Ontogenesis of GABA receptor sites in chick embryo cerebellum.

The time-course of the development of GABA receptor sites in chick embryo cerebellum was correlated with the appearance of synaptic junctions in the cerebellar cortex. At 13 days of incubation, the earliest stage examined, specific [3H]GABA binding was only 19% of that found in cerebella of adult chicks. Between 15 days of incubation and hatching, specific [3H]GABA binding increased 3-fold, already reaching at birth adult values. During this period the number of synaptic junctions also increased. Scatchard analysis of the binding data obtained at birth revealed two binding sites of KdS 40 and 174 nM and a maximal number of binding sites (n) of about 1.6 and 4.0 pmol/mg protein, respectively. The high-affinity binding site for [3H]GABA was inhibited by muscimol, GABA, imidazoleacetic acid and bicuculline (IC50: 0.007, 0.020, 0.1 and 10 microM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X-100 enhanced [3H]GABA binding depending on the developmental stage studied, suggesting that GABA-modulin that inhibits the binding also appears during that period.

Development of GABA binding sites in chick embryo optic lobe: effect of triton X-100.

The temporal course of the development of GABA receptor sites in chick optic lobe was studied as a parameter of neuronal differentiation in the central nervous system. At 10 days of incubation, specific [3H]GABA binding was of 0.08 pmol/optic lobe and increased 7-8 fold between 12 and 16 days of incubation, reaching at 16 days a value of 0.60 pmol/optic lobe. This coincides with the period of arrival of the retinal fibers to the optic lobe. After this stage, the number of GABA binding sites decreased to a value of 0.35 pmol/optic lobe at hatching. After hatching a new increase appeared which reached at 5 days post-hatching a value of 0.87 pmol/optic lobe. Scatchard analysis of the saturation binding data obtained at 16 days of incubation and at hatching revealed the presence of two binding sites: one with high affinity and the other with low affinity, while at 12 days of incubation, the earliest stage examined, only the low-affinity binding site appeared. The high-affinity binding site for [3H]GABA was inhibited by muscimol, GABA, and bicuculline (IC50: 0.006, 0.002 and 10 microM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X-100 showed a marked increase in the amount of specific [3H]GABA binding after 16 days of incubation reaching a 3-fold increase at hatching. These results suggest that endogenous inhibitors of the higher affinity binding site, probably appear during this period.

Methods for removing endogenous factors from CNS membrane preparations: differences in [3H]GABA binding parameters and developmental-related effects.

The present report describes a systematic study comparing and combining methods currently used for the removal of endogenous factors known to affect the interaction of GABA with its receptor. The effects of these methods were analyzed by performing [3H]GABA binding studies, and by measuring the amount of residual GABA left in the different membrane preparations. The effectiveness of these methods were also applied to different developmental stages. The results show that: 1) an exhaustive buffer washing procedure is necessary to accurately measure the maximal binding capacity (Bmax) of the low-affinity GABA binding site, and 2) the use of more drastic methods, including freeze-thawing and Triton treatment allows a clear demonstration of receptor heterogeneity and a precise measurement of the Bmax of the high-affinity GABA binding site as well as increases the affinity of the low-affinity site. The analysis of the Bmax values obtained with these different procedures in relation to the values of GABA removal, strongly indicates that the exhaustive washing procedure removes some unknown endogenous substances required for Triton treatment to exhibit its maximal effectiveness. Finally, a detailed analysis of Kd and Bmax values obtained with these three methods in the developing nervous tissue shows the existence of significant differences with regard to their effectiveness in removing endogenous substances when applied in different developmental stages.

Current Triton X-100 treatments do not allow a complete plasminogen activator extraction from developing nervous tissue.

Determinations of plasminogen activator (PA) activity are usually performed in Triton X-100-treated tissue homogenates or crude membrane fractions. Such preparations usually involve a single Triton X-100 treatment. In the present paper we describe the pattern of variability of PA activity measured in different fractions obtained from the developing chick CNS by a repetitive procedure of Triton X-100 treatment and ultracentrifugation. To further characterize this PA activity we have also performed zymographic analyses during the embryonic development and the early postnatal life. Our results show that: a) a single Triton X-100 treatment does not completely extract the enzyme and this lead to an underestimation of the total PA activity; b) the PA activity is associated with the particulate component of the total tissue homogenate requiring its complete solubilization more drastic Triton X-100 treatments; c) better estimations of total and specific activities are obtained by using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions; d) the developing chick optic lobe expresses only one kind of PA molecule along the entire development; e) the level of PA activity vary characteristically during the ontogeny and the early postnatal life indicating the existence of a developmentally regulated mechanism of PA expression.

The incorporation of hydrophobic protein receptors and artificial lipid membranes.

The mechanism of chemical synaptic transmission implies: 1) the existence of a specific protein receptor at the postsynaptic membrane, and 2) the interaction between the transmitter released and the receptor, thus producing a change in ionic permeability. Previous studies from our laboratory have shown that special hydrophobic proteins extracted from postsynpatic membranes of different tissues showed a high affinity binding for the different pharmacological agents. The present paper describes experiments in which different hydrophobic protein binding acetylcholine, noradrenaline, gamma-aminobutyric acid, and glutamate were incorporated into artificial lipid membranes, similar to those first described by Mueller et al. (19). The effect of the different pharmacological agents was tested under experimental conditions of voltage clamp and the d.c. current changes measured. The results were then compared for the different lipid-protein membranes employed during the steady state and during transient conductance changes. The specificity of the responses indicate that artificial lipid membranes containing these hydrophobic proteins from electroplax, myocardium, spleen capsule and shrimp muscle can be used as a model to study pharmacologic receptors.