleus were found. These neuronsformed a distinct and dense band over the dorsal aspect of theinterpeduncular nucleus with no clear break between left andright halves around the midline (corresponding to theparabrachial pigmental nucleus in the rat). A cluster ofTH+ neurons loosely associated with the midline of the brain,between the A10c nucleus and the oculomotor nucleus, weredesignated as the A10d nucleus. The bipolar neurons withinthis nucleus were larger than those found in the previouslydescribed nuclei of the ventral tegmental area and werepiriform in shape. The dendrites emanating from these neuronswere oriented in a roughly dorsoventral plane. Within theperiaqueductal grey matter, in close proximity to the lowerhalf of the cerebral aqueduct was a cluster of TH+ neurons thatwere assigned to the A10dc nucleus. This nucleus extendedfrom the anterior most level of the periaqueductal grey matter

through to the beginning of the fourth ventricle. The neuronsof A10dc were bipolar, fusiform or ovoid in shape withdendrites oriented parallel to the wall of the cerebral aqueduct(Fig. 4D).

3.1.4. A9—Substantia nigra nuclear complexA large and distinct arc of TH+ neurons located dorsal to the

cerebral peduncle in the ventral lateral aspect of the midbraintegmentum was assigned to the various nuclei of the substantianigra (Figs. 2A and 3D–F). Within this arc of neurons we couldidentify the A9m (medial), A9pc (pars compacta), A9v(ventral, pars reticulata) and A9l (lateral) nuclei of thesubstantia nigra complex. The high density of ovoid, bipolar,TH+ neurons located between the oculomotor nerve and thedorsomedial edge of the cerebral peduncle were assigned to theA9m nucleus (Fig. 4E). The dendrites were oriented parallel tothe edge of the cerebral peduncle. Lateral to this nucleus, theTH+ neurons formed a distinct band of fusiform shapedneurons overlying the dorsal aspect of the cerebral peduncle.These neurons were designated as those forming the A9pc

Fig. 2. Composite reconstructions of the sagittal series of sections of the brain showing the locations of the major components of the (A) catecholaminergic and (B)serotonergic systems of the brain of the highveld gerbil. See list for abbreviations.

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144 137

nucleus had a low density and exhibited dendrites orientedparallel to the dorsal limit of the cerebral peduncle. Lateral tothe A9pc nucleus was a loosely aggregated cluster of neuronsfound lateral to the lateral edge of the cerebral peduncle in a lowdensity. These piriform shaped bipolar neurons had dendritesthat exhibited a weak mediolateral orientation and were thoughtto form the A9l nucleus. Ventral to the A9pc, intermingled withthe fasciculi of the cerebral peduncle, were a loosely aggregatedgroup of TH+ neurons that formed the A9v nucleus. Theseneurons were triangular in shape and multipolar and evinced nospecific dendritic orientation.

3.1.5. A8—Retrorubral cell groupDorsal to the A9pc, caudal to the magnocellular subdivi-

sion of the red nucleus, and within the ventral half of themidbrain tegmentum, was a group of TH+ neurons that wererecognizable as those forming the A8 nucleus (Figs. 2A and3F). The neurons of this nucleus were ovoid to fusiform inshape and were a mixture of bipolar and multipolar forms.The orientation of the dendrites was not strongly biased, but aweak ventrolateral to dorsomedial orientation could bediscerned.

3.1.6. A7–A4—The locus coeruleus nuclear complexWithin the pontine region of the highveld gerbil a large

number of TH+ neurons that could be assigned to various nucleiwithin the locus coeruleus complex were found (Figs. 2A and3H and I). Within the locus coeruleus complex we identified theA7sc (subcoeruleus, compact portion), A7d (subcoeruleus,diffuse portion), A6d (locus coeruleus, diffuse portion), A5(fifth arcuate nucleus) and A4 (dorsomedial) nuclei. Within thedorsal middle pontine tegmentum a loosely coalesced group ofTH+ neurons located medial, lateral and inferior to the superiorcerebellar peduncle and dorsal to the trigeminal motor nucleuswere classified as the A7d nucleus. The neurons of this nucleuswere multipolar with no clear dendritic orientation andexhibiting a variety of shapes. Within the most dorsal part ofthe pontine tegmentum, a dense cluster of TH+ neuronsadjacent to the ventrolateral border of the periventricular greymatter was identified as the A7sc nucleus (Dahlstrom and Fuxe,1964), or what is often referred to as the subcoeruleus (Olsonand Fuxe, 1972). These neurons exhibited a similar morphol-ogy to that seen for the A7d nucleus.

Within the ventrolateral periventricular grey matter of thepons a strongly stained and moderately densely packed cluster

Fig. 3. Diagrammatic reconstructions of a series of coronal sections through the brain of the highveld gerbil showing the location of neurons immunohistochemically

reactive for tyrosine hydroxylase (black circles) and serotonin (open squares). Figurine A represents the most rostral section, K the most caudal. Each section isapproximately 1 mm apart. See list for abbreviations.

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144138

of TH+ neurons represents the A6d nucleus of the locuscoeruleus complex (Fig. 3H). We term this the diffuse portionof the locus coeruleus to differentiate the packing of neurons inthis nucleus in comparison to the tightly packed neuronalcluster seen in the laboratory rat (Dahlstrom and Fuxe, 1964).All the neurons in this cluster were multipolar and appeared tohave dendrites oriented in a ventrolateral to dorsomedialdirection.Within the ventrolateral pontine tegmentum, adjacentto the lateral aspect of the facial nerve nucleus a looselyarranged column of TH+ neurons represents the A5 nucleus.The neurons of the A5 nucleus evinced a range of somatalshapes and were more numerous in the caudal half of thisnucleus. The neurons had a rough dendritic orientation in aventrolateral to dorsomedial plane. The last nucleus recognizedin the highveld gerbil locus coeruleus complex was the A4nucleus. This tightly packed cluster of round, bipolar, TH+neurons (Fig. 4F) was located in the dorsolateral periventriculargrey matter, adjacent to the floor of the fourth ventricle(Fig. 3H).

3.1.7. C1, C2, C3, A1, A2, area postrema—Caudalrhombencephalic nuclei

Within the caudal rhombencephalon we identified sixcatecholaminergic nuclei (Figs. 2A and 3J and K), all ofwhich correspond to nuclei previously described in thelaboratory rat. Rostrally within the ventrolateral medullary

tegmentum in the region of the nucleus ambiguus, a caudal TH+neuronal column was evident and appeared to be the C1 (rostralventrolateral tegmental cell group) nucleus. A variety ofmultipolar somatal shapes were evident in the neurons of thisnucleus, and a rough ventrolateral to mediodorsal orientation ofthe dendrites was observed. This column appeared to continuecaudally to the nucleus ambiguus, and became the A1 (caudalventrolateral tegmental cell group) nucleus, terminating at thespinomedullary junction. The neuronal morphology did notchange at the C1/A1 border. Immunohistochemistry forphenylethanolamine-N-methyltransferase would be requiredto determine the precise border between these two nuclei(Smeets and Gonzalez, 2000).

A densely packed cluster of small, round, bipolar andmultipolar TH+ neurons located at the midline dorsal to thedorsal motor vagus nucleus and the nucleus tractus solitariusrevealed the area postrema (Fig. 3K). Lateral to the areapostrema a moderately densely packed cluster of TH+ neuronslocated adjacent to the floor of the fourth ventricle representedthe dorsal strip of the C2 (rostral dorsomedial group) nucleus ofthe nucleus tractus solitarius (Kalia et al., 1985a). The neuronsof this nucleus were not particularly numerous and were allsmall, round and bipolar in form, with dendrites orientedparallel to the floor of the fourth ventricle. A small number ofloosely arranged TH+ neurons were located anterior to thedorsal strip of C2, representing the remainder of the C2 nucleus

Fig. 3. (Continued ).

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144 139

in the nucleus tractus solitarius (Kalia et al., 1985a). A clusterof TH+ neurons anterior to the area postrema, clustered aroundthe dorsal midline of the medulla adjacent to the floor of thefourth ventricle represented the C3 nucleus. These neuronswere small, ovoid and bipolar, with dendrites oriented parallelto the floor of the fourth ventricle. The final catecholaminergicnucleus identified was represented by a loosely arrangedmediolateral column of TH+ neurons located above the dorsalmotor vagus nucleus in the nucleus tractus solitarius andbetween the dorsal motor vagus nucleus and the hypoglossalnucleus. This nucleus, comprised of ovoid and fusiform shapedbipolar forms extended a small way into the caudal medullarytegmentum, and represented the A2 (caudal dorsomedial group)nucleus (Kalia et al., 1985b).

3.2. Serotonergic nuclei

3.2.1. CLi—Caudal linear nucleusThe most rostrally located serotonergic nucleus was that

identified as the caudal linear nucleus. The neurons forming thisnucleus were strongly serotonin immunoreactive (5-HT+) andwere located near the midline, anterior and ventral to thedecussation of the brachium conjunctivum, but dorsal to theinterpeduncular nucleus (Figs. 2B and 3F). Within this location,

the density of neurons was quite high and all neurons weremultipolar with a soma that was round in shape.

3.2.2. B9—Supralemniscal serotonergic clusterFanning out in a lateral direction from the ventral portion of

the CLi in a position posterior to the substantia nigra, was asmall cluster of serotonergic neurons that correspond to thepreviously described B9 group in the rat (Figs. 2B and 3F). The5-HT+ neurons in this region were bipolar and round in shape.The dendrites were oriented in a mediolateral plane.

3.3. Dorsal raphe nuclear complex

The majority of 5-HT+ neurons in the rostral cluster of theserotonergic system were found within, or associated with, thedorsal raphe (DR). The dorsal raphe began at the level of theoculomotor nuclei and was found through to the end of thepontine periventricular grey matter (Figs. 2B and 3E–H).Withinthis regionwe could identify six subdivisions,DRd (dorsal),DRv(ventral), DRif (interfascicular), DRl (lateral), DRp (peripheral)and DRc (caudal). The neurons classified as belonging to theDRd division were found within the periaqueductal grey matter,close to the midline, dorsal to the oculomotor nucleus andadjacent to the inferior aspect of the cerebral aqueduct (Fig. 3F).

Fig. 4. Photomicrographs of neurons immunohistochemically reactive for tyrosine hydroxylase in the brain of the highveld gerbil. (A) A14, rostral periventricular

cell group; (B) A15d, anterior hypothalamic group, dorsal division; (C) A12, tuberal cell group; (D) A10dc, dorsal caudal division of the ventral tegmental area; (E)

A9m, medial division of the substantia nigra; (F) A4, dorsomedial division of locus coeruleus. Scale bar in D = 250 mm and applies to all. In all photomicrographsdorsal is up and medial is to the right.

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144140

Immediately below this division, dorsal and medial to theoculomotor nuclei at the midline were the neurons classified asbelonging to the DRv division. In both these nuclei, the 5-HT+neurons were ovoid in shape and multipolar with the dendritesoriented in a roughly dorsoventral plane. Below the neurons ofthe DRvwas another midline cluster of 5-HT+ neurons that werefound between the posterior poles of the oculomotor nuclei andthe paired medial longitudinal fasciculi. These were located inthe most ventral portion of the midline periaqueductal greymatter andwere assigned to the DRif division of the dorsal raphe(Fig. 3G). The neurons were similar in morphology to thosefound in the DRd and DRv, but the dendrites were clearlyoriented in a dorsoventral plane (Fig. 5A).

Three nuclear subdivisions of the dorsal raphe were found ata small distance from the midline. The DRl division wasevident in a location dorsal and lateral to the DRv and DRddivisions, within the periaqueductal grey matter and adjacent tothe ventrolateral walls of the cerebral aqueduct (Fig. 3E–G).The 5-HT+ neurons of this division were large and ovoid inshape and multipolar in form. The dendrites of these neuronswere oriented roughly parallel to the wall of the cerebralaqueduct, but this did not occur for all neurons (Fig. 5B).Caudally, the DRl appeared to continue from the periaqueductalgrey matter into the periventricular grey matter and wasevidenced as a small number of loosely scattered neurons in and

around the midline region of the periventricular grey. While theneural morphology is similar to that of the DRl, we assignedthese periventricular grey matter 5-HT+ neurons to the DRcdivision. These were the most caudally located neurons of thedorsal raphe. Lateral to the DRv, above the oculomotor nucleiwithin the periaqueductal grey matter and extending into thedorsal anterior pontine tegmentum was the final nucleus of thedorsal raphe, the DRp. Within the periaqueductal grey matterthese 5-HT+ neurons were densely packed, but were scatteredand loosely packed in the adjacent tegmentum. The neuronswere similar in morphology to those of the DRl, but smaller insize and exhibited less dendritic branching. This nucleus is theonly one of the dorsal raphe nuclei that has some neuronslocated outside of the central grey matter.

3.4. MnR—Median raphe nucleus

At the midline of the pontine tegmentum, ventral to theDRif, two columns of intensely 5-HT+ neurons were observed.These paired pararaphe columns were found from the level ofthe decussation of the brachium conjunctivum to the trigeminalmotor nucleus and represent the MnR (Figs. 2B and 3G). Thecolumns were up to four cells wide and consisted of mostlyovoid shaped bipolar neurons with dendrites oriented in amediolateral plane (Fig. 5C).

Fig. 5. Photomicrographs of neurons immunohistochemically reactive for serotonin in the brain of the highveld gerbil. (A) DRif, Dorsal raphe interfasciculardivision; (B) DRl, dorsal raphe lateral division; (C) MnR, median raphe nucleus; (D) ROb, raphe obscurus nucleus; (E) RPa, raphe pallidus nucleus; (F) CVL, caudal

ventrolateral serotonergic cell column. Scale bar in F = 250 mm and applies to all. In all photomicrographs except for those at the midline (A and C) dorsal is up and

medial is to the right.

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144 141

3.5. RMg—Raphe magnus nucleus

Within the anterior half of the medulla, in a pararapheposition, a bilateral pair of columns of 5-HT+ neurons wasfound to represent the RMg (Figs. 2B and 3H and I). Thenumber of neurons within the RMg was low, but the somatawere large and triangular in shape and exhibited both bipolarand multipolar forms. There was no clear orientation to thedendrites emanating from these neurons.

3.6. RPa—Raphe pallidus nucleus

From the level of the facial nerve nucleus to thespinomedullary junction a small number of 5-HT+ neuronswere found at the midline close to and surrounding the dorsaledges of the pyramidal tract (Figs. 2B and 3I–K). Theseneurons were limited in their mediolateral extent by the inferiorolivary nuclei. The neurons were fusiform in shape andmultipolar, with the dendrites oriented either dorsoventrally orparallel to the dorsal surface of the pyramidal tracts (Fig. 5E).

3.7. RVL and CVL—Rostral and caudal ventrolateralserotonergic columns

Within the ventrolateral medullary tegmentum, from thelevel of the facial nerve nucleus to the spinomedullary junctiona column of 5-HT+ neurons were observed (Figs. 2A and3I–K). These neurons form the RVL and CVL, with the RVLbeing found to the mid level of the nucleus ambiguus and theCVL from this level to the spinomedullary junction. Thenumber and density of neurons showed some lesseningcaudally, but this was not significant. The neurons were ovoidin shape and exhibited a mixture of bipolar and multipolarforms, all with dendrites oriented parallel to the floor of themedulla (Fig. 5F).

3.8. ROb—Raphe obscurus nucleus

The 5-HT+ neurons forming the ROb were evinced aspararaphe columns from the level of the caudal pole of thefacial nerve nucleus to the spinomedullary junction (Figs. 2Band 3J and K). The neurons of this nucleus were ovoid andfusiform in shape, those closest to the midline being fusiform,and were bipolar and multipolar in form (Fig. 5D). Themajorityof the dendrites were oriented dorsoventrally, but the occasionaldendrite was seen to project laterally in the caudal medullarytegmentum.

4. Discussion

The central finding of the current study is the full homologyof all nuclei of the putative catecholaminergic and serotonergicsystems of the highveld gerbil with those previously describedin the laboratory rat. We did not find any nuclei of these systemsin the highveld gerbil that have not been previously reported forthe laboratory rat, nor were there any nuclei in the earlierreports of the laboratory rat that were not present in the

highveld gerbil. This finding indicates a strong conservatism, orphylogenetic constraint, in the evolution of the nuclearparcellation these systems, minimally within the familyMuridae and maximally within the order Rodentia. The currentstudy was designed to specifically examine the nuclearparcellation of these systems and does not investigate thepotential changes in the terminal networks and receptorssystems involved with these systems. It is possible that at theselevels of neural organization changes associated with differ-ences in species may be noted. This would be a fruitful area forfuture research given the conservative pattern of evolution ofthe nuclear parcellation of these systems.

4.1. Catecholaminergic system

Within the highveld gerbil putative catecholaminergicnuclei were found throughout the brain from the olfactorybulbs through to the spinomedullary junction. Within theolfactory bulbs the number and density of periglomerular TH+neurons appeared, in a qualitative appraisal, to be morenumerous and densely packed than previously reported for thelaboratory rat (Lichtensteiger, 1966; Lidbrink et al., 1974;Lindvall and Bjorklund, 1974; Hokfelt et al., 1977; Bjorklundand Lindvall, 1984). They represent periglomerular DAneurons but the amine levels are too low to be seen with theCA fluorescence technique of Falck and Hillarp (Dahlstromet al., 1965), unless they are pharmacologically increased withexogenous CA and l-dihydroxyphenylalanine (Lichtensteiger,1966; Lidbrink et al., 1974). It may be speculated that with arelatively large olfactory size bulb there is an increased need foran increased dopaminergic modulation of the olfactory odourprocessing in the glomeruli. Such a mechanism could explainthe increased density of the periglomerular TH positive cellsthat release DA from dendrites onto DA receptors located onthe mitral dendrites in the glomerulus (Pignatella et al., 2005).

All nuclei reported for the catecholaminergic systems of thehypothalamus, midbrain, pons and medulla in the laboratory rat(Dahlstrom and Fuxe, 1964; Fuxe et al., 1969; Lindvall andBjorklund, 1974; Bjorklund and Lindvall, 1984; Hokfelt et al.,1984), grass rat (Mahoney et al., 2007), guinea pig (Muldersand Roberston, 2005) and inbred mice (D’Este et al., 2007)were found in the highveld gerbil and did not appear to bequalitatively different in any significant aspects. Of specificinterest in comparative terms is the identification of the C3group within the medulla of the highveld gerbil. This group hasbeen reported in rat and hamster (Hokfelt et al., 1984; Vincent,1988; Kitahama et al., 1994), and in the highveld gerbil (presentpaper), but has not been found in any other mammalian species(Smeets and Gonzalez, 2000), making it a rodent specificnucleus. While only the midbrain and pons has beeninvestigated in the highveld molerat (Da Silva et al., 2006),all nuclei identified in that study are present in the laboratoryrat and highveld gerbil. The similarity in the overallcomplement of catecholaminergic nuclei in these rodentspecies that have been mapped in detail for the most partindicate a rodent specific complement of nuclei, but one that isdifferent in overall composition from other mammalian orders

D.-J. Moon et al. / Journal of Chemical Neuroanatomy 34 (2007) 134–144142

(Maseko et al., 2007). However, it should be noted that the C2group in the gerbil is substantially fewer in neuronal number inits rostral component compared with the laboratory rat. Alsothat part of the A2 group of the gerbil in the nucleus tractussolitarius is markedly diminished compared with the laboratoryrat. This is also true of the pars compacta (A9pc) and lateralsubgroup of the substantia nigra (A9l). Thus, the size of someCA cell groups can vary among species of the same orderprobably related to differences in functional demands althoughthe principal nuclear architecture remains very similar in thesame order. For example, the A2 and C2 groups may reflect achange in the functional demand of these groups in theirregulation of the central autonomic and neuroendocrinemechanisms in view of their projections to the hypothalamusand autonomic centers of the medulla (NTS and dmnX) andspinal cord (lateral horn with the sympathetic lateral column). Itmay therefore be that the A2 and C2 have an altered centralautonomic function in the highveld gerbil. The study byVincent (1988) in the hamster has delineated additional nucleithat were not found in the present study, nor in those studies ofthe laboratory rat or highveld molerat. It is possible that thehamster may have additional catecholaminergic nuclei notidentified in other rodent species. However, it may also be dueto methodological differences, such as different primary andsecondary antibodies, as the extra nuclei identified in thehamster are not found in any other mammalian species (Smeetsand Gonzalez, 2000; Maseko et al., 2007).

4.2. Serotonergic system

The nuclei and cellular groups delineated in the presentstudy are identical to those previously described for thelaboratory rat (Dahlstrom and Fuxe, 1964; Fuxe et al., 1969;Steinbusch, 1981; Lidov and Molliver, 1982; Waterhouse et al.,1993) and the portion of the brain studied in the highveldmolerat (Da Silva et al., 2006) and Mongolian gerbil (Janusoniset al., 1999, 2003; Janusonis and Fite, 2001). The nuclearparcellation of the serotonergic system across all mammalsstudied to date is very similar (Bjarkam et al., 1997; Masekoet al., 2007), the one major exception being the presence ofserotonergic neurons in the hypothalamus of the monotremes(Manger et al., 2002). These previous observations indicate thatit is not surprising that all rodents studied to date evince a nearidentical appearance of the serotonergic system.

4.3. Evolutionary considerations

The current study examined a rodent that is, in evolutionaryterms, not substantially divergent from the laboratory rat, bothspecies sharing a recent common ancestor approximately 20million years ago (Jansa and Weksler, 2004). Despite this, thereare some significant phenotypic differences in these species inthat the highveld gerbil possesses comparative large eyes andolfactory bulbs. Moreover, the animals used in this study werewild caught, and not specifically bred captive strains. In agradualistic framework of evolution, it may be predicted thatthese differences would be sufficient to engage major changes in

the brain, but this was not the case. From the 26 putativecatecholaminergic and 14 serotonergic nuclei normallydescribed in the laboratory rat, all (no more, no less) nucleiwere found in the highveld gerbil. This represents a strong degreeof homology, and is representative of a phylogenetic constraint,at least within the familyMuridae. Given the added observationsmade within the highveld molerat (Da Silva et al., 2006),Mongolian gerbil (Janusonis et al., 1999, 2003; Janusonis andFite, 2001) and hamster (Vincent, 1988) this phylogeneticconstraint in the nuclear parcellation of the catecholaminergicand serotonergic systems may be extended to the level of theorder as proposed by Manger (2005). This represents aninteresting trend in the evolution of the neuromodulatory systemsthat has yet to be fully tested. Further studies on large and small-brained rodents will test the consistency of the nuclearparcellation within the rodents including the variations of thesize and architecture of the various monoamine subgroups.Parallel studies in species of other orders (e.g. Maseko et al.,2007) will test for consistency within these orders and thedifferences between orders.

Acknowledgments

This study was supported by a grant from the South AfricanNational Research Foundation (Gun: 2054204) to PRM, and aSwedish Research Partnership Programme Bilateral Agreementbetween the Swedish International Development CooperationAgency and the South African National Research Foundation toKF and PRM. The authors wish to thanks Tracey Aemmey andNeville Pillay for the animals used in this study.

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