Prolonged exposure to hypobaric hypoxia transiently reduces GABA(A) receptor number in mice cerebral cortex.

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.

Synaptic membrane freezing affects modulatory sites in avian central nervous system GABA(A) receptor.

Studies were carried out to determine whether barbiturates and neurosteroids share common recognition sites at the GABA(A) receptor complex in avian CNS. To achieve this, differentially prepared fresh and frozen synaptic membranes were used. Both the barbiturate, pentobarbital, and the neurosteroid, 3alpha-hydroxy-5alpha-pregnan-20-one, were able to stimulate GABA binding in both types of membranes. Stimulation differed markedly when both drugs were added jointly to different treated tissue. In frozen membranes drugs acted synergistically and were differentially displaced by picrotoxinin, while in fresh ones, where both compounds were inhibited by the convulsant, this additivity was absent. Post-freezing wash supernatants were collected and used as a source of putative endogenous factors involved in the above mentioned membrane differences. Addition of a high molecular weight fraction from supernatants to frozen synaptic membranes led to an inhibition of barbiturate and neurosteroid potentiation, as well as a loss of their additive effect. Our results indicate that GABA(A) receptor modulation by barbiturates and neurosteroids is affected by synaptic membrane treatment, with a common modulatory site in fresh membranes and separate recognition sites after a freeze-thawing procedure. There may also be endogenous factors involved in overlapping of modulatory sites, which would thus regulate GABA(A) receptor functionality by direct interaction with the complex.

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.

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.

GABA-stimulated chloride uptake during avian CNS development: modulation by neurosteroids.

In the present report we studied the GABA-stimulated 36Cl- uptake during chick optic lobe development in order to establish the ontogenetic profile of the functional GABAA receptor complex. A concentration-dependent stimulation of 36Cl- influx by GABA was demonstrated, starting at developmental stages as early as 10 days of incubation. The maximal GABA-induced 36Cl- uptake changed significantly during ontogeny with highest values near hatching. However, GABA potency to stimulate ion influx remained unchanged. We also examined the effect of two neurosteroids, allopregnanolone and epipregnanolone, on GABA-stimulated 36Cl- influx at three developmental stages (embryonic day 14, post-hatching day 1 and adult stage). Both steroids enhanced ion uptake in a concentration-dependent manner, exerting greater stimulatory effects at early developmental stages. Allopregnanolone displayed EC50 values lower than epipregnanolone at all three time points and was also more potent at post-hatching stages. Analysis of the GABA concentration-effect curve disclosed that both steroid decreased EC50 values for GABA stimulation while Emax levels were unaffected. In conclusion, our results showed an early appearance of the GABA-associated chloride channel together with the ability of neurosteroids to modulate GABA-gating of such channel.