Experience-dependent decrease in synaptically localized Fra-1.

The Fos family of transcription factors has been repeatedly shown to participate in the long-term neural responses associated with a variety of physiological stimuli, including activity-dependent plastic processes. Quite recently, several transcription factors have been found in synaptic regions, localized in dendrites and presynaptic terminals. Here we show that the transcription factor Fos-related antigen-1 (Fra-1) was detected in synaptosomes (Syn) and synaptic plasma membrane (SPM) fractions from the rat cerebral cortex and hippocampus as a single band migrating with M(r) 42-43 kDa. The 55-kDa c-Fos protein was also detected in syn and SPM fractions. Conversely, the inducible 62-65-kDa c-Fos is present in nuclear fractions from metrazole-treated animals (positive control), but not in Syn or SPM fractions. Furthermore, no Fra-2, Fos B or c-Jun immunoreactivities were detected in these same synaptic regions. DNA-mobility shift assays showed the presence of specific AP-1 binding activity in synaptic protein extracts. Immunoelectronmicroscopic analysis of cortical and hippocampal tissues revealed that Fra-1 and Fos-like immunoreactivities are localized in association with presynaptic plasma membranes. One trial inhibitory avoidance training, a hippocampal-dependent task, is associated with a time-dependent decrease (-31%) in Fra-1, but not in 55-kDa c-Fos, levels in hippocampal SPM fractions. In hippocampal homogenates, we do not detect significant changes in Fra-1 immunoreactivity, suggesting that this behavioural experience is probably accompanied by a subcellular redistribution of Fra-1 protein. These results suggest that Fra-1 may participate in the communication between synapse and the nucleus and in experience-dependent hippocampal plasticity.

Histochemical localization of cyclic 3',5'-AMP phosphodiesterase in rat pineal gland.

Female Wistar rats maintained in a light:dark 12:12 photoperiod cycle were used to investigate the histochemical localization of the cAMP phosphodiesterase activity in the pineal gland of rats killed in the light period or in the dark period of the diurnal lighting regimen. Enzyme activity was identified along the plasma membrane of pinealocytes. The distribution of phosphodiesterase activity for cyclic guanosine monophosphate was similar to that found for cyclic adenosine monophosphate, suggesting that the same enzyme hydrolyzes both nucleotides. In the theophylline control, the reaction product along the plasma membrane of pinealocytes disappeared almost totally. In control sections without substrate, no reaction product was found. The animals decapitated in the dark period of the diurnal cycle showed a distribution of phosphodiesterase activity similar to that found in animals killed in the light period.

Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient.

A method for the isolation of oligodendroglial cells from young and adult whole rat brains, using a Percoll density gradient is presented. The minced tissue, incubated in a balanced salt solution containing 0.1% trypsin is further dissociated by forcing it through nylon screens to 145 and 74 ?m pore size. The crude suspension is then mixed with an isosmotic Percoll solution and centrifuged for 15 min. An in situ generated density gradient allows the separation of five bands, only one of which (Band C) lying between ?1.050 and ?1.062 contains cellular elements. The isolated cells show the typical morphological characteristics of oligodendroglia. A detailed morphological study of the cells isolated from whole brains of 10-, 30- and 120-day old rats is presented for the first time in the literature and immunocytochemical characterization is carried out using specific (antigalactocerebroside) and non specific (anti-glial fibrillary acidic protein) anti-sera. The method is simple and rapid and isosmotic conditions are maintained throughout, resulting in a better preservation of cell integrity. It represents an improvement over the two previous methods described in the literature and will be useful for studying different developmental events (biochemical and morphological) occurring in oligodendroglial cells at early stages of myelin formation.

Significance of the maillet method (ZIO) for cytochemical studies of subcellular structures.

The present knowledge on the methodology, specificity, localization, cytochemical significance and experimental variations of the zinc iodide-osmium tetroxide (ZIO) staining are reviewed. Some new findings supporting the concept that ZIO reacts with -SH groups in some structures under specific conditions are also communicated. In this respect the effect of in vitro pretreatment with -SH reagents on ZIO staining, already described in different kinds of synaptic vesicles, was studied in rod photoreceptor outer segments. It was observed that the intra- and extradiskal electron opaque deposits, present in untreated rod outer segments directly stained with ZIO at 4 degrees C for 2 h, were enhanced by previous incubation with dithioerythrytol (DTE), which protects -SH groups and reduce -S-S-bridges; when N-ethyl-maleimide (NEM), a -SH blocking agent was used directly or after DTE, the electron opaque deposits were considerably reduced or abolished. Furthermore the influence of previous incubation with cysteine on ZIO staining was studied in the rat pineal gland. In the controls ZIO made at 4 degrees C for 2 h, reacts strongly with the matrix of the small granulated synaptic vesicles in the pineal nerves; no electron opaque deposits are seen in the membranes of the vacuolar system. Previous incubation with cysteine 5 mM in phosphate buffer makes negative ZIO reaction in the matrix of the synaptic vesicles while with a concentration of 50 mM cysteine, a partial recovery is observed. In both concentrations the membranes of the vacuolar system appeared covered by patchy electron opaque deposits.

Morphological changes induced by -SH reagents in rod photoreceptor outer segments.

Rat retinas were treated in vitro with -SH reagents and "stained" with zinc iodide-osmium tetroxide (ZIO). Dithioerythritol (DTE), an -S-S-reducing agent, increased the electron opaque deposits observed after ZIO staining in the intra- and extradiskal spaces of the rods. N-ethyl-maleimide (nem), an -SH blocking agent, applied directly or after DTE, blocks the ZIO reaction. Furthermore, after treatment with NEM, distorted tubular and vesicular structures are substituted for the stacks of disks. These results strongly suggest that ZIO reacts with -SH groups in rod outer segments. They also indicate that SH-groups play an important role in the structural organization of rod outer segments.

Localizing -SH groups in monoaminergic synaptic vesicles with the mixture of zinc iodide-osmium tetroxide (ZIO).

In monoaminergic granulated vesicles the mixture of zinc iodide-osmium tetroxide (ZIO) stains two compartments: the core and the matrix, the latter being more intensively stained than the core. Rat pineal glands were incubated in 0.1 M N-ethylmaleimide (NEM). It was observed that NEM blocks ZIO reaction both in the matrix and the core, whereas the controls were fully reactive. This finding strongly suggests that ZIO reaction is due to -SH groups and can be correlated with the effect of NEM on uptake processes.