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In mammalian females, both adolescence and motherhood are critical periods modulating behavioural changes to favour evolutionary survival. In mice, olfaction is a key driver of social behaviours, and adult neurogenesis in the olfactory bulb is an important form of neural plasticity underlying adequate behavioural responses towards conspecifics. In this study, we sought to investigate whether transition to adulthood and motherhood would affect the populations of immature neurons described in other areas of the olfactory system. To do so, we analysed the expression of doublecortin (DCX), a marker of immature neurons, and Ki67, a marker of proliferation, in the olfactory tubercle and piriform cortex of pre-pubertal and adult CD1 female mice. Our results revealed sustained proliferation of neuroblasts in the olfactory tubercle in both pre-pubertal and adult females, with Ki67 nuclei located in close apposition to clusters of DCX-immunoreactive cells. However, the density of DCX-immunoreactive cells in this area was not affected by either puberty, pregnancy, or lactation. In contrast, lactating females had significantly less DCX-immunoreactive cells than pup-sensitised virgin females at the piriform cortex, with a similar non-significant tendency observed in the group of pregnancy alone. This suggests that lactation can act on top of pregnancy to promote the maturation of these embryonically generated DCX-immunoreactive neurons at the piriform cortex. Our results expand our current knowledge on how motherhood remodels the brain and reveal the olfactory tubercle as an unexplored niche for adult neurogenesis.

Achieving precise control over quantum systems has groundbreaking ap-plications, such as simulating complex quantum systems, or implementing quantum algorithms with an improved efficiency over classical algorithms. Quantum computing has been under development for the last decades, re-sulting in the so-called Noisy Intermediate-Scale Quantum (NISQ) devices with about 50 qubits. While these devices are built with the purpose of achieving practical advantages over classical processors, they are vulnerable to noise sources. In order to account for error sources, one needs to im-plement Quantum Error Correction (QEC) techniques. These techniques protect quantum information by encoding a logical qubit into several phys-ical ones. For the implementation of NISQ devices and QEC techniques, it is vital to keep improving the quantum operations and states involved. Here we develop calibration protocols based on the application of Bayesian inference. We show their advantages compared to the more traditional frequentist approaches, such as their ability to maximise the information gained with each measurement, or their straightforward automation. In our first line of research, we develop a Bayesian protocol for the correction of unwanted phases appearing in the experimental preparation of Steane code states. This protocol requires 13 times less measurements than the frequentist approach. In our second line of research, we develop a proto-col for locking a laser to the qubit transition frequency in a trapped-ion architecture. This calibration is vital, since the correct implementation of single-qubit gates depends on it. In our final line of research, we study the Mølmer-Sørensen entangling gate, used in trapped-ion systems. First we introduce the calibration parameters, and derive a semianalytical model of the effects of one of them, for which there was no previous analytical understanding. Finally, we develop a Bayesian protocol for the calibration of these parameters, which was successfully implemented and tested in an experimental trapped-ion quantum processor.

A major challenge for the realization of useful universal quantum computers is achieving high fidelity two-qubit entangling gate operations. However, calibration errors can affect the quantum gate operations and limit their fidelity. To reduce such errors it is desirable to have an analytical understanding and quantitative predictions of the effects that miscalibrations of gate parameters have on the gate performance. In this work, we study a systematic perturbative expansion in miscalibrated parameters of the Mølmer-Sørensen entangling gate, which is widely used in trapped-ion quantum processors. Our analytical treatment particularly focuses on systematic center-line detuning miscalibrations. Via a unitary Magnus expansion, we compute the gate evolution operator, which allows us to obtain relevant key properties such as relative phases, electronic populations, quantum state purity and fidelities. These quantities, subsequently, are used to assess the performance of the gate using the fidelity of entangled states as performance metric. We verify the predictions from our model by benchmarking them against measurements in a trapped-ion quantum processor. The method and the results presented here can help design and calibrate high-fidelity gate operations of large-scale quantum computers.

Contains fulltext : 286841.pdf (Publisher’s version ) (Closed access) BACKGROUND: Resistant hypertension is associated with increased cardiovascular risk. The endothelin pathway has been implicated in the pathogenesis of hypertension, but it is currently not targeted therapeutically, thereby leaving this relevant pathophysiological pathway unopposed with currently available drugs. The aim of the study was to assess the blood pressure lowering efficacy of the dual endothelin antagonist aprocitentan in patients with resistant hypertension. METHODS: PRECISION was a multicentre, blinded, randomised, parallel-group, phase 3 study, which was done in hospitals or research centres in Europe, North America, Asia, and Australia. Patients were eligible for randomisation if their sitting systolic blood pressure was 140 mm Hg or higher despite taking standardised background therapy consisting of three antihypertensive drugs, including a diuretic. The study consisted of three sequential parts: part 1 was the 4-week double-blind, randomised, and placebo-controlled part, in which patients received aprocitentan 12·5 mg, aprocitentan 25 mg, or placebo in a 1:1:1 ratio; part 2 was a 32-week single (patient)-blind part, in which all patients received aprocitentan 25 mg; and part 3 was a 12-week double-blind, randomised, and placebo-controlled withdrawal part, in which patients were re-randomised to aprocitentan 25 mg or placebo in a 1:1 ratio. The primary and key secondary endpoints were changes in unattended office systolic blood pressure from baseline to week 4 and from withdrawal baseline to week 40, respectively. Secondary endpoints included 24-h ambulatory blood pressure changes. The study is registered on ClinicalTrials.gov, NCT03541174. FINDINGS: The PRECISION study was done from June 18, 2018, to April 25, 2022. 1965 individuals were screened and 730 were randomly assigned. Of these 730 patients, 704 (96%) completed part 1 of the study; of these, 613 (87%) completed part 2 and, of these, 577 (94%) completed part 3 of the study. The least square mean (SE) change in office systolic blood pressure at 4 weeks was -15·3 (SE 0·9) mm Hg for aprocitentan 12·5 mg, -15·2 (0·9) mm Hg for aprocitentan 25 mg, and -11·5 (0·9) mm Hg for placebo, for a difference versus placebo of -3·8 (1·3) mm Hg (97·5% CI -6·8 to -0·8, p=0·0042) and -3·7 (1·3) mm Hg (-6·7 to -0·8; p=0·0046), respectively. The respective difference for 24 h ambulatory systolic blood pressure was -4·2 mm Hg (95% CI -6·2 to -2·1) and -5·9 mm Hg (-7·9 to -3·8). After 4 weeks of withdrawal, office systolic blood pressure significantly increased with placebo versus aprocitentan (5·8 mm Hg, 95% CI 3·7 to 7·9, p

During lactation, adult female mice display aggressive responses toward male intrud-ers, 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 ex-pressing maternal aggression compared to pup- sensitized virgin females in the me-dial 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 hor-mone, 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 expres-sion 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 lev-els 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 ma-ternal behavior.

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. Funding for open access charge: CRUE-Universitat Jaume I

This is a pre-print of an article published in Brain Structure and Function. The final authenticated version is available online at: https://doi.org/10.1007/s00429-019-01860-6 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.

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.

Evolution selects those adaptive features that increase reproductive probabilities and facilitate survival. Analysing the brain circuits mediating risk-avoidance (e.g. defense) and those allowing reward-seeking (motivated) behaviours in different vertebrates leads to several main conclusions. First, circuits mediating risk-avoidance are similar in all studied vertebrates, where they include amygdala homologues located in the posterior half of the cerebral hemispheres, in close relationship with the chemosensory systems. Second, in all vertebrates, reward-seeking behaviours involve the activity of tegmento-striatal dopaminergic pathways, plus other inputs to the ventral striatum, including amygdalo-striatal glutamatergic projections. Third, output structures in these forebrain circuits for both risk-avoidance and reward-seeking behaviours occupy the caudal and rostral poles of the ventral striato-pallidum, namely the central amygdala and nucleus accumbens-olfactory tubercle respectively. This brain configuration was already present in at least the ancestral amniote, likely also in anamniotes. Finally, social behaviours (sexual, agonistic-territorial, parental) are fundamental for reproduction and survival. Consequently, the so-called socio-sexual brain network that governs these conducts is closely related with brain centres mediating motivation (maybe also risk-avoidance). Central nonapeptidergic circuits are apparently required for endowing social stimuli with rewarding (attractive) properties. More studies in non-mammals are required to further test and expand these ideas.

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.

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.

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.

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.

As server densities increase to support the rising demand for high density computing, traditional room cooling infrastructure is struggling to keep up. Rack-mounted cooling units are increasingly being deployed in data centers. These cooling units are located closer to the heat sources (the servers) which allow them to cool more efficiently than traditional cooling infrastructure. In this work, a Multi-Input Single-Output (MISO) Adaptive Predictive Controller (APC) for rack-mounted cooling units is investigated and implemented using a low-cost general-purpose microcontroller. The proposed APC is implemented using Weighted Recursive Least Squares (WRLS) and a sub-optimal but fast algorithm based on the General Predictive Controller (GPC) approach. These are combined with a variable forgetting factor and variable prediction horizon algorithms. In addition, methods are proposed to handle stability issues arising due to practical hardware limitations. The controller is implemented on a real single rack system and challenges for practical implementation are addressed and illustrated. The proposed APC is also compared (via simulation) to a standard APC with equivalent complexity and a split-range PI controller. The results show that the proposed controller outperforms both the standard APC and the split-range PI controllers with respect to Mean Squared Error (MSE).

Aquest assaig proposa una reflexió urgent sobre el risc de l’ús de psicofàrmacs per al tractament de trastorns del comportament infantil. Amb aquest fi, compararé l’ús de neurolèptics en malalties mentals greus (esquizofrènia), dels quals revisarem la història del seu descobriment, amb la utilització de psicoestimulants per a tractar el trastorn amb dèficit d’atenció i hiperactivitat (TDAH) en infants. Estudis experimentals en rosegadors demostren efectes duradors dels psicoestimulants que incrementen la vulnerabilitat a l’addicció i l’aparició d’estereotípies semblants al trastorn obsessivocompulsiu. Acabarem discutint el probable sobrediagnòstic del TDAH, els interessos en joc i la responsabilitat de pares, mestres i metges en aquest preocupant fenomen social. Este ensayo propone una reflexión urgente sobre el riesgo del uso de psicofármacos para tratar trastornos del comportamiento infantil. Con este fin compararé el uso de neurolépticos en enfermedades mentales graves (esquizofrenia), de los que revisaremos la historia de su descubrimiento, con el uso de psicoestimulantes para tratar el trastorno con déficit de atención e hiperactividad (TDAH) en niños. Estudios de laboratorio demuestran efectos a largo plazo de los psicoestimulantes que incrementan la vulnerabilidad a la adicción y la aparición de estereotipias, una especie de trastorno obsesivo-compulsivo. Acabaremos discutiendo el probable sobrediagnóstico del TDAH, los intereses en juego y la responsabilidad de padres, maestros y médicos en este preocupante fenómeno social. Cet essai propose une réflexion urgente sur le risque de l’emploi de psychomédicaments pour le traitement des troubles du comportement enfantin. Dans ce but, je compare l’emploi des neuroleptiques sur les maladies mentales graves (schyzophrénie), dont l’histoire de leur decouverte est révisée, avec l’emploi de psychostimulants pour le traitement du trouble du déficit de l’attention avec ou sans hyperactivité (TDAH) chez les enfants. Certaines études de laboratoire démontrent des effets à long terme des psychostimulants capables d’accroître la vulnérabilité à l’addiction et la parution de stéréotypyes, une sorte de trouble obsessionnel-compulsif. Je finis par examiner le probable surdiagnostic du TDAH, les intérêts mis en jeux et la responsabilité des parents, des instituteurs et des médecins sur cet inquiétant phénomène social. This article presents a critical reflection on the risks of using psychiatric medication for the treatment of behavioural disorders in children. With this in mind, the use of neuroleptics (including a brief account of their discovery) in the treatment of a severe mental disorder, schizophrenia, is compared with that of psycho-stimulant drugs as treatment for Attention Deficit Hyperactivity Disorder (ADHD) in children. Experimental studies in rodents indicate that there may be long-term effects including increased addiction vulnerability and the appearance of stereotypies associated with obsessive-compulsive disorder. This leads on to a discussion of the likely over-diagnosis of ADHD, the interests at stake, and the responsibility of parents, teachers and doctors in this worrying social phenomenon.

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.

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.

This article is part of a Special Issue “Chemosignals and Reproduction”. This paper reviews the role of chemosignals in the socio-sexual interactions of female mice, and reports two experiments testing the role of pup-derived chemosignals and the male sexual pheromone darcin in inducing and promoting maternal aggression. Female mice are attracted to urine-borne male pheromones. Volatile and non-volatile urine fractions have been proposed to contain olfactory and vomeronasal pheromones. In particular, the male-specific major urinary protein (MUP) MUP20, darcin, has been shown to be rewarding and attractive to females. Non-urinary male chemosignals, such as the lacrimal protein ESP1, promote lordosis in female mice, but its attractive properties are still to be tested. There is evidence indicating that ESP1 and MUPs are detected by vomeronasal type 2 receptors (V2R). When a female mouse becomes pregnant, she undergoes dramatic changes in her physiology and behaviour. She builds a nest for her pups and takes care of them. Dams also defend the nest against conspecific intruders, attacking especially gonadally intact males. Maternal behaviour is dependent on a functional olfactory system, thus suggesting a role of chemosignals in the development of maternal behaviour. Our first experiment demonstrates, however, that pup chemosignals are not sufficient to induce maternal aggression in virgin females. In addition, it is known that vomeronasal stimuli are needed for maternal aggression. Since MUPs (and other molecules) are able to promote intermale aggression, in our second experiment we test if the attractive MUP darcin also promotes attacks on castrated male intruders by lactating dams. Our findings demonstrate that the same chemosignal, darcin, promotes attraction or aggression according to female reproductive state

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.

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.

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 b-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. Funded by the Spanish Ministry of Education and Science- FEDER (BFU2007-67912-C02-01/BFI and BFU2016-77691-C2- 1-P and PROMETEO/2016/076).

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. Funded bytheSpanishMinistryofEconomyand Competitiveness-FEDER(BFU2013-47688-P),theGeneralitat Valenciana(PROMETEO/2016/076)andtheJuntade ComunidadesdeCastilla-LaMancha(PEIC-2014-006-P).BC-M is apredoctoralfellowofthe“BecasChile”programofthe Government ofChile.MA-Aisafellowofthe“Programade Empleo Joven”PEJ-2014-A-63220oftheSpanishGovernment. CP-Bisapredoctoralfellowofthe“AtracciódeTalent”program of theUniversityofValència.

Most mammals possess a vomeronasal system that detects predominantly chemical signals of biological relevance. Vomeronasal information is relayed to the accessory olfactory bulb (AOB), whose unique cortical target is the posteromedial cortical nucleus of the amygdala. This cortical structure should therefore be considered the primary vomeronasal cortex. In the present work, we describe the afferent and efferent connections of the posteromedial cortical nucleus of the amygdala in female mice, using anterograde (biotinylated dextranamines) and retrograde (Fluorogold) tracers, and zinc selenite as a tracer specific for zinc-enriched (putative glutamatergic) projections. The results show that the posteromedial cortical nucleus of the amygdala is strongly interconnected not only with the rest of the vomeronasal system (AOB and its target structures in the amygdala), but also with the olfactory system (piriform cortex, olfactory-recipient nuclei of the amygdala and entorhinal cortex). Therefore, the posteromedial cortical nucleus of the amygdala probably integrates olfactory and vomeronasal information. In addition, the posteromedial cortical nucleus of the amygdala shows moderate interconnections with the associative (basomedial) amygdala and with the ventral hippocampus, which may be involved in emotional and spatial learning (respectively) induced by chemical signals. Finally, the posteromedial cortical nucleus of the amygdala gives rise to zinc-enriched projections to the ventrolateral septum and the ventromedial striatum (including the medial islands of Calleja). This pattern of intracortical connections (with the olfactory cortex and hippocampus, mainly) and cortico-striatal excitatory projections (with the olfactory tubercle and septum) is consistent with its proposed nature as the primary vomeronasal cortex.

Some chemicals elicit innate emotionally laden behavioral responses. Pheromones mediate sexual attraction, parental care or agonistic confrontation, whereas predators' kairomones elicit defensive behaviors in their preys. This essay explores the hypothesis that the detection of these semiochemicals relies on highly specific olfactory and/or vomeronasal receptors. The V1R, V2R, and formyl-peptide vomeronasal receptors bind their ligands in highly specific and sensitive way, thus being good candidates for pheromone- or kairomone-detectors (e.g., secreted and excreted proteins, peptides and lipophilic volatiles). The olfactory epithelium also expresses specific receptors, for example trace amine-associated receptors (TAAR) and guanylyl cyclase receptors (GC-D and other types), some of which bind kairomones and putative pheromones. However, most of the olfactory neurons express canonical olfactory receptors (ORs) that bind many ligands with different affinity, being not suitable for mediating responses to pheromones and kairomones. In this respect, trimethylthiazoline (TMT) is considered a fox-derived kairomone for mice and rats, but it seems to be detected by canonical ORs. Therefore, we have reassessed the kairomonal nature of TMT by analyzing the behavioral responses of outbred (CD1) and inbred mice (C57BL/J6) to TMT. Our results confirm that both mouse strains avoid TMT, which increases immobility in C57BL/J6, but not CD1 mice. However, mice of both strains sniff at TMT throughout the test and show no trace of TMT-induced contextual conditioning (immobility or avoidance). This suggests that TMT is not a kairomone but, similar to a loud noise, in high concentrations it induces aversion and stress as unspecific responses to a strong olfactory stimulation.

Quantitative analysis of the immunoreactivity for arginine-vasopressin (AVP-ir) in the telencephalon of male (intact and castrated) and female CD1 mice allows us to precisely locate two sexually dimorphic (more abundant in intact than castrated males and females) AVP-ir cell groups in the posterior bed nucleus of the stria terminalis (BST) and the amygdala. Chemoarchitecture (NADPH diaphorase) reveals that the intraamygdaloid AVP-ir cells are located in the intra-amygdaloid BST (BSTIA) rather than the medial amygdala (Me), as previously thought. Then, we have used for the first time tract tracing (combined with AVP immunofluorescence) and fiber-sparing lesions of the BST to analyze the projections of the telencephalic AVP-ir cell groups. The results demonstrate that the posterior BST originates the sexually dimorphic innervation of the lateral septum, the posterodorsal Me and a substance P-negative area in the medioventral striato-pallidum (mvStP).The BSTIA may also contribute to some of these terminal fields. Our material also reveals non-dimorphic AVP-ir processes in two locations of the amygdala. First, the ventral Me shows dendrite-like AVP-ir processes apparently belonging supraoptic neurons, whose possible functions are discussed. Second, the Ce shows sparse, thick AVP-ir axons with high individual variability in density and distribution, whose possible influence on stress coping in relation to the affiliative or agonistic behaviors mediated by the Me are discussed. Finally, we propose that the region of the mvStP showing sexually dimorphic AVP-ir innervation is part of the brain network for socio-sexual behavior, in which it would mediate motivational aspects of chemosensory-guided social interactions.

Nitric oxide is known to take part in the control of sexual and agonistic behaviours. This is usually attributed to its role in neural transmission in the hypothalamus and other structures of the limbic system. However, socio-sexual behaviours in rodents are mainly directed by chemical signals detected by the vomeronasal system, and nitric oxide is abundant in key structures along the vomeronasal pathway. Thus, here we check whether pharmacological treatments interfering with nitrergic transmission could affect socio-sexual behaviour by impairing the processing of chemical signals. Treatment with an inhibitor of nitric oxide synthesis (Nomega-Nitro-l-arginine methyl ester hydrochloride, L-NAME, 100mg/kg) blocks the innate preference displayed by female mice for sexual pheromones contained in male-soiled bedding, with a lower dose of the drug (50mg/kg) having no effect. Animals treated with the high dose of L-NAME show no reduction of olfactory discrimination of male urine in a habituation-dishabituation test, thus suggesting that the effect of the drug on the preference for male pheromones is not due to an inability to detect male urine. Alternatively, it may result from an alteration in processing the reinforcing value of pheromones as sexual signals. These results add a new piece of evidence to our understanding of the neurochemistry of intraspecific chemical communication in rodents, and suggest that the role of nitric oxide in socio-sexual behaviours should be re-evaluated taking into account the involvement of this neuromodulator in the processing of chemical signals.

In rodents, sexual advertisement and gender recognition are mostly (if not exclusively) mediated by chemosignals. Specifically, there is ample evidence indicating that female mice are ‘innately’ attracted by male sexual pheromones that have critical non-volatile components and are detected by the vomeronasal organ. These pheromones can only get access to the vomeronasal organ by active pumping mechanisms that require close contact with the source of the stimulus (e.g. urine marks) during chemoinvestigation. We have hypothesised that male sexual pheromones are rewarding to female mice. Indeed, male-soiled bedding can be used as a reinforcer to induce conditioned place preference, provided contact with the bedding is allowed. The neural mechanisms of pheromone reward seem, however, different from those employed by other natural reinforcers, such as the sweetness or postingestive effects of sucrose. In contrast to vomeronasal-detected male sexual pheromones, male-derived olfactory stimuli (volatiles) are not intrinsically attractive to female mice. However, after repeated exposure to male-soiled bedding, intact female mice develop an acquired preference for male odours. On the contrary, in females whose accessory olfactory bulbs have been lesioned, exposure to male-soiled bedding induces aversion to male odorants. These considerations, together with data on the different properties of olfactory and vomeronasal receptors, lead us to make a proposal for the complementary roles that the olfactory and vomeronasal systems play in intersexual attraction and in other forms of intra- or inter-species communication.

Endogenous opioids mediate some reward processes involving both natural (food, sweet taste) and artificial (morphine, heroin) rewards. In contrast, sexual behavior (which is also reinforcing) is generally inhibited by opioids. To establish the role of endogenous opioids for a newly described natural reinforcer, namely male sexual pheromones for female mice, we checked the effects of systemic injections of the general opioid antagonist naloxone (1-10 mg/kg) and the agonist fentanyl (0.1- 0.5 mg/kg) in a number of behavioral tests. Naloxone affected neither the innate preference for male-soiled bedding (vs. female-soiled bedding) in 2-choice tests nor the induction of place conditioning using male pheromones as rewarding stimuli, although it effectively blocked the preference for consuming a sucrose solution. In contrast, fentanyl inhibited the preference for male chemosignals without altering sucrose preference. These results suggest that, in macrosmatic animals such as rodents, opioidergic inhibition of sexual behavior might be due, at least partially, to an impaired processing of pheromonal cues and that the hedonic value of sweet-tasting solutions and sexual pheromones are under different opioid modulation.

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. This work was funded by the Spanish Ministry of Science—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 Marcos Otero-García, granted by the FPU (Formación de Profesorado Universitario) programme of the Spanish Ministry of Education and Science. The authors gratefully acknowledge Dr. Harold Gainer for his generous gift of antibodies.

Virgin adult female mice display nearly spontaneous maternal care towards foster pups after a short period of sensitization. This indicates that maternal care is triggered by sensory stimulation provided by the pups and that its onset is largely independent on the physiological events related to gestation, parturition and lactation. Conversely, the factors influencing maternal aggression are poorly understood. In this study, we sought to characterize two models of maternal sensitization in the outbred CD1 strain. To do so, a group of virgin females (godmothers) were exposed to continuous cohabitation with a lactating dam and their pups from the moment of parturition, whereas a second group (pup-sensitized females), were exposed 2 h daily to foster pups. Both groups were tested for maternal behavior on postnatal days 2–4. Godmothers expressed full maternal care from the first test. Also, they expressed higher levels of crouching than dams. Pup-sensitized females differed from dams in all measures of pup-directed behavior in the first test, and expressed full maternal care after two sessions of contact with pups. However, both protocols failed to induce maternal aggression toward a male intruder after full onset of pup-directed maternal behavior, even in the presence of pups. Our study confirms that adult female mice need a short sensitization period before the onset of maternal care. Further, it shows that pup-oriented and non-pup-oriented components of maternal behavior are under different physiological control. We conclude that the godmother model might be useful to study the physiological and neural bases of the maternal behavior repertoire.

The amygdala is considered a key center in managing emotional information and its dysfunction is at the base of disorders ranging through anxiety, depression, PTSD and autism. However, the amygdala seems heterogeneous both structurally (with pallial and supallial components) and functionally. Thus, whereas cortical and thalamic multimodal sensory inputs enter the basolateral complex, the corticomedial amygdala is dominated by olfactory and vomeronasal inputs. Intrinsic amygdaloid circuitry, connects these two amygdaloid divisions and convey processed information to the main amygdala outputs. The pallial amygdala is the main source for telencephalic outputs to associative cortical areas (e.g., frontal lobe and hippocampus) and to the ventral striatum. The two subdivisions of the extended amygdala, central and medial, originate descending projections to the hypothalamus and brainstem centers for emotional responses and behavioral control. The amygdala is the key node of the neural network where sensory information acquires emotional value through Pavlovian associations and where fear memories may be extinguished. It is also a fundamental piece of the neural machinery controlling socio-sexual and defensive behaviors.

Copyright © 2015 Fortes-Marco, Lanuza, Martínez-García and Agustín-Pavón., Chemosignals mediate both intra- and inter-specific communication in most mammals. Pheromones elicit stereotyped reactions in conspecifics, whereas kairomones provoke a reaction in an allospecific animal. For instance, predator kairomones elicit anticipated defensive responses in preys. The aim of this work was to test the behavioral responses of female mice to two chemosignals: 2-heptanone (2-HP), a putative alarm pheromone, and 2,4,5-trimethylthiazoline (TMT), a fox-derived putative kairomone, widely used to investigate fear and anxiety in rodents. The banana-like odorant isoamyl acetate (IA), unlikely to act as a chemosignal, served as a control odorant. We first presented increasing amounts of these odorants in consecutive days, in a test box in which mice could explore or avoid them. Female mice avoided the highest amounts of all three compounds, with TMT and IA eliciting avoidance at lower amounts (3.8 pmol and 0.35 μmol, respectively) than 2-HP (35 μmol). All three compounds induced minimal effects in global locomotion and immobility in this set up. Further, mice detected 3.5 pmol of TMT and IA in a habituation–dishabituation test, so avoidance of IA started well beyond the detection threshold. Finally, both TMT and IA, but not 2-HP, induced conditioned place avoidance and increased immobility in the neutral compartment during a contextual memory test. These data suggest that intense odors can induce contextual learning irrespective of their putative biological significance. Our results support that synthetic predator-related compounds (like TMT) or other intense odorants are useful to investigate the neurobiological basis of emotional behaviors in rodents. Since intense odorants unlikely to act as chemosignals can elicit similar behavioral reactions than chemosignals, we stress the importance of using behavioral measures in combination with other physiological (e.g., hormonal levels) or neural measures (e.g., immediate early gene expression) to establish the ethological significance of odorants., This study was funded by the Spanish MINECO-FEDER (BFU2010-16656; BFU2013-47688-P), Junta de Comunidades de Castilla-La Mancha/FEDER (PEIC11-0045-4490), and Vicerrectorado de Investigación, Universitat Jaume I de Castelló.