Mapping of tyrosine hydroxylase in the diencephalon of alpaca

Journal of Chemical Neuroanatomy 50–51 (2013) 66–74

Mapping of tyrosine hydroxylase in the diencephalon of alpaca(Lama pacos) and co-distribution with somatostatin-28 (1-12)

P. Marcos a,*, M.M. Arroyo-Jimenez a, G. Lozano a, J. Gonzalez-Fuentes a,M.J. Lagartos-Donate a, L.A. Aguilar b, R. Covenas c

a Laboratorio de Neuroanatomıa Humana, Centro Regional de Investigaciones Biomedicas (CRIB), Universidad de Castilla-La Mancha, Facultad de Medicina,

Avenida de Almansa 14, 02006 Albacete, Spainb Peruvian University Cayetano Heredia, School of Medicine ‘‘Alberto Hurtado’’, Lima, Peruc Universidad de Salamanca, Institute of Neurosciences of Castilla and Leon (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems (Lab. 14),

Salamanca, Spain

A R T I C L E I N F O

Article history:

Received 25 January 2013

Received in revised form 21 February 2013

Accepted 21 February 2013

Available online 6 March 2013

Keywords:

Catecholaminergic cell groups

Diencephalon

Hypothalamus

Somatostatin

Artiodactyla

Immunohistochemistry

A B S T R A C T

Based on previous work describing the distribution of somatostatin-28 (1-12) in the male alpaca (Lama

pacos) diencephalon, and owing to the well known interactions between this peptide and the

catecholaminergic system, the aims of this work are (1) to describe the distribution of putative

catecholaminergic cell groups in the alpaca diencephalon and (2) to study the possible morphological

basis of the interactions between these substances in the diencephalon of the alpaca by using double

immunohistochemistry methods. Thus, the distribution of catecholaminergic cell groups in the alpaca

diencephalon agrees with that previously described in the diencephalon of other mammalian species of

the same order: the A11, A12, A13, A14 and A15d cell groups have been identified; however, we have

observed an additional hitherto undescribed cell group containing tyrosine hydroxylase in the medial

habenula. In addition, double-labelling procedures did not reveal neurons containing tyrosine

hydroxylase and somatostatin, suggesting that the hypothalamic interactions between catecholamines

and somatostatin at intra-cellular level must be carried out by a somatostatin molecule other than

fragment (1-12). Otherwise, the overlapping distribution patterns of these substances would suggest

some interconnections between groups of chemospecific neurons. These results could be the starting

point for future studies on hypothalamic functions in alpacas, for example those concerning

reproductive control, since other physiological studies have suggested that this species could have

different regulatory mechanisms from other mammalian species. Our results support the Manger

hypothesis that the same nuclear complement of neural systems exists in the brain of species of the same

order.

� 2013 Elsevier B.V. All rights reserved.

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Journal of Chemical Neuroanatomy

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1. Introduction

Knowledge of the distribution of catecholamines and otherneuroactive substances in the brain of different mammalian

Abbreviations: 3V, third ventricle; A9, substantia nigra; A10, ventral tegmental area;

A11, caudal diencephalic group; A12, tuberal cell group; A13, zona incerta; A14,

rostral periventricular nucleus; A15d?, incipient anterior hypothalamic group,

dorsal division?; A15v, anterior hypothalamic group, ventral division; Arc, arcuate

nucleus; Ci, capsula interna; D6?, D6-type cells?; Fx, fornix; LH, lateral hypothalamic

area; LHb, lateral habenular nucleus; LM, lateral mammillary nucleus; MHb, medial

habenular nucleus; Opt, optic tract; PC, cerebral peduncle; PeVH, periventricular

hypothalamic nucleus; PVH, paraventricular hypothalamic nucleus; Sch, supra-

chiasmatic nucleus; so, supraoptic nucleus; VMH, ventromedial hypothalamic

nucleus.

* Corresponding author. Tel.: +34 967599200; fax: +34 967599340.

E-mail address: Pilar.Marcos@uclm.es (P. Marcos).

0891-0618/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.jchemneu.2013.02.006

species has been available for some years (for a review, see Smeetsand Gonzalez, 2000). However, despite the large number of studiesperformed on the rodent brain (Manger et al., 2008), in manyspecies there is a lack of sufficient data concerning the location ofthese cellular groups in this organ. One of these species is thealpaca (Lama pacos), an ungulate included in the family ofcamelidae and a member of the order Artiodactyla. Although thisspecies has been studied because of its importance for theeconomy of South American countries such as Peru owing to thehigh quality of the wool (De Souza et al., 2007), such studies havemainly focused on physiological reproductive aspects andhormonal mechanisms (for review, see De Souza et al., 2008).These animals have a unique physiology, because they are able tolive at altitudes ranging from sea level to more than 5000 m andhave a long neck (similar to other camelidae members), suggestinginteresting and specific regulatory mechanisms, for example those

P. Marcos et al. / Journal of Chemical Neuroanatomy 50–51 (2013) 66–74 67

implicated in pumping blood against the force of gravity to enablesufficient blood flow to the brain (see Bux et al., 2010). In addition,it has been described that alpacas and llamas, unlike othermembers of order Artiodactyla that ovulate spontaneously, areinduced ovulators (Adams et al., 2005); thus, ovulation is not aregular cyclic event. It is possible that these special physiologicalcharacteristics have a morphological correlation in the alpacabrain.

Catecholamines are organic compounds involved in a largevariety of functions and they have been located in several nuclei ofthe alpaca brainstem (Marcos et al., 2011). However, thedistribution of putative catecholaminergic neurons is unknownin other brain regions involved in the regulation of many importantmechanisms, such as in the diencephalon. For example, themammalian hypothalamus contains neuronal groups controllingintricate physiological mechanisms such as reproductive behav-iour and hence knowledge of the morphological characteristics ofthis region in domestic species may be important from aneconomic point of view. Catecholamines and other classicalneurotransmitters often exert their actions together with severalneuropeptides, as has been described for tyrosine hydroxylase (TH)and calcitonin gene-related peptide (CGRP) in the alpaca brainstem(Marcos et al., 2011). In the hypothalamus of different domesticmammals (e.g., guinea pig, see Marcos et al., 1996, 1997) TH andneuropeptides have been colocalized in the same neurons.Moreover, several forms of the neuropeptide somatostatin havebeen found in ovine hypothalamic neurons displaying oestrogenreceptors (Scanlan et al., 2003), suggesting very complex interac-tions between multiple neuroactive substances in the same region.

Recently, the distribution of somatostatin-28 (1-12) (Som-12)has been described in the alpaca diencephalon (Covenas et al.,2011). The neuropeptide somatostatin is the inhibitor of growthhormone and also inhibits the release of noradrenaline. In addition,somatostatin stimulates the release of serotonin and acetylcholine(Reichlin, 1983). The prosomatostatin precursor is cleaved intothree peptides: somatostatin-14 (first isolated from the hypothal-amus), somatostatin-28 (the entire sequence of somatostatin-14 atits carboxyl terminal plus a double pair of basic amino acids) andSom-12, corresponding to the first 12 amino acids of somatostatin-28 (Benoit et al., 1985). The presence of several forms ofsomatostatin in the mammalian brain has been widely reported(see Covenas et al., 2011 for a review).

Owing to the important actions of these neuroactive sub-stances, the aims of the present work are: (1) to describe thedistribution and nuclear parcellation of catecholaminergic cellgroups in the alpaca diencephalon, detecting TH – the rate limitingenzyme of catecholaminergic synthesis – by using immunohisto-chemical methods; (2) to compare this distribution pattern withsimilar data already published on rodent and human brains, themost studied species in neuroscience, but also taking into accountother previously studied mammals, mainly those included in thesame order as the alpaca; and (3) to determine whethermorphological relationships between Som-12 and TH could existin the alpaca diencephalon using double immunohistochemicalmethods.

2. Material and methods

Five male adult alpacas (L. pacos), weighing 70–80 kg were used. The animals

were obtained from the Faculty of Veterinary Medicine and Animal Sciences of the

Cayetano Heredia Peruvian University (Lima, Peru), and they were consistently

maintained at 0 m above sea level (from birth to perfusion). The experimental

design, protocols, and procedures of this work were performed under the principles

of laboratory animal care and under the guidelines of the ethical and legal

recommendations of Peruvian and Spanish legislation. This work was also approved

by the research commission of the Cayetano Heredia Peruvian University (Lima,

Peru). The animal storage conditions, perfusion and the brain dissection procedure,

as well as Som-12 immunohistochemistry, have been detailed previously (Covenas

et al., 2011).

Once Som-12 was detected with the DAB developing method, coronal sections

were prepared for double-labelling immunostaining, following a modified version

of the protocol described elsewhere (Marcos et al., 1996, 2011). Free-floating

sections were extensively rinsed in phosphate buffered saline (PBS) (6� 10 min)

and pre-incubated with a solution of PBS containing 1% normal horse serum and

0.3% Triton X-100. This solution was used for pre-incubation rinses and for the

dilution of all the antibodies as well as streptavidin conjugated with peroxidase,

used in the second immunohistochemical reaction. Sections were incubated

overnight at 4 8C with primary polyclonal antibody against TH (Sigma) raised in

rabbits, diluted 1:500. After rinses in PBS (3� 10 min) and in the pre-incubation

solution (5 min), sections were incubated at room temperature for 90 min in

biotinylated donkey anti-rabbit antibody (Jackson Laboratories) diluted 1:2000.

They were rinsed again in PBS (3� 10 min) and in the pre-incubation solution

(5 min) and then incubated in streptavidin coupled to peroxidase (Jackson

Laboratories), diluted 1:2000 for 90 min at room temperature. Sections were

rinsed in PBS (3� 10 min) and Tris–HCl buffer (TB, 0.05 M; pH 7.6) for 5 min. Then,

the product of the immunohistochemical reaction was revealed using 4-chloro-1-

napthol as the chromogen. This substance provides a blue precipitate easily

distinguishable from the brown product of DAB, which enables a second

immunohistochemical reaction to be carried out on the same section without

antibody removal even when both primary antibodies are obtained from the same

species. The DAB reaction product masks the antigen and catalytic sites of the first

sequence of immunoreagents, preventing interaction with the reagents of the

second sequence, as previously described (Marcos et al., 1997, 2011).

In order to check that no loss of TH-immunoreactivity had occurred, in some

sections a single reaction for the detection of TH was carried out. The procedure,

antibody dilutions and time of incubation were exactly the same as in the case of the

double-staining protocol, and the results obtained in the single TH immunohisto-

chemical reaction were identical to those observed in double-labelled (Som-12/TH)

sections.

When the second immunohistochemical reaction was completed, sections were

mounted on gelatine-coated slides and coverslipped with a mixture of glycerol and

PBS (3:1) since the chloronaphtol reaction does not allow dehydration.

The results were analyzed with a Nikon Eclipse 80i microscope. Images were

obtained with a digital camera attached to the microscope and viewed using Adobe

Photograph 6.0 Software. In order to improve the visualization of the results, only

the brightness and contrast of the images were adjusted using Adobe1 PhotoShop1

CS 8.0.1 and Canvas 11 Build 1173 software, without any further manipulation of

the photographs.

The distribution of the immunoreactive neurons was analyzed with the

Accustage MDPlot v5.2 computerized digital mapping system (MD3-Digitizer,

Accustage, Minnesota Datametrics, USA). For the nomenclature and location of the

catecholaminergic groups in the diencephalon we also followed the descriptions of

Hokfelt et al. (1976, 1984) as the main references, although the distribution of these

cell groups in other mammalian species was also checked (Bux et al., 2010; Dell

et al., 2010; Dwarika et al., 2008; Kitahama et al., 1990, 1998; Leshin et al., 1995a,

1995b; Manger et al., 2002, 2004; Maseko et al., 2007; Maseko and Manger, 2007;

Moon et al., 2007; Tillet and Kitahama, 1998; Tillet and Thibault, 1989). The

different diencephalic areas were identified with the aid of brain atlases for Lama

glama (obtained from the Mammalian Brain Collections of the University of

Wisconsin, Madison, USA) and non-camelid mammals (Jasper and Ajmone-Marsan,

1966; Paxinos and Watson, 1998), as previously described in the study performed

by Covenas et al. (2011).

3. Results

Positive cell bodies and fibres containing TH were observed inseveral diencephalic areas of the alpaca brain (Fig. 1). While wecannot say whether the neurons immunolabelled with TH aredopaminergic, noradrenergic or adrenergic, studies carried out inother mammals would suggest that they are dopaminergic (Smeetsand Gonzalez, 2000). No additional immunohistochemical reac-tions were performed in order to confirm the exact neurochemicalcontent of the TH-immunoreactive cell bodies, which could beneurons expressing TH without really producing other catechol-amine.

These TH-immunoreactive cell bodies have been detected indiencephalic regions displaying Som-12-positive fibres (seeCovenas et al., 2011), and in some cases TH-positive neuronshave been found in areas previously reported to contain Som-12-immunoreactive cell bodies. The distinct morphological features ofsomatostatinergic and TH-positive neurons confirm the observa-tion of non-double-labelled cell bodies (Fig. 2A). Neurons

Fig. 1. Distribution of TH and Som-12 in the alpaca diencephalon. Schematic drawings of coronal sections of the alpaca diencephalon from rostral (A) to caudal (F) levels. The

distribution of TH is marked by black circles, whereas Som-12-immunoreactive cell bodies are represented by red circles. Figures are represented as hemi-sections, coupled to

their corresponding Nissl-stained sections modified from images obtained from the atlas of Lama glama, available from the Mammalian Brain Collections of the University of

Wisconsin, Madison, USA. Scale bar: 1 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

P. Marcos et al. / Journal of Chemical Neuroanatomy 50–51 (2013) 66–7468

containing Som-12 were in general smaller than TH-immunoreac-tive cell bodies, displayed the typical granular cytoplasmicprecipitate, and had fusiform or round perikarya. By contrast, TH-positive neurons were uni- or bipolar, larger than the somatosta-tinergic perikarya, round or ovoid in shape, and their dark blue-stained cytoplasmatic precipitate was diffuse and non-granular. Insome cases, it was not possible to focus both neuronal populations inthe same plane, even when they were located in the same region.

The fibres displayed different profiles, depending on theirneurochemical content. Som-12-immunoreactive processes werevaricose and mainly short, whereas TH-positive fibres were non-varicose and in general thicker than somatostatinergic profiles.Some of TH-immunoreactive fibres were very long and could befollowed for a considerable distance. Puncta containing one of

the two substances were also observed. In general, immunos-tained fibres were arranged randomly throughout the entirediencephalon.

The results are described from rostral (Fig. 1A) to caudal(Fig. 1F) levels of the diencephalon. Since the mapping of Som-12-immunoreactive cell bodies and fibres has already been published(Covenas et al., 2011), only the distribution of TH-immunoreactiveprofiles was considered. The nomenclature of the putativecatecholaminergic cell groups is the same as that used by Buxet al. (2010) in giraffes since this species belongs to orderArtiodactyla, like the alpaca. In addition, some other mammalianspecies have also been considered for diencephalic nomenclature(Bux et al., 2010; Dell et al., 2010; Dwarika et al., 2008; Kitahamaet al., 1990, 1998; Leshin et al., 1995a, 1995b; Manger et al., 2002,

Fig. 2. Distribution of TH and Som-12 in the diencephalon of Lama pacos. The images show the appearance of neurons containing TH (dark blue precipitate) or Som-12 (brown

colour) in different regions. In (A) both neuronal populations are visible in the rostral-most part of A15v, close to the medial posterior edge of the optic tract. Black arrows

point to TH-immunoreactive neurons, larger than Som-12-positive cell bodies (white arrows). No double-labelled structures were found. Two hypothalamic regions are

shown in image (B): arcuate nucleus (A12) and a more caudal part of A15v than that shown in panel (A), lateral to the median eminence. Images (C) and (D) represent the

regions delimited by rectangles in image (B). In image (C) dark blue TH-positive cell bodies are seen in the supraoptic nucleus, in a region corresponding to the caudal level of

A15v. Neurons containing TH located in the arcuate nucleus, the A12 cell group, are shown in image (D). These neurons are smaller than other TH-positive neurons, but are

located more medially than Som-12-immunoreactive cell bodies; thus, no double-labelled profiles were found in this region. The A13 cell group is visible in image (E).

Neurons containing TH located more dorsally and medially, delimited by a rectangle, could be A15d, but their morphology and disposition are similar to that observed in A13,

as shown in image (F); thus they have been designated A15d?. Scale bar: 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to

the web version of this article.)

P. Marcos et al. / Journal of Chemical Neuroanatomy 50–51 (2013) 66–74 69

P. Marcos et al. / Journal of Chemical Neuroanatomy 50–51 (2013) 66–7470

2004; Maseko et al., 2007; Maseko and Manger, 2007; Moon et al.,2007; Tillet and Kitahama, 1998; Tillet and Thibault, 1989).

3.1. Anterior hypothalamic group (A15 cell group)

In the ventrolateral portion of the hypothalamus, close to thefloor of the brain, a TH-positive cellular group was detected(Fig. 1A). This group is the anterior hypothalamic group, ventraldivision (A15v), and coursed from the medial posterior edge of theoptic tract (Figs. 1A and 2A) to the lateral median eminence(Figs. 1B and 2B). This latter could be included within thesupraoptic nucleus (Figs. 1A–C and 2C) and is well separated fromneurons of the arcuate nucleus (A12 group, Fig. 2B and D), althoughsome other TH-positive cell bodies belonging to A15v located morerostrally were intermingled with Som-12-immunoreactive neu-rons (see Covenas et al., 2011, Fig. 1A). The putative catecholamin-ergic neurons found in the entire A15v were in general round orovoid in shape, uni- or bipolar, and larger in size than Som-12-positive cell bodies (Fig. 2A). Thus, no double-labelled neurons forTH/Som-12 were observed in this region. Fibres containing TH,arranged randomly, were also detected in the A15v region (Fig. 2C).

3.2. Arcuate/tuberal nucleus (A12 cell group)

Neurons containing densely packed TH were located in theventrolateral portion of the hypothalamus (Figs. 1A–C, 2B and 2D),surrounding and below the floor of the third ventricle, in an areacorresponding to the arcuate nucleus. These cell bodies wereconsidered the A12 cell group, and were smaller than the neuronsfound in A15v. In general, their shape was round or ovoid, and theirperikarya were uni- or bipolar. In the ventral-most part of thearcuate nucleus, the A12 neurons followed a ventrolateralorientation. These cell bodies were located more medially thanthe few Som-12-positive neurons observed in the arcuate nucleus(Figs. 1A, 1B and 2D), and no double-labelled neuronal profileswere observed in this region.

3.3. Zona incerta (A13 cell group)

Neurons containing TH were observed in the dorsolateral part ofthe hypothalamus (Fig. 1C and D). This cell group extended from thewalls of the third ventricle to the lateral hypothalamic area and thezona incerta, close to the internal capsule, and is referred to as theA13 cell group (Fig. 2E). The neurons of the A13 group formed atriangular area, with the base of the triangle directed towards thewall of the third ventricle. In this region, TH-immunoreactive cellbodies were mainly round or ovoid, uni- or bipolar, with differentsizes. In the dorsal part of this region (Fig. 1C and D) it seems that themajority of the neurons displayed large perikarya, similar to thoseobserved in the A15v cell group (Fig. 2E and 2F), whereas in theventral-most part a mixture of small and large neurons was found.These large TH-positive neurons located in the dorsal part of A13could correspond to a possible dorsal division of A15, and hence arereferred to as A15d? (Fig. 2F). In the lateral-most part of A13 (towardsthe lateral hypothalamic area), the number of TH-immunoreactiveneurons decreased (Fig. 1D); however, a high density of fibrescontaining TH was detected (Fig. 3A). In general, these fibres werefound running parallel to the internal capsule and seemed to be incontact with the Som-12-immunoreactive cell bodies detected in thelateral hypothalamic area (see Covenas et al., 2011). No double-labelled TH/Som-12 neurons were observed in this region.

3.4. Rostral periventricular nucleus (A14 cell group)

Parallel to the walls of the third ventricle, bilateral columns ofTH-immunoreactive neurons were observed (Fig. 1A–D). These cell

bodies had different sizes and shapes and corresponded to the A14cell group (Fig. 3B). They were located in a region with a highdensity of Som-12-positive fibres, and hence the blue precipitate ofthe TH-positive reaction seemed weaker than in other putativecatecholaminergic regions.

3.5. Caudal diencephalic group (A11 cell group)

Few neurons containing TH were observed in the caudal-mostpart of the diencephalon, adjacent to the posterior pole of the thirdventricle and within the grey matter, corresponding to the A11 cellgroup (Fig. 1E and F). They were round or ovoid and mainlyunipolar, although some bipolar cell bodies were also found.

3.6. Other areas

In the caudal diencephalon, neurons containing TH were foundin the medial habenular nucleus (Figs. 1E, 1F and 3C). They weresmall, similar to those observed in the A12 cell group, anddisplayed a round-ovoid morphology. These cell bodies werearranged in two groups: one more dorsal, with darkly stainedneurons, and another group located more ventromedially, withperikarya showing only weak TH immunostaining.

Regarding the Som-12-positive cell bodies, these were found inthe same areas as described previously (Covenas et al., 2011) andalso in the reticular thalamic nucleus (Figs. 1A–D and 3D). Thesesmall neurons had round perikarya and were closely appended tothe TH-immunoreactive fibres also found in this nucleus.

4. Discussion

4.1. Distribution of catecholaminergic cell groups

The description of the catecholaminergic cell groups found inthe alpaca diencephalon is in general in agreement with thatobserved in other members of the order Artiodactyla reportedpreviously (Bux et al., 2010; Dell et al., 2010; Leshin et al., 1995a,1995b, 1996; Tillet and Thibault, 1989) as well as in othermammals (Maseko et al., 2007; Smeets and Gonzalez, 2000; Tillet,1994; Tillet and Kitahama, 1998; Bhagwandin et al., 2008),including humans (Kitahama et al., 1998). In some species, dataabout certain catecholaminergic nuclei are lacking, and hence thewhole diencephalic nuclear complement remains unknown. Thus,in the opossum (Didelphis virginiana) only A11 has been described.No data on the two subdivisions of A15 are available for thebottlenose dolphin (Tursiops truncatus), pygmy marmoset (Cebuella

pigmaea) or baboon (Papio papio), and regarding this latter speciesthere is also a lack of references about A14 (see Dell et al., 2010).Thus, a complete description of the diencephalic catecholaminer-gic cell groups has been reported for 31 mammalian species.

The most constant catecholaminergic cell groups are A11 andA12, since they are present in all the species with available data.The catecholaminergic cell group A13 has not been found inbottlenose dolphin (Tursiops truncatus), and A14 is missing in thetree shrew (Tupaia glis). Regarding the A15 subdivisions, six of the31 species do not show the ventral part: Schreiber’s long fingeredbat (Miniopterus scheribersii), the little free-tailed bat (Chaerophon

pumilis), Commerson’s leaf-nosed bat (Hipposideros commersoni),the Persian trident bat (Triaenops persicus), the rabbit (Oryctolagus

cuniculus), and the tree shrew (T. glis). In two other species (heart-nosed bat, Cardioderma cor; and African sheath-tailed bat, Coleura

afra) this cellular group has only been observed occasionally.However, the most conspicuously absent part of the A15subdivisions was the dorsal part, which has not been detectedin these same species (except rabbits, which did show A15d) or inthe hedgehog (Erinaceus europaeus), pig (Sus scrofa), sheep (Ovis

Fig. 3. Distribution of TH and Som-12 in the diencephalon of Lama pacos. The most lateral part of A13 is shown in image (A). In this area, corresponding to the zona incerta, few

neurons containing TH (black arrows) but a high density of TH-immunoreactive fibres was found. The Som-12-positive neurons located in this region are easily

distinguishable from the dark blue precipitate of TH-immunoreactive profiles. These peptidergic neurons could be innervated by the numerous fibres containing TH detected

in this area. (B) Image showing the periventricular nucleus, A14 cell group. In this region, a high density of fibres containing Som-12 was found, suggesting that the neurons of

A14 could receive peptidergic influences. The medial habenular nucleus (C) displays neurons containing TH. This nucleus does not show TH labelling in any other species of

the order Artiodactyla. The reticular thalamic nucleus is shown in image (D). Small neurons containing Som-12 were detected in this nucleus, surrounded by TH-positive

fibres. Scale bar: 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

P. Marcos et al. / Journal of Chemical Neuroanatomy 50–51 (2013) 66–74 71

aries), giraffe (Giraffa camelopardalis), cattle (Bos taurus), andelephant shrew (Elephantulus myurus). In conclusion, 6 of the 31species (19.35%) displayed the same diencephalic catecholamin-ergic nuclear complement as the alpaca: namely hedgehog,elephant shrew, cattle, giraffe, sheep, and pig. The latter four areungulates, members of the order Artiodactyla. All these datasupport the hypothesis of Manger (2005): ‘‘within an order, allspecies exhibit the same complement of subdivisions of neuralsystems, no matter what the brain size, phenotype, or life history ofthe species’’. In fact, on comparing the data available about thewhole putative catecholaminergic system (Marcos et al., 2011), thealpaca brain shows the same nuclear complement as the pig, sheepand giraffe; since other species share some nuclei, but not all, withthe brainstem and diencephalon catecholaminergic cell groups ofthe alpaca.

In the diencephalon, the most variable catecholaminergicregion is A15d, since 13 of the 31 species lack this subdivision.However, rodents, primates and monotremes displayed a clearlyidentifiable A15d cellular group. In our study, as well as in thediencephalon of the giraffe and other Artiodactyla, some catechol-aminergic cell bodies were detected in the dorsal-most region ofA13 that could be similar to A15d, but these neurons could not be

clearly separated from the A13 group. Neuronal size is not a goodcriterion for the classification of these groups, since in the alpacathis area shows large and small neurons containing TH, althoughthis characteristic has previously been described in sheep A13(Tillet and Thibault, 1989). For this reason, these cell bodies havebeen designated A15d?, although they may be a dorsomedialextension of the A13 cellular group, as in giraffes (Bux et al., 2010).On comparing the morphology and disposition of the wholecatecholaminergic cell groups in giraffe and alpaca, some minordifferences can be found, mainly regarding the orientation ofdendrites and the density of A11 neurons containing TH. In thealpaca diencephalon, only a few neurons were detected in thisregion, whereas in giraffes A11 contains a moderate density of TH-positive cell bodies (Bux et al., 2010). Otherwise, from themorphological point of view the giraffe is the most similar speciesto the alpaca regarding the catecholaminergic system.

However, in the alpaca diencephalon a cellular group contain-ing TH was observed in the medial habenular nucleus, not detectedin other members of order Artiodactyla. We cannot rule out thepossibility of methodological explanations for the immunostainingof these cells, although their labelling was specific according to allthe antibody controls performed during this study. It is possible

P. Marcos et al. / Journal of Chemical Neuroanatomy 50–51 (2013) 66–7472

that these TH-positive neurons could produce TH without beingreally functional catecholaminergic cells. Although it has beendescribed that many catecholaminergic cell groups belonging tothe hypothalamus develop ontogenetically in prosomeres p4–p6,the dorsal portion of the alar plate of prosomeres p1–p3 alsocontains catecholaminergic cell groups. For example, in rats andsome cartilaginous fishes a catecholaminergic cell group has beenfound in the habenula, and in early human embryos a transientexpression of TH has been described in the habenula, whichdevelops as from p2 (Smeets and Gonzalez, 2000). Immunohisto-chemical studies performed in rats for the detection of thearomatic acid decarboxylase (AADC) enzyme allowed the identifi-cation of some neurons of the habenular region as the D6 cellgroup. Theoretically, D cell groups are AADC-positive and TH-negative, and hence our results may indicate that the TH-positiveneurons found in the habenular region of the alpaca brain do notbelong to D-type cells. Since in the present work no double-labelling procedures were performed, we cannot make compar-isons with the results obtained in rats. It is possible that the TH-positive habenular neurons found in the alpaca diencephalon maybe the remainder of some transient expression of neuronscontaining TH during the ontogeny, and this could be a specificmechanism in alpaca development, although more studies areneeded to fully elucidate this question.

The type of monoamine contained in TH-immunoreactiveneurons found in the alpaca diencephalon could not beenidentified, since in the present study no multiple labelling wasperformed. However, previous works carried out in ungulates(Tillet and Kitahama, 1998) and other mammalian species (Smeetsand Gonzalez, 2000) have reported that the sheep arcuate nucleus(A12) contains numerous AADC-positive neurons showing asimilar distribution to that of TH-immunoreactive cell bodies:thus, the catecholaminergic cell bodies observed here in this regioncould be dopaminergic. The same labelling pattern for enzymes ofthe biosynthetic pathway of catecholamines has been found inA15v in sheep. In contrast, some neurons located in these tworegions are TH-positive but AADC-immunonegative in rats andhumans, and they synthesize L-DOPA. Dopamine (DA) immuno-histochemistry performed in other mammal species reveals thatmost of the cells of the A11–A15 areas contain DA as aneurotransmitter (Smeets and Gonzalez, 2000). In pigs, nodopamine-b-hydroxylase (DBH) has been detected in hypotha-lamic neurons, although both TH and DBH have been observed infibres across areas containing TH-immunoreactive neurons (Leshinet al., 1996). In sheep, A11–A15 groups are considered asdopaminergic owing to the absence for TH-DBH double immuno-staining (Tillet and Thibault, 1989). Taken together, these resultssuggest that this could also be the case of the alpaca diencephalon.In order to confirm such a hypothesis, more studies are requiredusing antibodies against phenylethanolamine-N-methyltransfer-ase (PNMT) to detect adrenaline, DBH for noradrenaline identifi-cation, or AADC to visualize the immunostaining of dopamine.

4.2. Somatostatinergic distribution

Our results on the distribution of Som-12-immunoreactiveprofiles are fairly similar to those described previously by Covenaset al. (2011). The only difference is that in the present work thepresence of neurons containing Som-12 was observed in thereticular thalamic nucleus, where it has not been detectedpreviously. This could be due to some minor differences in themethodology employed, since the species used in both studies wasthe same. For example, Covenas et al. (2011) used the ABC complexfor the immunohistochemical detection of Som-12, whereas in thepresent study we incubated the sections with streptavidin. Thisstep could possibly explain the difference. In cats, monkeys and

humans (Bouras et al., 1987; Graybiel and Elde, 1983) somato-statin-28 and other prosomatostatin-derived peptides have beenfound in the neurons of the reticular thalamic nucleus, as weobserved in the alpaca for Som-12. However, no data on thedistribution of different forms of somatostatin in the alpacadiencephalon are available.

4.3. TH-Som-12 double labelling

Neuronal immunostaining for TH and Som-12 was observed inthree diencephalic areas of the alpaca brain. However, the differentmorphologies and locations of the TH and the somatostatinergicneuronal populations indicate that no double-labelled cell bodieswere detectable. This first impression has been confirmed by ourresults, obtained by double-labelling immunohistochemistry.

In the lateral hypothalamic area (partially coincident with theA15v cell group) both populations were detected in the samelocation, but TH-containing neurons were larger than Som-12-positive cell bodies. The arcuate nucleus, where Som-12-immu-noreactive neurons were observed, is the A12 cell group.Catecholaminergic neurons were densely packed more mediallythan the scattered Som-12-positive cell bodies found in this area.In addition, these Som-12-positive cell bodies were smaller thanthe TH-immunoreactive neurons. Finally, in the lateral-most partof zona incerta, part of the A13 cell group, neurons containing Som-12 were observed, but as in the other regions they were smallerthan TH-positive cell bodies and no double-labelled neurons weredetected. The interaction between TH and somatostatin has beendescribed previously in the mammalian brain (Thermos et al.,1996; Ceccatelli et al., 1989; Elde and Hokfelt, 1979). Our resultsshow TH and Som-12 overlapping distribution patterns suggestingphysiological interactions between groups of chemospecificneurons. It is possible that interactions at intra-cellular levelcould include molecules of somatostatin of different lengths fromthe (1-12) fragment, since we did not find double-labelled neurons.A good candidate could be somatostatin-14, first isolated from thehypothalamus (Bouras et al., 1987), and already described as amodulator of dopamine release in rat striatum (Thermos et al.,1996).

Although no interactions at intra-cellular level could bepossible for TH and Som-12 due to their separate distributionpatterns, the overlapping between these distributions suggeststhat they could influence each other functions (see Section 4.4).The high density of fibres containing TH detected running inparallel to the internal capsule seemed to be in contact with Som-12-positive neurons found in the lateral hypothalamic area,suggesting a possible catecholaminergic regulation of somatosta-tinergic neurons. This could also be possible in the reticularthalamic nucleus, where some cell bodies containing Som-12 weresurrounded by TH-immunoreactive fibres.

The opposite pattern, that is, a possible somatostatinergicregulation of the TH-positive neurons, could occur in A14(periventricular nucleus) since a very high density of fibrescontaining the peptide was observed in close relationship withcatecholaminergic cell bodies. However, more direct evidences areneeded to confirm these possible interactions.

4.4. Functional considerations

It is well known that catecholamines have a strong influenceover the control of many diencephalic functions, mainly thesecretion of hormones by the pituitary gland, and they playimportant roles in reproductive and neuroendocrine aspects. Forexample, dopamine secreted by A12 neurons is the major inhibitorof prolactin release from lactotrophs. It has also been describedthat dopamine inhibits the pulsed release of luteinizing hormone

P. Marcos et al. / Journal of Chemical Neuroanatomy 50–51 (2013) 66–74 73

(LH). Pituitary gland growth hormone (GH) secretion is stronglyinhibited by somatostatin. In the sheep hypothalamus it has beendescribed that two molecules of somatostatin (Som-12 andsomatostatin-14) are present in the same hypothalamic neurons,and that these substances are activated in response to oestradioltreatment, which induces both GH and LH surges (Scanlan et al.,2003), most likely mediated by an indirect interneuronal pathway.The authors reported that oestrogen receptors were only found inarcuate somatostatinergic cells. It should be noted that in sheep anadditional, seasonal control of reproduction has been described,involving seasonal inhibition of the pulsed LH release induced bysteroids and mediated by A15 dopaminergic neurons (for reviewsee Smeets and Gonzalez, 2000). In the alpaca diencephalon, Som-12-positive and catecholaminergic-independent populations havebeen observed, suggesting that the hypothalamic influence on GHand LH surges may be mediated by these two populationsseparately.

However, an induced ovulation mechanism has been demon-strated in alpacas and other camelids (see Adams et al., 2005), andthe presence of a potent ovulation-induced factor has beendescribed in the seminal plasma of alpacas and llamas that eliciteda surge in the circulating concentrations of LH and induced aluteotropic response (Kersahw-Young et al., 2012). The existenceof this factor may be a specific evolutionary mechanism, and it ispossible that the hypothalamic regulation of reproduction inalpacas could be different from that of other mammalian species.This difference could have a morphological correlation with thedistribution of several neuroactive substances in the hypothalamicregions involved in this function. From the economy point of view,it seems of interest gain a good understanding of the regulationmechanisms underlying the behavioural aspects of domesticanimals, since in some countries they represent importantresources.

5. Conclusions

The distribution of putative catecholaminergic cell groups inthe alpaca brain agrees with that found in other mammals,particularly those of the order Artiodactyla, since A11, A12, A13,A14, and A15v cell groups were observed in the alpacadiencephalon. In addition, cell bodies containing TH were observedin the medial habenular nucleus. Double-labelling immunohis-tochemistry for TH and Som-12 allowed the detection of differentlocations of neurons containing these substances. Thus, nocoexistence between them has been found in the alpacadiencephalon. The special characteristics of the reproductivebehaviour of L. pacos could have a morphological basis, mainlyin the hypothalamic region, and the aim of the present work was tocontribute to a better knowledge of the morphology of the alpacabrain.

Acknowledgements

This work has been supported by the Junta de Comunidades deCastilla-La Mancha, Consejerıa de Educacion y Ciencia (GrantsPAC08-0261-1581 and PCI08-0113) and Consejerıa de Sanidad andFISCAM (Grant PI-2006/14), Spain. The authors thank N. Skinner(University of Salamanca, Spain) for stylistic revision of the Englishtext.

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