Mapping of tyrosine hydroxylase in the alpaca (Lama pacos) brainstem and colocalization with CGRP

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Abstract

The distribution of tyrosine hydroxylase (TH) in the brainstem of alpaca (Lama pacos) has been analysed using immunohistochemical methods. The following catecholaminergic cell nuclei have been detected: A1, C1, A2, C2 and area postrema in the medulla oblongata; A5, A6d, A7sc and A7d in the pons; as have several mesencephalic groups: A8, A9l, A9m, A9v, A9pc, A10, A10c, A10d and A10dc. This nuclear parcellation differs from that found in rodents, but agrees with the results reported in other members of the Artiodactyla order, such as giraffe or pig, and with the catecholaminergic distribution detected in species of other mammalian orders. Thus, these findings support the hypothesis that the animals included in the same order show the same nuclear complement in the neuromodulatory systems. In addition, it seems that other species share the same catecholaminergic groups as the alpaca, suggesting that a specific nuclear disposition was important and worth maintaining throughout evolution. Moreover, the distribution of TH has been compared with that of CGRP by double immunohistochemistry. Double-labelled neurons were very isolated and observed only in a few catecholaminergic groups: A1 and C2 in the medulla oblongata, A6d, A7sc and A7d in the pons, and A9l in the mesencephalon. However, interaction between TH and CGRP may be possible in more brainstem regions, particularly the area postrema. This interaction may prove important in the regulation of the specific cardiovascular control of alpacas given their morphological characteristics.

Research highlights

▶ Distribution of TH and CGRP in Lama pacos brainstem.

Introduction

The alpaca (Lama pacos) is an ungulate in the family of camelidae and a member of the order Artiodactyla. This species is very important in the economy of some South American countries like Peru thanks to its excellent quality wool (de Souza et al., 2007). For this reason, those studies previously carried out on alpacas have mainly covered reproductive aspects; for example, hormonal mechanisms (see de Souza et al., 2008 for a review).

Besides, the physiology of alpacas is very particular for many aspects (de Souza et al., 2008). They are able to live at sea level and at more than 5000 m altitude. This characteristic, plus their special morphology in that they show a long neck with only seven cervical vertebrae like giraffes (Badlangana et al., 2009), make these mammals an interesting, unknown species that could show special and unique regulatory mechanisms.

Catecholamines are organic compounds involved in a wide variety of functions. They have been located in the brain of many animal species, but not in the alpaca (see Smeets and González, 2000 for a review). Several catecholaminergic groups have been detected more or less constantly in the brains of mammals by means of various techniques, including fluorescent methods or immunohistochemistry. Some of these cellular groups have been found in the brainstem, a region associated with the regulation of processes such as blood pressure or cardiac frequency (Saper, 2000).

Recently, the distributions of leucine-enkephalin (Leu-enk) and calcitonin gene-related peptide (CGRP) in the alpaca brainstem have been published (de Souza et al., 2007, de Souza et al., 2008). These two neuropeptides are implicated in multiple functions (see Marcos, 2007) and their distributions in the alpaca brainstem (particularly CGRP) suggest they can play some role, among others, in cardiovascular regulation (see Díaz-Cabiale et al., 2007). The distribution of these two neuropeptides is very similar in the alpaca brainstem. However, neurons containing CGRP were found in a larger amount than those immunolabelled for Leu-enk.

Thus the present work aims to: (1) describe the distribution and nuclear parcellation of the catecholaminergic cell groups in the brainstem of the alpaca using immunohistochemical techniques to detect tyrosine hydroxylase (TH, the limiting enzyme of catecholaminergic synthesis); (2) compare this distribution pattern with published data about other mammals since examining the brains of several species may lead to opportunities that reveal not readily discernable features in rodent and human brains, these being the most far studied species in neuroscience (Manger et al., 2008), and (3) study whether there is a morphological basis for possible interactions between CGRP and TH in the alpaca brainstem by means of double immunohistochemical methods.

Section snippets

Materials and methods

Five male adult alpacas (Lama pacos), weighing 70–80 kg, were used in this study. These animals were obtained from the Faculty of Veterinary Medicine and Animal Sciences of the Peruvian University Cayetano Heredia (Lima, Peru). All the animals used in this study were always maintained at 0 m sea level (from birth to perfusion). The experimental design, protocols and procedures of this work were performed according to the principles of laboratory animal care and the guidelines of ethics and legal

Results

Positive labelling of the cell bodies and fibres containing TH has been observed in several brainstem catecholaminergic groups. These nuclear groups located in the medulla oblongata, pons and mesencephalon are putatively adrenergic, noradrenergic or dopaminergic.

Double-labelling immunohistochemistry allowed the observation that some catecholaminergic regions also displayed CGRP-positive cell bodies and/or fibres (Fig. 1, Fig. 2, Fig. 3). TH-immunoreactive cell bodies were, in general,

Distribution of catecholaminergic cell groups

The description of the catecholaminergic cell groups found in the alpaca brainstem, as well as the morphological characteristics of the TH-immunoreactive neurons, are generally in agreement with those observed in other previously studied mammals (Badlangana et al., 2007a, Bhagwandin et al., 2008, Bux et al., 2010, Da Silva et al., 2006, Dell et al., 2010, Dwarika et al., 2008, Gravett et al., 2009, Kruger et al., 2010, Limacher et al., 2008, Manger et al., 2002, Manger et al., 2003, Manger et

Acknowledgements

This work has been supported by the Junta de Comunidades de Castilla-La Mancha, Consejería de Educación y Ciencia (Grants PAC08-0261-1581 and PCI08-0113) and Consejería de Sanidad and FISCAM (Grant PI-2006/14), Spain. The authors wish to thank Helen Warburton and Antonio Maravilla for checking the style of the English text.

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