Adjusting and validating a procedure for parenteral anaesthesia in neonatal mice.

For neonatal pups, parenteral anaesthesia is said to be not reliable as low doses induce no anaesthesia whereas high doses render high mortality rates. In this work we have adapted parenteral anaesthesia procedures approved for pups >7 days of age, to anaesthetize neonatal animals (postnatal days 3-4; P3-P4) for keeping them immobile for a long period. In our first experiment we analysed the behaviour of P3-P4 mouse pups for 70 min after intraperitoneal administration of low (37.5/3.75 mg/kg) or high (50/5) doses of a ketamine/xylazine anaesthetic mixture, both in the low range as compared with dosages employed in adults. Pups became immobile in ≈7 min and remained immobile for ≈45 min, irrespective of the age and dose of anaesthesia, younger pups (P3) being apparently more sensitive to the dosage. In the second experiment, we studied the response of P3 pups to mildly nociceptive stimulations, performed with a 4.0 g von Frey filament applied to the dorsal aspect of their paws. These stimuli elicited reaction in 100% of the cases in non-anaesthetized pups. The results indicate that the high dose significantly reduced responses as compared with the low dose of anaesthesia. With the low dose, <40% of the pups were unresponsive to nociceptive stimulation, whereas the high dose resulted in 50-60% of the animals not responding. Mortality was low irrespective of age or dose, suggesting that doses can be further increased if needed for invasive experimental procedures.

Untargeted metabolomics approach using UHPLC-IMS-QTOF MS for surface body samples to identify low-volatility chemosignals related to maternal care in mice.

The present study is focused on the determination of low-volatile chemosignals excreted or secreted by mouse pups in their early days of life involved in maternal care induction in mice adult females. Untargeted metabolomics was employed to differentiate between samples collected with swabs from facial and anogenital area from neonatal mouse pups receiving maternal care (first two weeks of life) and the elder mouse pups in the weaning period (4th week old). The sample extracts were analysed by ultra-high pressure liquid chromatography (UHPLC) coupled to ion mobility separation (IMS) in combination with high resolution mass spectrometry (HRMS). After data processing with Progenesis QI and multivariate statistical analysis, five markers present in the first two weeks of mouse pups life and putatively involved in materno-filial chemical communication were tentatively identified: arginine, urocanic acid, erythro-sphingosine (d17:1), sphingosine (d18:1) and sphinganine. The four-dimensional data and the tools associated to the additional structural descriptor obtained by IMS separation were of great help in the compound identification. The results demonstrated the great potential of UHPLC-IMS-HRMS based untargeted metabolomics to identity putative pheromones in mammals.

Inhibition of the medial amygdala disrupts escalated aggression in lactating female mice after repeated exposure to male intruders.

Virgin female laboratory mice readily express pup care when co-housed with dams and pups. However, pup-sensitized virgins fail to express intruder-directed aggression on a single session of testing. To study whether repeated testing would affect the onset and dynamics of maternal or intruder-directed aggression, we tested dams and their accompanying virgins from postpartum day 4 to 6. Repeated testing led to escalated aggression towards male intruders in dams, but virgins never developed aggression. In dams, inhibition of the medial amygdala using DREADD (designer receptors exclusively activated by designer drugs) vectors carrying the hM4Di receptor blocked the expected increase in maternal aggression on the second testing day. Our data support that the onset of maternal aggression is linked to physiological changes occurring during motherhood, and that medial amygdala, a key centre integrating vomeronasal, olfactory and hormonal information, enables the expression of escalated aggression induced by repeated testing. Future studies selectively targeting specific neuronal populations of the medial amygdala are needed to allow a deeper understanding of the control of experience-dependent aggression increase, a phenomenon leading to the high aggression levels found in violent behaviours.

Neural activity patterns in the chemosensory network encoding vomeronasal and olfactory information in mice.

Rodents detect chemical information mainly through the olfactory and vomeronasal systems, which play complementary roles to orchestrate appropriate behavioral responses. To characterize the integration of chemosensory information, we have performed electrophysiological and c-Fos studies of the bulbo-amygdalar network in freely behaving female mice exploring neutral or conspecific stimuli. We hypothesize that processing conspecifics stimuli requires both chemosensory systems, and thus our results will show shared patterns of activity in olfactory and vomeronasal structures. Were the hypothesis not true, the activity of the vomeronasal structures would be independent of that of the main olfactory system. In the c-Fos analysis, we assessed the activation elicited by neutral olfactory or male stimuli in a broader network. Male urine induced a significantly higher activity in the vomeronasal system compared to that induced by a neutral odorant. Concerning the olfactory system, only the cortex-amygdala transition area showed significant activation. No differential c-Fos expression was found in the reward system and the basolateral amygdala. These functional patterns in the chemosensory circuitry reveal a strong top-down control of the amygdala over both olfactory bulbs, suggesting an active role of the amygdala in the integration of chemosensory information directing the activity of the bulbs during environmental exploration.

Becoming a mother shifts the activity of the social and motivation brain networks in mice.

During pregnancy hormones increase motivated pup-directed behaviors. We here analyze hormone-induced changes in brain activity, by comparing cFos-immunoreactivity in the sociosexual (SBN) and motivation brain networks (including medial preoptic area, MPO) of virgin versus late-pregnant pup-naïve female mice exposed to pups or buttons (control). Pups activate more the SBN than buttons in both late-pregnant and virgin females. By contrast, pregnancy increases pup-elicited activity in the motivation circuitry (e.g. accumbens core) but reduces button-induced activity and, consequently, button investigation. Principal components analysis supports the identity of the social and motivation brain circuits, placing the periaqueductal gray between both systems. Linear discriminant analysis of cFos-immunoreactivity in the socio-motivational brain network predicts the kind of female and stimulus better than the activity of the MPO alone; this suggests that the neuroendocrinological basis of social (e.g. maternal) behaviors conforms to a neural network model, rather than to distinct hierarchical linear pathways for different behaviors.

Novel sampling strategy for alive animal volatolome extraction combined with GC-MS based untargeted metabolomics: Identifying mouse pup pheromones.

In this study, we identify 11 mouse pup volatiles putatively involved in maternal care induction in adult females. For this purpose, we have adapted the dynamic headspace methodology to extract the volatolome of whole alive animals. Untargeted metabolomic methodology was used to compare the volatolome of neonatal (4-6 days) with elder pups until the age of weaning (21-23 days old). Pup volatolome was analyzed by gas chromatography (GC) coupled to single quadrupole mass spectrometry (MS) using automated thermal desorption for sample introduction. After data processing and multivariate statistical analysis, comparison with NIST spectral library allowed identifying compounds secreted preferentially by neonatal pups: di(propylen glycol) methyl ether, 4-nonenal, di(ethylene glycol) monobutyl ether, 2-phenoxyethanol, isomethyl ionone, tridecanal, 1,3-diethylbenzene, 1,2,4,5-tetramethylbenzene, 2-ethyl-p-xylene and tri(propylene glycol) methyl ether. Palmitic acid was enriched in the volatolome of fourth week youngsters compared to neonatal pups. The results demonstrated the great potential of the new sampling procedure combined with GC-MS based untargeted volatolomics to identify volatile pheromones in mammals.

Motherhood-induced gene expression in the mouse medial amygdala: Changes induced by pregnancy and lactation but not by pup stimuli.

During lactation, adult female mice display aggressive responses toward male intruders, triggered by male-derived chemosensory signals. This aggressive behavior is not shown by pup-sensitized virgin females sharing pup care with dams. The genetic mechanisms underlying the switch from attraction to aggression are unknown. In this work, we investigate the differential gene expression in lactating females expressing maternal aggression compared to pup-sensitized virgin females in the medial amygdala (Me), a key neural structure integrating chemosensory and hormonal information. The results showed 197 genes upregulated in dams, including genes encoding hormones such as prolactin, growth hormone, or follicle-stimulating hormone, neuropeptides such as galanin, oxytocin, and pro-opiomelanocortin, and genes related to catecholaminergic and cholinergic neurotransmission. In contrast, 99 genes were downregulated in dams, among which we find those encoding for inhibins and transcription factors of the Fos and early growth response families. The gene set analysis revealed numerous Gene Ontology functional groups with higher expression in dams than in pup-sensitized virgin females, including those related with the regulation of the Jak/Stat cascade. Of note, a number of olfactory and vomeronasal receptor genes was expressed in the Me, although without differences between dams and virgins. For prolactin and growth hormone, a qPCR experiment comparing dams, pup-sensitized, and pup-naïve virgin females showed that dams expressed higher levels of both hormones than pup-naïve virgins, with pup-sensitized virgins showing intermediate levels. Altogether, the results show important gene expression changes in the Me, which may underlie some of the behavioral responses characterizing maternal behavior.

Corrigendum: Wired for motherhood: induction of maternal care but not maternal aggression in virgin female CD1 mice.

[This corrects the article DOI: 10.3389/fnbeh.2015.00197.].

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.

Male-specific features are reduced in Mecp2-null mice: analyses of vasopressinergic innervation, pheromone production and social behaviour.

Deficits in arginine vasopressin (AVP) and oxytocin (OT), two neuropeptides closely implicated in the modulation of social behaviours, have been reported in some early developmental disorders and autism spectrum disorders. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene are associated to Rett syndrome and other neuropsychiatric conditions. Thus, we first analysed AVP and OT expression in the brain of Mecp2-mutant mice by immunohistochemistry. Our results revealed no significant differences in these systems in young adult Mecp2-heterozygous females, as compared to WT littermates. By contrast, we found a significant reduction in the sexually dimorphic, testosterone-dependent, vasopressinergic innervation in several nuclei of the social brain network and oxytocinergic innervation in the lateral habenula of Mecp2-null males, as compared to WT littermates. Analysis of urinary production of pheromones shows that Mecp2-null males lack the testosterone-dependent pheromone darcin, strongly suggesting low levels of androgens in these males. In addition, resident-intruder tests revealed lack of aggressive behaviour in Mecp2-null males and decreased chemoinvestigation of the intruder. By contrast, Mecp2-null males exhibited enhanced social approach, as compared to WT animals, in a 3-chamber social interaction test. In summary, Mecp2-null males, which display internal testicles, display a significant reduction of some male-specific features, such as vasopressinergic innervation within the social brain network, male pheromone production and aggressive behaviour. Thus, atypical social behaviours in Mecp2-null males may be caused, at least in part, by the effect of lack of MeCP2 over sexual differentiation.

Pregnancy Changes the Response of the Vomeronasal and Olfactory Systems to Pups in Mice.

Motherhood entails changes in behavior with increased motivation for pups, induced in part by pregnancy hormones acting upon the brain. This work explores whether this alters sensory processing of pup-derived chemosignals. To do so, we analyse the expression of immediate early genes (IEGs) in the vomeronasal organ (VNO; Egr1) and centers of the olfactory and vomeronasal brain pathways (cFos) in virgin and late-pregnant females exposed to pups, as compared to buttons (socially neutral control). In pup-exposed females, we quantified diverse behaviors including pup retrieval, sniffing, pup-directed attack, nest building and time in nest or on nest, as well as time off nest. Pups induce Egr1 expression in the VNO of females, irrespective of their physiological condition, thus suggesting the existence of VNO-detected pup chemosignals. A similar situation is found in the accessory olfactory bulb (AOB) and posteromedial part of the medial bed nucleus of the stria terminalis (BSTMPM). By contrast, in the medial amygdala and posteromedial cortical amygdala (PMCo), responses to pups-vs-buttons are different in virgin and late-pregnant females, thus suggesting altered sensory processing during late pregnancy. The olfactory system also shows changes in sensory processing with pregnancy. In the main olfactory bulbs, as well as the anterior and posterior piriform cortex, buttons activate cFos expression in virgins more than in pregnant females. By contrast, in the anterior and especially posterior piriform cortex, pregnant females show more activation by pups than buttons. Correlation between IEGs expression and behavior suggests the existence of two vomeronasal subsystems: one associated to pup care (with PMCo as its main center) and another related to pup-directed aggression observed in some pregnant females (with the BSTMPM as the main nucleus). Our data also suggest a coactivation of the olfactory and vomeronasal systems during interaction with pups in pregnant females.

Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system.

The protein doublecortin is mainly expressed in migrating neuroblasts and immature neurons. The X-linked gene MECP2, associated to several neurodevelopmental disorders such as Rett syndrome, encodes the protein methyl-CpG-binding protein 2 (MeCP2), a regulatory protein that has been implicated in neuronal maturation and refinement of olfactory circuits. Here, we explored doublecortin immunoreactivity in the brain of young adult female Mecp2-heterozygous and male Mecp2-null mice and their wild-type littermates. The distribution of doublecortin-immunoreactive somata in neurogenic brain regions was consistent with previous reports in rodents, and no qualitative differences were found between genotypes or sexes. Quantitatively, we found a significant increase in doublecortin cell density in the piriform cortex of Mecp2-null males as compared to WT littermates. A similar increase was seen in a newly identified population of doublecortin cells in the olfactory tubercle. In these olfactory structures, however, the percentage of doublecortin immature neurons that also expressed NeuN was not different between genotypes. By contrast, we found no significant differences between genotypes in doublecortin immunoreactivity in the olfactory bulbs. Nonetheless, in the periglomerular layer of Mecp2-null males, we observed a specific decrease of immature neurons co-expressing doublecortin and NeuN. Overall, no differences were evident between Mecp2-heterozygous and WT females. In addition, no differences could be detected between genotypes in the density of doublecortin-immunoreactive cells in the hippocampus or striatum of either males or females. Our results suggest that MeCP2 is involved in neuronal maturation in a region-dependent manner.

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.

Synchronized Activity in The Main and Accessory Olfactory Bulbs and Vomeronasal Amygdala Elicited by Chemical Signals in Freely Behaving Mice.

Chemosensory processing in mammals involves the olfactory and vomeronasal systems, but how the activity of both circuits is integrated is unknown. In our study, we recorded the electrophysiological activity in the olfactory bulbs and the vomeronasal amygdala in freely behaving mice exploring a battery of neutral and conspecific stimuli. The exploration of stimuli, including a neutral stimulus, induced synchronic activity in the olfactory bulbs characterized by a dominant theta rhythmicity, with specific theta-gamma coupling, distinguishing between vomeronasal and olfactory structures. The correlated activation of the bulbs suggests a coupling between the stimuli internalization in the nasal cavity and the vomeronasal pumping. In the amygdala, male stimuli are preferentially processed in the medial nucleus, whereas female cues induced a differential response in the posteromedial cortical amygdala. Thus, particular theta-gamma patterns in the olfactory network modulates the integration of chemosensory information in the amygdala, allowing the selection of an appropriate behaviour.

Afferent and efferent projections of the anterior cortical amygdaloid nucleus in the mouse.

The anterior cortical amygdaloid nucleus (ACo) is a chemosensory area of the cortical amygdala that receives afferent projections from both the main and accessory olfactory bulbs. The role of this structure is unknown, partially due to a lack of knowledge of its connectivity. In this work, we describe the pattern of afferent and efferent projections of the ACo by using fluorogold and biotinylated dextranamines as retrograde and anterograde tracers, respectively. The results show that the ACo is reciprocally connected with the olfactory system and basal forebrain, as well as with the chemosensory and basomedial amygdala. In addition, it receives dense projections from the midline and posterior intralaminar thalamus, and moderate projections from the posterior bed nucleus of the stria terminalis, mesocortical structures and the hippocampal formation. Remarkably, the ACo projects moderately to the central nuclei of the amygdala and anterior bed nucleus of the stria terminalis, and densely to the lateral hypothalamus. Finally, minor connections are present with some midbrain and brainstem structures. The afferent projections of the ACo indicate that this nucleus might play a role in emotional learning involving chemosensory stimuli, such as olfactory fear conditioning. The efferent projections confirm this view and, given its direct output to the medial part of the central amygdala and the hypothalamic 'aggression area', suggest that the ACo can initiate defensive and aggressive responses elicited by olfactory or, to a lesser extent, vomeronasal stimuli.

Glutamate and Opioid Antagonists Modulate Dopamine Levels Evoked by Innately Attractive Male Chemosignals in the Nucleus Accumbens of Female Rats.

Sexual chemosignals detected by vomeronasal and olfactory systems mediate intersexual attraction in rodents, and act as a natural reinforcer to them. The mesolimbic pathway processes natural rewards, and the nucleus accumbens receives olfactory information via glutamatergic projections from the amygdala. Thus, the aim of this study was to investigate the involvement of the mesolimbic pathway in the attraction toward sexual chemosignals. Our data show that female rats with no previous experience with males or their chemosignals display an innate preference for male-soiled bedding. Focal administration of the opioid antagonist ß-funaltrexamine into the posterior ventral tegmental area does not affect preference for male chemosignals. Nevertheless, exposure to male-soiled bedding elicits an increase in dopamine efflux in the nucleus accumbens shell and core, measured by microdialysis. Infusion of the opioid antagonist naltrexone in the accumbens core does not significantly affect dopamine efflux during exposure to male chemosignals, although it enhances dopamine levels 40 min after withdrawal of the stimuli. By contrast, infusion of the glutamate antagonist kynurenic acid in the accumbens shell inhibits the release of dopamine and reduces the time that females spend investigating male-soiled bedding. These data are in agreement with previous reports in male rats showing that exposure to opposite-sex odors elicits dopamine release in the accumbens, and with data in female mice showing that the behavioral preference for male chemosignals is not affected by opioidergic antagonists. We hypothesize that glutamatergic projections from the amygdala into the accumbens might be important to modulate the neurochemical and behavioral responses elicited by sexual chemosignals in rats.

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.

Afferent and Efferent Connections of the Cortex-Amygdala Transition Zone in Mice.

The transitional zone between the ventral part of the piriform cortex and the anterior cortical nucleus of the amygdala, named the cortex-amygdala transition zone (CxA), shows two differential features that allow its identification as a particular structure. First, it receives dense cholinergic and dopaminergic innervations as compared to the adjacent piriform cortex and amygdala, and second, it receives projections from the main and accessory olfactory bulbs. In this work we have studied the pattern of afferent and efferent projections of the CxA, which are mainly unknown, by using the retrograde tracer Fluorogold and the anterograde tracer biotinylated dextranamine. The results show that the CxA receives a relatively restricted set of intratelencephalic connections, originated mainly by the olfactory system and basal forebrain, with minor afferents from the amygdala. The only relevant extratelencephalic afference originates in the ventral tegmental area (VTA). The efferent projections of the CxA reciprocate the inputs from the piriform cortex and olfactory amygdala. In addition, the CxA projects densely to the basolateral amygdaloid nucleus and the olfactory tubercle. The extratelencephalic projections of the CxA are very scarce, and target mainly hypothalamic structures. The pattern of connections of the CxA suggests that it is indeed a transitional area between the piriform cortex and the cortical amygdala. Double labeling with choline acetyltransferase indicates that the afferent projection from the basal forebrain is the origin of its distinctive cholinergic innervation, and double labeling with dopamine transporter shows that the projection from the VTA is the source of dopaminergic innervation. These connectivity and neurochemical features, together with the fact that it receives vomeronasal in addition to olfactory information, suggest that the CxA may be involved in processing olfactory information endowed with relevant biological meaning, such as odors related to reproductive or defensive behaviors.

Afferent projections to the different medial amygdala subdivisions: a retrograde tracing study in the mouse.

The medial amygdaloid nucleus (Me) is a key node in the socio-sexual brain, composed of anterior (MeA), posteroventral (MePV) and posterodorsal (MePD) subdivisions. These subdivisions have been suggested to play a different role in reproductive and defensive behaviours. In the present work we analyse the afferents of the three Me subdivisions using restricted injections of fluorogold in female outbred CD1 mice. The results reveal that the MeA, MePV and MePD share a common pattern of afferents, with some differences in the density of retrograde labelling in several nuclei. Common afferents to Me subdivisions include: the accessory olfactory bulbs, piriform cortex and endopiriform nucleus, chemosensory amygdala (receiving direct inputs from the olfactory bulbs), posterior part of the medial bed nucleus of the stria terminalis (BSTM), CA1 in the ventral hippocampus and posterior intralaminar thalamus. Minor projections originate from the basolateral amygdala and amygdalo-hippocampal area, septum, ventral striatum, several allocortical and periallocortical areas, claustrum, several hypothalamic structures, raphe and parabrachial complex. MeA and MePV share minor inputs from the frontal cortex (medial orbital, prelimbic, infralimbic and dorsal peduncular cortices), but differ in the lack of main olfactory projections to the MePV. By contrast, the MePD receives preferential projections from the rostral accessory olfactory bulb, the posteromedial BSTM and the ventral premammillary nucleus. In summary, the common pattern of afferents to the Me subdivisions and their interconnections suggest that they play cooperative instead of differential roles in the various behaviours (e.g., sociosexual, defensive) in which the Me has been shown to be involved.

Distribution of oxytocin and co-localization with arginine vasopressin in the brain of mice.

Oxytocin (OT) and vasopressin (AVP) play a major role in social behaviours. Mice have become the species of choice for neurobiology of social behaviour due to identification of mouse pheromones and the advantage of genetically modified mice. However, neuroanatomical data on nonapeptidergic systems in mice are fragmentary, especially concerning the central distribution of OT. Therefore, we analyse the immunoreactivity for OT and its neurophysin in the brain of male and female mice (strain CD1). Further, we combine immunofluorescent detection of OT and AVP to locate cells co-expressing both peptides and their putative axonal processes. The results indicate that OT is present in cells of the neurosecretory paraventricular (Pa) and supraoptic hypothalamic nuclei (SON). From the anterior SON, OTergic cells extend into the medial amygdala, where a sparse cell population occupies its ventral anterior and posterior divisions. Co-expression of OT and AVP in these nuclei is rare. Moreover, a remarkable OTergic cell group is found near the ventral bed nucleus of the stria terminalis (BST), distributed between the anterodorsal preoptic nucleus and the nucleus of anterior commissure (ADP/AC). This cell group, the rostral edge of the Pa and the periventricular hypothalamus display frequent OT + AVP double labelling, with a general dominance of OT over AVP immunoreactivity. Fibres with similar immunoreactivity profile innervate the accumbens shell and core, central amygdala and portions of the intervening BST. These data, together with data in the literature on rats, suggest that the projections of ADP/AC nonapeptidergic cells onto these brain centres could promote pup-motivated behaviours and inhibit pup avoidance during motherhood.