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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Journal of Chemical Neuroanatomy 40 (2010) 210–222 Contents lists available at ScienceDirect Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu Nuclear organization of cholinergic, putative catecholaminergic and serotonergic 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 b School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa Queensland Brain Institute, University of Queensland 4072, Australia A R T I C L E I N F O A B S T R A C T Article history: Received 26 March 2010 Received in revised form 28 May 2010 Accepted 28 May 2010 Available online 4 June 2010 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 microchiropterans 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. ß 2010 Elsevier B.V. All rights reserved. Keywords: Microbat Chiroptera Neuromodulatory systems Diphyly Evolution Mammalia 1. Introduction The proposed monophyletic order Chiroptera has been divided into two suborders: megachiroptera and microchiroptera; however, Linnaeus originally grouped the megachiropterans with primates. This classification was largely ignored until the finding that primates and megachiropterans share several advanced visual pathway characteristics, in particular the retinotectal pathway, that are not shared by other mammals (Pettigrew, 1986; Pettigrew et al., 1989, 2008). The ‘‘flying primate’’ hypothesis proposes that the megachiropterans, with the dermopterans, form a sister group to the primates, and is based on several derived neural features that are absent in microchiropterans and other mammals (Pettigrew et al., 1989, 2008; Manger et al., 2001; Maseko and Manger, 2007; Maseko 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). 0891-0618/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2010.05.007 Author's personal copy J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 et al., 2007). The logical conclusion of this hypothesis is that the Chiropteran order is actually diphyletic and flight evolved twice in mammals. A summary of the evidence on each side of the debate about whether bats are monophyletic or diphyletic is provided in the accompanying paper (Dell et al., 2010). Most studies that support the diphyletic origin of bats have concentrated on specific neuroanatomical structures in megachiropterans, with little work to date having been done in microchiropterans. Maseko and Manger (2007) and Maseko et al. (2007) undertook investigations into the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems in the microchiropteran Miniopterus schreibersii, and the megachiropteran Rousettus aegyptiacus, which defined several further differences between the mega- and microchiropterans lending further support for the diphyletic origin of the Chiroptera. In contrast, studies favouring Chiropteran monophyly are usually based 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 nuclear organization of the cholinergic, catecholaminergic and serotonergic systems of a range of mammalian species (see also Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008; Gravett et al., 2009; Pieters et al., 2010; Bux et al., 2010), that all species within an order will exhibit the same complement of homologous nuclei of these systems. This proposal infers that if mega- and microchiropterans belonged to the same mammalian order, they should 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 origin of the Chiropterans, it should be noted that the microchiropterans in particular are one of the most species-rich suborders of mammals, consisting of over 800 species (Nowack, 1999). Thus, before firm conclusions regarding differences between the two suborders can be made, further species should be investigated. The current study describes the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems in the brains of five previously unstudied microchiropteran species from a range of phylogenetically distant families (distant within the microchiropteran suborder). A speculative hypothesis proposed that the shared midbrain binocular circuitry of primates and megachiropterans represented two independent evolutionary events that were each driven by the selective forces of the ‘‘small branch niche’’ (Martin, 1986). It is important to note that the vast majority of the nuclei of the systems under investigation here do not play any direct role in the neural processes related to flight, vision or echolocation and as such the findings cannot be ignored on the basis of sensory or motor specialisations of the Chiroptera, an argument that has been previously levelled at the studies of Chiropteran neuroanatomy that 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 211 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 stereomicroscope 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 Dahlström and Fuxe (1964), Hökfelt et al. (1984), Törk (1990), Woolf (1991), Smeets and González (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, catecholaminergic and serotonergic neural systems generally followed that found by Maseko and Manger (2007) in their study of M. schreibersii, although there were some notable differences between species, specifically in the cholinergic and catecholaminergic systems, and these are explicitly described. The following description applies generally to all the microchiropteran species studied, except where specifically noted. 3.1. Cholinergic nuclei The cholinergic system is generally subdivided into the cortical cholinergic interneurons, the striatal region, basal forebrain, diencephalon, pontomesencephalon and the cranial nerve nuclei groups (Woolf, 1991). In the five species examined in the current study, we identified cholineacetyltransferase immunoreactive Author's personal copy 212 J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 (ChAT+) neurons in all these subdivisions, excluding the cortical cholinergic interneurons, which have a variable occurrence across mammalian species (e.g. Bhagwandin et al., 2006). Additionally, there was no evidence of any ChAT+ neurons in the medullary tegmental field. 3.1.1. Cholinergic striatal interneurons ChAT+ neurons were found in the Islands of Calleja, the olfactory tubercle, nucleus accumbens, the caudate/putamen complex and in the globus pallidus. The distribution, density, topography and nuclear organization of ChAT+ neurons in these areas was found to be similar across all five microchiropteran species, as well as following the distribution and morphology as described by Maseko and Manger (2007) in their study of M. schreibersii. tegmental (LDT) nuclei in all five species studied (Fig. 1). An unusual dense aggregation of ChAT+ neurons in the PPT of C. pumilus was observed in the postero-lateral portion of this nucleus in a position lateral to the superior cerebellar peduncle (Fig. 2); however, the remaining species evinced ‘‘typical’’ microchiropteran PPT and LDT morphology (Maseko and Manger, 2007). In contrast to the findings of Maseko and Manger (2007), evidence of the parabigeminal nucleus was found in C. cor and C. afra (Fig. 3). ChAT+ neurons in the parabigeminal nucleus were most readily observed in C. cor, with only weak staining occurring in this nucleus in C. afra. In both species, the location of the parabigeminal [(Fig._2)TD$IG] 3.1.2. Cholinergic basal forebrain nuclei The basal forebrain nuclei are composed of the medial septal nucleus, the Diagonal band of Broca and the nucleus basalis (Woolf, 1991; Maseko et al., 2007). ChAT+ neurons were observed in all of these nuclei across all five microchiropteran species, as found by Maseko and Manger (2007) in M. schreibersii. No notable interspecies differences were observed. 3.1.3. Diencephalic cholinergic nuclei ChAT+ neurons were found in the medial habenular nucleus, as well as in the three hypothalamic nuclei (dorsal, lateral and ventral) in all five microchiropteran species. In T. persicus there was very strong ChAT immunoreactivity in the medial habenular nucleus, as well as in the lateral hypothalamic cluster, while C. afra and C. cor showed only a weak reactivity in the lateral hypothalamic region. The ventral hypothalamic nucleus was most well expressed in C. cor. In general, our findings were similar across all five microchiropteran species and in terms of nuclear organization are identical to that found in M. schreibersii (Maseko and Manger, 2007). No evidence of ChAT+ neurons could be found in the anterior nuclei of the dorsal thalamus as seen in the rock hyrax (Gravett et al., 2009). 3.1.4. Midbrain/pontine cholinergic nuclei As with the observations made in M. schreibersii, we observed ChAT+ neurons in the pedunculopontine (PPT) and the laterodorsal [(Fig._1)TD$IG] 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. 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. Author's personal copy [(Fig._3)TD$IG] J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 213 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. nucleus was typical of that seen in other mammals. No evidence of ChAT+ neurons within the parabigeminal nucleus could be found in the other three species investigated, which is congruent with the findings for M. schreibersii (Maseko and Manger, 2007). No evidence of ChAT+ neurons could be found in the pedunculopontine parvocellular (PPTpc), the laterodorsal tegmental parvocellular (LDTpc) or the superior and inferior colliculus interneurons as occasionally observed in other species (Gravett et al., 2009; Pieters et al., 2010). The lack of ChAT+ neurons in these regions follows the findings of Maseko and Manger (2007) for M. schreibersii. observed in other mammalian species (e.g. Maseko et al., 2007; Bhagwandin et al., 2008; Gravett et al., 2009). All other typically ChAT immunoreactive cranial nerve nuclei were observed in all five species, in line with earlier findings for M. schreibersii (Maseko and Manger, 2007). ChAT+ expression was found to be particularly strong in the oculomotor neurons in C. cor, while in T. persicus the nucleus ambiguus was found to be particularly large. C. afra possessed a large ventral division of the facial nerve nucleus, as well as strong ChAT+ expression in pVII, pIX, the hypoglossal nucleus and the ventral horn of the spinal cord. 3.1.5. Cranial nerve nuclei As observed by Maseko and Manger (2007) in M. schreibersii, no evidence of ChAT+ neurons were observed in the cochlear nucleus or the medullary tegmental field in any of the five microchiropteran species investigated; however, in the current study evidence of ChAT+ neurons was found for the Edinger–Westphal nucleus (Fig. 4), the preganglionic superior salivatory nucleus (pVII) and the preganglionic inferior salivatory nucleus (pIX) (Fig. 5). These nuclei were present in all five microchiropteran species investigated and showed similar neuronal morphology, distributions and numbers as 3.2. Catecholaminergic nuclei The catecholaminergic nuclei were found throughout the brains of all five microchiropteran species investigated and were revealed using tyrosine hydroxylase (TH) immunoreactivity. Generally our findings mirrored those of Maseko and Manger (2007), although differences in TH+ immunoreactivity were noted in the A15v (anterior hypothalamic ventral group), A10d (dorsal ventral tegmental area), A10dc (dorsal caudal ventral tegmental area) and A9v (substantia nigra ventral) in specific species. Author's personal copy [(Fig._4)TD$IG] 214 J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 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$IG] Fig. 5. Photomicrographs of the ChAT immunoreactive neurons forming the preganglionic neurons of the superior salivatory (pVII) and inferior salivatory (pIX) nuclei in (A) Cardioderma cor, (B) Triaenops persicus and (C) Chaerophon pumilus in relation to the ChAT immunoreactive neurons of the ventral (VIIv) and dorsal (VIId) subdivisions of the facial nerve nucleus and the abducens nucleus (VI). Scale bar in C = 500 mm and applies to all. Author's personal copy J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 3.2.1. A16 – olfactory bulb These neurons, found in the stratum granulosum of the olfactory bulb, showed TH+ immunoreactivity across all five microchiropteran species investigated, which concurs with the findings for M. schreibersii (Maseko and Manger, 2007). 3.2.2. Hypothalamic nuclei (A11–A15) TH+ neurons were identified in the A11, A12, A13 and A14 nuclei (Fig. 6) in all species studied, in agreement with previous findings in M. schreibersii (Maseko and Manger, 2007). The A14 (rostral periventricular cell group) and A11 (caudal diencephalic group) nuclei were both strongly expressed in terms of neuronal number in H. commersoni (Fig. 6D). No evidence was found for the A15d (anterior hypothalamic group, dorsal division) nucleus in any of the five species studied, as seen in M. schreibersii; although this nucleus is present in many other mammals (e.g. Maseko et al., 2007; Bhagwandin et al., 2008; Gravett et al., 2009). TH+ neurons were found in the A15v (anterior hypothalamic group, ventral [(Fig._6)TD$IG] 215 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 contrast to 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+ immunoreactivity in many of the midbrain catecholaminergic nuclei typically described for mammals (Fig. 7; Smeets and González, 2000), these include the A8, A9m, A9pc, A9l, A10, A10c, and A10d nuclei, all of which were found in the locations typically seen across all mammals. 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 the periaqueductal grey matter near the ventrolateral edge of the cerebral aqueduct (Fig. 7); however, no evidence of this nucleus could be found in the other microchiropteran species, in line with the findings of Maseko and Manger (2007). Additionally, partial 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. Author's personal copy [(Fig._7)TD$IG] 216 J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 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. evidence for A9v could be found in all five microchiropteran species under investigation, which differs from the findings for M. schreibersii (Maseko and Manger, 2007). It should be noted though that in all species studied herein, that the number of neurons that could be assigned to the A9v nucleus was small, varied between individuals 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 (locus coeruleus diffuse), A7sc (subcoeruleus compact) and A7d (subcoeruleus diffuse) nuclei in all five microchiropteran species investigated in this study, as well as in M. schreibersii (Maseko and Manger, 2007). The location, densities, and morphology of these neurons were similar across all species. No evidence for an A6c or A4 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 in terms of neuronal numbers in C. pumilus. For the remaining nuclei a strong homogeneity in the expression of these nuclei across species was observed. No evidence of the C3 nucleus, a nucleus that has only been found in rodents (e.g. Bhagwandin et al., 2008), was observed. 3.3. Serotonergic neurons In mammals, the serotonergic neurons are typically subdivided into rostral and caudal nuclear clusters, with both being located entirely the brainstem (Törk, 1990; Jacobs and Azmitia, 1992). In mammals in general, the rostral cluster consists of the CLi (caudal linear), B9 (supralemniscal), MnR (median raphe) and the dorsal raphe nuclei, while the caudal cluster is composed of the RMg (raphe magnus), RPa (raphe pallidus), RVL (rostral ventrolateral cell column), CVL (caudal ventrolateral cell column) and ROb (raphe obscurus) nuclei. Author's personal copy [(Fig._8)TD$IG] J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 217 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$IG] 3.3.1. Rostral cluster All serotonergic nuclei typically assigned to the rostral cluster showed specific serotonergic immunoreactivity across all five microchiropteran species investigated. This is consistent with the previous findings for M. schreibersii (Maseko and Manger, 2007) and for all Eutherian mammals (Maseko et al., 2007). The CLi nucleus was strongly expressed in T. persicus, with the B9 nucleus being strongly expressed in C. afra, and C. cor (Fig. 9) and C. pumilus. The dorsal raphe cluster could be divided into dorsal (DRd), ventral (DRv), interfascicular (DRif), lateral (DRl), caudal (DRc) and peripheral (DRp) nuclei, all of which were observed in all five species, all evincing a similar appearance (Fig. 10). As with all prior observations, only a few neurons of the peripheral nucleus of the dorsal raphe were located outside the periaqueductal grey matter. 3.3.2. Caudal cluster All five microchiropteran species studied had 5-HT+ neurons in the RMg, RPa, RVL, CVL and ROb nuclei, as was observed in M. schreibersii (Maseko and Manger, 2007) and all other Eutherian mammals studied to date. It was noted that RMg possessed few 5HT+ neurons in C. afra, and CVL was weakly expressed in terms of Fig. 9. A photomicrograph showing the most rostral serotonergic nuclei in Cardioderma cor, the caudal linear nucleus (CLi) and the supralemniscal serotonergic neurons (B9) at the level of the interpeduncular nucleus (IP). Scale bar = 100 mm. Author's personal copy [(Fig._10)TD$IG] 218 J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 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. neuronal numbers in T. persicus. In all other respects these nuclei were similar to previous observations in other mammals. 4. Discussion Within the cholinergic, catecholaminergic and serotonergic systems many similarities in nuclear organization were found in the five microchiropterans investigated in this study and that reported for M. schreibersii (Maseko and Manger, 2007); however, some notable differences in nuclear organization were found in the cholinergic and catecholaminergic systems. Several differences were observed when comparing the nuclear organization of these systems with observations made on the megachiropterans previously studied (Maseko et al., 2007; Dell et al., 2010), the most notable of which occurred, again, in the cholinergic and catecholaminergic systems. When making a broader comparison across mammals, the general organization of these systems in the microchiropterans were similar to that seen in other mammals, but notable 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 show the greatest number of similarities in terms of nuclear organization of these systems with that observed in microchiropterans (see also Dell et al., 2010), suggesting a close phylogenetic link between the microchiropterans and the insectivores. 4.1. Comparisons amongst microchiropterans 4.1.1. Cholinergic nuclei The nuclear organization of the cholinergic system was generally found to be similar in all species; however there were notable 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 microchiropterans studied herein or in M. schreibersii (Maseko and Manger, 2007). While a strongly expressed parabigeminal nucleus was observed in C. cor, in C. afra the immunoreactivity of these neurons was weak. ChAT+ immunoreactivity also occurred in the Author's personal copy J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 Edinger–Westphal, pVII and pIX cholinergic nuclei across all five microchiropteran species studied, whereas no evidence for ChAT immunoreactivity of the neurons within these nuclei was found in M. schreibersii (Maseko and Manger, 2007). These latter differences may be attributed to the antibody used in the current study – Maseko and Manger (2007) utilised the ChAT antibody AB143 (Chemicon), while the current study employed AB144P (Chemicon). A study by Bhagwandin et al. (2006) looked at the revelation of cortical cholinergic neurons in a range of rodent species using three different antibodies (AB143, AB144P and vChAT). It was shown that AB144P consistently revealed the most cholinergic neurons, while the revelation was limited using AB143 and vChAT. It is possible that these antibodies bind to different regions of the cholineacetyltransferase molecule and thus the molecule involved in producing acetylcholine may differ in structure in different parts of the central nervous system and differ within specific phylogenies (Bhagwandin et al., 2006). The cholinergic neurons revealed in this study, but not in the study by Maseko and Manger (2007), are mostly neurons that appear to be involved with the autonomic nervous system (except for those seen in the parabigeminal nucleus); thus, there may be, at least in the microchiropterans and rodents, a differing morphology of the cholineacetyltransferase molecule in the parts of the brain associated with the ‘‘classical’’ cholinergic system and that part of the cholinergic system associated with the autonomic nervous system. 4.1.2. Catecholaminergic nuclei The most notable difference in the catecholaminergic nuclei was the presence of the A15v nucleus in all five microchiropteran species in this study, as well as the possible existence of the A9v nucleus; 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 and Manger, 2007). Some evidence was also found for A10dc but only in C. cor and C. afra, with no other species expressing this nucleus, yet all were observed to have the A10d nucleus which was reported as absent in M. schreibersii. It is important to note that C. cor and C. afra are also the only two microchiropteran species that showed the presence of the parabigeminal nucleus, as well as a very weak expression of A15v. These findings group these two species, from the families Megadermatidae and Emballonuridae, together, but slightly separate them from the other 17 or so microchiropteran families, which is not in agreement with the recently published molecular, paraphyletic phylogenies of the microchiropterans, where megadermatids are aligned with rhinolophoids and split from all other microbats (Van Den Bussche and Hoofer, 2004; Jones et al., 2005; Teeling et al., 2005; Gu et al., 2008). On the other hand, morphological studies have consistently placed these two Old World microchiropteran families together (e.g. Smith, 1976; Van Valen, 1979; Pierson, 1986). All other catecholaminergic nuclei were found to be similar across all six microchiropteran species. 4.1.3. Serotonergic nuclei The nuclear organization of the serotonergic system is homogenous across all six microchiropteran species studied to date, i.e. the five species in this study together with M. schreibersii (Maseko and Manger, 2007). There were a few differences in expression patterns of individual nuclei observed between the species studied. C. afra had a notably small RMg nucleus, while CVL was particularly small in T. persicus. As specific functions have not been ascribed to these nuclei beyond the modulation of certain portions of the neurons within the grey matter of the spinal cord (Törk, 1990) it is difficult to speculate if these differences have any particular functional implications. 219 4.2. Comparisons with megachiropterans 4.2.1. Cholinergic nuclei In the cholinergic system, the five microchiropterans examined in this study and the three megachiropterans that have been studied (Rousettus aegyptiacus, Eidolon helvum, Epomophorus wahlbergi) have the expression of almost all nuclei in common, including the Edinger–Westphal, pVII and pIX nuclei which were reported as absent in M. schreibersii (Maseko et al., 2007; Dell et al., 2010). The only difference in organization of the cholinergic nuclei is found in the pontine region, this being the variable presence of ChAT immunoreactivity in the neurons of the parabigeminal nucleus. ChAT immunoreactivity of neurons within this nucleus occurs in all megachiropterans studied (Maseko et al., 2007; Dell et al., 2010), as well as in two of the microchiropterans C. cor and C. afra; however, ChAT immunoreactivity the parabigeminal nucleus was absent in the four other microchiropteran species that have been examined, and in C. afra the ChAT immunoreactivity was weak. The variability in the occurrence of ChAT immunoreactivity of the neurons of the parabigeminal nucleus is difficult to explain at present, and further examination of the presence or absence of this immunoreactivity in other microchiropteran species may elucidate this issue, determining whether this is a phylogenetic or functional variation. It may also be possible that the parabigeminal nucleus is actually absent in the species where we have not detected any ChAT immunoreactivity. Connectional studies from the superior colliculi of those species without ChAT immunoreactivity would determine the presence or absence of this nucleus. 4.2.2. Catecholaminergic nuclei The major differences noted between megachiropterans previously studied (Maseko et al., 2007; Dell et al., 2010) and the five microchiropteran species examined in this study occur in the presence or absence of nuclei assigned to the catecholaminergic system. The A15d, A6c and A4 nuclei were not present in the microchiropterans, including M. schreibersii (Maseko and Manger, 2007); however these nuclei were found in the three megachiropteran species that have been studied (Maseko et al., 2007; Dell et al., 2010). The A10dc nucleus is possibly present in C. cor and C. afra, but not in the other three microchiropteran species investigated, nor M. schreibersii (Maseko and Manger, 2007). This nucleus is however found in the three megachiropteran species that have been studied (Maseko et al., 2007; Dell et al., 2010). The A9v nucleus was very weakly expressed in all five microchiropteran species, while it is strongly expressed in all megachiropteran species (Maseko et al., 2007; Dell et al., 2010). The A15v nucleus is only weakly expressed in C. cor and C. afra, while it is strongly expressed in the other three microchiropterans studied and in the megachiropterans. All remaining catecholaminergic nuclei were consistent across microchiropterans and megachiropterans. 4.2.3. Serotonergic nuclei There were no differences between microchiropterans and megachiropterans in terms of the nuclear organization of the serotonergic system (Maseko and Manger, 2007; Maseko et al., 2007; Dell et al., 2010). 4.3. Comparisons with other mammals 4.3.1. Cholinergic nuclei The presence of all cholinergic striatal and basal forebrain nuclei is 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). Author's personal copy 220 J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 Microchiropterans and all other Eutherian mammals have cholinergic neurons in the medial habenular nucleus and three nuclei within 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 the dorsal, ventral and lateral hypothalamic nuclei (Manger et al., 2002a). There are a number of differences and similarities within the pontine cholinergic nuclei across mammalian species. The parabigeminal nucleus was found in two of the microchiropteran species in this study: C. cor and C. afra. This nucleus has been found in the giraffe, the rodents, the afrotherians (in this study represented by the rock hyrax and elephant shrew), the carnivores, the megachiropterans 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 and the platypus (Manger et al., 2002a; Karasawa et al., 2003). The superior collicular interneurons have, to date, only been seen in the rodents, elephant shrew and tree shrew (Pieters et al., 2010), while microchiropterans and other mammals do not express these interneurons (Maseko and Manger, 2007; Gravett et al., 2009; Limacher et al., 2008; Maseko et al., 2007). The inferior collicular cholinergic interneurons do not occur in the microchiropterans or other 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 nerve nuclei generally follow a similar pattern throughout all mammals with a few notable exceptions (Maseko and Manger, 2007; Gravett et al., 2009; Limacher et al., 2008; Maseko et al., 2007; Bhagwandin et al., 2008). In this study, the Edinger–Westphal, pVII and pIX nuclei were present in all five microchiropteran species investigated. These nuclei are not present in the echidna, the platypus and the laboratory shrew (Manger et al., 2002a; Karasawa et al., 2003); however all other mammals studied to date have these nuclei in common (Maseko and Manger, 2007; 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). The medullary tegmental field is absent in microchiropterans, the elephant shrew, the rock hyrax, the megachiropterans and primates (Maseko and Manger, 2007; Maseko et al., 2007; Gravett et al., 2009; Pieters et al., 2008), while it has been seen in the monotremes, rodents and carnivores (Manger et al., 2002a; Maseko et al., 2007; Bhagwandin et al., 2008). 4.3.2. Catecholaminergic nuclei In general the hypothalamic catecholaminergic nuclei are similar across mammals (Skagerberg et al., 1988; Tillet and Thibault, 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; Bux et al., 2010). The A15v nucleus shows a variable presence in the microchiropterans studied and has also been reported to be absent in the rabbit and the tree shrew (Maseko et al., 2007; Dell et al., 2010). The A15d nucleus is absent in all microchiropterans, as well as the insectivores, artiodactyls, the elephant shrew and the tree shrew (Pieters et al., 2010; Bux et al., 2010; Dell et al., 2010). The midbrain nuclei are also, for the most part, homogenous across mammalian species (Skagerberg et al., 1988; Tillet and Thibault, 1989; Leshin et al., 1995; Tillet et al., 2000; Manger et 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, in that the A10dc and A9v nuclei have a variable occurrence. The A10dc nucleus was absent in C. pumilus, H. commersoni, T. persicus and M. schreibersii, while C. cor and C. afra possibly possess this nucleus but it is very weakly expressed in terms of neuronal number. In the microchiropterans A9v may be present but again, only weakly expressed in terms of neuronal number. The A9v nucleus shows a similar morphology in the hedgehog, and is absent in the rabbit and carnivores (Dell et al., 2010). The microchiropterans lack the A6c and A4 within the locus coeruleus complex. These two specific nuclei are also absent in the monotremes, insectivores, artiodactyls, rodents, afrotherians and carnivores, but are present in the rabbit, megachiropterans and primates (Maseko et al., 2007; Dell et al., 2010). In the caudal rhombencephalon nuclei, only C3 shows any variation across mammalian species. This nucleus has only been seen in the rodents (Skagerberg et al., 1988; Bhagwandin et al., 2008; Limacher et al., 2008), while it is absent in all other mammalian species. 4.3.3. Serotonergic nuclei The serotonergic system is homogenous, in terms of nuclear organization, across all Eutherian mammals that have been previously studied, including microchiropterans. No Eutherian mammals have been observed to possess serotonergic neurons within the periventricular organ, which have only been found in the monotremes (Manger et al., 2002c). In the rostral serotonergic cluster, all 9 nuclei are present in microchiropterans and other Eutherian mammals (Da Silva et al., 2006; Limacher et al., 2008; Moon et al., 2007; Dwarika et al., 2008; Gravett et al., 2009; Pieters et al., 2010; Bux et al., 2010; Dell et al., 2010). The monotremes have a similar nuclear organization, although the DRc (dorsal raphe, caudal, or B6) nucleus is not present in these mammals (Manger et al., 2002c). A similar distribution is seen for the caudal serotonergic cluster, with microchiropterans and other Eutherian mammals 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 not present (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 microchiropterans and megachiropterans, it is important to note that these similarities are those that are found to be common to most mammals studied (see the tables provided in Dell et al., 2010 for a full summary). The differences between the microchiropterans and megachiropterans are far more revealing, as, using an unbiased phylogenetic analysis (see Dell et al., 2010), megachiropterans align more closely with primates than any other group, while microchiropterans align more readily with the insectivores. It is also necessary to recall that the neural systems investigated in this study are, for the most part, unrelated to flight, olfaction, echolocation or vision and any differences can therefore be considered related to the phylogenetic history of the two chiropteran suborders and not related to adaptation associated with chiropteran specialisations. The placement of the megachiropterans as a sister group to the primates has become standard in the examination of the diphyletic origin of bats; however, our proposal that the microchiropterans form a sister group to the insectivores is a novel concept, with only Crosby and Woodburne (1943) briefly touching on this possibility from a neural perspective. Although further study is required, as the insectivora is a large and heterogeneous group (Symonds, 2005), it would seem that the microchiropteran – insectivoran link is one of particular interest in Author's personal copy J.-L. Kruger et al. / Journal of Chemical Neuroanatomy 40 (2010) 210–222 the debate surrounding chiropteran phylogenetic history (Siemers et al., 2009). In addition to the order level phylogeny examined here, the results of the current study are revealing in terms of internal phylogeny of the microchiroptera. It is clear that two species of the microchiropterans studied here share more in common that the other three species, these two species being C. cor and C. afra. In common, and to the exclusion of the other three microchiropterans studied, these two share the presence of the parabigeminal nucleus (PBg), the dorsal caudal nucleus of the ventral tegmental area (A10dc), and poor expression of the ventral division of the anterior hypothalamic group (A15v). At first glance, this might appear to support the ‘‘paraphyly of microbats’’, a new DNA-based phylogeny in which three families of microchiropterans are united with the megachiroptera in the Yinpterochiroptera, to the exclusion of the remaining families of microchiropterans that are lumped together into the Yangochiroptera (Teeling, 2009). Any similarities, however, are superficial and slight. To begin with, the brain data provide no support whatever for the most radical plank of ‘‘microbat paraphyly’’, which is the inclusion of megachiropterans in Yinpterochiroptera. According to this DNA-based hypothesis (from portions of 17 nuclear genes, Teeling et al., 2005), the three rhinolophoids whose brains we have studied should share neural features with the three megachiropteran species we studied (Maseko et al., 2007; Dell et al., 2010). There are no features of this kind, so there is no support from the brain data for this aspect of ‘‘microbat paraphyly’’. Secondly, our data do not conform to the composition of Yinpterochiroptera. While we have two outlying microbats based on the brain nuclei, the megadermatid Cardioderma and the emballonurid Coleura, only the megadermatid Cardioderma belongs to the Yinpterochiroptera as constituted, while the emballonurid Coleura belongs to Yangochiroptera. Moreover, two rhinolophoids that we studied, Hipposideros and Triaenops, should belong to Yinpterochiroptera as constituted, but instead show no brain specialisations that would set them apart from Yangochiropterans examined, such as the molossid, Chaerophon, and the vespertilionid relative, Miniopterus (Maseko and Manger, 2007). Our brain data appears to more in agreement with earlier morphological phylogenetic studies of microchiropteran relationships (Smith, 1976; Van Valen, 1979; Pierson, 1986); thus there appears to be a distinct DNA vs morphology difference regarding the phylogeny of the chiropterans. Acknowledgments This work was supported by funding from the South African National Research Foundation (PRM and JDP, South African Biosystematics Initiative, KFD2008052300005). The authors wish to extend their gratitude to the members of the National Museums of Kenya, especially Mr. Bernard ‘Risky’ Agwanda, without whom this work would not have been possible. 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