The calcium-dependent [3H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: effects of enucleation on A1 receptor density and cholinergic markers.

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A1 adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A1 receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [3H]ACh evoked by 30 mM KCl was Ca2+ dependent and it was dose-dependently inhibited by the A1 adenosine receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 microM. The maximum of inhibition was reached at 10 microM. The A1 receptor antagonist 8-cyclopentyltheopylline (CPT, 10 microM), reversed almost completely the inhibition induced by CCPA 10 microM. In Fura-2/AM loaded synaptosomes, K(+) depolarization raised [Ca2+](i) by about 64%. CCPA (10 microM) reduced the K(+)-evoked Ca2+ influx increase by about 48% and this effect was completely antagonised by CPT 10 microM. Synaptosome pretreatment with different Ca2+ channel blockers differently affected K(+)-evoked Ca2+ influx. This was not significantly modified by nifedipine (1 microM, L-type blocker) nor by omega-agatoxin IVA (0.3 microM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 microMomega-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A(1) adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A1 receptor density without changing the affinity. The K(+)-evoked release of [3H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 microMCCPA in decreasing [3H]ACh release was not apparently modified.

The effects of aromatase and 5 alpha-reductase inhibitors, antiandrogen, and sex steroids on Bidder's organs development and gonadal differentiation in Bufo bufo tadpoles.

Embryos of toads (Bufo bufo) were treated with aromatase (4-OHA) and 5 alpha-reductase (17 beta C) inhibitors, antiandrogen (CPA), estradiol-17 beta, testosterone, and 5 alpha-dihydrotestosterone in order to study the role played by sex steroids in the development and sex differentiation of gonads. Test compounds were administered to tadpoles in water and morphometric and cytometric analyses were carried out on histological sections of the cephalic Bidder's organ (a rudimentary ovary) and of the gonadal region. In Bidder's organs, the number and size of oogonia and oocytes were modified by the treatments. However, the female commitment of the Bidder's organ occurs independently from steroid treatments that lead to an acceleration or slackening of the processes of proliferation and differentiation of oogonia. 4-OHA and androgens caused various degrees of inhibition of ovarian differentiation, with gonads maintaining an undifferentiated condition. Estrogen provoked a shift of the sex ratio towards the female sex, yet slackened gonadal growth. 17 beta C accelerated ovarian differentiation in females while CPA enhanced gonadal differentiation in both sexes by promoting the germ and somatic cell proliferation. We suggest that sex hormones may have a local regulatory role in gonadal differentiation during early developmental stages. Furthermore, the strong tendency of Bidderian germ cells to develop in the oogenetic way regardless of sex genotype and steroid treatments, and the quantitative sex differences found in the control Bidder's organs and gonads, suggest that other factors (such as intracellular mechanisms) may be involved in the initial steps of the process of germ cell differentiation.

Comparative analysis of glucosephosphate isomerase, lactate dehydrogenase and malate dehydrogenase isozymes in 9 cyprinid species from Italy.

The developmental and the tissue-specific expression of glucosephosphate isomerase (GPI), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) multilocus isozymes were analyzed in samples of Leuciscus cephalus and the adult patterns compared with those of 8 additional Italian cyprinid species: Alburnus alburnus alborella, Chondrostoma genei, L. lucumonis, L. souffia, Rutilus rubilio, R. erythrophthalmus, Scardinius erythrophthalmus and Tinca tinca, the taxonomic status of many of them being uncertain and highly debated. The spatial and temporal patterns of expression obtained generally agree with literature data. Main exceptions are the single expression of GPI-A* and MDH-A* loci of the liver in L. cephalus and the GPI pattern of the eye in all species examined. Since delayed appearence of the subunits coded by the GPI-B* locus and the very early ontogenetic expression of the sMDH-B* locus were found in L. cephalus, the onset of expression of orthologous loci can vary in related species. Genetic structure comparisons support a high genetic divergence of T. tinca from all other species.

Early aspects of sex differentiation in the gonads of chick embryos.

In chick embryos whose sex had been previously identified by cytokaryologic methods, a light-microscope study of the number and dimensions of the germ cells (GCs) has been made from 2 to 7 days of incubation. Early differences between the sexes have been found. In females the GCs were larger and increased in number earlier than in males. This suggests an earlier differentiation of GCs in females. On the other hand, ultrastructural observations on GCs at 70 h incubation (colonization stage of the genital ridges) have revealed that male and female GCs differ from each other mainly in the amount of rough and smooth endoplasmic reticulum, mitochondria, glycogen particles and lipid droplets. This suggests early morpho-functional differences between male and female GCs.

Differences between male and female protogonia in chick embryos before sex differentiation of the gonads.

The sex of chick embryos is diagnosed by cyto-karyological methods on skin fragments of 2-7 days' incubation before gonadal sex differentiation. In 44 males and 42 females statistical analyses have been made of the number and dimension of the germ cells, and of the volume of the gonadal primordia. Moreover an ultrastructural study has been made on the germ cells colonizing the genital ridges (70-hours of incubation). Early differences between the sexes have been found regarding: earlier numerical increase of CGs in the left gonadal primordium of the females; larger primordial germ cells in the female; the same cytological characteristics at ultrastructural level.