The medial preoptic area (MPOA) of the hypothalamus drives the expression of maternal behaviors. Although prolactin action in the MPOA is essential for the onset of postpartum maternal behavior, it is still unknown how prolactin acts on underlying neural circuitry to modulate different aspects of maternal behavior. Here, we have identified a prolactin-receptive projection from the MPOA to the ventral tegmental area (VTA) that is specifically activated during interactions with pups. Activation of this prolactin-receptive MPOA-VTA projection stimulated increased maternal interactions with pups in virgin female mice, and the removal of prolactin action within this projection prevented the postpartum increase in maternal-pup interactions. Prolactin-receptive MPOA-VTA neurons modulate dopamine release into the nucleus accumbens (NAc), with stimulation of this projecting inducing dopamine release and increased pup interactions and inhibition blocking pup-induced dopamine release into the NAc. These data define a prolactin-receptive neural circuit that engages with reward pathways to drive increased motivation in mothers to interact with newborn offspring.

The adaptation of stress responses during motherhood is critical for the well-being of the mother and the offspring. Stress responses are controlled by corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus. Although it is commonly believed that the activity of CRH neurons is dampened during motherhood, this hypothesis has not been directly tested. Using fibre photometry, we obtain optical recordings of PVN CRH neuron activity in freely behaving mice. Contrary to expectations, CRH neuron excitation in response to external stressors is not suppressed during lactation compared to virgin mice. However, we observe significant reductions in Crh mRNA expression and corticosterone release following stress in the lactating mice. In a pup retrieval task, there are large reductions of CRH neuron activity upon pup retrieval only in lactating mice. When lactating mice are in the presence of an inaccessible pup that could not be physically retrieved, this induces an amplified level of CRH neuron activity. These data show that during motherhood, CRH neuron activity is not globally suppressed. More importantly, we show for the first time that during lactation, CRH neuron responses to pup cues are amplified. These adaptations likely facilitate maternal behaviours that promote offspring survival.

Pregnancy and lactation are reproductive periods that require major energy and nutrient investment by the mother. Dietary perturbations over reproduction can impair offspring development and increase the risk of metabolic disease for the mother. However, how the intake of specific macronutrients, independent of total calorie intake, influence maternal reproductive investment and metabolic health remains poorly understood. To understand the role of protein, carbohydrate, and fat intake in influencing these parameters, we fed mice one of ten isocaloric diets that differed systematically in their macronutrient make-up. We allowed females to breed and observed striking effects of different macronutrients on fetal development, with protein intake having strong positive effects on offspring survival, accompanied by major shifts in the morphological structure of the placenta and placental lactogen production. However, maternal glucose tolerance was strongly impaired by high protein intake during pregnancy, with reproductive females more susceptible to the effects of these macronutrients than nonpregnant animals. Strikingly, metabolic effects were reversed after lactation, with mothers developing a resilience to the chronic effects of protein and fat intake on glucose tolerance observed in virgin animals. During lactation, we also observed that offspring development was optimized by a different ratio of macronutrients compared to during pregnancy. These results highlight the importance of optimizing macronutrient, particularly protein, intake to specific levels during pregnancy, ensuring a balance that maintains maternal glucose tolerance while providing sufficient nutrients to ensure healthy offspring growth and survival.

First-line stimulant therapies for attention deficit hyperactivity disorder (ADHD) implicate the forebrain monoaminergic neurotransmitter systems of dopamine, noradrenaline and serotonin. However, there is no curative treatment. Investigation of neurochemical monoaminergic changes, followed by curative treatments, requires sub-type specific animal models. In a novel rat model of the hyperactive-impulsive and attentive subtype of ADHD, specific changes in forebrain monoamines, and correlations with ADHD-like hyperactivity/impulsivity after delayed reward, are unknown. To address this, we investigated the monoaminergic systems using high performance liquid chromatography at ~2 and 7 months-of-age. We correlated the latter findings with ADHD-like hyperactivity and impulsivity that was measured after delayed reward. We observed long-term effects on the serotonergic, dopaminergic and noradrenergic systems in the forebrain of ADHD-like, hyperactive-impulsive male rats exposed to repeated hypoxia during postnatal day 1-3, the equivalent of extreme prematurity. These long-term effects were strikingly correlated with ADHD-like impulsive, and mostly with ADHD-like hyperactive, behaviour after delayed reward. The correlation was stronger for the dopaminergic and noradrenergic systems and weaker for the serotonergic system. The findings strongly implicate the dopaminergic system in the right prefrontal cortex, the noradrenergic system in the left prefrontal cortex and the serotonergic system in the right posterior caudate-putamen in ADHD-like hyperactivity and ADHD-like impulsivity. Earlier reports of forebrain lateralisation and lifespan changes of monoamines and their metabolites, and of marked differences between the anterior versus posterior caudate-putamen bilaterally, were confirmed and extended. New treatments to collectively cure the neurochemical changes can now be investigated in this unique, ADHD-like subtype-specific animal model.

Pregnancy represents a significant challenge to thermal homeostasis, with adaptive changes required to cope with the increased generation of metabolic heat. We have investigated the hypothesis that prolactin action in the preoptic area (POA) of the hypothalamus contributes to adaptive changes in thermoregulation during pregnancy. Prolactin receptors (Prlr) are expressed in warm-responding neurons (WRNs) in the POA, and chemogenetic activation of this subpopulation induces profound hypothermia. Adeno-associates virus (AAV)-Cre-mediated deletion of Prlr in the POA results in significant hyperthermia throughout pregnancy, suggesting that prolactin action in the POA is required for appropriate thermoregulation during pregnancy. Moreover, deletion of Prlr in glutamate neurons (including WRNs) results in markedly impaired pup survival when animals were housed at mildly elevated environmental temperatures, demonstrating that prolactin action in the POA provides resilience to thermal challenges in pregnancy. Thus, prolactin has a key role in regulating the thermoregulatory circuits, providing optimal conditions for successful pregnancy.

During the post-partum period, new mothers are vulnerable to mood disorders. In adults, impairments in neurogenesis commonly associate with anxiety and depressive behaviors. Insulin-like growth factor 2 (IGF2) is expressed in the choroid plexus (CP) within the subventricular zone (SVZ) neurogenic niche, and global loss of IGF2 leads to increased anxiety. Previously, we have shown that Igf2 expression in CP tissue increases 6-fold during lactation but returns to baseline on suppression of prolactin present in lactation, suggesting it is induced by high levels of prolactin. To gain more insight into the role of prolactin-induced Igf2 expression in the CP, we have measured IGF2 levels in cerebrospinal fluid across reproductive states and developed mice in which Igf2 is conditionally removed from the CP. Using CP-derived IGF2 knockout mouse models, we have measured Prlr expression in CP tissue, SVZ mitogenesis, olfaction, and anxiety-like behavior using an elevated plus maze (EPM) and light/dark transition test (LDTT). Interestingly, we observed a reduction in Prlr expression in CP tissue in one of our Igf2 knockout mouse models, suggesting Igf2 may also act upstream to regulate Prlr expression in CP tissue. No changes were detected in SVZ proliferation rates between Igf2 knockout and controls. Using a buried food test (BFT), however, we show mice with conditional loss of Igf2 in the CP take longer to find a buried fruit loop as compared to controls, indicating olfaction deficits. Overall anxiety levels, however, were comparable between knockout and controls in the EPM and LDTT. Together, our findings reveal loss of CP-derived IGF2 leads to hyposmia in the absence of detectable changes to SVZ mitogenesis. We propose that CP-derived IGF2 may be acting directly in the olfactory bulb to elicit changes to improve olfaction, which may become particularly important during the post-partum period to facilitate mother-pup interactions.

Growth hormone (GH) receptor (GHR) and prolactin (PRL) receptor (PRLR) are transmembrane class I cytokine receptors that co-exist in various normal and cancerous cells. Both receptors respond to their associated ligands predominantly by activating the Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways, and both are also known to initiate receptor-specific JAK2-independent signaling. Together with their cognate ligands, these receptors have been associated with pro-tumorigenic effects in various cancers, including breast cancer (BC). Human GH is known to bind GHR and PRLR, while PRL can only bind PRLR. A growing body of work suggests that GHR and PRLR can form heteromers in BC cells, modulating GH signal transduction. However, the dynamics of PRLR and GHR on the plasma membrane and how these could affect their respective signaling still need to be understood.To this end, we set out to unravel the spatiotemporal dynamics of GHR and PRLR on the surface of human T47D breast cancer cells and γ2A-JAK2 cells. We applied direct stochastic optical reconstruction microscopy (dSTORM) and quantified the colocalization and availability of both receptors on the plasma membrane at the nanometer scale at different time points following treatment with GH and PRL. In cells co-expressing GHR and PRLR, we surprisingly observed that not only GH but also PRL treatment induces a significant loss of surface GHR. In cells lacking PRLR or expressing a mutant PRLR deficient in JAK2 binding, we observed that GH induces downregulation of membrane-bound GHR, but PRL no longer induces loss of surface GHR. Colocalizations of GHR and PRLR were confirmed by proximity ligation (PL) assay.Our results suggest that PRLR-GHR interaction, direct or indirect, is indispensable for PRL-but not GH-induced loss of surface GHR and for both GH-induced and PRL-induced increase of surface PRLR, with potential consequences for downstream signaling. Furthermore, our results suggest that JAK2 binding via the receptor intracellular domain’s Box1 element is crucial for the observed regulation of one class I cytokine receptor’s cell surface availability via ligand-induced activation of another class I cytokine receptor. Our findings shed new light on the reciprocal and collective role that PRLR and GHR play in regulating cell signaling.

The specific role that prolactin plays in lactational infertility, as distinct from other suckling or metabolic cues, remains unresolved. Here, deletion of the prolactin receptor (Prlr) from forebrain neurons or arcuate kisspeptin neurons resulted in failure to maintain normal lactation-induced suppression of estrous cycles. Kisspeptin immunoreactivity and pulsatile LH secretion were increased in these mice, even in the presence of ongoing suckling stimulation and lactation. GCaMP fibre photometry of arcuate kisspeptin neurons revealed that the normal episodic activity of these neurons is rapidly suppressed in pregnancy and this was maintained throughout early lactation. Deletion of Prlr from arcuate kisspeptin neurons resulted in early reactivation of episodic activity of kisspeptin neurons prior to a premature return of reproductive cycles in early lactation. These observations show dynamic variation in arcuate kisspeptin neuronal activity associated with the hormonal changes of pregnancy and lactation, and provide direct evidence that prolactin action on arcuate kisspeptin neurons is necessary for suppressing fertility during lactation in mice.

Prolactin is a polypeptide hormone secreted from the lactotrophs in the anterior pituitary gland. It is recognized as a pleiotropic hormone with multiple biological actions, which seem to have evolved to provide physiological adaptations required for the success of reproduction. Among its numerous roles, lactation is one to which prolactin is essential, being the primary hormone responsible for the synthesis of milk. The neuroendocrine control of prolactin secretion is unique among the anterior pituitary hormones. The lactotrophs have a high spontaneous capacity for proliferation and secretion of prolactin that is suppressed by dopamine. Neuroendocrine dopaminergic (NEDA) neurons release dopamine into the hypothalamic-pituitary portal blood system to inhibit prolactin secretion. Prolactin exerts a short-loop negative feedback effect stimulating several aspects of NEDA neuronal activity, resulting in inhibition of its own secretion. The nipple suckling by the offspring during lactation is the most powerful stimulus to increase prolactin secretion. Thus, lactation is a hyperprolactinemic state, characterized by suckling-induced prolactin surges and chronically elevated basal levels of prolactin. Phenotypic and functional modifications in the NEDA neurons associated with changes in the number and organization of lactotrophs are responsible for the remarkable increase in prolactin secretion during lactation. Hypothalamic prolactin-releasing factors seem to also stimulate the rise in prolactin secretion, but their identity remains to be determined. In this chapter, we review the classical concepts and recent advances in our understanding of the neuroendocrine control of prolactin secretion and how it changes to promote the physiological state of hyperprolactinemia during lactation.

Abstract Disclosure: P. Papaioannou: None. T. Georgescu: None. D.R. Grattan: None. S. Bunn: None. S. Yip: None. The tuberoinfundibular dopaminergic (TIDA) neurons play a crucial role in regulating prolactin secretion. Their synchronized network activity with slow but highly rhythmic firing pattern is important for dopamine release in rats[1]. While this unique TIDA network activity is vital for prolactin negative feedback in non-lactating conditions, its behaviour during lactation, when prolactin demand is high, remains unknown. Our hypothesis posited that the TIDA neuronal network in lactating rats becomes desynchronized, disrupting the negative feedback loop. To test this, we utilized ex-vivo Ca2+ imaging to simultaneously monitor population-wide TIDA neuron activity in non-lactating (NL; n=13) and lactating (L; n=10) rats injected with a cre-inducible adeno-associated virus (AAV) containing the Ca2+ indicator, GCaMP6s, into their arcuate nucleus. Analysis of neuronal network synchronicity revealed significant desynchronization in the TIDA network during lactation, as indicated by a markedly lower mean correlation coefficient matrix (CM) compared to non-lactating conditions (NL: 0.87±0.02, n= 26 sections vs L: 0.22±0.03, n=29 sections; p<0.001, Student’s t-test). Furthermore, the oscillatory activity patterns of these desynchronized TIDA neurons displayed a significantly lower cell rhythmicity index (RI) in lactating animals compared to non-lactating ones (NL: 0.17±0.01, n=186 cells vs L: 0.70±0.02, n=77 cells; p<0.001, Student’s t-test). Interestingly, within these low rhythmic neurons, some exhibited higher while others showed lower firing frequencies compared to non-lactating TIDA neurons. After 7 days post-weaning (n=4 animals), the TIDA network activity returned to non-lactating behaviour (CM = 0.85 ± 0.04, n=6 sections and RI = 0.17±0.01; n=47 cells). In summary, our findings demonstrate a reversible reconfiguration of the TIDA neuronal networks during lactation, characterized by low rhythmic oscillations and individual unique firing patterns, leading to diminished intercellular synchrony that may impede dopamine release, ultimately facilitating prolactin release to support lactation. Once weaned, the TIDA neuronal network reset to NL state to allow a new reproductive cycle. [1] Lyons, D. J., Horjales-Araujo, E. & Broberger, C. Synchronized network oscillations in rat tuberoinfundibular dopamine neurons: switch to tonic discharge by thyrotropin-releasing hormone. Neuron 65, 217-229, doi:10.1016/j.neuron.2009.12.024 (2010). Presentation: 6/2/2024

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/jne.13416. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

beta-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, beta-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether beta-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female beta-catenin(flox) mice, to specifically delete beta-catenin expression in the mediobasal hypothalamus (MBH-beta-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-beta-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-beta-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-beta-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for beta-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.

Suppression of the hypothalamic-pituitary-adrenal (HPA) axis is a well-characterised maternal adaptation that limits the exposure of the offspring to maternally-derived stress hormones. This current study has investigated the possible involvement of the lactogenic hormone, prolactin, in this physiologically important adaptation. As expected, circulating prolactin levels were higher in unstressed lactating mice compared to their virgin counterparts. Interestingly however, the ability of an acute period of restraint stress to further elevate prolactin levels was diminished in the former group. The stress-induced rise in prolactin levels in the virgin animals was concurrent with an increase in prolactin receptor activation within the adrenal cortical cells. This adrenal response was not seen in either the stressed or control lactation group, an observation that may be in part explained by the observed downregulation of prolactin receptor mRNA expression within this tissue. Further evidence of suppression of the HPA axis during lactation was revealed using in situ hybridisation to demonstrate that while acute restraint stress increased corticotrophin releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus in both virgin and lactating mice, the magnitude of this response was reduced in the latter group. This potentially adaptive response did not, however, appear to result from the altered prolactin profile during lactation because it was not affected by the pharmacological suppression of prolactin secretion from the pituitary. This study therefore suggests that during lactation the response of the HPA axis to stress is suppressed at multiple physiological levels which are mediated by both prolactin-dependent and prolactin-independent mechanisms.

The prolactin receptor (Prlr) is widely expressed in the brain, particularly in the hypothalamus. Prolactin also has an increasing range of well-characterised effects on central nervous system function. Because of this, over many years, there has been interest in whether the hormone itself is also expressed within the brain, perhaps acting as a neuropeptide to regulate brain function via its receptor in neurons. The aim of this invited review is to critically evaluate the evidence for brain production of prolactin. Unlike the evidence for the Prlr, evidence for brain prolactin is inconsistent and variable. A range of different antibodies have been used, each characterising a different distribution of prolactin-like immunoreactivity. Prolactin mRNA has been detected in the brain, but only at levels markedly lower than seen in the pituitary gland. Importantly, it has largely only been detected by highly sensitive amplification-based techniques, and the extreme sensitivity means there is a risk of false-positive data. Modern in situ hybridisation methods and single-cell RNA sequencing have not provided supporting evidence, but it is hard to prove a negative! Finally, I acknowledge and discuss the possibility that prolactin might be produced in the brain under specific circumstances, such as to promote a neuroprotective response to cell damage. Collectively, however, based on this analysis, I have formed the opinion that brain production of prolactin is unlikely, and even if occurs, it is of little physiological consequence. Most, if not all of the brain actions of prolactin can be explained by pituitary prolactin gaining access to the brain. While prolactin receptor (Prlr) is widely expressed in the brain (right), the equivalent evidence fo prolactin expression is lacking (left). image

Journal of NeuroendocrinologyVolume 35, Issue 11 e13345 EDITORIAL Advances in peptide modulation in systems physiology Lee E. Eiden, Lee E. Eiden orcid.org/0000-0001-7524-944X Section on Molecular Neuroscience, National Institute of Mental Health, NIH, Bethesda, Maryland, USA Contribution: Writing - original draft, Writing - review & editingSearch for more papers by this authorDavid Grattan, David Grattan orcid.org/0000-0001-5606-2559 Centre for Neuroendocrinology, Division of Health Sciences, University of Otago, Dunedin, New Zealand Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorXiao-Dong Wang, Xiao-Dong Wang orcid.org/0000-0001-7730-3710 School of Brain Science and Brain Medicine, Zhejiang University, Hangzhou, China Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorLimei Zhang, Corresponding Author Limei Zhang [email protected] orcid.org/0000-0002-7422-5136 Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico Correspondence Limei Zhang, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico. Email: [email protected] Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this author Lee E. Eiden, Lee E. Eiden orcid.org/0000-0001-7524-944X Section on Molecular Neuroscience, National Institute of Mental Health, NIH, Bethesda, Maryland, USA Contribution: Writing - original draft, Writing - review & editingSearch for more papers by this authorDavid Grattan, David Grattan orcid.org/0000-0001-5606-2559 Centre for Neuroendocrinology, Division of Health Sciences, University of Otago, Dunedin, New Zealand Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorXiao-Dong Wang, Xiao-Dong Wang orcid.org/0000-0001-7730-3710 School of Brain Science and Brain Medicine, Zhejiang University, Hangzhou, China Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorLimei Zhang, Corresponding Author Limei Zhang [email protected] orcid.org/0000-0002-7422-5136 Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico Correspondence Limei Zhang, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico. Email: [email protected] Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this author First published: 03 October 2023 https://doi.org/10.1111/jne.13345 Special Issue of papers from the 24th International Symposium on Regulatory Peptides (RegPep24 World Conference), 1st–5th August 2022, University of Stirling, Scotland, UK. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Open Research PEER REVIEW The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/jne.13345. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were created or analyzed in this study. REFERENCES 1Hernandez-Perez OR, Hernández VS, Zetter MA, Eiden LE, Zhang L. Nucleus of the lateral olfactory tract: a hub linking the water homeostasis-associated supraoptic nucleus-arginine vasopressin circuit and neocortical regions to promote social behavior under osmotic challenge. J Neuroendocrinol. 2022;223:e13202. 2Peles G, Swaminathan A, Levkowitz G. Glucocorticoid-sensitive period of corticotroph development-implications for mechanisms of early life stress. J Neuroendocrinol. 2022;e13229. 3Sabban EL, Serova L, Nahvi RJ, Liu X. Potential benefits of intranasal neuropeptide Y include sustained extinction of fear memory. J Neuroendocrinol. 2023;223:e13279. 4Chen X, Coffman BL, Brindley RL, et al. Phospholipase C-epsilon defines a PACAP-stimulated pathway for secretion in the chromaffin cell. J Neuroendocrinol. 2023;223:e13255. 5Bakalar D, Gavrilova O, Jiang SZ, et al. Constitutive and conditional deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of the neuropeptide PACAP. J Neuroendocrinol. 2023;223:e13286. 6Kozlova EV, Bishay AE, Denys ME, et al. Gene deletion of the PACAP/VIP receptor, VPAC2R, alters glycemic responses during metabolic and psychogenic stress in adult female mice. J Neuroendocrinol. 2023;223. 7Paes-Leme B, Monteiro LRN, Gholami K, et al. Fasting increases circulating angiotensin levels and brain Agtr1a expression in male rats. J Neuroendocrinol. 2023;223:e13334. 8Chen D, Hagen SJ, Boyce M, Zhao CM. Neuroendocrine mechanism of gastric acid secretion: historical perspectives and recent developments in physiology and pharmacology. 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Maternal interactions with offspring are highly rewarding, which reinforces expression of essential caregiving behaviours that promote offspring survival. In rats, the rewarding effect of pups depends on reproductive state, with lactating females specifically developing strong preferences for pup-associated contexts. Whether this also occurs in mice is unknown, hence we aimed to characterise pup-related preference across reproductive states in female mice. In a conditioned place preference (CPP) test, pups were a rewarding stimulus to female mice prior to lactation, with virgin and pregnant females developing a preference for a pup-associated context. We have previously shown that lactogenic hormones, acting through the prolactin receptor (Prlr), play an important role in maternal motivation. Here, we aimed to investigate whether Prlr action is important for pup-related reward behaviour in mice. We showed that prolactin itself had a reinforcing effect in a CPP test, and that exposure to pups increased blood prolactin levels in virgin female mice. Prlr expression in CamKIIα-expressing neurons and GABAergic neurons has previously been shown to be important for different aspects of parental behaviour. However, we found that conditional Prlr deletion from either of these neuronal populations did not disrupt the development of a preference for pup-associated contexts in pregnant female mice, indicating that lactogenic action on these populations is not necessary for the rewarding effect of pups. Together, these data show that while lactogenic hormones likely contribute to a rewarding effect of pups, their action on two key neuronal populations is not necessary for this effect in female mice.

Lactation in mammals is associated with a period of infertility, which serves to direct maternal metabolic resources toward caring for the newborn offspring rather than supporting another pregnancy. This lactational infertility is characterized by reduced pulsatile luteinizing hormone (LH) secretion and lack of ovulation. The mechanisms mediating suppression of LH secretion during lactation are unclear. There are potential roles for both hormonal cues such as prolactin and progesterone, and pup-derived cues such as suckling, on the inhibition of reproduction. To enable future studies using transgenic animals to investigate these mechanisms, in the present study our aim was to characterize lactational infertility in mice, and to investigate the effect of removing pup-derived cues on LH secretion, time to ovulation, and kisspeptin immunoreactivity. We first confirmed that C57BL/6J mice experience prolonged anestrus during lactation, which is dependent on establishment of lactation, as removal of pups the day of parturition led to immediate resumption of pulsatile LH secretion and normal estrous cycles. Once lactation is established, however, the lactational anestrus persisted for several days even after premature removal of pups. Pharmacological suppression of prolactin following premature weaning significantly reduced this period of lactational infertility. Progesterone does not appear to play a significant role in the suppression of fertility during lactation in mice, as levels measured during lactation were not different from nonpregnant mice. These data suggest that prolactin plays a key role in mediating anestrus during early lactation in mice, even in the absence of the suckling stimulus.

Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal precision. Using liquid chromatography-mass spectrometry, now considered the gold standard for steroid hormone analysis, we aimed to generate a detailed profile of gonadal steroid levels across the estrous cycle of C57BL/6J mice. For reference, luteinizing hormone (LH) and prolactin concentrations were measured in the same samples by sandwich enzyme-linked immunosorbent assay. Terminal blood samples were collected at 8-hour intervals (10 Am, 6 Pm, 2 Am) throughout the 4 stages of the estrous cycle. As expected, the LH surge was detected at 6 Pm on proestrus with a mean (+/- SEM) concentration of 11 +/- 3 ng/mL and occurred coincident with the peak in progesterone levels (22 +/- 4 ng/mL). Surprisingly, estradiol concentrations peaked at 10 Am on diestrus (51 +/- 8 pg/mL), with levels on proestrus 6 Pm reaching only two-thirds of this value (31 +/- 5 pg/mL). We also observed a proestrus peak in prolactin concentrations (132.5 +/- 17 ng/mL) that occurred earlier than expected at 2 Am. Estrone and androstenedione levels were often close to the limit of detection (LOD) and showed no consistent changes across the estrous cycle. Testosterone levels were rarely above the LOD (0.01 ng/mL). These observations provide the first detailed assessment of fluctuating gonadal steroid and reproductive hormone levels across the mouse estrous cycle and indicate that species differences exist between mice and other spontaneously ovulating species.