Study of the glial cytoarchitecture of the developing olfactory bulb of a shark using immunochemical markers of radial glia.

During development of the olfactory bulb (OB), glial cells play key roles in axonal guiding/targeting, glomerular formation and synaptic plasticity. Studies in mammals have shown that radial glial cells and peripheral olfactory glia (olfactory ensheathing cells, OECs) are involved in the development of the OB. Most studies about the OB glia were carried out in mammals, but data are lacking in most non-mammalian vertebrates. In the present work, we studied the development of the OB glial system in the cartilaginous fish Scyliorhinus canicula (catshark) using antibodies against glial markers, such as glial fibrillary acidic protein (GFAP), brain lipid-binding protein (BLBP), and glutamine synthase (GS). These glial markers were expressed in cells with radial morphology lining the OB ventricle of embryos and this expression continues in ependymal cells (tanycytes) in early juveniles. Astrocyte-like cells were also observed in the granular layer and surrounding glomeruli. Numerous GS-positive cells were present in the primary olfactory pathway of embryos. In the developmental stages analysed, the olfactory nerve layer and the glomerular layer were the regions with higher GFAP, BLBP and GS immuno-reactivity. In addition, numerous BLBP-expressing cells (a marker of mammalian OECs) showing proliferative activity were present in the olfactory nerve layer. Our findings suggest that glial cells of peripheral and central origin coexist in the OB of catshark embryos and early juveniles. These results open the path for future studies about the differential roles of glial cells in the catshark OB during embryonic development and in adulthood.

Comparative analysis of Met-enkephalin, galanin and GABA immunoreactivity in the developing trout preoptic-hypophyseal system.

We studied the organization of Met-enkephalin-containing cells and fibers in the developing preoptic-hypophyseal system of the brown trout (Salmo trutta fario) by immunohistochemistry and determined the relationship of these cells and fibers to the galaninergic and GABAergic systems. Met-enkephalin immunoreactivity was observed in cells in the preoptic area, the hypothalamus and the pituitary of late larvae. In the hypophysis, a few Met-enkephalin-containing cells were present in all divisions of the adenohypophysis, and some immunoreactive fibers were present in the interdigitations of the neural lobe with the proximal pars distalis. Concurrently, GABAergic fibers innervated the anterior and posterior neural lobe. Galanin cells coexisted with Met-enkephalin cells in neuronal groups of the preoptic-hypophyseal system. Galaninergic and GABAergic fibers innervated the preoptic and hypothalamic areas, but GABAergic fibers containing galanin were not observed. These results indicate that Met-enkephalin, galanin and GABA may modulate neuroendocrine activities in the preoptic area, hypothalamus and pituitary during the transition from larval to juvenile period. To better know how the development of the trout preoptic-hypophyseal system takes place, we studied the patterns of cell proliferation and expression of Pax6, a conserved transcription factor involved in the hypophysis development. Pax6 expressing cells and proliferating cells were present in the Rathke's pouch, the hypothalamus and the hypophysis of early larvae. In late larvae, Pax6 expression was no longer observed in these areas, and the density of proliferating cells largely decreased throughout development, although they remained in the hypophysis of late larvae and juveniles, suggesting that Pax6 might play an important role in the early regionalization of the pituitary in the trout.

Expression of radial glial markers (GFAP, BLBP and GS) during telencephalic development in the catshark (Scyliorhinus canicula).

Radial glial cells (RGCs) are the first cell populations of glial nature to appear during brain ontogeny. They act as primary progenitor (stem) cells as well as a scaffold for neuronal migration. The proliferative capacity of these cells, both in development and in adulthood, has been subject of interest during past decades. In contrast with mammals where RGCs are restricted to specific ventricular areas in the adult brain, RGCs are the predominant glial element in fishes. However, developmental studies on the RGCs of cartilaginous fishes are scant. We have studied the expression patterns of RGCs markers including glial fibrillary acidic protein (GFAP), brain lipid binding protein (BLBP), and glutamine synthase (GS) in the telencephalic hemispheres of catshark (Scyliorhinus canicula) from early embryos to post-hatch juveniles. GFAP, BLBP and GS are first detected, respectively, in early, intermediate and late embryos. Expression of these glial markers was observed in cells with radial glia morphology lining the telencephalic ventricles, as well as in their radial processes and endfeet at the pial surface and their expression continue in ependymal cells (or tanycytes) in early juveniles. In addition, BLBP- and GS-immunoreactive cells morphologically resembling oligodendrocytes were observed. In late embryos, most of the GFAP- and BLBP-positive RGCs also coexpress GS and show proliferative activity. Our results indicate the existence of different proliferating subpopulations of RGCs in the embryonic ventricular zone of catshark. Further investigations are needed to determine whether these proliferative RGCs could act as neurogenic and/or gliogenic precursors.

Mitral cell development in the olfactory bulb of sharks: evidences of a conserved pattern of glutamatergic neurogenesis.

In mammals, the development of the olfactory bulb (OB) relies in part on the expression of transcription factors involved in the specifications/differentiation of glutamatergic cells. In a previous study from our group, a high molecular similarity was reported between mammals and cartilaginous fishes regarding the neurogenic mechanisms underlying the development of glutamatergic cells in the telencephalon. However, information about the transcriptional program operating in the development of the glutamatergic system (mainly represented by mitral cells) in the OB is lacking in the catshark Scyliorhinus canicula, a cartilaginous fish. Using immunohistochemistry and in situ hybridization techniques, we have found that, previously to the appearance of the olfactory primordium (OP), proliferating cells expressing Pax6 with molecular hallmarks of progenitor radial glia were located in the ventrolateral pallial ventricular zone. Later in development, when the OP is recognizable, a stream of Pax6-positive cells were observed between the ventricular zone and the OP, where transcription factors involved in mitral cell development in mammals (ScTbr2, ScNeuroD, Tbr1) are expressed. Later in development, these transcription factors became expressed in a layered-like structure where ScVglut1, a marker of mitral cells, is also present. Our data suggest that the transcriptional program related with the specification/differentiation of glutamatergic cells in the telencephalon has been conserved throughout the evolution of vertebrates. These results, in combination with previous studies concerning GABAergic neurogenesis in sharks, have evidenced that the OB of mammals and sharks shares similarities in the timing and molecular programs of development.

Study of pallial neurogenesis in shark embryos and the evolutionary origin of the subventricular zone.

The dorsal part of the developing telencephalon is one of the brain areas that has suffered most drastic changes throughout vertebrate evolution. Its evolutionary increase in complexity was thought to be partly achieved by the appearance of a new neurogenic niche in the embryonic subventricular zone (SVZ). Here, a new kind of amplifying progenitors (basal progenitors) expressing Tbr2, undergo a second round of divisions, which is believed to have contributed to the expansion of the neocortex. Accordingly, the existence of a pallial SVZ has been classically considered exclusive of mammals. However, the lack of studies in ancient vertebrates precludes any clear conclusion about the evolutionary origin of the SVZ and the neurogenic mechanisms that rule pallial development. In this work, we explore pallial neurogenesis in a basal vertebrate, the shark Scyliorhinus canicula, through the study of the expression patterns of several neurogenic markers. We found that apical progenitors and radial migration are present in sharks, and therefore, their presence must be highly conserved throughout evolution. Surprisingly, we detected a subventricular band of ScTbr2-expressing cells, some of which also expressed mitotic markers, indicating that the existence of basal progenitors should be considered an ancestral condition rather than a novelty of mammals or amniotes. Finally, we report that the transcriptional program for the specification of glutamatergic pallial cells (Pax6, Tbr2, NeuroD, Tbr1) is also present in sharks. However, the segregation of these markers into different cell types is not clear yet, which may be linked to the lack of layering in anamniotes.

Effect of latanoprost as adjunctive therapy.

BACKGROUND: Latanoprost may be a useful adjunct in some patients receiving maximum tolerated medical therapy. We report our clinical experience with latanoprost when added to one or two other glaucoma medications. METHODS: Review of the charts of 53 patients with open-angle glaucoma whose intraocular pressure (IOP) was uncontrolled with one or two glaucoma medications and who had latanoprost added as a second or third drug. Patients whose IOP decreased by 3 mm Hg or more were considered to be responders. RESULTS: The shortest length of follow-up was 2.3 months (median 5.8 months). Latanoprost was given as a second medication to 35 patients, of whom 22 (63%) responded, with a mean IOP reduction of 6.1 mm Hg (standard deviation [SD] 2.73 mm Hg) (28.7% [SD 12.10%]). Of the 18 patients to whom latanoprost was given as a third medication, 10 (56%) responded, with a mean IOP reduction of 6.3 mm Hg (SD 3.86 mm Hg) (24.5% [SD 10.12%]). INTERPRETATION: Latanoprost provides additional IOP reduction in some patients with open-angle glaucoma when added to one or two other glaucoma medications.