Central kisspeptin does not affect ERK1/2 or p38 phosphorylation in oxytocin neurons of late-pregnant rats

Central kisspeptin does not affect ERK1/2 or p38 phosphorylation in oxytocin neurons of late-pregnant rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Central kisspeptin does not affect ERK1/2 or p38 phosphorylation in oxytocin neurons of late-pregnant rats Mehwish Abbasi, Rachael Augustine, Karl Iremonger, Colin Brown This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1386498/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oxytocin is secreted by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) oxytocin neurons to induce uterine contractions during parturition. Increased activation of oxytocin neurons at parturition involves a network of afferent inputs that increase oxytocin neuron excitability. Kisspeptin fibre density increases around oxytocin neurons during pregnancy, and central kisspeptin administration excites oxytocin neurons only in late pregnancy. Kisspeptin signals via extracellular regulated kinase 1/2 (ERK1/2) and p38. Therefore, to determine whether kisspeptin excites oxytocin neurons via ERK1/2-p38 signalling in late-pregnant rats, we performed immunohistochemistry for phosphorylated ERK1/2 (pERK1/2) and phosphorylated p38 (p-p38) in oxytocin neurons of non-pregnant and late-pregnant rats. Intracerebroventricular (ICV) kisspeptin administration (2 µg) did not affect pERK1/2 or p-p38 expression in SON and PVN oxytocin neurons of non-pregnant or late-pregnant rats. Furthermore, ICV kisspeptin did not affect pERK1/2 or p-p38 expression in brain areas with major projections to the SON and PVN: the nucleus tractus solitarius, rostral ventrolateral medulla, locus coeruleus, dorsal raphe nucleus, organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, anteroventral periventricular nucleus, periventricular nucleus and arcuate nucleus. Hence, kisspeptin-induced excitation of oxytocin neurons in late pregnancy does not appear to involve ERK1/2 or p38 activation in oxytocin neurons or their afferent inputs. kisspeptin oxytocin supraoptic nucleus paraventricular nucleus pregnancy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The hormone, oxytocin, induces uterine contractions for delivery of the offspring during birth. While oxytocin is not essential for birth (Nishimori et al. 1996 ), it is necessary for the normal progression of birth because oxytocin receptor antagonism delays the onset of, and prolongs the duration of, parturition in rats (Antonijevic et al. 1995 ). Oxytocin is synthesised by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) oxytocin neurons that each send a single axon to the posterior gland where oxytocin secretion is triggered by action potential invasion of the axon terminal (Brown et al. 2013 ). Increased activation of oxytocin neurons at parturition is triggered by afferent inputs that relay peripheral signals from cervical stretch receptors (Brown et al. 2013 ). The best characterised excitatory afferent input to oxytocin neurons arises from the A2 noradrenergic cell group of the nucleus tractus solitarius (NTS), which is robustly activated at parturition (Meddle et al. 2000 ). However, acute activation of central noradrenergic receptors alone is not sufficient to trigger parturition in late-pregnant rats (Lipschitz et al. 2004 ), indicating that other mechanisms are likely also involved. Kisspeptin neurons also project to the SON (Desroziers et al. 2010 ) and kisspeptin fibre density increases around oxytocin neurons during pregnancy (Seymour et al. 2017 ). Furthermore, intracerebroventricular (ICV) kisspeptin excites oxytocin neurons only in late pregnancy (Seymour et al. 2017 ) and we have recently shown that this excitation is mediated by direct effects on oxytocin neurons as well as by enhancement of excitatory afferent signalling (Abbasi et al. 2022 ). Kisspeptin principally signals via kisspeptin receptor 1 (Kiss1R) (Kotani et al. 2001 ) but also has high affinity for neuropeptide FF receptors (NPFFR) (Oishi et al. 2011 ). Classically, Kiss1R activation increases phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) (Kotani et al. 2001 ; Ohtaki et al. 2001 ; Masui et al. 2004 ; Kim et al. 2010 ; Peng et al. 2013 ) but also increases phosphorylation of p38 in some cell lines (Masui et al. 2004 ; Kim et al. 2010 ). Similarly to Kiss1R, NPFFR activation also increases phosphorylation of ERK1/2 (Anko and Panula 2006 ; Sun et al. 2012 ; Yu et al. 2016 ; Karnosova et al. 2021 ) and p38 (Karnosova et al. 2021 ). While ERK1/2 and p38 signalling pathways share upstream regulators (Hu et al. 2018 ), p38 phosphorylation has been observed without measurable changes in ERK1/2 phosphorylation, suggesting that p38 can also be activated via a different, as yet unidentified, pathway (Lemonnier et al. 2004 ; Samuvel et al. 2005 ). Furthermore, phosphorylated p38 (p-p38) can directly suppress ERK1/2 phosphorylation (Zhang et al. 2001 ). To test the hypothesis that kisspeptin directly activates oxytocin neurons at the end of pregnancy via ERK1/2 and/or p38 signalling, we measured phosphorylated ERK1/2 (pERK1/2) and p-p38 expression in oxytocin neurons after ICV kisspeptin administration to non-pregnant and late-pregnant rats. Furthermore, to determine which afferent inputs might be activated by kisspeptin to excite oxytocin neurons at the end pregnancy, we also measured pERK1/2 and p-p38 expression in brain areas that express Kiss1R and/or NPFFR and project to the SON and/or PVN. We found that ICV kisspeptin did not affect expression of pERK1/2 or p-p38 in oxytocin neurons or in any of the brain areas examined, suggesting that kisspeptin activates oxytocin neurons at the end of pregnancy via an alternative signalling pathway. Materials And Methods Ethical Approval All experimental procedures were approved by the University of Otago Animals Ethics Committee (approval number: D56/17) and carried out in accordance with the New Zealand Animal Welfare Act and associated guidelines. Animals Adult female Sprague-Dawley rats (6 ­– 12 weeks of age) were purchased from the University of Otago Animal Facility and housed in controlled temperature and lighting (22 – 24ºC; 12 h light / 12 h dark), with free access to standard laboratory rodent food and water. Non-pregnant rats were freely-cycling virgin rats and were housed in groups of 3 – 5 until after surgery. Primiparous pregnant rats were used on gestation day 21 (G21, the expected day of parturition). For timed mating, oestrous cycle stage was assessed by vaginal cytology. At pro-oestrus, rats were placed overnight with a male for mating and the next morning was considered to be G0 after confirmation of the presence of sperm in the vaginal smear. Intracerebroventricular cannulation and kisspeptin administration Under isoflurane anaesthesia (2 / 2.5% in 1 L min -1 O 2 ), non-pregnant and G13/14 rats were implanted with an ICV guide cannula (22-gauge, Plastics One Inc, Roanoke, USA) into the lateral cerebral ventricle (co-ordinates relative to bregma, in mm: right lateral = 1.3; rostral/caudal = 0: ventral = 3.0) using stereotaxic surgical procedures, as previously described (Augustine and Grattan 2008). The guide cannula was anchored to screws (1 mm) inserted into the dorsal surface of the skull using light cure adhesive transbond (Henry Schein Shalfoon, Auckland, New Zealand). All rats were individually housed post-surgery. The clinical condition of each rat was monitored daily until the day of experiment. On day seven or eight following surgery, non-pregnant and G21 rats were anaesthetised with intraperitoneal pentobarbitone (60 mg kg -1 ; 300 mg ml -1 ). Upon cessation of the flexor withdrawal reflex, an internal cannula (28-guage, Plastics One Inc, Roanoke, USA) was inserted into the lateral cerebral ventricle through the guide cannula and attached to a Hamilton syringe (2 µl, Hamilton Company, Reno, USA) via a polyethylene tube (PE-10, 0.5 mm diameter). Rats were injected ICV with either aCSF (artificial cerebrospinal fluid) or 2 µg kisspeptin (1 µg µl -1 dissolved in aCSF, Merck, USA) over 1 min, 60 min after induction of anaesthesia. Brain collection and sectioning 15 min after ICV kisspeptin or aCSF administration, rats were perfused transcardially with ̴ 50 ml of 0.9% saline followed by 250 – 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.6) under continued pentobarbitone anaesthesia. This timeframe was selected because kisspeptin-induced pERK1/2 and p-p38 levels peak 10 – 15 min after incubation with kisspeptin in various cell lines (Masui et al. 2004; Novaira et al. 2009; Kim et al. 2010). Brains were post-fixed for 24 h in 4% paraformaldehyde solution and then 30% sucrose in 0.1 M phosphate buffer (pH 7.6) at 4ºC for 72 h. 30 µm coronal sections were cut on a freezing microtome (Leica SM2400, Wetzlar, Germany) from +0.60 to -3.00 mm relative to bregma to capture the SON, PVN, medial septum (MS), organum vasculosum of the lamina terminalis (OVLT), MnPO (median preoptic nucleus), subfornical organ (SFO), anteroventral periventricular nucleus (AVPe), periventricular nucleus (PeN) and arcuate nucleus (ARC) in the forebrain, from -7.56 to -8.04 mm for the dorsal raphe nucleus (DRN), -9.60 to -10.08 mm for the locus coeruleus (LC), and from -12.00 mm to -14.64 mm for the NTS and rostral ventrolateral medulla (RVLM) in the brainstem (Paxinos and Watson 2007). Sections containing forebrain areas were collected in 4 series and brainstem areas in 3 series; and each series contained 6 – 8 sections. Sections were stored in fresh cryoprotectant (pH 7.6, 0.05 M phosphate buffer saline, 0.9% sodium chloride, 30% sucrose, 1% polyvinylpyrrolidone, 30% ethylene glycol) at -20ºC until use. Immunohistochemistry For fluorescent immunohistochemistry, sections were washed in Tris-buffered saline (TBS) and endogenous aldehydes were blocked by incubating in 0.1% sodium borohydride in TBS for 20 min at room temperature (RT). Sections were washed in TBS and placed in incubation solution (0.3% Triton X-100 and 0.25% bovine serum albumin in TBS) containing host serum in which secondary antibody was raised for 60 min, to avoid non-specific binding. Next, sections were incubated in incubation solution containing a cocktail of primary antibodies for 48 h on an orbital shaker. After washing in TBS, sections were incubated for 3 h at RT in fluorescent-tagged secondary antibodies diluted in incubation solution. Primary antibodies used for fluorescent staining of pERK1/2 and p-p38 with oxytocin and tyrosine hydroxylase (TH, a marker for identification of noradrenaline-containing neurons) were rabbit anti-pERK1/2 antibody (p-44/42 MAPK (T 202/Y 204), 9101S, Cell Signalling; 1:1000), rabbit anti-p-p38 antibody (Thr 180/Tyr, 182, 9211, Cell Signalling; 1:2000), mouse oxytocin (MAB-5296, Millipore; 1:5000) and mouse anti-TH (MAB 318, Millipore; 1:2,000). The secondary antibodies used were goat anti-mouse Alexa fluor 488 antibody (A11029, Thermofisher Scientific; 1:500) and goat anti-rabbit Alexa fluor 568 antibody (ab 175471, Abcam; 1:500). For chromogenic immunohistochemistry, sections were washed in Tris-buffered saline (TBS) and endogenous aldehydes were blocked by incubating in TBS containing methanol and 30% H 2 O 2 for 10-15 min at RT. Brain sections were washed and incubated in rabbit anti-pERK1/2 (1: 5000) or rabbit anti-p-p38 (1:2000) antibody with 4% normal goat serum for 48 h. Sections were then incubated in a secondary antibody solution containing a goat anti-rabbit biotinylated secondary antibody (BA-1000, Vector Laboratories; 1:500) for 90 min. After washing again in TBS, sections were incubated in an avidin-biotin-peroxidase solution (Vector Laboratories, California) for 90 min. Finally, pERK1/2-p-p38 staining was visualised by immersing sections in a nickel-diaminobenzidine (Ni DAB) peroxidase solution (Vector Laboratories, California) prepared in distilled water. Some sections were randomly mounted on slides and regularly examined under the bright field microscope. When staining became visible against background (usually after 2 – 10 min), the Ni DAB reaction was stopped by washing the sections in TBS. Sections were mounted on gelatinized slides, cleared, dehydrated (only DAB stained sections pass through increasing concentrations of ethanol series followed by xylene) and dried. Fluorescent stained sections were coverslipped using Fluoromout-G ® (Southern Biotech, Birmingham, USA), whereas DAB stained sections were coverslipped using DPX mounting medium (VWR International Limited, England). Stained sections were examined using Olympus bright-field/fluorescence microscope (BX51- NAOS) attached to a digital camera (GRYPHAX) to capture photomicrographs. The brain areas of interest were photographed at magnifications of 500x, 200x and 100x and quantification was completed manually using the cell counter plugin on Fiji (NIH, v. 1.47) software. For all analyses, slides were randomly coded to avoid experimenter bias in counting. Successful delivery of ICV kisspeptin was confirmed by double labelling for gonadotrophin releasing hormone (GnRH) and pERK1/2 in the MS. First, DAB staining of pERK1/2 was performed as described above, and sections were co-stained using guinea pig anti-GnRH primary antibody (GA02, a generous gift from Professor Allan Herbison; 1: 10,000) with 4% normal goat serum for 48 h. Sections were then incubated in secondary antibody solution containing a goat anti-guinea pig (BA-1000, Vector Laboratories; 1:500). Finally, the DAB solution without nickel was added to label GnRH-expressing neurons. Similarly to our previous study using Fos protein as a marker of activation (Augustine et al. 2018), the number of GnRH neurons expressing pERK1/2 was significantly higher in kisspeptin-treated non-pregnant rats than in aCSF-treated non-pregnant rats, but was not different between kisspeptin-treated late-pregnant rats and aCSF-treated late-pregnant rats (Reproductive status (RS): F (1,1) = 1.064, p = 0.314; Treatment (T): F (1,1) = 4.461, p = 0.047; RS x T interaction: F (1,2) = 6.77, p = 0.017, two-way ANOVA; Supplementary Figure 1). Previous studies validated the specificity of oxytocin (Dabrowska et al. 2011), pERK1/2 and p-p38 (Worsley et al. 2014), TH (Tagliaferro and Morales 2008) and GnRH (Rizwan et al. 2012) antibodies. Specificity controls were performed by omitting primary antibodies. No non-specific staining was evident in any section with primary antibody omitted. Statistical Analysis Data were analysed on GraphPad Prism version 8 for Windows (GraphPad Software Inc, San Diego, CA, USA). Statistical significance between groups was determined by two-way analysis of variance (ANOVA) followed by post hoc Holm Sidak's test, where the F ratio was significant. All values are presented as mean ± standard error of mean (SEM). Pearson product moment correlations were run to determine correlations. Probabilities ( p ) < 0.05 were considered significant. Results Kisspeptin does not affect pERK1/2 expression in SON or PVN oxytocin neurons While there was a higher number of oxytocin-positive neurons co-expressing pERK1/2 in the SON and PVN of G21 rats than non-pregnant rats, there was no effect of ICV kisspeptin on the number of oxytocin-positive neurons co-expressing pERK1/2 in the SON (RS: F (1,1) = 33.17, p ˂ 0.0001; T: F (1,1) = 4.20, p = 0.050; RS x T: F (1,2) = 0.050, p = 0.824, two-way ANOVA; Figure 1e-g) or PVN (RS: F (1,1) = 28.52, p ˂ 0.0001; T: F (1,1) = 0.0009, p = 0.975; RS x T: F (1,2) = 0.039, p = 0.844, two-way ANOVA; Figure 1l-n) of non-pregnant and G21 rats. Subdividing the PVN into the magnocellular PVN (mPVN) and parvocellular PVN (pPVN) revealed similar results for both regions as was found for the PVN as whole, with a higher number of oxytocin-positive neurons co-expressing pERK1/2 in the mPVN and pPVN of G21 rats than non-pregnant rats, but no effect of ICV kisspeptin on the number of oxytocin-positive neurons co-expressing pERK1/2 in the mPVN (RS: F (1,1) = 26.27, p < 0.0001; T: F (1,1) = 0.028, p = 0.866; RS x T: F (1,2) = 0.0006, p = 0.980; Figure 2a-c) or pPVN (RS: F (1,1) = 23.15, p < 0.0001; T: F (1,1) = 0.076, p = 0.783; RS x T: F (1,2) = 0.186, p = 0.669; Figure 2d-f) of non-pregnant and G21 rats. Kisspeptin does not affect p-p38 expression in SON or PVN oxytocin neurons Similarly to pERK1/2, there was a higher number of oxytocin-positive neurons co-expressing p-p38 in the SON of G21 rats than non-pregnant rats, but no effect of ICV kisspeptin on the number of oxytocin-positive neurons co-expressing p-p38 in the SON of non-pregnant or G21 rats (RS: F (1,1) = 9.90, p = 0.004; T: F (1,1) = 0.007, p = 0.930; RS x T: F (1,2) = 0.052, p = 0.821, two-way ANOVA; Figure 3e-g). By contrast to the SON, there was no effect of reproductive status or ICV kisspeptin on p-p38 expression in oxytocin neurons in the PVN as a whole (RS: F (1,1) = 3.39, p = 0.077 ; T: F (1,1) = 0.939, p = 0.341; RS x T: F (1,2) = 4.23, p = 0.050; Figure 3l-n), or in the mPVN (RS: F (1,1) = 2.90, p = 0.100; T: F (1,1) = 0.99, p = 0.32; RS x T: F (1,2) = 3.55, p = 0.071; Figure 4a-c) or pPVN (RS: F (1,1) = 3.35, p = 0.078; T: F (1,1) = 0.353, p = 0.557; RS x T: F (1,2) = 3.35, p = 0.078; Figure 4d-f) of non-pregnant and G21 rats. Kisspeptin does not affect pERK1/2 or p-p38 expression in brainstem noradrenergic neurons in the NTS, RVLM or LC To determine whether ICV kisspeptin might excite oxytocin neurons at the end of pregnancy by activation of NTS noradrenergic neurons, which project to SON and PVN oxytocin neurons, express NPFFR (Liu et al. 2001), and are robustly activated at parturition (Meddle et al. 2000), pERK1/2 and p-p38 were each double-labelled with TH. pERK1/2 and p-p38 were also each double-labelled with TH in the RVLM to determine whether excitatory effects of ICV kisspeptin might be mediated via RVLM because RVLM noradrenergic neurons are also activated at parturition (Meddle et al. 2000), express Kiss1R (Herbison et al. 2010) and project catecholaminergic neurons (containing dopamine, noradrenaline, adrenaline) to SON and PVN oxytocin (and vasopressin) neurons (Cunningham Jr et al. 1990). There was no effect of reproductive status or ICV kisspeptin on the number of TH-positive neurons co-expressing pERK1/2 in the NTS (RS: F (1,1) = 0.580, p = 0.453; T: F (1,1) = 0.501, p = 0.485; RS x T (F (1,2) = 0.900, p = 0.351, two-way ANOVA; Figure 5e-g). Irrespective of kisspeptin treatment, pERK1/2 expression in TH-positive NTS neurons correlated with pERK1/2 expression in oxytocin-positive SON neurons (r = 0.536, p = 0.039) but there was no correlation between pERK1/2 expression in TH-positive NTS neurons and oxytocin-positive PVN neurons (r = 0.098, p = 0.726), or mPVN neurons (r = 0.221, p = 0.428) or pPVN neurons (r = -0.192, p = 0.492) in G21 rats. While there was a higher number of TH-positive neurons co-expressing pERK1/2 in the RVLM of aCSF-treated G21 rats than aCSF-treated non-pregnant rats, there was no effect of ICV kisspeptin on the number of TH-positive neurons co-expressing pERK1/2 in the RVLM of non-pregnant or G21 rats (RS: F (1,1) = 5.27, p = 0.029; T: F (1,1) = 0.667, p = 0.421; RS x T: F (1,2) = 6.35, p = 0.018; Figure 5l-n). Similarly to pERK1/2, there was no effect of reproductive status or ICV kisspeptin on the number of TH-positive neurons co-expressing p-p38 in the NTS (RS: F (1,1) = 0.086, p = 0.771 ; T: F (1,1) = 0.344, p = 0.562; RS x T: F (1,2) = 0.086, p = 0.771; Figure 6e-g). Also, there was no correlation between p-p38 expression in TH-positive NTS neurons and oxytocin-positive SON neurons (r = -0.287, p = 0.392), PVN neurons (r = 0.181, p = 0.572), mPVN neurons (r = 0.169, p = 0.598) or pPVN neurons (r = 0.198, p = 0.536) in G21 rats. By contrast to pERK1/2 expression in TH-positive RVLM neurons, there was no effect of reproductive status or ICV kisspeptin on p-p38 expression in TH-positive RVLM neurons (RS: F (1,1) = 1.44, p = 0.241; T: F (1,1) = 0.005, p = 0.944; RS x T: F (1,2) = 0.005, p = 0.944; Figure 6l-n). Also, there was no correlation between p-p38 expression in TH-positive RVLM neurons and oxytocin-positive SON neurons (r = -0.199, p = 0.607) in G21 rats. pERK1/2 was labelled in the LC to serve as a brain area control for the NTS because LC express Kiss1R and NPFFR (Lee et al. 1999; Liu et al. 2001) and LC noradrenergic neurons project to the PVN but principally to non-magnocellular neurons (Schreihofer and Guyenet 2002; Berridge and Waterhouse 2003). As expected, there was no effect of reproductive status or ICV kisspeptin on the number of pERK1/2-positive neurons in the LC (RS: F (1,1) = 1.19, p = 0.286; T: F (1,1) = 0.563, p = 0.460; RS x T: F (1,2) = 0.292, p = 0.594; Supplementary Figure 2e). Also, there was no correlation between pERK1/2 expression in the LC and oxytocin-positive neurons of SON in G21 rats (r = -0.266, p = 0.401). Kisspeptin does not affect pERK1/2 expression in the DRN neurons DRN neurons express Kiss1R and NPFFR (Liu et al. 2001; Higo et al. 2016) and project to SON and PVN oxytocin neurons (Sawchenko et al. 1983) but DRN inputs to oxytocin neurons are not involved in parturition (Herbison et al. 1997). Therefore, pERK1/2 was labelled in the DRN to serve as brainstem control for inputs to oxytocin neurons that are activated at parturition. While there was a higher number of pERK1/2-positive neurons in the DRN of G21 rats than non-pregnant rats, there was no effect of ICV kisspeptin on the number of pERK1/2-positive neurons in the DRN of non-pregnant or G21 rats (RS: F (1,1) = 4.38, p = 0.048; T: F (1,1) = 0.077, p = 0.783; RS x T: F (1,2) = 0.015, p = 0.901, two-way ANOVA; Supplementary Figure 2j). Also, there was no correlation between pERK1/2 expression in DRN neurons and SON oxytocin-positive neurons in G21 rats (r = -0.522, p = 0.099). Kisspeptin does not affect pERK1/2 or p-p38 expression in the OVLT, MnPO or SFO To determine whether ICV kisspeptin activates OVLT, MnPO and/or SFO neurons, which each project to the SON and PVN (Weiss and Hatton 1990; McKinley et al. 1992; Westerhaus and Loewy 1999) and express Kiss1R and/or NPFFR (Lee et al. 1999; Herbison et al. 2010; Higo et al. 2016; Higo et al. 2021), pERK1/2 and p-p38 were labelled in each brain area. There was no effect of reproductive status or ICV kisspeptin on the number of pERK1/2-positive OVLT neurons (RS: 0.194, p = 0.664; T: F (1,1) = 0.021, p = 0.884;RS x T: F (1,2) = 1.62, p = 0.218, two-way ANOVA; Figure 7e), MnPO neurons (RS: F (1,1) = 1.63, p = 0.215; T: F (1,1) = 0.003, p = 0.956; RS x T: F (1,2) = 0.0009, p = 0.976; Figure 7j) or SFO neurons (RS: F (1,1) = 0.00001, p = 0.999; T: F (1,1) = 0.035, p = 0.851; RS x T: F (1,2) = 0.073, p = 0.789; Figure 7o) in non-pregnant or G21 rats. Also, there was no correlation between pERK1/2 expression in oxytocin-positive SON neurons and pERK1/2 expression in OVLT neurons (r = -0.065, p = 0.866), MnPO neurons(r = -0.259, p = 0.415) or SFO neurons (r = -0.105, p = 0.772) in G21 rats. Similarly to pERK1/2, there was no effect of reproductive status or ICV kisspeptin on the number of p-p38-positive OVLT neurons (RS: F (1,1) = 0.601, p = 0.448; T: F (1,1) = 0.231, p = 0.636; RS x T: F (1,2) = 0.019, p = 0.890; Figure 8e), MnPO neurons (RS: F (1,1) = 0.00007, p = 0.993; T: F (1,1) = 0.003, p = 0.952; RS x T: F (1,2) = 0.408, p = 0.534; Figure 8j) or SFO neurons (RS: F (1,1) = 1.31, p = 0.271; T: F (1,1) = 1.50, p = 0.240; RS x T: F (1,2) = 1.32, p = 0.268, Figure 8o) in non-pregnant or G21 rats. Also, there was no correlation between p-p38 expression in oxytocin-positive SON neurons and pERK1/2-positive OVLT neurons (r = 0.112, p = 0.809), MnPO neurons (r = -0.648, p = 0.115) or SFO neurons (r = 0.189, p = 0.684) in G21 rats. Kisspeptin does not affect pERK1/2 or p-p38 expression in the AVPe, Pe or pERK1/2 expression in the ARC pERK1/2 and p-p38 were also labelled in the AVPe, PeN and ARC, which contain kisspeptin neurons (Clarkson and Herbison 2009; Lehman et al. 2010), express Kiss1R and/or NPFFR (Herbison et al. 2010; Higo et al. 2016; Higo et al. 2021). Kisspeptin PeN neurons project to the SON but AVPe and ARC kisspeptin neurons do not (Seymour et al. 2017). There was no effect of reproductive status or ICV kisspeptin on the number of pERK1/2-positive AVPe neurons (RS: F (1,1) = 0.270, p = 0.609; T: F (1,1) = 0.126, p = 0.726; RS x T: F (1,2) = 0.090, p = 0.767, two-way ANOVA; Figure 9e), PeN neurons (F (1,1) = 0.046, p = 0.834; T: F (1,1) = 0.159, p = 0.698; RS x T: F (1,2) = 0.498, p = 0.496; Figure 9j) or ARC neurons (RS: F (1,1) = 0.764, p = 0.389; T: F (1,1) = 0.719, p = 0.403; RS x T: F (1,2) = 0.358, p = 0.554; Figure 9o) in non-pregnant or G21 rats. Also, there was no correlation between pERK1/2 expression in oxytocin-positive SON neurons and pERK1/2-positive AVPe neurons (r = 0.069, p = 0.882), PeN neurons (r = -0.143, p = 0.735) or ARC neurons (r = -0.074, p = 0.809) in G21 rats. Similarly to pERK1/2, there was no effect of reproductive status or ICV kisspeptin on the number of p-p38-positive AVPe neurons (RS: F (1,1) = 0.231, p = 0.637; T: F (1,1) = 0.882, p = 0.362; RS x T: F (1,2) = 0.225, p = 0.641; Figure 10e) or PeN neurons (RS: F (1,1) = 0.455, p = 0.510; T: F (1,1) = 0.0008, p = 0.977; RS x T: F (1,2) = 0.559, p = 0.467; Figure 10j) in non-pregnant or G21 rats. Also, there was no correlation between p-p38 expression in oxytocin-positive SON neurons and pERK1/2-positive AVPe neurons (r = -0.378, p = 0.459) or PeN neurons (r = -0.178, p = 0.672) in G21 rats. There was insufficient tissue to label p-p38 in the ARC. Discussion We have recently shown that kisspeptin excites oxytocin neurons in late pregnancy, in part, by a direct action on oxytocin neurons (Abbasi et al. 2022). The present study revealed that ICV kisspeptin did not induce ERK1/2 or p38 phosphorylation in oxytocin neurons of non-pregnant or late-pregnant rats when administered at a dose that increases oxytocin neuron firing rate in late-pregnant rats (Seymour et al. 2017). Nevertheless, consistent with previous findings (Chandaka et al. 2016), ERK1/2 (and p38) phosphorylation was higher in oxytocin neurons of late-pregnant rats than in non-pregnant rats. Hence, it appears likely that kisspeptin excitation of oxytocin neurons in late-pregnant rats is not mediated by Kiss1R and/or NPFFR activation of ERK1/2 or p38 signalling. Local kisspeptin activation of oxytocin neurons in late pregnancy The failure of kisspeptin to induce ERK1/2 (or p38) phosphorylation in oxytocin neurons of late-pregnant rats probably does not reflect a failure to deliver sufficient kisspeptin to activate these signalling pathways because kisspeptin administration increased ERK1/2 phosphorylation in GnRH neurons of the same non-pregnant rats in which ERK1/2 and p38 phosphorylation were unchanged in oxytocin neurons. While kisspeptin did not induce ERK1/2 phosphorylation in GnRH neurons of late-pregnant rats, this likely reflects downregulation of GnRH neuron responsiveness to kisspeptin during pregnancy, as we have previously reported using Fos protein as a marker of activation (Augustine et al. 2018). Elevated ERK1/2 phosphorylation in oxytocin neurons at the end of pregnancy might have occluded the ability of exogenous kisspeptin to induce further phosphorylation. However, ≤50% of oxytocin neurons expressed pERK1/2 in late-pregnant rats. Hence, occlusion also appears unlikely to account for the failure of kisspeptin to induce ERK1/2 phosphorylation in oxytocin neurons of late-pregnant rats. Taken together, these observations suggest that the lack of kisspeptin-induced ERK1/2 (and p38) phosphorylation in oxytocin neurons in late pregnancy was not due to a technical failure. Rather, it appears that kisspeptin excitation of oxytocin neurons in late pregnancy is not mediated by Kiss1R-mediated phosphorylation of ERK1/2 or p38. If kisspeptin does not activate Kiss1R/NPFFR-ERK1/2/p38 signalling in oxytocin neurons, another signalling pathway must mediate the direct effects of kisspeptin of oxytocin neurons in late pregnancy. Kiss1R activates neuronal nitric oxide synthase in preoptic neurons via phosphatidylinositol-3-kinase(PI3K)-Akt (Hanchate et al. 2012). Hence, PI3K-Akt might mediate the direct effects of kisspeptin on oxytocin neurons in late pregnancy. While kisspeptin excites GnRH neurons by activating non-specific cation channels and inhibiting potassium channels (Liu et al. 2008), it does not appear to excite oxytocin neurons via these channels because baseline holding currents are unchanged by kisspeptin superfusion in SON brain slices (Abbasi et al. 2022). Alternatively, or additionally, kisspeptin activation of oxytocin neurons might not be mediated by Kiss1R. Indeed, SON Kiss1R mRNA expression does not change during pregnancy (Seymour et al. 2017), suggesting that upregulation of Kiss1R does not underpin kisspeptin excitation of oxytocin neurons in late pregnancy, although the possibility of increased Kiss1R surface expression and/or sensitivity cannot be discounted. Oxytocin neurons also express NPFFR (Kim et al. 2016), which couples to Gs and Gi as well as to Gq (Bonini et al. 2000; Liu et al. 2001; Mollereau et al. 2002) to increase phosphorylation of cAMP response element-binding protein (CREB) and c-Jun N terminal kinase (JNK) (Karnosova et al. 2021). Hence, NPFFR-CREB/JNK signalling might mediate the direct effects of kisspeptin on oxytocin neurons in late pregnancy. Further work will be required to determine which, if any, of these signalling pathways mediate kisspeptin excitation of oxytocin neurons at the end of pregnancy. Lack of kisspeptin induction of ERK1/2 or p38 phosphorylation in afferent inputs to the oxytocin system While our recent findings showed that kisspeptin excites oxytocin neurons in late pregnancy, in part, by a direct action of kisspeptin on oxytocin neurons, our findings also suggested that activation of afferent inputs might also contribute to the excitation (Abbasi et al. 2022). Therefore, we mapped kisspeptin-induced ERK1/2 and p38 phosphorylation in brain areas that project to the oxytocin system and express Kiss1R and NPFFR (LC, DRN, OVLT, SFO, AVPe, ARC) (Lee et al. 1999; Herbison et al. 2010; Higo et al. 2016; Higo et al. 2021), only NPFFR (NTS, PeN) (Higo et al. 2021) or only Kiss1R (RVLM, MnPO) (Irwig et al. 2004; Herbison et al. 2010). However, neither ERK1/2 nor p38 phosphorylation was affected by kisspeptin in any of the brain areas studied in non-pregnant or late-pregnant rats. Hence, it appears that any indirect kisspeptin excitation of oxytocin neurons in late pregnancy is not mediated by activation of ERK1/2-p38 signalling in these afferent inputs. While kisspeptin did not induce ERK1/2 or p38 phosphorylation in afferent inputs to the oxytocin system in non-pregnant or late-pregnant rats, there was a positive correlation of ERK1/2 phosphorylation in late pregnancy between SON oxytocin neurons and NTS noradrenergic neurons, which project to oxytocin neurons and are activated during parturition (Meddle et al. 2000); the correlation with ERK1/2 phosphorylation in oxytocin neurons was specific to NTS noradrenergic neurons, further implicating NTS noradrenergic neurons in driving oxytocin neuron activity for parturition. While the same caveats apply to the lack of ERK1/2 or p38 phosphorylation in their afferent inputs as apply to the oxytocin neuron themselves, a further possibility is that kisspeptin might enhance excitatory synaptic transmission via presynaptic actions on oxytocin neurons afferent inputs. If active, this mechanism does not involve local glutamatergic or GABAergic inputs to oxytocin neurons, which are unaffected by kisspeptin in brain slices from non-pregnant and late-pregnant rats (Abbasi et al. 2022). Therefore, the most likely candidate might be the NTS noradrenergic input, which is activated at parturition (Meddle et al. 2000), releasing noradrenaline (Herbison et al. 1997) to excite oxytocin neurons via α 1 -adrenoreceptors (Douglas et al. 2001). Concluding remarks Taken together, the current results show that kisspeptin-induced excitation of oxytocin neurons in late pregnancy is not mediated by phosphorylation of canonical Kiss1R (or NPFFR)-activated second messengers, ERK1/2 (or p38), and further work will be required to determine which signalling pathway mediates kisspeptin excitation of oxytocin neurons in late pregnancy. Abbreviations aCSF, artificial cerebrospinal fluid; ARC, arcuate nucleus; AVPe, anteroventral periventricular nucleus; ERK1/2, extracellular regulated kinase 1/2; ICV, intracerebroventricular; Kiss1R, kisspeptin-1 receptor; MnPO, median preoptic nucleus; OVLT, organum vasculosum lamina terminalis; NPFFR, Neuropeptide FF receptor; pERK1/2, phosphorylated ERK1/2; p-p38, phosphorylated p38; SFO, subfornical organ; SON, supraoptic nucleus; PVN, paraventricular nucleus; PeN, periventricular nucleus. Declarations Funding This study was funded by Manatu Hauora | Health Research Council of New Zealand (CHB) and a University of Otago Postgraduate Scholarship (MA). Competing interests The authors have no competing financial interests to disclose. Author contributions All authors contributed to the experimental design. Experiments were performed by Mehwish Abbasi and Rachael Augustine. Data collection, analysis and figures were prepared by Mehwish Abbasi. The first draft of the manuscript was written by Mehwish Abbasi. All authors provided revised the manuscript and approved the submitted version. Data availability All data acquired during this study are available from corresponding author upon reasonable request. References Abbasi M, Perkinson MR, Seymour AJ, Piet R, Campbell RE, Iremonger KJ, Brown CH (2022) Local kisspeptin excitation of rat oxytocin neurones in late pregnancy. J Physiol. https://doi.org/10.1113/JP282531 Anko ML, Panula P (2006) Regulation of endogenous human NPFF2 receptor by neuropeptide FF in SK-N‐MC neuroblastoma cell line. J Neurochem 96(2):573–584. doi: https://doi.org/10.1111/j.1471-4159.2005.03581.x Antonijevic IA, Douglas AJ, Dye S, Bicknell RJ, Leng G, Russell JA (1995) Oxytocin antagonists delay the initiation of parturition and prolong its active phase in rats. J Endocrinol 145(1):97–103. doi: https://doi.org/10.1677/joe.0.1450097 Augustine RA, Grattan DR (2008) Induction of central leptin resistance in hyperphagic pseudopregnant rats by chronic prolactin infusion. Endocrinology 149(3):1049–1055. doi: https://doi.org/10.1210/en.2007-1018 Augustine RA, Seymour AJ, Campbell RE, Grattan DR, Brown CH (2018) Integrative neuro-humoral regulation of oxytocin neuron activity in pregnancy and lactation. J Neuroendocrinol 30(8):12569–12572. doi: https://doi.org/10.1111/jne.12569 Berridge CW, Waterhouse BD (2003) The locus coeruleus–noradrenergic system: modulation of behavioral state and state-dependent cognitive processes. Brain Res Rev 42(1):33–84. doi: https://doi.org/10.1016/S0165-0173(03)00143-7 Bonini JA, Jones KA, Adham N, Forray C, Artymyshyn R, Durkin MM, Smith KE, Tamm JA, Boteju LW, Lakhlani PP, Raddatz R, Yao W-J, Ogozalek KL, Noel B, Kouranova EV, Quan Y, Vaysse PJ, Wetzel JM, Branchek TA, Gerald C, Borowsky B (2000) Identification and characterization of two G protein-coupled receptors for neuropeptide FF. J Biol Chem 275(50):39324–39331. doi: https://doi.org/10.1074/jbc.M004385200 Brown CH, Bains JS, Ludwig M, Stern JE (2013) Physiological regulation of magnocellular neurosecretory cell activity: integration of intrinsic, local and afferent mechanisms. J Neuroendocrinol 25(8):678–710. doi: https://doi.org/10.1111/jne.12051 Chandaka GK, Wang L, Senogles S, Armstrong WE (2016) Late pregnancy is a critical period for changes in phosphorylated mitogen-activated protein kinase/extracellular signal‐regulated kinase 1/2 in oxytocin neurones. J Neuroendocrinol 28(9):e12398. doi: https://doi.org/10.1111/jne.12398 Clarkson J, Herbison AE (2009) Oestrogen, Kisspeptin, GPR54 and the Pre-Ovulatory Luteinising Hormone Surge. J Neuroendocrinol 21(4):305–311. doi: https://doi.org/10.1111/j.1365-2826.2009.01835.x Cunningham ET Jr, Bohn MC, Sawchenko PE (1990) Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J Comp Neurol 292(4):651–667. doi: https://doi.org/10.1002/cne.902920413 Dabrowska J, Hazra R, Ahern TH, Guo JD, McDonald AJ, Mascagni F, Muller JF, Young LJ, Rainnie DG (2011) Neuroanatomical evidence for reciprocal regulation of the corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed nucleus of the stria terminalis of the rat: implications for balancing stress and affect. Psychoneuroendocrinology 36(9):1312–1326. doi: https://doi.org/10.1016/j.psyneuen.2011.03.003 Desroziers E, Mikkelsen J, Simonneaux V, Keller M, Tillet Y, Caraty A, Franceschini I (2010) Mapping of kisspeptin fibres in the brain of the pro-oestrous rat. J Neuroendocrinol 22(10):1101–1112. doi: https://doi.org/10.1111/j.1365-2826.2010.02053.x Douglas A, Scullion S, Antonijevic I, Brown D, Russell J, Leng G (2001) Uterine contractile activity stimulates supraoptic neurons in term pregnant rats via a noradrenergic pathway. Endocrinology 142(2):633–644 Hanchate NK, Parkash J, Bellefontaine N, Mazur D, Colledge WH, de d'Anglemont X, Prevot V (2012) Kisspeptin-GPR54 signaling in mouse NO-synthesizing neurons participates in the hypothalamic control of ovulation. J Neurosci 32(3):932–945. doi: https://doi.org/10.1523/JNEUROSCI.4765-11.2012 Herbison AE, d'Anglemont de Tassigny X, Doran J, Colledge WH (2010) Distribution and postnatal development of Gpr54 gene expression in mouse brain and gonadotropin-releasing hormone neurons. Endocrinology 151(1):312–321. doi: https://doi.org/10.1210/en.2009-0552 Herbison AE, Voisin DL, Douglas AJ, Chapman C (1997) Profile of monoamine and excitatory amino acid release in rat supraoptic nucleus over parturition. Endocrinology 138(1):33–40. doi: https://doi.org/10.1210/endo.138.1.4859 Higo S, Honda S, Iijima N, Ozawa H (2016) Mapping of kisspeptin receptor mRNA in the whole rat brain and its co-localisation with oxytocin in the paraventricular nucleus. J Neuroendocrinol 28(4):1–8. doi: https://doi.org/10.1111/jne.12356 Higo S, Kanaya M, Ozawa H (2021) Expression analysis of neuropeptide FF receptors on neuroendocrine-related neurons in the rat brain using highly sensitive in situ hybridization. Histochem Cell Biol 155(4):465–475. doi: https://doi.org/10.1007/s00418-020-01956-9 Hu KL, Zhao H, Chang HM, Yu Y, Qiao J (2018) Kisspeptin/kisspeptin receptor system in the ovary. Front Endocrinol 8:365. doi: https://doi.org/10.3389/fendo.2017.00365 Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA (2004) Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. J Neuroendocrinol 80(4):264–272. doi: https://doi.org/10.1159/000083140 Karnosova A, Strnadova V, Hola L, Zelezna B, Kunes J, Maletínska L (2021) Palmitoylation of Prolactin-Releasing Peptide Increased Affinity for and Activation of the GPR10, NPFF-R2 and NPFF-R1 Receptors: In Vitro Study. Int J Mol Sci 22(16):8904. doi: https://doi.org/10.3390/ijms22168904 Kim GL, Dhillon SS, Belsham DD (2010) Kisspeptin directly regulates neuropeptide Y synthesis and secretion via the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways in NPY-secreting hypothalamic neurons. Endocrinology 151(10):5038–5047. doi: https://doi.org/10.1210/en.2010-0521 Kim JS, Brown CH, Anderson GM (2016) Anti-opioid effects of RFRP-3 on magnocellular neuron activity in morphine-naïve and morphine-treated female rats. Endocrinology 157(10):4003–4011 Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, C B, Schiffmann SN, Vassart G, Parmentier M (2001) The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276(37):34631–34636. doi: https://doi.org/10.1074/jbc.M104847200 Lee DK, Nguyen T, O'Neill GP, Cheng R, Liu Y, Howard AD, Coulombe N, Tan CP, Tang-Nguyen AT, George SR, O'Dowd BF (1999) Discovery of a receptor related to the galanin receptors. FEBS lett 446(1):103–107. doi: https://doi.org/10.1016/S0014-5793(99)00009-5 Lehman MN, Merkley CM, Coolen LM, Goodman RL (2010) Anatomy of the kisspeptin neural network in mammals. Brain Res 1364:90–102. doi: https://doi.org/10.1016/j.brainres.2010.09.020 Lemonnier J, Ghayor C, Guicheux J, Caverzasio J (2004) Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation. J Biol Chem 279(1):259–264. doi: https://doi.org/10.1074/jbc.M308665200 Lipschitz DL, Crowley WR, Bealer SL (2004) Differential sensitivity of intranuclear and systemic oxytocin release to central noradrenergic receptor stimulation during mid-and late gestation in rats. Am J Physiol Endocrinol Metab 287(3):E523–E528. doi: https://doi.org/10.1152/ajpendo.00572.2003 Liu Q, Guan XM, Martin WJ, McDonald TP, Clements MK, Jiang Q, Zeng Z, Jacobson M, Williams DL, Yu H, Bomford D, Figueroa D, Mallee J, Wang R, Evans J, Gould R, Austin CP (2001) Identification and characterization of novel mammalian neuropeptide FF-like peptides that attenuate morphine-induced antinociception. J Biol Chem 276(40):36961–36969. doi: https://doi.org/10.1074/jbc.M105308200 Liu X, Lee K, Herbison AE (2008) Kisspeptin excites gonadotropin-releasing hormone neurons through a phospholipase C/calcium-dependent pathway regulating multiple ion channels. Endocrinology 149(9):4605–4614. doi: https://doi.org/10.1210/en.2008-0321 Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S, Niida A, Fujii N, Imamura M (2004) Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 315(1):85–92. doi: https://doi.org/10.1016/j.bbrc.2004.01.021 McKinley MJ, Bicknell RJ, Hards D, McAllen RM, Vivas L, Weisinger RS, Oldfield BJ (1992) Efferent neural pathways of the lamina terminalis subserving osmoregulation. Prog Brain Res 91:395–402. doi: https://doi.org/10.1016/S0079-6123(08)62358-4 Meddle SL, Leng G, Selvarajah JR, Bicknell RJ, Russell JA (2000) Direct pathways to the supraoptic nucleus from the brainstem and the main olfactory bulb are activated at parturition in the rat. Neuroscience 101(4):1013–1021. doi: https://doi.org/10.1016/S0306-4522(00)00300-6 Mollereau C, Mazarguil H, Marcus D, Quelven I, Kotani M, Lannoy V, Dumont Y, Quirion R, Detheux M, Parmentier M, Zajac JM (2002) Pharmacological characterization of human NPFF1 and NPFF2 receptors expressed in CHO cells by using NPY Y1 receptor antagonists. Eur J Pharmacol 451(3):245–256. doi: https://doi.org/10.1016/S0014-2999(02)02224-0 Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM (1996) Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci 93(21):11699–11704. doi: https://doi.org/10.1073/pnas.93.21.11699 Novaira HJ, Ng Y, Wolfe A, Radovick S (2009) Kisspeptin increases GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines. Mol Cell Endocrinol 311(1–2):126–134. doi: https://doi.org/10.1016/j.mce.2009.06.011 Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M (2001) Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411(6837):613–617. doi: https://doi.org/10.1038/35079135 Oishi S, Misu R, Tomita K, Setsuda S, Masuda R, Ohno H, Naniwa Y, Ieda N, Inoue N, Ohkura S, Uenoyama Y, Tsukamura H, Maeda K, Hirasawa A, Tsujimoto G, Fujii N (2011) Activation of neuropeptide FF receptors by kisspeptin receptor ligands. ACS Med Chem Lett 2(1):53–57. doi: https://doi.org/10.1021/ml1002053 Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates, 6 edn. Elsevier Academic Press, Amsterdam Peng J, Tang M, Zhang BP, Zhang P, Zhong T, Zong T, Yang B, Kuang HB (2013) Kisspeptin stimulates progesterone secretion via the Erk1/2 mitogen-activated protein kinase signaling pathway in rat luteal cells. Fertil Steril 99(5):1436–1443. doi: https://doi.org/10.1016/j.fertnstert.2012.12.008 Rizwan MZ, Poling MC, Corr M, Cornes PA, Augustine RA, Quennell JH, Kauffman AS, Anderson GM (2012) RFamide-related peptide-3 receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action. Endocrinology 153(8):3770–3779. doi: https://doi.org/10.1210/en.2012-1133 Samuvel DJ, Jayanthi LD, Bhat NR, Ramamoorthy S (2005) A role for p38 mitogen-activated protein kinase in the regulation of the serotonin transporter: evidence for distinct cellular mechanisms involved in transporter surface expression. J Neurosci 25(1):29–41. doi: https://doi.org/10.1523/JNEUROSCI.3754-04.2005 Sawchenko PE, Swanson LW, Steinbusch HW, Verhofstad AA (1983) The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat. Brain Res 277(2):355–360. doi: https://doi.org/10.1016/0006-8993(83)90945-9 Schreihofer AM, Guyenet PG (2002) The baroreflex and beyond: control of sympathetic vasomotor tone by GABAergic neurons in the ventrolateral medulla. Clin Exp Pharmacol Physiol 29(5–6):514–521. doi: https://doi.org/10.1046/j.1440-1681.2002.03665.x Seymour AJ, Scott V, Augustine RA, Bouwer GT, Campbell RE, Brown CH (2017) Development of an excitatory kisspeptin projection to the oxytocin system in late pregnancy. J Physiol 595(3):825–838. doi: https://doi.org/10.1113/JP273051 Sun YL, Zhang XY, He N, Sun T, Zhuang Y, Fang Q, Wang KR, Wang R (2012) Neuropeptide FF activates ERK and NF kappa B signal pathways in differentiated SH-SY5Y cells. Peptides 38(1):110–117. doi: https://doi.org/10.1016/j.peptides.2012.08.019 Tagliaferro P, Morales M (2008) Synapses between corticotropin-releasing factor‐containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic. J Comp Neurol 506(4):616–626. doi: https://doi.org/10.1002/cne.21576 Weiss ML, Hatton GI (1990) Collateral input to the paraventricular and supraoptic nuclei in rat. I. Afferents from the subfornical organ and the anteroventral third ventricle region. Brain Res Bull 24(2):231–238. doi: https://doi.org/10.1016/0361-9230(90)90210-Q Westerhaus MJ, Loewy AD (1999) Sympathetic-related neurons in the preoptic region of the rat identified by viral transneuronal labeling. J Comp Neurol 414(3):361–378. doi: https://doi.org/10.1002/(SICI)1096-9861 Worsley MA, Allen CE, Billinton A, King AE, Boissonade FM (2014) Chronic tooth pulp inflammation induces persistent expression of phosphorylated ERK (pERK) and phosphorylated p38 (pp38) in trigeminal subnucleus caudalis. Neuroscience 269(100):318–330. doi: https://doi.org/10.1016/j.neuroscience.2014.03.056 Yu HP, Zhang N, Zhang T, Wang ZL, Li N, Tang HH, Zhang R, Zhang MN, Xu B, Fang Q (2016) Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway. Peptides 86:24–32. doi: https://doi.org/10.1016/j.peptides.2016.09.012 Zhang H, Shi X, Hampong M, Blanis L, Pelech S (2001) Stress-induced inhibition of ERK1 and ERK2 by direct interaction with p38 MAP kinase. J Biol Chem 276(10):6905–6908. doi: https://doi.org/10.1074/jbc.C000917200 Supplementary Files Suppl.Fig.1.png Suppl. Fig. 1 Kisspeptin increases pERK1/2 expression in GnRH neurons of non-pregnant rats. Photomicrographs of coronal MS sections (a-d) showing GnRH-positive neurons (white arrows) and GnRH-positive neurons co-expressing pERK1/2 (black arrows point pERK1/2 and GnRH co-labelling) in non-pregnant rats (a, b) and G21 rats (c, d) after ICV administration of aCSF (a, c) or kisspeptin (2 µg in 2 µl; b, d). Mean (± SEM) number of (e) GnRH-positive neurons (RS: F (1,1) = 0.228, p = 0.637; T: F (1,1) = 0.057, p = 0.813; RS x T: F (1,2) = 0.915, p = 0.350, two-way ANOVA), (f) pERK1/2-positive neurons (RS: F (1,1) = 2.14, p = 0.158; T: F (1,1) = 2.98, p = 0.099; RS x T: F (1,2) = 3.94, p = 0.060, two-way ANOVA) and (g) GnRH-positive neurons co-expressing pERK1/2 (RS: F (1,1) = 1.064, p = 0.314; T: F (1,1) = 4.461, p = 0.047; RS x T: F (1,2) = 6.77, p = 0.017, two-way ANOVA) per section in the MS of non-pregnant rats and G21 rats. Scale bar = 100 µm. * p < 0.05 Holm-Sidak post hoc test Suppl.Fig.2.png Suppl. Fig. 2 Kisspeptin does not affect pERK1/2 expression in brainstem LC and DRN neurons of non-pregnant and G21 rats. Photomicrographs of coronal sections containing LC (a-d) and DRN (f-i), within red dotted area, showing pERK1/2-positive neurons in non-pregnant rats (a, b, f, g) and G21 rats (c, d, h, i) after ICV administration of aCSF (a, c, f, h) or kisspeptin (2 µg in 2 µl; b, d, g, i). Red arrows point to representative pERK1/2 labelling. Mean (± SEM) number pERK1/2-positive neurons per section in the (e) LC (RS: F (1,1) = 1.19, p = 0.286; T: F (1,1) = 0.563, p = 0.460; RS x T: F (1,2) = 0.292, p = 0.594, two-way ANOVA) and (j) DRN (RS: F (1,1) = 4.38, p = 0.048; T: F (1,1) = 0.077, p = 0.783; RS x T: F (1,2) = 0.015, p = 0.901, two-way ANOVA) of non-pregnant rats and G21 rats. Aq aqueduct, DRN dorsal raphe nucleus, LC locus coeruleus, mlf medial longitudinal fasciculus, 4V fourth ventricle, scale bar = 500 µm (on a, b, c, d) and 200 µm (on f, g, h, i). * p < 0.05 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1386498","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":94584136,"identity":"f7c1ec57-58aa-42ef-b8c7-2a4489289461","order_by":0,"name":"Mehwish Abbasi","email":"","orcid":"","institution":"University of Otago","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mehwish","middleName":"","lastName":"Abbasi","suffix":""},{"id":94584137,"identity":"fcefb77d-72ff-4494-90eb-4906cdc2d62c","order_by":1,"name":"Rachael Augustine","email":"","orcid":"","institution":"University of Otago","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rachael","middleName":"","lastName":"Augustine","suffix":""},{"id":94584138,"identity":"7c3b2568-3125-496c-b7cd-0c83f43205ff","order_by":2,"name":"Karl Iremonger","email":"","orcid":"","institution":"University of Otago","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karl","middleName":"","lastName":"Iremonger","suffix":""},{"id":94584139,"identity":"961d858c-8799-4dab-a9fe-390196755abc","order_by":3,"name":"Colin Brown","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYFAC5oYDDAwHQCzGB0RqYYRrYTZgSCBSCwNUC5sEUVr42xsbDxcw3Mnnl8g9VvHxh51dPwPzww+MO+pwapE4c7Dh8AyGZ5YzZ+Sl3ZyRkJw8s4HNWILxDBtOLQYSiQ2HeRgOGxjczjG7zZPAnGxwgMGMgbGNhzgtxTwJ9UAt7N+AWiSI08LMk3DYzuAAD8gWA/x+4TF4ZiA5/42x5Iy04wmSzTzFEoltCTi18Lc3H/7MU3HHgJ/njOGHDzbV9vzs7Rs/fGzDHWJQ5yGYiQ3MQBK3HViAPSmKR8EoGAWjYGQAAE48UGfC4DlnAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2305-846X","institution":"University of Otago","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Colin","middleName":"","lastName":"Brown","suffix":""}],"badges":[],"createdAt":"2022-02-22 20:45:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1386498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1386498/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19794507,"identity":"4e812bb9-dd05-4e9a-a2ea-ed624dffebfd","added_by":"auto","created_at":"2022-03-30 20:01:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10055953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect pERK1/2 expression in SON or PVN oxytocin neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal SON (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and PVN (\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003esections showing oxytocin-positive neurons (green) and pERK1/2-positive neurons (red) in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e). Yellow arrows point to representative oxytocin and pERK1/2 co-labelling. Mean (± SEM) number of (\u003cstrong\u003ee\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eoxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.395, \u003cem\u003ep\u003c/em\u003e = 0.534; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 3.81, \u003cem\u003ep\u003c/em\u003e = 0.061; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.025, \u003cem\u003ep\u003c/em\u003e = 0.874, two-way ANOVA), (\u003cstrong\u003ef\u003c/strong\u003e) pERK1/2-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.23, \u003cem\u003ep\u003c/em\u003e = 0.275; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.410, \u003cem\u003ep\u003c/em\u003e = 0.527; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.0002, \u003cem\u003ep\u003c/em\u003e = 0.986, two-way ANOVA) and (\u003cstrong\u003eg\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eoxytocin-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 33.17,\u003cem\u003e p\u003c/em\u003e ˂ 0.0001; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 4.20, \u003cem\u003ep\u003c/em\u003e = 0.050; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.050, \u003cem\u003ep\u003c/em\u003e = 0.824, two-way ANOVA) per section in the SON of non-pregnant and G21 rats. Mean (± SEM) number of (\u003cstrong\u003el\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eoxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.703, \u003cem\u003ep\u003c/em\u003e = 0.408; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.0003, \u003cem\u003ep\u003c/em\u003e = 0.986; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.138, \u003cem\u003ep\u003c/em\u003e = 0.294, two-way ANOVA), (\u003cstrong\u003em\u003c/strong\u003e) pERK1/2-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 5.52, \u003cem\u003ep\u003c/em\u003e = 0.025; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.21, \u003cem\u003ep\u003c/em\u003e = 0.279; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.65, \u003cem\u003ep\u003c/em\u003e = 0.208, two-way ANOVA) and (\u003cstrong\u003en\u003c/strong\u003e) oxytocin-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 28.52, \u003cem\u003ep\u003c/em\u003e ˂ 0.0001; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.0009, \u003cem\u003ep\u003c/em\u003e = 0.975; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.039, \u003cem\u003ep\u003c/em\u003e = 0.844, two-way ANOVA) per section in the PVN of non-pregnant rats and G21 rats. \u003cem\u003eOC\u003c/em\u003e optic chiasma, \u003cem\u003e3V\u003c/em\u003e\u0026nbsp;third ventricle, scale bar = 100 µm (on \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) and 200 µm (on \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e). ****\u003cem\u003ep\u003c/em\u003e ˂ 0.0001, *\u003cem\u003ep\u003c/em\u003e ˂ 0.05\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/7db9dee5a7367b7e06c79b33.png"},{"id":19794506,"identity":"c6de55fe-79ff-485b-be25-8f2d3548d1c7","added_by":"auto","created_at":"2022-03-30 20:01:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect pERK1/2 expression in mPVN or pPVN oxytocin neurons of non-pregnant and G21 rats. \u003c/strong\u003eMean (± SEM) number of (\u003cstrong\u003ea\u003c/strong\u003e) oxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.282, \u003cem\u003ep\u003c/em\u003e = 0.599; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.668, \u003cem\u003ep\u003c/em\u003e = 0.420; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.221,\u003cem\u003e p\u003c/em\u003e = 0.641, two-way ANOVA), (\u003cstrong\u003eb\u003c/strong\u003e) pERK1/2-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 3.82, \u003cem\u003ep\u003c/em\u003e = 0.060; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.431, \u003cem\u003ep\u003c/em\u003e = 0.516; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 2.35,\u003cem\u003e p\u003c/em\u003e = 0.136, two-way ANOVA) and (\u003cstrong\u003ec\u003c/strong\u003e) oxytocin-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 26.27, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.028, \u003cem\u003ep\u003c/em\u003e = 0.866; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.0006, \u003cem\u003ep\u003c/em\u003e = 0.980, two-way ANOVA) per section in the mPVN of non-pregnant rats and G21 rats after ICV administration of aCSF/kisspeptin (2 µg in 2 µl). Mean (± SEM) number of (\u003cstrong\u003ed\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eoxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.95, \u003cem\u003ep\u003c/em\u003e = 0.172; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.343, \u003cem\u003ep\u003c/em\u003e = 0.562; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.09,\u003cem\u003e p\u003c/em\u003e = 0.305, two-way ANOVA), (\u003cstrong\u003ee\u003c/strong\u003e) pERK1/2-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 5.97, \u003cem\u003ep\u003c/em\u003e = 0.020; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.46, \u003cem\u003ep\u003c/em\u003e = 0.235; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.748, \u003cem\u003ep\u003c/em\u003e = 0.393, two-way ANOVA) and (\u003cstrong\u003ef\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eoxytocin-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 23.15, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.076, \u003cem\u003ep\u003c/em\u003e = 0.783; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.186, \u003cem\u003ep\u003c/em\u003e = 0.669, two-way ANOVA) per section in the pPVN of non-pregnant rats and G21 rats after ICV administration of aCSF/kisspeptin (2 µg in 2 µl). ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/de1fad14c8fc00337d67eeec.png"},{"id":19794829,"identity":"39f25ab3-ae40-412f-81d3-9d7fb5b906b9","added_by":"auto","created_at":"2022-03-30 20:06:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9949527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect p-p38 expression in SON or PVN oxytocin neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal SON (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and PVN (\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003esections showing oxytocin-positive neurons (green) and p-p38-positive neurons (red) in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e). Yellow arrows point to representative p-p38 and oxytocin co-labelling. Mean (± SEM) number of (\u003cstrong\u003ee\u003c/strong\u003e) oxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.263, \u003cem\u003ep\u003c/em\u003e = 0.612; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.090, \u003cem\u003ep\u003c/em\u003e = 0.765; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.796, \u003cem\u003ep \u003c/em\u003e= 0.380, two-way ANOVA), (\u003cstrong\u003ef\u003c/strong\u003e) p-p38-positive neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.118, \u003cem\u003ep\u003c/em\u003e = 0.734; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.020, \u003cem\u003ep \u003c/em\u003e= 0.886; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.028,\u003cem\u003e p\u003c/em\u003e = 0.867, two-way ANOVA) and (\u003cstrong\u003eg\u003c/strong\u003e) oxytocin-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 9.90,\u003cem\u003e p\u003c/em\u003e = 0.004; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.007,\u003cem\u003e p\u003c/em\u003e = 0.930; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.052, \u003cem\u003ep\u003c/em\u003e = 0.821, two-way ANOVA) per section in the SON of non-pregnant rats and G21 rats. Mean (± SEM) number of (\u003cstrong\u003el\u003c/strong\u003e) oxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.149, \u003cem\u003ep\u003c/em\u003e = 0.702; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 2.78, \u003cem\u003ep\u003c/em\u003e = 0.107; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 2.58, \u003cem\u003ep\u003c/em\u003e = 0.120, two-way ANOVA), (\u003cstrong\u003em\u003c/strong\u003e) p-p38-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.194, \u003cem\u003ep\u003c/em\u003e = 0.662; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.019, \u003cem\u003ep\u003c/em\u003e = 0.889; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.29,\u003cem\u003e p\u003c/em\u003e = 0.266, two-way ANOVA) and (\u003cstrong\u003en\u003c/strong\u003e) oxytocin-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 3.39, \u003cem\u003ep\u003c/em\u003e = 0.077; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.939, \u003cem\u003ep\u003c/em\u003e = 0.341; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 4.23, \u003cem\u003ep\u003c/em\u003e = 0.050, two-way ANOVA) per section in the PVN of non-pregnant rats and G21 rats. \u003cem\u003eOC\u003c/em\u003e optic chiasma, \u003cem\u003e3V\u003c/em\u003e third ventricle, scale bar = 100 µm (on \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) and 200 µm (on \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e). **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/247e13e81f06cd26c3e6a74e.png"},{"id":19794508,"identity":"cab6bccd-f763-47cc-aff8-1d0202e7a2b3","added_by":"auto","created_at":"2022-03-30 20:01:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":341373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect p-p38 expression in mPVN or pPVN oxytocin neurons of non-pregnant and G21 rats.\u003c/strong\u003e Mean (± SEM) number of (\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eoxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.15, \u003cem\u003ep\u003c/em\u003e = 0.292; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 2.69, \u003cem\u003ep\u003c/em\u003e = 0.113; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.30,\u003cem\u003e p\u003c/em\u003e = 0.264, two-way ANOVA), (\u003cstrong\u003eb\u003c/strong\u003e) p-p38-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.083, \u003cem\u003ep\u003c/em\u003e = 0.775; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.145, \u003cem\u003ep\u003c/em\u003e = 0.706; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.726, \u003cem\u003ep\u003c/em\u003e = 0.402, two-way ANOVA) and (\u003cstrong\u003ec\u003c/strong\u003e) oxytocin-positive neurons co-expressing p-p38 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 2.90, \u003cem\u003ep\u003c/em\u003e = 0.100; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.99, \u003cem\u003ep\u003c/em\u003e = 0.32; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 3.55, \u003cem\u003ep\u003c/em\u003e = 0.071, two-way ANOVA) per section in the mPVN of non-pregnant rats and G21 rats after ICV administration of aCSF/kisspeptin (2 µg in 2 µl). Mean (± SEM) number of\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ed\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eoxytocin-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.527, \u003cem\u003ep\u003c/em\u003e = 0.474; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.74, \u003cem\u003ep\u003c/em\u003e = 0.199; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 3.58,\u003cem\u003e p\u003c/em\u003e = 0.070, two-way ANOVA), (\u003cstrong\u003ee\u003c/strong\u003e) p-p38-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 2.56, \u003cem\u003ep\u003c/em\u003e = 0.122; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.001, \u003cem\u003ep\u003c/em\u003e = 0.967; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 2.06, \u003cem\u003ep\u003c/em\u003e = 0.163. two-way ANOVA) and (\u003cstrong\u003ef\u003c/strong\u003e) oxytocin-positive neurons co-expressing p-p38 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 3.35, \u003cem\u003ep\u003c/em\u003e = 0.078; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.353, \u003cem\u003ep\u003c/em\u003e = 0.557; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 3.35, \u003cem\u003ep\u003c/em\u003e = 0.078) per section in the pPVN of non-pregnant rats and G21 rats after ICV administration of aCSF/kisspeptin (2 µg in 2 µl)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/f5582a7220a763e9b83abedc.png"},{"id":19794511,"identity":"84aacfda-4075-4ecb-838c-76efaafaab51","added_by":"auto","created_at":"2022-03-30 20:01:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7438210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect pERK1/2 expression in NTS or RVLM noradrenergic neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal NTS (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and RVLM (\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003esections, within white dotted area, showing TH-positive neurons (green) and pERK1/2-positive neurons (red) in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e). Yellow arrows point to representative pERK1/2 and TH co-labelling. Mean (± SEM) number of (\u003cstrong\u003ee\u003c/strong\u003e) TH-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.058, \u003cem\u003ep\u003c/em\u003e = 0.810; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.039, \u003cem\u003ep\u003c/em\u003e = 0.843; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.126, \u003cem\u003ep\u003c/em\u003e = 0.724, two-way ANOVA), (\u003cstrong\u003ef\u003c/strong\u003e) pERK1/2-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.335, \u003cem\u003ep\u003c/em\u003e = 0.567; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.018, \u003cem\u003ep\u003c/em\u003e = 0.892; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.288, \u003cem\u003ep\u003c/em\u003e = 0.595, two-way ANOVA) and (\u003cstrong\u003eg\u003c/strong\u003e) TH-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.580,\u003cem\u003e p\u003c/em\u003e = 0.453; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.501, \u003cem\u003ep\u003c/em\u003e = 0.485; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.900, \u003cem\u003ep\u003c/em\u003e = 0.351, two-way ANOVA) per section in the NTS of non-pregnant rats and G21 rats. Mean (± SEM) number of (\u003cstrong\u003el\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eTH-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 5.73, \u003cem\u003ep\u003c/em\u003e = 0.024; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.036, \u003cem\u003ep\u003c/em\u003e = 0.849; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.47, \u003cem\u003ep\u003c/em\u003e = 0.236, two-way ANOVA), (\u003cstrong\u003em\u003c/strong\u003e) pERK1/2-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.002, \u003cem\u003ep\u003c/em\u003e = 0.959; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.740, \u003cem\u003ep\u003c/em\u003e = 0.397; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.274, \u003cem\u003ep\u003c/em\u003e = 0.605, two-way ANOVA) and (\u003cstrong\u003en\u003c/strong\u003e) TH-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 5.27,\u003cem\u003e p\u003c/em\u003e = 0.029; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.667, \u003cem\u003ep\u003c/em\u003e = 0.421; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 6.35, \u003cem\u003ep\u003c/em\u003e = 0.018, two-way ANOVA) per section in the RVLM of non-pregnant rats and G21 rats. \u003cem\u003eAP\u003c/em\u003e area postrema, \u003cem\u003ecc\u003c/em\u003e central canal, \u003cem\u003eNTS\u003c/em\u003e nucleus tractus solitarius, \u003cem\u003eRVLM \u003c/em\u003erostral ventrolateral medulla, scale bar = 200 µm. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 Holm-Sidak \u003cem\u003epost hoc\u003c/em\u003e test \u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/0c390f824a2cbb09dd7bc5e0.png"},{"id":19794514,"identity":"a1306af0-c6b8-4a60-a794-20b933b7f329","added_by":"auto","created_at":"2022-03-30 20:01:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10511314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect p-p38 expression in NTS or RVLM noradrenergic neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal NTS (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and RVLM (\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003esections, within white dotted area, showing TH-positive neurons (green) and p-p38-positive neurons (red) in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e). Yellow arrows point to representative p-p38 and TH co-labelling. Mean (± SEM) number of (\u003cstrong\u003ee\u003c/strong\u003e) TH-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.005, \u003cem\u003ep\u003c/em\u003e = 0.942; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.024, \u003cem\u003ep\u003c/em\u003e = 0.877; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.52, \u003cem\u003ep\u003c/em\u003e = 0.229, two-way ANOVA), (\u003cstrong\u003ef\u003c/strong\u003e) p-p38-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.25, \u003cem\u003ep\u003c/em\u003e = 0.272; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.0007, \u003cem\u003ep\u003c/em\u003e = 0.978; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.129, \u003cem\u003ep\u003c/em\u003e = 0.721, two-way ANOVA) and (\u003cstrong\u003eg\u003c/strong\u003e) TH-positive neurons co-expressing p-p38 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.086,\u003cem\u003e p\u003c/em\u003e = 0.771; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.344, \u003cem\u003ep\u003c/em\u003e = 0.562; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.086, \u003cem\u003ep\u003c/em\u003e = 0.771, two-way ANOVA) per section in the NTS of non-pregnant rats and G21 rats. Mean (± SEM) number of (\u003cstrong\u003el\u003c/strong\u003e) TH-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 4.48, \u003cem\u003ep\u003c/em\u003e = 0.045; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.532, \u003cem\u003ep\u003c/em\u003e = 0.473; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.071, \u003cem\u003ep\u003c/em\u003e = 0.792, two-way ANOVA), (\u003cstrong\u003em\u003c/strong\u003e) p-p38-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.238, \u003cem\u003ep\u003c/em\u003e = 0.630; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.873, \u003cem\u003ep\u003c/em\u003e = 0.360; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.52, \u003cem\u003ep\u003c/em\u003e = 0.229, two-way ANOVA) and (\u003cstrong\u003en\u003c/strong\u003e) TH-positive neurons co-expressing p-p38 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.44,\u003cem\u003e p\u003c/em\u003e = 0.241; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.005, \u003cem\u003ep\u003c/em\u003e = 0.944; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.005, \u003cem\u003ep\u003c/em\u003e = 0.944, two-way ANOVA) per section in the RVLM of non-pregnant rats and G21 rats. \u003cem\u003eAP\u003c/em\u003e area postrema, \u003cem\u003ecc\u003c/em\u003e central canal, \u003cem\u003eNTS\u003c/em\u003e nucleus tractus solitarius, \u003cem\u003eRVLM\u003c/em\u003e rostral ventrolateral medulla,\u0026nbsp;scale bar = 200 µm (on \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) and 100 µm (on \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, , \u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 Holm-Sidak \u003cem\u003epost hoc\u003c/em\u003e test\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/6b72e32217319b581b520d6a.png"},{"id":19794517,"identity":"93f5c768-5a30-4401-8106-8007c9541717","added_by":"auto","created_at":"2022-03-30 20:01:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4745413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect pERK1/2 expression in OVLT, MnPO or SFO neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal sections containing OVLT (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e), MnPO (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e) and SFO (\u003cstrong\u003ek\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e), within the red dotted area, showing pERK1/2-positive neurons in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e b\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e,\u003cstrong\u003e l\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e,\u003cstrong\u003e n\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e g\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e,\u003cstrong\u003e l\u003c/strong\u003e,\u003cstrong\u003e n\u003c/strong\u003e). Red arrows point to representative pERK1/2 labelling. Mean (± SEM) number pERK1/2-positive neurons per section in the (\u003cstrong\u003ee\u003c/strong\u003e) OVLT (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.194, \u003cem\u003ep \u003c/em\u003e= 0.664; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.021, \u003cem\u003ep \u003c/em\u003e= 0.884; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.62, \u003cem\u003ep \u003c/em\u003e= 0.218, two-way ANOVA), (\u003cstrong\u003ej\u003c/strong\u003e) MnPO (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.63, \u003cem\u003ep \u003c/em\u003e= 0.215; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.003, \u003cem\u003ep \u003c/em\u003e= 0.956; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.0009, \u003cem\u003ep \u003c/em\u003e= 0.976, two-way ANOVA) and (\u003cstrong\u003eo\u003c/strong\u003e) SFO (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.00001, \u003cem\u003ep \u003c/em\u003e= 0.999; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.035, \u003cem\u003ep \u003c/em\u003e= 0.851; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.073, \u003cem\u003ep \u003c/em\u003e= 0.789, two-way ANOVA) of non-pregnant and G21 rats. \u003cem\u003eac\u003c/em\u003e anterior commissure, \u003cem\u003efx\u003c/em\u003e fornix, \u003cem\u003eMnPO\u003c/em\u003e median preoptic nucleus, \u003cem\u003eOVLT\u003c/em\u003e organum vasculosum lamina terminalis, \u003cem\u003eSFO\u003c/em\u003e subfornical organ, \u003cem\u003e3V\u003c/em\u003e third ventricle, scale bar = 100 µm (on \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and 200 µm (on \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei \u003c/strong\u003eand\u003cstrong\u003e k\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/5868b60f9853ea13b1936343.png"},{"id":19794510,"identity":"612c4f1f-4eb4-4254-8ecb-cc813bb0ede3","added_by":"auto","created_at":"2022-03-30 20:01:33","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6877654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect p-p38 expression in OVLT, MnPO or SFO neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal sections containing OVLT (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e), MnPO (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e) and SFO (\u003cstrong\u003ek\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e), within the red dotted area, showing p-p38-positive neurons in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e b\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e,\u003cstrong\u003e l\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e,\u003cstrong\u003e n\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e g\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e,\u003cstrong\u003e l\u003c/strong\u003e,\u003cstrong\u003e n\u003c/strong\u003e). Red arrows point to representative p-p38 labelling. Mean (± SEM) number p-p38-positive neurons per section in the (\u003cstrong\u003ee\u003c/strong\u003e) OVLT (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.601, \u003cem\u003ep \u003c/em\u003e= 0.448; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.231, \u003cem\u003ep \u003c/em\u003e= 0.636; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.019, \u003cem\u003ep \u003c/em\u003e= 0.890, two-way ANOVA), (\u003cstrong\u003ej\u003c/strong\u003e) MnPO (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.00007, \u003cem\u003ep \u003c/em\u003e= 0.993; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.003, \u003cem\u003ep \u003c/em\u003e= 0.952; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.408, \u003cem\u003ep \u003c/em\u003e= 0.534, two-way ANOVA) and (\u003cstrong\u003eo\u003c/strong\u003e) SFO (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.31, \u003cem\u003ep \u003c/em\u003e= 0.271; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.50, \u003cem\u003ep \u003c/em\u003e= 0.240; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 1.32, \u003cem\u003ep \u003c/em\u003e= 0.268, two-way ANOVA) of non-pregnant and G21 rats. \u003cem\u003eac\u003c/em\u003e anterior commissure, \u003cem\u003efx \u003c/em\u003efornix, \u003cem\u003eMnPO\u003c/em\u003e median preoptic nucleus, \u003cem\u003eOC\u003c/em\u003e optic chiasma, \u003cem\u003eOVLT\u003c/em\u003e organum vasculosum lamina terminalis, \u003cem\u003eSFO\u003c/em\u003e subfornical organ, \u003cem\u003e3V\u003c/em\u003e third ventricle, scale bar = 200 µm\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/665f6537bd73fb7c12761191.png"},{"id":19794515,"identity":"7d7eecfc-a93c-45d7-9afa-2f53534bf879","added_by":"auto","created_at":"2022-03-30 20:01:33","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":5506884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect pERK1/2 expression in AVPe, PeN or ARC neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal sections containing AVPe (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e), PeN (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e), and ARC (\u003cstrong\u003ek\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e), within the red dotted area, showing pERK1/2-positive neurons in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e g\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e,\u003cstrong\u003e l\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e,\u003cstrong\u003e n\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e g\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e, \u003cstrong\u003en\u003c/strong\u003e). Red arrows point to representative pERK1/2 labelling. Mean (± SEM) number pERK1/2-positive neurons per section in the (\u003cstrong\u003ee\u003c/strong\u003e) AVPe (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.270, \u003cem\u003ep \u003c/em\u003e= 0.609; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.126, \u003cem\u003ep \u003c/em\u003e= 0.726; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.090, \u003cem\u003ep \u003c/em\u003e= 0.767, two-way ANOVA), (\u003cstrong\u003ej\u003c/strong\u003e) PeN (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.046, \u003cem\u003ep \u003c/em\u003e= 0.834; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.159, \u003cem\u003ep \u003c/em\u003e= 0.698; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.498, \u003cem\u003ep \u003c/em\u003e= 0.496, two-way ANOVA) and (\u003cstrong\u003eo\u003c/strong\u003e) ARC (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.764, \u003cem\u003ep \u003c/em\u003e= 0.389; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.719, \u003cem\u003ep \u003c/em\u003e= 0.403; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.358, \u003cem\u003ep \u003c/em\u003e= 0.554, two-way ANOVA)\u003cstrong\u003e \u003c/strong\u003eof non-pregnant and G21 rats. \u003cem\u003eARC\u003c/em\u003e arcuate nucleus, \u003cem\u003eAVPe\u003c/em\u003e anteroventral periventricular nucleus, \u003cem\u003eOC\u003c/em\u003e optic chiasma, \u003cem\u003ePeN \u003c/em\u003eperiventricular nucleus, \u003cem\u003e3V\u003c/em\u003e third ventricle, scale bar = 200 µm\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/1b1c3196da381a32083885a1.png"},{"id":19795471,"identity":"18ba6478-bb2a-4abb-b635-a5298cfa1fbb","added_by":"auto","created_at":"2022-03-30 20:11:33","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":4326199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKisspeptin does not affect p-p38 expression in AVPe or PeN neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal sections containing AVPe (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and PeN (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e), within the red dotted area, showing p-p38-positive neurons in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e b\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e g\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e). Red arrows point to representative p-p38 labelling. Mean (± SEM) number p-p38-positive neurons per section in the (\u003cstrong\u003ee\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eAVPe (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.231, \u003cem\u003ep \u003c/em\u003e= 0.637; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.882, \u003cem\u003ep \u003c/em\u003e= 0.362; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.225, \u003cem\u003ep \u003c/em\u003e= 0.641, two-way ANOVA) and (\u003cstrong\u003ej\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePeN (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.455, \u003cem\u003ep \u003c/em\u003e= 0.510; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.0008, \u003cem\u003ep \u003c/em\u003e= 0.977; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.559, \u003cem\u003ep \u003c/em\u003e= 0.467, two-way ANOVA) of non-pregnant and G21 rats. \u003cem\u003eAVPe\u003c/em\u003e anteroventral periventricular nucleus, \u003cem\u003eOC\u003c/em\u003e optic chiasma, \u003cem\u003ePeN\u003c/em\u003e periventricular nucleus, \u003cem\u003e3V\u003c/em\u003e third ventricle, scale bar = 200 µm\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/ec92ef4f843272eafe827502.png"},{"id":20848143,"identity":"740d1d8b-4f45-4c5e-ab89-65f8dc346439","added_by":"auto","created_at":"2022-04-27 20:34:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7308629,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/de34a174-c89a-48c8-bb01-521fd416521c.pdf"},{"id":19794828,"identity":"bd0d1c47-a644-4517-865f-5c329d9a1a43","added_by":"auto","created_at":"2022-03-30 20:06:33","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5199708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppl. Fig. 1 Kisspeptin increases pERK1/2 expression in GnRH neurons of non-pregnant rats. \u003c/strong\u003ePhotomicrographs of coronal MS sections (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) showing GnRH-positive neurons (white arrows) and GnRH-positive neurons co-expressing pERK1/2 (black arrows point pERK1/2 and GnRH co-labelling) in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e b\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e). Mean (± SEM) number of (\u003cstrong\u003ee\u003c/strong\u003e) GnRH-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.228, \u003cem\u003ep\u003c/em\u003e = 0.637; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.057, \u003cem\u003ep\u003c/em\u003e = 0.813; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.915, \u003cem\u003ep\u003c/em\u003e = 0.350, two-way ANOVA), (\u003cstrong\u003ef\u003c/strong\u003e) pERK1/2-positive neurons (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 2.14, \u003cem\u003ep\u003c/em\u003e = 0.158; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 2.98, \u003cem\u003ep\u003c/em\u003e = 0.099; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 3.94, \u003cem\u003ep\u003c/em\u003e = 0.060, two-way ANOVA) and (\u003cstrong\u003eg\u003c/strong\u003e) GnRH-positive neurons co-expressing pERK1/2 (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.064, \u003cem\u003ep\u003c/em\u003e = 0.314; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 4.461, \u003cem\u003ep\u003c/em\u003e = 0.047; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 6.77, \u003cem\u003ep\u003c/em\u003e = 0.017, two-way ANOVA) per section in the MS of non-pregnant rats and G21 rats. Scale bar = 100 µm. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 Holm-Sidak \u003cem\u003epost hoc\u003c/em\u003e test\u003c/p\u003e","description":"","filename":"Suppl.Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/59b802f84b7bf952adaffdff.png"},{"id":19794831,"identity":"ba20ab13-dda4-4d12-a8ae-2ea796606070","added_by":"auto","created_at":"2022-03-30 20:06:33","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2928076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppl. Fig. 2 Kisspeptin does not affect pERK1/2 expression in brainstem LC and DRN neurons of non-pregnant and G21 rats. \u003c/strong\u003ePhotomicrographs of coronal sections containing LC (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) and DRN (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e),\u003cstrong\u003e \u003c/strong\u003ewithin red dotted area, showing pERK1/2-positive neurons in non-pregnant rats (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ef, g\u003c/strong\u003e) and G21 rats (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e) after ICV administration of aCSF (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e) or kisspeptin (2 µg in 2 µl; \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e). Red arrows point to representative pERK1/2 labelling. Mean (± SEM) number pERK1/2-positive neurons per section in the (\u003cstrong\u003ee\u003c/strong\u003e) LC (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 1.19, \u003cem\u003ep \u003c/em\u003e= 0.286; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.563, \u003cem\u003ep \u003c/em\u003e= 0.460; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.292, \u003cem\u003ep \u003c/em\u003e= 0.594, two-way ANOVA) and (\u003cstrong\u003ej\u003c/strong\u003e) DRN (RS: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 4.38, \u003cem\u003ep \u003c/em\u003e= 0.048; T: F\u003csub\u003e(1,1)\u0026nbsp;\u003c/sub\u003e= 0.077, \u003cem\u003ep \u003c/em\u003e= 0.783; RS x T: F\u003csub\u003e(1,2)\u0026nbsp;\u003c/sub\u003e= 0.015, \u003cem\u003ep \u003c/em\u003e= 0.901, two-way ANOVA) of non-pregnant rats and G21 rats. \u003cem\u003eAq\u003c/em\u003e aqueduct, \u003cem\u003eDRN\u003c/em\u003e dorsal raphe nucleus, \u003cem\u003eLC\u003c/em\u003e locus coeruleus, \u003cem\u003emlf \u003c/em\u003emedial longitudinal fasciculus, \u003cem\u003e4V\u003c/em\u003e fourth ventricle, scale bar = 500 µm (on \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) and 200 µm (on \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Suppl.Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-1386498/v1/13fa7b309fdeaaa25498c919.png"}],"financialInterests":"","formattedTitle":"Central kisspeptin does not affect ERK1/2 or p38 phosphorylation in oxytocin neurons of late-pregnant rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe hormone, oxytocin, induces uterine contractions for delivery of the offspring during birth. While oxytocin is not essential for birth (Nishimori et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), it is necessary for the normal progression of birth because oxytocin receptor antagonism delays the onset of, and prolongs the duration of, parturition in rats (Antonijevic et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Oxytocin is synthesised by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) oxytocin neurons that each send a single axon to the posterior gland where oxytocin secretion is triggered by action potential invasion of the axon terminal (Brown et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Increased activation of oxytocin neurons at parturition is triggered by afferent inputs that relay peripheral signals from cervical stretch receptors (Brown et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The best characterised excitatory afferent input to oxytocin neurons arises from the A2 noradrenergic cell group of the nucleus tractus solitarius (NTS), which is robustly activated at parturition (Meddle et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). However, acute activation of central noradrenergic receptors alone is not sufficient to trigger parturition in late-pregnant rats (Lipschitz et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), indicating that other mechanisms are likely also involved.\u003c/p\u003e \u003cp\u003eKisspeptin neurons also project to the SON (Desroziers et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and kisspeptin fibre density increases around oxytocin neurons during pregnancy (Seymour et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, intracerebroventricular (ICV) kisspeptin excites oxytocin neurons only in late pregnancy (Seymour et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and we have recently shown that this excitation is mediated by direct effects on oxytocin neurons as well as by enhancement of excitatory afferent signalling (Abbasi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eKisspeptin principally signals via kisspeptin receptor 1 (Kiss1R) (Kotani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) but also has high affinity for neuropeptide FF receptors (NPFFR) (Oishi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Classically, Kiss1R activation increases phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) (Kotani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Ohtaki et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Masui et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) but also increases phosphorylation of p38 in some cell lines (Masui et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Similarly to Kiss1R, NPFFR activation also increases phosphorylation of ERK1/2 (Anko and Panula \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Karnosova et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and p38 (Karnosova et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile ERK1/2 and p38 signalling pathways share upstream regulators (Hu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), p38 phosphorylation has been observed without measurable changes in ERK1/2 phosphorylation, suggesting that p38 can also be activated via a different, as yet unidentified, pathway (Lemonnier et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Samuvel et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Furthermore, phosphorylated p38 (p-p38) can directly suppress ERK1/2 phosphorylation (Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo test the hypothesis that kisspeptin directly activates oxytocin neurons at the end of pregnancy via ERK1/2 and/or p38 signalling, we measured phosphorylated ERK1/2 (pERK1/2) and p-p38 expression in oxytocin neurons after ICV kisspeptin administration to non-pregnant and late-pregnant rats. Furthermore, to determine which afferent inputs might be activated by kisspeptin to excite oxytocin neurons at the end pregnancy, we also measured pERK1/2 and p-p38 expression in brain areas that express Kiss1R and/or NPFFR and project to the SON and/or PVN. We found that ICV kisspeptin did not affect expression of pERK1/2 or p-p38 in oxytocin neurons or in any of the brain areas examined, suggesting that kisspeptin activates oxytocin neurons at the end of pregnancy via an alternative signalling pathway.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the University of Otago Animals Ethics Committee (approval number: D56/17) and carried out in accordance with the New Zealand Animal Welfare Act and associated guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdult female Sprague-Dawley rats (6 \u0026shy;\u0026ndash; 12 weeks of age) were purchased from the University of Otago Animal Facility and housed in controlled temperature and lighting (22 \u0026ndash; 24\u0026ordm;C; 12 h light / 12 h dark), with free access to standard laboratory rodent food and water. Non-pregnant rats were freely-cycling virgin rats and were housed in groups of 3 \u0026ndash; 5 until after surgery. Primiparous pregnant rats were used on gestation day 21 (G21, the expected day of parturition). \u003c/p\u003e\n\u003cp\u003eFor timed mating, oestrous cycle stage was assessed by vaginal cytology. At pro-oestrus, rats were placed overnight with a male for mating and the next morning was considered to be G0 after confirmation of the presence of sperm in the vaginal smear.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIntracerebroventricular cannulation and kisspeptin administration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eUnder isoflurane anaesthesia (2 / 2.5% in 1 L min\u003csup\u003e-1\u003c/sup\u003e O\u003csub\u003e2\u003c/sub\u003e), non-pregnant and G13/14 rats were implanted with an ICV guide cannula (22-gauge, Plastics One Inc, Roanoke, USA) into the lateral cerebral ventricle (co-ordinates relative to bregma, in mm: right lateral = 1.3; rostral/caudal = 0: ventral = 3.0) using stereotaxic surgical procedures, as previously described (Augustine and Grattan 2008). The guide cannula was anchored to screws (1 mm) inserted into the dorsal surface of the skull using light cure adhesive transbond (Henry Schein Shalfoon, Auckland, New Zealand). All rats were individually housed post-surgery. The clinical condition of each rat was monitored daily until the day of experiment.\u003c/p\u003e\n\u003cp\u003eOn day seven or eight following surgery, non-pregnant and G21 rats were anaesthetised with intraperitoneal pentobarbitone (60 mg kg\u003csup\u003e-1\u003c/sup\u003e; 300 mg ml\u003csup\u003e-1\u003c/sup\u003e). Upon cessation of the flexor withdrawal reflex, an internal cannula (28-guage, Plastics One Inc, Roanoke, USA) was inserted into the lateral cerebral ventricle through the guide cannula and attached to a Hamilton syringe (2 \u0026micro;l, Hamilton Company, Reno, USA) via a polyethylene tube (PE-10, 0.5 mm diameter). Rats were injected ICV with either aCSF (artificial cerebrospinal fluid) or 2 \u0026micro;g kisspeptin (1 \u0026micro;g \u0026micro;l\u003csup\u003e-1\u003c/sup\u003e dissolved in aCSF, Merck, USA) over 1 min, 60 min after induction of anaesthesia.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBrain collection and sectioning\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e15 min after ICV kisspeptin or aCSF administration, rats were perfused transcardially with ̴ 50 ml of 0.9% saline followed by 250 \u0026ndash; 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.6) under continued pentobarbitone anaesthesia. This timeframe was selected because kisspeptin-induced pERK1/2 and p-p38 levels peak 10 \u0026ndash; 15 min after incubation with kisspeptin in various cell lines (Masui et al. 2004; Novaira et al. 2009; Kim et al. 2010). Brains were post-fixed for 24 h in 4% paraformaldehyde solution and then 30% sucrose in 0.1 M phosphate buffer (pH 7.6) at 4\u0026ordm;C for 72 h. \u003c/p\u003e\n\u003cp\u003e30 \u0026micro;m coronal sections were cut on a freezing microtome (Leica SM2400, Wetzlar, Germany) from +0.60 to -3.00 mm relative to bregma to capture the SON, PVN, medial septum (MS), organum vasculosum of the lamina terminalis (OVLT), MnPO (median preoptic nucleus), subfornical organ (SFO), anteroventral periventricular nucleus (AVPe), periventricular nucleus (PeN) and arcuate nucleus (ARC) in the forebrain, from -7.56 to -8.04 mm for the dorsal raphe nucleus (DRN), -9.60 to -10.08 mm for the locus coeruleus (LC), and from -12.00 mm to -14.64 mm for the NTS and rostral ventrolateral medulla (RVLM) in the brainstem (Paxinos and Watson 2007). Sections containing forebrain areas were collected in 4 series and brainstem areas in 3 series; and each series contained 6 \u0026ndash; 8 sections. Sections were stored in fresh cryoprotectant (pH 7.6, 0.05 M phosphate buffer saline, 0.9% sodium chloride, 30% sucrose, 1% polyvinylpyrrolidone, 30% ethylene glycol) at -20\u0026ordm;C until use.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunohistochemistry\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor fluorescent immunohistochemistry, sections were washed in Tris-buffered saline (TBS) and endogenous aldehydes were blocked by incubating in 0.1% sodium borohydride in TBS for 20 min at room temperature (RT). Sections were washed in TBS and placed in incubation solution (0.3% Triton X-100 and 0.25% bovine serum albumin in TBS) containing host serum in which secondary antibody was raised for 60 min, to avoid non-specific binding. Next, sections were incubated in incubation solution containing a cocktail of primary antibodies for 48 h on an orbital shaker. After washing in TBS, sections were incubated for 3 h at RT in fluorescent-tagged secondary antibodies diluted in incubation solution. Primary antibodies used for fluorescent staining of pERK1/2 and p-p38 with oxytocin and tyrosine hydroxylase (TH, a marker for identification of noradrenaline-containing neurons) were rabbit anti-pERK1/2 antibody (p-44/42 MAPK (T 202/Y 204), 9101S, Cell Signalling; 1:1000), rabbit anti-p-p38 antibody (Thr 180/Tyr, 182, 9211, Cell Signalling; 1:2000), mouse oxytocin (MAB-5296, Millipore; 1:5000) and mouse anti-TH (MAB 318, Millipore; 1:2,000). The secondary antibodies used were goat anti-mouse Alexa fluor 488 antibody (A11029, Thermofisher Scientific; 1:500) and goat anti-rabbit Alexa fluor 568 antibody (ab 175471, Abcam; 1:500). \u003c/p\u003e\n\u003cp\u003eFor chromogenic immunohistochemistry, sections were washed in Tris-buffered saline (TBS) and endogenous aldehydes were blocked by incubating in TBS containing methanol and 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10-15 min at RT. Brain sections were washed and incubated in rabbit anti-pERK1/2 (1: 5000) or rabbit anti-p-p38 (1:2000) antibody with 4% normal goat serum for 48 h. Sections were then incubated in a secondary antibody solution containing a goat anti-rabbit biotinylated secondary antibody (BA-1000, Vector Laboratories; 1:500) for 90 min. After washing again in TBS, sections were incubated in an avidin-biotin-peroxidase solution (Vector Laboratories, California) for 90 min. Finally, pERK1/2-p-p38 staining was visualised by immersing sections in a nickel-diaminobenzidine (Ni DAB) peroxidase solution (Vector Laboratories, California) prepared in distilled water. Some sections were randomly mounted on slides and regularly examined under the bright field microscope. When staining became visible against background (usually after 2 \u0026ndash; 10 min), the Ni DAB reaction was stopped by washing the sections in TBS. \u003c/p\u003e\n\u003cp\u003eSections were mounted on gelatinized slides, cleared, dehydrated (only DAB stained sections pass through increasing concentrations of ethanol series followed by xylene) and dried. Fluorescent stained sections were coverslipped using Fluoromout-G\u003csup\u003e\u0026reg;\u003c/sup\u003e (Southern Biotech, Birmingham, USA), whereas DAB stained sections were coverslipped using DPX mounting medium (VWR International Limited, England). Stained sections were examined using Olympus bright-field/fluorescence microscope (BX51- NAOS) attached to a digital camera (GRYPHAX) to capture photomicrographs. The brain areas of interest were photographed at magnifications of 500x, 200x and 100x and quantification was completed manually using the \u003cem\u003ecell counter\u003c/em\u003e plugin on Fiji (NIH, v. 1.47) software. For all analyses, slides were randomly coded to avoid experimenter bias in counting. \u003c/p\u003e\n\u003cp\u003eSuccessful delivery of ICV kisspeptin was confirmed by double labelling for gonadotrophin releasing hormone (GnRH) and pERK1/2 in the MS. First, DAB staining of pERK1/2 was performed as described above, and sections were co-stained using guinea pig anti-GnRH primary antibody (GA02, a generous gift from Professor Allan Herbison; 1: 10,000) with 4% normal goat serum for 48 h. Sections were then incubated in secondary antibody solution containing a goat anti-guinea pig (BA-1000, Vector Laboratories; 1:500). Finally, the DAB solution without nickel was added to label GnRH-expressing neurons. Similarly to our previous study using Fos protein as a marker of activation (Augustine et al. 2018), the number of GnRH neurons expressing pERK1/2 was significantly higher in kisspeptin-treated non-pregnant rats than in aCSF-treated non-pregnant rats, but was not different between kisspeptin-treated late-pregnant rats and aCSF-treated late-pregnant rats (Reproductive status (RS): F\u003csub\u003e(1,1) \u003c/sub\u003e= 1.064, \u003cem\u003ep\u003c/em\u003e = 0.314; Treatment (T): F\u003csub\u003e(1,1) \u003c/sub\u003e= 4.461, \u003cem\u003ep\u003c/em\u003e = 0.047; RS x T interaction: F\u003csub\u003e(1,2) \u003c/sub\u003e= 6.77, \u003cem\u003ep\u003c/em\u003e = 0.017, two-way ANOVA; Supplementary Figure 1). \u003c/p\u003e\n\u003cp\u003ePrevious studies validated the specificity of oxytocin (Dabrowska et al. 2011), pERK1/2 and p-p38 (Worsley et al. 2014), TH (Tagliaferro and Morales 2008) and GnRH (Rizwan et al. 2012) antibodies. Specificity controls were performed by omitting primary antibodies. No non-specific staining was evident in any section with primary antibody omitted. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData were analysed on GraphPad Prism version 8 for Windows (GraphPad Software Inc, San Diego, CA, USA). Statistical significance between groups was determined by two-way analysis of variance (ANOVA) followed by \u003cem\u003epost hoc\u003c/em\u003e Holm Sidak\u0026apos;s test, where the F ratio was significant. All values are presented as mean \u0026plusmn; standard error of mean (SEM). Pearson product moment correlations were run to determine correlations. Probabilities (\u003cem\u003ep\u003c/em\u003e) \u0026lt; 0.05 were considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eKisspeptin does not affect pERK1/2 expression in SON or PVN oxytocin neurons\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWhile there was a higher number of oxytocin-positive neurons co-expressing pERK1/2 in the SON and PVN of G21 rats than non-pregnant rats, there was no effect of ICV kisspeptin on the number of oxytocin-positive neurons co-expressing pERK1/2 in the SON (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 33.17,\u003cem\u003e p\u003c/em\u003e ˂ 0.0001; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 4.20, \u003cem\u003ep\u003c/em\u003e = 0.050; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.050, \u003cem\u003ep\u003c/em\u003e = 0.824, two-way ANOVA; Figure 1e-g) or PVN (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 28.52, \u003cem\u003ep\u003c/em\u003e ˂ 0.0001; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.0009, \u003cem\u003ep\u003c/em\u003e = 0.975; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.039, \u003cem\u003ep\u003c/em\u003e = 0.844, two-way ANOVA; Figure 1l-n) of non-pregnant and G21 rats. \u003c/p\u003e\n\u003cp\u003eSubdividing the PVN into the magnocellular PVN (mPVN) and parvocellular PVN (pPVN) revealed similar results for both regions as was found for the PVN as whole, with a higher number of oxytocin-positive neurons co-expressing pERK1/2 in the mPVN and pPVN of G21 rats than non-pregnant rats, but no effect of ICV kisspeptin on the number of oxytocin-positive neurons co-expressing pERK1/2 in the mPVN (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 26.27, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.028, \u003cem\u003ep\u003c/em\u003e = 0.866; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.0006, \u003cem\u003ep\u003c/em\u003e = 0.980; Figure 2a-c) or pPVN (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 23.15, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.076, \u003cem\u003ep\u003c/em\u003e = 0.783; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.186, \u003cem\u003ep\u003c/em\u003e = 0.669; Figure 2d-f) of non-pregnant and G21 rats. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKisspeptin does not affect p-p38 expression in SON or PVN oxytocin neurons\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSimilarly to pERK1/2, there was a higher number of oxytocin-positive neurons co-expressing p-p38 in the SON of G21 rats than non-pregnant rats, but no effect of ICV kisspeptin on the number of oxytocin-positive neurons co-expressing p-p38 in the SON of non-pregnant or G21 rats (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 9.90,\u003cem\u003e p\u003c/em\u003e = 0.004; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.007,\u003cem\u003e p\u003c/em\u003e = 0.930; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.052, \u003cem\u003ep\u003c/em\u003e = 0.821, two-way ANOVA; Figure 3e-g). By contrast to the SON, there was no effect of reproductive status or ICV kisspeptin on p-p38 expression in oxytocin neurons in the PVN as a whole (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 3.39, \u003cem\u003ep\u003c/em\u003e = 0.077 ; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.939, \u003cem\u003ep\u003c/em\u003e = 0.341; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 4.23, \u003cem\u003ep\u003c/em\u003e = 0.050; Figure 3l-n), or in the mPVN (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 2.90, \u003cem\u003ep\u003c/em\u003e = 0.100; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.99, \u003cem\u003ep\u003c/em\u003e = 0.32; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 3.55, \u003cem\u003ep\u003c/em\u003e = 0.071; Figure 4a-c) or pPVN (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 3.35, \u003cem\u003ep\u003c/em\u003e = 0.078; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.353, \u003cem\u003ep\u003c/em\u003e = 0.557; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 3.35, \u003cem\u003ep\u003c/em\u003e = 0.078; Figure 4d-f) of non-pregnant and G21 rats. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKisspeptin does not affect pERK1/2 or p-p38 expression in brainstem noradrenergic neurons in the NTS, RVLM or LC \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether ICV kisspeptin might excite oxytocin neurons at the end of pregnancy by activation of NTS noradrenergic neurons, which project to SON and PVN oxytocin neurons, express NPFFR (Liu et al. 2001), and are robustly activated at parturition (Meddle et al. 2000), pERK1/2 and p-p38 were each double-labelled with TH. pERK1/2 and p-p38 were also each double-labelled with TH in the RVLM to determine whether excitatory effects of ICV kisspeptin might be mediated via RVLM because RVLM noradrenergic neurons are also activated at parturition (Meddle et al. 2000), express Kiss1R (Herbison et al. 2010) and project catecholaminergic neurons (containing dopamine, noradrenaline, adrenaline) to SON and PVN oxytocin (and vasopressin) neurons (Cunningham Jr et al. 1990). \u003c/p\u003e\n\u003cp\u003eThere was no effect of reproductive status or ICV kisspeptin on the number of TH-positive neurons co-expressing pERK1/2 in the NTS (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.580,\u003cem\u003e p\u003c/em\u003e = 0.453; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.501, \u003cem\u003ep\u003c/em\u003e = 0.485; RS x T (F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.900, \u003cem\u003ep\u003c/em\u003e = 0.351, two-way ANOVA; Figure 5e-g). Irrespective of kisspeptin treatment, pERK1/2 expression in TH-positive NTS neurons correlated with pERK1/2 expression in oxytocin-positive SON neurons (r = 0.536, \u003cem\u003ep\u003c/em\u003e = 0.039) but there was no correlation between pERK1/2 expression in TH-positive NTS neurons and oxytocin-positive PVN neurons (r = 0.098, \u003cem\u003ep\u003c/em\u003e = 0.726), or mPVN neurons (r = 0.221, \u003cem\u003ep\u003c/em\u003e = 0.428) or pPVN neurons (r = -0.192, \u003cem\u003ep\u003c/em\u003e = 0.492) in G21 rats. \u003c/p\u003e\n\u003cp\u003eWhile there was a higher number of TH-positive neurons co-expressing pERK1/2 in the RVLM of aCSF-treated G21 rats than aCSF-treated non-pregnant rats, there was no effect of ICV kisspeptin on the number of TH-positive neurons co-expressing pERK1/2 in the RVLM of non-pregnant or G21 rats (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 5.27,\u003cem\u003e p\u003c/em\u003e = 0.029; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.667, \u003cem\u003ep\u003c/em\u003e = 0.421; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 6.35, \u003cem\u003ep\u003c/em\u003e = 0.018; Figure 5l-n). \u003c/p\u003e\n\u003cp\u003eSimilarly to pERK1/2, there was no effect of reproductive status or ICV kisspeptin on the number of TH-positive neurons co-expressing p-p38 in the NTS (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.086, \u003cem\u003ep\u003c/em\u003e = 0.771 ; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.344, \u003cem\u003ep\u003c/em\u003e = 0.562; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.086, \u003cem\u003ep\u003c/em\u003e = 0.771; Figure 6e-g). Also, there was no correlation between p-p38 expression in TH-positive NTS neurons and oxytocin-positive SON neurons (r = -0.287, \u003cem\u003ep\u003c/em\u003e = 0.392), PVN neurons (r = 0.181, \u003cem\u003ep\u003c/em\u003e = 0.572), mPVN neurons (r = 0.169, \u003cem\u003ep\u003c/em\u003e = 0.598) or pPVN neurons (r = 0.198, \u003cem\u003ep\u003c/em\u003e = 0.536) in G21 rats. By contrast to pERK1/2 expression in TH-positive RVLM neurons, there was no effect of reproductive status or ICV kisspeptin on p-p38 expression in TH-positive RVLM neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 1.44, \u003cem\u003ep\u003c/em\u003e = 0.241; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.005, \u003cem\u003ep\u003c/em\u003e = 0.944; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.005, \u003cem\u003ep\u003c/em\u003e = 0.944; Figure 6l-n). Also, there was no correlation between p-p38 expression in TH-positive RVLM neurons and oxytocin-positive SON neurons (r = -0.199, \u003cem\u003ep\u003c/em\u003e = 0.607) in G21 rats. \u003c/p\u003e\n\u003cp\u003epERK1/2 was labelled in the LC to serve as a brain area control for the NTS because LC express Kiss1R and NPFFR (Lee et al. 1999; Liu et al. 2001) and LC noradrenergic neurons project to the PVN but principally to non-magnocellular neurons (Schreihofer and Guyenet 2002; Berridge and Waterhouse 2003). As expected, there was no effect of reproductive status or ICV kisspeptin on the number of pERK1/2-positive neurons in the LC (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 1.19, \u003cem\u003ep \u003c/em\u003e= 0.286; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.563, \u003cem\u003ep \u003c/em\u003e= 0.460; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.292, \u003cem\u003ep \u003c/em\u003e= 0.594; Supplementary Figure 2e). Also, there was no correlation between pERK1/2 expression in the LC and oxytocin-positive neurons of SON in G21 rats (r = -0.266, \u003cem\u003ep\u003c/em\u003e = 0.401).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKisspeptin does not affect pERK1/2 expression in the DRN neurons \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDRN neurons express Kiss1R and NPFFR (Liu et al. 2001; Higo et al. 2016) and project to SON and PVN oxytocin neurons (Sawchenko et al. 1983) but DRN inputs to oxytocin neurons are not involved in parturition (Herbison et al. 1997). Therefore, pERK1/2 was labelled in the DRN to serve as brainstem control for inputs to oxytocin neurons that are activated at parturition. While there was a higher number of pERK1/2-positive neurons in the DRN of G21 rats than non-pregnant rats, there was no effect of ICV kisspeptin on the number of pERK1/2-positive neurons in the DRN of non-pregnant or G21 rats (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 4.38, \u003cem\u003ep\u003c/em\u003e = 0.048; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.077, \u003cem\u003ep \u003c/em\u003e= 0.783; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.015, \u003cem\u003ep \u003c/em\u003e= 0.901, two-way ANOVA; Supplementary Figure 2j). Also, there was no correlation between pERK1/2 expression in DRN neurons and SON oxytocin-positive neurons in G21 rats (r = -0.522, \u003cem\u003ep\u003c/em\u003e = 0.099).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKisspeptin does not affect pERK1/2 or p-p38 expression in the OVLT, MnPO or SFO\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether ICV kisspeptin activates OVLT, MnPO and/or SFO neurons, which each project to the SON and PVN (Weiss and Hatton 1990; McKinley et al. 1992; Westerhaus and Loewy 1999) and express Kiss1R and/or NPFFR (Lee et al. 1999; Herbison et al. 2010; Higo et al. 2016; Higo et al. 2021), pERK1/2 and p-p38 were labelled in each brain area. \u003c/p\u003e\n\u003cp\u003eThere was no effect of reproductive status or ICV kisspeptin on the number of pERK1/2-positive OVLT neurons (RS: 0.194, \u003cem\u003ep \u003c/em\u003e= 0.664; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.021, \u003cem\u003ep \u003c/em\u003e= 0.884;RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 1.62, \u003cem\u003ep \u003c/em\u003e= 0.218, two-way ANOVA; Figure 7e), MnPO neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 1.63, \u003cem\u003ep \u003c/em\u003e= 0.215; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.003, \u003cem\u003ep \u003c/em\u003e= 0.956; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.0009, \u003cem\u003ep \u003c/em\u003e= 0.976; Figure 7j) or SFO neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.00001, \u003cem\u003ep \u003c/em\u003e= 0.999; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.035, \u003cem\u003ep \u003c/em\u003e= 0.851; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.073, \u003cem\u003ep \u003c/em\u003e= 0.789; Figure 7o) in non-pregnant or G21 rats. Also, there was no correlation between pERK1/2 expression in oxytocin-positive SON neurons and pERK1/2 expression in OVLT neurons (r = -0.065, \u003cem\u003ep\u003c/em\u003e = 0.866), MnPO neurons(r = -0.259, \u003cem\u003ep\u003c/em\u003e = 0.415) or SFO neurons (r = -0.105, \u003cem\u003ep\u003c/em\u003e = 0.772) in G21 rats. \u003c/p\u003e\n\u003cp\u003eSimilarly to pERK1/2, there was no effect of reproductive status or ICV kisspeptin on the number of p-p38-positive OVLT neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.601, \u003cem\u003ep \u003c/em\u003e= 0.448; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.231, \u003cem\u003ep \u003c/em\u003e= 0.636; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.019, \u003cem\u003ep \u003c/em\u003e= 0.890; Figure 8e), MnPO neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.00007, \u003cem\u003ep \u003c/em\u003e= 0.993; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.003, \u003cem\u003ep \u003c/em\u003e= 0.952; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.408, \u003cem\u003ep \u003c/em\u003e= 0.534; Figure 8j) or SFO neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 1.31, \u003cem\u003ep \u003c/em\u003e= 0.271; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 1.50, \u003cem\u003ep \u003c/em\u003e= 0.240; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 1.32, \u003cem\u003ep \u003c/em\u003e= 0.268, Figure 8o) in non-pregnant or G21 rats. Also, there was no correlation between p-p38 expression in oxytocin-positive SON neurons and pERK1/2-positive OVLT neurons (r = 0.112, \u003cem\u003ep\u003c/em\u003e = 0.809), MnPO neurons (r = -0.648, \u003cem\u003ep\u003c/em\u003e = 0.115) or SFO neurons (r = 0.189, \u003cem\u003ep\u003c/em\u003e = 0.684) in G21 rats. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKisspeptin does not affect pERK1/2 or p-p38 expression in the AVPe, Pe or pERK1/2 expression in the ARC\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003epERK1/2 and p-p38 were also labelled in the AVPe, PeN and ARC, which contain kisspeptin neurons (Clarkson and Herbison 2009; Lehman et al. 2010), express Kiss1R and/or NPFFR (Herbison et al. 2010; Higo et al. 2016; Higo et al. 2021). Kisspeptin PeN neurons project to the SON but AVPe and ARC kisspeptin neurons do not (Seymour et al. 2017). \u003c/p\u003e\n\u003cp\u003eThere was no effect of reproductive status or ICV kisspeptin on the number of pERK1/2-positive AVPe neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.270, \u003cem\u003ep \u003c/em\u003e= 0.609; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.126, \u003cem\u003ep \u003c/em\u003e= 0.726; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.090, \u003cem\u003ep \u003c/em\u003e= 0.767, two-way ANOVA; Figure 9e), PeN neurons (F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.046, \u003cem\u003ep \u003c/em\u003e= 0.834; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.159, \u003cem\u003ep \u003c/em\u003e= 0.698; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.498, \u003cem\u003ep \u003c/em\u003e= 0.496; Figure 9j) or ARC neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.764, \u003cem\u003ep \u003c/em\u003e= 0.389; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.719, \u003cem\u003ep \u003c/em\u003e= 0.403; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.358, \u003cem\u003ep \u003c/em\u003e= 0.554; Figure 9o) in non-pregnant or G21 rats. Also, there was no correlation between pERK1/2 expression in oxytocin-positive SON neurons and pERK1/2-positive AVPe neurons (r = 0.069, \u003cem\u003ep\u003c/em\u003e = 0.882), PeN neurons (r = -0.143, \u003cem\u003ep\u003c/em\u003e = 0.735) or ARC neurons (r = -0.074, \u003cem\u003ep\u003c/em\u003e = 0.809) in G21 rats. \u003c/p\u003e\n\u003cp\u003eSimilarly to pERK1/2, there was no effect of reproductive status or ICV kisspeptin on the number of p-p38-positive AVPe neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.231, \u003cem\u003ep \u003c/em\u003e= 0.637; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.882, \u003cem\u003ep \u003c/em\u003e= 0.362; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.225, \u003cem\u003ep \u003c/em\u003e= 0.641; Figure 10e) or PeN neurons (RS: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.455, \u003cem\u003ep \u003c/em\u003e= 0.510; T: F\u003csub\u003e(1,1) \u003c/sub\u003e= 0.0008, \u003cem\u003ep \u003c/em\u003e= 0.977; RS x T: F\u003csub\u003e(1,2) \u003c/sub\u003e= 0.559, \u003cem\u003ep \u003c/em\u003e= 0.467; Figure 10j) in non-pregnant or G21 rats. Also, there was no correlation between p-p38 expression in oxytocin-positive SON neurons and pERK1/2-positive AVPe neurons (r = -0.378, \u003cem\u003ep\u003c/em\u003e = 0.459) or PeN neurons (r = -0.178, \u003cem\u003ep\u003c/em\u003e = 0.672) in G21 rats. There was insufficient tissue to label p-p38 in the ARC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have recently shown that kisspeptin excites oxytocin neurons in late pregnancy, in part, by a direct action on oxytocin neurons\u0026nbsp;(Abbasi et al. 2022). The present study revealed that ICV kisspeptin did not induce ERK1/2 or p38 phosphorylation in oxytocin neurons of non-pregnant or late-pregnant rats when administered at a dose that increases oxytocin neuron firing rate in late-pregnant rats\u0026nbsp;(Seymour et al. 2017). Nevertheless, consistent with previous findings\u0026nbsp;(Chandaka et al. 2016), ERK1/2 (and p38)\u0026nbsp;phosphorylation was higher in oxytocin neurons of late-pregnant rats\u0026nbsp;than in non-pregnant rats. Hence, it appears likely that kisspeptin excitation of oxytocin neurons in late-pregnant rats is not mediated by Kiss1R and/or NPFFR activation of ERK1/2 or p38 signalling.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLocal kisspeptin activation of oxytocin neurons in late pregnancy\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe failure of kisspeptin to induce ERK1/2 (or p38) phosphorylation in oxytocin neurons of late-pregnant rats probably does not reflect a failure to deliver sufficient kisspeptin to activate these signalling pathways because kisspeptin administration increased ERK1/2 phosphorylation in GnRH neurons of the same non-pregnant rats in which ERK1/2 and p38 phosphorylation were unchanged in oxytocin neurons. While kisspeptin did not induce ERK1/2 phosphorylation in GnRH neurons of late-pregnant rats, this likely reflects downregulation of GnRH neuron responsiveness to kisspeptin during pregnancy, as we have previously reported using Fos protein as a marker of activation\u0026nbsp;(Augustine et al. 2018). Elevated ERK1/2 phosphorylation in oxytocin neurons at the end of pregnancy might have occluded the ability of exogenous kisspeptin to induce further phosphorylation. However, \u0026le;50% of oxytocin neurons expressed pERK1/2 in late-pregnant rats. Hence, occlusion also appears unlikely to account for the failure of kisspeptin to induce ERK1/2 phosphorylation in oxytocin neurons of late-pregnant rats. Taken together, these observations suggest that the lack of kisspeptin-induced ERK1/2 (and p38) phosphorylation in oxytocin neurons in late pregnancy was not due to a technical failure. Rather, it appears that kisspeptin excitation of oxytocin neurons in late pregnancy is not mediated by Kiss1R-mediated phosphorylation of ERK1/2 or p38.\u003c/p\u003e\n\u003cp\u003eIf kisspeptin does not activate Kiss1R/NPFFR-ERK1/2/p38 signalling in oxytocin neurons, another signalling pathway must mediate the direct effects of kisspeptin of oxytocin neurons in late pregnancy. Kiss1R activates neuronal nitric oxide synthase in preoptic neurons via\u0026nbsp;phosphatidylinositol-3-kinase(PI3K)-Akt\u0026nbsp;(Hanchate et al. 2012). Hence, PI3K-Akt might mediate the direct effects of kisspeptin on oxytocin neurons in late pregnancy. While kisspeptin excites\u0026nbsp;GnRH neurons by\u0026nbsp;activating non-specific cation channels and inhibiting potassium channels\u0026nbsp;(Liu et al. 2008), it does not appear to excite oxytocin neurons via these channels because baseline holding currents are unchanged by kisspeptin superfusion in SON brain slices\u0026nbsp;(Abbasi et al. 2022).\u0026nbsp;Alternatively, or additionally, kisspeptin activation of oxytocin neurons might not be mediated by Kiss1R. Indeed, SON Kiss1R mRNA expression does not change during pregnancy\u0026nbsp;(Seymour et al. 2017), suggesting that upregulation of Kiss1R does not underpin kisspeptin excitation of oxytocin neurons in late pregnancy, although the possibility of increased Kiss1R surface expression and/or sensitivity cannot be discounted. Oxytocin neurons also express NPFFR\u0026nbsp;(Kim et al. 2016), which\u0026nbsp;couples to Gs and Gi as well as to Gq\u0026nbsp;(Bonini et al. 2000; Liu et al. 2001; Mollereau et al. 2002)\u0026nbsp;to increase phosphorylation of cAMP response element-binding protein (CREB) and c-Jun N terminal kinase (JNK)\u0026nbsp;(Karnosova et al. 2021). Hence, NPFFR-CREB/JNK signalling might mediate the direct effects of kisspeptin on oxytocin neurons in late pregnancy. Further work will be required to determine which, if any, of these signalling pathways mediate kisspeptin excitation of oxytocin neurons at the end of pregnancy.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLack of kisspeptin induction of ERK1/2 or p38 phosphorylation in afferent inputs to the oxytocin system\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWhile our recent findings showed that kisspeptin excites oxytocin neurons in late pregnancy, in part, by a direct action of kisspeptin on oxytocin neurons, our findings also suggested that activation of afferent inputs might also contribute to the excitation\u0026nbsp;(Abbasi et al. 2022). Therefore, we mapped kisspeptin-induced ERK1/2 and p38 phosphorylation in brain areas that project to the oxytocin system and express Kiss1R and NPFFR (LC, DRN, OVLT, SFO, AVPe, ARC)\u0026nbsp;(Lee et al. 1999; Herbison et al. 2010; Higo et al. 2016; Higo et al. 2021),\u0026nbsp;only NPFFR (NTS, PeN)\u0026nbsp;(Higo et al. 2021)\u0026nbsp;or only Kiss1R (RVLM, MnPO)\u0026nbsp;(Irwig et al. 2004; Herbison et al. 2010). However, neither ERK1/2 nor p38 phosphorylation was affected by kisspeptin in any of the brain areas studied in non-pregnant or late-pregnant rats. Hence, it appears that any indirect kisspeptin excitation of oxytocin neurons in late pregnancy is not mediated by activation of ERK1/2-p38 signalling in these afferent inputs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile kisspeptin did not induce ERK1/2 or p38 phosphorylation in afferent inputs to the oxytocin system in non-pregnant or late-pregnant rats, there was a positive correlation of ERK1/2 phosphorylation in late pregnancy between SON oxytocin neurons and NTS noradrenergic neurons, which project to oxytocin neurons and are activated during parturition\u0026nbsp;(Meddle et al. 2000); the correlation with\u0026nbsp;ERK1/2 phosphorylation in oxytocin neurons\u0026nbsp;was specific to\u0026nbsp;NTS noradrenergic neurons, further implicating NTS noradrenergic neurons in driving oxytocin neuron activity for parturition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile the same caveats apply to the lack of ERK1/2 or p38 phosphorylation in their afferent inputs as apply to the oxytocin neuron themselves, a further possibility is that kisspeptin might enhance excitatory synaptic transmission via presynaptic actions on oxytocin neurons afferent inputs. If active, this mechanism does not involve local glutamatergic or GABAergic inputs to oxytocin neurons, which are unaffected by kisspeptin in brain slices from non-pregnant and late-pregnant rats\u0026nbsp;(Abbasi et al. 2022). Therefore, the most likely candidate might be the\u0026nbsp;NTS noradrenergic input, which is activated at parturition\u0026nbsp;(Meddle et al. 2000), releasing noradrenaline\u0026nbsp;(Herbison et al. 1997)\u0026nbsp;to excite oxytocin neurons via \u0026alpha;\u003csub\u003e1\u003c/sub\u003e-adrenoreceptors\u0026nbsp;(Douglas et al. 2001).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConcluding remarks\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTaken together, the current results show that kisspeptin-induced excitation of oxytocin neurons in late pregnancy is not mediated by phosphorylation of canonical Kiss1R (or NPFFR)-activated second messengers, ERK1/2 (or p38), and further work will be required to determine which signalling pathway mediates kisspeptin excitation of oxytocin neurons in late pregnancy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eaCSF, artificial cerebrospinal fluid; ARC, arcuate nucleus; AVPe, anteroventral periventricular nucleus; ERK1/2, extracellular regulated kinase 1/2; ICV, intracerebroventricular; Kiss1R, kisspeptin-1 receptor; MnPO, median preoptic nucleus; OVLT, organum vasculosum lamina terminalis; NPFFR, Neuropeptide FF receptor; pERK1/2, phosphorylated ERK1/2; p-p38, phosphorylated p38; SFO, subfornical organ; SON, supraoptic nucleus; PVN, paraventricular nucleus; PeN, periventricular nucleus.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Manatu Hauora | Health Research Council of New Zealand (CHB) and a University of Otago Postgraduate Scholarship (MA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the experimental design. Experiments were performed by Mehwish Abbasi and Rachael Augustine. Data collection, analysis and figures were prepared by Mehwish Abbasi. The first draft of the manuscript was written by Mehwish Abbasi. All authors provided revised the manuscript and approved the submitted version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data acquired during this study are available from corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbasi M, Perkinson MR, Seymour AJ, Piet R, Campbell RE, Iremonger KJ, Brown CH (2022) Local kisspeptin excitation of rat oxytocin neurones in late pregnancy. J Physiol. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1113/JP282531\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnko ML, Panula P (2006) Regulation of endogenous human NPFF2 receptor by neuropeptide FF in SK-N‐MC neuroblastoma cell line. J Neurochem 96(2):573\u0026ndash;584. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1471-4159.2005.03581.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntonijevic IA, Douglas AJ, Dye S, Bicknell RJ, Leng G, Russell JA (1995) Oxytocin antagonists delay the initiation of parturition and prolong its active phase in rats. J Endocrinol 145(1):97\u0026ndash;103. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1677/joe.0.1450097\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAugustine RA, Grattan DR (2008) Induction of central leptin resistance in hyperphagic pseudopregnant rats by chronic prolactin infusion. Endocrinology 149(3):1049\u0026ndash;1055. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/en.2007-1018\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAugustine RA, Seymour AJ, Campbell RE, Grattan DR, Brown CH (2018) Integrative neuro-humoral regulation of oxytocin neuron activity in pregnancy and lactation. J Neuroendocrinol 30(8):12569\u0026ndash;12572. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jne.12569\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerridge CW, Waterhouse BD (2003) The locus coeruleus\u0026ndash;noradrenergic system: modulation of behavioral state and state-dependent cognitive processes. Brain Res Rev 42(1):33\u0026ndash;84. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0165-0173(03)00143-7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonini JA, Jones KA, Adham N, Forray C, Artymyshyn R, Durkin MM, Smith KE, Tamm JA, Boteju LW, Lakhlani PP, Raddatz R, Yao W-J, Ogozalek KL, Noel B, Kouranova EV, Quan Y, Vaysse PJ, Wetzel JM, Branchek TA, Gerald C, Borowsky B (2000) Identification and characterization of two G protein-coupled receptors for neuropeptide FF. J Biol Chem 275(50):39324\u0026ndash;39331. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M004385200\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown CH, Bains JS, Ludwig M, Stern JE (2013) Physiological regulation of magnocellular neurosecretory cell activity: integration of intrinsic, local and afferent mechanisms. J Neuroendocrinol 25(8):678\u0026ndash;710. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jne.12051\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChandaka GK, Wang L, Senogles S, Armstrong WE (2016) Late pregnancy is a critical period for changes in phosphorylated mitogen-activated protein kinase/extracellular signal‐regulated kinase 1/2 in oxytocin neurones. J Neuroendocrinol 28(9):e12398. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jne.12398\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClarkson J, Herbison AE (2009) Oestrogen, Kisspeptin, GPR54 and the Pre-Ovulatory Luteinising Hormone Surge. J Neuroendocrinol 21(4):305\u0026ndash;311. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2826.2009.01835.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCunningham ET Jr, Bohn MC, Sawchenko PE (1990) Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J Comp Neurol 292(4):651\u0026ndash;667. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cne.902920413\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDabrowska J, Hazra R, Ahern TH, Guo JD, McDonald AJ, Mascagni F, Muller JF, Young LJ, Rainnie DG (2011) Neuroanatomical evidence for reciprocal regulation of the corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed nucleus of the stria terminalis of the rat: implications for balancing stress and affect. Psychoneuroendocrinology 36(9):1312\u0026ndash;1326. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.psyneuen.2011.03.003\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesroziers E, Mikkelsen J, Simonneaux V, Keller M, Tillet Y, Caraty A, Franceschini I (2010) Mapping of kisspeptin fibres in the brain of the pro-oestrous rat. J Neuroendocrinol 22(10):1101\u0026ndash;1112. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2826.2010.02053.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDouglas A, Scullion S, Antonijevic I, Brown D, Russell J, Leng G (2001) Uterine contractile activity stimulates supraoptic neurons in term pregnant rats via a noradrenergic pathway. Endocrinology 142(2):633\u0026ndash;644\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanchate NK, Parkash J, Bellefontaine N, Mazur D, Colledge WH, de d'Anglemont X, Prevot V (2012) Kisspeptin-GPR54 signaling in mouse NO-synthesizing neurons participates in the hypothalamic control of ovulation. J Neurosci 32(3):932\u0026ndash;945. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/JNEUROSCI.4765-11.2012\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbison AE, d'Anglemont de Tassigny X, Doran J, Colledge WH (2010) Distribution and postnatal development of Gpr54 gene expression in mouse brain and gonadotropin-releasing hormone neurons. Endocrinology 151(1):312\u0026ndash;321. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/en.2009-0552\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbison AE, Voisin DL, Douglas AJ, Chapman C (1997) Profile of monoamine and excitatory amino acid release in rat supraoptic nucleus over parturition. Endocrinology 138(1):33\u0026ndash;40. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/endo.138.1.4859\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHigo S, Honda S, Iijima N, Ozawa H (2016) Mapping of kisspeptin receptor mRNA in the whole rat brain and its co-localisation with oxytocin in the paraventricular nucleus. J Neuroendocrinol 28(4):1\u0026ndash;8. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jne.12356\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHigo S, Kanaya M, Ozawa H (2021) Expression analysis of neuropeptide FF receptors on neuroendocrine-related neurons in the rat brain using highly sensitive in situ hybridization. Histochem Cell Biol 155(4):465\u0026ndash;475. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00418-020-01956-9\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu KL, Zhao H, Chang HM, Yu Y, Qiao J (2018) Kisspeptin/kisspeptin receptor system in the ovary. Front Endocrinol 8:365. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fendo.2017.00365\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIrwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA (2004) Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. J Neuroendocrinol 80(4):264\u0026ndash;272. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1159/000083140\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarnosova A, Strnadova V, Hola L, Zelezna B, Kunes J, Malet\u0026iacute;nska L (2021) Palmitoylation of Prolactin-Releasing Peptide Increased Affinity for and Activation of the GPR10, NPFF-R2 and NPFF-R1 Receptors: In Vitro Study. Int J Mol Sci 22(16):8904. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22168904\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim GL, Dhillon SS, Belsham DD (2010) Kisspeptin directly regulates neuropeptide Y synthesis and secretion via the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways in NPY-secreting hypothalamic neurons. Endocrinology 151(10):5038\u0026ndash;5047. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/en.2010-0521\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JS, Brown CH, Anderson GM (2016) Anti-opioid effects of RFRP-3 on magnocellular neuron activity in morphine-na\u0026iuml;ve and morphine-treated female rats. Endocrinology 157(10):4003\u0026ndash;4011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, C B, Schiffmann SN, Vassart G, Parmentier M (2001) The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276(37):34631\u0026ndash;34636. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M104847200\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee DK, Nguyen T, O'Neill GP, Cheng R, Liu Y, Howard AD, Coulombe N, Tan CP, Tang-Nguyen AT, George SR, O'Dowd BF (1999) Discovery of a receptor related to the galanin receptors. FEBS lett 446(1):103\u0026ndash;107. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0014-5793(99)00009-5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLehman MN, Merkley CM, Coolen LM, Goodman RL (2010) Anatomy of the kisspeptin neural network in mammals. Brain Res 1364:90\u0026ndash;102. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.brainres.2010.09.020\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLemonnier J, Ghayor C, Guicheux J, Caverzasio J (2004) Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation. J Biol Chem 279(1):259\u0026ndash;264. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M308665200\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLipschitz DL, Crowley WR, Bealer SL (2004) Differential sensitivity of intranuclear and systemic oxytocin release to central noradrenergic receptor stimulation during mid-and late gestation in rats. Am J Physiol Endocrinol Metab 287(3):E523\u0026ndash;E528. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/ajpendo.00572.2003\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Q, Guan XM, Martin WJ, McDonald TP, Clements MK, Jiang Q, Zeng Z, Jacobson M, Williams DL, Yu H, Bomford D, Figueroa D, Mallee J, Wang R, Evans J, Gould R, Austin CP (2001) Identification and characterization of novel mammalian neuropeptide FF-like peptides that attenuate morphine-induced antinociception. J Biol Chem 276(40):36961\u0026ndash;36969. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M105308200\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Lee K, Herbison AE (2008) Kisspeptin excites gonadotropin-releasing hormone neurons through a phospholipase C/calcium-dependent pathway regulating multiple ion channels. Endocrinology 149(9):4605\u0026ndash;4614. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/en.2008-0321\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S, Niida A, Fujii N, Imamura M (2004) Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 315(1):85\u0026ndash;92. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2004.01.021\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcKinley MJ, Bicknell RJ, Hards D, McAllen RM, Vivas L, Weisinger RS, Oldfield BJ (1992) Efferent neural pathways of the lamina terminalis subserving osmoregulation. Prog Brain Res 91:395\u0026ndash;402. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0079-6123(08)62358-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeddle SL, Leng G, Selvarajah JR, Bicknell RJ, Russell JA (2000) Direct pathways to the supraoptic nucleus from the brainstem and the main olfactory bulb are activated at parturition in the rat. Neuroscience 101(4):1013\u0026ndash;1021. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0306-4522(00)00300-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMollereau C, Mazarguil H, Marcus D, Quelven I, Kotani M, Lannoy V, Dumont Y, Quirion R, Detheux M, Parmentier M, Zajac JM (2002) Pharmacological characterization of human NPFF1 and NPFF2 receptors expressed in CHO cells by using NPY Y1 receptor antagonists. Eur J Pharmacol 451(3):245\u0026ndash;256. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0014-2999(02)02224-0\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM (1996) Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci 93(21):11699\u0026ndash;11704. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.93.21.11699\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovaira HJ, Ng Y, Wolfe A, Radovick S (2009) Kisspeptin increases GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines. Mol Cell Endocrinol 311(1\u0026ndash;2):126\u0026ndash;134. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mce.2009.06.011\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M (2001) Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411(6837):613\u0026ndash;617. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/35079135\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOishi S, Misu R, Tomita K, Setsuda S, Masuda R, Ohno H, Naniwa Y, Ieda N, Inoue N, Ohkura S, Uenoyama Y, Tsukamura H, Maeda K, Hirasawa A, Tsujimoto G, Fujii N (2011) Activation of neuropeptide FF receptors by kisspeptin receptor ligands. ACS Med Chem Lett 2(1):53\u0026ndash;57. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ml1002053\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaxinos G, Watson C (2007) The rat brain in stereotaxic coordinates, 6 edn. Elsevier Academic Press, Amsterdam\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng J, Tang M, Zhang BP, Zhang P, Zhong T, Zong T, Yang B, Kuang HB (2013) Kisspeptin stimulates progesterone secretion via the Erk1/2 mitogen-activated protein kinase signaling pathway in rat luteal cells. Fertil Steril 99(5):1436\u0026ndash;1443. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fertnstert.2012.12.008\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRizwan MZ, Poling MC, Corr M, Cornes PA, Augustine RA, Quennell JH, Kauffman AS, Anderson GM (2012) RFamide-related peptide-3 receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action. Endocrinology 153(8):3770\u0026ndash;3779. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/en.2012-1133\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamuvel DJ, Jayanthi LD, Bhat NR, Ramamoorthy S (2005) A role for p38 mitogen-activated protein kinase in the regulation of the serotonin transporter: evidence for distinct cellular mechanisms involved in transporter surface expression. J Neurosci 25(1):29\u0026ndash;41. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/JNEUROSCI.3754-04.2005\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSawchenko PE, Swanson LW, Steinbusch HW, Verhofstad AA (1983) The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat. Brain Res 277(2):355\u0026ndash;360. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0006-8993(83)90945-9\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchreihofer AM, Guyenet PG (2002) The baroreflex and beyond: control of sympathetic vasomotor tone by GABAergic neurons in the ventrolateral medulla. Clin Exp Pharmacol Physiol 29(5\u0026ndash;6):514\u0026ndash;521. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1440-1681.2002.03665.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeymour AJ, Scott V, Augustine RA, Bouwer GT, Campbell RE, Brown CH (2017) Development of an excitatory kisspeptin projection to the oxytocin system in late pregnancy. J Physiol 595(3):825\u0026ndash;838. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1113/JP273051\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun YL, Zhang XY, He N, Sun T, Zhuang Y, Fang Q, Wang KR, Wang R (2012) Neuropeptide FF activates ERK and NF kappa B signal pathways in differentiated SH-SY5Y cells. Peptides 38(1):110\u0026ndash;117. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.peptides.2012.08.019\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTagliaferro P, Morales M (2008) Synapses between corticotropin-releasing factor‐containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic. J Comp Neurol 506(4):616\u0026ndash;626. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cne.21576\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiss ML, Hatton GI (1990) Collateral input to the paraventricular and supraoptic nuclei in rat. I. Afferents from the subfornical organ and the anteroventral third ventricle region. Brain Res Bull 24(2):231\u0026ndash;238. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0361-9230(90)90210-Q\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWesterhaus MJ, Loewy AD (1999) Sympathetic-related neurons in the preoptic region of the rat identified by viral transneuronal labeling. J Comp Neurol 414(3):361\u0026ndash;378. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/(SICI)1096-9861\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorsley MA, Allen CE, Billinton A, King AE, Boissonade FM (2014) Chronic tooth pulp inflammation induces persistent expression of phosphorylated ERK (pERK) and phosphorylated p38 (pp38) in trigeminal subnucleus caudalis. Neuroscience 269(100):318\u0026ndash;330. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neuroscience.2014.03.056\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu HP, Zhang N, Zhang T, Wang ZL, Li N, Tang HH, Zhang R, Zhang MN, Xu B, Fang Q (2016) Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway. Peptides 86:24\u0026ndash;32. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.peptides.2016.09.012\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Shi X, Hampong M, Blanis L, Pelech S (2001) Stress-induced inhibition of ERK1 and ERK2 by direct interaction with p38 MAP kinase. J Biol Chem 276(10):6905\u0026ndash;6908. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.C000917200\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"kisspeptin, oxytocin, supraoptic nucleus, paraventricular nucleus, pregnancy","lastPublishedDoi":"10.21203/rs.3.rs-1386498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1386498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxytocin is secreted by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) oxytocin neurons to induce uterine contractions during parturition. Increased activation of oxytocin neurons at parturition involves a network of afferent inputs that increase oxytocin neuron excitability. Kisspeptin fibre density increases around oxytocin neurons during pregnancy, and central kisspeptin administration excites oxytocin neurons only in late pregnancy. Kisspeptin signals via extracellular regulated kinase 1/2 (ERK1/2) and p38. Therefore, to determine whether kisspeptin excites oxytocin neurons via ERK1/2-p38 signalling in late-pregnant rats, we performed immunohistochemistry for phosphorylated ERK1/2 (pERK1/2) and phosphorylated p38 (p-p38) in oxytocin neurons of non-pregnant and late-pregnant rats. Intracerebroventricular (ICV) kisspeptin administration (2 \u0026micro;g) did not affect pERK1/2 or p-p38 expression in SON and PVN oxytocin neurons of non-pregnant or late-pregnant rats. Furthermore, ICV kisspeptin did not affect pERK1/2 or p-p38 expression in brain areas with major projections to the SON and PVN: the nucleus tractus solitarius, rostral ventrolateral medulla, locus coeruleus, dorsal raphe nucleus, organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, anteroventral periventricular nucleus, periventricular nucleus and arcuate nucleus. Hence, kisspeptin-induced excitation of oxytocin neurons in late pregnancy does not appear to involve ERK1/2 or p38 activation in oxytocin neurons or their afferent inputs.\u003c/p\u003e","manuscriptTitle":"Central kisspeptin does not affect ERK1/2 or p38 phosphorylation in oxytocin neurons of late-pregnant rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-30 20:01:30","doi":"10.21203/rs.3.rs-1386498/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"634b5fcc-76f2-4b70-948c-82a8a183017c","owner":[],"postedDate":"March 30th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-04-27T20:34:04+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-30 20:01:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1386498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1386498","identity":"rs-1386498","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}