Chick Optic Lobe Research Articles

Programmed cell death and differential JNK, p38 and ERK response in a prenatal acute hypoxic hypoxia model

We previously found that prenatal hypoxia induces a significant increase in the levels of active Caspase 3 at 60 min post-hypoxia (p-h) and in the number of TUNEL-positive pyknotic cells, which peaks at 6 h p-h. The aim of this work was to study alterations in MAPKs pathways and the effect of specific inhibitors of the JNK (SP600125) and p38 (SB203580) pathways following acute hypoxia in chick optic lobe at embryonic day (ED) 12. To this end, JNK, p38 and ERK1-2 protein kinase expression levels were determined by Western blot in both their active and inactive forms, evaluated at successive p-h times. At 10 and 30 min p-h the P-JNK/JNK ratio was 1.912 ± 0.341 and 1.920 ± 0.304, respectively. Concomitantly, at 0 min p-h the P-p38/p38 ratio was 1.657 ± 0.203. Lastly, the P-ERK/ERK ratio proving non-significant throughout. When inhibitors for JNK and p38 were used, we observed a decrease in the values of active Caspase 3 at 60 min p-h, which correlated with the control values in the parameters of TUNEL-positive cells at 6 h p-h. Analysis for P-ATF-2 demonstrated an increase in hypoxic embryos compared to control ones which was reverted in a dose-dependent manner with the use of both inhibitors. All these results indicate that at ED 12, acute hypoxia might be differentially activating JNK and p38 pathways, without affecting the ERK pathway, which in turn would be activating Caspase 3, thus leading to cell death by apoptosis. Furthermore, JNK and p38 activation precede in time the programmed cell death induced by hypoxia.

Hypoxia-induced Cell Death and Activation of Pro- and Anti-apoptotic Proteins in Developing Chick Optic Lobe

Exposure of the CNS to hypoxia is associated with cell death. Our aim was to establish a temporal correlation between cellular and molecular alterations induced by an acute hypoxia evaluated at different post-hypoxia (p-h) times and at two stages of chick optic lobe development: embryonic days (ED) 12 and 18. TUNEL assays at ED12 disclosed a significant increase (300%) in pyknotic cells at 6 h p-h, while at ED18 no morphological changes were observed in hypoxic versus controls. At ED12 there was a significant increase (48%) in Bcl-2 levels at the end of the hypoxic treatment, followed by a significant increase of active caspase-9 (49%) and active caspase-3 (58%) at 30 and 60 min p-h, respectively, while at ED18 no significant changes were observed. These findings indicate that prenatal hypoxia produces an equilibrated imbalance in both pro- and anti-apoptotic proteins that culminates in a process of cell death, present at earlier stages of development.

Hypoxia differentially reduces GABA(A) receptor density during embryonic chick optic lobe development.

It has been demonstrated that the CNS is severely affected by hypoxic-ischemic insults during the prenatal-perinatal period, including imbalance in excitatory and inhibitory neurotransmitter release. Using a previously developed model of acute normobaric hypoxic hypoxia on chick embryos, we studied alterations observed both on [3H]GABA binding saturation parameters and on lactate concentration on successive embryonic days (ED). While maximal density of GABA binding sites (Bmax) from the low-affinity site was significantly reduced in an age-dependent manner, earlier stages of development (ED12 and 16) proving more vulnerable (ED12: control = 5.48 +/- 0.20, hypoxia = 3.90 +/- 0.39 pmol/mg prot, P < .05; ED16: control = 3.89 +/- 0.26, hypoxia = 2.80 +/- 0.28 pmol/mg prot, P < .05), ligand affinity (Kd) values and kinetic constants of the high-affinity site remained unaltered. Not unlikely, a physiological hypoxic state prevailing from ED17 up to hatching time rendered the whole embryo less sensitive to an externally induced hypoxic state (ED17: control = 2.93 +/- 0.06, hypoxia = 2.38 +/- 0.04 pmol/mg prot, P < .05; ED18: control = 2.97 +/- 0.12, hypoxia = 2.87 +/- 0.27 pmol/mg prot). Lactate levels in chick optic lobe homogenates were constant during development. The increase observed after hypoxic treatment compared to control value was significant at all stages studied, but increased percentage changes proved similar, indicating that all days of development equally perceive externally induced hypoxia. In conclusion, the present work demonstrates that after normobaric hypoxic hypoxia at different embryonic days, the embryo senses the externally induced hypoxic state as from ED12, but the GABA(A) receptor is differentially affected. It may be speculated that a different subunit composition of GABA(A) receptor is assembled in order to build a more stable receptor capable of resisting the physiological hypoxic state observed during the last few days before hatching.

Differential and irreversible CNS ontogenic reduction in maximal MK-801 binding site number in the NMDA receptor after acute hypoxic hypoxia

CNS exposure to hypoxia impairs excitatory and inhibitory neurotransmission. Our aim was to determine variations induced by normobaric acute hypoxic hypoxia (8% O 2 for 60 min) on the NMDA receptor complex, as well as their potential reversibility after normoxic recovery. To this end, [ 3H]MK-801 binding assays to a synaptic membrane fraction isolated from chick optic lobes were performed. Previous studies throughout development had disclosed a characteristic age-dependent pattern. Results at embryonic day (ED) 12 and 18 indicated two distinct MK-801 binding sites. Hypoxic treatment failed to alter either the high affinity site dissociation constant ( K d) or its maximal binding capacity ( B max), whereas the low affinity site B max was significantly decreased (50% and 30% at ED12 and 18, respectively), without alteration in its K d values. Hypoxic embryos restored for 48 h at ED12 to normoxic conditions displayed unchanged MK-801 binding reduction, unlike those treated likewise at ED18 whose values fully recovered control levels. To conclude, hypoxic treatment reduces low affinity MK-801 B max in the NMDA receptor which proves irreversible up to ED12. Such early neuronal vulnerability may be due to post-transcriptional changes, to endocytosis followed by receptor degradation, or alternatively to cell death.

Acute hypoxic hypoxia alters GABA A receptor modulation by allopregnanolone and pentobarbital in embryonic chick optic lobe

Using a previously developed model of acute normobaric hypoxic hypoxia on chick embryos, here we studied at embryonic day 12 the in vitro effect of two positive allosteric modulators of GABA binding, the barbiturate sodium pentobarbital and the neurosteroid allopregnanolone. In both cases an increase in E max values in membranes obtained from hypoxic embryos was observed. Studies of GABA-gated chloride influx showed that there were no differences in maximal chloride uptake between hypoxic and control membranes. We have already demonstrated that maximal density of GABA binding sites was decreased after hypoxia, suggesting that each of the remaining GABA A receptors display a greater chloride flux than controls. To further characterize GABA A receptor alterations, GABA-gated chloride influx modulated by the above barbiturate and neurosteroid was determined, finding that E max values were increased 60% and 42%, respectively. The increase in Cl − influx per receptor subsequent to hypoxic trauma, and the enhancement in the modulatory properties studied, may mediate neuronal damage by potential changes in subunit interaction at the GABA A receptor level.

In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABA A receptor modulatory sites

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5β-pregnan-3β-ol-20-one) on the GABA A receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [ 3H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 μM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 μM epipregnanolone decreased allopregnanolone EC 50 and E max values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K d and B max values for [ 3H]flunitrazepam and [ 3H]GABA binding. [ 3H]GABA binding modulation disclosed an increase in allopregnanolone EC 50 value with a decrease in its E max value. Diazepam EC 50 and E max values were enhanced, while low affinity sodium pentobarbital EC 50 value was reduced by epipregnanolone treatment. The investigation of the GABA A receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce 36Cl − influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [ 3H]flunitrazepam and [ 3H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA A receptor sites and function during chick optic lobe development.

Acute hypoxic hypoxia transiently reduces GABA A binding site number in developing chick optic lobe

The Central Nervous System is known to be critically affected in the prenatal–perinatal period by hypoxic–ischemic insults, which produce several disorders such as loss of neural projections, increased susceptibility to seizures, apoptosis and an imbalance in normal activity of glutamatergic and GABAergic neurones, resulting in acute cell excitotoxicity. The aim of the present work was to establish a chick embryo model of normobaric acute hypoxic hypoxia as well as to evaluate modifications in GABA A receptor complex from chick optic lobe, that may result from this injury. Fertile chicken ( Gallus gallus domesticus) eggs from White Leghorn were incubated and at embryonic days (ED) 12 to 18, subjected to a stream of 8%O 2/92%N 2 during1 h, and then were either returned to their shelves in the incubator for recovery, or immediately processed for biochemical studies. Hypoxic treatment produced a significant age dependent reduction in GABA binding sites showing the greatest decrease at the earliest stages studied (ED12–ED16). Saturation curves of GABA binding performed at ED12 showed a decrease in B max, (control, 5.48±0.20, hypoxic, 3.90±0.39 pmol/mg protein), but no significant change in K d. Following 48 h in normoxic atmosphere post-hypoxia reduction in [ 3H]GABA binding was reversed. Pharmacological properties of GABA A receptor at ED12 showed that positive allosteric modulation effects of the steroid 3α-hydroxy-5α-pregnan-20-one and the barbiturate pentobarbital sodium were enhanced by the treatment. This model of acute prenatal hypoxic hypoxia produced marked alterations in inhibitory CNS neurotransmission that proved reversible and age dependent.

Chick optic lobe contains a developmentally regulated alpha2alpha5beta2 nicotinic receptor subtype.

The most widely expressed neuronal nicotinic acetylcholine receptor subtype in chick brain is that containing the alpha4 and beta2 subunits. However, immunoprecipitation and localization studies have shown that some brain areas also contain the alpha2 and/or alpha5 subunits, whose role in the definition of receptor properties is still intriguing. Using subunit-specific polyclonal antibodies, we found that the optic lobe is the chick central nervous system region that expresses the highest level of alpha2-containing receptors. Immunoprecipitation studies of these immunopurified alpha2-containing receptors labeled with the nicotinic agonist [(3)H]epibatidine showed that almost all of them contained the beta2 subunit and that more than 66% contained the alpha5 subunit. Western blot analyses of the purified receptors confirmed the presence of the alpha2, alpha5, and beta2 subunits and the absence of the alpha3, alpha4, alpha6, alpha7, alpha8, beta3, and beta4 subunits. The alpha2-containing receptors are developmentally regulated: their expression increases 25 times from embryonic day 7 to posthatching day 1 in the optic lobe, compared with an increase of only 5-fold in the forebrain. The alpha2-containing optic lobe receptors bind [(3)H]epibatidine (K(d) = 29 pM) and a number of other nicotinic agonists with very high affinity and have a pharmacological profile very similar to that of the alpha4beta2 subtype. They form functional cationic channels when reconstituted in lipid bilayers, with pharmacological and biophysical properties different from those of the alpha4beta2 subtype. These channels are activated by nicotinic agonists in a dose-dependent manner and are blocked by the nicotinic antagonist d-tubocurarine.

Modulatory action of zinc on GABAA 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.

Modulatory action of zinc on GABAA receptor complex during avian CNS development

In the present work, we studied the effect of zinc on GABAA 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 μM 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 (Bmax) from 7.53 ± 1.06 pmol/mg protein to 4.63 ± 0.53 pmol/mg protein, in the absence and presence of 100 μM zinc, respectively, while the dissociation constant (Kd) remained unchanged. Analysis of the GABA concentration-effect curve at the embryonic stage revealed that the addition of 100 μM zinc decreased Emax values for GABA stimulation of chloride uptake from 26.46 ± 2.64% to 16.40 ± 1.96%, while EC50 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. J. Neurosci. Res. 57:536–540, 1999. © 1999 Wiley-Liss, Inc.

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.

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 [[14]C]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 5β-pregnanedione, 5β-pregan-3β-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5β-pregnanedione was converted to 5β-pregnan-3β-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 [[3]H]flunitrazepam binding by 5β-pregnan-3β-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5β-pregnan-3β-ol-20-one EC50 values were 0.195±0.049, 0.101±0.017, 0.147±0.009, and 0.569±0.114 μM, 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 5β-pregnan-3β-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.

4-Oxystilbene compounds are selective ligands for neuronal nicotinic alphaBungarotoxin receptors.

1. Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes. 2. MG 624, F3, F3A and F3B inhibited of 125I-alphaBungarotoxin (alphaBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-alphaBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations. 3. We immobilized the alpha7, beta2 and beta4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-alphaBgtx binding to the alpha7-containing receptors with nM affinity, but inhibited 3H-Epi binding to beta2-containing receptors only at very high concentrations (more than 35 microM); their affinity for the beta4-containing receptors was ten times more than for the beta2-containing subtype. 4. Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick alpha7 receptors with an IC50 of respectively 94 and 119 nM. 5. High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 microM) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 microM). 6. In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic alphaBgtx receptors containing the alpha7 subunit.

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

Our aim was to examine the effect of the potent neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-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 3α,5α-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 3α,5α-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 3α,5α-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.

Developmental pattern of plasminogen activator activity in chick brain hemispheres.

Plasminogen activators play key roles in several developmental events. In previous works we demonstrated the existence of typical developmental patterns of protease activity in the chick optic lobe and cerebellum. The aim of this work is to study the temporal pattern of development of plasminogen activator activity in the brain hemispheres. Plasminogen activator activity was assayed in soluble fractions derived by ultracentrifugation from Triton X-100 treated membrane fractions by using a radial fibrinolytic assay. Employing different inhibitors and anti-plasminogen activators antibodies we showed that developing brain hemispheres express only one type of enzyme which corresponds to the urokinase-type. Other results indicate that the protease activity displays a temporal pattern which completely differs from those of general parameters of development. This suggests that the plasminogen activator activity is developmentally regulated and could display specific functions during particular stages of development.

Presence of an endogenous factor in avian CNS with agonistic action on benzodiazepine receptors.

In the present paper we describe the presence in avian CNS of an endogenous inhibitor of [3H]flunitrazepam binding. This compound was extracted from a synaptic membrane fraction isolated from chick optic lobe and brain using an exhaustive aqueous washing procedure, then purified by means of solid-phase extraction with C18 cartridges and several HPLC steps until an homogeneous peak was obtained. Its chemical structure was studied by size-exclusion chromatography of the purified material which indicated that it possesses a molecular weight below 1350. Although its inhibitory activity was lost by HCl treatment, its peptidic nature was ruled out by an amino acid and N-terminal sequence analyses. Ultraviolet absorption spectrum showed two main peaks at 230 and 280 nm. The endogenous compound was found to inhibit competitively [3H]flunitrazepam binding to its recognition site without affecting [3H]GABA binding to the same receptor complex. The behavior of the endogenous factor in an "in vitro" GABA "shift" test and GABA-dependent chloride flux experiments were similar to that of benzodiazepine receptor agonists. In conclusion, these results demonstrate the existence in avian CNS of a competitive endogenous inhibitor of benzodiazepine binding with agonistic action on benzodiazepine receptors.

Comparative modulation by 3 alpha,5 alpha and 3 beta,5 beta neurosteroids of GABA binding sites during avian central nervous system development.

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABAA receptor complex. The presence of a 3 alpha-hydroxyl group and a 5 alpha-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [3H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3 alpha-hydroxy,5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 beta-hydroxy,5 beta-pregnan-20-one (3 beta,5 beta-P). Both steroids were found able to enhance [3H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3 alpha,5 alpha-P had greater potency (EC50 0.22 microM) and enhancing effect (Emax: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABAA receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3 alpha,5 alpha-P by its 3 beta,5 beta isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5 alpha-reduced progesterone metabolites present since early development, while 3 alpha,5 alpha-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5 beta-steroids in early avian development, we propose that 3 beta,5 beta-P, instead of the classical potent 3 alpha,5 alpha-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.

Developmental pattern of plasminogen activator activity in chick optic lobe

Plasminogen activators are serine proteases which play a key role in morphogenesis and tissue remodelling. Two different molecular types, tissue-type and urokinase-type, were identified and they were postulated to play a role in neural development. The developing chick optic lobe plays a central role in processing visual information. In previous studies we demonstrated the occurrence of high levels of plasminogen activator activity in this model. The aim of the present paper is to study the temporal pattern of expression of this activity and characterize the type of plasminogen activator expressed in the developing optic lobe.Using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions we measured the protease activity with a radial fibrinolytic assay. Employing different inhibitors of fibrinolytic activity and a zymographic assay, we showed that the developing optic lobe expresses only one type of plasminogen activator which corresponds to an urokinase-type of 70 kDa. Our results indicate that peaks of protease activity temporally correlate with massive neuronal migration, neurite outgrowth and synapse formation and maturation. This suggests that a plasminogen activator could play a role in these developmental events. This consistent pattern of variability strongly suggests that it is developmentally regulated and, if so, it could be a reliable parameter to study neural plastic changes induced by modifications in the environmental stimulation.

3β-OH-5β-Pregnan-20-One Enhances [3H]Gaba Binding in Developing Chick Optic Lobe

The neurosteroids are synthesized in the CNS and act mainly through allosteric modulation of the GABAA receptor. Structure-activity relationship studies in mammalian CNS have shown that a 3α-hydroxyl group and a 5α-reduced A-ring are striking features for their biological activity, while the 3β,5β structures as in 3β,5β-P are completely inactive. In this work we report the enhancing activity of epipregnanolone on [3H]GABA binding to its receptor sites in the chick optic lobe. Concentration-effect curves for this neurosteroid showed a concentration-dependent activity with different potencies at the three developmental stages studied, the hatching stage being the most sensitive to the steroid stimulatory effect. The displacement of a potent 3α,5α steroid by epipregnanolone indicated that this steroid behaved as a partial agonist of the steroid recognition site. Considering the developmental profile for steroidogenesis in avian tissues and the biological relevance of 5β-reduced steroids in early development, we propose that 3 or its 3α-epimer, pregnanolone, instead of the potent 3α,5α neurosteroids, modulates GABAA receptors in the chick optic lobe during development.

Epipregnanolone acts as a partial agonist on a common neurosteroid modulatory site of the GABA(A) receptor complex in avian CNS.

Neurosteroid modulatory sites present in the GABA(A) receptor complex in chick optic lobe were investigated, in order to evaluate whether allopregnanolone and alphaxalone act through a common site of action. Results showed that either allopregnanolone or alphaxalone present a single-component enhancement of [3H]flunitrazepam binding with EC50 of 1.18 +/- 0.12 and 6.56 +/- 0.86 microM and Emax of 82.18 +/- 5.80 and 62.98 +/- 3.73%, respectively. Epipregnanolone behaved as a partial agonist of these steroid modulatory sites with EC50 of 0.49 +/- 0.15 microM and Emax 12.34 +/- 1.03%. Moreover, the addition of 16 microM epipregnanolone to either allopregnanolone or alphaxalone decreased EC50 values to 0.54 +/- 0.09 and 1.24 +/- 0.25 microM respectively, while Emax values were not significantly affected. On the other hand, additivity experiments disclosed that a maximal concentration (16 microM) of alphaxalone in the presence of allopregnanolone failed to enhance [3H]flunitrazepam binding in excess of that produced by allopregnanolone alone. Results indicate that not only allopregnanolone and alphaxalone act through a common site of action, but such site is highly stereospecific with regard to the neurosteroid spatial configuration.