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Nuclear organization of cholinergic, putative catecholaminergic andserotonergic systems in the brains of five microchiropteran species

Jean-Leigh Kruger a, Leigh-Anne Dell a, Adhil Bhagwandin a, Ngalla E. Jillani a,John D. Pettigrew b, Paul R. Manger a,*a School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africab Queensland Brain Institute, University of Queensland 4072, Australia

1. Introduction

The proposed monophyletic order Chiroptera has been dividedinto two suborders: megachiroptera and microchiroptera; however,Linnaeus originally grouped the megachiropterans with primates.This classification was largely ignored until the finding that primatesand megachiropterans share several advanced visual pathway

characteristics, in particular the retinotectal pathway, that are notshared by other mammals (Pettigrew, 1986; Pettigrew et al., 1989,2008). The ‘‘flying primate’’ hypothesis proposes that the mega-chiropterans, with the dermopterans, form a sister group to theprimates, and is based on several derived neural features that areabsent in microchiropterans and other mammals (Pettigrew et al.,1989, 2008; Manger et al., 2001; Maseko and Manger, 2007; Maseko

Journal of Chemical Neuroanatomy 40 (2010) 210–222

A R T I C L E I N F O

Article history:

Received 26 March 2010

Received in revised form 28 May 2010

Accepted 28 May 2010

Available online 4 June 2010

Keywords:

Microbat

Chiroptera

Neuromodulatory systems

Diphyly

Evolution

Mammalia

A B S T R A C T

The current study describes, using immunohistochemical methods, the nuclear organization of the

cholinergic, catecholaminergic and serotonergic systems within the brains of five microchiropteran

species. For the vast majority of nuclei observed, direct homologies are evident in other mammalian

species; however, there were several distinctions in the presence or absence of specific nuclei that

provide important clues regarding the use of the brain in the analysis of chiropteran phylogenetic

affinities. Within the five species studied, three specific differences (presence of a parabigeminal nucleus,

dorsal caudal nucleus of the ventral tegmental area and the absence of the substantia nigra ventral)

found in two species from two different families (Cardioderma cor; Megadermatidae, and Coleura afra;

Emballonuridae), illustrates the diversity of microchiropteran phylogeny and the usefulness of brain

characters in phylogenetic reconstruction. A number of distinct differences separate the microchir-

opterans from the megachiropterans, supporting the diphyletic hypothesis of chiropteran phylogenetic

origins. These differences phylogenetically align the microchiropterans with the heterogenous grouping

of insectivores, in contrast to the alignment of megachiropterans with primates. The consistency of the

changes and stasis of neural characters with mammalian phylogeny indicate that the investigation of the

microchiropterans as a sister group to one of the five orders of insectivores to be a potentially fruitful

area of future research.

� 2010 Elsevier B.V. All rights reserved.

Abbreviations: III, oculomotor nucleus; Vmot, motor division of trigeminal nucleus; VI, abducens nucleus; VIId, facial nerve nucleus, dorsal division; VIIv, facial nerve nucleus,

ventral division; A1, caudal ventrolateral medullary tegmental nucleus; A2, caudal dorsomedial medullary nucleus; A4, dorsal medial division of locus coeruleus; A5, fifth

arcuate nucleus; A6c, compact portion of locus coeruleus; A6d, diffuse 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 or

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; A14, rostral periventricular nucleus; A15d, anterior hypothalamic group, dorsal division; A15v, anterior

hypothalamic group, ventral division; A16, catecholaminergic neurons of the olfactory bulb; AP, area postrema; B9, supralemniscal serotonergic nucleus; C1, rostral

ventrolateral medullary tegmental group; C2, rostral dorsomedial medullary nucleus; ca, cerebral aqueduct; CLi, caudal linear nucleus; CVL, caudal ventrolateral serotonergic

group; DRc, dorsal raphe nucleus, caudal division; DRd, dorsal raphe nucleus, dorsal division; DRif, dorsal raphe nucleus, interfascicular division; DRl, dorsal raphe nucleus,

lateral division; DRp, dorsal raphe nucleus, peripheral division; DRv, dorsal raphe nucleus, ventral division; EW, Edinger–Westphal nucleus; Fr, fasciculus retroflexus; GC,

periaqueductal grey matter; IC, inferior colliculus; IP, interpeduncular nucleus; LDT, laterodorsal tegmental nucleus; MnR, median raphe nucleus; PC, cerebral peduncle; pVII,

preganglionic motor neurons of the superior salivatory nucleus or facial nerve; pIX, preganglionic motor neurons of the inferior salivatory nucleus; PBg, parabigeminal

nucleus; PPT, pedunculopontine 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.

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

E-mail address: [email protected] (P.R. Manger).

Contents lists available at ScienceDirect

Journal of Chemical Neuroanatomy

journal homepage: www.e lsev ier .com/ locate / jchemneu

0891-0618/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.jchemneu.2010.05.007


et al., 2007). The logical conclusion of this hypothesis is that theChiropteran order is actually diphyletic and flight evolved twice inmammals. A summary of the evidence on each side of the debateabout whether bats are monophyletic or diphyletic is provided in theaccompanying paper (Dell et al., 2010).

Most studies that support the diphyletic origin of bats haveconcentrated on specific neuroanatomical structures in mega-chiropterans, with little work to date having been done inmicrochiropterans. Maseko and Manger (2007) and Masekoet al. (2007) undertook investigations into the nuclear organizationof the cholinergic, catecholaminergic and serotonergic systems inthe microchiropteran Miniopterus schreibersii, and the megachir-opteran Rousettus aegyptiacus, which defined several furtherdifferences between the mega- and microchiropterans lendingfurther support for the diphyletic origin of the Chiroptera. Incontrast, studies favouring Chiropteran monophyly are usuallybased on DNA and molecular findings (e.g. Murphy et al., 2001;Teeling et al., 2005; Van Den Bussche and Hoofer, 2004).

Manger (2005) proposed, based on studies of the nuclearorganization of the cholinergic, catecholaminergic and serotoner-gic systems of a range of mammalian species (see also Masekoet al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008; Gravettet al., 2009; Pieters et al., 2010; Bux et al., 2010), that all specieswithin an order will exhibit the same complement of homologousnuclei of these systems. This proposal infers that if mega- andmicrochiropterans belonged to the same mammalian order, theyshould have the same nuclear organization of these systems;however this is not the case as shown by Maseko and Manger(2007) and Maseko et al. (2007).

While these previous studies lend support to the diphyletic originof the Chiropterans, it should be noted that the microchiropterans inparticular are one of the most species-rich suborders of mammals,consisting of over 800 species (Nowack, 1999). Thus, before firmconclusions regarding differences between the two suborders can bemade, further species should be investigated. The current studydescribes the nuclear organization of the cholinergic, catecholamin-ergic and serotonergic systems in the brains of five previouslyunstudied microchiropteran species from a range of phylogeneti-cally distant families (distant within the microchiropteran subor-der). A speculative hypothesis proposed that the shared midbrainbinocular circuitry of primates and megachiropterans representedtwo independent evolutionary events that were each driven by theselective forces of the ‘‘small branch niche’’ (Martin, 1986). It isimportant to note that the vast majority of the nuclei of the systemsunder investigation here do not play any direct role in the neuralprocesses related to flight, vision or echolocation and as such thefindings cannot be ignored on the basis of sensory or motorspecialisations of the Chiroptera, an argument that has beenpreviously levelled at the studies of Chiropteran neuroanatomythat support the diphyletic hypothesis (Martin, 1986; Allman, 1999).

2. Materials and methods

Three brains of each of the following microchiropteran species were used in this

study: Cardioderma cor (average body mass = 26 g; average brain mass = 670 mg),

Chaerophon pumilus (average body mass = 5.4 g; average brain mass = 122 mg),

Coleura afra (average body mass = 11.5 g; average brain mass = 257 mg), Hipposideros

commersoni (average body mass = 101.9 g; average brain mass = 750 mg) and

Triaenops persicus (average body mass = 13.7 g; average brain mass = 271 mg). All

animals were captured from wild populations in Kenya and were treated and used

according to the guidelines of the University of the Witwatersrand Animal Ethics

Committee, the Kenya National Museums and the Kenyan Wildlife Services. The

animals were euthanazed (Euthanaze, 1 ml/kg, i.p.) and upon cessation of respiration,

perfused intracardially with an initial rinse of 0.9% saline solution at 4 8C (1 ml/g body

mass) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4 8C (1 ml/g

body mass). After removal from the skull, each brain was post-fixed overnight in the

paraformaldehyde solution and subsequently stored in an anti-freeze solution at

�20 8C. Before sectioning, the brains were allowed to equilibrate in 30% sucrose in

0.1 M PB at 4 8C. Each brain was then frozen in crushed dry ice and sectioned into

50 mm thick serial coronal sections on a freezing microtome. A one in four series of

stains was made for Nissl substance, choline-acyltransferase (ChAT), tyrosine

hydroxylase (TH) and serotonin (5-HT). Sections for Nissl staining were first mounted

on 0.5% gelatine coated slides, cleared in a solution of 1:1 absolute alcohol and

chloroform and then stained with 1% cresyl violet.

The sections used for immunohistochemical staining were treated for 30 min in

an endogenous peroxidase inhibitor (49.2% methanol:49.2% 0.1 M PB:1.6% of 30%

hydrogen peroxide) followed by three 10 min rinses in 0.1 M PB. Sections were then

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

serum, NRS, for ChAT sections or 3% normal goat serum, NGS, for TH and 5-HT

sections, 2% bovine serum albumin, BSA, and 0.25% Triton X-100 in 0.1 M PB).

Thereafter sections were incubated in the primary antibody solution in blocking

buffer for 48 h at 4 8C under gentle agitation. Anti-cholineacetyltransferase

(AB144P, Millipore, raised in goat) at a dilution of 1:3000 was used to reveal

cholinergic neurons. Anti-tyrosine hydroxylase (AB151, Millipore, raised in rabbit)

at a dilution of 1:7500 revealed the catecholaminergic neurons. Serotonergic

neurons were revealed using anti-serotonin (AB938, Millipore, raised in rabbit) at a

dilution of 1:10,000. This incubation was followed by three 10 min rinses in 0.1 M

PB and the sections were then incubated in a secondary antibody solution (1:750

dilution of biotinylated anti-goat IgG, BA 5000, Vector Labs, for ChAT sections or a

1:750 dilution of biotinylated anti-rabbit IgG, BA 1000, Vector Labs, for TH and 5-HT

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 AB solution (Vector Labs), followed by

three 10 min rinses in 0.1 M PB. Sections were then placed in a solution containing

0.05% diaminobenzidine (DAB) in 0.1 M PB for 5 min, followed by the addition of

3 ml of 30% hydrogen peroxide per 0.5 ml of solution. Chromatic precipitation was

visually monitored and verified under a low power stereomicroscope. Staining

continued until such time as the background stain was at a level that would allow

for accurate architectonic reconstruction without obscuring the immunopositive

neurons. Development was arrested by placing sections in 0.1 M PB, 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. The controls employed in this experiment included the omission of the

primary antibody and the omission of the secondary antibody in selected sections.

As a further control for the cholinergic immunohistochemistry, we used choline

acetyltransferase (AG220, Millipore) at a dilution of 5 mg/ml in the primary

antibody solution (see above) as an inhibition assay. This solution was incubated for

3 h at 4 8C prior to being used on the sections. We also reacted adjacent sections that

were not inhibited. In the sections where the primary antibody had been inhibited,

no staining was evident. Sections were examined under a low power stereomicro-

scope and using a camera lucida the architectonic borders of the sections were

traced following the Nissl stained sections. Sections containing the immunopositive

neurons were then matched to the drawings and the neurons were marked. Select

drawings were then scanned and redrawn using the Canvas 8 drawing program.

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.

All architectonic nomenclature was taken from the atlas of a Microchiropteran

brain (Baron et al., 1996), while the nomenclature used to describe the

immunohistochemically revealed systems was based on Dahlstrom and Fuxe

(1964), Hokfelt et al. (1984), Tork (1990), Woolf (1991), Smeets and Gonzalez

(2000), Manger et al. (2002a,b,c), Maseko and Manger (2007), Maseko et al. (2007),

Moon et al. (2007), Dwarika et al. (2008), Limacher et al. (2008), Bhagwandin et al.

(2008), Gravett et al. (2009) and Pieters et al. (2010).

3. Results

The nuclear organization of the cholinergic, catecholaminergicand serotonergic neural systems generally followed that found byMaseko and Manger (2007) in their study of M. schreibersii,although there were some notable differences between species,specifically in the cholinergic and catecholaminergic systems, andthese are explicitly described. The following description appliesgenerally to all the microchiropteran species studied, except wherespecifically noted.

3.1. Cholinergic nuclei

The cholinergic system is generally subdivided into the corticalcholinergic interneurons, the striatal region, basal forebrain,diencephalon, pontomesencephalon and the cranial nerve nucleigroups (Woolf, 1991). In the five species examined in the currentstudy, we identified cholineacetyltransferase immunoreactive

J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 211


(ChAT+) neurons in all these subdivisions, excluding the corticalcholinergic interneurons, which have a variable occurrence acrossmammalian species (e.g. Bhagwandin et al., 2006). Additionally,there was no evidence of any ChAT+ neurons in the medullarytegmental field.

3.1.1. Cholinergic striatal interneurons

ChAT+ neurons were found in the Islands of Calleja, the olfactorytubercle, nucleus accumbens, the caudate/putamen complex and inthe globus pallidus. The distribution, density, topography andnuclear organization of ChAT+ neurons in these areas was found tobe similar across all five microchiropteran species, as well asfollowing the distribution and morphology as described by Masekoand Manger (2007) in their study of M. schreibersii.

3.1.2. Cholinergic basal forebrain nuclei

The basal forebrain nuclei are composed of the medial septalnucleus, the Diagonal band of Broca and the nucleus basalis (Woolf,1991; Maseko et al., 2007). ChAT+ neurons were observed in all ofthese nuclei across all five microchiropteran species, as found byMaseko and Manger (2007) in M. schreibersii. No notableinterspecies differences were observed.

3.1.3. Diencephalic cholinergic nuclei

ChAT+ neurons were found in the medial habenular nucleus, aswell as in the three hypothalamic nuclei (dorsal, lateral andventral) in all five microchiropteran species. In T. persicus there wasvery strong ChAT immunoreactivity in the medial habenularnucleus, as well as in the lateral hypothalamic cluster, while C. afra

and C. cor showed only a weak reactivity in the lateralhypothalamic region. The ventral hypothalamic nucleus was mostwell expressed in C. cor. In general, our findings were similar acrossall five microchiropteran species and in terms of nuclearorganization are identical to that found in M. schreibersii (Masekoand Manger, 2007). No evidence of ChAT+ neurons could be foundin the anterior nuclei of the dorsal thalamus as seen in the rockhyrax (Gravett et al., 2009).

3.1.4. Midbrain/pontine cholinergic nuclei

As with the observations made in M. schreibersii, we observedChAT+ neurons in the pedunculopontine (PPT) and the laterodorsal

tegmental (LDT) nuclei in all five species studied (Fig. 1). Anunusual dense aggregation of ChAT+ neurons in the PPT of C.

pumilus was observed in the postero-lateral portion of this nucleusin a position lateral to the superior cerebellar peduncle (Fig. 2);however, the remaining species evinced ‘‘typical’’ microchirop-teran PPT and LDT morphology (Maseko and Manger, 2007). Incontrast to the findings of Maseko and Manger (2007), evidence ofthe parabigeminal nucleus was found in C. cor and C. afra (Fig. 3).ChAT+ neurons in the parabigeminal nucleus were most readilyobserved in C. cor, with only weak staining occurring in thisnucleus in C. afra. In both species, the location of the parabigeminal

[(Fig._1)TD$FIG]

Fig. 1. Photomicrograph of ChAT immunopositive neurons forming the laterodorsal

tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT) nuclei

in Cardioderma cor. The large inferior colliculus (IC) gives a slightly different

impression of the topological relations of LDT and PPT, although these nuclei still

maintain the same basic features as in all other mammals observed to date. Scale

bar = 500 mm. ca – cerebral aqueduct.

[(Fig._2)TD$FIG]

Fig. 2. Photomicrographs of three adjacent sections (250 mm apart, A the most

rostral, C the most caudal) through the caudal portion of the pedunculopontine

tegmental nucleus (PPT) in Chaerophon pumilus. Note the major density of ChAT

immunopositive neurons lateral to the superior cerebellar peduncle (scp). This is an

unusual feature of the PPT and was only seen in this species. Scale bar in C = 100 mm

and applies to all. Vmot – motor division of the trigeminal nucleus.

J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222212


nucleus was typical of that seen in other mammals. No evidence ofChAT+ neurons within the parabigeminal nucleus could be foundin the other three species investigated, which is congruent with thefindings for M. schreibersii (Maseko and Manger, 2007). Noevidence of ChAT+ neurons could be found in the pedunculopon-tine parvocellular (PPTpc), the laterodorsal tegmental parvocel-lular (LDTpc) or the superior and inferior colliculus interneurons asoccasionally observed in other species (Gravett et al., 2009; Pieterset al., 2010). The lack of ChAT+ neurons in these regions follows thefindings of Maseko and Manger (2007) for M. schreibersii.

3.1.5. Cranial nerve nuclei

As observed by Maseko and Manger (2007) in M. schreibersii, noevidence of ChAT+ neurons were observed in the cochlear nucleus orthe medullary tegmental field in any of the five microchiropteranspecies investigated; however, in the current study evidence ofChAT+ neurons was found for the Edinger–Westphal nucleus (Fig. 4),the preganglionic superior salivatory nucleus (pVII) and thepreganglionic inferior salivatory nucleus (pIX) (Fig. 5). These nucleiwere present in all five microchiropteran species investigated andshowed similar neuronal morphology, distributions and numbers as

observed in other mammalian species (e.g. Maseko et al., 2007;Bhagwandin et al., 2008; Gravett et al., 2009). All other typicallyChAT immunoreactive cranial nerve nuclei were observed in all fivespecies, in line with earlier findings for M. schreibersii (Maseko andManger, 2007). ChAT+ expression was found to be particularlystrong in the oculomotor neurons in C. cor, while in T. persicus thenucleus ambiguus was found to be particularly large. C. afra

possessed a large ventral division of the facial nerve nucleus, as wellas strong ChAT+ expression in pVII, pIX, the hypoglossal nucleus andthe ventral horn of the spinal cord.

3.2. Catecholaminergic nuclei

The catecholaminergic nuclei were found throughout the brainsof all five microchiropteran species investigated and were revealedusing tyrosine hydroxylase (TH) immunoreactivity. Generally ourfindings mirrored those of Maseko and Manger (2007), althoughdifferences in TH+ immunoreactivity were noted in the A15v(anterior hypothalamic ventral group), A10d (dorsal ventraltegmental area), A10dc (dorsal caudal ventral tegmental area)and A9v (substantia nigra ventral) in specific species.

[(Fig._3)TD$FIG]

Fig. 3. Five low power photomicrographs at the level of the ChAT immunoreactive oculomotor nucleus (III) and interpeduncular nucleus (IP) showing the strongly ChAT

immunoreactive parabigeminal nucleus (PBg) in Cardioderma cor (A), a weakly ChAT immunoreactive PBg in Coleura afra (B), and its complete absence in Chaerophon pumilus

(C), Hipposideros commersoni (D), and Triaenops persicus (E). (F) Higher power photomicrograph of the parabigeminal nucleus in Cardioderma cor. Scale bar in E = 500 mm and

applies to A–E. Scale bar in F = 250 mm. III – oculomotor nucleus; ca – cerebral aqueduct; EW – Edinger–Westphal nucleus.



[(Fig._4)TD$FIG]

Fig. 4. Photomicrographs of ChAT immunoreactive neurons within the Edinger–Westphal nucleus (EW) of three microchiropteran species: (A) Triaenops persicus; (B)

Cardioderma cor; and (C) Hipposideros commersoni. Note the ChAT immunoreactive fasciculus retroflexus (fr) on either side of the midline. Scale bar in B = 500 mm and applies

to all.[(Fig._5)TD$FIG]

Fig. 5. Photomicrographs of the ChAT immunoreactive neurons forming the preganglionic neurons of the superior salivatory (pVII) and inferior salivatory (pIX) nuclei in (A)

Cardioderma cor, (B) Triaenops persicus and (C) Chaerophon pumilus in relation to the ChAT immunoreactive neurons of the ventral (VIIv) and dorsal (VIId) subdivisions of the

facial nerve nucleus and the abducens nucleus (VI). Scale bar in C = 500 mm and applies to all.



3.2.1. A16 – olfactory bulb

These neurons, found in the stratum granulosum of theolfactory bulb, showed TH+ immunoreactivity across all fivemicrochiropteran species investigated, which concurs with thefindings for M. schreibersii (Maseko and Manger, 2007).

3.2.2. Hypothalamic nuclei (A11–A15)

TH+ neurons were identified in the A11, A12, A13 and A14nuclei (Fig. 6) in all species studied, in agreement with previousfindings in M. schreibersii (Maseko and Manger, 2007). The A14(rostral periventricular cell group) and A11 (caudal diencephalicgroup) nuclei were both strongly expressed in terms of neuronalnumber in H. commersoni (Fig. 6D). No evidence was found for theA15d (anterior hypothalamic group, dorsal division) nucleus in anyof the five species studied, as seen in M. schreibersii; although thisnucleus is present in many other mammals (e.g. Maseko et al.,2007; Bhagwandin et al., 2008; Gravett et al., 2009). TH+ neuronswere found in the A15v (anterior hypothalamic group, ventral

division) in C. pumilus, H. commersoni and T. persicus, but not in C.

cor or C. afra. The occurrence of A15v in three species is in contrastto the findings of Maseko and Manger (2007) for M. schreibersii.

3.2.3. Midbrain catecholaminergic nuclei (A8–A10)

In the present study, there was evidence of TH+ immunoreac-tivity in many of the midbrain catecholaminergic nuclei typicallydescribed for mammals (Fig. 7; Smeets and Gonzalez, 2000), theseinclude the A8, A9m, A9pc, A9l, A10, A10c, and A10d nuclei, all ofwhich were found in the locations typically seen across allmammals. In the prior study of M. schreibersii (Maseko and Manger,2007) no evidence for the A10dc, A10d, or A9v nuclei was observed.In the current study partial evidence of A10dc was found in C.

pumilus and C. cor in the form of occasional TH+ neurons in theperiaqueductal grey matter near the ventrolateral edge of thecerebral aqueduct (Fig. 7); however, no evidence of this nucleuscould be found in the other microchiropteran species, in line withthe findings of Maseko and Manger (2007). Additionally, partial

[(Fig._6)TD$FIG]

Fig. 6. Photomicrographs of various TH immunoreactive neurons in the hypothalamus of different species of microchiropterans. (A) The A13 nucleus in Coleura afra, (B) A14

neurons in Cardioderma cor, (C) A13 neurons in Hipposideros commmersoni, (D) A11 neurons in Hipposideros commmersoni. Scale bar in A = 100 mm. Scale bar in B = 50 mm and

applies to B–D.



evidence for A9v could be found in all five microchiropteranspecies under investigation, which differs from the findings for M.

schreibersii (Maseko and Manger, 2007). It should be noted thoughthat in all species studied herein, that the number of neurons thatcould be assigned to the A9v nucleus was small, varied betweenindividuals of the same species and varied across species.

3.2.4. Rostral rhombencephalon (locus coeruleus complex, A4–A7)

TH+ neurons were observed in the A5 (fifth arcuate), A6d (locuscoeruleus diffuse), A7sc (subcoeruleus compact) and A7d (sub-coeruleus diffuse) nuclei in all five microchiropteran speciesinvestigated in this study, as well as in M. schreibersii (Maseko andManger, 2007). The location, densities, and morphology of theseneurons were similar across all species. No evidence for an A6c orA4 nucleus was found (Fig. 8), as was also the case in M. schreibersii

(Maseko and Manger, 2007).

3.2.5. Caudal rhombencephalon (A1, A2, C1, C2, area postrema)

Evidence was found for all five caudal rhombencephalon nuclei,across all five microchiropteran species studied, which concurs

with previous findings in M. schreibersii (Maseko and Manger,2007) and across all mammals studied to date. The C1 nucleus(rostral ventrolateral tegmental group) was weakly expressed interms of neuronal numbers in C. pumilus. For the remaining nuclei astrong homogeneity in the expression of these nuclei acrossspecies was observed. No evidence of the C3 nucleus, a nucleus thathas only been found in rodents (e.g. Bhagwandin et al., 2008), wasobserved.

3.3. Serotonergic neurons

In mammals, the serotonergic neurons are typically subdividedinto rostral and caudal nuclear clusters, with both being locatedentirely the brainstem (Tork, 1990; Jacobs and Azmitia, 1992). Inmammals in general, the rostral cluster consists of the CLi (caudallinear), B9 (supralemniscal), MnR (median raphe) and the dorsalraphe nuclei, while the caudal cluster is composed of the RMg(raphe magnus), RPa (raphe pallidus), RVL (rostral ventrolateralcell column), CVL (caudal ventrolateral cell column) and ROb(raphe obscurus) nuclei.

[(Fig._7)TD$FIG]

Fig. 7. Diagrammatic reconstructions of the midbrain catecholaminergic nuclei in four species of microchiropteran. Note the similarity between species, as well as the

possible A10dc in C. pumilus and C. cor. See list for abbreviations.



3.3.1. Rostral cluster

All serotonergic nuclei typically assigned to the rostral clustershowed specific serotonergic immunoreactivity across all fivemicrochiropteran species investigated. This is consistent with theprevious findings for M. schreibersii (Maseko and Manger, 2007)and for all Eutherian mammals (Maseko et al., 2007). The CLinucleus was strongly expressed in T. persicus, with the B9 nucleusbeing strongly expressed in C. afra, and C. cor (Fig. 9) and C. pumilus.The dorsal raphe cluster could be divided into dorsal (DRd), ventral(DRv), interfascicular (DRif), lateral (DRl), caudal (DRc) andperipheral (DRp) nuclei, all of which were observed in all fivespecies, all evincing a similar appearance (Fig. 10). As with all priorobservations, only a few neurons of the peripheral nucleus of thedorsal raphe were located outside the periaqueductal grey matter.

3.3.2. Caudal cluster

All five microchiropteran species studied had 5-HT+ neurons inthe RMg, RPa, RVL, CVL and ROb nuclei, as was observed in M.

schreibersii (Maseko and Manger, 2007) and all other Eutherianmammals studied to date. It was noted that RMg possessed few 5-HT+ neurons in C. afra, and CVL was weakly expressed in terms of

[(Fig._8)TD$FIG]

Fig. 8. Photomicrographs of TH immunoreactive neurons in the locus coeruleus of different species of microchiropterans. (A) The A6d and A7d nuclei in Coleura afra, (B) A6d

and A7sc neurons in Triaenops persicus, (C) A6d neurons in Chaerophon pumilus, (D) A6d and A7d neurons in Cardioderma cor. Scale bar in D = 500 mm and applies to all.[(Fig._9)TD$FIG]

Fig. 9. A photomicrograph showing the most rostral serotonergic nuclei in

Cardioderma cor, the caudal linear nucleus (CLi) and the supralemniscal

serotonergic neurons (B9) at the level of the interpeduncular nucleus (IP). Scale

bar = 100 mm.



neuronal numbers in T. persicus. In all other respects these nucleiwere similar to previous observations in other mammals.

4. Discussion

Within the cholinergic, catecholaminergic and serotonergicsystems many similarities in nuclear organization were found inthe five microchiropterans investigated in this study and thatreported for M. schreibersii (Maseko and Manger, 2007); however,some notable differences in nuclear organization were found in thecholinergic and catecholaminergic systems. Several differenceswere observed when comparing the nuclear organization of thesesystems with observations made on the megachiropteranspreviously studied (Maseko et al., 2007; Dell et al., 2010), themost notable of which occurred, again, in the cholinergic andcatecholaminergic systems. When making a broader comparisonacross mammals, the general organization of these systems in themicrochiropterans were similar to that seen in other mammals, butnotable differences in the presence or absence of specific nuclei

within the cholinergic and catecholaminergic systems were found.Previous studies of these systems in insectivores appear to showthe greatest number of similarities in terms of nuclear organizationof these systems with that observed in microchiropterans (see alsoDell et al., 2010), suggesting a close phylogenetic link between themicrochiropterans and the insectivores.

4.1. Comparisons amongst microchiropterans

4.1.1. Cholinergic nuclei

The nuclear organization of the cholinergic system wasgenerally found to be similar in all species; however there werenotable differences in the pontine and cranial nerve nuclei.ChAT+ neurons were identified in the parabigeminal nucleus of C.

cor and C. afra, which were not seen in the other microchir-opterans studied herein or in M. schreibersii (Maseko and Manger,2007). While a strongly expressed parabigeminal nucleus wasobserved in C. cor, in C. afra the immunoreactivity of theseneurons was weak. ChAT+ immunoreactivity also occurred in the

[(Fig._10)TD$FIG]

Fig. 10. Photomicrographs of the nuclear subdivisions of dorsal raphe nuclear complex in four microchiropteran species: (A) Chaerophon pumilus; (B) Hipposideros

commersoni; (C) Coleura afra; and (D) Triaenops persicus. Scale bar in D = 500 mm and applies to all. See list for abbreviations.



Edinger–Westphal, pVII and pIX cholinergic nuclei across all fivemicrochiropteran species studied, whereas no evidence for ChATimmunoreactivity of the neurons within these nuclei was foundin M. schreibersii (Maseko and Manger, 2007). These latterdifferences may be attributed to the antibody used in the currentstudy – Maseko and Manger (2007) utilised the ChAT antibodyAB143 (Chemicon), while the current study employed AB144P(Chemicon). A study by Bhagwandin et al. (2006) looked at therevelation of cortical cholinergic neurons in a range of rodentspecies using three different antibodies (AB143, AB144P andvChAT). It was shown that AB144P consistently revealed themost cholinergic neurons, while the revelation was limited usingAB143 and vChAT. It is possible that these antibodies bind todifferent regions of the cholineacetyltransferase molecule andthus the molecule involved in producing acetylcholine may differin structure in different parts of the central nervous system anddiffer within specific phylogenies (Bhagwandin et al., 2006). Thecholinergic neurons revealed in this study, but not in the study byMaseko and Manger (2007), are mostly neurons that appear to beinvolved with the autonomic nervous system (except for thoseseen in the parabigeminal nucleus); thus, there may be, at least inthe microchiropterans and rodents, a differing morphology of thecholineacetyltransferase molecule in the parts of the brainassociated with the ‘‘classical’’ cholinergic system and that partof the cholinergic system associated with the autonomic nervoussystem.

4.1.2. Catecholaminergic nuclei

The most notable difference in the catecholaminergic nucleiwas the presence of the A15v nucleus in all five microchiropteranspecies in this study, as well as the possible existence of the A9vnucleus; however A15v was only poorly expressed in C. cor and C.

afra, while it was strongly expressed in the other three species.Neither of these nuclei were present in M. schreibersii (Maseko andManger, 2007). Some evidence was also found for A10dc but onlyin C. cor and C. afra, with no other species expressing this nucleus,yet all were observed to have the A10d nucleus which wasreported as absent in M. schreibersii. It is important to note that C.

cor and C. afra are also the only two microchiropteran species thatshowed the presence of the parabigeminal nucleus, as well as avery weak expression of A15v. These findings group these twospecies, from the families Megadermatidae and Emballonuridae,together, but slightly separate them from the other 17 or somicrochiropteran families, which is not in agreement with therecently published molecular, paraphyletic phylogenies of themicrochiropterans, where megadermatids are aligned withrhinolophoids and split from all other microbats (Van DenBussche and Hoofer, 2004; Jones et al., 2005; Teeling et al.,2005; Gu et al., 2008). On the other hand, morphological studieshave consistently placed these two Old World microchiropteranfamilies together (e.g. Smith, 1976; Van Valen, 1979; Pierson,1986). All other catecholaminergic nuclei were found to be similaracross all six microchiropteran species.

4.1.3. Serotonergic nuclei

The nuclear organization of the serotonergic system ishomogenous across all six microchiropteran species studied todate, i.e. the five species in this study together with M. schreibersii

(Maseko and Manger, 2007). There were a few differences inexpression patterns of individual nuclei observed between thespecies studied. C. afra had a notably small RMg nucleus, while CVLwas particularly small in T. persicus. As specific functions have notbeen ascribed to these nuclei beyond the modulation of certainportions of the neurons within the grey matter of the spinal cord(Tork, 1990) it is difficult to speculate if these differences have anyparticular functional implications.

4.2. Comparisons with megachiropterans


In the cholinergic system, the five microchiropterans examinedin this study and the three megachiropterans that have beenstudied (Rousettus aegyptiacus, Eidolon helvum, Epomophorus

wahlbergi) have the expression of almost all nuclei in common,including the Edinger–Westphal, pVII and pIX nuclei which werereported as absent in M. schreibersii (Maseko et al., 2007; Dell et al.,2010). The only difference in organization of the cholinergic nucleiis found in the pontine region, this being the variable presence ofChAT immunoreactivity in the neurons of the parabigeminalnucleus. ChAT immunoreactivity of neurons within this nucleusoccurs in all megachiropterans studied (Maseko et al., 2007; Dellet al., 2010), as well as in two of the microchiropterans C. cor and C.

afra; however, ChAT immunoreactivity the parabigeminal nucleuswas absent in the four other microchiropteran species that havebeen examined, and in C. afra the ChAT immunoreactivity wasweak. The variability in the occurrence of ChAT immunoreactivityof the neurons of the parabigeminal nucleus is difficult to explainat present, and further examination of the presence or absence ofthis immunoreactivity in other microchiropteran species mayelucidate this issue, determining whether this is a phylogenetic orfunctional variation. It may also be possible that the parabigeminalnucleus is actually absent in the species where we have notdetected any ChAT immunoreactivity. Connectional studies fromthe superior colliculi of those species without ChAT immunoreac-tivity would determine the presence or absence of this nucleus.


The major differences noted between megachiropteranspreviously studied (Maseko et al., 2007; Dell et al., 2010) andthe five microchiropteran species examined in this study occur inthe presence or absence of nuclei assigned to the catecholaminer-gic system. The A15d, A6c and A4 nuclei were not present in themicrochiropterans, including M. schreibersii (Maseko and Manger,2007); however these nuclei were found in the three megachir-opteran species that have been studied (Maseko et al., 2007; Dellet al., 2010). The A10dc nucleus is possibly present in C. cor and C.

afra, but not in the other three microchiropteran speciesinvestigated, nor M. schreibersii (Maseko and Manger, 2007). Thisnucleus is however found in the three megachiropteran speciesthat have been studied (Maseko et al., 2007; Dell et al., 2010). TheA9v nucleus was very weakly expressed in all five microchir-opteran species, while it is strongly expressed in all megachir-opteran species (Maseko et al., 2007; Dell et al., 2010). The A15vnucleus is only weakly expressed in C. cor and C. afra, while it isstrongly expressed in the other three microchiropterans studiedand in the megachiropterans. All remaining catecholaminergicnuclei were consistent across microchiropterans and megachir-opterans.


There were no differences between microchiropterans andmegachiropterans in terms of the nuclear organization of theserotonergic system (Maseko and Manger, 2007; Maseko et al.,2007; Dell et al., 2010).

4.3. Comparisons with other mammals


The presence of all cholinergic striatal and basal forebrain nucleiis homogenous across all mammalian species studied thus far,including the microchiropterans (Ferreira et al., 2001; Manger et al.,2002a; Maseko et al., 2007; Limacher et al., 2008; Bhagwandin et al.,2008; Gravett et al., 2009; Pieters et al., 2010; Dell et al., 2010).



Microchiropterans and all other Eutherian mammals have cholin-ergic neurons in the medial habenular nucleus and three nucleiwithin the hypothalamus (Ferreira et al., 2001; Manger et al., 2002a;Maseko et al., 2007; Limacher et al., 2008; Bhagwandin et al., 2008;Gravett et al., 2009; Pieters et al., 2010; Bux et al., 2010; Dell et al.,2010); however, the monotremes do not show this reactivity in thedorsal, ventral and lateral hypothalamic nuclei (Manger et al.,2002a). There are a number of differences and similarities within thepontine cholinergic nuclei across mammalian species. The para-bigeminal nucleus was found in two of the microchiropteran speciesin this study: C. cor and C. afra. This nucleus has been found in thegiraffe, the rodents, the afrotherians (in this study represented by therock hyrax and elephant shrew), the carnivores, the megachiropter-ans and the primates (Jones and Cuello, 1989; Maseko et al., 2007;Bhagwandin et al., 2008; Limacher et al., 2008; Gravett et al., 2009;Pieters et al., 2010; Bux et al., 2010); however, it is absent in C.

pumilus, H. commersoni and T. persicus, as well as M. schreibersii

(Maseko and Manger, 2007), the laboratory shrew, the echidna andthe platypus (Manger et al., 2002a; Karasawa et al., 2003). Thesuperior collicular interneurons have, to date, only been seen in therodents, elephant shrew and tree shrew (Pieters et al., 2010), whilemicrochiropterans and other mammals do not express theseinterneurons (Maseko and Manger, 2007; Gravett et al., 2009;Limacher et al., 2008; Maseko et al., 2007). The inferior collicularcholinergic interneurons do not occur in the microchiropterans orother mammals (Maseko and Manger, 2007; Gravett et al., 2009;Limacher et al., 2008; Maseko et al., 2007; Bhagwandin et al., 2008),except for the elephant shrew (Pieters et al., 2010). The cranial nervenuclei generally follow a similar pattern throughout all mammalswith a few notable exceptions (Maseko and Manger, 2007; Gravettet al., 2009; Limacher et al., 2008; Maseko et al., 2007; Bhagwandinet al., 2008). In this study, the Edinger–Westphal, pVII and pIX nucleiwere present in all five microchiropteran species investigated.These nuclei are not present in the echidna, the platypus and thelaboratory shrew (Manger et al., 2002a; Karasawa et al., 2003);however all other mammals studied to date have these nuclei incommon (Maseko and Manger, 2007; Maseko et al., 2007; Limacheret al., 2008; Bhagwandin et al., 2008; Gravett et al., 2009; Pieterset al., 2010; Bux et al., 2010; Dell et al., 2010). The medullarytegmental field is absent in microchiropterans, the elephant shrew,the rock hyrax, the megachiropterans and primates (Maseko andManger, 2007; Maseko et al., 2007; Gravett et al., 2009; Pieters et al.,2008), while it has been seen in the monotremes, rodents andcarnivores (Manger et al., 2002a; Maseko et al., 2007; Bhagwandinet al., 2008).


In general the hypothalamic catecholaminergic nuclei aresimilar across mammals (Skagerberg et al., 1988; Tillet andThibault, 1989; Leshin et al., 1995; Tillet et al., 2000; Manger et al.,2002b, 2004; Maseko et al., 2007; Bhagwandin et al., 2008;Limacher et al., 2008; Gravett et al., 2009; Pieters et al., 2010; Buxet al., 2010). The A15v nucleus shows a variable presence in themicrochiropterans studied and has also been reported to beabsent in the rabbit and the tree shrew (Maseko et al., 2007; Dellet al., 2010). The A15d nucleus is absent in all microchiropterans,as well as the insectivores, artiodactyls, the elephant shrew andthe tree shrew (Pieters et al., 2010; Bux et al., 2010; Dell et al.,2010). The midbrain nuclei are also, for the most part, homoge-nous across mammalian species (Skagerberg et al., 1988; Tilletand Thibault, 1989; Leshin et al., 1995; Tillet et al., 2000; Mangeret al., 2002b; Bhagwandin et al., 2008; Limacher et al., 2008;Gravett et al., 2009; Pieters et al., 2010; Bux et al., 2010; Dell et al.,2010); however differences occur in the microchiropterans, inthat the A10dc and A9v nuclei have a variable occurrence. TheA10dc nucleus was absent in C. pumilus, H. commersoni, T. persicus

and M. schreibersii, while C. cor and C. afra possibly possess thisnucleus but it is very weakly expressed in terms of neuronalnumber. In the microchiropterans A9v may be present but again,only weakly expressed in terms of neuronal number. The A9vnucleus shows a similar morphology in the hedgehog, and isabsent in the rabbit and carnivores (Dell et al., 2010). Themicrochiropterans lack the A6c and A4 within the locus coeruleuscomplex. These two specific nuclei are also absent in themonotremes, insectivores, artiodactyls, rodents, afrotheriansand carnivores, but are present in the rabbit, megachiropteransand primates (Maseko et al., 2007; Dell et al., 2010). In the caudalrhombencephalon nuclei, only C3 shows any variation acrossmammalian species. This nucleus has only been seen in therodents (Skagerberg et al., 1988; Bhagwandin et al., 2008;Limacher et al., 2008), while it is absent in all other mammalianspecies.


The serotonergic system is homogenous, in terms of nuclearorganization, across all Eutherian mammals that have beenpreviously studied, including microchiropterans. No Eutherianmammals have been observed to possess serotonergic neuronswithin the periventricular organ, which have only been found inthe monotremes (Manger et al., 2002c). In the rostral serotonergiccluster, all 9 nuclei are present in microchiropterans and otherEutherian mammals (Da Silva et al., 2006; Limacher et al., 2008;Moon et al., 2007; Dwarika et al., 2008; Gravett et al., 2009; Pieterset al., 2010; Bux et al., 2010; Dell et al., 2010). The monotremeshave a similar nuclear organization, although the DRc (dorsalraphe, caudal, or B6) nucleus is not present in these mammals(Manger et al., 2002c). A similar distribution is seen for the caudalserotonergic cluster, with microchiropterans and other Eutherianmammals having all 6 nuclei in common (Da Silva et al., 2006;Badlangana et al., 2007; Moon et al., 2007; Dwarika et al., 2008;Bhagwandin et al., 2008; Limacher et al., 2008; Gravett et al., 2009;Pieters et al., 2010). The monotremes are again similar, however,the CVL (caudal ventrolateral serotonergic group) nucleus is notpresent (Manger et al., 2002c). CVL is also absent in the opossum(Crutcher and Humbertson, 1978).

4.4. Bat phylogeny

Although there are many similarities in the cholinergic,catecholaminergic and serotonergic systems between microchir-opterans and megachiropterans, it is important to note that thesesimilarities are those that are found to be common to mostmammals studied (see the tables provided in Dell et al., 2010 for afull summary). The differences between the microchiropterans andmegachiropterans are far more revealing, as, using an unbiasedphylogenetic analysis (see Dell et al., 2010), megachiropteransalign more closely with primates than any other group, whilemicrochiropterans align more readily with the insectivores. It isalso necessary to recall that the neural systems investigated in thisstudy are, for the most part, unrelated to flight, olfaction,echolocation or vision and any differences can therefore beconsidered related to the phylogenetic history of the twochiropteran suborders and not related to adaptation associatedwith chiropteran specialisations. The placement of the megachir-opterans as a sister group to the primates has become standard inthe examination of the diphyletic origin of bats; however, ourproposal that the microchiropterans form a sister group to theinsectivores is a novel concept, with only Crosby and Woodburne(1943) briefly touching on this possibility from a neural perspec-tive. Although further study is required, as the insectivora is a largeand heterogeneous group (Symonds, 2005), it would seem that themicrochiropteran – insectivoran link is one of particular interest in



the debate surrounding chiropteran phylogenetic history (Siemerset al., 2009).

In addition to the order level phylogeny examined here, theresults of the current study are revealing in terms of internalphylogeny of the microchiroptera. It is clear that two species of themicrochiropterans studied here share more in common that theother three species, these two species being C. cor and C. afra. Incommon, and to the exclusion of the other three microchiropter-ans studied, these two share the presence of the parabigeminalnucleus (PBg), the dorsal caudal nucleus of the ventral tegmentalarea (A10dc), and poor expression of the ventral division of theanterior hypothalamic group (A15v). At first glance, this mightappear to support the ‘‘paraphyly of microbats’’, a new DNA-basedphylogeny in which three families of microchiropterans areunited with the megachiroptera in the Yinpterochiroptera, to theexclusion of the remaining families of microchiropterans that arelumped together into the Yangochiroptera (Teeling, 2009). Anysimilarities, however, are superficial and slight. To begin with, thebrain data provide no support whatever for the most radical plankof ‘‘microbat paraphyly’’, which is the inclusion of megachir-opterans in Yinpterochiroptera. According to this DNA-basedhypothesis (from portions of 17 nuclear genes, Teeling et al.,2005), the three rhinolophoids whose brains we have studiedshould share neural features with the three megachiropteranspecies we studied (Maseko et al., 2007; Dell et al., 2010). Thereare no features of this kind, so there is no support from the braindata for this aspect of ‘‘microbat paraphyly’’. Secondly, our data donot conform to the composition of Yinpterochiroptera. While wehave two outlying microbats based on the brain nuclei, themegadermatid Cardioderma and the emballonurid Coleura, onlythe megadermatid Cardioderma belongs to the Yinpterochiropteraas constituted, while the emballonurid Coleura belongs toYangochiroptera. Moreover, two rhinolophoids that we studied,Hipposideros and Triaenops, should belong to Yinpterochiropteraas constituted, but instead show no brain specialisations thatwould set them apart from Yangochiropterans examined, such asthe molossid, Chaerophon, and the vespertilionid relative, Mini-

opterus (Maseko and Manger, 2007). Our brain data appears tomore in agreement with earlier morphological phylogeneticstudies of microchiropteran relationships (Smith, 1976; VanValen, 1979; Pierson, 1986); thus there appears to be a distinctDNA vs morphology difference regarding the phylogeny of thechiropterans.

Acknowledgments

This work was supported by funding from the South AfricanNational Research Foundation (PRM and JDP, South AfricanBiosystematics Initiative, KFD2008052300005). The authors wishto extend their gratitude to the members of the National Museumsof Kenya, especially Mr. Bernard ‘Risky’ Agwanda, without whomthis work would not have been possible.

Ethical statement: The microchiropterans used in the presentstudy were caught from wild populations in Kenya underpermission and supervision from the appropriate wildlife direc-torates. All animals were treated and used according to theguidelines of the University of the Witwatersrand Animal EthicsCommittee, which parallel those of the NIH for the care and use ofanimals in scientific experimentation.

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