Supplementary Material for: GABAergic Neurotransmission in the Premammillary Nucleus of the Turkey Hypothalamus Regulates Reproductive Seasonality and the Onset of Photorefractoriness

<br><strong><em>Background/Aims:</em></strong> Photoperiod is a major environmental cue in temperate-zone birds which synchronizes breeding with the time of year that offers the optimal environment for offspring survival. Despite continued long photoperiods, these birds eventually become refractory to the stimulating photoperiod and their reproductive systems regress. In this study, we characterized the role of γ-aminobutyric acid (GABA)ergic neurotransmission in modulating the response of the premammillary nucleus (PMM) to a gonad stimulatory photoperiod and the onset of photorefractoriness. <b><i>Methods and Results:</i></b> Bilateral ablation of the PMM blocked the light-induced neuroendocrine response from occurring in photosensitive turkeys. Microarray analyses revealed an increase in GABAergic activity in the PMM of photorefractory birds as opposed to photosensitive ones, and this enhanced GABAergic activity appeared to inhibit the photoperiodic signal. Additionally, GABA<sub>A</sub> and GABA<sub>B</sub> receptors were expressed by dopamine-melatonin neurons in the PMM, and the administration of the GABA receptor agonist baclofen blocked the photoperiodic reproductive neuroendocrine responses. <b><i>Conclusions:</i></b> Consistent with the present findings, we propose that the long-sought-after mechanism underlying photorefractoriness is linked to the inhibitory actions of GABA. We suggest that (1) GABAergic interference with photoperiodic entrainment in the PMM initiates the photorefractory state and terminates the annual breeding season in temperate-zone birds, and (2) the PMM is a site of photoreception and photorefractoriness that controls the initiation and termination of avian reproductive seasonality.