Journal of Chemical Neuroanatomy 61–62 (2014) 94–106

Nuclear organization of cholinergic, catecholaminergic, serotonergicand orexinergic systems in the brain of the Tasmanian devil(Sarcophilus harrisii)

Nina Patzke a, Mads F. Bertelsen b, Kjell Fuxe c, Paul R. Manger a,*a School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africab Centre for Zoo and Wild Animal Health, Copenhagen Zoo, Frederiksberg, Denmarkc Department of Neuroscience, Karolinska Institutet, Retzius vag 8, S-171 77 Stockholm, Sweden

A R T I C L E I N F O

Article history:

Received 28 February 2014

Received in revised form 12 August 2014

Accepted 12 August 2014

Available online 20 August 2014

Keywords:

Choline acetyltransferase

Tyrosine hydroxylase

Serotonin

Orexin

Evolution

Mammal

A B S T R A C T

This study investigated the nuclear organization of four immunohistochemically identifiable neural

systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male

Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6 g. We found that the nuclei

generally observed for these systems in other mammalian brains were present in the brain of the

Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic,

catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic

system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal

subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of

the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4

nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of

the serotonergic system. The organization of the orexinergic system was similar to that seen in many

mammals previously studied. Overall, while showing strong similarities to the organization of these

Contents lists available at ScienceDirect

Journal of Chemical Neuroanatomy

jo ur n al ho mep ag e: www .e lsev ier . c om / lo cate / jc h emn eu

Marsupial

Abbreviations: III, oculomotor nucleus; IV, trochlear nucleus; Vmot, motor division of the trigeminal nerve nucleus; Vs, sensory division of the trigeminal nerve nucleus; VI, abducens

nucleus; VIId, dorsal division of facial nerve nucleus; VIIv, ventral division of facial nerve nucleus; X, dorsal motor vagus nucleus; XII, hypoglossal nucleus; 3n, oculomotor nerve; 3V,

third ventricle; 4V, fourth ventricle; 7n, facial nerve; 8n, vestibulocochlear nerve; A1, caudal ventrolateral medullary tegmental nucleus; A2, caudal dorsomedial medullary nucleus; A5,

fifth arcuate nucleus; A6c, compact portion of locus coeruleus; A7d, nucleus subcoeruleus, diffuse portion; A7sc, nucleus subcoeruleus, compact portion; A8, retrorubral nucleus; A9l,

substantia nigra, lateral; A9m, substantia nigra, medial; A9pc, substantia nigra, pars compacta; A9v, substantia nigra, ventral, pars reticulata; A10, ventral tegmental area; A10c, ventral

tegmental area, central; A10d, ventral tegmental area, dorsal; A10dc, ventral tegmental area, dorsal caudal; A11, caudal diencephalic group; A12, tuberal cell group; A13, zona incerta

cell group; A14, rostral periventricular nucleus; A15d, anterior hypothalamic group, dorsal division; A15v, anterior hypothalamic group, ventral division; A16, catecholaminergic

neurons of the olfactory bulb; ac, anterior commissure; Amyg, amygdaloid body; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; AP, area postrema; arc, arcuate nucleus

of the hypothalamus; B9, supralemniscal serotonergic nucleus; C, caudate nucleus; C1, rostral ventrolateral medullary tegmental group; C2, rostral dorsomedial medullary nucleus; ca,

cerebral aqueduct; Cb, cerebellum; Chat IN, cholinergic interneurons of the gelatinous layer of the caudal spinal trigeminal nucleus; cic, commissure of the inferior colliculus; Cl,

claustrum; CLi, caudal linear nucleus; Cu, cuneate nucleus; DCN, deep cerebellar nuclei; dfu, dorsal funiculus; Diag.B, diagonal band of Broca; DR, dorsal raphe; DRc, dorsal raphe, caudal

division; DRd, dorsal raphe, dorsal division; DRif, dorsal raphe, interfascicular division; DRl, dorsal raphe, lateral division; DRp, dorsal raphe, peripheral division; DRv, dorsal raphe,

ventral division; DT, dorsal thalamus; ER, entorhinal cortex; EW, Edinger–Westphal nucleus; f, fornix; fr, fasciculus retroflexus; GC, central grey matter; Ge5, gelatinous layer of the

caudal spinal trigeminal nucleus; GiCRt, gigantocellular reticular column; GP, globus pallidus; Gr, gracilis nucleus; Hbl, lateral habenular nucleus; Hbm, medial habenular nucleus; Hc,

hippocampal commissure; Hip, hippocampus; Hyp, hypothalamus; Hyp.d, dorsal hypothalamic cholinergic nucleus; IC, inferior colliculus; ic, internal capsule; icp, inferior cerebellar

peduncle; IGL, intergeniculate leaflet; io, inferior olivary nucleus; IP, interpeduncular nucleus; Is.Call/TOL, islands of calleja/olfactory tubercle; LDT, laterodorsal tegmental nucleus;

LDTd, laterodorsal tegmental nucleus, dorsal division; lfp, longitudinal fasciculus of the pons; lfu, lateral funiculus; LGd, dorsal lateral geniculate nucleus; LGv, ventral lateral geniculate

nucleus; LOT, lateral olfactory tract; LRt, lateral reticular nucleus; LV, lateral ventricle; Mc, main orexinergic cluster; mcp, middle cerebellar peduncle; mlf, median longitudinal

fasciculus; MOB, main olfactory bulb; MnR, medial raphe nucleus; mtf, medullary tegmental field; N.Acc, nucleus accumbens; N.Amb, nucleus ambiguus; N.Bas, nucleus basalis; NEO,

neocortex; OT, optic tract; OTc, orexinergic optic tract cluster; OV, olfactory ventricle; P, putamen nucleus; PBg, parabigeminal nucleus; PC, cerebral peduncle; PCRt, parvocellular

reticular column; PIR, piriform cortex; PPT, pedunculopontine nucleus; PV, paraventricular thalamic nucleus; py, pyramidal tract; pyx, decussation of the pyramidal tract; pVII,

preganglionic motor neurons of the superior salivatory nucleus or facial nerve; pIX, preganglionic motor neurons of the inferior salivatory nucleus; R, thalamic reticular nucleus; Rmc,

red nucleus, magnocellular division; Rmg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; RVL, rostral ventrolateral serotonergic group; SC, superior

colliculus; scp, superior cerebellar peduncle; Sep.M, medial septal nucleus; SON, supraoptic nucleus; Sp5, spinal trigeminal tract; Sp5c, spinal trigeminal nucleus, caudal part; Stn,

subthalamic nucleus; sub, subiculum; vfu, ventral funiculus; vh, ventral horn; VPO, ventral pontine nucleus; zi, zona incerta; Zic, orexinergic zona incerta cluster.

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

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

http://dx.doi.org/10.1016/j.jchemneu.2014.08.005

0891-0618/� 2014 Elsevier B.V. All rights reserved.

systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order

specific, or class specific, features of these systems. Further studies will reveal the extent of change in the

nuclear organization of these systems in marsupials and how these potential changes may affect

functionality.

� 2014 Elsevier B.V. All rights reserved.

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106 95

Introduction

The Tasmanian devil (Sarcophilus harrisii), a member of theDasyuridae family, order Dasyuromorphia, is a marsupial carnivorecurrently found only on the Australian island of Tasmania. It is thelargest of the dasyurids, reaches a length of 57–65 cm and malescan weigh as much as 14 kg, while females weigh around 9 kg(Jones et al., 2008). The Tasmanian devil is a scavenger, feedingupon the carcasses of large mammals such as kangaroos, but it canalso catch and eat small possums and pademelons (Ashwell, 2010).The Tasmanian devil is nocturnal, solitary and has a home range of8 to 20 square kilometres (Ashwell, 2010). Once widespread acrossTasmania, the population of wild Tasmanian devils is rapidlydeclining due to facial tumour disease, a unique and lethalinfectious cancer (Keeley et al., 2012).

In the present study we detail the nuclear organization ofseveral specific neural systems (cholinergic, catecholaminergic,serotonergic and orexinergic) within the brain of the Tasmaniandevil; systems which have not been previously studied in thisspecies. To date only a few descriptions of the systems examined inthe present study are available for the marsupial brain. Thecatecholaminergic system was described in the North Americanopossum (Didelphis virginiana) (Crutcher and Humbertson, 1978),while the serotonergic system has been described in the NorthAmerican opossum (Crutcher and Humbertson, 1978; Martin et al.,1985) and the tammar wallaby (Macropus eugenii) (Ferguson et al.,1999). The distribution of orexinergic cells has been reported in theEastern grey kangaroo (Macropus giganteus) (Yamamoto et al.,2006). A full characterization of the cholinergic system in themarsupial brain is absent from the literature, but a partialdescription was provided by Ashwell (2010). While for the mostpart the nuclear organization of these systems were similar tothose reported in placentals, several species-specific differenceswere observed in prior studies of marsupials. A detailed list of thepresence/absence of specific nuclei within these systems formammals can be found in Dell et al. (2010). The basic similarity ofthese systems in monotremes, marsupials and placentals indicatesthat the structure and function of these systems are probablybroadly similar in all mammals (Manger et al., 2002a–c; Ashwell,2010).

The present study provides, for the first time in the samemarsupial species, a detailed chemoarchitectural characterizationof the cholinergic, catecholaminergic, serotonergic and orexinergicsystems in the Tasmanian devil. In addition to the availablechemoarchitectural descriptions of the marsupial brain, the datafrom the current study provide a broader base for comparisonwithin the marsupials and across mammals in general. This mayhelp to further elucidate evolutionary significant differences in thebrain between mammalian groups and lead to a better under-standing of the phylogenetic and functional signals they carry(Manger, 2005).

Materials and methods

Brains from three male Tasmanian devils (S. harrisii) (brain masses of 11.2. 11.4

and 12.2 g, body masses of 9.5, 8.2 and 10.1 kg) were used in this study (Fig. 1). The

specimens were obtained from the Copenhagen zoo following the euthanasia of

these animals due to veterinary reasons (metastatic squamous cell carcinoma,

prolapsed thoracic intervertebral disc, senescence and dilated cardiomyopathy,

respectively). No issues concerning the brains of these animals were noted prior to

euthanasia, or during specimen preparation and analysis (e.g. Holz and Little, 2005).

The harvesting and use of the specimens was approved by the University of the

Witwatersrand Animal Ethics Committee (2008/36/1). The animals were

anesthetized with sevoflurane delivered by facemask, euthanized (200 mg

sodium pentobarbital/kg, i.v.) and, upon cessation of respiration, perfused

intracardially with 0.9% saline solution followed by 4% paraformaldehyde in

0.1 M phosphate buffer (PB, pH 7.4), both solutions having a temperature of

approximately 4 8C. The brains were removed and post-fixed in 4% paraformal-

dehyde overnight, equilibrated in 30% sucrose in 0.1 M PB at 4 8C and stored in an

antifreeze solution at �20 8C until sectioning. Before sectioning, the tissue was

allowed to equilibrate in 30% sucrose in 0.1 M PB at 4 8C. The specimens were

frozen in crushed dry ice and sectioned in the frontal plane into 50-mm-thick

sections on a sliding microtome.

A one in six series of sections was used for Nissl and myelin staining, and choline-

acyltransferase (ChAT), tyrosine hydroxylase (TH), serotonin (5-HT) and orexin-A

(hypocretin/OxA) immunohistochemistry. Sections for Nissl staining were first

mounted on 0.5% gelatine coated slides, cleared overnight in a solution of 1:1

absolute alcohol and chloroform and then stained with 1% cresyl violet. The series of

sections used for myelin staining were refrigerated for two weeks in 5% formalin

then mounted on 1% gelatine coated slides and stained with a modified silver stain

(Gallyas, 1979).

The sections used for immunohistochemical staining were incubated in a

solution containing 1.6% of 30% H2O2, 49.2% methanol and 49.2% 0.1 M PB solution,

for 30 min to reduce endogenous peroxidase activity, which was followed by three

10 min rinses in 0.1 M PB. To block unspecific binding sites the sections were then

pre-incubated for 2 h, at room temperature, in blocking buffer (3% normal goat

serum, NGS, for TH, 5HT and OxA; 3% normal rabbit serum, NRS, for ChAT, 2% bovine

serum albumin, BSA, and 0.25% Triton-X in 0.1 M PB). Thereafter, the sections were

incubated for 48 h at 4 8C in the primary antibody solutions that contained the

appropriate diluted primary antibody in blocking buffer under gentle agitation.

Goat anti-cholineacetyltransferase antibody (AB144P, Millipore) at a dilution of

1:3000 was used to reveal cholinergic neurons. Rabbit anti-tyrosine hydroxylase

antibody (AB151, Millipore) at a dilution of 1:7500 revealed the putative

catecholaminergic neurons. Serotonergic neurons were revealed using rabbit

anti-serotonin antibody (AB938, Millipore) at a dilution of 1:7500. Orexinergic

neurons were revealed using rabbit anti-Orexin-A antibody (AB3704, Millipore) at a

dilution of 1:3000. The primary antibody incubation was followed by three 10-min

rinses in 0.1 M PB and the sections were then incubated in a secondary antibody

solution (1:1000 dilution of biotinylated goat anti-rabbit IgG, BA-1000, Vector Labs,

for TH, 5-HT and OxA sections, or a 1:1000 dilution of biotinylated rabbit anti-goat

IgG, BA-5000, Vector Labs, for ChAT sections, in a blocking buffer containing 3%NGS/

NRS and 2% BSA in 0.1 M PB) for 2 h at room temperature. This was followed by

three 10 min rinses in 0.1 M PB, after which sections were incubated for 1 h in an

avidin–biotin solution (1:125; Vector Labs), followed by three 10 min rinses in

0.1 M PB. Sections were then placed in a solution containing 0.05% diaminobenzi-

dine (DAB) in 0.1 M PB for 5 min, followed by the addition of 3.3 ml of 30% hydrogen

peroxide per 1 ml of DAB solution. Chromatic precipitation was visually monitored

under a low power stereomicroscope. Staining continued until such time as the

background stain was at a level that would allow for accurate architectonic

matching to the Nissl and myelin sections without obscuring the immunopo-

sitive structures. Development was arrested by placing sections in 0.1 M PB for

10 min, followed by two more rinses in this solution. Sections were then

mounted on 0.5% gelatine-coated glass slides, dried overnight, dehydrated in a

graded series of alcohols, cleared in xylene and coverslipped with Depex. To

ensure that there was no non-specific staining of the immunohistochemical

protocol, we ran tests on sections where we omitted the primary antibody, and

sections where we omitted the secondary antibody. In both cases no staining was

observed. All sections were examined under low power using a stereomicroscope

and the architectonic borders of the sections were traced according to the Nissl

and myelin stained sections using a camera lucida. The immunostained sections

were then matched to the traced drawings, adjusted slightly for any differential

shrinkage of the stained sections and immunopositive neurons were marked. The

drawings were then scanned and redrawn using the Canvas 8TM (Deneba) drawing

programme. Digital photomicrographs were captured using a Zeiss Axioskop and

the Axiovision software. No adjustments of pixels, or manipulation of the captured

images were undertaken, except for the adjustment of contrast, brightness, and

levels using Adobe Photoshop 7.

Fig. 1. Dorsal (A), lateral (B) and ventral (C) photographs of the brain of the Tasmanian devil (Sarcophilus harrisii). Note the large olfactory bulbs and associated piriform cortex,

the mostly lissencephalic neocortex, and the clearly visible corpora quadrigemina. Scale bar = 5 mm.

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–10696

Results

The present study describes the nuclear organization ofcholinergic, catecholaminergic, serotonergic and orexinergic sys-tems in the brain of the Tasmanian devil (S. harrisii). The overallappearance and organization of the brains of the Tasmanian devilsstudied herein could be thought of as typical of marsupials (Ashwell,2010). In general, the systems investigated exhibited a similarorganization to that previously reported in placental and mono-treme mammals, but several specific differences were observed. Theaverage brain mass of the Tasmanian devils investigated in thecurrent study was 11.6 g, with the gross morphology of the brainbelonging to the type I classification of Haight and Murray (1981).

Thus, the brain of the Tasmanian devil has a trapezoid appearancewhen examined from either above or below, and shows little to nodorsoventral flexion when viewed from the lateral aspect (Fig. 1).The cerebral cortex was mostly lissencephalic, and while lacking thecorpus callosum, the anterior commissure was very large. Thecorpora quadrigemina were also evident in dorsal view. In addition,the large size of the olfactory bulbs and piriform cortex wereprominent features of the Tasmanian devil brain.

Cholinergic nuclei

The cholinergic immunoreactive neurons (ChAT+) found inthe Tasmanian devil formed several distinct nuclei that were

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106 97

organized in five distinct groups: the striatal, basal forebrain,diencephalic, pontomesencephalic, and the cranial motor nervenuclei. No evidence of cortical cholinergic interneurons was found.

Striatal cholinergic interneurons

ChAT+ neurons were found in the caudate/putamen complex,the globus pallidus, the nucleus accumbens, the Islands of Callejaand the olfactory tubercle (Fig. 2A–J). The caudate (C) and theputamen (P) displayed a moderate number of ChAT+ neurons,whereas the globus pallidus (GP) had a slightly higher number. Amoderate number of ChAT+ neurons were also found in thenucleus accumbens (N.Acc), and at the caudoventral pole of thisnucleus, they appear to intermingle with the most dorsal ChAT+neurons of the olfactory tubercle (TOL). The TOL is situated at themost ventral portion of the rostral telencephalon. A moderatenumber of ChAT+ neurons were present in the TOL, these neuronswere predominantly located in the dorsal aspect of this structure,and within the most ventral portion of this region clusters of ChAT+neurons were observed to form the Islands of Calleja. In terms ofthe relative densities reported herein, the reader is referred to thefigures to establish our estimates of density.

Cholinergic nuclei of the basal forebrain

In the basal forebrain the medial septal nucleus, the Diagonalband of Broca and the nucleus basalis were found to contain ChAT+neurons (Fig. 2F–J). A moderate number of ChAT+ neurons werefound within the medial septal nucleus (Sep.m) that was locatedwithin the rostral half of the medial wall of the septal nuclearcomplex, rostral to the anterior commissure. Located in theventromedial corner of the cerebral hemisphere, rostral to thehypothalamus, a band of densely packed ChAT+ neurons wasidentified as the diagonal band of Broca (Diag.B) (Fig. 3A). A high tomoderate density of ChAT+ neurons, located medial and rostral tothe globus pallidus (GP), caudal to the nucleus accumbens anddorsal to the TOL, formed the nucleus basalis (N.Bas) (Fig. 3B).

Diencephalic cholinergic nuclei

In the diencephalon of the Tasmanian devil ChAT+ neuronswere observed to form the medial habenular nucleus and thedorsal hypothalamic cluster (Fig. 2J–L). The medial habenularnucleus (Hbm) exhibited a high density of ChAT+ neurons and wassituated in the dorsomedial aspect of the diencephalon adjacent tothe third ventricle. In the hypothalamus of the Tasmanian devilonly the dorsal hypothalamic cluster (Hyp.d) exhibited a few pale-stained ChAT+ neurons. This cluster was located in a dorsal andmedial location within the hypothalamus adjacent to the thirdventricle, at the middle rostrocaudal level of the hypothalamus.Other hypothalamic clusters of cholinergic neurons, seen ineutherian mammals, were not evident in the Tasmanian devil.

Pontomesencephalic cholinergic nuclei

Within the pontomesencephalon three nuclei exhibited ChAT+neurons: the parabigeminal nucleus (PBg), the pedunculopontinetegmental nucleus (PPT) and the laterodorsal tegmental nucleus(LDT) (Fig. 2O and P). ChAT+ neurons forming the parabigeminalnucleus (PBg) were found in a high density at the lateral wall of themidbrain tegmentum, dorsal to the lateral lemniscus andventrolateral to the inferior colliculus. The pedunculopontinetegmental nucleus (PPT) exhibited a moderate to high density ofChAT+ neurons and was situated within the dorsal pontinetegmentum, between the lateral lemniscus and the periaqueductalgrey matter. The laterodorsal tegmental nucleus (LDT) was locatedin the ventrolateral portion of the periaqueductal grey andperiventricular grey of the rostral pons. In addition to the standardLDT a dorsal cluster of cholinergic neurons (LDTd) was evident. TheLDT and LDTd were bridged by a small number of cholinergic

neurons and a high density of cholinergic dendrites (Fig. 3C). LDTdwas located close to the central aqueduct, whereas the ChAT+neurons of the LDT were located closer to the ventrolateral borderof the periaqueductal grey matter in a position typical of allmammals previously studied. The transition to the pontinetegmentum was contiguous with the ChAT+ neurons of the PPTnucleus. The Tasmanian devil is the first animal reported to evincea distinct dorsal subdivision of the LDT.

Cholinergic cranial nerve motor nuclei

The cholinergic cranial nerve motor nuclei in the Tasmaniandevil were found in positions typical of all mammals (Fig. 2N–W).The ChAT+ nuclei identified included: the oculomotor (III),trochlear (IV), motor division of the trigeminal (Vmot), adbucens(VI), dorsal and ventral subdivisions of the facial (VIId and VIIv),nucleus ambiguus, dorsal motor vagus (X), hypoglossal (XII),Edinger–Westphal (EW), medullary tegmental field (mtf) and thepreganglionic motor neurons of the salivatory (pVII) and theglossopharyngeal (pIX) nerves. Within the spinomedullary junc-tion at the level of the decussation of the pyramidal tract ChAT+neurons could be identified in the ventral horn of the cervicalspinal cord. In addition to these nuclei and regions, ChAT+ neurons,presumably interneurons, were observed in the gelatinous layer ofthe caudal spinal trigeminal nucleus (Ge5) (Fig. 3D–F), a featurenot previously observed in other mammals.

Catecholaminergic nuclei

Immunoreactivity to the tyrosine hydroxylase antibody visual-ized catecholaminergic (TH+) neurons. These TH+ neurons formeda range of identifiable nuclear complexes that could be divided intothe olfactory bulb, diencephalic, midbrain, pontine and medullarynuclear clusters. The TH+ neurons found in the Tasmanian devilwere typical of the pattern seen in many other mammals, but againsome exceptions were noted.

Olfactory bulb neurons (A16)

As previously reported in other marsupials the olfactory bulbsof the Tasmanian devil were large with a distinct olfactoryventricle; however, the laminar organization of this structure issimilar to that seen in other mammals and this structure includesboth a main and accessory olfactory bulb. A large number of TH+neurons were found in the glomerular layer of the olfactory bulbwith their dendrites forming a mesh around the glomeruli (Fig. 2A–D).These neurons are presumably dopaminergic periglomerular inter-neurons and were small in size. This appearance and location is typicalfor all mammals studied to date.

Diencephalic nuclei (A15–A11)

In the diencephalon TH immunohistochemistry revealed sixcatecholaminergic nuclei (A15–A11), all located in the hypothala-mus (Fig. 2J–L). The anterior hypothalamic group (A15) located inthe rostral portion of the hypothalamus could be subdivided into adorsal (A15d) and a ventral (A15v) portion. TH+ neurons formingthe A15d were located dorsolateral to the third ventricle (Fig. 4A),whereas A15v TH+ neurons were found in the ventrolateral portionof the anterior hypothalamus just medial to the optic tract (OT)close to the floor of the brain. Two dorsoventral columns of TH+cells were located lateral to the walls of the third ventricle, situatedat the hypothalamic periventricular zone, forming the rostralperiventricular cell group (A14). In the dorsolateral hypothalamusbetween the A15d and zona incerta of the ventral thalamus amoderate number of TH+ neurons was observed forming the zonaincerta nucleus (A13) (Fig. 4B). TH+ cells forming the tuberal cellgroup (A12) were located in the ventromedial portion of thehypothalamus, at the floor of the hypothalamus medial to the

Fig. 2. Serial drawings of coronal sections through one half ofthe Tasmaniandevil brain from the olfactorybulb through tothespinomedullary junction. (A) is the most rostral section,

(W) the most caudal. The outlines of the architectonic regions were drawn using Nissl and myelin stains and immunoreactive cells marked on the drawings. Solid black circles depict

cholinergic neurons, solid triangles depict catecholaminergic neurons (those immunoreactive for tyrosine hydroxylase), open squares depict serotonergic neurons and closed stars

represent orexinergic neurons. Each circle, triangle, square or star represents an individual neuron. The figures are approximately 1500 mm apart. See list for abbreviations.

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–10698

Fig. 2. (Continued ).

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106 99

Fig. 2. (Continued ).

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106100

Fig. 3. Photomicrographs of cholinergic neurons within the brain of the Tasmanian devil. (A) diagonal band of Broca, (B) nucleus basalis, (C) laterodorsal tegmental nucleus

and (D) the gelatinous layer of the caudal spinal trigeminal nucleus. While the diagonal band of Broca and the nucleus basalis show an organization that can be considered

typically mammalian, the laterodorsal tegmental nucleus is clearly divided into dorsal (LDTd) and ventral (LDTv) subdivisions. The LDTv appears to correspond to the LDT

described in other mammals previously, while the LDTd appears to be either unique to the Tasmanian devil, or may be a feature found in more marsupials when they are

studied. Cholinergic interneurons were also present in the gelatinous layer of the caudal spinal trigeminal nucleus ((D)–(F)), which is a feature not previously seen in other

mammals. Again, this may be a feature specific to the Tasmanian devil, or may be found in other marsupials when examined. Scale bar in D = 500 mm and applies to (A)–(D).

Scale bar in E = 250 mm, scale bar in F = 100 mm. In all images medial is to the left and dorsal to the top, see list for abbreviations.

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106 101

arcuate nucleus. A moderate number of TH+ neurons making upthe caudal diencephalic group (A11), were situated ventral to theposterior pole of the third ventricle. This organization ofcatecholaminergic nuclei is similar to that seen in many mammals.

Midbrain nuclei (A10–A8)

In the midbrain TH+ neurons were present in the ventraltegmental area (the A10 complex, including the A10, A10c, A10d,A10dc nuclei), the substantia nigra (the A9 complex, including theA9pc, A9l, A9v, A9m nuclei) and the retrorubral nucleus (A8)within the midbrain tegmentum (Fig. 2L–O). A high density of TH+neurons was located lateral to the interpeduncular nucleus (IP) andventromedial to the magnocellular division of the red nucleus(Rmc), forming the specific A10 nucleus of the A10 nuclearcomplex. TH+ neurons forming the A10 central (A10c) nucleuswere situated at the midline medial to A10 and dorsorostral to the

interpeduncular nucleus. Dorsal to A10c and ventral to theoculomotor nucleus (III) TH+ cells lying along the midline curtailedlaterally by the oculomotor nerve (3n), were assigned to A10 dorsalnucleus (A10d). The TH+ neurons of the A10 dorsocaudal nucleus(A10dc) were located within the periaqueductal grey matter,surrounding the ventral half of the cerebral aqueduct (Fig. 4C).

The substantia nigra nuclear complex formed a horizontallyelongated band of TH+ cells in the ventrolateral portion of themidbrain tegmentum, situated between the A10 nuclear complexand the caudal pole of the dorsal thalamus, in a position dorsal tothe cerebral peduncles (Fig. 2M and N). Lateral to A10 and betweenthe magnocellular division of the red nucleus and the medialportion of the cerebral peduncle (pc) TH+ neurons forming A9medial nucleus (A9m) were observed. The A9 pars compacta(A9pc) nucleus was situated dorsolateral to A9m and superior tothe medial part of the cerebral peduncles. TH+ cells forming the A9

Fig. 4. Photomicrographs of catecholaminergic neurons within the brain of the Tasmanian devil. Note the different locations within the hypothalamus of the A15d (A) and A13

(B) nuclei. A moderately extensive A10dc (C) was observed in the periaqueductal grey matter, as well as several subdivisions of the locus coeruleus complex (D) in the pons.

Scale bar in C = 250 mm, scale bar in D = 500 mm and applies to (A), (B) and (D). In all images medial is to the left and dorsal to the top, see list for abbreviations.

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106102

ventral nucleus (A9v) were located ventral to the A9pc nucleus andwere intermingled with the fibres that form the cerebral peduncle.At the dorsolateral edge of the A9pc nucleus, the A9 lateral (A9l)nucleus was observed as a moderately dense cluster of TH+neurons. A group of loosely packed TH+ neurons forming theretrorubral nucleus (A8) were scattered throughout the midbraintegmentum, located caudal to the magnocellular division of the rednucleus and dorsocaudal to the A9 complex.

Rostral rhombencephalon—The locus coeruleus complex, A7–A5

Within the pontine region a large number of TH+ neuronsforming the locus coeruleus complex were readily observed(Fig. 2P–R). The locus coeruleus complex could be readilysubdivided into four nuclei, these being: the subcoeruleus compactportion (A7sc), subcoeruleus diffuse portion (A7d), locus coeruleusdiffuse portion (A6d), and the fifth arcuate nucleus (A5). Noevidence of the compact division of the locus coeruleus (A6d) orthe dorsolateral division of locus coeruleus (A4) was found in theTasmanian devil. A densely packed cluster of TH+ neurons withinthe pontine tegmentum was located lateral to the periventriculargrey and ventromedial to the superior cerebellar peduncle,forming the A7 compact portion (A7sc, subcoeruleus compact)of the locus coeruleus (Fig. 4D). Surrounding the A7sc on its ventraland lateral aspects was the more diffusely localized TH+ neuronsthat formed the diffuse portion of the subcoeruleus (A7d,subcoeruleus diffuse). The TH+ neurons of the A7d were clearlyless dense packed that of A7sc (Fig. 4D). In the ventrolateral aspectof the pontine periventricular grey matter a moderately denselypacked cluster of TH+ neurons was assigned to the A6 diffusenucleus (A6d, locus coeruleus diffuse portion) (Fig. 4D). An A6compact nucleus (A6c, locus coeruleus compact portion) aspreviously observed in certain placental mammals (primates,megachiropterans and murid rodents, Dahlstrom and Fuxe, 1964;

Dell et al., 2010) could not be detected in the Tasmanian devil. Aloosely associated ventrolateral column of TH+ neurons formed thefifth arcuate nucleus (A5). The neurons were found ventromedialto the motor division of trigeminal nerve nucleus (Vmot) andlateral to the facial nerve nucleus (VIIv).

Medullary nuclei (C1, C2, C3, A1, A2, area postrema)

Within the medulla oblongata five catecholaminergic nucleiwere observed (A2, A1, C2, C1 and the area postrema) (Fig. 2R–W).TH+ neurons located in the central grey surrounding the dorsalmotor vagus nucleus (X) were designated to the caudal dorsome-dial medullary group (A2), mainly located in the nucleus tractussolitarius. A column of TH+ neurons forming the caudalventrolateral medullary tegmental group (A1) was located lateralto the nucleus ambiguus (N.Amb) and dorsal to lateral reticularnucleus (LRt), extending from the rostral part of the nucleusambiguus to the level of the decussation of the pyramidal tract.Parallel to A1 a column of TH+ neurons forming the rostralventrolateral medullary tegmental group (C1) was situated medialto nucleus ambiguus, having the same rostrocaudal extent as thenucleus ambiguus. The rostral dorsomedial medullary group (C2)was found in the medullary periventricular grey matter dorsal tothe dorsal motor vagus nucleus close to the floor of the fourthventricle. A dense cluster of TH+ neurons located in the medullaryperiventricular grey matter dorsal to the dorsal motor vagusnucleus and hypoglossal nucleus (XIIn) was assigned to the areapostrema (AP) nucleus.

Serotonergic nuclei

The serotonergic nuclei were all located within the brainstemand can be divided into a rostral and a caudal cluster. Both of theseclusters contained a number of distinct nuclei that are found

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106 103

throughout the brainstem from the level of the decussation of thesuperior cerebellar peduncle through to the spinomedullaryjunction (Fig. 2N–W).

Rostral cluster

The rostral cluster of serotonergic neurons was located at thelevel of the brainstem ranging between the decussation of thesuperior cerebellar peduncle to the trigeminal motor nucleus.Within the rostral cluster we found evidence for the caudal linearnucleus (CLi), the supralemniscal nucleus (B9), the median raphenucleus (MnR) and the dorsal raphe complex formed of six distinctnuclei (Fig. 2N–Q). 5HT+ neurons forming the caudal linear nucleus(CLi) were situated along the midline immediately dorsal to theinterpeduncular nucleus (IP), intermingled with the catecholamin-ergic neurons of the A10c and A10d nuclei. 5HT+ neurons of thesupralemniscal group (B9) were found in a low density in theventromedial midbrain, between A10 and A9m of the catechol-aminergic system, lateral to ventral serotonergic neurons of the CLinucleus, with this nucleus appearing to form a continuation of theCLi nucleus. The median raphe nucleus (MnR) was identified as twocolumns of densely packed 5HT+ neurons on either side of themidline in a para-raphe position. The two dorsoventral columnsextended from just caudal to the decussation of the superiorcerebellar peduncle through to the level of the trigeminal motornucleus.

Within the 5HT+ neuronal region designated as the dorsal raphenuclear complex there were six distinct nuclei, these being: thedorsal raphe interfascicular (DRif) nucleus, dorsal raphe ventral(DRv) nucleus, dorsal raphe dorsal (DRd) nucleus, dorsal raphelateral (DRl) nucleus, dorsal raphe peripheral (DRp) nucleus andthe dorsal raphe caudal (DRc) nucleus (Figs. 2N–P and 5). These sixnuclei were found, for the most part, within the periaqueductal andperiventricular grey matter from the level of the oculomotornucleus to the trigeminal motor nucleus. The DRif was locatedbetween the two medial longitudinal fasciculi and exhibited a highdensity of 5HT+ neurons. The DRv was found immediately dorsal tothe DRif between and just caudal to the oculomotor nuclei. TheDRv exhibited a high density of 5HT+ neurons. Immediately dorsalto DRv and ventral to the inferior border of the cerebral aqueduct ahigh-density cluster of 5HT+ neurons was designated as the DRdnucleus. A moderate density of 5HT+ neurons representing theDRp, were located in the ventrolateral portion of the periaque-ductal grey matter lateral to the DRd and DRv. Some neurons of theDRp were found in the adjacent tegmentum and are the only onesfound outside the periaqueductal grey matter. The 5HT+ neurons

Figure 5. Photomicrographs of serotonergic neurons within the dorsal raphe of the

brain of the Tasmanian devil. Note the presence of several subdivisions of the dorsal

raphe that conform to that seen in most other mammals. Scale bar = 500 mm. See

list for abbreviations.

of the DRl were located dorsolateral to the DRd and adjacent to theventrolateral edges of the cerebral aqueduct in a low to moderatedensity. The neurons of this nucleus were readily distinguishablefrom the remainder of the dorsal raphe nuclei since they weresubstantially larger. As we followed the DRl caudally, where thecerebral aqueduct opened into the fourth ventricle and the DRd,DRv and DRif disappeared, the neurons of the DRl formed an arcacross the midline of the dorsal portion of the periventricular greymatter. This caudal arc of the DRl was classified as the DRc nucleus.We classified this as an independent nucleus due to the lack of5HT+ neurons in this region in the brain of monotremes (Mangeret al., 2002a–c; Dell et al., 2010).

Caudal cluster

The caudal cluster was composed of four nuclei, the raphemagnus nucleus (RMg), the raphe obscurus nucleus (ROb), theraphe pallidus nucleus (RPa) and the rostral ventrolateral nucleus(RVL). No evidence of the caudal ventrolateral nucleus (CVL), astypically observed in placental mammals (Dell et al., 2010, Calveyet al., 2013), was found in the Tasmanaian devil. This clusterextended caudally from the level of the trigeminal motor nucleus(mlf) to the spinomedullary junction (Fig. 2P–W). The two columnsof 5HT+ neurons forming the raphe magnus nucleus (RMg) weresituated on either side of the midline from the level of the caudalpole of the trigeminal motor nucleus to the rostral pole of nucleusambiguus. The RVL began as a lateroventral continuation of 5HT+neurons from the lower portion of the RMg extending over thepyramidal tracts and consolidating as a distinct column lateral tothe inferior olives. The inferior olive topologically distinguishes leftand right RVL, and at the level of nucleus ambiguus the neuronsforming the RVL were no longer observed. The raphe obscurusnucleus (ROb) was composed of two loosely arranged columns of5HT+ neurons located either side of the midline from the level ofthe caudal end of the facial nerve nucleus (VII) to thespinomedullary junction. Ventral to the ROb 5HT+ neuronssituated at the midline between the pyramidal tracts wereassigned to the raphe pallidus nucleus (RPa). The RPa extendedfrom the caudal end of the facial nerve nucleus (VII) to the caudalpole of the nucleus ambiguus.

Orexinergic (hypocretinergic) nuclei

Orexin-A immunopositive (OrX+) neurons found in thehypothalamus of the Tasmanian devil, formed three distinctclusters: a main cluster (Mc), a zona incerta cluster (Zic) and anoptic tract cluster (OTc) (Figs. 2(K and L) and 6). A large group ofdensely packed OrX+ neurons forming the main cluster (Mc) wasidentified lateral to the third ventricle in the perifornical region,with a moderate number of neurons extending dorsal towards thecatecholaminergic A15d nucleus. From the main cluster a group ofOrX+ neurons extended laterally into the region of the zona incerta(Zic) cluster located immediately ventral to the catecholaminergicA13 nucleus. Loosely packed OrX+ neurons forming the optic tractcluster (OTc) were situated ventral and lateral to the main clusterand extended into the ventral hypothalamus adjacent to the optictract.

Discussion

The current study has outlined the organization of thecholinergic, catecholaminergic, serotonergic and orexinergic neu-ronal systems in the brain of the Tasmanian devil, a marsupialbelonging to the order Dasyuromorphia. While previous reportshave outlined certain aspects of these systems in differentmarsupial species (Crutcher and Humbertson, 1978; Martinet al., 1985; Ferguson et al., 1999; Yamamoto et al., 2006; Ashwell,

Fig. 6. Photomicrographs of orexinergic neurons within the brain of the Tasmanian

devil. Note the presence of three clusters of orexinergic neurons, the main cluster

(Mc) located in the perifornical region, the zona incerta cluster (Zic) located in the

dorsolateral hypothalamus, and the optic tract cluster (Otc) located in the

ventrolateral hypothalamus (A). Note also the dense orexinergic innervation

throughout the hypothalamus (B), especially so in the same region as the location of

the neurons of the main cluster (C). Scale bar in A = 500 mm, scale bar in B = 250 mm,

scale bar in C = 100 mm. In all images medial is to the left and dorsal to the top, see

list for abbreviations.

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106104

2010), no previous studies have provided a comprehensiveorganizational description of these neural systems in the samespecies, making the current study interesting in terms ofunderstanding the evolution of these systems across mammalsas a whole. While for the most part the overall organization ofthese systems was quite similar to that seen in other mammalspreviously studied, including monotremes and eutherians, therewere differences in some of these systems that may be related tothe phylogenetic history and evolution of the examined neuronal

systems in mammals. Thus, the basic features of the marsupialbrain, such as the absence of the corpus callosum, had no clearimpact on the fundamental parcellation of the cholinergic,catecholaminergic, serotonergic and orexinergic neuronal systems.

Cholinergic nuclei

Across mammals as a whole, the cholinergic system shows aquite conservative pattern of organization. For example, thecholinergic nuclei found in the basal forebrain and cranial nervemotor nuclei of the Tasmanian devil are identical to those seen inall mammals studied to date, including monotremes (Manger et al.,2002a–c), other marsupials (Ashwell, 2010) and a range ofeutherian mammals (Dell et al., 2010; Calvey et al., 2013). Despitethis consistency, there are variations in the presence and absenceof specific cholinergic nuclei, and three variants were observed inthe Tasmanian devil. Within the hypothalamus of eutherianmammals, 3 distinct cholinergic clusters of neurons (dorsal, lateraland ventral) have been noted (Dell et al., 2010; Calvey et al., 2013;Maseko et al., 2013); however, in the monotremes no hypothalamiccholinergic neurons were observed (Manger et al., 2002a–c). In theTasmanian devil, a single cluster of cholinergic neurons, which wepropose to be homologous to the dorsal hypothalamic cholinergicnucleus of eutherian mammals, was observed. A second variant ofnote was the observation of a clear dorsal subdivision of thelaterodorsal tegmental nucleus (LDTd) in the pontine periventri-cular grey matter of the Tasmanian devil. While in most mammalsthe LDT is composed of a single nucleus located in the ventrolateralportion of the pontine periventricular grey matter (Manger et al.,2002a–c; Dell et al., 2010; Calvey et al., 2013; Maseko et al., 2013), avariation on this pattern has also been observed in the rock hyrax(Gravett et al., 2009), where a shell of small cholineacetyltransferaseimmunopositive neurons surrounded the typically mammalian LDTin this species. We propose that what we have labelled as the LDT inthe Tasmanian devil is homologous to the LDT of other mammals,while the dorsal division of the LDT, LDTd, in the Tasmanian devilappears to be a novel nucleus in this species. Further work onmarsupial species will determine whether the dorsal LDT observedherein is a nucleus restricted in its phylogenetic occurrence to theorder Dasyuromorphia (Manger, 2005), or whether this type ofsubdivided LDT is a feature of the marsupials as a whole. Given thefunction of the LDT nucleus in the sleep-wake cycle of mammals,being involved in the production of the desynchronized EEG ofwaking and REM sleep (Steriade et al., 1990), the presence of thisnucleus in the Tasmanian devil and perhaps other marsupials, maybe an important consideration in understanding the physiology ofsleep in the Tasmanian devil (Nicol and Maskrey, 1980) and moregenerally sleep in marsupials, although at present it is difficult tospeculate what effect such a novel nucleus may have on sleepphysiology. As a last variant of note for the cholinergic neurons in theTasmanian devil is the observation of a moderate number of whatappear to be cholinergic interneurons in the gelatinous layer of thecaudal spinal trigeminal nucleus. Such cholinergic interneuronshave not been reported in other mammals studied; however, theappearance of cholinergic interneurons across species in regionsoutside the ‘‘typical’’ cholinergic system have been observed in thegolden mole and elephant shrews (Pieters et al., 2010; Calvey et al.,2013) and murid rodents (Bhagwandin et al., 2006; Kruger et al.,2012). Speculation as to the functional role of these neurons isbeyond the current study.

Catecholaminergic nuclei

The neurons classified as representing catecholaminergicneurons in the current study (those neurons immunoreactive fortyrosine hydroxylase) did not form any specific nuclei that appear

N. Patzke et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 94–106 105

to be different from those generally observed across mammals(Manger et al., 2004; Dell et al., 2010; Calvey et al., 2013). The onevariance observed in the Tasmanian devil was the lack of thedorsomedial division of the locus coeruleus (A4), which is alsoabsent in the North American opossum (Crutcher and Humbertson,1978) and the monotremes (Manger et al., 2002a–c) and has avariable occurrence in the eutherian mammals (Dell et al., 2010).The potential functional significance of the lack of this nucleus isdifficult to speculate upon, but as the A4 group appears to be acontinuation of the A6 group (Smeets and Gonzalez, 2000), itwould appear that any functional impairment caused by a lack ofthis nucleus should readily be accommodated for by the A6nucleus. Thus, as with the cholinergic system, the catecholamin-ergic system has a conservative pattern of change in regards to thenuclear organization across mammals.

Serotonergic nuclei

The distribution and parcellation of the serotonergic neurons inthe brain of the Tasmanian devil was identical to that reportedpreviously in the North American opossum (Crutcher andHumbertson, 1978), as both lacked the caudal ventrolateralserotonergic group reported in the Tammar wallaby (Fergusonet al., 1999) and all eutherian mammals studied to date exceptthe laboratory rat (Dahlstrom and Fuxe, 1964; Dell et al., 2012;Calvey et al., 2013). Thus, within the marsupials, two orders,the Dasyuromorphia and the Didelphimorphia, appear to share theabsence of this nucleus, while it appears to be present in theDiprotodonta. The lack of this nucleus in the monotremes(Manger et al., 2002a–c) indicates that this particular seroto-nergic nucleus has a variable occurrence, with some phylogeneticcontinuity, but further species of marsupials from different ordersneed to be examined to determine the specific phylogeneticboundaries of the occurrence of this nucleus. Additionally, theTasmanian devil, and other marsupials that have been studied,lacked the periventricular serotonergic neurons found in thehypothalamus of monotremes (Manger et al., 2002a–c). Overall,the nuclear organization of the serotonergic system acrossmammals appears to be very conservative, with only twovariances being noted across the species studied. While thesevariances appear to correlate to phylogenetic relationships, theexact boundaries are not yet clear due to the paucity of speciesstudied. The functional implication of the lack of the caudalventrolateral serotonergic group is difficult to speculate upon atthe moment due to a lack of understanding of the function of theseneurons in the species in which they are present.

Orexinergic nuclei

The only prior study of orexinergic neurons in the brain of amarsupial was undertaken in the eastern grey kangaroo (Macropus

giganteus) (Yamamoto et al., 2006). In this case, as in the Tasmaniandevil studied here, the three clusters of orexinergic neuronstypically reported for a variety of mammals, was observed. Thus, inthe two marsupials studied to date, no apparent absence orpresence of additional orexinergic neurons was observed. Ineutherian mammals, microchiropterans and hamsters appear tolack the optic tract cluster (Kruger et al., 2010), while in the giraffe,harbour porpoise and African elephant an additional mediallylocated parvocellular cluster has been reported (Dell et al., 2013;Maseko et al., 2013). It would appear that the parcellation of theorexinergic system across mammals is quite conservative inevolutionary terms and for the most part is restricted to the threeclusters as reported herein for the Tasmanian devil, although thevariants mentioned above may have interesting functionalconsequences.

Summary

In general, the nuclear organization of the four systems studiedin the Tasmanian devil show many similarities to previous reportsin other marsupials, monotremes and eutherian mammals. Thisglobal similarity indicates that the nuclear parcellation of thesystems studied are not particularly plastic in their evolution,suggesting that they likely provide somewhat invariant functionsacross mammalian species. Despite this, the variations that havebeen observed in different mammals do appear to have an inherentphylogenetic signal, most typically at the level of the order(Manger, 2005), but also within orders (Bhagwandin et al., 2006;Kruger et al., 2010), and within classes (Dell et al., 2010). As furtherspecies are studied and the full range of the nuclear organization ofthese systems revealed, a meaningful interpretation of thephylogenetic and functional implications of both the stasis andvariance of the nuclei that compose these systems will becomeclearer.

Acknowledgments

This study was supported by funding from the South AfricanNational Research Foundation (PRM) and a fellowship within thePostdoctoral Programme of the German Academic ExchangeService, DAAD (NP).

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