Galo Ramirez

Spatial and Temporal Patterns of Neurogenesis in the Chick Retina.

Chick embryo retinas were labelled in ovo by single injections of [3H]thymidine at selected times between days 2 and 12 of incubation. Embryos were later removed, at different stages of development, and the retinas processed for autoradiography of either serial sections or dissociated cell preparations. Analysis of unlabelled cells shows that neurogenesis starts, on day 2 of incubation, in a dorsotemporal area of the central retina, close to the posterior pole and to the optic nerve head. A gradient of neurogenesis spreads from this central area to the periphery, where neurogenesis ends, shortly after day 12, when the last few bipolar cells withdraw from the cell cycle. Additional dorsal‐to‐ventral and temporal‐to‐nasal gradients can be discerned in our autoradiographs. In all retinal sectors, ganglion cells start first to withdraw from the cell cycle, followed, with substantial overlapping, by amacrine, horizontal, photoreceptor plus Müller, and bipolar neuroblasts. Ganglion cells are also the first to reach the 50% level of unlabelled cells, followed this time by horizontal, photoreceptor, amacrine, Müller and bipolar cells. Finally, 100% levels of unlabelled cell populations are attained simultaneously by ganglion, horizontal and photoreceptor cells, followed by amacrine, then by Müller, and last by bipolar cells. Although all classes of neurons, in varying proportions, are being produced most of the time, our results also demonstrate that, in any given retinal area, the first cells leaving the cycle are determined to become ganglion cells, and the last ones bipolar cells, and not other types.

Effect of orally administered guanosine on seizures and death induced by glutamatergic agents.

Intraperitoneal guanosine has been shown to prevent quinolinic acid-induced seizures in mice. In this study, we investigated the effect of orally administered guanosine on seizures induced by the glutamate agonists quinolinic acid and kainate, and the endogenous glutamate releaser alpha-dendrotoxin. Guanosine (7.5 mg/kg, per os), administered 75 min in advance, prevented 70% of seizures induced by i.c.v. quinolinic acid, being as efficient as the NMDA channel blocker MK-801 administered intraperitoneally. Guanosine was ineffective against kainate-induced seizures, but significantly reversed the potentiation of seizures and death caused by the concomitant injection of MK-801. Guanosine also significantly prevented seizures and death induced by i.c.v. alpha-dendrotoxin, whereas MK-801 and phenobarbital only prevented death. Altogether, our findings underscore the therapeutic potential of oral administration of guanosine for treating diseases involving glutamatergic excitotoxicity, including epilepsy.

Effects of guanine nucleotides on kainic acid binding and on adenylate cyclase in chick optic tectum and cerebellum.

Adenylate cyclase activity and binding of neurotransmitters to some receptors can be modulated simultaneously by guanine nucleotides. Furthermore it has been shown, in different neurotransmitter systems, that the ability of GTP to inhibit agonist binding is related to the capacity of the transmitter to modulate adenylate cyclase activity. In the present report we show that in chick optic tectum and cerebellum the effects of guanine nucleotides on kainic acid binding and on adenylate cyclase activity can be dissociated. In lysed membrane preparations, GTP, GDP, and GMP, or their analogs, displace binding of kainic acid with the same efficiency, whereas only GTP stimulates adenylate cyclase. In vesicle preparations, all three nucleotides inhibit binding of kainic acid without modifying adenylate cyclase activity. The present results suggest that, if adenylate cyclase is indeed coupled to this particular type of excitatory amino acid receptor, the coupling mechanism would be probably different from those operating in other neurotransmitter systems and also that the displacement of kainic acid by GDP and GMP (and even perhaps by GTP) is not likely to depend on the interaction between the receptor and a Gs-protein-mediated effector system.

Structural proteins of bacteriophage ∅29

Bacteriophage ∅29, specific for some species of Bacillus, is formed of a head with fibers, a neck, and a short tail. Complete particles were dissociated by treatment with dimethyl sulfoxide or EDTA into heads, heads with neck, and necks with tail. The head contains at least three polypeptides (molecular weights 54,000, 48,000, and 28,000). The neck consists of two collars and twelve appendages, and it is formed by three polypeptides (molecular weights, 80,000, 40,000, and 36,000). The tail contains one polypeptide (molecular weight, 71,000). Heads isolated from lysates contain, besides the two major polypeptides present in the head of normal particles, two additional polypeptide chains. The seven structural polypeptides of ∅29 account for about 60% of the genetic information of phage DNA (molecular weight, 11 × 106 daltons).

Guanine nucleotides inhibit the stimulation of GFAP phosphorylation by glutamate.

Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by guanine nucleotides. Recent work has shown that guanine nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that guanine nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.

Differential effects of guanine nucleotides on kainic acid binding and on adenylate cyclase activity in chick optic tectum

In G protein‐coupled receptors, neurotransmitter‐induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor—G protein complex. In the present study we show that, in the chick optic tectum, guanine nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP‐PNP, a non‐hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP‐binding sites are pre‐saturated with GMP‐PNP. Experiments carried out with these membranes show that GMP‐PNP, GDP‐S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP‐PNP activates adenylate cyclase activity by binding to G proteins.

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand‐binding core in the presence of glutamate and kainate

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three‐dimensional structure of the extracellular ligand‐binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand‐binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand‐binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate‐bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand‐binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed. Proteins 2001;44:460–469.

Effects of Guanine Nucleotides on Glutamate-Induced Chemiluminescence in Rat Hippocampal Slices Submitted to Hypoxia

Glutamate significantly increased levels of spontaneous chemiluminescence (CL) in rat hippocampal slices incubated under hypoxic conditions. Although it has been previously shown that guanine nucleotides (GN) displace glutamate from several of its receptors, in our study only GMP, as well as the glutamate antagonist MK-801, was able to reverse the increase in CL provoked by glutamate. On the other hand, not only GTP or Gpp(NH)p failed to reverse the action of glutamate, but they increased CL production like glutamate. This effect of GTP/Gpp(NH)p was also reversed by GMP. We concluded that, under neurotoxic conditions, GMP acted as an antagonist and GTP or Gpp(NH)p acted as agonists of glutamate. These results reinforced the evidence of the existence of extracellular site(s) for GN and indicated a possible role for GN in excitotoxicity.

Guanine nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation

Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti‐excitotoxic potency of guanine nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.

Two modes of free migration of amacrine cell neuroblasts in the chick retina

SummaryThe migration of amacrine neuroblasts toward the prospective amacrine cell layer in the chick embryo retina has been studied, in Golgi-stained sections, between days 5 and 9 of embryogenesis.Two distinct populations of presumptive amacrine neuroblasts have been identified on the basis of their shape and migratory behavior. One population (smooth amacrine neuroblasts) display smooth, monopolar or bipolar contours, moving freely across the retina without major changes in the original postmitotic shape, and give processes only after reaching the primitive inner plexiform layer. The second population (multipodial amacrine neuroblasts) includes multipolar neuroblasts with abundant filiform and/or lamelliform processes sprouting in various directions; these highly plastic cells begin modifying their shapes at the time of release from the ventricular lining and continue to do so as they move toward their definitive location.Thus, the well-known heterogeneity of adult amacrine cells seems to be preluded by differences in neuroblastic migratory patterns, suggesting the existence of at least two different subsets of amacrine cell precursors.