Fernando Martínez-García

A new stabilizing agent for the tetramethyl benzidine (TMB) reaction product in the histochemical detection of horseradish peroxidase (HRP)

In this paper, an alternative procedure for the histochemical detection of HRP using amonium heptamolybdate (AHM) as a stabilizing agent and tetramethyl benzidine (TMB) as a chromogen is reported. This procedure avoids the two main problems that occur in previous methods using sodium nitroferricyanide (SNF) as the stabilizer, namely, the appearance of needle-shaped crystals at non-specific anatomical sites, and intensive tissue shrinkage. A comparative study of both, the TMB-AHM and TMB-SNF methods, was performed in the analysis of cerebral cortex afferent connections of the lizard Podarcis hispanica. This study demonstrates that the two methods are of similar sensitivity. The TMB-AHM reaction can be carried out at physiological pH (from 6 to 8), thus, avoiding tissue contraction. The reaction product is of an intense blue-green colour and, as with the TMB-SNF method, shows granulation. The appearance of non-specific precipitates is completely avoided when the incubation medium is maintained at a pH in excess of 5.

The pallial amygdala of amniote vertebrates: evolution of the concept, evolution of the structure

Embryological studies indicate that the amygdala includes pallial structures, namely the cortical amygdala (olfactory and vomeronasal) and the basolateral complex deep to it. In squamate reptiles, the cortical amygdala includes secondary olfactory (the ventral anterior amygdala) and vomeronasal centres (the nucleus sphericus). In birds, the situation is far less clear, due to the relative underdevelopment of the chemosensory systems. The basolateral amygdala of squamate reptiles includes two ventropallial structures: the posterior dorsal ventricular ridge and the lateral amygdala. Like their mammalian counterparts, these centres give rise to glutamatergic projections to the striatal (centromedial) amygdala and the ventromedial hypothalamus. Using the same criteria, the caudal neostriatum and the ventral intermediate archistriatum may represent the ventral pallial amygdala of birds. The basal nucleus of the mammalian amygdala is a lateropallial territory. In reptiles, the lateral pallium includes the dorsolateral amygdala, which, like the mammalian basal nucleus, projects bilaterally to the striatum/accumbens and receives distinct cholinergic and dopaminergic innervations. In the avian brain, the same embryological, hodological, and histochemical criteria are met by the area temporo-parieto-occipitalis, the caudolateral neostriatum and the dorsal intermediate archistriatum. Therefore, the projections from these structures to the paleostriatum and the lobus paraolfactorius are amygdalostriatal, rather than corticostriatal connections.

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.

Identification of the reptilian basolateral amygdala: an anatomical investigation of the afferents to the posterior dorsal ventricular ridge of the lizard Podarcis hispanica

The presence of multimodal association in the telencephalon of reptiles has been investigated by tracing the afferent connections to the posterior dorsal ventricular ridge (PDVR) of the lizard Podarcis hispanica. The PDVR receives telencephalic afferents from the lateral (olfactory) and dorsal cortices, and from the three unimodal areas of the anterior dorsal ventricular ridge, in a convergent manner. From the diencephalon, it receives afferents from the dorsomedial anterior and medial posterior thalamic nuclei, and from several hypothalamic nuclei. Brainstem afferents to the PDVR originate in the dorsal interpeduncular nucleus, the nucleus of the lateral lemniscus and parabrachial nucleus.

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.

Amygdalo-hypothalamic projections in the lizard Podarcis hispanica: a combined anterograde and retrograde tracing study.

The cells of origin and terminal fields of the amygdalo‐hypothalamic projections in the lizard Podarcis hispanica were determined by using the anterograde and retrograde transport of the tracers, biotinylated dextran amine and horseradish peroxidase. The resulting labeling indicated that there was a small projection to the preoptic hypothalamus, that arose from the vomeronasal amygdaloid nuclei (nucleus sphericus and nucleus of the accessory olfactory tract), and an important projection to the rest of the hypothalamus, that was formed by three components: medial, lateral, and ventral. The medial projection originated mainly in the dorsal amygdaloid division (posterior dorsal ventricular ridge and lateral amygdala) and also in the centromedial amygdaloid division (medial amygdala and bed nucleus of the stria terminalis). It coursed through the stria terminalis and reached mainly the retrochiasmatic area and the ventromedial hypothalamic nucleus. The lateral projection originated in the cortical amygdaloid division (ventral anterior and ventral posterior amygdala). It coursed via the lateral amygdalofugal tract and terminated in the lateral hypothalamic area and the lateral tuberomammillary area. The ventral projection originated in the centromedial amygdaloid division (in the striato‐amygdaloid transition area), coursed through the ventral peduncle of the lateral forebrain bundle, and reached the lateral posterior hypothalamic nucleus, continuing caudally to the hindbrain.

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.

Connections of the lateral cortex in the lizard Podarcis hispanica

The connections of the lateral cortex of the lizard Podarcis hispanica have been traced using horseradish peroxidase transport techniques. After injections, restricted to the lateral cortex, labelled neurons can be observed bilaterally in the main olfactory bulbs and the diagonal band, contralaterally in the lateral cortex and ipsilaterally in the nucleus of the lateral olfactory tract, the ventral amygdaloid nucleus and also in the area triangularis. An efferent has also been shown on the ipsilateral medial cortex. This pattern of connections supports the hypothesis that the reptilian lateral cortex is comparable to the entorhinal and piriform cortex of mammals.