Salvador Guirado

Expression of calcium-binding proteins in the diencephalon of the lizard Psammodromus algirus.

This work is a study of the distribution pattern of calbindin–D28k, calretinin, and parvalbumin in the diencephalic alar plate of a reptile, the lizard Psammodromus algirus, by using the prosomeric model (Puelles [1995] Brain Behav Evol 46:319–337), which divides the alar plate of the diencephalon into the caudorostrally arranged pretectum (p1), dorsal thalamus plus epithalamus (p2), and ventral thalamus (p3). Calbindin and calretinin are more extensively expressed in the dorsal thalamus than in the neighboring alar regions, and therefore these calcium–binding proteins are particularly suitable markers for delimiting the dorsal thalamus/epithalamus complex from the ventral thalamus and the pretectum . Conversely, parvalbumin is more intensely expressed in the pretectum and ventral thalamus than in the dorsal thalamus/epithalamus complex. Within the dorsal thalamus , calcium–binding protein immunoreactivity reveals a three–tiered division. The pretectum displays the most intense expression of parvalbumin within the diencephalon. Virtually all nuclei in the three sectors of the pretectum (commissural, juxtacommissural, and precommissural) present strong to moderate expression of parvalbumin. We compare the distribution of calcium–binding proteins in the diencephalon of Psammodromus with other vertebrates, with mammals in particular, and suggest that the middle and ventral tiers of the reptilian dorsal thalamus may be comparable to nonspecific or plurimodal posterior/intralaminar thalamic nuclei in mammals, on the basis of the calcium–binding protein expression patterns, as well as the hodological and embryological data in the literature. J. Comp. Neurol. 427:67–92, 2000.

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.

Expression of calcium-binding proteins in the mouse claustrum

The present paper describes the distribution of three calcium-binding proteins (calbindin D28k, calretinin, and parvalbumin) in the mouse dorsal claustrum and endopiriform nucleus. The three calcium-binding proteins were distinctly expressed in structures of both the claustrum and the endopiriform nucleus. Calbindin was the calcium-binding protein showing the highest expression in the claustrum and the endopiriform nucleus. In contrast, calretinin-immunoreactive structures, particularly cell bodies, were very scarce in these regions. Both calbindin-immunoreactive and parvalbumin-immunoreactive neurons were more abundant in the claustrum than in the endopiriform nucleus, and more in rostral than in caudal levels. Nevertheless, calcium-binding protein immunoreactive neurons constitute a minority population of claustral neurons. The colocalization study of calbindin and parvalbumin immunoreactivities has demonstrated that both calcium-binding proteins are mostly expressed by separate claustral neurons in the mouse. On the other hand, our results on parvalbumin and calretinin immunoreactivity match a novel subdivision of the mouse claustrum mostly based on the pattern of cadherin expression [Neuroscience 106 (2001) 505]. In this sense, we propose that a specific zone of the dorsal claustrum with cell bodies that strongly express Rcad and cadherin-8 would be the selective target for parvalbumin-expressing fibers, and that they would be mostly avoided by calretinin-expressing axons.

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.

Expression of somatostatin and neuropeptide Y in the embryonic, postnatal, and adult mouse amygdalar complex.

Recent developmental studies indicate that distinct neuronal subpopulations in the amygdala, including somatostatin (SOM)‐containing neurons, originate from progenitor domains in the anterior entopeduncular area, thus suggesting a different origin from subpallial territories for amygdalar versus cortical SOM‐expressing interneurons, the latter derived from the dorsal part of the medial ganglionic eminence. In this context, we carried out an immunohistochemical study analyzing spatiotemporal expression patterns for SOM‐ and neuropeptide Y (NPY)‐containing neurons in the embryonic, postnatal, and adult mouse amygdala. Our results indicate that SOM‐ and NPY‐immunoreactive cells are present in the amygdalar complex from embryonic day (E)12.5, and that these peptidergic cells seem to arise from the anterior entopeduncular area progenitor domain. From E12.5 on there was a notable increase in the number and immunoreactivity of cells containing these peptides in distinct territories of the amygdalar complex, reaching a peak around birth. The distribution pattern for NPY neurons was very similar to that of SOM neurons in most nuclei of the amygdala, although the number of NPY neurons was always lower than that of SOM. At postnatal ages a reduction in the number of immunoreactive cells is observed in most amygdalar nuclei, remaining then similar from P14 to the adult. We interpret this reduction of the number of immunoreactive neurons in relation to the increased immunoreactivity for axons that occurs postnatally. We also suggest that the anterior entopeduncular area‐derived SOM‐ and NPY‐containing neurons in pallial and subpallial amygdaloid nuclei become local interneurons and projection neurons, respectively. J. Comp. Neurol. 513:335–348, 2009.

The ascending tectofugal visual system in amniotes: New insights

Ascending tectal axons carrying visual information constitute a fiber pathway linking the mesencephalon with the dorsal thalamus and then with a number of telencephalic centers. The sauropsidian nucleus rotundus and its mammalian homologue(s) occupy a central position in this pathway. The aim of this study was analyzing the rotundic connections in reptiles and birds in relation with comparable connections in mammals, by using biotinylated dextran amines and the lipophilic carbocyanine dye DiI as tracing molecules. In general, rotundic connections in reptiles and birds are quite similar, especially with regards to pretectal and tectal afferences; as a novel finding, we describe varicose fibers arising from nucleus rotundus that reached the developing chick striatum. In addition, this study described the dorsal claustrum as a novel telencephalic target for the suprageniculate nucleus in mammals. Overall, telencephalic projections from the posterior/intralaminar complex of the mammalian thalamus can be compared with the telencephalic projections of the reptilian nucleus rotundus. With the exception of the isocortical connections, the mouse suprageniculate nucleus shares a number of afferent and efferent connections with the sauropsidian nucleus rotundus. Especially significant were the suprageniculate fibers reaching the striatum and then following to reach pallial derivatives such as the lateral amygdala (ventral pallium) and the dorsal claustrum (lateral pallium). These connections can be compared with the rotundic fibers reaching the ventromedial part of the anterior dorsal ventricular ridge in reptiles/entopallium in birds (ventral pallium) and the dorsolateral part of the anterior dorsal ventricular ridge in reptiles (lateral pallium), and probably the mesopallium in birds.

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.

Thalamo-telencephalic connections: New insights on the cortical organization in reptiles

Tracer injections into the dorsal tier of the lacertilian dorsal thalamus revealed an extensive innervation of the cerebral cortex. The medial cortex, the dorsomedial cortex, and the medial part of the dorsal cortex received a bilateral projection, whereas the lateral part of dorsal cortex and the dorsal part of the lateral cortex received only an ipsilateral thalamic projection. Thalamocortical fibers were found superficially in all cortical regions, but in the dorsal part of the lateral cortex, varicose axons within the cellular layer were also observed. The bilateral thalamocortical projection originates from a cell population located throughout the dorsolateral anterior nucleus, whereas the ipsilateral input originates mainly from a rostral neuronal subpopulation of the nucleus. This feature suggests that the dorsolateral anterior nucleus consists of various parts with different projections. The dorsal subdivision of the lateral cortex displayed hodological and topological (radial glia processes) features of a dorsal pallium derivative. After tracer injections into the dorsal cortex of lizards, we found long descending projections that reached the striatum, the diencephalic basal plate, and the mesencephalic tegmentum, which suggests that it may represent a sensorimotor cortex.