Manuel J. Andreu

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.

Calbindin-D28k in Cortical Regions of the Lizard Psammodromus algirus

The morphology, distribution, and ultrastructural features of calbindin‐D28k–immunoreactive neurons and fibers in the cortical regions of the lizard Psammodromus algirus, considered homologues to the mammalian hippocampal formation, were analyzed by using the peroxidase anti‐peroxidase technique at the light and electron microscopic level. On the basis of staining properties and localization, two distinct populations of calbindin‐D28k–immunoreactive neurons were observed in both the medial and dorsal cortices. Those located in the cell layer, namely principal neurons, were weakly immunostained, whereas a number of Golgi‐like stained neurons were observed in plexiform layers. Double immunocytochemistry showed that all calbindin immunoreactive neurons in the deep plexiform layers were also γ‐aminobutyric acid immunoreactive. We consider them as a population of nonprincipal neurons different from those containing the calcium‐binding proteins parvalbumin and calretinin. Two types of immunoreactive Boutons were revealed by electron microscopy on the basis of the synaptic specialization: Boutons making asymmetrical synapses were generally smaller in size and contacted on small dendritic profiles or cell bodies, whereas larger boutons established symmetrical synapses mainly on dendritic shafts. We propose that the first type of boutons arises from principal neurons and that the second type arises from nonprincipal ones. Finally, the staining pattern, localization, and the circuit in which nonprincipal calbindin‐immunoreactive neurons and other neurochemically defined neurons could be involved in cortical regions of Psammodromus are compared with those of mammalian hippocampus. J. Comp. Neurol. 405:61–74, 1999.

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.

Calcium-Binding Proteins in the Dorsal Ventricular Ridge of the Lizard Psammodromus algirus

The aim of the present work was to study further the intrinsic organization of the dorsal ventricular ridge of lizards. For that purpose, the morphology and distribution of cells and fibers containing the calcium‐binding proteins calbindin‐D28k, parvalbumin, and calretinin were investigated by using immunohistochemical methods. Colocalization of calcium‐binding proteins with the neurotransmitter γ‐aminobutyric acid (GABA) was also studied because they are shown to coexist in many areas of the telencephalon where they define distinct subpopulations of GABAergic local circuit neurons. Neurons containing calcium‐binding proteins are limited to the anterior part of the dorsal ventricular ridge (ADVR), whereas the posterior or caudal portion of the ridge is devoid of immunoreactive cells. This result gives further evidence for defining both regions of the dorsal ventricular ridge. Calcium‐binding proteins mark three distinct populations of neurons within the ADVR. Two of them, parvalbumin‐ and calretinin‐expressing cells, are GABAergic. On the other hand, calbindin‐containing neurons do not express GABA, and the possibility is discussed that these cells are projection neurons. The distribution and overall density of fibers immunoreactive to calcium‐binding proteins suggests that most fibers are of extrinsic origin, the thalamic nuclei projecting to the ADVR and the lateral amygdala being good candidates for their origin. The comparison of data on the populations of calcium‐binding protein‐containing neurons in the reptilian ADVR with those of mammals illustrate the difficulty in finding a mammalian homologue for this controversial region of the reptilian telencephalon. J. Comp. Neurol. 405:32–44, 1999.

Monoaminergic innervation patterns in the anterior dorsal ventricular ridge of a lacertid lizard, Psammodromus algirus.

In contrast to the view of a diffuse monoaminergic innervation of the telencephalon, studies on the monoaminergic innervation in certain mammalian isocortical regions have shown a high degree of regional and laminar specificity. The present study was designed to examine the distribution patterns of dopamine, noradrenaline and serotonin in a telencephalic structure, the anterior dorsal ventricular ridge, of the sand lizard Psammodromus algirus (Lacertidae) using specific antibodies against each monoamine. The anterior dorsal ventricular ridge receives an abundant monoaminergic innervation compared to that of cortical telencephalic regions. The distribution of the different monoamines presented zonal and regional patterns throughout the ridge. The cell cluster zone was profusely innervated by catecholamines, whereas no serotoninergic fibers innervated the cell bodies in the cluster zone. On the other hand, the periventricular zone was heavily innervated by serotonin, but catecholaminergic fibers were almost lacking. With regard to regional patterns, dopamine exhibited major differences in the mediolateral axis of the anterior dorsal ventricular ridge: dopaminergic innervation was densest in the lateral region, which in other reptiles is described as a target of visual thalamic projections. Whereas the zonal pattern of the monoaminergic innervation of the anterior dorsal ventricular ridge seems to be a constant feature in the reptiles studied to date, the regional pattern varies among reptilian groups, especially taking into account the density of monoaminergic innervation.

A putative striato-dorsal thalamic pathway in lizards

Striatal targets related to the dorsal thalamus were studied in reptiles. The lateral striatum projects to globus pallidus and to three cellular groups associated to the lateral forebrain bundle: the anterior entopeduncular nucleus, the suprapeduncular nucleus, and the ventromedial thalamic nucleus. The projection is heavier on the suprapeduncular nucleus, which in turn projects on nucleus rotundus in the dorsal thalamus. Nucleus rotundus is the origin of a prominent projection to the lateral striatum among other forebrain areas. The intermediomedial striatum projects also to globus pallidus and to the three cellular groups associated with the lateral forebrain bundle, but in this case, the projection is heavier on the ventromedial thalamic nucleus. The latter nucleus targets a number of nuclear aggregates in the ventral tier of the dorsal thalamus, which in turn project to the intermediomedial striatum. As in mammals, the striatum in reptiles may influence through these pathways its input from the dorsal thalamus.

Cholecystokinin Innervation of the Cerebral Cortex in a Reptile, the Lizard Psammodromus algirus

We used light and electron microscopic and immunohistochemical methods to map the distribution of cholecystokinin (CCK) in the cerebral cortex of a lizard, Psammodromus algirus. At light microscopy, the CCK immunoreactivity was limited to fibers and terminals densely innervating all cortical regions except for the lateral (pyriform) cortex which was very slightly immunostained. The CCK-positive terminals were almost restricted to the cell layers in every cortical region where they surrounded immunonegative cell bodies and proximal dendrites of neurons within the layer. No CCK-containing neurons were observed within the cerebral cortex. At the electron microscopic level, most positive structures were presynaptic boutons contacting cell bodies and proximal dendrites. All contacts appeared to form symmetric junctions. both the distribution and type of synaptic contacts of CCK fibers in the cerebral cortex of Psammodromus are very similar to the corresponding features in the hippocampus of mammals, although in this lizard the CCK cortical innervation, unlike that in mammals, is probably of extrinsic origin. Double HRP-retrograde labeling and CCK immunohistochemistry show that part of the CCK in the cerebral cortex of Psammodromus arises from the hypothalamic supramammillary nuclei.

MULTIVARIATE STATISTICAL ANALYSIS OF GOLGI STAINED NEURONS

The multivariate statistical analysis provides a useful method to study neuronal populations. It allows both the objective classification of neuronal types and the study of the morphological variation within a neuronal population. We report a particular example of the use of these techniques on a Golgi study of a complex telencephalic structure, the reptilian anterior dorsal ventricular ridge (aDVR). We present a R-mode factor analysis and a cluster analysis on the results of the factor analysis. Sixteen original variables were chosen for the study in order to obtain the greatest information about the cell body, the dendritic field and the location of 96 Golgi-stained cells. Six factors were obtained after the R-mode factor analysis, the interpretation of which was clear in five of them. This contributed to explain the morphological variation of the neuronal types within the aDVR. The cluster analysis classified the 96 cells into eight groups. Some groups could be ascribed to specific regions of the aDVR.