Two interconnected functional systems in the amygdala of amniote vertebrates.

The amygdala shows ventropallial and lateropallial derivatives that can be compared among vertebrates according to their topological position, either superficial (cortical amygdala) or deep (basolateral amygdala and amygdalo-hippocampal area), connections and histochemical features. On the other hand, the subpallial amygdala, also called extended amygdala, is composed of medial and central divisions. In mammals, both divisions consist of an intra-amygdaloid portion and a part of the bed nucleus of the stria terminalis. In non-mammals, the intratelencephalic trajectory of the stria terminalis is short and both poles of the extended amygdala are close together. Like its mammalian counterpart, the medial extended amygdala of non-mammals receives an olfactory input (reduced in birds), projects to the medial hypothalamus and shows a sexually dimorphic vasotocinergic (vasopressinergic) cell group. Thus, the medial extended amygdala is part of the forebrain circuitry for the expression of defensive and reproductive behaviours. In turn, the central extended amygdala of amniotes shows a prominent CGRP innervation and a medially located CRF/neurotensin-expressing cell group, and projects to the lateral hypothalamus and to the midbrain and brainstem centres involved in fear/anxiety expression. The projections from the pallial amygdala to the central and medial extended amygdala are similarly organized in the mammals and non-mammals. These circuits, which have not changed substantially in birds despite the disappearance of the vomeronasal system, delineate two functional divisions within the amygdala that, together, orchestrate the expression of species-specific behaviours with a strong emotional component.

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.

Projections from the posterolateral olfactory amygdala to the ventral striatum: neural basis for reinforcing properties of chemical stimuli.

BACKGROUND: Vertebrates sense chemical stimuli through the olfactory receptor neurons whose axons project to the main olfactory bulb. The main projections of the olfactory bulb are directed to the olfactory cortex and olfactory amygdala (the anterior and posterolateral cortical amygdalae). The posterolateral cortical amygdaloid nucleus mainly projects to other amygdaloid nuclei; other seemingly minor outputs are directed to the ventral striatum, in particular to the olfactory tubercle and the islands of Calleja. RESULTS: Although the olfactory projections have been previously described in the literature, injection of dextran-amines into the rat main olfactory bulb was performed with the aim of delimiting the olfactory tubercle and posterolateral cortical amygdaloid nucleus in our own material. Injection of dextran-amines into the posterolateral cortical amygdaloid nucleus of rats resulted in anterograde labeling in the ventral striatum, in particular in the core of the nucleus accumbens, and in the medial olfactory tubercle including some islands of Calleja and the cell bridges across the ventral pallidum. Injections of Fluoro-Gold into the ventral striatum were performed to allow retrograde confirmation of these projections. CONCLUSION: The present results extend previous descriptions of the posterolateral cortical amygdaloid nucleus efferent projections, which are mainly directed to the core of the nucleus accumbens and the medial olfactory tubercle. Our data indicate that the projection to the core of the nucleus accumbens arises from layer III; the projection to the olfactory tubercle arises from layer II and is much more robust than previously thought. This latter projection is directed to the medial olfactory tubercle including the corresponding islands of Calleja, an area recently described as critical node for the neural circuit of addiction to some stimulant drugs of abuse.

Short-term effect of acute and repeated urinary bladder inflammation on thigmotactic behaviour in the laboratory rat.

Understanding the non-sensory components of the pain experience is crucial to developing effective treatments for pain conditions. Chronic pain is associated with increased incidence of anxio-depressive disorders, and patients often report feelings of vulnerability which can decrease quality of life. In animal models of pain, observation of behaviours such as thigmotaxis can be used to detect such affective disturbances by exploiting the influence of nociceptive stimuli on the innate behavioural conflict between exploration of a novel space and predator avoidance behaviour. This study investigates whether acute and repeated bladder inflammation in adult female Wistar rats increases thigmotactic behaviour in the open field paradigm, and aims to determine whether this correlates with activation in the central amygdala, as measured by c-Fos immunoreactivity. Additionally, up-regulation of inflammatory mediators in the urinary bladder was measured using RT-qPCR array featuring 92 transcripts to examine how local mediators change under experimental conditions. We found acute but not repeated turpentine inflammation of the bladder increased thigmotactic behaviour (decreased frequency of entry to the inner zone) in the open field paradigm, a result that was also observed in the catheter-only instrumentation group. Decreases in locomotor activity were also observed in both models in turpentine and instrumentation groups. No differences were observed in c-Fos activation, although a general increased in activation along the rostro-caudal axis was seen. Inflammatory mediator up-regulation was greatest following acute inflammation, with CCL12, CCL7, and IL-1ß significantly up-regulated in both conditions when compared to naïve tissue. These results suggest that acute catheterisation, with or without turpentine inflammation, induces affective alterations detectable in the open field paradigm accompanied by up-regulation of multiple inflammatory mediators.

Amygdalostriatal projections in reptiles: a tract-tracing study in the lizard Podarcis hispanica.

Whereas the lacertilian anterior dorsal ventricular ridge contains unimodal sensory areas, its posterior part (PDVR) is an associative center that projects to the hypothalamus, thus being comparable to the amygdaloid formation. To further understand the organization of the reptilian cerebral hemispheres, we have used anterograde and retrograde tracing techniques to study the projections from the PDVR and adjoining areas (dorsolateral amygdala, DLA; deep lateral cortex, dLC; nucleus sphericus, NS) to the striatum in the lizard Podarcis hispanica. This information is complemented with a detailed description of the organization of the basal telencephalon of Podarcis. The caudal aspect of the dorsal ventricular ridge projects nontopographically mainly (but not exclusively) to the ventral striatum. The NS projects bilaterally (with strong ipsilateral dominance) to the nucleus accumbens, thus recalling the posteromedial cortical amygdala of mammals. The PDVR (especially its lateral aspect) and the dLC project massively to a continuum of structures connecting the striatoamygdaloid transition area (SAT) and the nucleus accumbens (rostrally), the projection arising from the dLC being probably bilateral. Finally, the DLA projects massively and bilaterally to both the ventral and dorsal striatum, including the SAT. Our findings lend further support to the view that the PDVR and neighboring structures constitute the reptilian basolateral amygdala and indicate that an emotional brain was already present in the ancestral amniote. These results are important to understand the comparative significance of the caudal aspect of the amniote cerebral hemispheres, and specifically challenge current views on the nature of the avian caudal neostriatum.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the lizard Podarcis hispanica.

The present work studies the distribution of calcitonin gene-related peptide-immunoreactive (CGRP-li) neurons and fibers in the brain of a reptile, the lizard Podarcis hispanica. CGRP-li perikarya were not present in the telencephalon. In the thalamus, CGRP-li perikarya were restricted to the posteromedial and posterolateral nuclei. In the hypothalamus, CGRP-li cells were found mainly in the supramammillary and mammillary nuclei. In the midbrain and brainstem, CGRP-li cells appeared in the ventral tegmental area, the parabrachial nucleus, and the motor nuclei of the III-VII, IX, X, and XII cranial nerves. Motoneurons of the ventral horn of the spinal cord were also immunoreactive for CGRP. CGRP-li fibers were seen in the telencephalic hemispheres, where a dense plexus of reactive fibers appeared in the septum and in the lateral striatoamygdaloid transition area. From the latter, CGRP-li fibers entered the posterior dorsal ventricular ridge, the cell layer and deep stratum of the ventral lateral cortex, and various amygdaloid nuclei. Parts of the striatum (nucleus accumbens) and pallidum also displayed CGRP-li innervation. In the diencephalon, CGRP-li innervation was observed in parts of the dorsal thalamus and in the periventricular and medial hypothalamus. The pretectum and deep layers of the optic tectum also showed CGRP-li fibers, and numerous CGRP-li fibers were observed in the midbrain central gray, tegmentum, and pons. Some of the sensory fibers of the trigeminal, vagal, and spinal nerves were also CGRP-li. These results show that the distribution of CGRP-li structures in the reptilian brain is similar to that described for other vertebrates and suggest that the thalamotelencephalic CGRPergic projections appear to be conserved among amniote vertebrates.

Understanding the basic circuitry of the cerebral hemispheres: the case of lizards and its implications in the evolution of the telencephalon.

The organization of the cerebral hemispheres of mammals is characterized by corticostriatal glutamatergic projections and striatopallidal GABAergic ones, plus the descending projections of the pallium and subpallium to extratelencephalic targets. The present review of the available neuroanatomical data on the forebrain of lizards suggests that the telencephalon of reptiles also follows this basic pattern of connectivity. In addition, we show that this basic circuitry includes a pallido-cortical projection, therefore forming a cortico-striato-pallido-cortical circuit. The analysis of this circuitry for the medial, dorsal, lateral, and ventral pallial divisions in reptiles and mammals leads to the following conclusions: (1) The medial and dorsal cortices of lizards together appear to be equivalent to the medial pallium of mammals. (2) The projection from the lacertilian dorsal cortex to the striatum proper resembles the subiculo-striatal projection of mammals, rather than the isocortical projection to the caudatus-putamen. (3) Most of the dorsal striatum of reptiles is engaged in the corticostriatal circuit corresponding to the ventral pallium (the anterior dorsal ventricular ridge), and therefore, it is not equivalent to the mammalian caudatus-putamen, which is involved in the circuit of the dorsal pallium. (4) The main and accessory olfactory bulbs also follow this pattern of connections.

Attractive properties of sexual pheromones in mice: innate or learned?

It is generally assumed that chemical signals (sexual pheromones) constitute the primary stimulus for sexual attraction in many mammals. However, it is unclear whether these pheromones are volatile or nonvolatile and which sensory systems are involved in their detection (vomeronasal and/or olfactory). Moreover, it has been demonstrated that experience influences the behavioral response to sexual pheromones and the sensory systems implicated. In order to clarify this issue, the attractive properties of volatile and nonvolatile components of the male-soiled bedding have been analyzed in female mice that had no previous experience with adult male-derived chemical signals (chemically naïve females) using two-choice preference tests. The results indicate that some nonvolatile male-derived substances exert an innate attraction to females, but volatiles derived from male-soiled bedding do not attract chemically nai;ve females. Therefore, the primary attractive sexual pheromone includes a nonvolatile compound (e.g. major urinary proteins, MUPs). On the other hand, male-derived volatiles become attractive to females because of repeated exposure to male-soiled bedding. This represents a Pavlovian-like associative learning in which previously neutral volatiles (very likely odorants) acquire attractive properties by association with the nonvolatile, innately attractive pheromone(s). These findings indicate that not only the sexual but also the 'chemical' experience (previous experience with sexual pheromones) has to be taken into account to interpret the role of chemicals as releaser or primer pheromones. The sensory systems involved in the detection of these stimuli and the neural basis of the odor-pheromone association are discussed.

Differential efferent projections of the anterior, posteroventral, and posterodorsal subdivisions of the medial amygdala in mice.

The medial amygdaloid nucleus (Me) is a key structure in the control of sociosexual behavior in mice. It receives direct projections from the main and accessory olfactory bulbs (AOB), as well as an important hormonal input. To better understand its behavioral role, in this work we investigate the structures receiving information from the Me, by analysing the efferent projections from its anterior (MeA), posterodorsal (MePD) and posteroventral (MePV) subdivisions, using anterograde neuronal tracing with biotinylated and tetrametylrhodamine-conjugated dextranamines. The Me is strongly interconnected with the rest of the chemosensory amygdala, but shows only moderate projections to the central nucleus and light projections to the associative nuclei of the basolateral amygdaloid complex. In addition, the MeA originates a strong feedback projection to the deep mitral cell layer of the AOB, whereas the MePV projects to its granule cell layer. The Me (especially the MeA) has also moderate projections to different olfactory structures, including the piriform cortex (Pir). The densest outputs of the Me target the bed nucleus of the stria terminalis (BST) and the hypothalamus. The MeA and MePV project to key structures of the circuit involved in the defensive response against predators (medial posterointermediate BST, anterior hypothalamic area, dorsomedial aspect of the ventromedial hypothalamic nucleus), although less dense projections also innervate reproductive-related nuclei. In contrast, the MePD projects mainly to structures that control reproductive behaviors [medial posteromedial BST, medial preoptic nucleus, and ventrolateral aspect of the ventromedial hypothalamic nucleus], although less dense projections to defensive-related nuclei also exist. These results confirm and extend previous results in other rodents and suggest that the medial amygdala is anatomically and functionally compartmentalized.

Amygdaloid projections to the ventral striatum in mice: direct and indirect chemosensory inputs to the brain reward system.

Rodents constitute good models for studying the neural basis of sociosexual behavior. Recent findings in mice have revealed the molecular identity of the some pheromonal molecules triggering intersexual attraction. However, the neural pathways mediating this basic sociosexual behavior remain elusive. Since previous work indicates that the dopaminergic tegmento-striatal pathway is not involved in pheromone reward, the present report explores alternative pathways linking the vomeronasal system with the tegmento-striatal system (the limbic basal ganglia) by means of tract-tracing experiments studying direct and indirect projections from the chemosensory amygdala to the ventral striato-pallidum. Amygdaloid projections to the nucleus accumbens, olfactory tubercle, and adjoining structures are studied by analyzing the retrograde transport in the amygdala from dextran amine and fluorogold injections in the ventral striatum, as well as the anterograde labeling found in the ventral striato-pallidum after dextran amine injections in the amygdala. This combination of anterograde and retrograde tracing experiments reveals direct projections from the vomeronasal cortex to the ventral striato-pallidum, as well as indirect projections through different nuclei of the basolateral amygdala. Direct projections innervate mainly the olfactory tubercle and the islands of Calleja, whereas indirect projections are more widespread and reach the same structures and the shell and core of nucleus accumbens. These pathways are likely to mediate innate responses to pheromones (direct projections) and conditioned responses to associated chemosensory and non-chemosensory stimuli (indirect projections). Comparative studies indicate that similar connections are present in all the studied amniote vertebrates and might constitute the basic circuitry for emotional responses to conspecifics in most vertebrates, including humans.

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.

Attraction to sexual pheromones and associated odorants in female mice involves activation of the reward system and basolateral amygdala.

Adult female mice are innately attracted to non-volatile pheromones contained in male-soiled bedding. In contrast, male-derived volatiles become attractive if associated with non-volatile attractive pheromones, which act as unconditioned stimulus in a case of Pavlovian associative learning. In this work, we study the chemoinvestigatory behaviour of female mice towards volatile and non-volatile chemicals contained in male-soiled bedding, in combination with the analysis of c-fos expression induced by such a behaviour to clarify: (i) which chemosensory systems are involved in the detection of the primary attractive non-volatile pheromone and of the secondarily attractive volatiles; (ii) where in the brain male-derived non-volatile and volatile stimuli are associated to induce conditioned attraction for the latter; and (iii) whether investigation of these stimuli activates the cerebral reward system (mesocorticolimbic system including the prefrontal cortex and amygdala), which would support the view that sexual pheromones are reinforcing. The results indicate that non-volatile pheromones stimulate the vomeronasal system, whereas air-borne volatiles activate only the olfactory system. Thus, the acquired preference for male-derived volatiles reveals an olfactory-vomeronasal associative learning. Moreover, the reward system is differentially activated by the primary pheromones and secondarily attractive odorants. Exploring the primary attractive pheromone activates the basolateral amygdala and the shell of nucleus accumbens but neither the ventral tegmental area nor the orbitofrontal cortex. In contrast, exploring the secondarily attractive male-derived odorants involves activation of a circuit that includes the basolateral amygdala, prefrontal cortex and ventral tegmental area. Therefore, the basolateral amygdala stands out as the key centre for vomeronasal-olfactory associative learning.