Modulatory action of zinc on GABA(A) receptor complex during avian CNS development.

In the present work, we studied the effect of zinc on GABA(A) receptor complex at three developmental stages of chick optic lobe (embryonic day 14, post-hatching day 1, and adulthood), in order to investigate the role of this cation in central nervous system (CNS) functional maturation. It was demonstrated that zinc exerts an inhibitory modulation of both GABA binding and GABA-gated chloride flux in a concentration-dependent manner with maximal effects at 100 microM zinc concentration. Maximal inhibition was higher at the embryonic stage and declined thereafter, disclosing minimal values at the adult stage. The effect of zinc on saturation GABA binding experiments performed at embryonic day 14 demonstrated that the cation decreased the maximal number of binding sites (B(max)) from 7. 53 +/- 1.06 pmol/mg protein to 4.63 +/- 0.53 pmol/mg protein, in the absence and presence of 100 microM zinc, respectively, while the dissociation constant (K(d)) remained unchanged. Analysis of the GABA concentration-effect curve at the embryonic stage revealed that the addition of 100 microM zinc decreased E(max) values for GABA stimulation of chloride uptake from 26.46 +/- 2.64% to 16.40 +/- 1. 96%, while EC(50) values were unaffected. In conclusion, our results suggest that zinc acts as a non-competitive inhibitor of both GABA binding and GABA responses during avian CNS development, with its effect inversely related to age.

Biosynthesis of progesterone derived neurosteroids by developing avian CNS: in vitro effects on the GABAA receptor complex.

It has been demonstrated in different vertebrate species that the GABAA receptor complex is modulated by certain steroids. Theses results prompted work on the synthesis of these neurosteroids in the Central Nervous System. However, there are scarcely any studies analyzing their production or their modulatory effects on this receptor during development. In this work, the biosynthesis of [14C]progesterone metabolites as well as the characterization of their in vitro effects on the GABAA receptor complex in developing chick optic lobe were investigated. Studies on progesterone metabolism indicated that this steroid was converted to 5 beta-pregnanedione, 5 beta-pregan-3 beta-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5 beta-pregnanedione was converted to 5 beta-pregnan-3 beta-ol-20-one. Thus, it seems that some of the steroidogenic activities present in chick optic lobe are age-dependent, though greater at embryonic stages. Results from in vitro modulation of [3H]flunitrazepam binding by 5 beta-pregnan-3 beta-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5 beta-pregnan-3 beta-ol-20-one EC50 values were 0.195 +/- 0.049, 0.101 +/- 0.017, 0.147 +/- 0.009, and 0.569 +/- 0.114 microM, and Emax were 22.37 +/- 1.57, 23.67 +/- 4.02, 29.01 +/- 1.08, and 15.11 +/- 2.67% at embryonic days 11, 14, hatching, and postnatal day 21, respectively. In conclusion, the biosynthesis of 5 beta-pregnan-3 beta-ol-20-one from progesterone in developing chick optic lobe, together with its ability to modulate the GABAA receptor present in such tissues, suggests a physiological role of this neurosteroid in developing avian Central Nervous System.

Neurosteroid modulation of GABA binding sites in developing avian central nervous system.

Our aim was to examine the effect of the potent neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) on [3H]-GABA binding to its receptor sites in the chick optic lobe. Binding was performed on synaptic membranes isolated at different stages of development and two different membrane preparation procedures were applied to expose high and low affinity GABA binding sites. The addition of 3 alpha, 5 alpha-P was shown to increase [3H]-GABA binding in an age- and concentration-dependent manner. Maximal stimulation for low affinity GABA binding sites was observed at hatching (130% enhancement), in fresh-washed as well as in frozen membranes. Saturation analysis performed on both membrane types disclosed that 3 alpha, 5 alpha-P increases the affinity of low affinity GABA binding sites without altering their maximal binding capacity. On the other hand, the augmenting effect at high affinity sites, displayed only in frozen membranes, was roughly 50% for all developmental stages. However, their saturation binding parameters remained unaltered in the presence of the steroid, suggesting that stimulation of such sites seems due to interference exerted by the low affinity site population. Findings indicate that 3 alpha, 5 alpha-P acts as an allosteric modulator only for low affinity GABA binding sites, displaying an age-dependent profile probably related to plastic events during visual pathway development.

Ontogeny of two different benzodiazepine binding sites in the chick optic lobe.

The developmental time-course of type I and type II benzodiazepine receptors in the chick optic lobe was determined using a triazolopyridazine, CL 218872. At embryonic day 13 most of the binding sites corresponded to type II (98.23%), while type I represented only a minor proportion (1.77%). During development there was an increase in type I binding sites which reached 62.88% in adulthood, while type II binding sites decreased to 37.12%. These results demonstrate a differential ontogeny of two benzodiazepine receptor subtypes. Changes in the benzodiazepine binding population may account for the variability in the GABA-benzodiazepine receptor interaction during chick optic lobe development.

Effect of diazepam on the low affinity GABA binding sites at different developmental stages of the chick optic lobe.

The developmental profile of GABA receptor sites was studied under experimental conditions where the low affinity binding site is mainly measured. The in vitro effects of nanomolar concentrations of diazepam upon low affinity GABA binding sites during development were also examined. The addition of 50 nM diazepam enhanced specific [(3)H]GABA binding at all stages of development, this stimulation being greater at early stages (10th-14th embryonic day). Scatchard analysis performed at the 14th embryonic day revealed only the binding sites of low affinity with a K(d) of 520 Nm and a maximal number of binding sites (B(max)) of 7.60 pmol/mg protein. At hatching two populations of GABA binding sites were present: one with high affinity (K(d) = 10.47 nM and B(max) = 0.22 pmol/mg protein) and the other with low affinity (K(d) = 686.51 nM and B(max) = 13.28 pmol/mg protein). The addition of 50 nM diazepam increased the affinity of the low affinity GABA binding sites in both stages of development, while that of the high affinity ones remained unchanged. The apparent K(d)s of [H(3)]GABA binding at the 14th embryonic day and at hatching were 120.50 and 452.06 nM, respectively, while the densities of receptor sites were unchanged. Our results show that diazepam increases low affinity GABA binding in an age-dependent manner, this effect being higher in tissue obtained from embryonic chicken optic lobe as compared to the adult one. The binding data obtained in this study suggest a tighter coupling of the GABA-benzodiazepine complex at early stages of development.