Perspectives on Endogenous Opioids in Birds

The present review summarizes the state of knowledge of endogenous opioids in birds. Endogenous opioid peptides acts in a neuromodulatory, hormonal and paracrine manner to mediate analgesic and other physiological functions. These peptides act through specific G-protein coupled receptors. Opioid receptors consist of a family of four closely-related proteins. The three types of opioid receptors are the mu (MOR or μ), delta (DOR or δ), and kappa (KOR or κ) opioid receptor proteins. The role of the fourth member of the opioid receptor family, the nociceptin or orphanin FQ receptor (ORL), is not clear. The ligands for opioid receptors are: β –endorphin (MOR), Met- enkephalin, Leu-enkephalin (DOR) and dynorphin (KOR), together with probably endomorphins 1 and 2. In spite of long history of research on endogenous opioid peptides, there are no studies of endogenous opioids per se in wild birds and few in poultry species. β-endorphin is present in all birds investigated and there is close agreement between the structures of β-endorphin in different birds. Plasma concentrations of β-endorphin are increased by ether stress in geese. There is evidence that β-endorphin plays a role in the control of luteinizing hormone release in chickens. Met-enkephalin is present in tissues such as the retina, hypothalamus, pituitary gland, and adrenals together with circulation of birds. Stresses such as crowding and withholding water increase circulating concentrations of Met-enkephalin in chickens. The structures of chicken dynorphin A and B have been deduced from cDNA. What is missing are comprehensive studies of plasma concentrations and expression of the full array of endogenous opioids in multiple avian species under different situations. Also, what is not known is the extent to which circulating or locally released or intra-cellular Met-enkephalin influence physiological process in birds. Thus, there is considerable scope for investigation of the physiology of endogenous opioids in birds.


INTRODUCTION
The ligands for opioid receptors are endogenous opioid peptides, specifically: Met-enkephalin, Leu-enkephalin, β -endorphin and dynorphin, together with probably endomorphins 1 and 2. These are the physiological signaling peptides that mimic the effects of synthetic and natural opioids such as morphine and codeine.
The opioid/orphanin gene family contains the following: • Proenkephalin A (PENK) gene-encoding proenkephalin which is processed to the endogenous opioids-Met-enkephalin and Leu-enkephalin (Figure 1).
The opioid receptors are members of the G-protein coupled receptors (GPCR) gene family and the rhodopsin-like superfamily of GPCR (reviewed Stevens, 2009): • Delta (δ) opioid peptide receptor (DOR or DOPr or OP 1 receptors) (d as in vas deferens). Endogenous ligand: Met enkephalin and Leu enkephalin together with probably βendorphin.
These endogenous opioid receptor agonists have analgesic (relieving pain) or antinociceptive (inhibiting the sensation of pain) properties. There are other peptides that bind to opioid receptors. For instance, the tetrapeptide, cytochrophin-4, is a breakdown product of cytochrome-b and influences memory formation in chicks (Freeman and Young, 2000). The carboxyamidated tetrapeptides, endomorphins 1 and 2, have been isolated from bovine brain tissue (Zadina et al., 1999). However, the endomorphins have received little attention in avian species. It is thought that the four opioid receptor genes and the four members of the opioid/orphanin family of genes are the result of two genome duplications leading to quadruplication of the ancestral genes; these occurring prior to the radiation of the Gnathostomes (Figure 2) (Khalap et al., 2005;Dreborg et al., 2008;Sundström et al., 2010). It has not been possible to identify pro-enkephalin genes in primitive vertebrates (Agnathans) or basal chordate (Dreborg et al., 2008;Sundström et al., 2010).
The present review summarizes the state of knowledge of endogenous opioids in birds. In addition, areas ripe for research are covered. The communication discusses endogenous opioids in the following order: β-endorphin, Met-enkephalin and finally, products of the prodynorphin.

β-ENDORPHIN IN BIRDS
There is arguably more information on β-endorphin than of any other endogenous opioid in birds. This is particularly the case with the deduced structures of POMC from multiple species of birds together with mammals and reptiles.
Evolutionary Aspects of β-Endorphin β-endorphin contains the Met-enkephalin pentapeptide motif (Figure 1). The deduced structures of β-endorphin in reptiles and birds together with evolutionary relationships are summarized in Figures 3, 4 (Shen et al., 2003). Despite the last common ancestor of reptiles and birds being about 250 million years ago, there is very close similarities between the structures of β-endorphin in reptiles and birds (Naudé et al., 2006;Shoureshi et al., 2007;Dores and Baron, 2011). A tentative structure of ancestral avian βendorphin in the common ancestor of both birds and Crocodilia (i.e., within the Archosauromorpha, the clade includes birds, crocodyles and dinosaurs) is the following: YGGFMXSEHSQTPLV TLFKNAIVKSAYKKGQ (Where X is S or T) (Endo and Park, 2004). This is identical to β-endorphin in both alligator and pigeon (Kobayashi et al., 2007). Within the class Aves, there are close similarities for β-endorphin. Excluding residue 6, there are two or fewer amino-acid residue differences between the ancestral form and that in multiple birds including chicken, egret, ibis, pelican, rifleman, and ruff (Figures 3, 4). This argues strongly that there is strong evolutionary pressure to maintain the structure of βendorphin and, therefore, also of the physiological importance of β-endorphin in birds. In contrast, there are multiple differences in the Oscines (song birds) (Figures 3, 4).

Circulating Concentrations of β-Endorphin
There is limited information on circulating concentrations of β-endorphin in either wild or domesticated birds. In plasma from adult chickens, two-thirds of β-endorphin immune-reactivity measured by radioimmunoassay followed by Sephadex G-75 chromatography has an identical size as β-endorphin but one-third of β-endorphin is the same size as β-lipotropin (Hylka and Thommes, 1991).
The later research on domestic gander proved that plasma concentrations of immunoreactive β-endorphin measured by specific radioimmunoassay are about 30 pmoles · ml −1 (Barna et al., 1998a).
It might be assumed that circulating concentrations of βendorphin would change in parallel with those of ACTH as they are both products of POMC. However, this does not seem to be necessarily the case. While, plasma concentrations of ACTH increased following castration in domestic geese, there is no effect on circulating concentrations of β-endorphin (Barna et al., 1998a). Moreover, while plasma concentrations of ACTH in domestic geese are increased by both ether stress or LPS endotoxin, plasma concentrations of β-endorphin are reported to be elevated following ether stress but not LPS endotoxin (Barna et al., 1998b). Thus, there is some evidence of independent control of synthesis and/or release and/or degradation of βendorphin and ACTH.

Physiological Roles of β-Endorphin
There is evidence that β-endorphin is involved in the inhibitory control of gonadotropin releasing hormone (GnRH) release from the hypothalamus at least in chickens. β-endorphin cell bodies in the periarcuate area project to the median eminence FIGURE 2 | A schema for the evolution of the opioid/orphanin gene family based on two genome duplications and the insertion of melanocortin sequence to ancestral beta-endorphin gene (based on Sundström et al., 2010;Navarro et al., 2016). (Contijoch et al., 1993). Intra-ventricular administration of βendorphin blocks the pre-ovulatory LH surge (Sakurai et al., 1986). Moreover, β-endorphin depresses in vitro GnRH release from hypothalamic tissue from hens during pre-ovulatory surge of luteinizing hormone (LH) (Contijoch et al., 1993)

Circulating Concentrations of Met-Enkephalin
Plasma concentrations of Met-enkephalin in chickens are 28 pg · ml −1 (∼50 fmoles · ml −1 ) (Pierzchala and Van Loon, 1990). There is a larger form of Met-enkephalin called cryptic ([Met 5 ]-enkephalin) that found both in the circulation and in tissues; this being enzymatically cleaved (processed) to the pentapeptide (Pierzchala and Van Loon, 1990). Cryptic Met-enkephalin appears to be not only the storage of Met-enkephalin (free pentapeptide) but is also quickly processed under different stressors such as withholding of food and water, overcrowding, cold and restraint (Pierzchala-Koziec et al., 1999). Cryptic Met-enkephalin concentration in blood plasma of growing chickens was ∼4.2 pmoles · ml −1 (Pierzchała-Koziec and Mazurkiewicz-Karasińska, 1997; Pierzchala-Koziec et al., Submitted). Enkephalins have short half-lives in the circulation. For instance, Leuenkephalin is rapidly degraded by aminopeptidase M in chicken plasma with a half-life of 0.7-1.0 min in vitro (Shibanoki et al., 1991). Thus, aminopeptidases inhibitors need to be present in tubes and syringes when sampling birds.
Some stressors have been demonstrated to increase plasma concentrations of Met enkephalin in chickens. This is in addition to the stress induced activation of the hypothalamopituitary-adrenocortical (HPA) axis. Plasma concentrations of Met-enkephalin were increased by short term crowding in chickens with the effect being attenuated in the presence of the opioid antagonist, naltrexone (Pierzchała-Koziec et al., 1996;Pierzchala-Koziec et al., Submitted). Fasting increased plasma concentrations of Met enkephalin in female chickens but decreased those in male chickens Pierzchala-Koziec et al., Submitted). Moreover, acute withholding of water was accompanied by increased plasma concentrations of Metenkephalin (Pierzchala-Koziec et al., Submitted). What are not known are the effects of stress on Met-enkephalin in wild birds or the physiological relevance of increased plasma concentrations of Met-enkephalin during stress.

Control of Met-Enkephalin Release
There is very limited information on control of Met-enkephalin release. In chickens, release of Met-enkephalin from the hypothalamus and pituitary gland in vitro is depressed in tissues taken from birds subjected to water deprivation stress (Mazurkiewicz-Karasińska and Pierzchała-Koziec, 1997; Pierzchala-Koziec et al., Submitted). There are increases in release of Met-enkephalin and cryptic [Met 5 ]-enkephalin from the retina during the night/darkness in chickens (Dowton et al., 1990).
There is evidence that endogenous opioids depress in vitro release of Met-enkephalin from the hypothalamus. In vitro release of Met-enkephalin is increased when chicken hypothalamic fragments are incubated in the presence of naltrexone Pierzchala-Koziec et al., Submitted). There are changes in release of Met-enkephalin and cryptic [Met 5 ]-enkephalin from the retina during the night/darkness in chickens (Dowton et al., 1990).
There is some evidence of cross talk between the HPA axis and Met-enkephalin in birds. This is supported by the report that stress increases plasma concentrations of both Metenkephalin, and corticosterone in chickens (Pierzchala-Koziec et al., Submitted). In addition, corticotropin releasing hormone (CRH) increases release of both Met-enkephalin (Pierzchala-Koziec et al., Submitted) and ACTH in chickens (Nakayama et al., 2011). Moreover, there is evidence that glucocorticoids influence release of Met-enkephalin with dexamethasone increasing release of Met-enkephalin in vitro from either chicken hypothalamic or adrenal tissue (Pierzchala-Koziec et al., Submitted).

Organ Any details References
Adrenal gland Both norepinephrine and epinephrine producing chromaffin cells Ohmori et al., 1997 Gastrointestinal tract Gizzard-myenteric plexus and the outer circular muscle Jiménez et al., 1993 But not in proventriculus Martínez et al., 2000 Small intestine: myenteric and the deep muscular plexuses Jiménez et al., 1993 Ceca: neurones projecting into the ceca Ohmori et al., 2003 Posterior pituitary gland Mesotocin containing neurosecretory terminals Martin et al., 1992Retina Dowton et al., 1990 Spinal cord Nerve fiber-and terminal-like processes of the lumbar spinal cord Maderdrut et al., 1986 Thymus Endocrine cells Atoji et al., 1997 Distribution of Met-Enkephalin and Tissue Concentrations Table 1 summarizes the available information on the distribution of Met-enkephalin. The distribution of enkephalin neurons has been reported in the pigeon brain (Bayon et al., 1980). Enkephalin neurons are found, for instance, in the brainstem, limbic regions, organum vasculosum hypothalamic, paleostriatum, and pituitary stalk (Bayon et al., 1980). There are marked differences in the distribution of enkephalin neurons and those containing β-endorphin (Bayon et al., 1980). Tissue concentration of Met-enkephalin and PENK gene expression have been determined.
Tissue concentrations of Met-enkephalin change during avian embryonic development. There are large increases (>1,000 fold) in immuno-reactive (IR-) Met-enkephalin in the lumbar spinal cord between days 4.5 and 18 in chick of embryos (Maderdrut et al., 1986). Moreover, IR-Met-enkephalin is not observed in the circular smooth muscle until day 17 of embryonic development (Epstein et al., 1983).

Physiological Role of Met-Enkephalin
Met-enkephalin may act in neuromodulatory, and/or hormonal and/or paracrine manners. Met-enkephalin inhibits release of gonadotropin releasing hormone (GnRH) from hypothalamic tissue from male chickens in vitro with the effect being via mu opioid receptors Cunningham, 1987, 1988). It is unclear whether these observations reflect an effect of Metenkephalin per se or exogenous Met-enkephalin acting as a surrogate for other endogenous opioids. Met-enkephalin has been reported to have other effects in birds. For instance, Metenkephalin acts as a gastric inhibitor in chickens via mu receptors (Jiménez et al., 1993).

DYNORPHIN IN BIRDS
There is very limited information on dynorphin in birds. The structures of chicken dynorphins can be deduced from the cDNA: • Dynorphin A -YGGFMRRIRPKLKWDN • Dynorphin B -YGGFLRRQFKVTT (based on Figure 1). An additional peptide product is α-neoendorphin.

Circulating Concentrations of Products of the Prodynorphin
There are no reports on circulating concentrations of dynorphin A or B in birds. However, overcrowding stress was associated with increased plasma concentrations of α-neo-endorphin (Pierzchała-Koziec et al., 1996).

Distribution of Dynorphin
The distribution of dynorphin neurons in the pigeon brain has been reported (Reiner, 1986). Outside of the hypothalamus, there are neurons containing both substance P and dynorphin (Reiner, 1986).

Physiological Effects of Dynorphin
There is evidence that dynorphin plays a role in the neuroendocrine control of prolactin release in birds. Intraventricular infusion of dynorphin into the II ventricle is followed by increases in circulating concentrations of prolactin in turkeys with the effect mediated via kappa (κ) opioid receptors as demonstrated by κ opioid receptor antagonists to block the effect (Youngren et al., 1993(Youngren et al., , 1999.

OTHER NEUROENDOCRINE EFFECTS OF ENDOGENOUS OPIOIDS IN BIRDS
There is evidence that endogenous opioids influence release of arginine vasotocin (AVT) in birds. Plasma concentrations of AVT, but not those of mesotocin, are increased by morphine and a specific mu receptor agonist in chickens (Saito et al., 1999;Sasaki et al., 2000). Moreover, AVT release is depressed by the opioid receptor antagonist, naloxone, in hypertonic saline treated chickens (Saito et al., 1999). It is not clear which endogenous opioid influences AVT release. It is reasonable to exclude dynorphin as it is not present in the pars nervosa of the chicken (Martin et al., 1992). Opioid agonists appear to inhibit whereas antagonists stimulate socio-sexual interactions in starlings (Riters, 2011). Moreover, it was suggested that enkephalin in medial preoptic area is involved in reward associated with both feeding and sexual behavior (Riters, 2011).

CONCLUSIONS AND FUTURE DIRECTIONS
There are few studies of endogenous opioids in poultry species and none in wild birds. The available evidence that both β-endorphin and Met-enkephalin are present across the class Aves. The structure of β-endorphin is very similar across birds suggesting its importance. There have been some physiological studies and all support a relationship between both β-endorphin and Met-enkephalin and stress. What is missing are comprehensive studies of plasma concentrations, expression and receptors of the full array of endogenous opioids in multiple avian species under the following situations: • Temporal changes during annual, daily/circadian and ovulatory cycles.
• Acute responses in response to activators of the HPA axis, other stressors (e.g., disease), neuropeptides and hormones.
• Interactions between the different endogenous opioids and their receptors.
There is considerable scope for investigation of the physiology of endogenous opioids in birds.

AUTHOR CONTRIBUTIONS
KP-K and CS: conceived, drafted and wrote the review. It is based in part on research from KP-K's laboratory. CS and KP-K: re-analyzed the research and together discussed their relevance.