C. Yanes

Radial glial cells, proliferating periventricular cells, and microglia might contribute to successful structural repair in the cerebral cortex of the lizard Gallotia galloti.

Reptiles are the only amniotic vertebrates known to be capable of spontaneous regeneration of the central nervous system (CNS). In this study, we analyzed the reactive changes of glial cells in response to a unilateral physical lesion in the cerebral cortex of the lizard Gallotia galloti, at 1, 3, 15, 30, 120, and 240 days postlesion. The glial cell markers glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), S100 protein, and tomato lectin, as well as proliferating cell nuclear antigen (PCNA) were used to evaluate glial changes occurring because of cortical lesions. A transitory and unilateral upregulation of GFAP and GS in reactive radial glial cells were observed from 15 to 120 days postlesion. In addition, reactive lectin-positive macrophage/microglia were observed from 1 to 120 days postlesion, whereas the expression of S100 protein remained unchanged throughout the examined postlesion period. The matricial zones closest to the lesion site, the sulcus lateralis (SL) and the sulcus septomedialis (SSM), showed significantly increased numbers of dividing cells at 30 days postlesion. At 240 days postlesion, the staining pattern for PCNA, GFAP, GS, and tomato lectin in the lesion site became similar to that observed in unlesioned controls. In addition, ultrastructural data of the lesioned cortex at 240 days postlesion indicated a structural repair process. We conclude that restoration of the glial framework and generation of new neurons and glial cells in the ventricular wall play a key role in the successful structural repair of the cerebral cortex of the adult lizard.

Cytoarchitectonic subdivisions in the subtectal midbrain of the lizard Gallotia galloti.

Contemporary study of molecular patterning in the vertebrate midbrain is handicapped by the lack of a complete topological map of the diverse neuronal complexes differentiated in this domain. The relatively less deformed reptilian midbrain was chosen for resolving this fundamental issue in a way that can be extrapolated to other tetrapods. The organization of midbrain centers was mapped topologically in terms of longitudinal columns and cellular strata on transverse, Nissl-stained sections in the lizard Gallotia galloti. Four columns extend along the whole length of the midbrain. In dorsoventral order: 1) the dorsal band contains the optic tectum, surrounded by three ventricularly prominent subdivisions, named griseum tectale, intermediate area and torus semicircularis, in rostrocaudal order; 2) a subjacent region is named here the lateral band, which forms the ventral margin of the alar plate and also shows three rostrocaudal divisions; 3) the basal band forms the basal plate or tegmentum proper; it appears subdivided into medial and lateral parts: the medial part contains the oculomotor and accessory efferent neurons and the medial basal part of the reticular formation, which includes the red nucleus rostrally; the lateral part contains the lateral basal reticular formation, and includes the substantia nigra caudally; 4) the median band contains the ventral tegmental area, representing the mesencephalic floor plate. The alar regions (dorsal and lateral) show an overall cellular stratification into periventricular, central and superficial strata, with characteristic cytoarchitecture for each part. The lateral band contains two well developed superficial nuclei, one of which is commonly misidentified as an isthmic formation. The basal longitudinal subdivisions are simpler and basically consist of periventricular and central strata.

Tenascin‐R and axon growth‐promoting molecules are up‐regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down‐regulation of axon growth‐inhibitory proteins, and to what extent outgrowth‐promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth‐inhibiting extracellular matrix molecules tenascin‐R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth‐promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin‐R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin‐R and CSPG were expressed in an extracellular matrix‐, oligodendrocyte/myelin‐ and neuron‐associated pattern and up‐regulated in the regenerating optic pathway. The expression pattern of tenascin‐R was not indicative of a role in channeling or restriction of re‐growing RGC axons. Up‐regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin‐R expression. Moreover, we analyzed the influence of substrates containing tenascin‐R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin‐R, and further improved on mixed substrates containing tenascin‐R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down‐regulation of tenascin‐R or CSPG. Presence of tenascin‐R is insufficient to prevent RGC axon growth, and concomitant up‐regulation of axon growth‐promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up‐regulation of contactin in RGCs suggests that tenascin‐R may have an instructive function during axon regeneration in the lizard optic pathway.

Heterogeneous Immunoreactivity of Glial Cells in the Mesencephalon of a Lizard: A Double Labeling Immunohistochemical Study

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star‐shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear‐shaped cells that are therefore astrocytes with unusual morphology.

Expression of neuronal markers, synaptic proteins, and glutamine synthetase in the control and regenerating lizard visual system

Spontaneous regrowth of retinal ganglion cell (RGC) axons occurs after optic nerve (ON) transection in the lizard Gallotia galloti. To gain more insight into this event we performed an immunohistochemical study on selected neuron and glial markers, which proved useful for analyzing the axonal regrowth process in different regeneration models. In the control lizards, RGCs were beta‐III tubulin‐ (Tuj1) and HuCD‐positive. The vesicular glutamate transporter‐1 (VGLUT1) preferentially stained RGCs and glial somata rather than synaptic layers. In contrast, SV2 and vesicular GABA/glycine transporter (VGAT) labeling was restricted to both plexiform layers. Strikingly, the strong expression of glutamine synthetase (GS) in both Müller glia processes and macroglial somata revealed a high glutamate metabolism along the visual system. Upregulation of Tuj1 and HuCD in the surviving RGCs was observed at all the timepoints studied (1, 3, 6, 9, and 12 months postlesion). The significant rise of Tuj1 in the optic nerve head and optic tract (OTr) by 1 and 6 months postlesion, respectively, suggests an increase of the beta‐III tubulin transport and incorporation into newly formed axons. Persistent Tuj1+ and SV2+ puncta and swellings were abnormally observed in putative degenerating/dystrophic fibers. Unexpectedly, neuron‐like cells of obscure significance were identified in the control and regenerating ON‐OTr. We conclude that: 1) the persistent upregulation of Tuj1 and HuCD favors the long‐lasting axonal regrowth process; 2) the latter succeeded despite the ectopia and dystrophy of some regrowing fibers; and 3) maintenance of the glutamate‐glutamine cycle contributes to the homeostasis and plasticity of the system. J. Comp. Neurol. 518:4067–4087, 2010.

Anterior dorsal ventricular ridge in the lizard: Embryonic development

In lacertids the telencephalic vesicle starts its development at stage E = 30, at which time it is lined by a homogeneous nucleated zone in which particular ventricular zone territories or sulci cannot be distinguished. At stage E = 32 coinciding with the initial development of the anterior dorsal ventricular ridge (ADVR), one may distinguish the ventricular zone b in the dorsolateral wall of the ventricle adjacent to the sulcus lateralis. The ADVR continues growing by incorporation of cells produced in two proliferative zones (zone b and wall of the sulcus lateralis) and appears fully developed in postnatal lizards. Ultrastructural characteristics of young ADVR neurons between stages E‐32 and E‐33 are typical of those in immature cells. Beginning at stage E‐34, some of these neurons appear to be degenerating (pycnotic). Thereafter, neurons of the ADVR develop abundant cytoplasmic organelles and the neuropile grows quickly. Myelination starts in the ADVR between stages E‐38 and E‐40, but is not observed in other striatal masses in the same period. Vascularization begins and is well developed at E‐40. The first synaptic contacts were observed in embryos of stage E=38; they are chiefly axo‐dendritic, although some are axo‐somatic. Degenerating neurons were found in the ADVR up to hatching. From stage E‐40 onward, the ADVR shows a greater and more rapid differentiation than all other striatal nuclei, including the ventral and amygdaloid complex.

Neuronal differentiation patterns in the optic tectum of the lizard Gallotia galloti.

This study examines in detail the sequences of morphological differentiation and deduces mode of migration into specific layers of all types of neurons present in the optic tectum of the lizard Gallotia galloti. It complements previous similar work on tectal histogenesis in the chick. It was found that the neuronal population diversity in the lizard tectum can be reduced by developmental analysis to three neuroblast classes, called Types I, II and III. These classes correspond closely to those present in the developing avian tectum. Neurons belonging to each developmental class were characterized by their initial polarity, mode of translocation into the mantle layer and pattern of sprouting of primary axonal and dendritic processes. Each class produced along time a subset of the cell types distinguished in the mature tectum. Some aspects of sauropsidian tectal histogenesis are also common of other vertebrates, suggesting that fundamental mechanisms of tectal neuronal differentiation are conserved in tetrapods. Analysis of evolutive differences of tectal structure points to changes affecting the layering and perhaps the population size of specific cell types. Whereas tectal cell-type homology can be easily fundamented on embryological evidence and seems to be consistent with hodological and, to some extent, functional homology, the periventricular, central and superficial strata of the tectum are heterogeneous in cellular composition in different species and therefore represent analogous, rather than homologous entities.

Golgi study of the anterior dorsal ventricular ridge in a lizard. I. neuronal typology in the adult

Using Golgi techniques we have studied neuronal cell types in the anterior dorsal ventricular ridge (ADVR) of the adult lizard Gallotia galloti. Multipolar, bitufted, and juxtaependymal neuronal forms were found. The multipolar and bitufted neurons are present in both the periventricular and central ADVR zones. Multipolar neurons can be subdivided into multipolar neurons with polygonal somata and four to six main dendritic trunks and multipolar neurons with pyramidal somata and three or more dendritic trunks. The former are the cells most frequently impregnated in the ADVR. In the population of bitufted neurons, we distinguish subtypes I, II, and III according to the number of dendritic trunks that emerge from the somata. Juxtaependymal neurons are restricted to a cell‐poor zone, adjacent to ependymal cells. Their dendrites either are orientated parallel to the ventricular surface or extend into the periventricular zone. The dendrites of ADVR neurons have pedunculated spines with knob‐like tips. However, such spines do not appear on the somata or on the primary dendritic trunks. The number of spines is scarce or moderate. The periventricular neuronal clusters contain two to five cells. The morphology of these neurons is mainly multipolar, but we also found some bitufted neurons.

Neuronal and glial differentiation during lizard (Gallotia galloti) visual system ontogeny

We studied the histogenesis of the lizard visual system (E30 to adulthood) by using a selection of immunohistochemical markers that had proved relevant for other vertebrates. By E30, the Pax6+ pseudostratified retinal epithelium shows few newborn retinal ganglion cells (RGCs) in the centrodorsal region expressing neuron‐ and synaptic‐specific markers such as betaIII‐tubulin (Tuj1), synaptic vesicle protein‐2 (SV2), and vesicular glutamate transporter‐1 (VGLUT1). Concurrently, pioneer RGC axons run among the Pax2+ astroglia in the optic nerve and reach the superficial optic tectum. Between E30 and E35, the optic chiasm and optic tract remain acellular, but the latter contains radial processes with subpial endfeet expressing vimentin (Vim). From E35, neuron‐ and synaptic‐specific stainings spread in the retina and optic tectum, whereas retinal Pax6, and Tuj1/SV2 in RGC axons decrease. Müller glia and abundant optic nerve glia express a variety of glia‐specific markers until adulthood. Subpopulations of optic nerve glia are also VGLUT1+ and cluster differentiation‐44 (CD44)‐positive but cytokeratin‐negative, unlike the case in other regeneration‐competent species. Specifically, coexpression of CD44/Vim and glutamine synthetase (GS)/VGLUT1 reflects glial specialization, insofar as most CD44+ glia are GS−. In the adult optic tract and tectum, radial glia and free astroglia coexist. The latter show different immunocharacterization (Pax2−/CD44−/Vim−) compared with that in the optic nerve. We conclude that upregulation of Tuj1 and SV2 is required for axonal outgrowth and search for appropriate targets, whereas Pax2+ optic nerve astroglia and Vim+ radial glia may aid in early axonal guidance. Spontaneous axonal regrowth seems to succeed despite the heterogeneous mammalian‐like glial environment in the lizard optic nerve. J. Comp. Neurol. 520:2163–2184, 2012.

Expression of BDNF and NT-3 During the Ontogeny and Regeneration of the Lacertidian (Gallotia galloti) Visual System

Retinal ganglion cell (RGC) axons regrow spontaneously after optic nerve (ON) transection in G. galloti. Because brain‐derived neurotrophic factor (BDNF) is considered the major neurotrophin participating in vertebrate visual system development and promotes RGC survival, we investigated its distribution using dual‐labeling immunohistochemistry for neuronal and glial markers. We examined the developing and regenerating lizard visual system at 1, 3, 6, 9, and 12 months postlesion to comparatively evaluate BDNF expression patterns. BDNF was detected from midembryonic stages (E35) in both retinal plexiform layers, and in radial glial processes in the tectum. Moreover, RGC axon staining was detected at late prenatal stages (E39), showing a transient punctate staining which progressed in a temporo‐spatial pattern that was similar to myelination. Strong expression in RGC axons was maintained in adults. However, transient downregulation of BDNF staining occurred on the experimental side one month after ON transection followed by a gradual recovery with extensive punctate/swelling distribution and persistent upregulation at 12 months. Conversely, quantitative PCR analysis for 1 and 12 months regenerate lizards showed downregulation of the ratio of BDNF mRNA expression at 12 months and nonsignificant changes of NT‐3 transcripts. In summary, we demonstrate that BDNF and NT‐3 are abundantly expressed during lizard visual system ontogeny and regeneration suggesting their participation inthe development, maintenance and plasticity of the system.