No abstract

No abstract

The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual-oscillation in dopamine (DA)/melatonin (MEL) as well as c-fos mRNA expression that is associated with the daily photo-inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase-related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase-delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light-induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase-shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.

Serotonin and catecholamines (dopamine, norepinephrine, epinephrine) have important roles as neurotransmitters in avian reproduction, but their anatomical relationship to the neuroendocrine circuitry that regulates reproduction is poorly understood. Our previous studies have shown that co-localised dopamine-melatonin (DA-MEL) neurones in the avian premammillary nucleus (PMM) are active during periods of photoresponsiveness and, therefore, are potentially photosensitive neurones. Because serotonergic and catecholaminergic neurotransmitters are important regulators of reproductive function in the female turkey, we hypothesised that the serotonergic/catecholaminergic neurones within the brainstem might interact with PMM DA-MEL neurones and constitute an important circuit for reproductive function. To examine this possible interaction, the retrograde fluorescent tract tracer, 1,1'dioctadecyl-3,3,3'3'-tetramethyleindocarbocyanine perchlorate (DiI) was injected into the PMM, and combined with serotonin, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and phenyl N-methyltransferse (PNMT) immunocytochemistry to reveal neuroanatomical connections. Changes in the activities of serotonergic, dopaminergic, adrenergic and noradrenergic neuronal systems projecting to the PMM were measured at different reproductive states with in situ hybridisation (ISH) techniques, using tryptophan hydroxylase 2 (TPH2) and TH mRNA expression, respectively. Cells labelled with DiI were found in anatomically discrete areas in or near the hypothalamus and the brainstem. Double immunocytochemistry confirmed that there were serotonin, DBH and PNMT fibres in close apposition to DA-MEL neurones. TPH2 mRNA expression in serotonin neurones was found in several nuclei, and its most abundant mRNA expression was seen in the nucleus Locus ceruleus of laying and incubating hens. TH mRNA expression levels in the six catecholaminegic areas labelled with DiI was measured across the different reproductive states. In the nucleus tractus solitarius (adrenergic), the highest level of TH mRNA expression was found in photorefractory hens and the lowest level in incubating hens. These observed patterns of serotonin/catecholamine neuronal distribution and their variable interactions with PMM DA-MEL neurones during different reproductive states may offer a significant neuroanatomical basis for understanding the control of avian reproductive seasonality.

No abstract