Nuclear organization of cholinergic, putative catecholaminergic and serotonergic nuclei in the brain of the eastern rock elephant shrew, Elephantulus myurus

Journal of Chemical Neuroanatomy 39 (2010) 175–188

Contents lists available at ScienceDirect

Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu

Nuclear organization of cholinergic, putative catecholaminergic and serotonergic nuclei in the brain of the eastern rock elephant shrew, Elephantulus myurus Raymond P. Pieters a, Nadine Gravett a, Kjell Fuxe 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 Department of Neuroscience, Karolinska Institutet, Retzius va¨g 8, S-171 77 Stockholm, Sweden

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 June 2009 Received in revised form 23 December 2009 Accepted 1 January 2010 Available online 11 January 2010

The organization of the nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems of the brain of the elephant shrew (Elephantulus myurus) were determined following immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin, respectively. This was done in order to determine if differences in the nuclear organization of these systems in comparison to other mammals were evident and how any noted differences may relate to specialized behaviours of the elephant shrew. The elephant shrew belongs to the order Macroscelidea, and forms part of the Afrotherian mammalian cohort. In general, the organization of the nuclei of these systems resembled that described in other mammalian species. The cholinergic system showed many features in common with that seen in the rock hyrax, rodents and primates; however, specific differences include: (1) cholinergic neurons were observed in the superior and inferior colliculi, as well as the cochlear nuclei; (2) cholinergic neurons were not observed in the anterior nuclei of the dorsal thalamus as seen in the rock hyrax; and (3) cholinergic parvocellular nerve cells forming subdivisions of the laterodorsal and pedunculopontine tegmental nuclei were not observed at the midbrain/pons interface as seen in the rock hyrax. The organization of the putative catecholaminergic system was very similar to that seen in the rock hyrax and rodents except for the lack of the rodent specific C3 nucleus, the dorsal division of the anterior hypothalamic group (A15d) and the compact division of the locus coeruleus (A6c). The nuclear organization of the serotonergic system was identical to that seen in all eutherian mammals studied to date. The additional cholinergic neurons found in the cochlear nucleus and colliculi may relate to a specific acoustic signalling system observed in elephant shrews expressed when the animals are under stress or detect a predator. These neurons may then function to increase attention to this type of acoustic signal termed foot drumming. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Evolution Acetylcholine Dopamine Noradrenalin Adrenalin Serotonin Afrotheria

Abbreviations: III, oculomotor nucleus; IV, trochlear nucleus; Vmot, motor division of trigeminal nucleus; VI, abducens nucleus; VIId, facial nerve nucleus, dorsal division; VIIn, facial nerve; VIIv, facial nerve nucleus, ventral division; X, dorsal motor vagus nucleus; XII, hypoglossal nucleus; 3V, third ventricle; 4V, fourth ventricle; A1, caudal ventrolateral medullary tegmental nucleus; A2, caudal dorsomedial medullary nucleus; A4, dorsal medial division of locus coeruleus; A5, fifth arcuate nucleus; 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?, possible 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; A15v, anterior hypothalamic group, ventral division; A16, catecholaminergic neurons of the olfactory bulb; ac, anterior commissure; Amyg, amygdala; AP, area postrema; B9, supralemniscal serotonergic nucleus; C, caudate nucleus; C1, rostral ventrolateral medullary tegmental group; C2, rostral dorsomedial medullary nucleus; ca, cerebral aqueduct; Cb, cerebellum; cc, corpus callosum; CLi, caudal linear nucleus; CN, deep cerebellar nuclei; CO, cochlear nucleus; C/P, caudate and putamen nuclei; CP, cerebral peduncle; CVL, caudal ventrolateral serotonergic group; Diag.B, diagonal band of Broca; 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; DT, dorsal thalamus; EW, Edinger–Westphal nucleus; f, fornix; GC, periaqueductal grey matter; GLD, dorsal lateral geniculate nucleus; GP, globus pallidus; Hbl, lateral habenular nucleus; Hbm, medial habenular nucleus; Hip, hippocampus; Hyp, hypothalamus; Hyp.d, dorsal hypothalamic cholinergic nucleus; Hyp.l, lateral hypothalamic cholinergic nucleus; Hyp.v, ventral hypothalamic cholinergic nucleus; IC, inferior colliculus; ic, internal capsule; io, inferior olivary nuclei; Is.Call/TOL., Islands of Calleja/olfactory tubercle; LDT, laterodorsal tegmental nucleus; LV, lateral ventricle; MnR, median raphe nucleus; mtf, cholinergic medullary tegmental field; N.Acc, nucleus accumbens; N.Amb, nucleus ambiguus; N.Bas, nucleus basalis; NEO, neocortex; OB, olfactory bulb; OC, optic chiasm; OLS, superior olivary nucleus; OT, optic tract; P, putamen; pVII, preganglionic motor neurons of the superior salivatory nucleus or facial nerve; pIX, preganglionic motor neurons of the inferior salivatory nucleus; PBg, parabigeminal nucleus; pc, posterior commissure; PIR, piriform cortex; PPT, pedunculopontine nucleus; py, pyramidal tract; R, thalamic reticular nucleus; Rmc, red nucleus, magnocellular division; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; RVL, rostral ventrolateral serotonergic group; SC, superior colliculus; Sep.M, medial septal nucleus; trl, lateral olfactory tract; vh, ventral horn of spinal cord; VPO, ventral pontine nucleus; ZI, zona incerta. * 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.2010.01.001

176

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

1. Introduction

2. Materials and methods

The Supercohort Afrotheria is an unusual mammalian group that includes a variety of very morphologically and ecologically diverse mammals, and is comprised of the elephant shrews, elephants, tenrecs, golden moles, aardvarks, hyraxes and sea cows (Malia et al., 2002; Skinner and Chimimba, 2005). The elephant shrews form the order Macroscelidea (Woodall and Mackie, 1987; Malia et al., 2002; Murata et al., 2003; Skinner and Chimimba, 2005), and are the smallest members, in terms of body size, of this Supercohort. To avoid confusion and due to the fact that the elephant shrew is not closely related to the true shrews, the name ‘‘sengi’’ has now been attributed to the elephant shrew (Skinner and Chimimba, 2005). It is of interest to reveal the organization of the cholinergic, catecholaminergic and serotonergic neural systems of the elephant shrews to determine whether they display a unique complement of these systems (Manger, 2005), have features found only in the Afrotheria (Gravett et al., 2009), or display a nuclear organization common to many mammals (Maseko et al., 2007). The Eastern rock elephant shrew, Elephantulus myurus, which is the subject of this study, has an elongated mobile snout that encloses the nostrils, large eyes and a fairly long tail. It has broad upright ears that are just as mobile as the snout and appear to reflect the elephant shrew’s mood and it can be differentiated from other elephant shrews by the colour of its coat (Skinner and Chimimba, 2005). E. myurus is found in the north-western regions of southern Africa, its habitat is in rocky areas with overhanging boulders, and it has an omnivorous diet (Skinner and Chimimba, 2005). The elephant shrew has an unusual acoustic signal process in which it taps the ground with its hind limbs producing what is known as foot drumming. These signals are used, in the wild, by the elephant shrew in the presence of a predator or to express stress (Skinner and Chimimba, 2005). The nuclei of the cholinergic system can be divided into five main groups, according to their location, connections and morphology (Woolf, 1991; Maseko et al., 2007). The cholinergic system is thought to be involved in many functions related to modulation of behaviour, attention, learning and memory, the sleep–wake cycle, and even in generating conscious experiences (Vanderwolf, 1987; Woolf, 1991; Reiner and Fibiger, 1995; Woolf and Hameroff, 2001). Catecholaminergic nuclei are found throughout the CNS of all vertebrates (Smeets and Gonza´lez, 2000), while serotonergic nuclei are found in the raphe nuclear regions within the brainstem (To¨rk, 1990; Bjarkam et al., 1997; Leger et al., 2001). The catecholaminergic and serotonergic neurons are associated with emotional behaviour, sleep–wake cycle, mood, cognition, temporal analysis and sequencing, and sensory-motor control amongst other functions (Carlsson et al., 1968; Jouvet, 1969, 1972; Fuxe et al., 1970, 2007a,b; Kianmaa and Fuxe, 1977; To¨rk, 1990; Chalmers and Pilowsky, 1991; Jacobs and Azmitia, 1992; Tillet and Kitahama, 1998; Previc, 1999; Pompeiano, 2001; Siegel, 2006). Brains of species within the Order Macroscelidea have not been previously studied using immunohistochemical techniques to reveal the aforementioned neural systems. The study of these systems should lead to a clearer understanding of the Afrotherian Supercohort’s neuroanatomy and may aid in the understanding of the groups phylogenetic relationships (e.g. Maseko et al., 2007; Asher et al., 2009). The aim of the current study is to reveal the total complement of cholinergic, catecholaminergic and serotonergic nuclei within the elephant shrew. It may be that certain nuclei are unique to the elephant shrew, or alternatively, shared, absent or in common with the other members of the Afrotheria to the exclusion of other mammals (Manger, 2005; Gravett et al., 2009). The current study represents the second study of these systems in a member of the Afrotheria (Gravett et al., 2009).

Six eastern rock elephant shrews, Elephantulus myurus, were used in this study. All animals were adult males and had an average body mass of 45.08 g and an average brain mass of 1.27 g. All guidelines for the treatment and housing of animals, from the University of the Witwatersrand Animal Ethics Committee, whose guidelines parallel those of the National Institutes of Health (NIH), were followed. The animals were euthanazed using deep barbiturate anaesthesia (Euthanaze, 200 mg sodium pentobarbital/kg, i.p.). Upon cessation of breathing the animals were perfused intracardially, initially with a rinse solution of 0.9% saline at 4 8C, thereafter a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4 8C was used (approximately 50 ml of each solution). The brain was then removed from the skull and post-fixed in 4% paraformaldehyde in 0.1 M PB overnight at 4 8C. The brains were then allowed to equilibrate in 30% sucrose in 0.1 M PB at 4 8C, following which the brains were frozen and sectioned into either coronal (n = 3) or sagittal (n = 3) sections of 50 mm thickness using a sliding microtome. Sections were stained in adjacent series for Nissl substance, myelin, choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT). Nissl substance sections were mounted on 0.5% gelatine coated glass slides, cleared in a 1:1 solution of chloroform and absolute alcohol, after which staining was carried out using a 1% cresyl violet solution. Sections used for the myelin series were stored for 2 weeks in a 5% formalin solution at 4 8C, mounted on 1.5% gelatine coated glass slides, and stained using a modified Gallyas (1979) silver stain. The procedure for immunohistochemical staining commenced with the treatment of the sections with an endogenous peroxidase inhibitor (49.2% methanol, 49.2% of 0.1 M PB: 1.6% of 30% H2O2) for 30 min, followed by a series of three 10 min rinses in 0.1 M PB. This was followed by a 2-h pre-incubation, at room temperature, in a solution containing 3% normal goat serum (NGS, Chemicon), or 3% normal rabbit serum for the anti-choline acetyltransferase (NRS, Chemicon), plus 2% bovine serum albumin (BSA, Sigma), and 0.25% Triton X-100 (Merck) in 0.1 M PB (blocking buffer). The sections were then placed in a primary antibody solution containing the appropriately diluted antibody in blocking buffer, for 48 h at a temperature of 4oC under gentle shaking. Anti-choline acetyltransferase (AB144P, Chemicon, raised in goat), at a dilution of 1:2000, was used to reveal cholinergic neurons. To reveal catecholaminergic neurons, anti-tyrosine hydroxylase (TH) (AB151, Chemicon, raised in rabbit) was used at a dilution of 1:7500. To reveal serotonergic neurons, anti-serotonin (AB938, Chemicon, raised in rabbit) was used at a dilution of 1:10,000. Following this the sections were passed through a series of three 10 min rinses in 0.1 M PB, followed by incubation in a secondary antibody at room temperature for 2 h. The secondary antibody solution contained a 1:500 dilution of biotinylated anti-rabbit IgG (BA-1000, Vector Labs) in 3% NGS or NRS, and 2% BSA in 0.1 M PB (or anti-goat IgG, BA-5000 in 3% NRS for the ChAT sections). Following a series of three 10 min rinses in 0.1 M PB, the sections underwent a 1-h incubation in AB solution (Vector Labs), and were again rinsed. The sections were then placed in a solution of 0.05% diaminobenzidine (DAB) in 0.1 M PB for 5 min, after this time 3 ml of 30% H2O2 was added to each 0.5 ml of solution. Development was monitored visually and checked under a low power stereomicroscope. Development was allowed to continue until the background staining was at a level at which it could assist reconstruction without making immunopositive neurons indistinguishable. The development process was halted by removing the sections from the DAB solution and placing them into a clean rinse of 0.1 M PB. To ensure cessation of development, two further rinses were carried out in 0.1 M PB. Omission of the primary or secondary antibody in selected sections was employed as negative controls, for which no staining was evident. In addition to this we ran a peptide inhibition assay specifically for the ChAT antibody (AB144P), as in our previous studies the cholinergic system has shown the greatest variability amongst species (Bhagwandin et al., 2006; Maseko et al., 2007; Gravett et al., 2009), and thus the novel findings of cholinergic neurons in the inferior colliculus and cochlear nuclei of the elephant shrew (see below) needed to be verified. We used choline acetyltransferase (AG220, Millipore) at a dilution of 5 mg/ ml in the primary antibody solution (see above). 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 latter case, cholinergic neurons were observed in the basal forebrain, inferior colliculus and cochlear nucleus of the elephant shrew. In the sections where the primary antibody had been inhibited, no staining was evident. Moreover, to further validate our results we used similar sections from the laboratory rat and rock hyrax (Gravett et al., 2009), with similar results. The immunohistochemically stained sections were mounted on 0.5% gelatine coated glass slides. The sections were then dehydrated in a series of alcohols, cleared in xylene, and cover slipped using Depex. Observation of the sections was carried out using a low power stereomicroscope, and a camera lucida was used for the tracing of the architectonic borders from both the myelin and Nissl series. The immunopositive neurons were marked on the drawings after they had been matched to the architectonic outlines. The drawings were scanned and redrawn using the Canvas 8 drawing program. The nomenclature used for the various systems investigated were adopted from Dahlstro¨m and Fuxe (1964), Ho¨kfelt et al. (1984), To¨rk (1990), Woolf (1991), Smeets and Gonza´lez (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), and Gravett et al. (2009). While we use the standard nomenclature for the catecholaminergic system in this paper, we realize that the neuronal groups we revealed with tyrosine hydroxylase immunohistochemistry may not correspond directly with these nuclei

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188 as has been described in previous studies by Dahlstro¨m and Fuxe (1964), Ho¨kfelt et al. (1976), Meister et al. (1988), Kitahama et al. (1990, 1996), and Ruggiero et al. (1992); however, given the striking similarity of the results of the tyrosine hydroxylase immunohistochemistry to that seen in other mammals we feel this terminology is appropriate. Clearly further studies in the elephant shrew with a wider range of antibodies, such as those to phenylethanolamine-N-methyltransferase (PNMT), dopamine-b-hydroxylase (DBH) and aromatic L-amino acid decarboxylase (AADC) would be required to fully determine the implied homologies ascribed in this study. We address this potential problem with the caveat of putative catecholaminergic neurons where appropriate in the text.

3. Results Immunohistochemistry was used in the current study to reveal the cholinergic, putative catecholaminergic and serotonergic systems in Eastern rock elephant shrew brains. These systems in

177

the elephant shrew show a generalized mammalian pattern, but also show certain features not previously, or rarely described in other mammalian species, and lack some features typically described for other mammals. These variances include aspects of both the cholinergic and putative catecholaminergic systems. 3.1. Cholinergic neurons The general nuclear organization for the cholinergic system of mammals consists of five main groups: the striatal, basal forebrain, diencephalic, and pontomesencephalic groups, and the cranial motor nerve nuclei (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008); however, there are cases of cholinergic neurons existing outside these groups, such

Fig. 1. Diagrams of serial coronal sections through the brain of the elephant shrew showing the location of neurons reactive for choline acetyltransferase (ChAT, black circles), tyrosine hydroxylase (TH, black triangles) and serotonin (open squares). The outlines of architectonic regions were made using Nissl and myelin stains and immunoreactive neurons marked on these drawings. A represents the most rostral section, T the most caudal. The figurines are approximately 1000 mm apart. See list for abbreviations.

178

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

Fig. 1. (Continued ).

as in the cerebral cortex (e.g. Bhagwandin et al., 2006). The cholinergic system found in the elephant shrew extends from the level of the anterior horn of the lateral ventricle to the spinomedullary junction (Figs. 1 and 2). The cholinergic system of the elephant shrew displayed the typical mammalian condition with one exception unique to this species, this being choline acetyltransferase immunopositive (ChAT+) interneurons in the inferior colliculus, and rarely described ChAT+ neurons

being found in the superior colliculus and the cochlear nuclear complex. 3.1.1. Striatal cholinergic interneurons 3.1.1.1. Nucleus accumbens. This cluster of ChAT+ neurons was found ventral to the dorsal striatopallidal complex (caudate, putamen and globus pallidus, see below), in a position similar

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

179

Fig. 2. Diagrams of an ideal sagittal section through the elephant shrew brain showing (A) cholinergic, (B) putative catecholaminergic, and (C) serotonergic nuclei or nuclear complexes. See list for abbreviations.

to that seen in all mammals (e.g. Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008). The anterior border of this cluster corresponded with that of the anterior horn of the lateral ventricle, while the posterior border was seen at the same level as the anterior commissure (Figs. 1C–E and 2). The neurons showed an ovoid, multipolar soma with no specific dendritic orientation. The ChAT+ neurons in this nucleus had a moderate to low density throughout and a moderate ChAT immunoreactivity with the exception of a row of strongly immunoreactive nerve cell bodies outlining the lateral border of a dorsomedial zone with a richer ChAT+ neuropil (Fig. 3B). 3.1.1.2. Dorsal striatopallidal complex (caudate, putamen and globus pallidus). The caudate/putamen extended from the anterior horn of the lateral ventricle, lateral to this space, to the level of the habenular nuclei (Figs. 1C–G and 2), a position that conforms to that of all mammals (e.g. Woolf, 1991; Manger et al., 2002a;

Maseko et al., 2007; Limacher et al., 2008). The distinction between the caudate and putamen was not readily definable as no clear internal capsule was observed. The globus pallidus was found medially and just ventral to the caudate/putamen posterior to the level of the anterior commissure. The complex showed a moderate to low density of ChAT+ neurons, with the globus pallidus having a less intensely ChAT immunoreactive neuropil than the caudate/ putamen and showing a lower ChAT+ neuronal density overall. The morphology of the neurons seen in the complex was the same for both the caudate/putamen and globus pallidus, consisting of ovoid shaped cell bodies with a mixture of bipolar and multipolar neurons. The ChAT+ neurons within the complex showed no specific dendritic orientation. 3.1.2. Cholinergic nuclei of the basal forebrain 3.1.2.1. Islands of Calleja and the olfactory tubercle. The ChAT+ neurons of these nuclei were found in the ventral most portions of

180

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

Fig. 3. Photomicrographs of adjacent sections showing the basal forebrain region immediately rostral to the anterior commissure (ac). Nissl stain (A), cholineacetyletransferase (ChAT) immunoreactivity (B), and myelin stain (C). Note the strongly ChAT+ densely packed neurons of the medial septal nucleus (Sep.M), the scattered ChAT+ neurons in nucleus accumbens (N.Acc), caudate (C) and putamen (P), and the clusters of ChAT+ neurons representing the islands of Calleja (Is.Call). A weakly to moderately intense ChAT+ neuropil is found in these regions. Scale bar in C = 1 mm and applies to all.

the cerebral hemisphere ventral to the nucleus accumbens in a position that resembles that seen in all mammals studied to date (Figs. 1C–E and 2) (e.g. Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008). A moderate to low density of ChAT+ cells were present with occasional dense ChAT+ neuronal clusters representing the Islands of Calleja (Fig. 3B). The neurons exhibited ovoid cell bodies, with the majority being of the bipolar type showing no specific dendritic orientation. 3.1.2.2. Medial septal nuclei. The overall size of the septal nuclear complex in the elephant shrew was large in relation to brain size, and a high density of numerous ChAT+ neurons was observed in the medial septal nucleus. The position of the complex, in particular the medial septal nucleus, was the same as has previously been described in other mammals (Figs. 1D and 2) (e.g. Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008). These neurons were of the multipolar type with large, ovoid shaped cell bodies with strong ChAT immunoreactivity, showing no specific orientation of the dendrites (Fig. 3B). 3.1.2.3. Diagonal band of Broca. The diagonal band of Broca was found in a position typical of all mammals (e.g. Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008), being located in the ventromedial corner of the cerebral hemisphere, anterior to the hypothalamus (Figs. 1D and 2). A moderate density of ChAT+ neurons with a similar morphology to that of the medial septal nuclei was observed, the neurons were of the multipolar type, exhibiting large, ovoid shaped soma and no specific dendritic orientation. 3.1.2.4. Nucleus basalis. The nucleus basalis was found ventral and ventromedial to the globus pallidus complex and contained a low to moderate density of ChAT+ neurons (Figs. 1F and 2). The nucleus basalis appears to be almost continuous with the globus pallidus, but displays differences in cell morphology. These cells have ovoid to round shaped soma, with a mixture of bipolar and multipolar cell types. The ChAT+ neurons within this nucleus were mostly of the bipolar type and showed no specific dendritic orientation.

3.1.3. Diencephalic nuclei 3.1.3.1. Medial habenular nucleus. This nucleus was found adjacent to the third ventricle and dorsal to the midline portion of the dorsal thalamus in a position similar to that of all mammals (Figs. 1G and 2) (e.g. Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008). The medial habenular nucleus consists of a dense aggregation of small round ChAT+ neurons with an undistinguishable dendritic orientation (due to the high density of the ChAT+ neurons). The clearly defined ChAT+ fasciculus retroflexus was formed by the axons of this nucleus and seen to terminate in the interpeduncular nucleus. 3.1.3.2. Hypothalamic cholinergic nuclei. Three clusters of ChAT+ neurons were observed within the hypothalamus: the dorsal, lateral and ventral hypothalamic cholinergic nuclei (Figs. 1H and 2). The cellular morphology of the neurons in all three groups was similar and had an ovoid somal shape, being a mixture of both multipolar and bipolar cell types and the orientation of the dendrites presented with no specific orientation. The ChAT+ neurons of all three groups were clearly seen but were stained palely. The dorsal nucleus was found in a dorsomedial position within the hypothalamus, between the fornix and the third ventricle. The lateral nucleus was situated in a dorsolateral position within the hypothalamus, lateral to the fornix, while the ventral group was located close to the inferior border of the third ventricle in a ventromedial position within the hypothalamus. 3.1.4. Pontomesencephalic nuclei 3.1.4.1. Collicular interneurons. ChAT+ neurons were found within the superficial layers of the superior colliculus (Figs. 1I–K and 2). These ChAT+ neurons presented in a low density and were of the bipolar type. The dendrites were seen to be oriented in a plane orthogonal to the pial surface. Occasional bipolar ChAT+ neurons with no specific dendritic orientation were found within the ventrolateral aspect of the inferior colliculus (Figs. 1K–N, 2 and 4B). 3.1.4.2. Parabigeminal nucleus. This strongly stained dense cluster of ChAT+ neurons was found in the lateral aspect of the midbrain

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

181

Fig. 4. Photomicrographs of ChAT immunoreactive neurons in sections through the dorsal midbrain/pontine region of the elephant shrew brain. (A) The pedunculopontine nucleus (PPT), laterodorsal tegmental nucleus (LDT) and the parabigeminal nucleus (PBg). (B) The laterodorsal (LDT) and pedunculopontine (PPT) nuclei at a level slightly caudal to that shown in (A) Note the occasional ChAT+ neuron (arrowheads) found in the ventrolateral quadrant of the inferior colliculus (IC). Scale bar in (B) = 500 mm and applies to (A) and (B). 4 V: fourth ventricle, ca: cerebral aqueduct.

tegmentum ventral to the inferior colliculus (Figs. 1K, 2). The cells were seen to be round in shape and appeared to be bipolar, but due to the high density of ChAT+ neurons in this nucleus, any specific dendritic orientation was impossible to distinguish (Fig. 4A). 3.1.4.3. Pedunculopontine tegmental nucleus (PPT). The PPT nucleus was distinguishable as a group of scattered ChAT+ neurons positioned in the region of the superior cerebellar peduncle and anterior to the trigeminal motor nucleus within the dorsal aspect of the isthmic and pontine tegmenta (Figs. 1K–L and 2). A low to moderate density of strongly ChAT+ neurons were noted. These neurons were of a multipolar type and showed a variety of somal shapes, having no specific dendritic orientation (Fig. 4). 3.1.4.4. Laterodorsal tegmental nucleus (LDT). This high-density cluster of ChAT+ neurons showed a similar neuronal morphology as that of the PPT ChAT+ neurons, but was positioned within the ventrolateral portions of the periaqueductal and periventricular grey matter (Figs. 1L–M and 2). The neurons of the LDT did not show any specific dendritic orientation (Fig. 4). 3.1.4.5. Cochlear nucleus. A small number of ChAT+ neurons were found within deeper aspects of the cochlear nuclei (Figs. 1O and 2). The morphology presented was that of a mixture of multipolar and bipolar types with moderately sized ovoid or fusiform soma (Fig. 5C). The dendrites of these nuclei showed no specific orientation. Within the more superficial aspects of the cochlear nucleus a moderately ChAT+ terminal network was observed (Fig. 5C). 3.1.5. Cholinergic cranial nerve motor nuclei All the typical mammalian cranial nerve motor nuclei were seen in the elephant shrew, all containing large multipolar type ChAT+ motor neurons and in positions typical for all mammals (e.g. Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008). These nuclei were: oculomotor (III), trochlear (IV), trigeminal motor nucleus (Vmot), abducens (VI), dorsal and ventral divisions of the facial (VIId and VIIv), dorsal division of the vagus (X), hypoglossal (XII), preganglionic neurons of the superior salivatory nucleus or facial nerve (pVII) and the inferior salivatory nucleus (pIX), Edinger–Westphal nucleus (EW) and nucleus ambiguus (Figs. 1K–T and 2). The EW neurons were found to be smaller and in a lower density and palely stained compared to the closely associated neurons of the oculomotor nucleus. The EW ChAT+ neurons were found intermingled with the most medial of

the oculomotor neurons with some being slightly anterior to the oculomotor nucleus, within the periaqueductal grey matter alongside the midline. The soma of the EW neurons was of the bipolar type and had a dorsoventral dendritic orientation. The pIX ChAT+ neurons stained intensely and were well expressed in terms of number and density (Fig. 5A and B), more so than in most other mammals, a feature the elephant shrew shares with the rock hyrax (Gravett et al., 2009). 3.2. Putative catecholaminergic nuclei Putative catecholaminergic neurons within the brain of the elephant shrew were found from the olfactory bulb to the level of spinomedullary junction. The tyrosine hydroxylase immunopositive (TH+) nuclei found in the elephant shrew were similar to that seen in a range of other mammals (e.g. Smeets and Gonza´lez, 2000; Maseko et al., 2007; Limacher et al., 2008), with the exception of the A15d nucleus that was absent. The catecholaminergic system has been divided into 5 nuclear complexes: the olfactory bulb, diencephalic, midbrain, pontine and medullary nuclear complexes. As discussed earlier the A1–A17 and C1–C3 nomenclature has been used for simplicity, as additional nuclei not defined by this nomenclature were not found as previously described for some other vertebrates (Smeets and Gonza´lez, 2000). 3.2.1. Olfactory bulb (A16) TH+ neurons of a moderate density were found throughout stratum granulosum of the olfactory bulb, in the typical mammalian position (Figs. 1A,B and 2) (Smeets and Gonza´lez, 2000; Maseko et al., 2007; Limacher et al., 2008). Ovoid shaped neurons of the bipolar type projected dendrites that were seen to form a mesh-like network around the glomeruli. 3.2.2. Diencephalic nuclei (A15–A11) This cluster consisted of five TH+ nuclei within the hypothalamus. These nuclei were: the anterior hypothalamic nucleus, ventral division (A15v), the rostral periventricular nucleus (A14), the zona incerta (A13), the tuberal nucleus (A12), and the caudal diencephalic nucleus (A11) (Figs. 1F–I and 2). A small group of very few TH+ neurons was found within the ventrolateral portion of the hypothalamus next to the optic tract and near the base of the brain. These neurons were assigned to the A15v group and were of a bipolar type with ovoid soma and showed a dendritic orientation parallel to the floor of the brain. TH+ neurons forming the A14 nucleus were located close to the lateral walls of the third ventricle

182

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

Fig. 5. Photomicrographs of ChAT immunoreactive neurons in sections through the periventricular grey matter in the dorsal caudal medullary region and cochlear nucleus of the elephant shrew brain. (A) The greatly expanded preganglionic neurons of the inferior salivatory nucleus (pIX) at a rostral level through this nucleus where it is found lateral to the abducens nucleus (VI) and the facial nerve (VIIn). (B) The same nucleus (pIX) at a more caudal level where it has expanded in size. (C) Strongly ChAT immunoreactive neurons (arrowheads) exist in the deeper aspects of the cochlear nucleus (CO) and moderately ChAT immunoreactive band of terminal networks were observed in the superficial aspects of the CO. The scale bar in (C) = 500 mm, applies to all.

and had morphology similar to that of the A15v group, with the exception that these neurons had no specific dendritic orientation. Located within the dorsal hypothalamus but dorsal and lateral to the fornix was a cluster of TH+ neurons that extended into and surrounded the zona incerta (Fig. 6A). This cluster of neurons was classified as the A13 group, and once again had the same morphology as the neurons of the A15v nucleus, but showed a rough mediolateral dendritic orientation. The A12 nucleus consisted of a cluster of TH+ neurons found surrounding the floor

Fig. 6. Photomicrographs of selected neuronal groups demonstrating tyrosine hydroxylase immunoreactivity within the brain of the elephant shrew. (A) The rostral periventricular nucleus (A14) located adjacent to the walls of the third ventricle, and the zona incerta nucleus (A13) in the dorsolateral aspect of the hypothalamus. (B) The ventral tegmental (A10, A10c, A10d) and substantia nigra (A9m, A9pc, A9l) nuclear complexes in the ventral lateral midbrain tegmentum located around the cerebral peduncle and the root of the oculomotor nerve (IIIn). (C) Few TH+ neurons are found in the diffuse portion of the locus coeruleus (A6d) within the ventrolateral periaqueductal grey matter, while the compact portion of the nucleus subcoeruleus (A7sc) in the adjacent dorsal pontine tegmentum is rich in strongly TH+ neurons. Scattered TH+ neurons in the more peripheral pontine tegmentum form the diffuse portion of the subcoeruleus (A7d). Scale bar in (A) = 500 mm. Scale bar in (C) = 1 mm, applies to (B) and (C). scp: superior cerebellar peduncle.

of the third ventricle near the midline and was incorporated into or was found to be in close proximity to the arcuate nucleus. The morphology of the neurons of A12 was of the bipolar type with ovoid shaped cell bodies and that showed no specific dendritic orientation. A cluster consisting of a few TH+ neurons was assigned to the A11 nucleus and was easily distinguished from the nearby

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

TH+ neurons of the A10dc nucleus (see below) due to their larger somatal size. The A11 nucleus was found in the most caudal portion of the hypothalamus in a column on each side of the midline near the posterior pole of the third ventricle. The A11 nucleus consisted of ovoid to polygonal shaped soma with the dendrites exhibiting no specific orientation. 3.2.3. Midbrain nuclei (A10–A8) 3.2.3.1. Ventral tegmental area (VTA, A10 complex). The medial portion of the midbrain tegmentum nucleus housed the A10 nuclear complex. Four nuclei comprise this complex: the ventral tegmental area, central nucleus (A10c); the ventral tegmental area nucleus (A10); the ventral tegmental area, dorsal nucleus (A10d); and the ventral tegmental area, dorsal caudal nucleus (A10dc). These TH+ neurons were found dorsal, dorsolateral and lateral to the interpeduncular nucleus (A10, A10c, A10d) and within the periaqueductal grey matter (A10dc) (Figs. 1I–K, 2 and 6B). The TH+ neurons forming the A10 nucleus were found dorsolateral and lateral to the interpeduncular nucleus, between it and the root of the oculomotor nerve. A moderate to high density of TH+ neurons of the bipolar type with ovoid soma were found in this nucleus. These neurons displayed a dendritic orientation running parallel to the lateral border of the interpeduncular nucleus. The TH+ neurons forming the A10c nucleus were found dorsal and medial to A10, the nucleus showing a rough triangular shape, spanning the midline above the interpeduncular nucleus. A10c consisted of a moderate density of TH+ neurons that tapered toward the dorsal limits of the formation. The morphology of A10c neurons were similar to that of A10 but displayed a dorsolateral dendritic orientation. Between the A10c nucleus and the periaqueductal grey matter, in a region medial and ventral to the oculomotor nucleus, a few TH+ neurons were designated to the A10d nucleus. These bipolar ovoid shaped neurons exhibited a dorsoventral dendritic orientation. The A10dc nucleus was represented by a small number of TH+ neurons found within the periaqueductal grey matter close to the ventral border of the cerebral aqueduct. These neurons were bipolar in type, small in size and ovoid in shape with the dendrites running parallel to the edge of the cerebral aqueduct. 3.2.3.2. Substantia nigra nuclear complex (A9). This complex was located in the ventrolateral aspect of the midbrain tegmentum, dorsal to the cerebral peduncles and consisted of three distinct nuclei: the substantia nigra, intermediate part of pars compacta (A9pc); the substantia nigra, pars medialis of zona compacta (A9m); and the substantia nigra, pars lateralis of the zona compacta (A9l) (Figs. 1I–K and 2). The moderately dense mediolaterally arranged band of TH+ neurons found just dorsal to the cerebral peduncle were designated as the A9pc nucleus. This nucleus was made up of a mixture of bipolar and multipolar neurons that were ovoid to triangular in shape (Fig. 6B). The dendritic orientation of the neurons of the A9pc group was that of a mediolateral direction, parallel to the lower border of the medial lemniscus. The A9l nucleus was located immediately lateral to A9pc, towards the dorsolateral margin of the cerebral peduncle, in the ventrolateral part of the midbrain tegmentum (Fig. 6B). A9l exhibited large multipolar neurons with triangular and polygonal shaped soma that exhibited no specific dendritic orientation. TH+ neurons assigned to the A9m nucleus were located between the root of the oculomotor nerve and the medial edge of the band of neurons forming the A9pc. A9m exhibited a high density of TH+ neurons that were of similar morphology to that of A9pc but that exhibited no specific dendritic orientation (Fig. 6B). The substantia nigra, ventral or pars reticulata (A9v) nucleus consisted of a very low number of TH+ neurons and these neurons were located just ventral to the A9pc nucleus and appeared to have a very similar

183

morphology to it. Due to the low density of the A9v nucleus it was thought to be incipient and the presence of the nucleus could be called into question. 3.2.3.3. Retrorubral nucleus (A8). TH+ neurons assigned to the A8 nucleus were found within the lower half of the midbrain tegmentum, dorsal to the A9 complex, and caudal to the magnocellular division of the red nucleus (Figs. 1K and 2). This nucleus was comprised of a small number of scattered TH+ neurons. The neuronal type was a mixture of bipolar and multipolar and the cell shape was predominantly ovoid. The neurons of the A8 nucleus showed no specific dendritic orientation. 3.2.4. Pontine nuclei—the locus coeruleus (LC) nuclear complex (A7–A4) The locus coeruleus complex consisted of five distinct nuclei comprised of a moderate number of TH+ neurons. These five nuclei were: the subcoeruleus compact (A7sc); subcoeruleus diffuse (A7d); locus coeruleus diffuse (A6d); fifth arcuate (A5); and the dorsal medial nucleus of locus coeruleus (A4) (Figs. 1M–N and 2). TH+ neurons located in the dorsal pontine tegmentum adjacent to the periventricular grey matter were designated as the A7sc nucleus (Fig. 6C). These neurons have been previously described as the subcoeruleus (Dahlstro¨m and Fuxe, 1964). This moderate to high-density group consisted of bipolar, ovoid TH+ neurons that exhibited a dorsomedial to ventrolateral dendritic orientation. Lateral, dorsolateral and ventral to the A7sc nucleus, in the pontine tegmentum, a scattered cluster of TH+ neurons were assigned to the A7d nucleus (Fig. 6C). The A7d nucleus shows a moderate to low neuronal density and was found anterior to and almost surrounding the trigeminal motor nucleus. A small number of these A7d neurons were also seen to be lateral and dorsal to the superior cerebellar peduncle. The morphology of the neurons of A7d was similar to that of A7sc, but the A7d neurons showed no specific orientation of the dendrites. TH+ neurons within the periventricular grey matter, in a location distinct to the cholinergic LDT nucleus were assigned to the A6d nucleus (Fig. 6C). The A6d nucleus had a moderate to high density of TH+ neurons that had the same morphology as the neurons of the A7d nucleus. The A5 nucleus was found in the ventrolateral aspect of the pontine tegmentum, anterior to the facial nerve nucleus and ventral to the trigeminal motor nerve nucleus, but lateral to the superior olive. It was made up of a small number of TH+ neurons with a similar morphology to those of the A7d nucleus, but of a multipolar type. The dendrites of these neurons exhibited a loosely arranged meshlike network in the surrounding tegmental region. A low density and low number of TH+ neurons situated near the wall of the fourth ventricle in the dorsolateral portion of the periventricular grey matter were assigned to the A4 nucleus. Once again the neuronal morphology was similar to that of the neurons of the A7d nucleus, but the neurons of this nucleus did not exhibit any specific dendritic orientation. 3.2.5. Medullary nuclei (C1, C2, A1, A2, area postrema) Five TH+ nuclei were observed within the medulla of the elephant shrew, namely: the rostral ventrolateral tegmental (C1); rostral dorsomedial (C2); caudal ventrolateral tegmental (A1); caudal dorsomedial (A2) nuclei and the area postrema (AP) (Fig. 1N–S). The C1 nucleus consisted of a small column of TH+ neurons in the rostral ventrolateral medulla located lateral to the superior olive and facial nerve nucleus, but medial to the nucleus ambiguus and the column of neurons projected from the edge of the medulla into the medullary tegmentum. The morphologies of the C1 neurons, as well as those of the A1 neurons, were multipolar in type with triangular soma with dendrites surrounding the

184

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

fasciculi in the medullary tegmentum. The A1 nucleus was found in the caudal ventrolateral aspect of the medullary tegmentum, from the nucleus ambiguus through to the spinomedullary junction. The column of neurons forming this nucleus was located in a position lateral to the lateral reticular nucleus and lateral to the nucleus ambiguus, in contrast to C1 that was found medial to nucleus ambiguus. The TH+ neurons assigned to the C2 nucleus were found in the dorsal portion of the medulla, anterior and dorsal to the vagus motor nerve nucleus. Above the vagus motor nerve nucleus an area known as the dorsal strip of C2 (Kalia et al., 1985a,b) was found near to the floor of the fourth ventricle; however, the rostral extension of C2 was absent. The C2 nucleus was comprised of a low density of TH+ neurons that were ovoid in shape and bipolar in type. The neurons of this nucleus showed no specific dendritic orientation. Surrounding the dorsal motor vagus nucleus and hypoglossal nucleus were a small number of TH+ neurons assigned to the A2 nucleus. Some of these neurons were found intermingled with those of the dorsal motor vagus nucleus. The A2 neurons were moderately sized ovoid bipolar neurons with dendrites that were orientated in a mediolateral direction. The area postrema consisted of a high density of small sized TH+ neurons located in the midline of the medulla, dorsal to the vagus motor nerve nucleus and central canal. This position was adjacent to the floor of the fourth ventricle and just anterior to the spinomedullary junction. No specific dendritic orientation was observed in these ovoid bipolar neurons. The area postrema appears to be quite large in the elephant shrew when qualitatively comparing it to that of other species. 3.3. Serotonergic nuclei The serotonergic system consisted of a number of nuclei that were found within the brainstem from the level of the decussation of the superior cerebellar peduncle to the spinomedullary junction. The neurons forming the serotonin immunoreactive (5HT+) nuclei could be grouped into a rostral and a caudal cluster. All serotonergic nuclei found in the elephant shrew have been described previously in other eutherian mammals (e.g. Maseko et al., 2007; Limacher et al., 2008), but no hypothalamic serotonergic neurons, as described in the monotremes (Manger et al., 2002c), were observed.

3.3.1. Rostral cluster 3.3.1.1. Caudal linear nucleus (CLi). A distinct cluster of 5HT+ neurons was seen in the midline of the midbrain tegmentum and was assigned to the CLi. The position of this nucleus was dorsocaudal to the interpeduncular nucleus and just anterior to the decussation of the superior cerebellar peduncle (Figs. 1K and 2). This most rostral serotonergic nucleus exhibited a moderate to low density of 5HT+ neurons that were of a bipolar type (Fig. 7A). The dendrites of the 5HT+ neurons were observed to show no specific orientation. 3.3.1.2. Supralemniscal nucleus (B9). The 5HT+ neurons of this nucleus appeared to be a ventrolateral continuation of the neurons forming the CLi nucleus (Figs. 1K and 2). The 5HT+ neurons of the B9 nucleus were few in number and stretched from the dorsolateral aspect of the interpeduncular nucleus laterally over the superior aspect of the caudal-most cerebral peduncle in a position caudal to the substantia nigra. Their morphology was similar to that of the CLi but the dendrites were found to be in an orientation parallel to the dorsal edge of the superior aspect of the cerebral peduncle. 3.3.1.3. Median raphe (MnR). This cluster of 5HT+ neurons was defined as two moderately densely packed columns of neurons found from the dorsal to ventral aspects adjacent to the midline within the midbrain and pontine tegmentum (Figs. 1K–N and 2). The MnR columns extended from a position just caudal to the decussation of the superior cerebellar peduncle to the posterior most portion of the trigeminal motor nucleus. The 5HT+ neurons were ovoid shaped, bipolar and the dendrites were oriented in a dorsoventral plane (Fig. 7). 3.3.1.4. Dorsal raphe nuclear complex (DR). The DR complex consisted of 6 distinct nuclei: the dorsal raphe interfascicular (DRif), dorsal raphe ventral (DRv), dorsal raphe dorsal (DRd), dorsal raphe lateral (DRl), dorsal raphe peripheral (DRp), and dorsal raphe caudal (DRc) nuclei (Figs. 1K–N, 2 and 7). The majority of the above nuclei were found within the periventricular and periaqueductal grey matter from the level of the oculomotor nucleus to the level of

Fig. 7. Photomicrographs of the neuronal groups showing serotonin immunoreactivity within the rostral serotonergic cluster of the elephant shrew brain. (A) Caudal linear nucleus (CLi), dorsal raphe dorsal nucleus (DRd), dorsal raphe ventral nucleus (DRv), dorsal raphe interfascicular nucleus (DRif), dorsal raphe peripheral nucleus (DRp) and the median raphe nucleus (MnR). (B) Dorsal raphe lateral nucleus (DRl), dorsal raphe dorsal nucleus (DRd), dorsal raphe ventral nucleus (DRv), dorsal raphe peripheral nucleus (DRp) and the median raphe nucleus (MnR). (C) Dorsal raphe caudal nucleus (DRc) and the median raphe nucleus (MnR). Scale bar in (B) = 1 mm, applies to (A) and (B). Scale bar (C) = 500 mm.

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

the trigeminal motor nucleus. The DRif cluster consisted of moderately dense, ovoid shaped, bipolar 5HT+ neurons that were located in the most ventral medial portion of the periventricular grey matter between the two medial longitudinal fasciculi. The dendrites of these neurons exhibited a dorsoventral orientation. The DRv was located just caudal to the oculomotor nuclei and dorsal to the DRif in the periaqueductal grey matter. The DRv consisted of a moderate density of 5HT+ neurons with a similar morphology to those of the DRif, with the exception of the dendritic orientation that showed no specificity. The DRd nucleus consisted of a moderate density of 5HT+ neurons that appeared similar to those of the DRif and DRv. This nucleus was found dorsal to the DRv but ventral to the inferior border of the cerebral aqueduct, and was located within the periaqueductal grey matter. The neurons within the DRd exhibited a mediolateral dendritic orientation. The DRp was located in the ventrolateral part of the periaqueductal grey mater, lateral to the DRd and DRv nuclei. The DRp was made up of only a few neurons within the periaqueductal grey matter and had a few neurons outside of the periaqueductal grey matter in the adjacent midbrain tegmentum. The 5HT+ neurons within the DRp were of the bipolar type with ovoid shaped soma and exhibited no specific dendritic orientation. Immediately dorsolateral to the DRd nucleus, adjacent to the ventrolateral edge of the cerebral aqueduct, a few 5HT+ neurons were assigned to the DRl nucleus. The DRl consisted of a small number of large multipolar neurons with no specific dendritic orientation. Posteriorly the DRl coalesced across the midline to form an arc of 5HT+ neurons within the dorsal part of the periventricular grey matter. These neurons were assigned to the DRc nucleus and had a similar morphology to that of the DRl neurons. The distinction between DRl and DRc is made herein, as the monotremes do not exhibit a cluster of 5HT+ neurons that can be defined as the DRc nucleus (Manger et al., 2002c). 3.3.2. Caudal cluster 3.3.2.1. Raphe magnus nucleus (RMg). A low density of 5HT+ neurons forming two columns on either side of the midline in the rostral aspect of the medullary tegmentum from the level of the facial nerve nucleus to the anterior border of nucleus ambiguus was designated as RMg (Figs. 1N–P and 2). The morphology of the 5HT+ neurons was that of a bipolar type with large soma that had a dorsoventral dendritic orientation. 3.3.2.2. Rostral and caudal ventrolateral serotonergic medullary columns (RVL and CVL). The rostral and caudal ventrolateral serotonergic medullary columns appear to be lateral continuations of the ventral portion of the RMg (Figs. 1O–Q and 2). Rostrally these columns were found in a position just dorsal to the pyramidal tracts, extending from the RMg. These columns bifurcated around the anterior pole of the inferior olivary nuclei to form two bilateral laterally located columns of 5HT+ neurons in the ventrolateral aspect of the medullary tegmentum. The columns continue caudally within the ventrolateral medulla lateral to the inferior olive. The portions of the columns located between the facial nerve nucleus and the rostral border of the nucleus ambiguus were assigned to the RVL, while the caudal portion, that was seen to extend from the nucleus ambiguus to the spinomedullary junction, was assigned to the CVL. The morphology of 5HT+ neurons forming both the RVL and CVL were very similar to that of the RMg, but these neurons did not display any specific dendritic orientation. The density of the 5HT+ neurons decreased caudally, from a moderate density rostrally to a low density caudally. The reason for the distinction between the two columns is that the opossum and monotremes lack 5HT+ neurons that could be considered to form the CVL (Crutcher and Humbertson, 1978; Manger et al., 2002c).

185

3.3.2.3. Raphe pallidus nucleus (RPa). 5HT+ neurons within the ventral midline between the two pyramidal tracts around the level of the facial nerve nucleus were designated as the RPa (Figs. 1O–Q and 2). These small ovoid cells of the bipolar type were easily distinguished from the RMg neurons due to the size difference. The neurons had a dorsoventral dendritic orientation that ran parallel to the adjacent border of the pyramidal tracts. 3.3.2.4. Raphe obscurus nucleus (ROb). This group was assigned to the two columns of 5HT+ neurons on either side of the midline found between the nucleus ambiguus and spinomedullary junction (Figs. 1P–R and 2). These loosely packed columns were arranged from dorsal to ventral along the midline. The ROb consisted of fusiform shaped neurons of the bipolar type that exhibited a dorsoventral dendritic orientation. 4. Discussion The cholinergic, putative catecholaminergic and serotonergic neuronal systems found in the brain of the elephant shrew revealed a few novel nuclei of interest, as well as a number of expected nuclei, following the typical mammalian complement (e.g. Maseko et al., 2007; Limacher et al., 2008; Gravett et al., 2009). The cholinergic system of the elephant shrew, as revealed by choline acetyltransferase immunohistochemistry, contained a single cluster of neurons that is unique for this mammal namely the cholinergic neurons found within the inferior colliculus, as well as a complement of major nuclei in line with that of all mammals. The putative catecholaminergic system revealed a typical mammalian pattern but lacked the A15d and C3 nuclei, the latter (C3) being only found in rodents (e.g. Smeets and Gonza´lez, 2000; Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008; Gravett et al., 2009). The serotonergic system consisted of a complement of nuclei identical to that seen for all eutherian mammals studied to date (e.g. Maseko et al., 2007; Limacher et al., 2008; Gravett et al., 2009). The nuclear organization of these neural systems reinforce the placement of this species within a separate order, the Macroscelidea, and aligns this species most closely with the rock hyrax in terms of similarities in nuclear organization (Gravett et al., 2009). 4.1. The cholinergic system The elephant shrew’s cholinergic system was found to consist of several distinct nuclei showing a global organization typical of most mammals (e.g. Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007; Limacher et al., 2008). The striatal cholinergic interneurons and basal forebrain nuclei appear homologous to those previously seen in other mammalian species (Woolf, 1991; Karasawa et al., 2003; Maseko et al., 2007; Maseko and Manger, 2007). The diencephalon of the elephant shrew housed the medial habenular nucleus as well as three hypothalamic nuclei (the dorsal, ventral and lateral hypothalamic nuclei) as seen in other mammals (Vincent and Reiner, 1987; Oh et al., 1992; Karasawa et al., 2003; Maseko and Manger, 2007; Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008). The hypothalamic nuclei have been found in all mammals to date (Tago et al., 1989), with the exception of the monotremes (Manger et al., 2002a). An interesting point to note here is that the rock hyrax, the only other member of the Afrotheria that has been studied, had cholinergic neurons in the dorsal thalamus (Gravett et al., 2009), but these were not observed in the elephant shrew. Thus, this feature of the cholinergic system appears to be quite limited in its phylogenetic occurrence, perhaps occurring only in the hyraxes, or more closely related members of the Afrotheria (such as elephants, manatees and dugongs). The brains of these

186

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

other Afrotherian species need to be studied in order to determine how common this feature is. The pontomesencephalic nuclei of the elephant shrew consisted of the laterodorsal tegmental (LDT), pedunculopontine tegmental (PPT), parabigeminal (PBg), cochlear nucleus cholinergic neurons as well as the collicular cholinergic interneurons. The LDT and PPT nuclei have been observed in all mammalian species studied to date (e.g. Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008). In the rock hyrax, the LDT and PPT could be divided into parvo- and magnocellular components (Gravett et al., 2009), a situation not seen in other mammals, or the elephant shrew. As with the dorsal thalamic cholinergic neurons of the rock hyrax, other members of the Afrotherian cohort need to be examined to see if this is a hyrax-specific feature, or occurs more commonly. The PBg nucleus, a nucleus that stains palely in most rodents (Woolf, 1991; Da Silva et al., 2006; Bhagwandin et al., 2008; one exception being the Cape porcupine, Limacher et al., 2008), had neurons that were intensely stained in the elephant shrew. The PBg nucleus has been noted in carnivores, rodents, tree shrews, megabats and primates (Murray et al., 1982; Vincent and Reiner, 1987; Maseko et al., 2007; Limacher et al., 2008), but has not been observed in the monotremes, laboratory shrew and microbat (Manger et al., 2002a; Karasawa et al., 2003; Maseko and Manger, 2007). The cochlear nucleus contained a small number of ChAT+ neurons, an observation previously only made in the cat and rat (Sherriff and Henderson, 1994). ChAT+ interneurons were found within the superficial layers of the superior colliculus and in the core of the inferior colliculus of the elephant shrew. These ChAT+ inferior collicular neurons have not been described in any other mammalian species; however the superficial superior colliculus of the mouse, rat, ferret, tree shrew and cat do exhibit a similar arrangement of cholinergic interneurons (Tan and Harvey, 1989). All the typical mammalian cranial nerve nuclei were observed within the elephant shrew (e.g. Woolf, 1991; Manger et al., 2002a; Limacher et al., 2008). The elephant shrew’s Edinger–Westphal (EW) nucleus had a low density of palely stained ChAT+ neurons. ChAT+ neurons within the EW nucleus have been reported in rodents, carnivores, megabats, and primates (Murray et al., 1982; Vincent and Reiner, 1987; Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008), but these ChAT+ neurons have not been reported in the monotremes, laboratory shrews and microbats (Manger et al., 2002a; Karasawa et al., 2003; Maseko and Manger, 2007). The preganglionic neurons of the superior and inferior salivatory nuclei have previously been found in many mammalian species (e.g. Maseko et al., 2007), but have not been found in the monotremes or microbats (Manger et al., 2002a; Maseko and Manger, 2007). Interestingly, in common with the rock hyrax (Gravett et al., 2009), the preganglionic neurons of the inferior salivatory nucleus (pIX) were also well expressed and numerous in the elephant shrew, potentially representing an Afrotherian specific feature. The nuclear complement of the cholinergic system of the elephant shrew has many similarities with those of the rock hyrax, megabat, primate, rodent and carnivore, but has a large number of differences to the cholinergic systems of the monotremes, laboratory shrews and microbats. This overall complement of cholinergic nuclei phylogenetically aligns the elephant shrew most closely with the rock hyrax, underlining their placement in the Afrotheria.

Maseko et al., 2007; Limacher et al., 2008). The catecholaminergic neurons found within the olfactory bulb (A16) are similar to those seen in all other mammals (Smeets and Gonza´lez, 2000; Maseko and Manger, 2007; Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008). The diencephalon of the elephant shrew exhibited the neuronal clusters of the A15 ventral, A14, A13, A12 and A11 nuclei, but lacked the A15 dorsal nucleus. Six catecholaminergic hypothalamic nuclei have been observed in many other mammals (Smeets and Gonza´lez, 2000). The exceptions to this general nuclear organization include the elephant shrew (lacking A15d), the tree shrews (which lack an A14), microbats (which lack both A15d and A15v), dolphins (that lack an A13), hedgehogs (which lack the A15d) and artiodactyls (pigs, sheep and cattle, which also lack the A15d) (Maseko et al., 2007). The A8 (retrorubral) nucleus, A9 (substantia nigra complex) and A10 (ventral tegmental area complex) neuronal clusters were found within the midbrain tegmentum of the elephant shrew. The A9 v nucleus (or pars reticulata) was not clearly defined within the elephant shrew’s midbrain; a feature similar in appearance to this nucleus in the midbrain of the hedgehog (Michaloudi and Papadopoulos, 1996). All other nuclear complexes were found to have a nuclear organization similar to the rodents, primates, megabats, tree shrews, opossums, monotremes and artiodactyls (Murray et al., 1982; Manger et al., 2002b, 2004; Da Silva et al., 2006; Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008). The carnivores, microbats and rabbits have been shown to lack the A9v group completely, but the microbats also lack the A10 and A10dc nuclei (Maseko and Manger, 2007). The locus coeruleus complex of the elephant shrew consisted of the A7d, A7sc, A6d, A5, and A4 nuclei. The elephant shrew lacks the A6 compact nucleus as do the rodents (with the exception of laboratory rats, Dahlstro¨m and Fuxe, 1964), hedgehogs, monotremes, opossums, carnivores, bottlenose dolphins, microbats and artiodactyls (Michaloudi and Papadopoulos, 1996; Manger et al., 2002b, 2004; Maseko and Manger, 2007; Bhagwandin et al., 2008; Limacher et al., 2008). The megabats, primates, tree shrews and rabbits, have all the nuclei found within the elephant shrew pons, plus the compact locus coeruleus (A6c) within their locus coeruleus complexes (Maseko et al., 2007). The medulla of the elephant shrew contained five TH+ nuclei (the area postrema (AP) A1, A2, C1 and C2 nuclei) that have been found within the medullary regions of all mammals studied to date (Badlangana et al., 2007; Maseko et al., 2007; Limacher et al., 2008). It is noteworthy that the elephant shrew lacks the rodent specific rostral dorsal midline medullary group (C3) nucleus (Smeets and Gonza´lez, 2000; Maseko et al., 2007; Bhagwandin et al., 2008; Limacher et al., 2008). The overall complement of the putative catecholaminergic nuclei of the elephant shrew phylogenetically aligns it most closely with the hyrax, rabbit, rodents, tree shrew, megabat and primates, but the lack of the A6 compact nucleus distinguishes the elephant shrew from the laboratory rat, rabbit, tree shrew, megabats and primates, and the lack of the C3 nucleus distinguishes it from rodents (Maseko et al., 2007; Gravett et al., 2009). While the elephant shrew shows many similarities with other mammalian species, the elephant shrew exhibits a unique complement of putative catecholaminergic nuclei not shared with other mammalian species studied to date (Manger, 2005; Maseko et al., 2007; Gravett et al., 2009). 4.3. The Serotonergic nuclei

4.2. Putative catecholaminergic system The catecholaminergic system of the elephant shrew exhibited nuclear complexes and nuclei in positions typical of most mammals studied to date (e.g. Smeets and Gonza´lez, 2000;

The serotonergic system of the elephant shrew consisted of a number of nuclei arranged in the typically mammalian rostral and caudal clusters (To¨rk, 1990; Fuxe et al., 2007b). The serotonergic nuclear organization within these clusters was found to be

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

identical to those of all eutherian mammals studied to date (e.g. Maseko and Manger, 2007; Gravett et al., 2009). The monotremes and the opossum (a marsupial) both lack the caudal ventrolateral cluster (Crutcher and Humbertson, 1978) and the monotremes possess hypothalamic serotonergic neurons (Manger et al., 2002c). As a result the elephant shrew is predictably phylogenetically aligned with eutherian mammals. 4.4. Evolutionary considerations This study has shown that the elephant shrew displays a unique complement of nuclei within the cholinergic, catecholaminergic and serotonergic neuronal systems (Manger, 2005). The nuclear organization of these systems support the classification of the elephant shrews as a separate mammalian order, and aligns them most closely with the only other Afrotherian studied to date, the rock hyrax (Gravett et al., 2009). Future comparative studies of the Afrotheria will highlight whether certain nuclei found only in the elephant shrew or rock hyrax are specific to these species or are common features of all Afrotherians. There are three differences within the cholinergic system of the elephant shrew in comparison to the rock hyrax that are of specific interest, as these may be related to a behavioural specialization of this species. In the elephant shrew we observed cholinergic neurons in both colliculi and in the cochlear nucleus. In elephant shrew, the foot-drumming acoustic communication system is specifically associated with the expression of stress, or to signal the presence of a predator (Skinner and Chimimba, 2005). Given that these audible signals are clearly of survival value, the additional cholinergic neurons found within the cochlear nuclei and the colliculi may play a role in attention to these signals (cochlear nucleus) and localization of the source of the signal and the source of the stress or predator (colliculi), as it is well known that the cholinergic system is involved in the processes related to spatial attention (Bellgrove and Mattingley, 2008). It would be of further interest to expand the study of the systems investigated here to members of the Xenarthra, which appear to be the closest extant sister-group to the Afrotheria (Arnason et al., 2008), as a recent study of the cerebral cortex has indicated many similarities not shared by other mammalian groups (Sherwood et al., 2009). The investigation of the cholinergic, catecholaminergic and serotonergic systems of a range of mammalian species may provide important clues regarding mammalian phylogeny (e.g. Maseko et al., 2007) and the evolution of behaviour. 4.5. Methodological considerations One of the essential criteria for determining phylogenetic differences of the systems under investigation across a range of novel species is the specificity of the antibodies being used – i.e. do the antibodies that we are using really recognize what we are looking for, or are we potentially examining false negatives or false positives? This question of false negatives and positives is important in that our work to date appears to be showing trends in the nuclear organization of these systems related to phylogeny amongst mammals. The serotonergic system is clearly quite conservative in terms of its nuclear organization across eutherian mammals, thus, the antibody used currently appears to be acceptable. A similar conclusion can be drawn for the antibody to tyrosine hydroxylase, where the variability across eutherian mammal species appears to be one of lacking of a small number of distinct nuclei rather than additional nuclei. However, in the case of the cholinergic system, our studies, including the current one, have explored the possibility of several previously undescribed cholinergic nuclei (e.g. Gravett et al., 2009) or variance in closely

187

related species with different cholinergic antibodies (e.g. Bhagwandin et al., 2006). In the current study, to ensure the accuracy of our results, we ran a peptide inhibition assay of the cholinergic antibody, which confirmed our findings. The necessity to undertake this inhibition assay for the cholinergic neurons is prompted by previous findings of peripheral and central splice variants of the choline acetyltransferase mRNA (Tooyama and Kimura, 2000), the differential expression patterns of these forms in the nervous system (Yasuhara et al., 2003), and that the AB144P antibody does not always recognize both splice variants (Matsumoto et al., 2007). Thus, while our findings to date appear to be consistent across species, we must be cautious in regards to the strength of interpretation, especially for novel cholinergic nuclei, in the various species under investigation. Ethical statement The elephant shrews used in the present study were caught from wild populations in South Africa under permission and supervision from the appropriate wildlife directorates. All animals were treated and used according to the guidelines of the University of the Witwatersrand Animal Ethics Committee, which parallel those of the NIH for the care and use of animals in scientific experimentation. References Arnason, U., Adegoke, J., Gullberg, A., Harley, E.H., Janke, A., Kullberg, M., 2008. Mitogenomic relationships of placental mammals and molecular estimates of their divergences. Gene 421, 37–51. Asher, R.J., Bennett, N., Lehmann, T., 2009. The new framework for understanding placental mammal evolution. BioEssays 31, 853–864. Badlangana, N.L., Bhagwandin, A., Fuxe, K., Manger, P.R., 2007. Distribution and morphology of putative catecholaminergic and serotonergic neurons in the medulla oblongata of a sub-adult giraffe, Giraffa camelopardalis. J. Chem. Neuroanat. 34, 69–79. Bellgrove, M.A., Mattingley, J.B., 2008. Molecular genetics of attention. Ann. N. Y. Acad. Sci. 1129, 200–212. Bhagwandin, A., Fuxe, K., Manger, P.R., 2006. Choline acetyltransferase immunoreactive cortical interneurons do not occur in all rodents: a study of the phylogenetic occurrence of this neural characteristic. J. Chem. Neuroanat. 32, 208–216. Bhagwandin, A., Fuxe, K., Bennett, N.C., Manger, P.R., 2008. Nuclear organization and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brains of two species of African mole-rat. J. Chem. Neuroanat. 35, 371–387. Bjarkam, C.R., Sorensen, J.C., Geneser, F.A., 1997. Distribution and morphology of serotonin-immunoreactive neurons in the brainstem of the New Zealand white rabbit. J. Comp. Neurol. 380, 507–519. Carlsson, A., Fuxe, K., Ungerstedt, U., 1968. The effect of imipramine on central 5hydroxytryptamine neurons. J. Pharm. Pharmacol. 20, 150–151. Chalmers, J., Pilowsky, P., 1991. Brainstem and bulbospinal neurotransmitter systems in the control of blood pressure. J. Hypertens. 9, 675–694. Crutcher, K.A., Humbertson, A.O., 1978. The organization of monoamine neurons within the brainstem of the North American opossum (Didelphis virginiana). J. Comp. Neurol. 179, 195–222. Dahlstro¨m, A., Fuxe, K., 1964. Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamine in the cell bodies of brainstem neurons. Acta Physiol. Scand. 62, 1–52. Da Silva, J.N., Fuxe, K., Manger, P.R., 2006. Nuclear parcellation of certain immunohistochemically identifiable neuronal systems in the midbrain and pons of the Highveld mole-rat (Cryptomys hottentotus). J. Chem. Neuroanat. 31, 37–50. Dwarika, S., Maseko, B.C., Ihunwo, A.O., Fuxe, K., Manger, P.R., 2008. Distribution and morphology of putative catecholaminergic and serotonergic neurons in the brain of the greater canerat, Thryonomys swinderianus. J. Chem. Neuroanat. 35, 108–122. Fuxe, K., Ho¨kfelt, T., Ungerstedt, U., 1970. Morphological and functional aspects of central monoamine neurons. Int. Rev. Neurobiol. 13, 93–126. Fuxe, K., Marcellino, D., Genedani, S., Agnati, L., 2007a. Adenosine A(2A) receptors, dopamine D(2) receptors and their interactions in Parkinson’s disease. Mov. Disord. 22, 1990–2017. Fuxe, K., Dahlstro¨m, A., Ho¨istad, M., Marcellino, D., Jansson, A., Rivera, A., DiazCabiale, Z., Jacobsen, K., Tinner-Staines, B., Hagman, B., Leo, G., Staines, W., Guidolin, D., Kehr, J., Genedani, S., Belluardo, N., Agnati, L.F., 2007b. From the Golgi-Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: wiring and volume transmission. Brain Res. Rev. 55, 17–54. Gallyas, F., 1979. Silver staining of myelin by means of physical development. Neurol. Res. 1, 203–209.

188

R.P. Pieters et al. / Journal of Chemical Neuroanatomy 39 (2010) 175–188

Gravett, N., Bhagwandin, A., Fuxe, K., Manger, P.R., 2009. Nuclear organization and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brain of the rock hyrax, Procavia capensis. J. Chem. Neuroanat. 38, 57–74. Ho¨kfelt, T., Johansson, O., Fuxe, K., Goldstein, M., Park, D., 1976. Immunohistochemical studies on the localization and distribution of monoamine neuron systems in the rat brain. I. Tyrosine hydroxylase in the mes- and diencephalon. Med. Biol. 54, 427–453. Ho¨kfelt, T., Martenson, R., Bjo¨rklund, A., Kleinau, S., Goldstein, M., 1984. Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In: Bjo¨rklund, A., Ho¨kfelt, T. (Eds.), Handbook of Chemical Neuroanatomy, vol. 2. Classical Neurotransmitters in the CNS, Part 1, Elsevier, Amsterdam, pp. 277–379. Jacobs, B.L., Azmitia, E.C., 1992. Structure and function of the brain serotonin system. Physiol. Rev. 72, 165–229. Jouvet, M., 1969. Biogenic amines and the states of sleep. Science 163, 32–41. Jouvet, M., 1972. The role of monoamines and acetylcholine-containing neurons in the regulation of the sleep–waking cycle. Ergeb. Physiol. 64, 166–307. Kalia, M., Fuxe, K., Goldstein, M., 1985a. Rat medulla oblongata. II. Noradrenergic neurons, nerve fibers and preterminal processes. J. Comp. Neurol. 233, 308–332. Kalia, M., Fuxe, K., Goldstein, M., 1985b. Rat medulla oblongata. III. Adrenergic (C1 and C2) neurons, nerve fibers and presumptive terminal processes. J. Comp. Neurol. 233, 333–349. Karasawa, N., Takeuchi, T., Yamada, K., Iwasa, M., Isomura, G., 2003. Choline acetyltransferase positive neurons in the laboratory shrew (Suncus murinus) brain: coexistence of ChAT/5-HT (Raphe dorsalis) and ChAT/TH (Locus ceruleus). Acta Histochem. Cytochem. 36 (4), 399–407. Kianmaa, K., Fuxe, K., 1977. The effects of 5,7-dihydroxytryptamine-induced lesions of the ascending 5-hydroxytryptamine pathways on the sleep wakefulness cycle. Brain Res. 131, 287–301. Kitahama, K., Geffard, M., Okamura, H., Nagatsu, I., Mons, N., Jouvet, M., 1990. Dopamine- and dopa-immunoreactive neurons in the cat forebrain with reference to tyrosine hydroxylase-immunohistochemistry. Brain Res. 518, 83–94. Kitahama, K., Sakamoto, N., Jouvet, A., Nagatsu, I., Pearson, J., 1996. Dopamine-betahydroxylase and tyrosine hydroxylase immunoreactive neurons in the human brainstem. J. Chem. Neuroanat. 10, 137–146. Leger, L., Charnay, Y., Hof, P.R., Bouras, C., Cespuglio, R., 2001. Anatomical distribution of serotonin-containing neurons and axons in the central nervous system of the cat. J. Comp. Neurol. 433, 157–182. Limacher, A.M., Bhagwandin, A., Fuxe, K., Manger, P.R., 2008. Nuclear organization and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brain of the Cape porcupine (Hystrix africaeaustralis): increased brain size does not lead to increased organizational complexity. J. Chem. Neuroanat. 36, 33–52. Malia, M.J., Adkins, R.M., Allarda, M.W., 2002. Molecular support for Afrotheria and the polyphyly of Lipotyphla based on analyses of the growth hormone receptor gene. Mol. Phylo. Evol. 24, 91–101. Manger, P.R., 2005. Establishing order at the systems level in mammalian brain evolution. Brain Res. Bull. 66, 282–289. Manger, P.R., Fahringer, H.M., Pettigrew, J.D., Siegel, J.M., 2002a. Distribution and morphology of cholinergic neurons in the brain of the monotremes as revealed by ChAT immunohistochemistry. Brain Behav. Evol. 60, 275–297. Manger, P.R., Fahringer, H.M., Pettigrew, J.D., Siegel, J.M., 2002b. Distribution and morphology of catecholaminergic neurons in the brain of monotremes as revealed by tyrosine hydroxylase immunohistochemistry. Brain Behav. Evol. 60, 298–314. Manger, P.R., Fahringer, H.M., Pettigrew, J.D., Siegel, J.M., 2002c. Distribution and morphology of serotonergic neurons in the brain of the monotremes. Brain Behav. Evol. 60, 315–332. Manger, P.R., Fuxe, K., Ridgway, S.H., Siegel, J.M., 2004. The distribution and morphological characteristics of catecholamine cells in the diencephalon and midbrain of the bottlenose dolphin (Tursiops truncatus). Brain Behav. Evol. 64, 42. Maseko, B.C., Manger, P.R., 2007. Distribution and morphology of cholinergic, catecholaminergic and serotonergic neurons in the brain of Schreiber’s longfingered bat, Miniopterus schreibersii. J. Chem. Neuroanat. 34, 80–94. Maseko, B.C., Bourne, J.A., Manger, P.R., 2007. Distribution and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brain of the Egyptian Rousette flying fox, Rousettus aegyptiacus. J. Chem. Neuroanat. 34, 108–127. Matsumoto, M., Xie, W., Inoue, M., Ueda, H., 2007. Evidence for the tonic inhibition of spinal pain by nicotinic cholinergic transmission through primary afferents. Mol. Pain. 3, 41.

Meister, B., Ho¨kfelt, T., Steinbusch, H.W., Skagerberg, G., Lindvall, O., Geffard, M., Joh, T.H., Cuello, A.C., Goldstein, M., 1988. Do tyrosine hydroxylase-immunoreactive neurons in the ventrolateral arcuate nucleus produce dopamine or only L-dopa? J. Chem. Neuroanat. 1, 59–64. Michaloudi, H.C., Papadopoulos, G.C., 1996. Atlas of serotonin-containing cell bodies and fibres in the central nervous system of the hedgehog. J. Brain Res. 1, 77–100. Moon, D.J., Maseko, B.C., Ihunwo, A., Fuxe, K., Manger, P.R., 2007. Distribution and morphology of catecholaminergic and serotonergic neurons in the brain of the highveld gerbil, Tatera brantsii. J. Chem. Neuroanat. 34, 134–144. Murata, Y., Nikaido, M., Sasaki, T., Cao, Y., Fukumoto, Y., Hasegawa, M., Okadaa, N., 2003. Afrotherian phylogeny as inferred from complete mitochondrial genomes. Mol. Phylo. Evol. 28, 253–260. Murray, H.M., Dominguez, W.F., Martinez, J.E., 1982. Catecholaminergic neurons in the brain stem of tree shrew (Tupaia). Brain Res. Bull. 9, 205–215. Oh, J.D., Woolf, N.J., Roghani, A., Edwards, R.H., Butcher, L.L., 1992. Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA. Neuroscience 47, 807–822. Pompeiano, O., 2001. Role of the locus coeruleus in the static and dynamic control of posture. Arch. Ital. Biol. 139, 109–124. Previc, F.H., 1999. Dopamine and the origins of human intelligence. Brain Cogn. 41, 299–350. Reiner, P.B., Fibiger, H.C., 1995. Functional heterogeneity of central cholinergic systems. In: Bloom, F.E., Kupfer, D.J. (Eds.), Psychopharmacology: The Fourth Generation of Progress. Raven, New York, pp. 147–153. Ruggiero, D.A., Anwar, M., Gootman, P.M., 1992. Presumptive adrenergic neurons containing phenylethanolamine N-methyltransferase immunoreactivity in the medulla oblongata of neonatal swine. Brain Res. 583, 105–119. Sherriff, F.E., Henderson, Z., 1994. Cholinergic neurons in the ventral trapezoid nucleus project to the cochlear nuclei in the rat. Neuroscience 58, 627–633. Sherwood, C.C., Stimpson, C.D., Butti, C., Bonar, C.J., Newton, A.L., Allman, J.M., Hof, P.R., 2009. Neocortical neuron types in Xenarthra and Afroteria: implications for brain evolution in mammals. Brain Struct. Funct. 213, 301–328. Siegel, J.M., 2006. The stuff dreams are made of anatomical substrates of REM sleep. Nat. Neurosci. 9, 721–722. Skinner, J.D., Chimimba, C.T., 2005. The Mammals of the Southern African Subregion, 3rd ed. Cambridge University Press, Cape Town. Smeets, W.J.A.J., Gonza´lez, A., 2000. Catecholamine systems in the brain of vertebrates: new perspectives through a comparative approach. Brain Res. Rev. 33, 308–379. Tago, H., McGeer, P.L., McGeer, E.G., Akiyama, H., Hersch, L.B., 1989. Distribution of choline acetyltransferase immunopositive structures in the rat brainstem. Brain Res. 495, 271–297. Tan, M.M.L., Harvey, A.R., 1989. The cholinergic innervation of normal and transplanted superior colliculus in the rat: an immunohistochemical study. Neuroscience 32, 511–520. Tillet, Y., Kitahama, K., 1998. Distribution of central catecholaminergic neurons: a comparison between ungulates, humans and other species. Histol. Histopathol. 13, 1163–1177. Tooyama, I., Kimura, H., 2000. A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers. J. Chem. Neuroanat. 18, 11–22. To¨rk, I., 1990. Anatomy of the serotonergic system. Ann. N. Y. Acad. Sci. 600, 9–35. Vanderwolf, C.H., 1987. Near-total loss of ‘learning’ and ‘memory’ as a result of combined cholinergic and serotonergic blockade in the rat. Behav. Brain Res. 23, 43–57. Vincent, S.R., Reiner, P.B., 1987. The immunohistochemical localization of choline acetyltransferase in the cat brain. Brain Res. Bull. 18, 371–415. Woodall, P.F., Mackie, R., 1987. Caecal size and function in the rock elephant shrew elephantulus myurus (Insectivora, Macroscelidiae) and the Namaqua rock mouse aethomys namaquensis (Rodentia, muridae). Comp. Biochem. Physiol. 87A, 311– 314. Woolf, N.J., 1991. Cholinergic systems in mammalian brain and spinal cord. Prog. Neurobiol. 37, 475–524. Woolf, N.J., Hameroff, S.R., 2001. A quantum approach to visual consciousness. Trends Cogn. Sci. 5, 472–478. Yasuhara, O., Tooyama, I., Aimi, Y., Bellier, J.P., Hisano, T., Matsuo, A., Park, M., Kimura, H., 2003. Demonstration of cholinergic ganglion cells in rat retina: expression of an alternative splice variant of choline acetyltransferase. J. Neurosci. 23, 2872–2881.