Amparo Novejarque

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.

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.

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

BackgroundVertebrates 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.ResultsAlthough 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.ConclusionThe 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.

Evolution of the Amygdala in Vertebrates

The main aim of this article is to identify the homologues of the different components of the mammalian amygdala in the cerebral hemispheres of non-mammals using, primarily, a topological/embryological perspective. Thus, we first consider two main divisions of the amygdala of mammals, namely the pallial and subpallial (striatopallidal) amygdala. The pallial amygdala includes derivatives of both the lateral and ventral embryonic pallium that in the adult conform layered, superficial areas usually called cortical amygdala, and deep nuclei that conform the basolateral division of the amygdala plus the amygdalohippocampal area (AHA). The components of the subpallial amygdala are usually grouped in two divisions known as central (central amygdala plus parts of the bed nucleus of the stria terminalis, BST) and medial (medial amygdala plus the posteromedial BST) extended amygdala (EA). We then characterize each of the pallial and subpallial components of the mammalian amygdala using neurochemical and hodological data from the literature. After dissecting out and characterizing the amygdaloid centers of mammals, we use the same criteria (topological/embryological, neurochemical, and hodological) to identify the different components of the reptilian amygdala. This approach reveals that the cortical amygdala of reptiles is composed of the nucleus sphericus and the ventral anterior amygdala, plus maybe portions of the caudal lateral cortex. The reptilian basolateral amygdala includes the posterior dorsal ventricular ridge and the dorsolateral amygdaloid nucleus. In addition, the ventral posterior amygdala seems the reptilian homologue of the mammalian AHA. As in mammals, centers in the subpallial amygdala of reptiles conform a central (striatoamygdaloid transition area and dorsolateral BST) and medial (medial amygdala plus the ventromedial BST) EA. The strong similarities between the avian and reptilian cerebral hemispheres allow us to make a proposal for the identity of the amygdala and its components in the avian telencephalon. This proposal, which nicely fits the embryological/topological, hodological, and neurochemical criteria used to define the divisions of the mammalian amygdala, suggests that the avian amygdala is much larger than previously believed. Whereas in birds the cortical amygdala is reduced to a small rim of olfacto-recipient tissue in the caudal cerebral hemispheres (posterior cortex piriformis plus the surface of the rostral arcopallium), the avian basolateral amygdala consists of the rest of the arcopallium and most of the caudal nidopallium. In addition, the posterior amygdala is the best candidate for the avian homologue of the AHA of mammals. Finally, the nonpallial centers of avian amygdala can also be grouped into a central (SpA and lateral BST) and a medial (nucleus teniae and medial BST) EA. This thorough comparative analysis suggests that the amygdala is an ancient component of the cerebral hemispheres of tetrapods that includes two functional subsystems, namely the central/basolateral and the medial subsystem (which includes the medial EA and the AHA), involved in managing two different, but closely related, functions. The central/basolateral subsystem coordinates innate and learned reactions of fear/anxiety/aversion (through the descending projections of the central EA) or of attraction/reward-directed behaviors (through its projections to the striatum) to virtually any stimulus. The medial subsystem is primarily involved in the coordination of species-specific behavioral responses to chemosensory stimuli (olfactory and vomeronasal) with a strong emotional component, such as reproductive behaviors, defensive/aggressive behaviors to conspecifics (agonistic behaviors), or to predator-derived chemosignals. The deep interconnections of both subsystems explain why reproductive-agonistic behaviors are strongly emotional and might mediate learned emotional responses to many odorants.

Distribution of CGRP‐like immunoreactivity in the chick and quail brain

Calcitonin gene‐related peptide (CGRP)‐containing neurones have been implicated in the transmission of visceral sensory information to the cortex and in the control of arterial blood pressure in mammals. However, little is known about its function in other vertebrates. As a first step toward investigating the function of CGRP in birds, its distribution was studied in the domestic chick and quail brain by means of immunocytochemistry, by using antibodies against rat CGRP. The distribution of CGRP immunoreactivity in the chick and quail central nervous system was found to be similar. CGRP‐immunoreactive (CGRPi) perikarya were not present in the telencephalon. In the diencephalon, CGRPi perikarya were present mainly in the shell of the thalamic nucleus ovoidalis, the nucleus semilunaris paraovoidalis, the nucleus dorsolateralis posterior thalami, and in the hypothalamic nucleus of the ansa lenticularis. In the brainstem, CGRPi perikarya were present in the nucleus mesencephalicus nervi trigemini, the nucleus tegmenti ventralis, the locus coeruleus, the nucleus linearis caudalis and in the parabrachial region. In addition CGRPi perikarya were found in the motor nuclei of the III, IV, V, VI, VII, IX, X, and XII cranial nerves. The telencephalon contained CGRPi fibres within the paleostriatal complex (mainly in the ventral paleostriatum), parts of the neostriatum and ventral hyperstriatum, parts of the archistriatum, and the septum. In the diencephalon, the densest plexus of CGRPi fibres was observed in the dorsal reticular thalamus. A less dense CGRPi innervation was present in some dorsal thalamic nuclei and in the medial and periventricular hypothalamus. The pretectum and midbrain tegmentum also contained CGRPi fibres, whereas the optic tectum was virtually devoid of immunolabelling. Scattered CGRPi fibres were observed in the central grey and neighbouring pontine areas. Some of the sensory fibres of the trigeminal, vagal, glossopharyngeal, and spinal nerves were also CGRPi. The results of comparative studies indicate that the presence of CGRP in some thalamo‐telencephalic projections is a primitive feature of the forebrain of amniotes. Therefore, the brain areas giving rise to and receiving such a projection in different vertebrates, are likely to be homologous. J. Comp. Neurol. 421:515–532, 2000.

Vomeronasal inputs to the rodent ventral striatum

Vertebrates sense chemical signals through the olfactory and vomeronasal systems. In squamate reptiles, which possess the largest vomeronasal system of all vertebrates, the accessory olfactory bulb projects to the nucleus sphericus, which in turn projects to a portion of the ventral striatum known as olfactostriatum. Characteristically, the olfactostriatum is innervated by neuropeptide Y, tyrosine hydroxylase and serotonin immunoreactive fibers. In this study, the possibility that a structure similar to the reptilian olfactostriatum might be present in the mammalian brain has been investigated. Injections of dextran-amines have been aimed at the posteromedial cortical amygdaloid nucleus (the putative mammalian homologue of the reptilian nucleus sphericus) of rats and mice. The resulting anterograde labeling includes the olfactory tubercle, the islands of Calleja and sparse terminal fields in the shell of the nucleus accumbens and ventral pallidum. This projection has been confirmed by injections of retrograde tracers into the ventral striato-pallidum that render retrograde labeling in the posteromedial cortical amygdaloid nucleus. The analysis of the distribution of neuropeptide Y, tyrosine hydroxylase, serotonin and substance P in the ventral striato-pallidum of rats, and the anterograde tracing of the vomeronasal amygdaloid input in the same material confirm that, similar to reptiles, the ventral striatum of mammals includes a specialized vomeronasal structure (olfactory tubercle and islands of Calleja) displaying dense neuropeptide Y-, tyrosine hydroxylase- and serotonin-immunoreactive innervations. The possibility that parts of the accumbens shell and/or ventral pallidum could be included in the mammalian olfactostriatum cannot be discarded.

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.

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.

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. J. Comp. Neurol. 479:287–308, 2004.

Striato-amygdaloid transition area lesions reduce the duration of tonic immobility in the lizard Podarcis hispanica

Neuroanatomical data suggest that the lizard striato-amygdaloid transition area is homologous with the mammalian central amygdala. In order to investigate possible functional similarities, tonic immobility was induced in adult lizards and its duration recorded. Each lizard was then randomly assigned to one of three treatments: (1) bilateral striato-amygdaloid transition area lesions, (2) bilateral dorsal cortex lesions or (3) untreated controls. Three days after trial 1, each lizard was subjected to a second trial and the tonic immobility duration recorded. The mean tonic immobility duration in lizards with striato-amygdaloid transition area lesions was significantly shorter (80.5%; p < 0.0033) in trial 2 than in trial 1. There were no inter-trial differences within dorsal cortex-lesioned lizards or untreated controls. There was a significant treatment effect on tonic immobility duration in trial 2 (p < 0.0001). The mean tonic immobility duration of lizards with striato-amygdaloid transition area lesions was significantly shorter than that of dorsal cortex-lesioned lizards (72.2%; p < 0.01) or untreated controls (78.2%; p < 0.01). There was no significant difference in mean tonic immobility duration between dorsal cortex-lesioned lizards and untreated controls. Tonic immobility is considered to be an anti-predator behaviour that reflects the underlying state of fear. Therefore, the reduced tonic immobility duration in lizards with striato-amygdaloid transition area lesions reflects a reduction of fear. These results provide the first data to indicate a functional similarity between the lizard striato-amygdaloid transition area and the mammalian central amygdala.