Melatonin receptor density in Area X of European starlings is correlated with reproductive state and is unaffected by plasma melatonin concentration

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Abstract

Several of the song control nuclei of songbirds, including HVc (higher vocal center) and Area X, contain melatonin receptor (MelR). In laboratory-housed male starlings, the densities of MelR in Area X change markedly according to reproductive state. MelR are down-regulated when starlings are photostimulated (in full breeding condition) and are subsequently up-regulated when starlings become photorefractory (reproductively quiescent). However, seasonal regulation of MelR densities in Area X has only been investigated during the light phase of the light:dark cycle. Variation in MelR densities are physiologically relevant only if they also occur during the dark phase, when melatonin is present in the circulation. Brains from male starlings that were in different reproductive states but exposed to the same 18L:6D photoperiod were collected during either the mid-point of the light phase or the dark phase. Melatonin receptor distribution was assessed in vitro by 125Iodomelatonin (IMEL) receptor autoradiography. All photostimulated birds exhibited down-regulation of MelR in Area X, and all photorefractory birds exhibited high MelR density in Area X, regardless of time of sampling or plasma melatonin concentration. Thus, within each reproductive state, MelR density in Area X did not differ over the course of a circadian cycle. The functional significance of seasonal regulation of MelR in this song control nucleus remains unclear, but it is likely to involve a release of cellular inhibition by melatonin during photostimulation, with possible consequences for song learning, memory consolidation or regulation of the context of song production.

Introduction

In contrast to mammals, the annual profile of nocturnal pineal melatonin secretion is not involved in the control of avian reproduction (Juss et al., 1993; Turek and Wolfson, 1978; Wilson, 1991). Until recently, the only clear role for melatonin in birds has been the regulation of circadian activity (Gwinner et al., 1997a; Gwinner et al., 1997b; Hau and Gwinner, 1995; Heigl and Gwinner, 1995; Hendel and Turek, 1978; Kumar et al., 2000; Turek et al., 1976). Despite its historically enigmatic role in seasonal processes in birds, it is now apparent that melatonin strongly influences physiological processes that are associated with reproductive success, such as immune function (Bentley et al., 1998).

Birdsong is a reproductive behavior, and the complex network of interconnected nuclei referred to collectively as the song control system is considered a secondary sexual characteristic (see MacDougall-Shackleton and Ball (1999) for review). Another example of the involvement of melatonin in physiological and behavioral processes involved in reproduction is the inhibitory effect of melatonin upon the volumes of specific telencephalic nuclei within the song control system of starlings (Bentley et al., 1999). As with gonadal steroid receptors (Balthazart et al., 1992; Bernard et al., 1999; Gahr, 1990; Gahr and Metzdorf, 1997; Gahr and Wild, 1997) there are melatonin receptors within the song control nuclei of songbirds (Bentley and Ball, 2000; Gahr and Kosar, 1996; Whitfield-Rucker and Cassone, 1996). Melatonin binding sites exist in Area X, the higher vocal center (HVc), nucleus robustus archistriatalis (RA) of the song control system of starlings, and are also distributed throughout areas involved in vision (e.g., ectostriatum (E) and the optic tectum (TeO) (Bentley and Ball, 2000). Thus, melatonin appears to be acting directly upon the song control system.

Unlike in other song control nuclei, the distribution of melatonin receptor (MelR) within Area X of starlings is regulated as a function of reproductive state, and not as a function of photoperiod (Bentley and Ball, 2000). Reproductive state in photoperiodic bird species such as starlings is regulated by changing day length throughout the annual cycle (Dawson et al., 2001; Nicholls et al., 1988). During short, winter-like photoperiods, starlings are photosensitive. The reproductive axis responds to increasing spring day lengths by becoming photostimulated and increasing the amount of gonadotrophin-releasing hormone (GnRH) that is released to the pituitary gland, thus increasing the peripheral circulating concentrations of the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Full reproductive condition is attained in this way. Once a critical day length has been exceeded, which in the case of starlings is approximately 11.5 h of light per day (Nicholls et al., 1988), photostimulated starlings become photorefractory. The onset of photorefractoriness is a centrally-mediated physiological process that is dependent upon the presence of thyroid hormones (Goldsmith and Nicholls, 1984; Wieselthier and van Tienhoven, 1972) and that affects a plethora of physiological processes that have evolved to prevent starlings from breeding at inopportune times of the year and to prepare them for the forthcoming winter (for reviews of starling photorefractoriness see Bentley, 1997; Dawson et al., 2001; Nicholls et al., 1988). Once they have become photorefractory, starlings remain so indefinitely unless they experience an extensive period of short days, as happens in the winter in the wild. The described changes in reproductive state are centrally-mediated and occur independently of the gonads, so that they occur even in gonadectomized starlings (Dawson and Goldsmith, 1984).

As previously mentioned, concurrent with the annual changes in reproductive state are changes in the density of MelR in Area X. There is a high density of MelR in Area X when starlings are exposed to short photoperiods and are photosensitive. Transfer from short to long photoperiods causes starlings to become photostimulated, and there is a marked down-regulation of MelR throughout Area X. Continued exposure to long photoperiods causes photorefractoriness to ensue, and in photorefractory starlings there is a marked up-regulation of MelR to levels seen in short-day, photosensitive starlings (Bentley and Ball, 2000). The down-regulation of MelR as starlings are photostimulated is independent of gonadal steroids, although there appears to be an interaction with gonadal steroids in the medial portion of Area X (mArea X) (Bentley and Ball, 2000). The subsequent up-regulation of MelR as starlings become photorefractory is independent of changes in photoperiod and is dependent upon reproductive state. In this way, the regulation of MelR in Area X appears to be another physiological event that is associated with the centrally-mediated onset of photorefractoriness.

Despite the robust seasonal regulation of MelR described in Bentley and Ball (2000), all of the birds in that study were sampled during the day time, when endogenous melatonin concentrations tend to be low. Thus, even though changes in the density of MelR in Area X of starlings in different reproductive states are considerable, one cannot conclude that they are physiologically relevant unless they also occur at night when circulating concentrations of melatonin are high. The present study was designed to determine whether or not the down-regulation of MelR in Area X of starlings occurs during the dark phase of the light:dark cycle in addition to the light phase. In more general terms, is the regulation of MelR in Area X physiologically relevant? To be physiologically relevant, it would have to occur at a time when melatonin is present within the circulation at night, independently of circulating melatonin concentration.

Section snippets

Birds and treatments

All treatments complied with the NIH Health Guide for the Care and Use of Laboratory Animals, and, for treatments at JHU, with the approval of The Johns Hopkins University Animal Care and Use Committee.

Twenty photorefractory male starlings were collected from the wild in the summer as juveniles (first-year birds can be identified easily by their brown plumage) and held on 8 h light and 16 h darkness per day (8L:16D) for 114 d to ensure the onset of photosensitivity. They were housed in cages (49 × 95

Results

In agreement with previous findings (Bentley and Ball, 2000), in starlings that were photostimulated on long days and killed during the light phase (Phstim DAY), Area X exhibited a substantial reduction in IMEL binding as compared to birds that were photorefractory and killed during the light phase (Phrefr DAY). These differences in IMEL binding were also observed in each group’s respective dark phase (NIGHT) comparison group. In other words, all Phstim birds exhibited the same pattern of low

Discussion

Photoperiod- and gonadal steroid-independent regulation of MelR in Area X of starlings occurs as a function of reproductive state. The present study confirms the earlier findings of Bentley and Ball (2000) and extends them with evidence that the regulation of MelR in Area X occurs in the dark phase as well as the light phase of the light:dark cycle. Thus, the seasonal regulation of MelR in this song control nucleus appears to be physiologically relevant. The down-regulation of MelR in Area X as

Acknowledgments

The author thanks Greg Ball and Verdun King for their assistance and advice. Rachel Finn, Kathleen Hanlon, First Piluek, Brian Spar, and Zaman Mirzadeh provided excellent technical support. Funding for this project was provided by NSF 9905401.

References (43)

  • E.D. Jarvis et al.

    For whom the bird sings: context-dependent gene expression

    Neuron

    (1998)
  • S.A. MacDougall-Shackleton et al.

    Comparative studies of sex differences in the song-control system of songbirds

    Trends Neurosci.

    (1999)
  • F.W. Turek et al.

    Lack of an effect of melatonin treatment via silastic capsules on photic-induced gonadal growth and the photorefractory condition in white-throated sparrows

    Gen. Comp. Endocrinol.

    (1978)
  • M.G. Whitfield-Rucker et al.

    Melatonin binding in the house sparrow song control system: sexual dimorphism and the effect of photoperiod

    Horm. Behav.

    (1996)
  • M. Adret-Hausberger et al.

    Individual life history and song repertoire changes in a colony of starlings (Sturnus vulgaris)

    Ethology

    (1990)
  • G.F. Ball et al.

    Changes in plasma levels of luteinizing hormone and sex steroid hormones in relation to multiple-broodedness and nest-site density in male starlings

    Physiol. Zool.

    (1987)
  • J. Balthazart et al.

    Immunocytochemical localization of androgen receptors in the male songbird and quail brain

    J. Comp. Neurol.

    (1992)
  • G.E. Bentley

    Thyroxine and photorefractoriness in starlings

    Poult. Avian Biol. Rev.

    (1997)
  • G.E. Bentley et al.

    Photoperiod-dependent and -independent regulation of melatonin receptors in the forebrain of songbirds

    J. Neuroendocrinol.

    (2000)
  • G.E. Bentley et al.

    Melatonin, immunity and cost of reproductive state in male European starlings

    Proc. R. Soc. Lond. B: Biol. Sci.

    (1998)
  • G.E. Bentley et al.

    Seasonal neuroplasticity in the songbird telencephalon: a role for melatonin

    Proc. Natl. Acad. Sci. USA

    (1999)
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