Synaptic alterations induced by perinatal asphyxia: 2-D and 3-D electron microscopy study

SARACENO, GUSTAVO EZEQUIEL; AON-BERTOLINO, LAURA; MADUREIRA DE OLIVERIA, DIÊGO; BARRETO, GEORGE; GALEANO, PABLO; GIRALDEZ-ALVAREZ, LISANDRO DIEGO; CAPANI, FRANCISCO

Rosario

Congreso; 10th Inter-American Congress of Electron Microscopy (CIASEM 2009) and 1st Congress of the Argentine Society of Microscopy (SAMIC 2009); 2009

Comité Interamericano de Sociedades de Microscopía Electrónica (CIASEM) y Sociedad Argentina de Microscopía (SAMIC)

Alterations in synaptic functions have been associated with neuronal cell death following perinatal asphyxia (PA). The lack of knowledge on the mechanisms underlying this dysfunction prompted us to investigate the morphological changes in the postsynaptic densities (PSDs) induced by PA. Also, we studied the actin cytoskeleton since it is highly concentrated in PSDs and is one of the most affected structures by PA. Full-term pregnant Spague dawley rats were rendered unconscious by CO2 inhalation, rapidly decapitated and immediately hysterectomized after their first pup delivered vaginally. One uterine horn, containing the fetuses, was placed in a water bath at 37 C for 10 min (slight PA), 15 min (moderate PA), 19 min (subsevere PA) and 20 min (severe PA). The other horn was placed in a bath at 15 C for 20 min (hypothermia during insult group). The different groups of pups were marked and mixed with surrogate´s normal litters. We maintained litters of 8 pups with each surrogate mother (for more details of this procedure see Capani et al., 1997). Once rats were 30 days and 6 months old were fixed for electron microscopy and ligth microscopic studies for actin and ubiquitin staining and E-PTA. Some sections were subjected to 3-D analysis for electron tomography (Capani et al., 2001, 2009). For western blot analysis, some brains were disected and processes as described in Capani et al. (2009). Using two dimensional (2D) electron microscopic analyses, an increase in the F-actin staining in neostriatum of 30 days old asphyctic rats was observed using immunoblot and serial sections reconstructions. After six months of PA, morphological changes of PSDs were studied by three dimensional (3D) electron microscopic analyses of synapses stained with ethanolic phosphotungstic acid. This methodology allowed us to observe an increment of PSD thickness dependent on the duration and severity of PA insult. Immunoblot for ubiquitin and immunoelectron microscopy showed an increment in the ubiquitinization of the PSDs. These results demonstrate that synaptic dysfunction following PA might be produced by early changes in the actin cytoskeleton organization and long-term misfolding and aggregation of proteins in the PSDs. We hypothesize that the early changes in the PSDs of the rat neostriatum might be correlated with the important modification and ubiquitization of the PSDs observed in asphyctic adult rats. Supported by PID 5784, UBACYT M407 and PICT 15001.