Feromones sexuals: un nou estímul reforçant en ratolins

RESUM En ratolins existeixen certes feromones no volàtils i de caràcter sexual contingudes en la borumballa embrutada per mascles i que resulten innatament atractives per a les femelles. Els senyals volàtils emanats de la borumballa de mascles, en canvi, no resulten atractius en la primera experiència amb els mateixos, però poden adquirir propietats atractives de manera secundària després de lexposició reiterada a aquests volàtils i als no volàtils innatament atractius de manera conjunta. Els dos primers objectius fonamentals daquesta tesi han estat estudiar si les feromones sexuals de mascle són detectades per lòrgan vomeronasal, o si pel contrari, constitueixen un estímul olfactiu, i comprovar, igualment, si latracció induïda per volàtils depèn de la integritat del sistema vomeronasal. Per abordar aquests objectius es va comparar la preferència cap a senyals químics de mascle que mostren les femelles amb la funció vomeronasal interrompuda (mitjançant la lesió electrolítica del bulb olfactiu accessori), amb la què mostren les femelles amb la funció vomeronasal intacta (sels practicà una lesió simulada) en diferents situacions experimentals. Els resultats daquests experiments mostraren que les feromones contingudes en la borumballa embrutada per mascles intactes que resulten innatament atractives per a les femelles de ratolí són detectades per lòrgan vomeronasal. També confirmaren que per tal que els volàtils puguen adquirir propietats atractives de manera secundària és necessària la seua associació amb un estímul detectat a través del sistema vomeronasal. A continuació comprovàrem si a més de ser innatament atractives, les feromones sexuals de mascle són reforçants per a les femelles, per a la qual cosa realitzàrem una sèrie dexperiments seguint un paradigma comportamental anomenat test dadquisició de preferència de lloc, utilitzar per comprovar les propietats reforçants dels estímuls. Els resultats mostraren que les femelles de ratolí són capaces de detectar el gènere i lestat de dominància dels conespecífics a través de senyals químics continguts en la borumballa embrutada pels mateixos. A més, revelaren que les feromones sexuals de mascle són reforçants per a les femelles de ratolí, però els senyals continguts en la borumballa embrutada per mascles castrat o altres femelles no. Per últim estudiàrem la via de transducció del reforç degut a feromones sexuals. Amb aquest objectiu, i donat que prèviament havíem comprovat que les feromones sexuals són detectades a través del sistema vomeronasal, analitzàrem lexpressió del proto-oncogen c-fos en distints nuclis vomeronasal (el nucli posteromedial de la amígdala cortical, els Illots de Calleja i lÀrea de transició amígdalo-hipocàmpica). Aquesta expressió la comparàrem entre dos grups experimentals, un al què les femelles solament tenien accés a borumballa neta, i un altre en què les femelles eren exposades a borumballa de mascle. Els resultats indicaren que la via responsable de mitjançar aquest reforç no és la via dopaminèrgica tegment-estriatal clàssica, sinó que es la via que va des del nucli posteromedial de la amígdala cortical i làrea amígdalo-hipocàmpica als Illots de Calleja més ventromedials. En resum els resultats daquesta tesis mostren lexistència dun nou estímul reforçant en ratolins, les feromones sexuals de mascle, que són detectades a través de lòrgan vomeronasal. A més, hem vist que existeixen altres regions de lestriat ventral, a part de les clàssiques, implicades en la codificació de les propietats reforçants dels estímuls naturals. __________________________________________________________________________________________________Male mice soiled bedding contains non volatile sexual pheromones that are innately attractive to females. Male volatile signals are, in contrast, not attractive to females in their very first contact with them, but can acquire secondary attractive properties by its association with the non volatile and innately attractive signals. In this work we analysed whether male sexual pheromones are detected through the accessory olfactory system, and if the association between non-volatiles and volatile signals is also dependent on the integrity of vomeronasal system. To do so, we carried out a battery of experiments in a group of females with a lesion in their accessory olfactory bulb (AOB) comparing their behaviour with that from another group of animals with a sham lesion in their AOB. In addition, we also analysed if male sexual pheromones are rewarding to female mice, using a behavioural paradigm named place preference test. Finally, we studied which would be the neural system that mediates these reinforcing properties. To do so, we compared c-fos expression in different vomeronasal nuclei in animals exposed to male-soiled bedding and animals exposed to clean bedding. The results of our experiments show that male sexual pheromones are detected through the vomeronasal organ and that the acquisition of secondary attractive properties by male volatile signals depends on its association with non volatiles signals also detected through the vomeronasal system. Furthermore we have demonstrated that male sexual pheromones have reinforcing properties to female mice, ant that the neural system that mediates these reinforcing properties is not the classical dopaminergic tegmento-estriatal projection, but the projections from some regions of the posteromedial cortical nucleus of the amygdala and the amygdalo-hypocampal area to the most ventromedial Islands of Calleja. The results from these experiments show the existence of a new reinforcing stimulus in mice, the sexual pheromones. In addition, these pheromones are detected through the vomeronasal organ. Moreover, we have shown that there are more regions a part from the classical ones in ventral striatum involved in mediating reinforcing properties of natural stimuli

Intraspecific Communication Through Chemical Signals in Female Mice: Reinforcing Properties of Involatile Male Sexual Pheromones

In rodents, social and reproductive behaviors critically depend on chemical signals, including sexual pheromones that have been suggested (but not demonstrated) to be rewarding. In this work, we analyze this issue by studying the chemoinvestigatory behavior of adult female mice (without experience with male-derived chemicals) toward 1) the synthetic odorant citralva, 2) bedding soiled by different conspecifics (females, males, and castrated males), and 3) volatiles derived from bedding soiled by males and castrated males (confronted in 2-choice tests). We also study whether these chemical signals are able to induce conditioned place preference, a reliable test for rewarding properties of stimuli. The results show that involatile, male-derived chemicals elicit an intense and sustained chemoinvestigation and, more importantly, are the only tested chemical signals that induce conditioned place preference. In contrast, volatile, male-derived chemicals are not significantly chemoinvestigated. Bedding soiled by castrated males induces a transient chemoinvestigation, likely directed to steroid-independent, biologically relevant chemical signals, whereas the intense chemoinvestigation of female-soiled bedding shows a slow habituation. Finally, females did not explore significantly citralva-odorized bedding. The present work constitutes the first demonstration of the unconditioned reinforcing properties of involatile (likely detected by the vomeronasal organ) steroid-dependent chemical signals in mammals

Role of the vomeronasal system in intersexual attraction in female mice

Although it is generally accepted that rodents' sociosexual behavior relies mainly on chemosignals, the specific roles played by the vomeronasal and olfactory systems in detecting these signals are presently unclear. This work reports the results of three experiments aimed at clarifying the role of the vomeronasal system on gender recognition and intersexual attraction, by analyzing the effects of lesions of the accessory olfactory bulbs (AOB) in chemically naïve female mice. The first experiment demonstrates that lesions of the AOB abolish the preference that females show for male-soiled bedding in tests in which the females can contact the bedding, thus having access to both volatile and involatile male chemosignals. The second experiment shows that airborne male-derived chemosignals are not attractive to intact, chemically naïve females but tend to be preferentially explored by females whose AOB has been lesioned. However, repeated exposure to male-soiled bedding has opposite effects in sham-operated and AOB-lesioned female mice. Whereas after this experience sham-operated females show an (acquired) attraction toward male airborne chemosignals, in AOB-lesioned females the same experience makes male-derived volatiles aversive. Finally, in the third experiment we have confirmed that our AOB-lesioned females are able to detect urine-borne male odorants, as well as to discriminate them from the synthetic terpene geraniol. These findings strongly suggest that in mice, the involatile male sexual pheromone that is intrinsically attractive is detected by the vomeronasal system of the females. In addition, the repeated experience of females with male-soiled bedding would probably allow the association of this pheromone, acting as unconditioned stimulus, with olfactory stimuli (odorants) that therefore would become conditioned attractors to the females

Role of nitric oxide in pheromone-mediated intraspecific communication in mice

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 (Nω-Nitro-l-arginine methyl ester hydrochloride, L-NAME, 100 mg/kg) blocks the innate preference displayed by female mice for sexual pheromones contained in male-soiled bedding, with a lower dose of the drug (50 mg/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

Sexual pheromones and the evolution of the reward system of the brain: The chemosensory function of the amygdala

The amygdala of all tetrapod vertebrates receives direct projections from the main and accessory olfactory bulbs, and the strong similarities in the organization of these projections suggest that they have undergone a very conservative evolution. However, current ideas about the function of the amygdala do not pay sufficient attention to its chemosensory role, but only view it as the core of the emotional brain. In this study, we propose that both roles of the amygdala are intimately linked since the amygdala is actually involved in mediating emotional responses to chemical signals. The amygdala is the only structure in the brain receiving pheromonal information directly from the accessory olfactory bulbs and we have shown in mice that males emit sexual pheromones that are innately attractive for females. In fact, sexual pheromones can be used as unconditioned stimuli to induce a conditioned attraction to previously neutral odorants as well as a conditioned place preference. Therefore, sexual pheromones should be regarded as natural reinforcers. Behavioural and pharmacological studies (reviewed here) have shown that the females’ innate preference for sexual pheromones is not affected by lesions of the dopaminergic cells of the ventral tegmental area, and that the systemic administration of dopamine antagonists do not alter neither the attraction nor the reinforcing effects of these pheromones. Anatomical studies have shown that the vomeronasal amygdala gives rise to important projections to the olfactory tubercle and the islands of Calleja, suggesting that these amygdalo-striatal pathways might be involved in the reinforcing value of sexual pheromones

Refining the dual olfactory hypothesis: Pheromone reward and odour experience

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

L'estimulació cerebral profunda al nucli accumbens i l'estudi pilot electrofisiològic d'escorça prefrontal medial, davant la resposta d'estrés

Aquest estudi es basa, conceptualment, en les investigacions relatives a l'estimulació cerebral profunda (ECP), que és una intervenció neuroquirúrgica en la que elèctrodes implantats alliberen impulsos elèctrics en zones «diana» de l'encèfal. Aquesta tècnica ha estat emprada per a tractar diferents patologies, entre altres les addiccions i les seues manifestacions comportamentals. Específicament, l'ECP al nucli accumbens, ha estat aplicada per a tractar, amb èxit, els trastorns addictius i el desig del consum craving. Així mateix destaquem com durant el proces addictiu i, concretament, en el craving s'ha comprovat, que l'estrés juga un paper significatiu. D'altra banda, banda, l'estudi electrofisiològic de l'escorça prefrontal medial i amígdala central és fonamental, per a l'obtenció de dades rellevants, en ambdues àrees involucrades, envers la gestió de l'estrés i les addiccions

Mating increases neuronal tyrosine hydroxylase expression and selectively gates transmission of male chemosensory information in female mice

Exposure to chemosensory signals from unfamiliar males can terminate pregnancy in recently mated female mice. The number of tyrosine hydroxylase-positive neurons in the main olfactory bulb has been found to increase following mating and has been implicated in preventing male-induced pregnancy block during the post-implantation period. In contrast, pre-implantation pregnancy block is mediated by the vomeronasal system, and is thought to be prevented by selective inhibition of the mate’s pregnancy blocking chemosignals, at the level of the accessory olfactory bulb. The objectives of this study were firstly to identify the level of the vomeronasal pathway at which selective inhibition of the mate’s pregnancy blocking chemosignals occurs. Secondly, to determine whether a post-mating increase in tyrosine hydroxylase-positive neurons is observed in the vomeronasal system, which could play a role in preventing pre-implantation pregnancy block. Immunohistochemical staining revealed that mating induced an increase in tyrosine-hydroxylase positive neurons in the arcuate hypothalamus of BALB/c females, and supressed c-Fos expression in these neurons in response to mating male chemosignals. This selective suppression of c-Fos response to mating male chemosignals was not apparent at earlier levels of the pregnancy-blocking neural pathway in the accessory olfactory bulb or corticomedial amygdala. Immunohistochemical staining revealed an increase in the number of tyrosine hydroxylase-positive neurons in the accessory olfactory bulb of BALB/c female mice following mating. However, increased dopamine-mediated inhibition in the accessory olfactory bulb is unlikely to account for the prevention of pregnancy block to the mating male, as tyrosine hydroxylase expression did not increase in females of the C57BL/6 strain, which show normal mate recognition. These findings reveal an association of mating with increased dopaminergic modulation in the pregnancy block pathway and support the hypothesis that mate recognition prevents pregnancy block by suppressing the activation of arcuate dopamine release

Neural oscillations in the infralimbic cortex after electrical stimulation of the amygdala. Relevance to acute stress processing

The stress system coordinates the adaptive reactions of the organism to stressors. Therefore, dysfunctions in this circuit may correlate to anxiety-related disorders, including depression. Comprehending the dynamics of this network may lead to a better understanding of the mechanisms that underlie these diseases. The central nucleus of the amygdala (CeA) activates the hypothalamic–pituitary–adrenal axis and brainstem nodes by triggering endocrine, autonomic and behavioral stress responses. The medial prefrontal cortex plays a significant role in regulating reactions to stressors, and is specifically important for limiting fear responses. Brain oscillations reflect neural systems activity. Synchronous neuronal assemblies facilitate communication and synaptic plasticity, mechanisms that cooperatively support the temporal representation and long-term consolidation of information. The purpose of this article was to delve into the interactions between these structures in stress contexts by evaluating changes in oscillatory activity. We particularly analyzed the local field potential in the infralimbic region of the medial prefrontal cortex (IL) in urethane-anesthetized rats after the electrical activation of the central nucleus of the amygdala by mimicking firing rates induced by acute stress. Electrical CeA activation induced a delayed, but significant, change in the IL, with prominent slow waves accompanied by an increase in the theta and gamma activities, and spindles. The phase-amplitude coupling of both slow waves and theta oscillations significantly increased with faster oscillations, including theta-gamma coupling and the nesting of spindles, theta and gamma oscillations in the slow wave cycle. These results are further discussed in neural processing terms of the stress response and memory formation.This work was supported by the Instituto de Salud Carlos III, and Subdirección General de Evaluación y Fomento de la Investigación (FIS Grant PI13–00038 and FIS Grant PI16–00217), which were co-funded by the European Regional Development Fund ('A way to build Europe'), and by the Generalitat Valenciana (Emergent groups grant GV/2016/103)