Conditional Deletion of ß-Catenin in the Mediobasal Hypothalamus Impairs Adaptive Energy Expenditure in Response to High-Fat Diet and Exacerbates Diet-Induced Obesity.

ß-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, ß-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 ß-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 ß-catenin flox mice, to specifically delete ß-catenin expression in the mediobasal hypothalamus (MBH-ß-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-ß-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-ß-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-ß-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 ß-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.

High fat diet-induced maternal obesity in mice impairs peripartum maternal behaviour.

Obesity during pregnancy represents a significant health issue and can lead to increased complications during pregnancy and impairments with breastfeeding, along with long-term negative health consequences for both mother and offspring. In rodent models, diet-induced obesity (DIO) during pregnancy leads to poor outcomes for offspring. Using a DIO mouse model, consisting of feeding mice a high fat diet for 8 weeks before mating, we recapitulate the effect of high pup mortality within the first 3 days postpartum. To examine the activity of the dam around the time of birth, late pregnant control and DIO dams were recorded in their home cages and the behaviour of the dam immediately before and after birth was analysed. Prior to giving birth, DIO dams spent less time engaging in nesting behaviour, while after birth, DIO dams spent less time in the nest with their pups compared to control dams, indicating reduced pup-engagement in the early postpartum period. We have previously reported that lactogenic hormone action, mediated by the prolactin receptor, in the medial preoptic area of the hypothalamus (MPOA) is critical for the onset of normal postpartum maternal behaviour. We hypothesized that DIO dams may have lower lactogenic hormone activity during late pregnancy, which would contribute to impaired onset of normal postpartum maternal behaviour. Day 16 lactogenic activity, transport of prolactin into the brain, and plasma prolactin concentrations around birth were all similar in control and DIO dams. Moreover, endogenous pSTAT5, a marker of prolactin receptor activity, in the MPOA was unaffected by DIO. Overall, these data indicate that lactogenic activity in late pregnancy of DIO dams is not different to controls and is unlikely to play a major role in impaired onset of normal postpartum maternal behaviour.

The role of prolactin in the suppression of the response to restraint stress in the lactating mouse.

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.

Mating-induced prolactin surge is not required for subsequent neurogenesis in male mice.

Parenting involves major behavioral transitions that are supported by coordinated neuroendocrine and physiological changes to promote the onset of novel offspring-directed behaviors. In comparison to maternal care, however, the mechanisms underlying the transition to paternal care are less understood. Male laboratory mice are predominantly infanticidal as virgins but show paternal responses 2 weeks after mating. Interestingly, males show a mating-induced surge of prolactin, which we hypothesized may be involved in initiating this behavioral transition. During pregnancy, prolactin stimulates olfactory bulb neurogenesis, which is essential for maternal behavior. Mating induces olfactory bulb neurogenesis in males, but it is unknown whether this is driven by prolactin or is important for subsequent paternal care. New olfactory neurons are generated from cells in the subventricular zone (SVZ) and take about 2 weeks to migrate to the olfactory bulb, which may account for the delayed behavioral change in mated males. We investigated whether mating increases cell proliferation at the SVZ. Males were either mated, exposed to receptive female cues, or left alone (control) and injected with Bromodeoxyuridine (BrdU, a marker of cell division). Contrary to our hypothesis, we found that mating decreased cell proliferation in the caudal lateral portion of the SVZ. Next, we tested whether prolactin itself mediates cell proliferation in the SVZ and/or new cell survival in the olfactory bulb by administering bromocriptine (prolactin inhibitor), vehicle, or bromocriptine + prolactin prior to mating. While suppressing prolactin had no effect on cell proliferation in the SVZ, administering exogenous prolactin resulted in significantly higher BrdU-labeled cells in mated but not virgin male mice. No effects of prolactin were observed on new olfactory cell survival. Taken together, prolactin may have context-dependent effects on new cell division in the SVZ, while other unknown mechanisms may be driving the effects on new olfactory cell survival following mating.

Unexpected Plasma Gonadal Steroid and Prolactin Levels Across the Mouse Estrous Cycle.

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.

Mechanisms of Lactation-induced Infertility 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.

Developmental Inhibitory Changes in the Primary Somatosensory Cortex of the Stargazer Mouse Model of Absence Epilepsy.

Childhood absence epilepsy seizures arise in the cortico-thalamocortical network due to multiple cellular and molecular mechanisms, which are still under investigation. Understanding the precise mechanisms is imperative given that treatment fails in ~30% of patients while adverse neurological sequelae remain common. Impaired GABAergic neurotransmission is commonly reported in research models investigating these mechanisms. Recently, we reported a region-specific reduction in the whole-tissue and synaptic GABAA receptor (GABAAR) α1 subunit and an increase in whole-tissue GAD65 in the primary somatosensory cortex (SoCx) of the adult epileptic stargazer mouse compared with its non-epileptic (NE) littermate. The current study investigated whether these changes occurred prior to the onset of seizures on postnatal days (PN) 17-18, suggesting a causative role. Synaptic and cytosolic fractions were biochemically isolated from primary SoCx lysates followed by semiquantitative Western blot analyses for GABAAR α1 and GAD65. We found no significant changes in synaptic GABAAR α1 and cytosolic GAD65 in the primary SoCx of the stargazer mice at the critical developmental stages of PN 7-9, 13-15, and 17-18. This indicates that altered levels of GABAAR α1 and GAD65 in adult mice do not directly contribute to the initial onset of absence seizures but are a later consequence of seizure activity.

Pups and prolactin are rewarding to virgin female and pregnant mice.

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.

Prolactin Action Is Necessary for Parental Behavior in Male Mice.

Parental care is critical for successful reproduction in mammals. Recent work has implicated the hormone prolactin in regulating male parental behavior, similar to its established role in females. Male laboratory mice show a mating-induced suppression of infanticide (normally observed in virgins) and onset of paternal behavior 2 weeks after mating. Using this model, we sought to investigate how prolactin acts in the forebrain to regulate paternal behavior. First, using c-fos immunoreactivity in prolactin receptor (Prlr) Prlr-IRES-Cre-tdtomato reporter mouse sires, we show that the circuitry activated during paternal interactions contains prolactin-responsive neurons in multiple sites, including the medial preoptic nucleus, bed nucleus of the stria terminalis, and medial amygdala. Next, we deleted Prlr from three prominent cell types found in these regions: glutamatergic, GABAergic, and CaMKIIα. Prlr deletion from CaMKIIα, but not glutamatergic or GABAergic cells, had a profound effect on paternal behavior as none of these KO males completed the pup-retrieval task. Prolactin was increased during mating, but not in response to pups, suggesting that the mating-induced secretion of prolactin is important for establishing the switch from infanticidal to paternal behavior. Pharmacological blockade of prolactin secretion at mating, however, had no effect on paternal behavior. In contrast, suppressing prolactin secretion at the time of pup exposure resulted in failure to retrieve pups, with exogenous prolactin administration rescuing this behavior. Together, our data show that paternal behavior in sires is dependent on basal levels of circulating prolactin acting at the time of interaction with pups, mediated through Prlr on CaMKIIα-expressing neurons.SIGNIFICANCE STATEMENT Parental care is critical for offspring survival. Compared with maternal care, however, the neurobiology of paternal care is less well understood. Here we show that the hormone prolactin, which is most well known for its female-specific role in lactation, has a role in the male brain to promote paternal behavior. In the absence of prolactin signaling specifically during interactions with pups, father mice fail to show normal retrieval behavior of pups. These data demonstrate that prolactin has a similar action in both males and females to promote parental care.

Multiple cell types in the oviduct express the prolactin receptor.

Little is known about the physiological role of prolactin in the oviduct. Examining mRNA for all four isoforms of the prolactin receptor (PRLR) in mice by functional oviduct segment and stage of the estrous cycle, we found short form 3 (SF3) to be the most highly expressed, far exceeding the long form (LF) in highly ciliated areas such as the infundibulum, whereas in areas of low ciliation, the SF3 to LF ratio was ~1. SF2 expression was low throughout the oviduct, and SF1 was undetectable. Only in the infundibulum did PRLR ratios change with the estrous cycle. Immunofluorescent localization of SF3 and LF showed an epithelial (both mucosal and mesothelial) distribution aligned with the mRNA results. Despite the high SF3/LF ratio in densely ciliated regions, these regions responded to an acute elevation of prolactin (30 min, intraperitoneal), with LF-tyrosine phosphorylated STAT5 seen within cilia. Collectively, these results show ciliated cells are responsive to prolactin and suggest that prolactin regulates estrous cyclic changes in ciliated cell function in the infundibulum. Changes in gene expression in the infundibulum after prolonged prolactin treatment (7-day) showed prolactin-induced downregulation of genes necessary for cilium development/function, a result supporting localization of PRLRs on ciliated cells, and one further suggesting hyperprolactinemia would negatively impact ciliated cell function and therefore fertility. Flow cytometry, single-cell RNAseq, and analysis of LF-td-Tomato transgenic mice supported expression of PRLRs in at least a proportion of epithelial cells while also hinting at additional roles for prolactin in smooth muscle and other stromal cells.

Elevated prolactin secretion during proestrus in mice: Absence of a defined surge.

Throughout the reproductive cycle in rodents, prolactin levels are generally low. In some species, including rats, a prolactin surge occurs on proestrus with peak concentrations coinciding with the preovulatory luteinizing hormone (LH) surge. In mice, however, there are conflicting reports relating to the occurrence and timing of a proestrous prolactin surge. To gain further insight into the incidence and characteristics of this surge in mice, we have used serial tail tip blood sampling and trunk blood collection from both C57BL/6J (inbred) and Swiss Webster (outbred) mouse strains to build a profile of prolactin secretion during proestrus in individual mice. A clearly defined LH surge was detected in most animals, suggesting the blood sampling approach was suitable for detecting patterns of hormone secretion on proestrus. Despite this, levels of prolactin were quite variable between individuals. Overall both mouse strains showed a generalized rise in prolactin levels on the day of proestrus compared with levels seen in diestrus. This pattern is quite distinct from the discreet, circadian-entrained surge observed in rats.

Leptin regulates glucose homeostasis via the canonical Wnt pathway in the zebrafish.

Leptin is best known for its role in adipostasis, but it also regulates blood glucose levels. The molecular mechanism by which leptin controls glucose homeostasis remains largely unknown. Here, we use a zebrafish model to show that Wnt signaling mediates the glucoregulatory effects of leptin. Under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis in zebrafish. In times of nutrient excess, however, we found that leptin also regulates body weight and size. Using a Wnt signaling reporter fish, we show that leptin activates the canonical Wnt pathway in vivo. Utilizing two paradigms for hyperglycemia, it is revealed that leptin regulates glucose homeostasis via the Wnt pathway, as pharmacological inhibition of this pathway impairs the glucoregulatory actions of leptin. Our results may shed new light on the evolution of the physiological function of leptin.

Prolactin-mediated restraint of maternal aggression in lactation.

Aggressive behavior is rarely observed in virgin female mice but is specifically triggered in lactation where it facilitates protection of offspring. Recent studies demonstrated that the hypothalamic ventromedial nucleus (VMN) plays an important role in facilitating aggressive behavior in both sexes. Here, we demonstrate a role for the pituitary hormone, prolactin, acting through the prolactin receptor in the VMN to control the intensity of aggressive behavior exclusively during lactation. Prolactin receptor deletion from glutamatergic neurons or specifically from the VMN resulted in hyperaggressive lactating females, with a marked shift from intruder-directed investigative behavior to very high levels of aggressive behavior. Prolactin-sensitive neurons in the VMN project to a wide range of other hypothalamic and extrahypothalamic regions, including the medial preoptic area, paraventricular nucleus, and bed nucleus of the stria terminalis, all regions known to be part of a complex neuronal network controlling maternal behavior. Within this network, prolactin acts in the VMN to specifically restrain male-directed aggressive behavior in lactating females. This action in the VMN may complement the role of prolactin in other brain regions, by shifting the balance of maternal behaviors from defense-related activities to more pup-directed behaviors necessary for nurturing offspring.

The Prolactin Family of Hormones as Regulators of Maternal Mood and Behavior.

Transition into motherhood involves profound physiological and behavioral adaptations that ensure the healthy development of offspring while maintaining maternal health. Dynamic fluctuations in key hormones during pregnancy and lactation induce these maternal adaptations by acting on neural circuits in the brain. Amongst these hormonal changes, lactogenic hormones (e.g., prolactin and its pregnancy-specific homolog, placental lactogen) are important regulators of these processes, and their receptors are located in key brain regions controlling emotional behaviors and maternal responses. With pregnancy and lactation also being associated with a marked elevation in the risk of developing mood disorders, it is important to understand how hormones are normally regulating mood and behavior during this time. It seems likely that pathological changes in mood could result from aberrant expression of these hormone-induced behavioral responses. Maternal mental health problems during pregnancy and the postpartum period represent a major barrier in developing healthy mother-infant interactions which are crucial for the child's development. In this review, we will examine the role lactogenic hormones play in driving a range of specific maternal behaviors, including motivation, protectiveness, and mother-pup interactions. Understanding how these hormones collectively act in a mother's brain to promote nurturing behaviors toward offspring will ultimately assist in treatment development and contribute to safeguarding a successful pregnancy.

Prolactin-Induced Adaptation in Glucose Homeostasis in Mouse Pregnancy Is Mediated by the Pancreas and Not in the Forebrain.

Adaptive changes in glucose homeostasis during pregnancy require proliferation of insulin-secreting beta-cells in the pancreas, together with increased sensitivity for glucose-stimulated insulin secretion. Increased concentrations of maternal prolactin/placental lactogen contribute to these changes, but the site of action remains uncertain. Use of Cre-lox technology has generated pancreas-specific prolactin receptor (Prlr) knockouts that demonstrate the development of a gestational diabetic like state. However, many Cre-lines for the pancreas also express Cre in the hypothalamus and prolactin could act centrally to modulate glucose homeostasis. The aim of the current study was to examine the relative contribution of prolactin action in the pancreas and brain to these pregnancy-induced adaptations in glucose regulation. Deletion of prolactin receptor (Prlr) from the pancreas using Pdx-cre or Rip-cre led to impaired glucose tolerance and increased non-fasting blood glucose levels during pregnancy. Prlrlox/lox /Pdx-Cre mice also had impaired glucose-stimulated insulin secretion and attenuated pregnancy-induced increase in beta-cell fraction. Varying degrees of Prlr recombination in the hypothalamus with these Cre lines left open the possibility that central actions of prolactin could contribute to the pregnancy-induced changes in glucose homeostasis. Targeted deletion of Prlr specifically from the forebrain, including areas of expression induced by Pdx-Cre and Rip-cre, had no effect on pregnancy-induced adaptations in glucose homeostasis. These data emphasize the pancreas as the direct target of prolactin/placental lactogen action in driving adaptive changes in glucose homeostasis during pregnancy.

Maternal adaptations to food intake across pregnancy: Central and peripheral mechanisms.

A sufficient and balanced maternal diet is critical to meet the nutritional demands of the developing fetus and to facilitate deposition of fat reserves for lactation. Multiple adaptations occur to meet these energy requirements, including reductions in energy expenditure and increases in maternal food intake. The central nervous system plays a vital role in the regulation of food intake and energy homeostasis and responds to multiple metabolic and nutrient cues, including those arising from the gastrointestinal tract. This review describes the nutrient requirements of pregnancy and the impact of over- and undernutrition on the risk of pregnancy complications and adult disease in progeny. The central and peripheral regulation of food intake is then discussed, with particular emphasis on the adaptations that occur during pregnancy and the mechanisms that drive these changes, including the possible role of the pregnancy-associated hormones progesterone, estrogen, prolactin, and growth hormone. We identify the need for deeper mechanistic understanding of maternal adaptations, in particular, changes in gut-brain axis satiety signaling. Improved understanding of food intake regulation during pregnancy will provide a basis to inform strategies that prevent maternal under- or overnutrition, improve fetal health, and reduce the long-term health and economic burden for mothers and offspring.

Prolactin-sensitive olfactory sensory neurons regulate male preference in female mice by modulating responses to chemosensory cues.

Chemosensory cues detected in the nose need to be integrated with the hormonal status to trigger appropriate behaviors, but the neural circuits linking the olfactory and the endocrine system are insufficiently understood. Here, we characterize olfactory sensory neurons in the murine nose that respond to the pituitary hormone prolactin. Deletion of prolactin receptor in these cells results in impaired detection of social odors and blunts male preference in females. The prolactin-responsive olfactory sensory neurons exhibit a distinctive projection pattern to the brain that is similar across different individuals and express a limited subset of chemosensory receptors. Prolactin modulates the responses within these neurons to discrete chemosensory cues contained in male urine, providing a mechanism by which the hormonal status can be directly linked with distinct olfactory cues to generate appropriate behavioral responses.

A reduction in voluntary physical activity in early pregnancy in mice is mediated by prolactin.

As part of the maternal adaptations to pregnancy, mice show a rapid, profound reduction in voluntary running wheel activity (RWA) as soon as pregnancy is achieved. Here, we evaluate the hypothesis that prolactin, one of the first hormones to change secretion pattern following mating, is involved in driving this suppression of physical activity levels during pregnancy. We show that prolactin can acutely suppress RWA in non-pregnant female mice, and that conditional deletion of prolactin receptors (Prlr) from either most forebrain neurons or from GABA neurons prevented the early pregnancy-induced suppression of RWA. Deletion of Prlr specifically from the medial preoptic area, a brain region associated with multiple homeostatic and behavioral roles including parental behavior, completely abolished the early pregnancy-induced suppression of RWA. As pregnancy progresses, prolactin action continues to contribute to the further suppression of RWA, although it is not the only factor involved. Our data demonstrate a key role for prolactin in suppressing voluntary physical activity during early pregnancy, highlighting a novel biological basis for reduced physical activity in pregnancy.

Changes in maternal motivation across reproductive states in mice: A role for prolactin receptor activation on GABA neurons.

The survival of newborn offspring in mammals is dependent on sustained maternal care. Mammalian mothers are highly motivated to interact with and care for offspring, however, it is unclear how hormonal signals act on neural circuitry to promote maternal motivation during the transition to motherhood. In this study we aimed to establish methods that enable us to evaluate change in maternal motivation across the reproductive life cycle in female mice. Using two behavioural testing paradigms; a novel T-maze retrieval test and a barrier climbing test, we found that pup retrieval behaviour was low in virgin and pregnant mice compared to lactating females, indicating that maternal motivation arises around the time of parturition. Furthermore, in reproductively experienced females, maternal motivation declined over time after weaning of pups. As we have previously shown that lactogenic action mediated through the prolactin receptor (Prlr) in the medial preoptic area (MPOA) is essential for the expression of maternal behaviour, we aimed to investigate the role of lactogenic hormones in promoting pup-related motivational behaviours. With GABAergic neurons expressing Prlr in multiple brain regions important for maternal behaviour, we conditionally deleted Prlr from GABA neurons. Compared to control females, lactating GABA neuron-specific Prlr knockout mice showed slower and incomplete pup retrieval behaviour in the T-maze test. Testing of anxiety behaviour on an elevated plus maze indicated that these mice did not have increased anxiety levels, suggesting that lactogenic action on GABA neurons is necessary for the full expression of motivational aspects of maternal behaviour during lactation.