M. Ángeles Real

Light and electron microscopic evidence for projections from the thalamic nucleus rotundus to targets in the basal ganglia, the dorsal ventricular ridge, and the amygdaloid complex in a lizard

To elucidate the organization and evolution of the tectorotundotelencephalic pathways in birds and reptiles, we reinvestigated at both light and electron microscopic levels the efferent projections of nucleus rotundus in a lizard, using the sensitive tracer biotinylated dextran amine. Our results indicate that nucleus rotundus projects to targets in the basal ganglia (lateral parts of striatum and olfactory tubercle and possibly the globus pallidus), the anterior dorsal ventricular ridge (ADVR), and the amygdaloid complex (the central and possibly lateral amygdaloid nuclei). In these targets, the rotundal axon terminals establish asymmetric, presumably excitatory synaptic contacts, usually with dendrites of local cells. In the ADVR, the rotundal projection terminates in two separate radial regions showing distinct cytoarchitecture: 1) a dorsolateral region that extends radially from the dorsolateral ADVR ventricular surface to the ventral part of the lateral cortex and 2) the lateral part of a ventromedial region that extends radially from the dorsomedial and medial ADVR ventricle to a superficial area interposed between the dorsolateral ADVR and the striatum. These two ADVR regions have different connections with the thalamus and telencephalon, which suggests that they may be involved in different degrees of integration. Our study also suggests that the rotundal projection to the ventromedial ADVR field of lizards may be comparable to the rotundoectostriatal/periectostriatal projection of birds. The connections and pathways involving nucleus rotundus suggest that this nucleus conveys visual information which may play a role in visuomotor, emotional, and visceral functions. J. Comp. Neurol. 424:216–232, 2000.

Calcium-Binding Proteins, Neuronal Nitric Oxide Synthase, and GABA Help to Distinguish Different Pallial Areas in the Developing and Adult Chicken. I. Hippocampal Formation and Hyperpallium

To better understand the formation and adult organization of the avian pallium, we studied the expression patterns of gamma‐aminobutyric acid (GABA), calbindin (CB), calretinin (CR), and neuronal nitric oxide synthase (nNOS) in the hippocampal formation and hyperpallium of developing and adult chicks. Each marker showed a specific spatiotemporal expression pattern and was expressed in a region (area)‐specific but dynamic manner during development. The combinatorial expression of these markers was very useful for identifying and following the development of subdivisions of the chicken hippocampal formation and hyperpallium. In the hyperpallium, three separate radially arranged subdivisions were present since early development showing distinct expression patterns: the apical hyperpallium (CB‐rich); the intercalated hyperpallium (nNOS‐rich, CB‐poor); the dorsal hyperpallium (nNOS‐poor, CB‐moderate). Furthermore, a novel division was identified (CB‐rich, CR‐rich), interposed between hyper‐ and mesopallium and related to the lamina separating both, termed laminar pallial nucleus. This gave rise at its surface to part of the lateral hyperpallium. Later in development, the interstitial nucleus of the apical hyperpallium became visible as a partition of the apical hyperpallium. In the hippocampal formation, at least five radial divisions were observed, and these were compared with the divisions proposed recently in adult pigeons. Of note, the corticoid dorsolateral area (sometimes referred as caudolateral part of the parahippocampal area) contained CB immunoreactivity patches coinciding with Nissl‐stained cell aggregates, partially resembling the patches described in the mammalian entorhinal cortex. Each neurochemical marker was present in specific neuronal subpopulations and axonal networks, providing insights into the functional maturation of the chicken pallium. J. Comp. Neurol. 497:751–771, 2006.

Development of neurons and fibers containing calcium binding proteins in the pallial amygdala of mouse, with special emphasis on those of the basolateral amygdalar complex.

We studied the development of neurons and fibers containing calbindin, calretinin, and parvalbumin in the mouse pallial amygdala, with special emphasis on those of the basolateral amygdalar complex. Numerous calbindin‐immunoreactive (CB+) cells were observed in the incipient basolateral amygdalar complex and cortical amygdalar area from E13.5. At E16.5, CB+ cells became more abundant in the lateral and basolateral nuclei than in the basomedial nucleus, showing a pattern very similar to that of γ‐aminobutyric acid (GABA)ergic neurons. Many CB+ cells observed in the pallial amygdala appeared to originate in the anterior entopeduncular area/ganglionic eminences of the subpallium. The density of CB+ cells gradually increased in the pallial amygdala until the first postnatal week and appeared to decrease later, coinciding with the postnatal appearance of parvalbumin cells and raising the possibility of a partial phenotypic shift. Calretinin (CR) immunoreactivity could be observed in a few cells and fibers in the pallial amygdala at E14.5, and by E16.5 it became a good marker of the different nuclei of the basolateral amygdalar complex. Numerous CB+ and CR+ varicosities, part of which have an intrinsic origin, were observed in the basolateral amygdalar complex from E16.5, and some surrounded unstained perikarya and/or processes before birth, indicating an early formation of inhibitory networks. Each calcium binding protein showed a distinct spatiotemporal expression pattern of development in the mouse pallial amygdala. Any alteration in the development of neurons and fibers containing calcium binding proteins of the pallial amygdala may result in important disorders of emotional and social behavior. J. Comp. Neurol. 488:492–513, 2005.

Distinct types of nitric oxide-producing neurons in the developing and adult mouse claustrum.

We studied at the light and electron microscopic levels the nitric oxide–producing neurons in the mouse claustrum. Nicotinamide adenine dinucleotide phosphate (NADPH)‐diaphorase histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemical staining were used to reveal putative nitrergic neurons. We also analyzed colocalization of nNOS with the inhibitory neurotransmitter γ‐aminobutyric acid (GABA) as well as the ontogenesis of the nNOS‐immunoreactive neurons, providing evidence for different populations of nitrergic neurons in the mouse claustrum. The general staining pattern was similar for the histochemical and the immunohistochemical methods, resulting in neuron and neuropil staining throughout the whole claustrum. We described two populations of nitric oxide–producing neurons in the mouse claustrum on the basis of a different level of nNOS expression. Densely nNOS‐stained neurons were mostly GABA immunoreactive, displayed ultrastructural features typically seen in aspiny neurons, and may originate in the subpallium; they were first seen in the claustrum at embryonic stage 17.5 and probably represent local inhibitory interneurons. Densely stained cells were found from rostral to caudal levels throughout the dorsal claustrum and the endopiriform nucleus. Lightly nNOS‐stained neurons, on the other hand, were more numerous than densely stained ones, especially in the dorsal claustrum. These claustral lightly stained cells, barely observed in the NADPH‐diaphorase reacted sections, were mostly non‐GABAergic, and appeared earlier during ontogenesis than densely stained cells (at embryonic stages 15.5–16.5). We suggest that these neurons are probably projection neurons. J. Comp. Neurol. 465:431–444, 2003.

Embryonic and Postnatal Development of GABA, Calbindin, Calretinin, and Parvalbumin in the Mouse Claustral Complex

We analyzed the development of immunoreactive expression patterns for the neurotransmitter γ‐aminobutyric acid (GABA) and the calcium‐binding proteins calbindin, calretinin, and parvalbumin in the embryonic and postnatal mouse claustral complex. Each calcium‐binding protein shows a different temporal and spatial pattern of development. Calbindin‐positive cells start to be seen very early during embryogenesis and increase dramatically until birth, thus becoming the most abundant cell type during embryonic development, especially in the ventral pallial part of the claustrum. The distribution of calbindin neurons throughout the claustrum during embryonic development partly parallels that of GABA neurons, suggesting that at least part of the calbindin neurons of the claustral complex are GABAergic and originate in the subpallium. Parvalbumin cells, on the other hand, start to be seen only postnatally, and their number then increases while the density of calbindin neurons decreases. Based on calretinin expression in axons, the core/shell compartments of the dorsal claustrum start to be clearly seen at embryonic day 18.5 and may be related to the development of the thalamoclaustral input. Comparison with the expression of Cadherin 8, a marker of the developing dorsolateral claustrum, indicates that the core includes a central part of the dorsolateral claustrum, whereas the shell includes a peripheral area of the dorsolateral claustrum, plus the adjacent ventromedial claustrum. The present data on the spatiotemporal developmental patterns of several subtypes of GABAergic neurons in the claustral complex may help for future studies on temporal lobe epilepsies, which have been related to an alteration of the GABAergic activity. J. Comp. Neurol. 481:42–57, 2005.

Mesencephalic and diencephalic afferent connections to the thalamic nucleus rotundus in the lizard, Psammodromus algirus

The present work is an analysis of the afferent projections to the thalamic nucleus rotundus in a lizard, both at the light‐ and electron‐microscopic level, using biotinylated dextran amine (BDA) as a neuroanatomical tracer. This study has confirmed previously reported afferent projections to nucleus rotundus in reptiles and has also identified a number of new cellular aggregates projecting to this dorsal thalamic nucleus. After BDA injections into nucleus rotundus, retrogradely labelled neurons were observed consistently within the following neuronal groups in the midbrain and the diencephalon: (i) the stratum griseum centrale of the optic tectum; (ii) the nucleus subpretectalis in the pretectum; (iii) the nucleus ansa lenticularis posterior, the posterior nucleus of the ventral supraoptic commissure, and the posteroventral nucleus, in the dorsal thalamus and (iv) the lateral suprachiasmatic nucleus and part of the reticular complex in the ventral thalamus. Tectal axons entering nucleus rotundus were fine and varicose and formed exclusively asymmetric synaptic contacts, mainly on small dendritic profiles. Rotundal neurons had symmetric synapses made by large boutons probably of nontectal origin. After comparing our results with those in other reptiles, birds and mammals, we propose that the sauropsidian nucleus rotundus forms part of a visual tectofugal pathway that conveys mesencephalic visual information to the striatum and dorsal ventricular ridge, and is similar to the mammalian colliculo‐posterior/intralaminar–striatoamygdaloid pathway, the function of which may be to participate in visually guided behaviour.

Distribution of nitric oxide-producing neurons in the developing and adult mouse amygdalar basolateral complex

We analysed the expression of neuronal nitric oxide synthase (nNOS) in the mouse amygdalar basolateral complex (BLC) from embryonic day 15.5 to adult, using standard immunohistochemical methods. Our results indicate that each nucleus of the amygdalar basolateral complex displays a distinct nNOS expression pattern, which is established during the ontogenesis with minor changes in the adult. The basomedial nucleus (BM) exhibited the highest nNOS immunoreactivity in the basolateral complex, observable from early embryonic stages, whereas the lateral nucleus displayed the lowest level of immunoreactivity. The expression pattern for nNOS in the basolateral nucleus differed substantially from that of the lateral and basomedial nuclei, showing a slightly increase in the number of nNOS cells and neuropil staining from intermediate developmental until early postnatal stages. Two distinct types of nitrergic neurons, densely and lightly stained neurons, were observed in the developing basolateral complex. Both types of putative nitrergic neurons were unevenly distributed in the basolateral complex. On the basis of previous data regarding the colocalization between nNOS and GABA in the mouse claustrum, we suggest that nNOS expressing neurons in the basolateral amygdalar complex are both GABAergic and non-GABAergic.

Intrinsic connections in the anterior dorsal ventricular ridge of the lizard Psammodromus algirus.

We have studied the intrinsic connections of the anterior dorsal ventricular ridge (ADVR) in the lacertid lizard Psammodromus algirus by means of retrograde transport of horseradish peroxidase (HRP) and fluorescent labeling with the lipophilic carbocyanine dye DiI. We injected HRP into different regions in the ADVR arrayed in a medial‐to‐lateral sequence, with each consisting of three distinct superficial‐to‐deep zones. When HRP was injected into a given region, many labeled neurons (always located ipsilateral to the injection site) were found at all mediolateral regions of ADVR in locations rostrally distant from the injection site. DiI crystals were applied on different superficial‐to‐deep zones within each region. Two patterns could be recognized: DiI crystals applied on the periventricular (most superficial) zone resulted in a labeling of cells widely distributed throughout the ADVR independently of the mediolateral region of the application site, whereas DiI crystals applied on deeper zones resulted in a staining of cells mostly restricted to a narrow radial area. Results from both types of labeling confirm that the ADVR has a prominent radial component in its intrinsic organization, but they also demonstrate that some areas of the ADVR receive projections from distant, rostrally located neurons in every ipsilateral region of the ridge itself, which establishes a clear non‐radial component. This organization may have important functional properties with regard to a putative integration of different sensory modalities conveyed by thalamic afferent fibers to the ADVR. Last, we analyzed some evolutionary implications of our results.

A proposed homology between the reptilian dorsomedial thalamic nucleus and the mammalian paraventricular thalamic nucleus

We have compared the reptilian dorsomedial thalamic nucleus with the mammalian paraventricular thalamic nucleus from a topographic, chemoarchitectonic, and hodological point of view. Both nuclei are localized to a similar position in the dorsalmost aspect of the dorsal thalamus (midline nuclei). They also are uniformly calretinin-immunoreactive, both their cells and neuropil are strongly immunostained for calretinin, and the dorsomedial nucleus presents a strong calbindin immunoreactivity as well. Finally, the reptilian and the mammalian nuclei share a set of afferent and efferent connections with a number of forebrain structures. On the whole, this set of data allows us to propose that the dorsomedial nucleus and the paraventricular thalamic nucleus are homologous. Both represent an important relay station in pathways connecting the hypothalamus with telencephalic areas involved in visceral and motivational aspects of behavior, such as nucleus accumbens and the central amygdala.

Males but not females show differences in calbindin immunoreactivity in the dorsal thalamus of the mouse model of fragile X syndrome

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of the Fmr1 gene product, fragile X mental retardation protein. Here we analyze the immunohistochemical expression of calcium‐binding proteins in the dorsal thalamus of Fmr1 knockout mice of both sexes and compare it with that of wildtype littermates. The spatial distribution pattern of calbindin‐immunoreactive cells in the dorsal thalamus was similar in wildtype and knockout mice but there was a notable reduction in calbindin‐immunoreactive cells in midline/intralaminar/posterior dorsal thalamic nuclei of male Fmr1 knockout mice. We counted the number of calbindin‐immunoreactive cells in 18 distinct nuclei of the dorsal thalamus. Knockout male mice showed a significant reduction in calbindin‐immunoreactive cells (range: 36–67% lower), whereas female knockout mice did not show significant differences (in any dorsal thalamic nucleus) when compared with their wildtype littermates. No variation in the calretinin expression pattern was observed throughout the dorsal thalamus. The number of calretinin‐immunoreactive cells was similar for all experimental groups as well. Parvalbumin immunoreactivity was restricted to fibers and neuropil in the analyzed dorsal thalamic nuclei, and presented no differences between genotypes. Midline/intralaminar/posterior dorsal thalamic nuclei are involved in forebrain circuits related to memory, nociception, social fear, and auditory sensory integration; therefore, we suggest that downregulation of calbindin protein expression in the dorsal thalamus of male knockout mice should be taken into account when analyzing behavioral studies in the mouse model of FXS. J. Comp. Neurol. 521:894–911, 2013.