Supplementary Material for: Medial Amygdala <b><i>Kiss1</i></b> Neurons Mediate Female Pheromone Stimulation of Luteinizing Hormone in Male Mice S.Aggarwal C.Tang K.Sing H.W.Kim R.P.Millar J.A.Tello 2019 <b><i>Background/Aims:</i></b> The medial amygdala (MeA) responds to olfactory stimuli and alters reproductive physiology. However, the neuronal circuit that relays signals from the MeA to the reproductive axis remains poorly defined. This study aimed to test whether MeA kisspeptin (MeA<sup>Kiss</sup>) neurons in male mice are sensitive to sexually relevant olfactory stimuli and transmit signals to alter reproductive physiology. We also investigated whether MeA<sup>Kiss</sup> neurons have the capacity to elaborate glutamate and GABA neurotransmitters and potentially contribute to reproductive axis regulation. <b><i>Methods:</i></b> Using female urine as a pheromone stimulus, MeA<sup>Kiss</sup> neuronal activity was analysed and serum luteinizing hormone (LH) was measured in male mice. Next, using a chemogenetic approach, MeA<sup>Kiss</sup> neurons were bi-directionally modulated to measure the effect on serum LH and evaluate the activation of the preoptic area. Lastly, using in situ<i></i> hybridization, we identified the proportion of MeA<sup>Kiss</sup> neurons that express markers for GABAergic (Vgat) and glutamatergic (Vglut2) neurotransmission. <b><i>Results:</i></b> Male mice exposed to female urine showed a two-fold increase in the number of c-Fos-positive MeA<sup>Kiss</sup> neurons concomitant with raised LH. Chemogenetic activation of MeA<sup>Kiss</sup> neurons significantly increased LH in the absence of urine exposure, whereas inhibition of MeA<sup>Kiss</sup> neurons did not alter LH. In situ<i></i> hybridization revealed that MeA<sup>Kiss</sup> neurons are a mixed neuronal population in which 71% express Vgat mRNA, 29% express Vglut2 mRNA, and 6% express both. <b><i>Conclusions:</i></b> Our results uncover, for the first time, that MeA<sup>Kiss</sup> neurons process sexually relevant olfactory signals to influence reproductive hormone levels in male mice, likely through a complex interplay of neuropeptide and neurotransmitter signalling.