Mapping the actions of prolactin in the brain: sexual dimorphism, steroid regulation and the neuroendocrinology of maternal behaviour

El comportamiento maternal cumple la función de proteger a la descendencia en su periodo de mayor vulnerabilidad y asegurar su supervivencia y bienestar. Esta colección de conductas sociales será expresada por la madre exclusivamente durante el periodo postparto, como resultado de la modulación del sustrato neural responsable de la expresión de la conducta social y reproductiva. Los agentes que llevan a cabo dicha modulación para inducir la expresión del comportamiento maternal incluyen, entre otros, señales endocrinas específicas de la gestación y lactancia. Entre éstas destaca la prolactina, hormona adenohipofisaria estrechamente vinculada con la adaptación del organismo a la maternidad. El objetivo principal de esta tesis ha sido la caracterización de las acciones de señalización de la prolactina en el cerebro del ratón y, más concretamente, en el contexto maternal. El primer capítulo de la misma se ha centrado en describir los patrones básicos de distribución de señalización mediada por prolactina en el cerebro de ratones machos y hembras no parentales, así como la influencia en los mismos de esteroides sexuales femeninos y masculinos, respectivamente. A este respecto, se ha hallado un dimorfismo sexual claro, en favor de hembras, en la extensión e intensidad de dichos patrones. Aun así, el cerebro del ratón macho también es sensible a prolactina. Por último, tanto el estradiol y progesterona en hembras, como la testosterona en machos, promueven la señalización mediada por prolactina en determinados núcleos cerebrales. En el segundo capítulo de esta tesis, se ha analizado los cambios producidos en los patrones basales de señalización mediada por prolactina en el cerebro femenino, como consecuencia de la maternidad, concretamente durante la gestación tardía y la lactancia. Los resultados de este capítulo indican que el cerebro femenino experimenta un incremento drástico en la señalización mediada por prolactina durante la gestación, indicativo de que esta hormona está ejerciendo una modulación a gran escala de la función cerebral en preparación a la maternidad. Este incremento afecta específicamente a las regiones cerebrales implicadas en la regulación del comportamiento social, lo que sugiere un papel clave de la prolactina en la inducción del comportamiento maternal. Además, se ha comprobado también como el input lactogénico incrementado hallado durante la gestación no es debido a prolactina de origen hipofisario, sino probablemente a lactógenos homólogos de origen placentario. Por último, este patrón incrementado persistirá con poca variación durante el periodo postparto, indicativo del mantenimiento de estos cambios o adaptaciones maternales inducidos por la prolactina durante el periodo postparto. En el capítulo final de esta tesis, se ha perseguido un objetivo doble. En primer lugar, se ha estudiado en profundidad el rasgo de la motivación maternal, en virtud del cual las crías adquieren carácter reforzante para la madre e impulsan la expresión de diversas conductas maternales proactivas. Para ello, se ha desarrollado y validado un nuevo test conductual para la medición específica de la motivación maternal, basado en la conducta de recogida de crías en el nido. Con ayuda de este test, se ha establecido que, si bien la exposición continua a estímulos derivados de crías puede promover un incremento parcial de motivación hacia las mismas en ratones hembra no maternales, la inducción plena de motivación maternal requiere de la acción de los agentes hormonales de la gestación, incluyendo probablemente a la prolactina. En segundo lugar, se ha estudiado también la interacción de la prolactina con el sistema neuroquímico de la oxitocina, nonapéptido con un papel esencial en la regulación del comportamiento maternal. Concretamente, se ha estudiado la acción de la prolactina sobre una población oxitocinérgica situada en una región clave en la integración del comportamiento maternal, la población del área preóptica dorsal/anterior comisural (AC/ADP). Si bien se ha hallado una acción incrementada de la prolactina sobre esta región durante el periodo postparto, este input estaría centrado no directamente en esta población oxitocinérgica, sino en neuronas no oxitocinérgicas del núcleo. Por último, se encontró correlaciones significativas entre el tamaño de la población de neuronas oxitocinérgicas en el AC/ADP y la expresión tanto de comportamiento maternal motivado como de agresión maternal (componente adicional del comportamiento maternal). El sentido de estas correlaciones sugiere que esta población oxitocinérgica estaría vinculada a la regulación del estrés y la ansiedad, ejerciendo a través de esta función un efecto indirecto sobre ambos componentes del comportamiento maternal. Los resultados obtenidos en esta tesis doctoral vinculan de manera sólida a la acción cerebral de la prolactina con la regulación del comportamiento maternal, sentando las bases para estudios posteriores que determinen con mayor exactitud el papel concreto de la prolactina en la regulación de este fenómeno conductual.Maternal behaviour subserves the function of protecting the offspring during the period of higher vulnerability, as well as to ensure its survival and welfare. This set of social behaviours will be expressed by the dam exclusively during the postpartum period, as a result of the maternal modulation of the neural substrate for the expression of social and reproductive behaviours. The agents responsible for this modulation include, among others endocrine signals of pregnancy and lactation. A relevant element among these endocrine signals is prolactin, adenohypophyseal hormone closely related to the physiological adaptation of the body to motherhood. The major aim of this work has been to characterise the actions of prolactin in the mouse brain, in general terms and specifically in the maternal context. The first chapter of this work has devoted to the description of the basic patterns of distribution of prolactin-derived signalling in the brain of non-parental female and male mice, as well as to evaluate how gonadal steroids contribute to shape these patterns. In this regard, we found a clear, female-biased sexual dimorphism in the extension and density of those patterns. However, the male mouse brain is indeed responsive to prolactin. In addition, estradiol and progesterone (in females) and testosterone (in males) all promote prolactin-derived signalling in specific brain regions. In the second chapter of this work, we have analysed the changes in the basal patterns of prolactin-derived signalling in the female mouse brain produced during motherhood, specifically during late pregnancy and lactation. Our findings indicate that the female mouse brain experiments a substantial increase in prolactin-derived signalling during pregnancy, which in turn suggests that this hormone is exerting an extensive modulation of brain function geared towards motherhood. This increase affects key areas in the regulation of sociosexual behaviour, which supports a central role for prolactin in the onset of maternal behaviour. Furthermore, we have also checked that this increased lactogenic input observed during pregnancy is not due to hypophyseal prolactin, but rather likely to placental lactogens, homologue lactogenic molecules secreted by the placenta during this period. Lastly, this increased prolactin signalling pattern will persist mostly unchanged during the postpartum period, which indicates that the maintenance of the major prolactin-induced maternal adaptations are likely maintained by this hormone during the postpartum period. In the last chapter of this work we have pursued a double aim. In the first place, we have studied the trait of maternal motivation, according to which pups and their derived stimuli acquire reinforcing properties for the dam and facilitate the expression of several proactive, pup-directed maternal behaviours. To this end, we have designed and validated a novel behavioural test to the specific assessment of maternal motivation, based on the maternal behaviour of pup retrieval. By means of this test, we have established that the complete and immediate acquisition of maternal motivation necessarily requires of the action of endocrine signals of motherhood, including most likely prolactin. However, prolonged exposition to pup-derived sensory cues among virgin female mice can also lead to a progressive and partial increase in motivation towards pups, in the context of a maternal sensitisation process. In the second part of this chapter, we have analysed the interaction of prolactin signalling onto oxytocin-secreting neuron populations. Oxytocin is a neuromodulatory nonapeptide with a key role in the regulation of maternal behaviour. Specifically, we will address the input of prolactin over a discrete oxytocinergic population located in the AC/ADP. This nucleus is located in the medial preoptic area of the hypothalamus, a key region in the expression of maternal behaviours. According to our results, we found an increased input of prolactin on the AC/ADP during postpartum period. However, this increased input would not be focused on the oxytocinergic population of the AC/ADP, but rather on other neurons of this nucleus. To conclude, we also found significant correlations associating AC/ADP oxutocin expression with motivated maternal behaviour and maternal aggression (a further hormone-dependent component of maternal behaviour). The direction of these correlations suggests that C/ADP oxytocinergic neurons might be involved in the downregulation of stress and anxiety during the maternal period, exerting an indirect and effect over the analysed components of maternal behaviour. The findings of this doctoral thesis consistently relate the central actions exerted by prolactin with the regulation of maternal behaviour in the mouse, which sets the foundations for future dedicated studies of the specific role of this hormone in the regulation of maternal behaviour

Tuning the brain for motherhood: prolactin-like central signalling in virgin, pregnant, and lactating female mice

Prolactin is fundamental for the expression of maternal behaviour. In virgin female rats, prolactin administered upon steroid hormone priming accelerates the onset of maternal care. By contrast, the role of prolactin in mice maternal behaviour remains unclear. This study aims at characterizing central prolactin activity patterns in female mice and their variation through pregnancy and lactation. This was revealed by immunoreactivity of phosphorylated (active) signal transducer and activator of transcription 5 (pSTAT5-ir), a key molecule in the signalling cascade of prolactin receptors. We also evaluated non-hypophyseal lactogenic activity during pregnancy by administering bromocriptine, which suppresses hypophyseal prolactin release. Late-pregnant and lactating females showed significantly increased pSTAT5-ir resulting in a widespread pattern of immunostaining with minor variations between pregnant and lactating animals, which comprises nuclei of the sociosexual and maternal brain, including telencephalic (septum, nucleus of the stria terminalis, and amygdala), hypothalamic (preoptic, paraventricular, supraoptic, and ventromedial), and midbrain (periaqueductal grey) regions. During late pregnancy, this pattern was not affected by the administration of bromocriptine, suggesting it to be elicited mostly by non-hypophyseal lactogenic agents, likely placental lactogens. Virgin females displayed, instead, a variable pattern of pSTAT5-ir restricted to a subset of the brain nuclei labelled in pregnant and lactating mice. A hormonal substitution experiment confirmed that estradiol and progesterone contribute to the variability found in virgin females. Our results reflect how the shaping of the maternal brain takes place prior to parturition and suggest that lactogenic agents are important candidates in the development of maternal behaviours already during pregnancy.This work has been funded by the Spanish MINECO-FEDER (BFU2013-47688-P), the Junta de Comunidades de Castilla-La Mancha/FEDER (PEIC11-0045-4490), and the Universitat Jaume I. This work is part of the Doctoral Thesis of Hugo Salais-López, granted by the FPU (Formación de Profesorado Universitario) programme of the Spanish Ministry of Education and Science

Madres, Madrinas y Vecinas. Neuroendocrinología del comportamiento maternal en el ratón.

La conducta maternal es algo más que la nutrición y protección de la descendencia. En humanos y otras especies, la desatención maternal provoca cambios epigenéticos que reducen la resistencia al estrés y perpetúan la desatención maternal. El estudio del sustrato neural de este comportamiento ayudará a romper este círculo vicioso y a promover la salud física y mental de futuras generaciones. Hemos investigado en el ratón cuáles son los factores clave que desencadenan la fiera defensa de las crías por parte de sus madres, la agresión maternal. Esta conducta debe estar promovida por señales hormonales de la gestación, puesto que las madrinas o comadres, hembras que han colaborado en el cuidado de las crías pero no han experimentado la maternidad, no son agresivas. Centrando nuestra atención sobre las hormonas de la maternidad, hemos caracterizado el efecto de la prolactina en el encéfalo. Los resultados indican que durante la gestación se produce una activación drástica, mediada por señales lactogénicas, de todo el cerebro sociosexual, incluyendo los núcleos responsables de la agresión. Esta activación continúa durante la lactancia, sustentada por los elevados niveles de prolactina propios de este periodo. Por último, hemos identificado las neuronas que generan circuitos centrales ricos en vasopresina y oxitocina, dos neuropéptidos clave en la regulación del comportamiento maternal. Neuronas que expresan ambos neuropéptidos, situadas entre la región amigdalina y el hipotálamo preóptico, parecen proyectar a centros responsables de la ansiedad (amígdala central) y la motivación (núcleo accumbens). Su modulación explicaría la reducción de la ansiedad durante la maternidad, que permite respuestas como la agresión maternal, así como el incremento de la motivación hacia las crías y su cuidado.Maternal behaviour is not limited to the nourishment and protection of the offspring. In humans as in other species, maternal neglect induces epigenetic modifications that compromise resilience to stress and perpetuate inattention. The study of the neural substrate of this behaviour will help to break this vicious cycle and to promote physical and mental health of future generations. We have explored which are the key factors, in the mouse, that trigger in the mother the fierce defence of the pups from external threats, maternal aggression. This behaviour must be promoted by the hormonal cues of the maternal period, since godmothers, females that shared pup care with the mother, but have not undergone motherhood, are not aggressive. Focusing on important hormonal signals during this period, we have characterised the central effect of prolactin, the maternal hormone. Our results indicate that, during pregnancy, lactogenic signals exert a drastic activation of the sociosexual brain, including centres responsible for aggression. These activation continues during lactation, hosted by the elevated levels of prolactin during this period. Lastly, we have identified the neurons generating central circuits rich in vasopressin and oxytocin, two neuropeptides with a key role in the regulation of maternal behaviour. Neurons expressing both neuropeptides and located between the amygdaloid region and preoptic hypothalamus, project to neural centres responsible for anxiety behaviour (central tonsil) and motivation (nucleus accumbens). The modulation of these sites would explain the reduction of anxiety during the motherhood, which ensures behaviours like maternal aggression, as well as the increase in pup-directed motivated behaviours

Maternal Motivation: Exploring the Roles of Prolactin and Pup Stimuli

Motherhood entails increased motivation for pups, which become strong reinforcers and guide maternal behaviours. This depends on steroids and lactogens acting on the brain of females during pregnancy and postpartum. Since virgin female mice exposed to pups are nearly spontaneously maternal, the specific roles of endocrine and pup-derived signals in the induction of maternal motivation remain unclear. This work investigates maternal motivation in dams and virgin female mice, using a novel variant of the pup retrieval paradigm, the motivated pup retrieval test. We also analyse the role of prolactin (PRL) and of stimuli derived from a litter of pups and its mother, in the acquisition of maternal motivation. Experimental design included female mice in 3 conditions: lactating dams, comothers (virgins housed and sharing pup care with dams) and pup-naïve virgins. Females underwent 3 motivated-pup-retrieval trials, with pups displaced behind a 10-cm-high wire-mesh barrier. Dams retrieved with significantly lower latencies than comothers or virgins, indicating that full maternal motivation appears only after pregnancy. Although initially comothers and virgins showed no retrieval, comothers significantly improved throughout the experiment, suggesting an induced sensitization process. Lengthening exposure of comothers to the dyad pups-dam (from 2 to 5 days at the beginning of testing) had no strong effects on maternal sensitization. PRL responsiveness was analysed in these animals using immunohistochemical detection of phosphorylated signal transducer and activator of transcription 5 (pSTAT5, PRL-derived signalling marker). As expected, dams showed significantly higher pSTAT5 expression in most of the analysed nuclei. Moreover, comothers displayed significantly higher PRL responsiveness than pup-naïve virgins in the medial preoptic nucleus, even if they display similar circulating PRL levels, which are significantly lower than those of dams. Given the instrumental role of this nucleus in the relay and integration of pup-derived stimuli to facilitate proactive maternal responses, this increase in PRL responsiveness likely reflects the mechanism underlying the maternal sensitization process reported in this work. Since the analyses of maternal motivation and PRL signalling in the brain were performed in the same animals, we were able to explore correlation between both set of data. The results shed light on the neuroendocrine mechanisms underlying maternal motivation and other aspects of maternal behaviour

Influencia del estrés en la conducta maternal en hembras de ratón

El comportamiento maternal asegura la supervivencia de la descendencia hasta alcanzar la madurez sexual. Se trata de una conducta regulada por el cerebro socio-sexual y diferentes factores (como el estrés) pueden alterar los núcleos neuronales que rigen su funcionamiento. Así, el objetivo del presente trabajo será revisar la bibliografía existente hasta la fecha sobre el efecto que producen diferentes tipos de estrés en la posterior conducta maternal en hembras de ratón, seleccionando principalmente por su importancia dos tipos de conductas: la motivación maternal y la agresión maternal (es decir, tanto conductas dirigidas como no dirigidas a las crías). Para ello, primero describiremos las diferentes metodologías utilizadas para estudiar ambos tipos de conductas y, después, mostraremos los principales estudios que muestran el efecto de diferentes tipos de estrés en dicha conducta. Los resultados observados indican que el estrés modifica la conducta maternal ya que se han observado cambios en motivación y agresión. Sin embargo, algunos datos son ambiguos y varían en función del tipo de estrés o período gestacional/postparto en el que se sufre. A pesar de que más estudios son necesarios en este campo, este trabajo contribuirá al avance del concomiendo en patologías asociadas al estrés y la maternidad.Maternal behaviour ensures offspring’s survival, until the progeny arrives to sexual maturity. This behavior is regulated by the socio-sexual brain and different factors (such as stress) can alter the neural cores that control its performance. The objective of the present study is to revise the existing bibliography till the date about the effect that different types of stress produce in the future maternal behavior on female mice, selecting by the relevance two types of behaviour: maternal motivation and maternal aggression (behavior directed and non-directed towards pups). First, we will describe the different methodologies used to study both kind of behavior, then we will present the principal studies that shows the effect of different types of stress on behavior. The observed results suggest that stress modifies maternal behavior, because stress changes maternal motivation and maternal aggression. However, some data are ambiguous and vary according to the type of stress or gestational/postpartum period. Although more studies are needed in this field, this work will contribute to the advancement of knowledge in pathologies associated with stress and motherhood

The maternal hormone in the male brain: Sexually dimorphic distribution of prolactin signalling in the mouse brain.

Research of the central actions of prolactin is highly focused on females, but this hormone has also documented roles in male physiology and behaviour. Here, we provide the first description of the pattern of prolactin-derived signalling in the male mouse brain, employing the immunostaining of phosphorylated signal transducer and activator of transcription 5 (pSTAT5) after exogenous prolactin administration. Next, we explore possible sexually dimorphic differences by comparing pSTAT5 immunoreactivity in prolactin-supplemented males and females. We also assess the role of testosterone in the regulation of central prolactin signalling in males by comparing intact with castrated prolactin-supplemented males. Prolactin-supplemented males displayed a widespread pattern of pSTAT5 immunoreactivity, restricted to brain centres showing expression of the prolactin receptor. Immunoreactivity for pSTAT5 was present in several nuclei of the preoptic, anterior and tuberal hypothalamus, as well as in the septofimbrial nucleus or posterodorsal medial amygdala of the telencephalon. Conversely, non-supplemented control males were virtually devoid of pSTAT5-immunoreactivity, suggesting that central prolactin actions in males are limited to situations concurrent with substantial hypophyseal prolactin release (e.g. stress or mating). Furthermore, comparison of prolactin-supplemented males and females revealed a significant, female-biased sexual dimorphism, supporting the view that prolactin has a preeminent role in female physiology and behaviour. Finally, in males, castration significantly reduced pSTAT5 immunoreactivity in some structures, including the paraventricular and ventromedial hypothalamic nuclei and the septofimbrial region, thus indicating a region-specific regulatory role of testosterone over central prolactin signalling