Immunohistochemical mapping of pro-opiomelanocortin- and pro-dynorphin-derived peptides in the alpaca (Lama pacos) diencephalon

https://doi.org/10.1016/j.jchemneu.2014.06.001Get rights and content

Highlights

  • ACTH-, alpha-MSH-immunoreactivity is reported in the alpaca diencephalon.

  • Immunoreactive fibres/cell bodies containing opioid peptides are described.

  • Peptidergic cell bodies were only found in the hypothalamus.

  • Peptidergic fibres were widely distributed throughout the diencephalon.

  • A close anatomical relationship was observed between the peptidergic fibres.

Abstract

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibres containing non-opioid peptides (adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone) and opioid peptides (beta-endorphin (1–27), alpha-neo-endorphin, leucine-enkephalin) in the alpaca diencephalon. No immunoreactive cell bodies containing ACTH were found. Perikarya containing the other four peptides were observed exclusively in the hypothalamus and their distribution was restricted. Perikarya containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin showed a more widespread distribution than those containing leucine-enkephalin or beta-endorphin (1–27). Cell bodies containing pro-opiomelanocortin-derived peptides were observed in the arcuate nucleus, anterior and lateral hypothalamic areas and in the ventromedial and supraoptic hypothalamic nuclei, whereas perikarya containing alpha-neo-endorphin (a pro-dynorphin-derived peptide) were found in the arcuate nucleus, dorsal and lateral hypothalamic areas, and in the paraventricular, ventromedial and supraoptic hypothalamic nuclei. Immunoreactive cell bodies containing leucine-enkephalin were found in the lateral hypothalamic area and in the paraventricular hypothalamic nucleus. Immunoreactive fibres expressing pro-opiomelanocortin-derived peptides were more numerous than those expressing pro-dynorphin-derived peptides. A close anatomical relationship was observed: in all the diencephalic nuclei in which beta-endorphin (1–27)-immunoreactive fibres were found, fibres containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were also observed. Fibres containing beta-endorphin (1–27), alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were widely distributed throughout the diencephalon, but fibres containing ACTH or leucine-enkephalin showed a moderate distribution. The distribution of the five peptides studied here is also compared with that reported previously in other mammalian species. The widespread distribution observed indicates that both the pro-dynorphin and the pro-opiomelanocortin systems are involved in multiple physiological actions (e.g., food intake, thermoregulation, neuroendocrine and reproductive mechanisms) in the alpaca diencephalon.

Introduction

Since 2007 knowledge of the chemical neuroanatomy of neuropeptides in South-American camelids has increased considerably. This is because, using immunocytochemical techniques, reports have been made about the distribution of calcitonin gene-related peptide in the alpaca brainstem and diencephalon (Coveñas et al., 2012, de Souza et al., 2008, Marcos et al., 2011, Marcos et al., 2013), the presence of somatostatin-28 (1–12) in the alpaca diencephalon (Coveñas et al., 2011) and the distribution of fibres and cell bodies containing neurotensin or leucine-enkephalin in the alpaca brainstem (de Souza et al., 2007, de Souza et al., 2014). Moreover, the colocalization of calcitonin gene-related peptide and somatostatin-28 (1–12) with tyrosine hydroxylase has been also reported in the alpaca brainstem and diencephalon respectively (Marcos et al., 2011, Marcos et al., 2013). In all cases, the studies were carried out in control animals (alpacas not treated with colchicine). To date, in the alpaca diencephalon, a region of the central nervous system involved in important functional mechanisms, the mapping of only two neuropeptides (calcitonin gene-related peptide and somatostatin-28 (1–12)) has been carried out (Coveñas et al., 2011, Coveñas et al., 2012, Marcos et al., 2013). Thus, no studies addressing the presence of adrenocorticotropin hormone-, alpha-melanocyte-stimulating hormone- and opioid peptide-immunoreactive structures in the alpaca diencephalon have been performed. Currently, the only opioid peptide mapped in the alpaca is leucine-enkephalin, and then only in the brainstem (de Souza et al., 2007).

Opioid peptides can be classified in three families according to their precursors: (1) beta-endorphin, alpha- and beta-melanocyte-stimulating hormone, adrenocorticotropin hormone, methionine-enkephalin, and alpha-, and gamma-endorphin are produced from pro-opiomelanocortin; (2) leucine-enkephalin, methionine-enkephalin, methionine-enkephalin-Arg-Gly-Leu and methionine-enkephalin-Arg-Phe from pro-enkephalin; and (3) alpha-neo-endorphin, leucine-enkephalin, dynorphin A and dynorphin B from pro-dynorphin. Our main aim in this work was to study and compare for the first time the distribution of the pro-opiomelanocortin and pro-dynorphin systems in the alpaca diencephalon. Thus, using an immunocytochemical technique we studied the distribution of fibres and cell bodies containing beta-endorphin (1–27), alpha-melanocyte-stimulating hormone and adrenocorticotropin hormone (exclusively produced from pro-opiomelanocortin), as well as the distribution of immunoreactive structures containing alpha-neo-endorphin (exclusively produced from pro-dynorphin). In addition, the distribution of leucine-enkephalin (derived from pro-enkephalin and from pro-dynorphin) was also studied, as the mapping of this opioid peptide has been reported previously in the alpaca brainstem (de Souza et al., 2007), but not in the alpaca diencephalon. A further aim was to compare the distribution of adrenocorticotropin hormone/alpha-melanocyte-stimulating hormone and the three opioid peptides studied here with that previously reported for calcitonin gene-related peptide/somatostatin-28 (1–12), in order to know whether a close anatomical relationship occurs or not among these hormones and peptides in the alpaca diencephalon (Coveñas et al., 2011, Coveñas et al., 2012). Another aim was to compare the distribution of the five peptides detected here with those described previously for the same peptides in the mammalian diencephalon. The peptides studied play important roles in the mammalian central nervous system. For example, they have been implicated in stress, sexual and feeding mechanisms, neuroendocrine, temperature and cardiovascular regulation, aggressive and defensive behaviour, and in aging (Coveñas et al., 2007). Alpacas are able to live at sea level and at more than 5000 m altitude, making this animal an interesting but relatively unknown species that could reveal the existence of special, unique regulatory mechanisms. In the future, the neuroanatomical findings from the alpaca described here should serve to better understand the involvement of adrenocorticotropin hormone/alpha-melanocyte-stimulating hormone and opioid peptides as regards the adaptation of the species to vastly different altitudes and their reproductive and social behaviour. This is important owing to the economic importance of the wool of these animals in some South American countries.

Section snippets

Animals and tissue preparation

The experimental design, protocols, and procedures of this work were performed under the principles of laboratory animal care and under the guidelines of the ethics 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). Four male adult alpacas (Lama pacos) (Huacaya breed) (5–8 years; 70–80 kg) were obtained from the Cayetano Heredia Peruvian University (Faculty of Veterinary

General considerations

Table 1 and Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 show the distribution and density of immunoreactive fibres and cell bodies containing ACTH/alpha-melanocyte-stimulating hormone and the opioid peptides studied in the alpaca diencephalon. In general, for each neuropeptide the distribution of the immunoreactive structures (fibres and cell bodies), as well as the density of such structures, were quite similar in the four diencephalons studied.

In general, cell bodies containing the peptides were

Pro-opiomelanocortin- and pro-dynorphin-derived peptides in the alpaca diencephalon

This is the first report describing the distribution of fibres and cell bodies containing opioid peptides and the non-opioid peptides ACTH and alpha-melanocyte-stimulating hormone in the alpaca diencephalon. Perikarya containing alpha-MSH or alpha-NE showed a more widespread distribution than those containing leu-enk or beta-endorphin (1–27). Cell bodies containing exclusively pro-dynorphin-derived peptides were observed in the dorsal hypothalamic area and those containing only

Acknowledgements

The authors thank Professor Gérard Tramu (University of Bordeaux I, France) for kindly providing the first antisera and N. Skinner (University of Salamanca, Spain) for stylistic revision of the English text. This work has been supported by the Ministerio de Educación y Ciencia (BFU2005-02241/BFI), Spain and the Ministerio de Ciencia e Innovación (BFU2008-03369/BFI), Spain and by the CONCYTEC: PROCYT project 2006, Peru.

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