Fig 2: Senktide administration directly in to the arcuate nucleus or preoptic area (at the level of the medial septum) stimulates LH release in adult WT but not Kiss1Cre/Cre (i.e., Kiss1 KO) female mice supplemented with estradiol.(A, B) Representative photomicrographs depicting dual label detection of NK3R (green) and GnRH (red) in the ARC (A) and ME (B) of ovariectomised WT (WTovx) and homozygous Kiss1Cre/Cre (i.e., Kiss1 KO animals). (A) Panels on the right are enlarged images (scale bar: 20 µm) from boxed areas on the left (scale bar: 100 µm) showing infrequent close appositions (arrowheads) between NK3R and GnRH in the ARC. (B) Enlarged images of the boxed area from the Kiss1 KO animal in (A) showing intense GnRH but lack of NK3R staining in the ME. (C) Dual-label detection of NK3R (green) and GnRH (red) in the POA and specifically the level of the MS of ovariectomized WT and homozygous Kiss1Cre/Cre (i.e., Kiss1 KO) female mice supplemented with estradiol [WTOVX+E2 and Kiss1 KO+E2, respectively; scale bar: 50 µm]. Arrowheads indicate infrequent sites of close apposition. (D, E) Mean ±SEM LH concentrations (ng/ml) and area under the curve (AUC) after an injection of senktide into the ARC (D) or POA (E) of ovariectomized WT (WTOVX+E2; blue line-blue bar) and homozygous Kiss1Cre/Cre (i.e., Kiss1 KO+E2; green line-green bar) female mice supplemented with estradiol (n = 5/group). 3V: third ventricle, ARC: arcuate nucleus, ME: median eminence, POA: preoptic area, MS: medial septum. Repeated LH concentrations at multiple time-points and between treatments were compared using a 2-WAY ANOVA and a Fishers posthoc test when appropriate. Area under the curve was compared with 2-tailed student t tests. **p < 0.0015, ****p < 0.0001.

Fig 3: Localization of neurokinin receptor 1 (NK1R), neurokinin receptor 2 (NK2R), neurokinin receptor 3 (NK3R) and contactin-associated protein 1 (CASPR) in relation to substance P (SP) in the rat trigeminal ganglion. (a) NK1R displayed robust immunoreactivity (ir) in the majority of observed neurons, neuron nuclei, Schwann cells and A-fibres (Asterix). The NK1R expression in SGCs and C-fibres was slightly weaker. NK1R was observed to co-localize with SP in C-fibres (arrow) and small to medium sized neurons (arrowhead). Insert: Three neurons displaying NK1R ir with one also expressing SP ir (arrowhead). Close-by a SP and NK1R immunoreactive C-fibre (arrow) can be observed. (b) NK2R was expressed in most observed neurons, neuron nuclei (arrow) and A-fibres. The expression in A-fibres was patchy and weaker than that of NK1R. NK2R ir co-localized with SP in neurons (arrowhead) but was not readily distinguished in associated C-fibres. Insert: An unevenly stained NK2R immunoreactive Ad-fibre (arrowhead) near a SP immunoreactive C-fibre (arrow). (c) In general, NK3R ir resembled NK1R ir, though weaker and without a clearly visible nuclear envelope. NK3R ir was observed in most neurons (arrowhead), A-fibres, SGCs and Schwann cells. When co-stained with SP a weak co-localization could be observed in C-fibres (arrow). Insert: Co-localization of NK3R and SP in a medium sized neuron (arrowhead). A weakly stained C-fibre (arrow) can be seen projecting from its associated neuron. (d) The nodes of Ranvier were robustly revealed utilizing the specific CASPR antibody. Double immunohistochemistry with SP displayed several SP positive C-fibre boutons aligning with the node of Ranvier (arrow). Insert: A close-up on the CASPR positive paranodal regions of the Ad-fibre axon flanking the node of Ranvier (arrowhead). A C-fibre bouton displaying SP ir (arrow) can be located nearby. The visible red ir in the C-fibre bouton is likely due to an unspecific binding of the polyclonal CASPR primary antibody.

Fig 4: Schematic representation of the neuronal pathways known to stimulate GnRH/LH release in the female mouse.The first pathway involves Kiss1 neurons located in the arcuate nucleus (ARC), which also express neurokinin B and dynorphin (thus, often referred to as KNDy neurons). These cells have been mainly implicated in the regulation of GnRH/LH pulsatile secretion in males and females. The second GnRH/LH stimulating mechanism, originates from Kiss1 cells located in the anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) and is mainly implicated in the generation of the GnRH/LH surge, which leads to ovulation, and is specific to females. The third and fourth stimulating mechanisms are described in this study and originate from the posterodorsal medial amygdala (MePD). Thus, the third mechanism known to stimulate GnRH/LH release involves Kiss1 neurons and the fourth NK3R expressing neurons of unknown phenotype. The latter are activated only in the presence of estradiol in a mechanism that is female specific. The precise neuronal pathway linking these MePD cells with the stimulation of GnRH/LH release, as well as the physiological relevance of this stimulation, remains to be elucidated. ARC: Arcuate Nucleus, AVPV/PeN: anteroventral periventricular/periventricular preoptic nucleus, MePD: posterodorsal medial amygdala, NK3R: neurokinin receptor 3, ?OR: ? opioid receptor, Kiss1r: kiss1 receptor, opt: optic tract.

Fig 5: Senktide administration into the posterodorsal medial amygdala stimulates LH release in WT and Kiss1 KO female mice supplemented with estradiol.(A, B) Representative photomicrographs depicting NK3R-immunoreactive cell bodies and fibers in the MePD of (A) ovariectomized WT (WTOVX; left panel) or ovariectomized and estradiol-supplemented WT (WTOVX+E2; right panel) and (B) homozygous Kiss1Cre/Cre (i.e., Kiss1 KO; left panel) or Kiss1 KO supplemented with estradiol (Kiss1 KO+E2; right panel) female mice. (C) Mean ±SEM number of NK3R-immunoreactive cells per 30 µm section in the MePD of WTOVX, WTOVX+E2, Kiss1 KO and Kiss1 KO+E2 female mice (as quantified from 6 to 8 sections from 4 to 5 animals/group). (D) Mean ± SEM LH concentrations (ng/ml) and area under the curve (AUC) after an injection of senktide into the MePD of ovariectomized WT (WTOVX+E2; blue line-blue bar) or homozygous Kiss1Cre/Cre (i.e., Kiss1 KO+E2; green line-green bar) supplemented with estradiol or hypogonadal (with low sex steroids) Kiss1 KO female mice (n = 5/group). (E) Schematic representation of the site of injection of an AAV encoding a Cre-dependent hM3Dq:mCherry. The left panel is a schematic representation of the location of the MePD injection site and its anatomical relationship to the optic tract (-1.82 mm from bregma). The right panel is a higher magnification of the boxed area on the left showing MePD kisspeptin neurons tagged with mCherry (red fluorescence), which indicates hM3Dq receptor expressing kisspeptin neurones. (F) Representative photomicrograph of a coronal brain section stained for GFP (green), mCherry (red) and merged GFP and mCherry immunoreactivity in the MePD of a Kiss1 KO female mouse >3 weeks after hM3Dq:mCherry injection (Scale bar 50 µm). In this animal model Cre and GFP have been targeted to the Kiss1 locus and are, therefore, expressed solely in Kiss1 neurons. Thus, colocalization of GFP and m-Cherry immunoreactivity represents Kiss1ARC cells that have been infected with the pAAV encoding a Cre-dependent hM3Dq DREADD. (G, H) Mean ± SEM LH responses and area under the curve (AUC) to an injection of saline (grey line-empty bar) or CNO (green line-green bar) of hM3Dq DREADD injected Kiss1Cre/+ (G) and Kiss1Cre/Cre (KO; H) female mice (n = 5/group). opt: optic tract. Repeated LH concentrations at multiple time-points and between treatments were compared using a 2-WAY ANOVA and a Fishers posthoc test when appropriate. Area under the curve was compared with 2-tailed student t tests. *p < 0.025, **p < 0.0014.