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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 3, 2005 as doi:10.1096/fj.04-2874fje. |
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* Department of Developmental and Behavioural Neuroscience, Institute of Neuroscience, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; and
Dipartimento di Fisiologia, Sezione di Neuroscienze e Fisiologia Applicata, Universita di Siena, Siena, Italy
2 Correspondience: Department of Developmental and Behavioral Neuroscience, Institute of Neuroscience, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam. 1087 De Boelelaan, Amsterdam 1081 HV, The Netherlands. E-mail: gahr{at}bio.vu.nl
SPECIFIC AIMS
The hormone melatonin is well known for its role in entrainment of circadian and circannual rhythms. In birds and mammals including humans, however, melatonin binding sites are also abundant in brain areas that have no known clock function. The visual system of birds, the cerebellum and striatum of birds and mammals, and the mammalian hippocampus and cortex all contain melatonin target sites. Despite this widespread occurrence of melatonin receptors in the brain, there is no clear evidence for the role of melatonin in behavioral patterning beyond circadian and circannual organization. We set out to provide experimental evidence for a direct function of melatonin in the neural control of a motor pattern, the song of the zebra finch. The song is controlled by a defined neural circuit, the vocal control system, and part of the sexual behavior of these birds.
PRINCIPAL FINDINGS
1. Melatonin receptors are expressed in the areas of the vocal control pathway
First, we cloned parts of three melatonin receptors of the zebra finch by standard PCR techniques. Sequence comparison of the cloned gene fragments with their chicken and human counterparts showed that the nucleotide sequence of the Mel1A (GenBank AY803771) receptor was 88% identical with the chicken sequence and 75.5% identical with the human sequence. The amino acid sequence of the zebra finch Mel1A protein was 92% identical with the chicken and 79% identical with the human sequence. The nucleotide sequence of the Mel1B (GenBank AY803772) was 93.5% and 69.8% identical with that of chicken and human, respectively, and its amino acid sequence was 96% (chicken) and 74% (human) identical. The nucleotide sequence of the Mel1C (GenBank AY803773) of the zebra finch was 92% similar to that of the chicken with 97.4% similarity for the amino acid sequence. No mammalian counterpart of the Mel1C receptor is known. In in situ hybridization procedures, we localized the mRNA of the melatonin receptors with cRNA probes of the cloned Mel1A, Mel1B, and Mel1C cDNAs in adjacent brain sections of adult male zebra finches. Among the areas of the descending song control motor system, only the premotor areas HVC (nucleus hyperstriatalis ventrale pars caudale) and RA (nucleus robustus archistriatalis) expressed the Mel1B type (Fig. 1
) but neither Mel1A nor Mel1C receptor mRNA. 17 ± 7% of the HVC neurons and 38 ± 6% of the RA neurons expressed Mel1B. In situ hybridization histochemistry of retrogradely labeled RA neurons showed that most (98±1.1%) of the Mel1B expressing RA neurons are interneurons. Neurons of the motor nucleus nXIIts (nucleus hypoglossus pars tracheosyringealis) expressed Mel1C but neither Mel1A nor Mel1B mRNA. The RAM (nucleus retroambigualis), a singing-related expiratory premotor area of the medulla, expressed Mel1C receptor mRNA but neither Mel1A nor Mel1B. These anatomical data suggested a cellular basis for direct receptor mediated action of melatonin on the song pattern.
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2. Melatonin inhibits spontaneously active RA neurons
To substantiate the hypothesis of a direct action of melatonin on neurons controlling the song pattern, we performed extracellular recordings of neurons of the vocal premotor area RA in acute slices of the forebrain of adult male zebra finches during their subjective day. Spontaneously active neurons in RA fired tonically at rates between 
2 and 5 Hz at room temperature. Melatonin significantly decreased the firing rate of RA-neurons and this effect persisted for as long as the duration of the recording, at least 1 h after application.
The median interspike-interval (ISI) before application was 0.5966 s in the melatonin group (n=21) and 0.4339 s in the controls (n=17). After application of melatonin, the median ISI had increased to 0.6790 s, whereas in the controls it remained virtually unchanged (median ISI: 0.4424 s). The difference between the median spike interval before and after application was larger after melatonin application than after vehicle application (Kruskal-Wallis, P<0.002). In controls, the median of the differences was –7.3 ms; in melatonin-treated slices it was 61.4 ms. The median coefficient of variation of the ISIs during the 30 min before application was 29.3% in the controls and 32.1% in the melatonin group. In the 30 min following application it was 26.0% in controls and 31.0% in the melatonin treated animals. The differences were not significant (post-pre difference; Kruskal-Wallis, P=0.52). This suggests that melatonin might modulate song pattern formation by direct action on the motor circuit that controls singing.
3. Melatonin antagonist transiently reduces song length
Since HVC and RA are required for the production of the song pattern but not for the motivation to sing, we expected changes of the song temporal organization following inhibition of the Mel1B receptor. We treated males systemically with the Mel1B specific ligand S20928 at the onset of the night. This procedure was preferred over pinealectomy in view of the multiple independent sources of melatonin and of the presence of Mel1C receptors in brainstem vocal control areas and areas of the forebrain auditory loop (this study).
Treatment of the males with S20928 led to a transient significant reduction of the length of undirected songs (ANOVA, P<0.001) of individual males during singing period of the following day (Fig. 2
). These songs were frequently interrupted either during or after the first motif. The following day, song length was back to pre-injection levels, which means that the effect lasted for one day of singing. Although we observed too few directed songs per male to allow statistical analysis, those also appeared reduced in length in the S20928 treated males. The transiently decreased song length coincided with a decreased motif length (ANOVA, P<0.001) and a changed length of syllables (ANOVA, P<0.001). The 8 males produced a total of 48 syllables. Of those, the length increased in 41.7%, decreased in 29.3%, and remained similar in 12.5%; 16.6% of the syllables at the end of the motifs were omitted after treatment. Besides the increased variability in syllable length there was no obvious change in spectral features of the syllables after treatment. Vehicle treatment affected neither song length, motif length, nor syllable length (ANOVA, P>0.1 for each). There was no effect of treatment on song activity.
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CONCLUSIONS AND SIGNIFICANCE
The vocal control pathway is a target of melatonin due to the area-specific presence of Mel1B (HVC and RA) and Mel1C (nXIIts and RAM) receptors in song control areas (Fig. 1)
. By combining this molecular work with electrophysiological and behavioral studies (Fig. 2)
, we provide for the first time evidence that melatonin has a direct role for the generation of a motor pattern (the song) by acting on a behavioral control circuit (song control neurons). Thus, melatonin not only affects circadian occurrence of behavior via its action on hypothalamic oscillators but also shapes the behavioral pattern itself by direct action on motor circuits (Fig. 3
). The latter process could be independent of circadian performance. Since the song pattern is relevant in the realm of reproduction, melatonin appears to affect a sexual behavior. So far, only gonadal hormones are thought to directly affect the patterning of sexual behaviors. This raises the question of whether the production of melatonin or the expression of melatonin receptors are under sexual selection pressure.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2874fje;
1 These authors contributed equally to this work. 
This article has been cited by other articles:
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  V. M. Cassone, P. A. Bartell, B. J. Earnest, and V. Kumar Duration of Melatonin Regulates Seasonal Changes in Song Control Nuclei of the House Sparrow, Passer domesticus: Independence from Gonads and Circadian Entrainment J Biol Rhythms, February 1, 2008; 23(1): 49 - 58. [Abstract] [PDF]
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 C. M. Glaze and T. W. Troyer Temporal Structure in Zebra Finch Song: Implications for Motor Coding J. Neurosci., January 18, 2006; 26(3): 991 - 1005. [Abstract] [Full Text] [PDF]
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