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

Journal of Chemical Neuroanatomy 36 (2008) 33–52

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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 Aude’Marie Limacher a, Adhil Bhagwandin 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 28 January 2008 Received in revised form 28 March 2008 Accepted 28 March 2008 Available online 7 April 2008

The distribution, morphology and nuclear organization of the cholinergic, putative catecholaminergic and serotonergic systems within the brain of the Cape porcupine (Hystrix africaeaustralis) were identified following immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin. The aim of the present study was to investigate possible differences in the complement of nuclear subdivisions of these systems in the Cape porcupine in comparison with previous studies of these systems in other rodents. The Cape porcupine is the largest rodent in which these systems have been examined and has an adult body mass of 10–24 kg and an average brain mass of approximately 37 g, around 15 times larger than the laboratory rat. The Cape porcupines were taken from the wild and while these differences, especially that of mass, may lead to the prediction of a significant difference in the nuclear organization or number within these systems, all the nuclei observed in all three systems in the laboratory rat and in other rodents had direct homologues in the brain of the Cape porcupine. Moreover, there were no additional nuclei in the brain of the Cape porcupine that are not found in the laboratory rat or other rodents studied and vice versa. It is noted that the medial septal nucleus of the Cape porcupine appeared qualitatively to have a reduced number of neurons in comparison to the laboratory rat and

Keywords: Acetylcholinesterase Tyrosine hydroxylase Serotonin Evolution Mammal Rodent Neural systems

* Corresponding author. Tel.: +27 11 717 2497; fax: +27 11 717 2422. E-mail address: [email protected] (P.R. Manger). Abbreviations: III, oculomotor nucleus; Vmot, motor nucleus of trigeminal nerve; VI, abducens nucleus; VIId, facial nerve nucleus, dorsal division; 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, 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; ac, anterior commissure; Amyg, amygdala; AP, area postrema; B9, supralemniscal serotonergic nucleus; C, caudate nucleus; C.tail, tail of caudate nucleus; C1, rostral ventrolateral medullary tegmental group; C2, rostral dorsomedial medullary nucleus; C3, rostral dorsal midline medullary group; C/P, caudate and putamen nuclei; ca, cerebral aqueduct; Cb, cerebellum; cc, corpus callosum; Cl, claustrum; CLi, caudal linear nucleus; CN, cerebellar nuclei; CVL, caudal ventrolateral serotonergic group; Cu, cuneate nucleus; CO, cochlear nuclei; Diag.B, diagonal band of Broca; DR, dorsal raphe; DRc, dorsal raphe nucleus, caudal division; DRd, dorsal raphe nucleus, dorsal division; DRif, dorsal raphe nucleus, interfascicular division; DRl, dorsal raphe nucleus, lateral division; DRv, dorsal raphe nucleus, ventral division; DRp, dorsal raphe nucleus, peripheral division; DT, dorsal thalamus; ECu, external cuneate nucleus; EW, Edinger–Westphal nucleus; f, fornix; fGr, fasciculus gracilis; fCu, fasciculus cuneatus; fr, fasciculus retroflexus; GC, periaqueductal grey matter; GP, globus pallidus; GLD, dorsal lateral geniculate nucleus; GLV, ventral lateral geniculate nucleus; Gr, gracile nucleus; Hbm, medial habenular nucleus; Hbl, lateral 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; icp, inferior cerebellar peduncle; io, inferior olivary nuclei; IP, interpeduncular nucleus; Is.Call/TOL, Islands of Calleja and olfactory tubercule; LDT, laterodorsal tegmental nucleus; LV, lateral ventricle; MnR, median raphe nucleus; N.Acc, nucleus accumbens; N.Amb, nucleus ambiguus; N.Bas, nucleus basalis; NEO, neocortex; OB, olfactory bulb; oc, optic chiasm; ot, optic tract; P, putamen nucleus; PBg, parabigeminal nucleus; PC, cerebral peduncle; pc, posterior commissure; pg, pineal gland; PIR, piriform cortex; PPT, pedunculopontine tegmental nucleus; pVII, preganglionic motor neurons of the superior salivatory nucleus or facial nerve; pIX, preganglionic motor neurons of the inferior salivatory nucleus; py, pyramidal tract; pyx, decussation of pyramidal tracts; R, thalamic reticular nucleus; REL, lateral reticular nucleus; Rmc, red nucleus, magnocellular division; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; RVL, rostral ventrolateral serotonergic group; SC, superior colliculus; scp, superior cerebellar peduncle; Sep.M, medial septal nucleus; sfo, subfornical organ; SON, superior olivary nucleus; Sub, subiculum; TOL, olfactory tubercle; vh, ventral horn; Vmes, fifth mesencephalic nucleus; Vn, trigeminal nerve; VPO, ventral pons. 0891-0618/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2008.03.007

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other rodents. The locus coeruleus of the laboratory rat differs in location to that observed for the Cape porcupine and several other rodent species. The Cape porcupine is distantly related to the laboratory rat, but still a member of the order Rodentia; thus, changes in the organization of these systems appears to demonstrate a form of constraint related to the phylogenetic level of the order. ß 2008 Elsevier B.V. All rights reserved.

1. Introduction Hystrix africaeaustralis, the Cape porcupine, is one of the largest rodents within an order that consists of over 2300 species (Jansa and Weksler, 2004). Reports detailing the neuroanatomy of the Cape porcupine brain have yet to be provided. The Cape porcupine has an adult body mass ranging between 10 and 24 kg and an average body length of 84 cm (van Aarde, 1987). This primarily nocturnal hystricomorph species has a rather unusual phenotype in that its back is covered in moveable long sharp spines and quills, up to 50 and 30 cm long, respectively (van Aarde, 1987; De Villiers et al., 1994). The Cape porcupine appears to possess an acute sense of hearing and when threatened initially freezes and rattles its tail, but with further menace becomes aggressive, raises its quills and rapidly backs into the adversary (van Aarde, 1987). The present study details our findings following immunohistochemical examination to reveal cholinergic, putative catecholaminergic and serotonergic systems in the brain of the Cape porcupine. Cholinergic neurons are widely distributed throughout the central nervous system (CNS) of mammals and have diffuse projections (Reiner and Fibiger, 1995; Manger et al., 2002a,b; Maseko et al., 2007); however, the catecholaminergic and serotonergic neurons while projecting to the entire CNS are mainly located within the brainstem (Dahlstro¨m and Fuxe, 1964; Ande´n et al., 1964, 1966; Fuxe et al., 1969, 1970, 2006, 2007a; Diksic and Young, 2001; Manger et al., 2002c; Maseko et al., 2007). The cholinergic system has been shown to globally innervate the entire brain from the olfactory bulb through to the spinal cord (Woolf, 1991). It has been suggested that these cholinergic cell groups are involved in learning and memory, are potentially involved in generating conscious experiences, and are known to be involved in the regulation of REM sleep (Woolf, 1991; Reiner and Fibiger, 1995; Woolf and Hameroff, 2001; Manger et al., 2002a). The catecholaminergic and serotonergic neurons have been found to play a significant role in large numbers of brain functions like neuroendocrine (Fuxe, 1964; Fuxe et al., 1970; Tillet and Kitahama, 1998), cognitive (Previc, 1999; Agnati et al., 2003), mood (Carlsson et al., 1968; Fuxe et al., 1977, 2007a), sleep-wake (Jouvet, 1969, 1972; Kiianmaa and Fuxe, 1977; Lidbrink and Fuxe, 1973; Siegel, 2006), sensory-motor (Pompeiano, 2001; Fuxe et al., 2007a), and central autonomic (Feldman and Ellenberger, 1988; Chalmers and Pilowsky, 1991; Fuxe et al., 1987; see also To¨rk, 1990; Jacobs and Azmitia, 1992; Fuxe et al., 2006, 2007b). Rattus norvegicus and Mus musculus are the two most frequently used animal models in neuroscience. Studies from these species yield information that is extrapolated to our understanding of the human brain, particularly the understanding of human mental illness; however, it remains unclear how representative the brains of rats and mice are of humans, and more particularly, if they are representative of the brains of rodents in general. Studies of the cholinergic, putative catecholaminergic and serotonergic systems within the subcortical regions of the brain of various rodents, such as the laboratory rat (Dahlstro¨m and Fuxe, 1964; Fuxe, 1964; Fuxe et al., 1969, 1970; Lindvall and Bjo¨rklund, 1974; Bjo¨rklund and Lindvall, 1984; Steinbusch, 1981; Ho¨kfelt et al., 1976, 1984; Meredith et al., 1989; To¨rk, 1990; Oh et al., 1992; Ichikawa et al., 1997), laboratory mouse (Ruggiero et al., 1984; Daszuta and Portalier, 1985; Ishimura et al., 1988; Mufson and Cunningham,

1988; Le´ger et al., 1998; Satoh et al., 1991; D’Este et al., 2007), grass rat (Mahoney et al., 2007), hamster (Vincent, 1988), Mongolian gerbil (Janusonis et al., 1999, 2003; Janusonis and Fite, 2001), highveld gerbil (Moon et al., 2007), greater canerat (Dwarika et al., 2008), and African molerats (Da Silva et al., 2006; Bhagwandin et al., 2008) have all demonstrated that the nuclear organization of these systems are the same despite phenotypic and life history differences and millions of years of independent evolutionary trajectories. One exception to this trend is a recent study of the phylogenetic occurrence of cortical cholinergic neurons of various species of rodents established that these neurons are present in the cortex of the subfamily Muridae but are not present in the cortex of other rodent species (Bhagwandin et al., 2006). It has been hypothesized by Manger (2005) that members within the same mammalian order irrespective of phenotype, life history, brain mass or evolutionary temporal distance will exhibit the same complement of nuclei within the brain; however, large changes in brain mass have not been tested in terms of this concept. It is often assumed that an increase in brain mass leads to increased nuclear complexity of the various systems (Stephan et al., 1981). The Cape porcupine brain is approximately 15 times larger in absolute mass than that of the laboratory rat and over 50 times larger than that of the laboratory mouse. Due to this enormous difference in absolute mass, examination of the Cape porcupine brain would provide a useful test to Manger’s (2005) hypothesis, as it probes changes in absolute brain mass (not relative brain size), phenotype, life history and evolutionary distance.

2. Methods and materials The brains of three adult Cape porcupines (H. africaeaustralis) were used for the purpose of this study (Fig. 1). The animals were caught from the wild in Limpopo Province, South Africa under permission of the Limpopo Provincial Government. The animals were treated and used according to the guidelines of the University of

Fig. 1. Photographs of the dorsal aspect of the brains of the Cape porcupine (left) and laboratory rat (middle) as well as a ventral view of the Cape porcupine brain (right). Note the dramatic difference in overall brain size between these two species and the overall typically rodent appearance of the porcupine brain. Scale bar = 1 cm.

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Fig. 2. Photographs of adjacent sagittal sections lying close to the midline of the Cape porcupine brain. (A) Nissl stained section; (B) myelin reacted section; and (C) section immunohistochemically reacted for cholineacetyletransferase. Scale bar in C = 1 cm and applies to all. the Witwatersrand Animal Ethics Committee, which parallel those set down by the NIH for use of animals in scientific experiments. The porcupines were placed under deep barbiturate anaesthesia (Euthanaze, 40 mg/kg, i.p) and then perfused intracardially following cessation of respiration. Initially they were perfused with a cold (4 8C) rinse of 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were removed from the skull and post-fixed overnight in 4% paraformaldehyde in 0.1 PB and then equilibrated in 30% sucrose in PB. The brains were then frozen and sectioned into coronal (n = 2) and sagittal (n = 1) planes (50 mm section thickness) (Fig. 2). A one in five series of stains was made for Nissl, myelin, choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin. The Nissl sections were mounted on 0.5% gelatine coated glass slides, cleared in a solution of 1:1 chloroform and absolute alcohol, then stained with 1% cresyl violet. Sections kept for the myelin series were stored for 2 weeks in 5% formalin solution, mounted on 1% gelatine coated slides, and then stained with a modified silver stain (Gallyas, 1979). The sections used for immunohistochemistry were first treated with an endogenous peroxidase inhibitor (49.2% methanol:49.2% 0.1 M PB:1.6% of 30% H2O2) for 30 min followed by three 10 min rinses in 0.1 M PB under gentle shaking. This was followed by a 2 h preincubation, at room temperature, in a blocking buffer solution made of 3% normal serum (normal rabbit serum for the cholineacetyltransferase and normal goat serum for tyrosine hydroxylase and serotonin), 2% bovine serum albumin (BSA) and 0.25% Triton X100 in 0.1 M PB. The sections were then placed in the primary antibody solution containing the appropriately diluted antibody in 0.25% Triton X100 in 0.1 M PB, 3% normal serum (see above), and 2% BSA for 48 h at 4 8C under gentle shaking. To reveal cholinergic neurons we used anti-cholineacetyltransferase (ChAT, AB144P, Chemicon, raised in goat) at a dilution of 1:2000. To reveal putative catecholaminergic neurons we used anti-tyrosine hydroxylase (TH; AB151, Chemicon, raised in rabbit) at a dilution of 1:6500, and to reveal serotonergic neurons we used anti-serotonin (5-HT, AB 938, Chemicon, raised in rabbit) at a dilution of

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1:10,000. This step was followed by three 10 min rinses in 0.1 M PB. The sections were then incubated in a secondary antibody solution for 2 h. The secondary antibody solution contained a 1:750 dilution of biotinylated anti-rabbit IgG (BA-1000, Vector Labs) in 3% NGS (or anti-goat IgG, BA-5000, Vector Labs, in 3% NRS for the ChAT sections), and 2% BSA in 0.1 M PB. After three 10 min rinses in 0.1 M PB the sections were placed in AB solution (Vector labs) for 1 h, and again rinsed three times in 0.1 M PB. The sections were then treated in 0.05% diaminobenzidine in 0.1 M PB for 5 min (1 ml/section), following which 3 ml of 30% H2O2 was added to the solution in which each section was immersed. The chromatic precipitation was monitored visually under a low power stereomicroscope until the background staining was at a level at which it could assist reconstruction without obscuring the immunopositive neurons. The precipitation was stopped by placing the sections in 0.1 M PB, and the sections were rinsed twice more in the same solution. Sections were mounted on glass slides coated with 0.5% gelatine and left to dry overnight. They were then dehydrated in a graded series of alcohols, cleared in xylene, and coverslipped with Depex. Two negative controls were used in the immunohistochemistry. The first omitted the primary antibody and the second omitted the secondary antibody, both of which resulted in no staining. A low power stereomicroscope was used to observe the sections and architectonic borders were traced according to the Nissl and myelin stained sections using a camera lucida. The immunoreacted sections were matched to the drawings, adjusted slightly for differential shrinkage, and the immunopositive neurons marked. Neurons were considered immunopositive when the cell bodies exhibited clearly stained primary dendrites. The drawings were then scanned and redrawn using the Canvas 8 (Deneba) drawing program. Digital photomicrographs were captured using a Zeiss Axioskop and Axiovision software. No pixilation adjustments, or manipulation of the captured images were undertaken, except for the adjustment of contrast, brightness, and levels using Adobe Photoshop 7. The nomenclature used for the cholinergic system was adopted from Woolf (1991), Manger et al. (2002a), Maseko and Manger (2007), and Maseko et al. (2007), the putative catecholaminergic system from Ho¨kfelt et al. (1984), Smeets and Gonza´lez (2000), Manger et al. (2002b), Maseko and Manger (2007), Maseko et al. (2007), Moon et al. (2007), and Dwarika et al. (2008) and for the serotonergic system from To¨rk (1990), Bjarkam et al. (1997), Manger et al. (2002c), Maseko and Manger (2007), Maseko et al. (2007), Moon et al. (2007), and Dwarika et al. (2008). While the standard nomenclature for the catecholaminergic system is used here, the neuronal groups revealed with tyrosine hydroxylase immunohistochemistry may not correspond directly with these nuclei as has been described in previous studies by Meister et al. (1988), Kitahama et al. (1990, 1996), and Ruggiero et al. (1992). However given the striking similarity of the results of the tyrosine hydroxylase immunohistochemistry to that seen in other rodents this terminology appears appropriate. Clearly further studies in the African porcupine 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 of catecholaminergic nuclei ascribed in this study.

3. Results In this study the cholinergic, putative catecholaminergic, and serotonergic systems of three Cape porcupine brains were revealed using immunohistochemical techniques. The Cape porcupines used in this study had brain and body masses of: (1) 36.7 g and 10.1 kg; (2) 39.1 g and 12.1 kg; and (3) 34.8 g and 13.3 kg. The Cape porcupine’s brain is approximately 15 times larger than that of the laboratory rat (Rattus norvegicus) and over 50 times larger than that of the laboratory mouse (Mus musculus), and represents one of the largest extant rodent brains (Figs. 1 and 2). Despite this large difference in brain mass, the cohort of nuclei described in the present study for the Cape porcupine does not differ from observations previously provided for these systems in other rodent species (see references listed in Section 1). 3.1. Cholinergic neurons The mammalian cholinergic system is generally divided into striatal, basal forebrain, diencephalic, and pontomesencephalic groups, as well as motor cranial nerve nuclei (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007). These groups are found throughout the brain from the level of the anterior horn of the lateral ventricle through to the spinomedullary junction (Figs. 2C, 3 and 4). Each group of cholinergic neurons in the brain of the Cape porcupine brain contained a cluster of distinct nuclei and this

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showed no specific difference to the general mammalian organizational plan. 3.1.1. Striatal cholinergic interneurons 3.1.1.1. Nucleus accumbens. Nucleus accumbens was demarcated as a loosely defined cluster of choline acetyltransferase immunopositive (ChAT+) neurons located ventral to the dorsal striatopallidal complex (caudate, putamen and globus pallidus, see below). The anterior border of this nucleus was coincident with the anterior horn of the lateral ventricle and the posterior border was

coincident with the anterior commissure (Figs. 3D–F and 4C). The location of the nucleus accumbens in the Cape porcupine was typical of all mammals (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007). There was a moderate to low density of ChAT+ neurons identified in this nucleus. The neurons were multipolar and the cell bodies ovoid in shape. No specific dendritic orientation was observed for the neurons in this nucleus. 3.1.1.2. Dorsal striatopallidal complex—caudate/putamen and globus pallidus. The caudate/putamen was located lateral to the lateral ventricle from the anterior horn of the lateral ventricle through to

Fig. 3. Diagrammatic reconstructions of a series of coronal sections through the brain of the Cape porcupine illustrating the location of neurons immunohistochemically reactive for cholineacetyltransferase (black circles), tyrosine hydroxylase (black triangles) and serotonin (open squares). The outlines of the architectonic regions were drawn using Nissl and myelin stains and immunopositive neurons marked on the drawings. Drawing A represents the most rostral section, Y the most caudal. The drawings are approximately 2500 mm apart. See list for abbreviations.

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Fig. 3. (Continued )

the level of the habenular nuclei within the cerebral hemisphere, as is typical of all mammals (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007); however, there was no clear distinction between the caudate and putamen nuclei formed by the internal capsule as seen in some other mammals (Figs. 3E–J and 4A–D). The globus pallidus was located medial and slightly ventral to the caudate/putamen from the level of the anterior commissure to the habenular nuclei. A moderate to low density of ChAT+ neurons were found in the caudate/putamen, while only a small number of ChAT+ neurons were found in the globus pallidus, which were located mostly on the lateral and ventral borders with the caudate/ putamen and nucleus basalis (see below). The neuropil of the globus pallidus was less intensely ChAT+ compared with that of the caudate/putamen. The neuronal morphology in the caudate/ putamen and globus pallidus was similar with the ChAT+ neurons exhibiting a variety of soma including large polygonal and ovoid shapes with a mixture of bipolar and multipolar types. No specific dendritic orientation was found for these neurons. 3.1.1.3. Islands of Calleja and olfactory tubercle. These nuclei were located in the ventral most part of the cerebral hemisphere, ventral to the nucleus accumbens, from the level of the anterior horn of the

lateral ventricle to the level of the anterior commissure (Figs. 3D–G and 4B, C). This is the typical position of these nuclei in all mammals (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007). A clustering of medium to moderate density of ChAT+ neurons was observed with occasional denser clusters in this region representing the islands of Calleja. The ChAT+ neurons within the olfactory tubercle were slightly more intensely immunoreactive than those of the adjacent nucleus basalis (see below) (Fig. 5D). The cell bodies of the neurons were ovoid in shape, multipolar in type and showed no specific dendritic orientation. 3.1.2. Cholinergic nuclei of the basal forebrain 3.1.2.1. Medial septal nucleus. The medial septal nucleus was located in the rostral half of the medial wall of the cerebral hemispheres within the septal nuclear complex, in a position below the rostrum of the corpus callosum and dorsal to the diagonal band of Broca (see below) (Fig. 3F–G). A low density of ChAT+ neurons was found throughout this nucleus. The neuronal numbers were not particularly strongly expressed in a qualitative comparison with other mammals (e.g. Manger et al., 2002a; Maseko and Manger, 2007; Maseko et al., 2007). The cell bodies of

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Fig. 3. (Continued )

the neurons were ovoid in shape, bipolar, and had a dorsoventral orientation of the dendrites. 3.1.2.2. Diagonal band of Broca. This nucleus was located in the ventromedial corner of the cerebral hemisphere in a position anterior to the hypothalamus (Figs. 3E, F and 4A). The nucleus

could be divided into horizontal and vertical bands but this was deemed to be unnecessary as the nucleus is continuous and this division would not add any value to the description. A strongly expressed high-density cluster of ChAT+ neurons was found throughout this nucleus. The neuronal cells of this nucleus were slightly larger than those of the adjacent olfactory tubercle and

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Fig. 3. (Continued ).

islands of Calleja. The ChAT+ neurons were ovoid to circular in shape, clearly multipolar and showed no specific dendritic orientation (Fig. 5A). 3.1.2.3. Nucleus basalis. ChAT+ neurons located ventral and ventromedial to anterior pole of globus pallidus at the level of the anterior commissure, and in a position caudal and dorsal to the olfactory tubercle were demarcated as nucleus basalis (Figs. 3G–I and 4A–D). A varying density of low to moderate ChAT+ neurons were scattered throughout this nucleus and these appeared to be continuous with those of the globus pallidus (see above). The cell bodies were ovoid in shape with a mixture of mostly bipolar and some multipolar types. The neurons in this nucleus exhibited a dorsolateral to ventromedial orientation of dendrites (Fig. 5D).

nuclei (Figs. 3H–J and 4A). The dorsal hypothalamic nucleus was located in the dorsomedial aspect of the hypothalamus between the wall of the third ventricle and the fornix. The ChAT+ neurons of this nucleus were palely stained. The lateral hypothalamic nucleus was located in the dorsolateral portion of the hypothalamus lateral to the fornix, with similarly palely stained low-density ChAT+ neurons. In the ventromedial portion of the hypothalamus, adjacent to the ventrolateral wall of the third ventricle, a cluster of palely stained ChAT+ neurons formed the ventral hypothalamic nucleus. The neurons of this nucleus were also palely stained. The neuronal morphology was similar in all three nuclei with the ChAT+ neurons being ovoid in shape, bipolar and showing no specific dendritic orientation (Fig. 5C). 3.1.4. Pontomesencephalic nuclei

3.1.3. Diencephalic cholinergic nuclei 3.1.3.1. Medial habenular nucleus. The medial habenular nucleus, part of the epithalamus, was located in the dorsomedial portion of the diencephalon adjacent to the third ventricle (Figs. 3J, K and 4A, B), which is typical of all mammals (Woolf, 1991). A dense aggregation of ChAT+ neurons was identified within this nucleus. The ChAT+ neurons were round and small, and due to the dense packing it was impossible to determine if there was any specific dendritic orientation. The axons emanating from these neurons formed the clearly ChAT+ fasciculus retroflexus, which formed a large swirling termination in the ChAT+ interpeduncular nucleus. 3.1.3.2. Hypothalamic cholinergic nuclei. Within the hypothalamus three distinct clusters of ChAT+ neurons were distinguished, these being the dorsal, lateral and ventral cholinergic hypothalamic

3.1.4.1. Parabigeminal nucleus. Located at the very lateral aspect of the midbrain tegmentum in a position ventral to the inferior colliculus, was a cluster of moderately stained ChAT+ neurons that were designated as the parabigeminal nucleus (Fig. 3O). The ChAT+ neurons of this nucleus were moderately densely packed and were smaller than the adjacent neurons of the pedunculopontine tegmental nucleus (see below) (Fig. 5B). The cell bodies were ovoid to circular in shape with a mixture of bipolar and multipolar types. A dorsoventral orientation of the dendrites was observed. 3.1.4.2. Pedunculopontine tegmental nucleus (PPT). The ChAT+ neurons comprising the PPT nucleus were scattered throughout the dorsal aspect of the isthmic and pontine tegmenta in a position anterior to trigeminal motor nucleus (Figs. 3O–R and 4A–C). In the

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Fig. 4. Diagrammatic reconstructions of a series of sagittal sections through the brain of the Cape porcupine. Conventions as in Fig. 3. Drawing A represents the most medial section, D the most lateral. The drawings are approximately 3500 mm apart. See list for abbreviations.

lateral aspect the neurons were observed to surround the superior cerebellar peduncle. A low to moderate density of ChAT+ neurons were found throughout this nucleus. The cell bodies of the ChAT+ neurons were a variety of somal shapes and the neurons themselves expressed a mixture of bipolar and multipolar types, and exhibited no specific dendritic orientation (Fig. 5B).

3.1.4.3. Laterodorsal tegmental nucleus (LDT). A moderate to highdensity cluster of ChAT+ neurons located within ventrolateral portion of the pontine periventricular grey matter was designated as the LDT nucleus (Figs. 3P, Q and 4A). These neurons were found to intermingle with the rostral-most neurons of the diffuse division of the locus coeruleus (A6d) neurons (see below). The neuronal

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Fig. 5. Photomicrographs of some selected neuronal groups immunohistochemically reactive for cholineacetyltransferase in the brain of the Cape porcupine. (A) Diagonal band of Broca; (B) parabigeminal (PBg) and pedunculopontine tegmental (PPT) nuclei; (C) ventral hypothalamic nucleus; and (D) olfactory tubercle (TOL) and adjacent nucleus basalis (N.Bas). Note that the ChAT+ neurons of the PBg nucleus forms a distinct cluster of small neurons compared to the nearby larger more diffuse neurons of the PPT nucleus. Scale bar in (D) = 500 mm and applies to (A), (B) and (D). Scale bar in (C) = 250 mm and applies to (C) only. In all photomicrographs, dorsal is to the top and medial to the left. 3V: third ventricle.

morphology was similar to that of the PPT nucleus with the cell bodies of the neurons exhibiting a variety of somal shapes, a mixture of bipolar and multipolar types, and showing no specific dendritic orientation. 3.1.5. Cholinergic cranial nerve motor nuclei A number of cranial nerve nuclei were found in positions typical of all mammals and all contained large multipolar ChAT+ motor neurons (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007). The ChAT+ nuclei identified were: oculomotor (III), trochlear (IV), motor division of the trigeminal (Vmot), abducens (VI), dorsal and ventral subdivisions of the facial (VIId and VIIv), nucleus ambiguus, dorsal motor vagus (X), hypoglossal (XII), Edinger–Westphal and the preganglionic motor neurons of the salivatory (pVII) and the glossopharyngeal (pIX) nerves (Figs. 3L–Y and 4A, B). The neurons of the Edinger–Westphal nucleus, a primarily visual structure, were small and few compared to the adjacent oculomotor neurons. These ChAT+ neurons were located between and slightly anterior to the oculomotor nuclei within the periaquaductal grey matter immediately adjacent to the midline. The cell bodies were palely stained, ovoid in shape, bipolar in type with dorsoventral dendritic

orientation. The pVII and pIX ChAT+ neurons were located dorsal to the facial (VII) nerve nuclei within the rostral medullary tegmentum. Both pVII and pIX contained a small number of scattered multipolar neurons that evinced the typical motor neuron morphology. These neurons were slightly smaller than those of the facial (VII) nerve nucleus. 3.2. Putative catecholaminergic nuclei Putative catecholaminergic neurons, those that were tyrosine hydroxylase immunoreactive (TH+), formed a number of identifiable nuclear complexes and nuclei located throughout the brain of the Cape porcupine, extending from the olfactory bulbs to the spinomedullary junction (Figs. 3 and 4). The location of the nuclear complexes and nuclei were typical of that seen in other rodents and mammals (Dahlstro¨m and Fuxe, 1964; Fuxe et al., 1969, 2007b; Smeets and Gonza´lez, 2000; Maseko et al., 2007), and were divisible into several clear clusters including the olfactory bulb, diencephalic, midbrain, pontine, and medullary nuclear clusters. For simplicity and ease of interspecies comparison, they are referred to using the nomenclature of Dahlstro¨m and Fuxe (1964),

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Fuxe et al. (1969) and Ho¨kfelt et al. (1984). No putative catecholaminergic nuclei were identified outside the bounds of the classically defined nuclei as sometimes described in other vertebrate orders (Smeets and Gonza´lez, 2000).

Gonza´lez, 2000) (Figs. 3A and 4A–C). The cell bodies of the TH+ neurons were ovoid in shape and found to be a mixture of bipolar and multipolar types. A dense dendritic network emanating from these TH+ neurons was found to surround the glomeruli.

3.2.1. Olfactory bulb (A16) A large number of periglomerular TH+ neurons were located in and around the stratum granulosum in a position typical of all mammals (Lichtensteiger, 1966; Lidbrink et al., 1974; Smeets and

3.2.2. Diencephalic nuclei Six distinct TH+ nuclei were observed within the hypothalamus, which include: the anterior hypothalamic nucleus, dorsal division (A15d); the anterior hypothalamic nucleus, ventral division

Fig. 6. Photomicrographs of selected neuronal groups immunohistochemically reactive for tyrosine hydroxylase within the hypothalamus and midbrain of the Cape porcupine. (A) Dorsal division of the anterior hypothalamic nucleus (A15d); (B) rostral periventricular nucleus (A14); (C) periaqueductal component of ventral tegmental area, dorsal caudal nucleus (A10dc), caudal diencephalic nucleus (A11); (D) ventral tegmental area, dorsal caudal nucleus (A10dc); (E) ventral tegmental area, dorsal nucleus (A10d), ventral tegmental area, central nucleus (A10c), ventral tegmental area nucleus (A10); and (F) substantia nigra, pars compacta nucleus (A9pc), substantia nigra, ventral or pars reticulata nucleus (A9v), substantia nigra, pars medialis nucleus (A9m) which is strongly labelled with its neurons forming many strongly labelled dendrites extending into the pars reticulata. Note the TH+ neurons of A11 nucleus are distinguished from those of the nearby anterior portion of the periaqueductal component of the A10dc nucleus by size and extensive dendritic arborization. Scale bar in (B) = 500 mm and applies to (A) and (B). Scale bar in (F) = 1 mm and applies to (C), (D), (E) and (F). In all the photographs, dorsal is to the top and medial to the left. 3V: third ventricle; ca: cerebral aqueduct; fr: fasciculus retroflexus; RMc: red nucleus, magnocellular division.

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(A15v); the rostral periventricular nucleus (A14); the zona incerta (A13); the tuberal nucleus (A12); and the caudal diencephalic nucleus (A11) (Figs. 3H–L and 4A). The A15d nucleus was located within the dorsal anterior portion of the hypothalamus between the third ventricle and the fornix. The TH+ neurons were found to be ovoid in shape, bipolar in type and exhibited a mediolateral dendritic orientation (Fig. 6A). The A15v nucleus was located within the ventrolateral hypothalamus close to the floor of the brain and adjacent to the optic tracts. A moderate to low density of TH+ neurons with similar neuronal morphology to A15d was found in this region. The cell bodies of the TH+ neurons were ovoid in shape, bipolar and exhibited dendrites oriented parallel to the floor of the brain. The TH+ neurons of the A14 nucleus were located in close proximity to the medial walls of the third ventricle. The TH+ neurons of this nucleus were ovoid in shape, bipolar, and exhibited no specific dendritic orientation (Fig. 6B). The A13 nucleus was found to be a continuation of the A15d nucleus located within the dorsal hypothalamus but lateral to the fornix. The TH+ neurons of this nucleus extended into and around the region of the zona incerta of the ventral thalamus. The TH+ neurons showed a similar morphology to those of the A15d nucleus with ovoid shaped cell bodies, bipolar morphology, and a mediolateral dendritic orientation. The TH+ neurons assigned to the A12 nucleus were located near the midline surrounding the floor of the third ventricle, ventral in the hypothalamus and within and in close proximity to the arcuate nucleus of the hypothalamus. These TH+ neurons were ovoid in shape, bipolar in type and exhibited no distinct dendritic orientation. The A11 nucleus was located in the most caudal part of the hypothalamus where a column of TH+ neurons was observed on either side of the midline around the posterior pole of the third ventricle. A moderate to low density of large multipolar TH+ neurons was found throughout this region. The TH+ neurons were ovoid to polygonal in shape and exhibited no specific dendritic orientation. The A11 nucleus TH+ neurons were distinguished from those of the proximate aqueductal component of the A10dc nucleus (see below) as the A11 neurons are larger than the A10dc neurons (Fig. 6C). 3.2.3. Midbrain nuclei 3.2.3.1. Ventral tegmental area nuclei (VTA, A10 complex). The A10 nuclear complex was found at the level of the oculomotor nucleus in the medial portion of the midbrain tegmentum. This complex was comprised of four nuclei: the ventral tegmental area nucleus (A10); ventral tegmental area, central nucleus (A10c); ventral tegmental area, dorsal nucleus (A10d); and ventral tegmental area, dorsal caudal nucleus (A10dc). These nuclei were seen to arise from the dorsal and dorsolateral areas surrounding the interpeduncular nucleus and extended dorsocaudally into the periaqueductal gray matter where its aqueductal component is formed (Figs. 3L–O and 4A, B). A10 was located dorsal and dorsolateral to the interpeduncular nucleus between this nucleus and the root of the oculomotor nerve. A moderate to high density of TH+ neurons were found throughout this nucleus. The cell bodies of the TH+ neurons were ovoid in shape, bipolar in type with a mostly mediolateral dendritic orientation (Fig. 6E). A moderate density of TH+ neurons immediately dorsal to the interpeduncular nucleus and A10, forming a rough triangular shape or pattern above the midline that tapered dorsally was designated as the A10c nucleus. The TH+ neurons were ovoid in shape, bipolar in type and exhibited a dorsolateral orientation of dendrites (Fig. 6E). The A10d nucleus was located dorsal to the A10c nucleus between the A10c nucleus and the periaqueductal grey matter medial to the oculomotor nucleus. A low density of TH+ neurons were found around the midline in this nucleus and these neurons were ovoid in shape,

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bipolar and exhibited a dorsoventral orientation of dendrites (Fig. 6E). TH+ neurons around the borders of the lower half of the cerebral aqueduct and within the periqueductal grey matter were assigned to the A10dc nucleus. The cell bodies of these TH+ neurons were ovoid in shape, bipolar in type and had dendrites oriented parallel to the edge of the aqueduct (Fig. 6C and D). 3.2.3.2. Substantia nigra nuclear complex (A9). The substantia nigra nuclear complex was comprised of four distinct nuclei located in the ventrolateral portions of the midbrain tegmentum just dorsal to the cerebral peduncles. The four nuclei identified were: the substantia nigra, pars compacta nucleus (A9pc); substantia nigra, pars lateralis nucleus (A9l); substantia nigra, ventral or pars reticulata nucleus (A9v); and substantia nigra, pars medialis nucleus (A9m) (Figs. 3L–O and 4A, B). The A9pc nucleus was seen to be a dense band of TH+ neurons oriented in a mediolateral plane lying immediately dorsal to the cerebral peduncle. A moderate to high density of TH+ neurons were found within this band, and these neurons were ovoid to triangular in shape (Figs. 6F and 7A). The neurons exhibited a mixture of bipolar and multipolar types and showed a predominantly mediolateral orientation of the dendrites. A moderately dense cluster of TH+ neurons located lateral to A9pc on the dorsolateral edge of the cerebral peduncle in ventrolateral portion of the midbrain tegmentum, were assigned to the A9l nucleus. The TH+ neurons of A9l were slightly larger than those of A9pc, all were multipolar in type, had soma that were triangular and polygonal in shape, and exhibited no specific dendritic orientation (Fig. 7A). A low to moderate density of TH+ neurons located ventral to A9pc within the cerebral peduncle was designated as the A9v nucleus. The neuronal cell bodies of this nucleus appeared to be slightly larger than those of A9pc and very similar in morphology to those of A9l, potentially being confluent with the neurons of A9l at the very lateral and dorsal margins of this nucleus. These TH+ neurons exhibited a rough dorsoventral orientation of dendrites (Figs. 6F and 7A). The A9m nucleus was located at the medial edge of the A9pc band between this band and the root of the oculomotor nerve nucleus. A very high-density cluster of TH+ neurons showing similar neuronal morphology to the A9pc nucleus was observed throughout this region. No clear orientation of dendrites was exhibited (Fig. 6F). 3.2.3.3. Retrorubal nucleus (A8). A fairly high number of scattered but sparsely packed TH+ neurons assigned to A8 nucleus were located in the lower half of the midbrain tegmentum (Figs. 3N, O and 4A, B). These were found caudal to the magnocellular division of the red nucleus and dorsal to the A9 complex. The TH+ neurons of this nucleus were ovoid in shape, a mixture of bipolar and multipolar in type and exhibited no specific dendritic orientation (Fig. 7A). 3.2.4. Pontine nuclei—the locus coeruleus (LC) nuclear complex Within the pontine region a large number of TH+ neurons form the locus coeruleus complex. The locus coeruleus complex could be readily subdivided into five nuclei: the subcoeruleus compact nucleus (A7sc); subcoeruleus diffuse nucleus (A7d); locus coeruleus diffuse nucleus (A6d); fifth arcuate nucleus (A5); and the dorsal medial nucleus of locus coeruleus (A4) (Figs. 3P–R and 4A– C). The A7sc contained a moderate to high-density aggregation of TH+ neurons lying within the dorsal portion of the pontine tegmentum adjacent to the periventricular grey matter. This nucleus is equivalent to what was previously described as the subcoeruleus (Dahlstro¨m and Fuxe, 1964). The TH+ neurons were ovoid in shape, a mixture of bipolar and multipolar in type and exhibited a predominantly dorsomedial to ventrolateral orientation of dendrites (Fig. 7B). A7d was located lateral and ventral to

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Fig. 7. Photomicrographs of selected neuronal groups immunohistochemically reactive for tyrosine hydroxylase within the midbrain, pons and medulla of the Cape porcupine. (A) Substantia nigra, pars compacta nucleus (A9pc), substantia nigra, pars lateralis (A9l), substantia nigra, ventral or pars reticulata (A9v), retrorubal nucleus (A8); (B) subcoeruleus compact nucleus (A7sc), subcoeruleus diffuse nucleus (A7d), locus coeruleus diffuse nucleus (A6d); (C) rostral ventrolateral tegmental nucleus (C1) extending from lateral to the superior olivary nucleus (a few small palely stained neurons at this level of the medulla) and forming a distinct cluster above this nucleus; (D) caudal ventrolateral tegmental nucleus (A1). Note how the TH+ neurons of A6d are not in close proximity to the wall of the fourth ventricle as previously noted in the laboratory rat (Dahlstro¨m and Fuxe, 1964). Scale bar in (D) = 1 mm and applies to all. In all the photographs, dorsal is to the top and medial to the left. REL: lateral reticular nucleus; scp: superior cerebellar peduncle; SON: superior olivary nucleus.

the A7sc nucleus and evinced a moderate to low density of scattered TH+ neurons in the lateral and laterodorsal portion of the pontine tegmentum anterior to the trigeminal motor nucleus. Occasionally some neurons of this nucleus were located lateral and dorsal to the superior cerebellar peduncle. The TH+ neurons within this nucleus decreased in density and number with distance from the A7sc nucleus. The TH+ neurons of the A7d nucleus were often mixed with the ChAT+ neurons of the PPT nucleus (see above). The morphology of the A7d neurons was similar to those of A7sc, but there was no specific dendritic orientation (Fig. 7B). Located within the periventricular grey matter, intermingled and caudal to the ChAT+ neurons of the LDT (see above), were the TH+ neurons that formed the A6d nucleus. A moderate to high density cluster of TH+ neurons were found throughout this region. The TH+ neurons of A6d were not found in close proximity to the wall of the fourth ventricle as previously noted in the laboratory rat (Dahlstro¨m and Fuxe, 1964), but were located in the ventrolateral portion of the periventricular grey matter as seen in other non-murine rodent species (e.g. Moon et al., 2007; Dwarika et al., 2008; Bhagwandin et al., 2008). The neurons within this nucleus exhibited a similar morphology to those found in the A7sc and A7d nuclei, but exhibited no clear dendritic orientation (Fig. 7B). A small number of TH+ neurons located in the ventrolateral portion of the pontine tegmentum, medial to superior olivary nucleus, anterior to the facial nerve nucleus and ventral to the trigeminal motor nerve nucleus were assigned to the A5 nucleus. The TH+ neurons were

sparsely packed and showed a similar morphology to the neurons found in the other nuclei of the LC nuclear complex, but they were all multipolar in type. The A4 nucleus was readily distinguished as a small number of TH+ neurons located in the dorsolateral aspect of caudal periventricular grey matter adjacent to the wall of the fourth ventricle and medial to the middle cerebellar peduncle. Again, the neuronal morphology was similar to the neurons found in the other nuclei of the LC complex, and the neurons evinced no specific dendritic orientation. 3.2.5. Medullary nuclei Within the medulla of the African porcupine six putative catecholaminergic nuclei were identified. These nuclei were: the rostral ventrolateral tegmental nucleus (C1); rostral dorsomedial nucleus (C2); rostral dorsal midline nucleus (C3); caudal ventrolateral tegmental nucleus (A1); caudal dorsomedial nucleus (A2); and area postrema (AP) (Figs. 3S–Y and 4A, B). The TH+ neurons of C1 form a substantive column in the ventrolateral medulla from the level of the superior olivary nucleus through to the level of the nucleus ambiguus. This distinct band of TH+ neurons projecting from the edge of the medulla into the tegmentum was found lateral to the superior olivary and facial nerve nuclei, but medial to the nucleus ambiguus. A low number of moderate densely packed TH+ neurons were found within this nucleus and these neurons had soma that were triangular in shape, multipolar in form and exhibited a mesh-like dendritic network

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surrounding the rostrocaudally oriented fasciculi in the medullary tegmentum (Fig. 7C). The TH+ neurons of the A1 nucleus were located in the ventrolateral portion of the medulla from the level of nucleus ambiguus through to the spinomedullary junction. The TH+ neurons of A1 were in a position lateral to the nucleus ambiguus and lateral to the lateral reticular nucleus. The morphology of the neurons of this nucleus was similar to that of the neurons of the C1 nucleus, and they exhibited a similar tegmental dendritic meshwork pattern (Fig. 7D). The A1 column was distinguished from the caudal portions of the C1 column by occupying a position lateral to the nucleus ambiguus, whilst the C1 column was located medial to this nucleus. In the dorsal part of the medulla, anterior and dorsal to the vagus motor nerve nucleus, a moderate number of TH+ neurons were designated as the C2 nucleus. Within this nucleus there was a clear region close to the floor of the fourth ventricle above the vagus motor nerve nucleus termed the dorsal strip (Kalia et al., 1985a,b) and a continuation of this cluster into the region of the tractus solitarius anterior to the rostral pole of the vagus motor nerve nucleus was termed the rostral subdivision of the C2 nucleus (Kalia et al., 1985a,b). The TH+ neurons within the C2 nucleus showed a moderate to low density and were ovoid in shape, bipolar and exhibited no specific dendritic orientation. A few TH+ neurons (less than 50) located in the dorsal part of the midline close to the floor of the fourth ventricle at the same level as the rostral portion of C2 represented the C3 nucleus. The cell bodies of the TH+ neurons of C3 were ovoid in shape, bipolar in type and exhibited a dorsolateral orientation of dendrites. The TH+ neurons of the A2 nucleus were located between and surrounding the dorsal motor vagus nucleus and the hypoglossal nucleus. Occasionally some TH+ neurons were mixed with neurons of the dorsal motor vagus nucleus. A low density of TH+ neurons was found throughout this region, although the number appeared to be more significant than seen in other rodents (e.g. Moon et al., 2007; Dwarika et al., 2008; Bhagwandin et al., 2008). The TH+ neurons of the A2 nucleus were moderate in size, ovoid in shape and bipolar in type. The dendritic orientation was generally mediolateral. Area postrema was located in the midline, dorsal to the central canal and the vagus motor nerve nucleus, and adjacent to the floor of the fourth ventricle in a position just anterior to the spinomedullary junction. A high density of small ovoid TH+ neurons was found throughout this region. The neurons were bipolar in appearance and exhibited no specific orientation of dendrites. 3.3. Serotonergic nuclei The serotonergic nuclei within the brain of the Cape porcupine were found to be the same as those previously identified in all eutherian mammals studied to date (e.g. Maseko et al., 2007). These serotonergic nuclei could be divided into rostral and caudal clusters and were all located within the brainstem. A number of distinct nuclei within these clusters were identified throughout the brainstem from the level of the decussation of the superior cerebellar peduncle through to the spinomedullary junction (Figs. 3 and 4). 3.3.1. Rostral cluster 3.3.1.1. Caudal linear nucleus (CLi). The serotonergic immunoreactive (5HT+) neurons of the CLi nucleus formed a distinct cluster in the ventral midline of the midbrain tegmentum immediately dorsal to the interpeduncular nucleus in a location just anterior to the decussation of the superior cerebellar peduncle (Fig. 3N and O). The neurons of this nucleus are also seen to extend along the lateral border of the interpeduncular nucleus towards the ventral surface

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of the brain (Fig. 9A). The 5HT+ neurons of this nucleus were the most rostral of the serotonergic nuclei in the brain of the Cape porcupine. A moderate density of 5HT+ neurons was identified throughout this region and these cell bodies were ovoid in shape, bipolar in type and exhibited a dorsoventral orientation of dendrites (Fig. 9A). 3.3.1.2. Supralemniscal nucleus (B9). The 5HT+ neurons of this nucleus appeared to be a lateral continuation of the neuronal cluster comprising the ventrolateral portion of the CLi nucleus (see above). The B9 nucleus was found on the dorsolateral aspect of the interpeduncular nucleus caudal to the A9pc nucleus (see above) in a position just above the cerebral peduncle and extended as a loosely packed arc of neurons into the ventrolateral most portion of the midbrain tegmentum (Figs. 3O and 4A, B). The morphology of the 5HT+ neurons of this nucleus was similar to that of the CLi 5HT+ neurons being ovoid in shape and bipolar in type; however, they exhibited a dendritic orientation parallel to the dorsal edge of the cerebral peduncle (Fig. 9A). 3.3.1.3. Median raphe (MnR). The median raphe was characterised by two clear, densely packed columns of 5HT+ neurons located either side of the midline in a pararaphe position (Fig. 3P–R). This nucleus was seen to extend from dorsal to ventral along the midline of the midbrain and pontine tegmentum. The median raphe nucleus was found to extend from a level just caudal to the decussation of the superior cerebellar peduncle to the anterior most level of the trigeminal motor nucleus. The 5HT+ neurons of this nucleus exhibited cell bodies that were ovoid in shape, bipolar in type, with a dorsoventral orientation of the dendrites. 3.3.1.4. Dorsal raphe nuclear complex (DR). Six distinct nuclei were identified within the dorsal raphe nuclear complex: the dorsal raphe interfascicular (DRif) nucleus; dorsal raphe ventral (DRv) nucleus; dorsal raphe dorsal (DRd) nucleus; dorsal raphe lateral (DRl) nucleus; dorsal raphe peripheral (DRp) nucleus; and the dorsal raphe caudal (DRc) nucleus. These six nuclei were for the most part located within the periaqueductal and periventricular grey matter from the level of the oculomotor nucleus to the level of the trigeminal motor nucleus (Figs. 3N–R, 4A, B and 8). The DRif nucleus was located between the two medial longitudinal fasciculi in the most ventral medial portion of the periventricular grey matter. This nucleus exhibited a dense cluster of 5HT+ neurons with cell bodies that were ovoid in shape, bipolar in type, and exhibited a dorsoventral orientation of the dendrites (Figs. 8 and 9C). The DRv nucleus was located immediately dorsal to the DRif nucleus in a position immediately caudal to the oculomotor nuclei. A high density of 5HT+ neurons was observed within the DRv, and these neurons exhibited a similar neuronal morphology to those found within the DRif nucleus, but they did not exhibit a specific dendritic orientation (Fig. 8). In a position immediately dorsal to the DRv nucleus and ventral to the inferior border of the cerebral aqueduct a high-density cluster of 5HT+ neurons with a similar morphology to the neurons of the DRif and DRv nuclei was designated as the DRd nucleus. The 5HT+ neurons of the DRd nucleus exhibited dendrites that had a mediolateral orientation (Figs. 8 and 9B). The 5HT+ neurons located in the ventrolateral portion of the periaqueductal grey matter, anterior to the ChAT+ neurons of the LDT and the TH+ neurons of the A6d nuclei, and lateral to 5HT+ neurons of the DRd and DRv nuclei, were designated as the DRp nucleus. Several 5HT+ neurons of this nucleus were found in the adjacent midbrain and anterior pontine tegmentum and were the only 5HT+ neurons of the dorsal raphe complex located outside the periaqueductal and periventricular grey matter. A moderate to low density of 5HT+ neurons were

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Fig. 8. Photomicrographic montage of the majority of the neuronal groups immunohistochemically reactive for serotonin within the dorsal raphe nuclear complex. Dorsal raphe lateral nucleus (DRl), dorsal raphe dorsal nucleus (DRd), dorsal raphe ventral nucleus (DRv), dorsal raphe interfascicular nucleus (DRif), dorsal raphe peripheral nucleus (DRp). Note the 5HT+ neurons of the DRl nucleus are readily distinguished from the other dorsal raphe nuclei as they are larger and multipolar. Scale bar = 1 cm. In the montage dorsal is to the top. ca: cerebral aqueduct.

identified in this nucleus as compared to the other nuclei of the dorsal raphe complex which have a high density of neurons. The 5HT+ neurons of the DRp nucleus were ovoid in shape, bipolar in type and exhibited no specific dendritic orientation (Fig. 8). The 5HT+ neurons of the DRl nucleus were located dorsolateral to the DRd nucleus and adjacent to the ventrolateral edge of the cerebral aqueduct. The 5HT+ neurons of the DRl nucleus were readily distinguished from those present in the remainder of the dorsal raphe nuclei as they were larger and multipolar in type. A moderate to low density of 5HT+ neurons with no specific dendritic orientation was observed within the DRl nucleus (Figs. 8 and 9D). The DRl neurons were found to intermingle with the TH+ neurons of the A10dc nucleus (see above). At the caudal level of the DRl, as the cerebral aqueduct opens into the fourth ventricle, the two lateralized clusters of the DRl nucleus were seen to coalesce across the midline in the dorsal part of the periventricular grey matter forming an arc of 5HT+ neurons across the midline. This arc of 5HT+ neurons was defined as the DRc nucleus. The neuronal morphology of the 5HT+ neurons of the DRc nucleus was identical to that of DRl. The DRc is classified as an independent nucleus due to the lack of 5HT+ neurons in this region of the brain in monotremes (Manger et al., 2002c; Maseko et al., 2007). 3.3.2. Caudal cluster 3.3.2.1. Raphe magnus nucleus (RMg). The raphe magnus nucleus exhibited a low density of 5HT+ neurons forming weakly coalesced columns on either side of the midline in the rostral medullary tegmentum occurring from the level of the anterior part of the facial nerve nucleus to the anterior border of nucleus ambiguus (Figs. 3S, T and 4A). The 5HT+ neurons were found to be moderate to large in size, bipolar in type with a dorsoventral orientation of dendrites.

3.3.2.2. Rostral and caudal ventrolateral serotonergic medullary columns (RVL and CVL). These columns appeared to be lateral extensions of the 5HT+ neurons that formed the most ventral portion of the RMg nucleus (see above) (Fig. 3S–W). Anteriorly, the neurons of the RVL column were found immediately dorsal to the pyramidal tracts and this neuronal cluster was seen to bifurcate around the anterior pole of the inferior olivary nuclei forming two distinct bilateral columns of 5HT+ neurons in the ventrolateral portion of the medullary tegmentum. These columns of 5HT+ neurons continued caudally in the ventrolateral medulla in a position just lateral to inferior olive. The RVL was designated as that part of the columns located between the level of the facial nerve nucleus and the rostral border of nucleus ambiguus. From this level to the spinomedullary junction the columns are termed CVL. The morphology of the 5HT+ neurons comprising the RVL and CVL columns was identical to that of RMg; however, for the most part they exhibited no specific dendritic orientation except in the very anterior portion of the RVL where the neurons overlying the pyramidal tracts had dendrites that were oriented in a mediolateral plane. The density of the 5HT+ neurons in these columns decreased from moderate to high density through to low density from rostral to caudal. The RVL and CVL columns were continuous in the African porcupines studied, which is similar to the situation seen in all other eutherian mammals previously studied (e.g. Maseko and Manger, 2007; Maseko et al., 2007; Moon et al., 2007; Dwarika et al., 2008; Bhagwandin et al., 2008). Despite this, a distinction is made between the two portions of these columns, as the CVL column has not been found in the opossum or the monotremes (Crutcher and Humbertson, 1978; Manger et al., 2002c). 3.3.2.3. Raphe pallidus nucleus (RPa). The 5HT+ neurons forming this nucleus were located in the ventral midline between the two pyramidal tracts at the level of the facial nerve nucleus (Figs. 3S–U

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Fig. 9. Photomicrographs of selected neuronal groups immunohistochemically reactive for serotonin in the brainstem of the Cape porcupine. (A) Caudal linear nucleus (CLi) with neurons extending along the lateral border of the interpeduncular nucleus, and the supralemniscal nucleus (B9); (B) dorsal raphe dorsal nucleus (DRd); (C) dorsal raphe interfascicular nucleus (DRif); (D) dorsal raphe lateral nucleus (DRl); (E) raphe obscurus nucleus (ROb). Note the two neuronal types identified within the ROb nucleus (see text for details). Scale bar in (A) = 1 mm and applies to (A). Scale bar in (E) = 500 mm and applies to (B), (C), (D) and (E). In all photographs, dorsal is to the top and for (A) and (D) medial is to the left. IP: interpeduncular nucleus.

and 4B). The 5HT+ neurons of the raphe pallidus were smaller than those of the RMg nucleus (see above), a feature that distinguished this nucleus from others. The 5HT+ neurons exhibited ovoid shaped cell bodies and were bipolar in type. The dendrites of this nucleus were found to have a dorsoventral orientation, running parallel to the medial border of the pyramidal tracts.

3.3.2.4. Raphe obscurus nucleus (ROb). The raphe obscurus nucleus was delineated as two loosely arranged bilateral columns of 5HT+ neurons located on either side of the midline from the level of nucleus ambiguus to the spinomedullary junction (Figs. 3U–W and 4A). The columns extended from dorsal to ventral along the midline and two neuronal types were identified within this

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nucleus. The loosely arranged columns of 5HT+ neurons near the midline were fusiform in shape, bipolar in type and exhibited a dorsoventral dendritic orientation. Occasional 5 HT+ neurons associated with this nucleus were located a short distance lateral to the midline. These laterally located 5HT+ neurons were triangular in shape, multipolar in type with two dendrites oriented dorsoventrally and one or more dendrites projecting laterally (Fig. 9E). 4. Discussion The central aim of this study was to test the effect of a greatly increased absolute brain mass on the nuclear organization of the cholinergic, putative catecholaminergic and serotonergic systems in comparison to other species within the order Rodentia. The Cape porcupines studied had brain masses of 36.5 g, 39.1 g, and 34.8 g, which are approximately 15 times larger than that of the laboratory rat and over 50 times larger than that of the laboratory mouse. We did not find any nuclei in these systems in the Cape porcupine that have not been previously described in the laboratory rat or mouse, nor were there any nuclei in earlier descriptions of the laboratory rat or mouse that were not found in the Cape porcupine. Despite this, a few qualitative differences were noted within certain nuclei in the Cape porcupine in comparison to the laboratory rat and other rodents studied. For example, the medial septal nucleus appeared to have decreased neuronal numbers, the parabigeminal nucleus was very distinct, and the locus coeruleus (A6d) nucleus was not positioned close to the fourth ventricle as found in the laboratory rat. The phenotype, life history and time since evolutionary divergence between the laboratory rat and mouse and the Cape porcupine are further factors that may contribute to the prediction that there will be differences in the nuclear organization of these systems in these species. We found that despite these differences and the larger absolute brain mass, our observations provide support for the hypothesis of Manger (2005) that proposes a phylogenetic constraint that acts at the level of the order that specifically restricts the parcellation (sensu Ebbesson, 1980) of the nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems. 4.1. Cholinergic nuclei The cholinergic neurons found in the region of the dorsal striatopallidal complex and basal forebrain of the Cape porcupine studied show no specific difference to that seen in previous studies of the laboratory rat and mouse (Mufson and Cunningham, 1988; Meredith et al., 1989; Oh et al., 1992; Ichikawa et al., 1997), or to the general mammalian organizational plan previously reported (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007); however, while not directly quantified, the number of cholinergic neurons in the medial septal nucleus appeared to be substantially lower than that generally seen in other mammals (Woolf, 1991; Manger et al., 2002a; Maseko et al., 2007). The medial septal nucleus may play a role in fear learning and memory due to its projections to the hippocampus, amygdala and cerebral cortex (Woolf, 1991; Calandreau et al., 2007), thus these behaviours may be processed differently in the African porcupine in comparison to other rodents (Elvander and Ogren, 2005). Within the diencephalon of the Cape porcupines studied, the medial habenular nucleus of the epithalamus and the dorsal, lateral and ventral hypothalamic cholinergic nuclei were found. The three hypothalamic neuronal groups have been reported for the laboratory rats, African mole-rats, laboratory shrews, cats, primates, microbat and megabat (Satoh et al., 1983; Tinner et al.,

1989; Meredith et al., 1989; Tago et al., 1989; Vincent and Reiner, 1987; Oh et al., 1992; Ichikawa et al., 1997; Karasawa et al., 2003; Maseko and Manger, 2007; Maseko et al., 2007; Bhagwandin et al., 2008); however, no hypothalamic cholinergic neurons are present in the monotremes (Manger et al., 2002a). The cholinergic pontomesencephalic nuclei observed within the Cape porcupine brain were the laterodorsal tegmental (LDT), pedunculopontine (PPT) and parabigeminal (PBg) nuclei. LDT and PPT have been located in all mammals studied to date (e.g. Maseko et al., 2007; Bhagwandin et al., 2008); however the monotremes, laboratory shrew and microbat lack the PBg nucleus (Manger et al., 2002a; Maseko and Manger, 2007; Maseko et al., 2007). The Cape porcupine evinced a very distinct cholinergic immunoreactive PBg nucleus, which is most often described as staining rather palely in other rodents (Woolf, 1991; Da Silva et al., 2006; Bhagwandin et al., 2008). A parabigeminal nucleus has been noted in rodents, carnivores, tree shrews, megabats and primates (Murray et al., 1982; Henderson and Sherriff, 1991; Mesulam et al., 1989; Da Silva et al., 2006; Maseko et al., 2007; Bhagwandin et al., 2008). The cranial nerve nuclei in the Cape porcupine were located in positions typical of all mammals previously studied (Woolf, 1991). The cholinergic Edinger–Westphal nucleus was found to be palely stained in the Cape porcupine and has been reported in the laboratory rat, cat, ferret, megabat and primates (Kimura et al., 1981; Armstrong et al., 1983; Satoh et al., 1983; Mizukawa et al., 1986; Henderson, 1987; Vincent and Reiner, 1987; Mesulam et al., 1989; Lavoie and Parent, 1994; Maseko et al., 2007); however, this nucleus has not been observed to be ChAT+ in monotremes, laboratory shrews or the microbat (Manger et al., 2002a; Karasawa et al., 2003; Maseko and Manger, 2007). Cholinergic preganglionic motor neurons of the salivatory and glossopharyngeal nerves were also found in the Cape porcupine. Similar neurons in the macaque monkey, baboon, human, megabat, laboratory rat, cat and ferret have been previously reported (Armstrong et al., 1983; Satoh and Fibiger, 1985; Mizukawa et al., 1986; Henderson, 1987; Shiromani et al., 1988; Mesulam et al., 1989; Maseko et al., 2007); however, these ChAT+ neurons have not been reported in monotremes or the microbat (Manger et al., 2002a; Maseko and Manger, 2007). Overall, the complement of cholinergic neuronal groups in the Cape porcupine exhibit many similarities to that seen in primates, carnivores and other rodents, but this complement is different from that seen in monotremes, microbats and the laboratory shrew. Since the Cape porcupine exhibited an identical complement of cholinergic nuclei to that previously noted in other rodents (Mufson and Cunningham, 1988; Meredith et al., 1989; Oh et al., 1992; Ichikawa et al., 1997; Bhagwandin et al., 2008), the present observations support the proposal of Manger (2005) and indicate that increases in absolute brain mass within an order do not correlate with increased nuclear complexity for the cholinergic system. 4.2. Putative catecholaminergic nuclei Putative catecholaminergic neurons formed a number of identifiable nuclear complexes and nuclei located throughout the brain of the Cape porcupine, extending from the olfactory bulb through to the spinomedullary junction. Periglomerular TH+ neurons within the olfactory bulb appear to be homologous to those seen in other rodents and mammals (Lichtensteiger, 1966; Lidbrink et al., 1974; Lindvall and Bjo¨rklund, 1974; Ho¨kfelt et al., 1977; Bjo¨rklund and Lindvall, 1984; Smeets and Gonza´lez, 2000; Maseko and Manger, 2007; Maseko et al., 2007; Moon et al., 2007; Dwarika et al., 2008; Bhagwandin et al., 2008). Within the diencephalon of the Cape porcupine the A15 dorsal, A15 ventral,

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A14, A13, A12 and A11 neuronal groups were all readily observed. These diencephalic nuclei have been identified in other rodent species, primates, cats, rabbits, megabats and monotremes (Fuxe et al., 1969; Cheung and Sladek, 1975; Blessing et al., 1978; Leshin et al., 1995; Tillet and Kitahama, 1998; Smeets and Gonza´lez, 2000; Manger et al., 2002b; Maseko et al., 2007; Moon et al., 2007; Dwarika et al., 2008). In comparison, the tree shrew seems to lack the A14, A15 dorsal and A15 ventral neuronal groups (Murray et al., 1982), pig, cattle, sheep and the hedgehog lack the A15 dorsal neuronal group (Tillet and Thibault, 1989; Leshin et al., 1995; Michaloudi and Papadopoulos, 1996), the bottlenose dolphin does not have an A13 neuronal group (Manger et al., 2004), and the microbat lacks the A15 dorsal and ventral subdivisions (Maseko and Manger, 2007). Throughout the midbrain of the Cape porcupine the A8 nucleus (retrorubral), and the A9 (substantia nigra) and A10 (ventral tegmental area) nuclear complexes were identified. The A9 and A10 complexes are further subdivided into a number of nuclei (A9pc, A9m, A9l, A9v, A10, A10c, A10d, A10dc). All these subdivisions are found in other rodents, primates, megabats, tree shrews, bottlenose dolphin, artiodactyls, opossum and monotremes (Fuxe et al., 1969; Felten et al., 1974; Crutcher and Humbertson, 1978; Murray et al., 1982; Østergaard et al., 1992; Tillet and Kitahama, 1998; Manger et al., 2002b, Manger et al., 2004; Da Silva et al., 2006; Moon et al., 2007; Maseko et al., 2007; Dwarika et al., 2008; Bhagwandin et al., 2008). Carnivores and rabbits lack the A9v nucleus (Blessing et al., 1978; Henderson, 1987; Dormer et al., 1993), while the microbat has been found to lack the A9v, A10d and A10dc nuclei (Maseko and Manger, 2007). The distinction of the A9v nucleus is not particularly clear in the hedgehog (Michaloudi and Papadopoulos, 1996). Five nuclear subdivisions of the locus coeruleus complex were found in the Cape porcupines studied (A4, A5 A6d, A7sc and A7d). The Cape porcupine lacks the A6 compact neuronal group as noted in previous studies of other rodents, monotremes, hedgehogs, carnivores, bottlenose dolphins, artiodactyls, microbats and opossums (Crutcher and Humbertson, 1978; Manger et al., 2002b, 2004; Michaloudi and Papadopoulos, 1996; Tillet and Kitahama, 1998; Moon et al., 2007; Maseko and Manger, 2007; Dwarika et al., 2008; Bhagwandin et al., 2008), whilst all six subdivisions of the locus coeruleus complex are seen in the megabat, primates, tree shrews and rabbits (Blessing et al., 1978; Murray et al., 1982; Bogerts, 1981; Schofield and Everitt, 1981; Maseko et al., 2007). The location of the TH+ neurons of the A6d nucleus in the laboratory rat (Dahlstro¨m and Fuxe, 1964; Bjo¨rklund and Lindvall, 1984; Ho¨kfelt et al., 1976, 1984) has been found to be different in comparison to that reported herein for the Cape porcupine and other rodent species such as the laboratory mouse (e.g. Ginovart et al., 1996; Von Coelln et al., 2004), hamster (Vincent, 1988), guinea pig (Mulders and Roberston, 2005), African mole-rats (Da Silva et al., 2006; Bhagwandin et al., 2008), highveld gerbil (Moon et al., 2007) and greater canerat (Dwarika et al., 2008). In the laboratory rat the TH+ neurons of the A6d nucleus are found within the periventricular grey matter, but in a medial position adjacent to the floor of the fourth ventricle. In other rodent species including the Cape porcupine the A6d nucleus is found in the ventrolateral portion of the periventricular grey matter and not in close proximity to the floor of the fourth ventricle. This distinct difference between the laboratory rat and other rodent species is worth understanding in more detail considering the amount of research dedicated to this region in the laboratory rat. In this sense, the laboratory rat may not be the most accurate model for extrapolation in understanding the human locus coeruleus as it is not representative of all rodent species or of any primate species studied to date. The putative catecholaminergic nuclei of the

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medulla of the Cape porcupine were comprised of the A1, A2, C1, C2, C3 and AP nuclei. A1, A2, C1, C2 and AP have been found in all mammals studied to date (Maseko et al., 2007; Badlangana et al., 2007); however, the C3 nucleus has only been reported in rodent species, making it a rodent specific nucleus (e.g. Ho¨kfelt et al., 1984; Vincent, 1988; Kitahama et al., 1994; Smeets and Gonza´lez, 2000; Moon et al., 2007; Dwarika et al., 2008; Bhagwandin et al., 2008). The overall complement of putative catecholaminergic nuclei within the Cape porcupine to a large extent resembles that seen in mammals, but the presence of the C3 nucleus aligns this complement specifically and exclusively with that of other rodents. 4.3. Serotonergic nuclei The complement of serotonergic nuclei within the brain of the Cape porcupine appears to be identical to those previously described for the laboratory rat (Dahlstro¨m and Fuxe, 1964; Fuxe et al., 1969; Steinbusch, 1981; Lidov and Molliver, 1982; Waterhouse et al., 1993), laboratory mouse (Daszuta and Portalier, 1985), the Mongolian gerbil (Janusonis et al., 1999, 2003; Janusonis and Fite, 2001), the portion of the brain studied in the Chilean degus (Fite and Janusonis, 2001), the African mole-rats (Da Silva et al., 2006; Bhagwandin et al., 2008), highveld gerbil (Moon et al., 2007) and greater canerat (Dwarika et al., 2008). The nuclear organization of the serotonergic system is very similar in all mammals studied to date (Bjarkam et al., 1997; Maseko et al., 2007); however, monotremes and opossums lack the caudal ventrolateral serotonergic neuronal group (Crutcher and Humbertson, 1978; Manger et al., 2002c). The monotremes also lack the dorsal raphe caudal nucleus and have hypothalamic serotonergic nuclei (Manger et al., 2002c). Thus the present observations of the nuclear subdivisions of this system in the Cape porcupine are consistent with all other rodents and indeed all eutherian mammals studied to date. 4.4. Evolutionary considerations The central aim of this study was to test the effect of a substantially larger absolute brain mass (not relative brain size) on the nuclear organization of three immunohistochemically identifiable neural systems, the cholinergic and biogenic amine systems, and to compare the observations made with species of the same mammalian order that have a substantially smaller brain. It has been reported in previous studies that species within the same mammalian order exhibit the same number of nuclear subdivisions; however species of different orders exhibit different numbers of nuclei (Manger et al., 2002a,b,c, 2003, 2004; see tables presented in Maseko et al., 2007). The Cape porcupine has a brain approximately 15 times larger in absolute mass than the laboratory rat and over 50 times larger than the laboratory mouse, and is to date the heaviest rodent brain in which the cholinergic, catecholaminergic and serotonergic systems have been mapped using immunohistochemical revelation. The current examination of these three systems in the Cape porcupine has demonstrated that increases in brain mass are not always accompanied by increases in the complexity of nuclear subdivisions when species being compared belong to the same mammalian order. The Cape porcupine supports the order specific pattern in that it has a catecholaminergic C3 nucleus that is found exclusively in rodents. Previous studies that have examined more closely related species of the rodent order (e.g. Moon et al., 2007) have revealed that differences in phenotype and life history do not lead to changes in nuclear complexity. The current study, that of the greater canerat

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(Dwarika et al., 2008) and that of the African mole-rats (Da Silva et al., 2006; Bhagwandin et al., 2008) demonstrate that time since evolutionary divergence does not alter the complexity of the neuromodulatory systems if the species belong to the same mammalian order. The study of the three neuronal systems in the brain of the Cape porcupine is a good test of Manger’s (2005) hypothesis due to its large absolute brain mass and very distinct phenotype. The Cape porcupines studied conform to Manger’s (2005) predictions and thus it is possible to conclude that irrespective of absolute brain mass, phenotype, life history and evolutionary distance, the nuclear complexity of the systems studied within the same mammalian order are constant (see tables presented in Maseko et al., 2007). It would be of interest in terms of future work to study the brains of very small rodents (such as Mus minutoides which has an adult body mass of 5 g) and those rodents with relatively large brains for their body mass (such as Pedetes capensis) to determine if major reductions in brain mass or increases in relative brain size are accompanied by changes in nuclear complexity; however, given the results of the current study one may predict that in both these cases the results would be similar. Ethical statement The Cape porcupines used in the present study were captured from the wild in Southern 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. Acknowledgements This study was supported by a grant from the South African National Research Foundation (Gun: 2054204) to PRM, and a Swedish Research Partnership Programme Bilateral Agreement between the Swedish International Development Cooperation Agency and the South African National Research Foundation to KF and PRM. References Agnati, L.F., Franzen, O., Ferre, S., Leo, G., Franco, R., Fuxe, K., 2003. Possible role of intramembrane receptor-receptor interactions in memory and learning via formation of long-lived heteromeric complexes: focus on motor learning in the basal ganglia. J. Neural. Transm. Suppl. 65, 1–28. Ande´n, N.E., Carlsson, A., Dahlstro¨m, A., Fuxe, K., Hillarp, N.A˚., Larsson, K., 1964. Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci. 15, 523–530. Ande´n, N.E., Dahlstro¨m, A., Fuxe, K., Larsson, K., Olson, L., Ungerstedt, U., 1966. Ascending monoamine neurons to the telencephalon and diencephalon. Acta Physiol. Scand. 67, 313–326. Armstrong, D.M., Saper, C.B., Levey, A.I., Wainer, H., Terry, R.D., 1983. Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyl transferase. J. Comp. Neurol. 216, 53–68. 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. 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.

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