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

SARACENO, GUSTAVO EZEQUIEL; AON-BERTOLINO, MARÍA LAURA; ROMERO, JUAN IGNACIO; GALEANO, PABLO; HOLUBIEC, MARIANA INÉS; BARRETO, GEORGE EMILIO; GIRALDEZ ÁLVAREZ, LISANDRO DIEGO; CAPANI, FRANCISCO

Buenos Aires

Congreso; II Congreso Argentino de Microscopía; 2012

Asociación 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. 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. The uterine horns, containing the fetuses, were placed in a water bath at 37 C for 19 min (subsevere PA). The different groups of pups were marked and mixed with surrogate´s normal litters. We maintained litters of 10 pups with each surrogate mother (for more details of this procedure see Saraceno et al., 2010). Once rats were 30 days and 4 months old were fixed for electron microscopy, light microscopic studies for actin, ubiquitin staining and E-PTA. Some sections were subjected to 3-D analysis for electron tomography (Capani et al., 2001 and 2009). For western blot analysis, some brains were dissected and processes as is described in Saraceno et al., 2011. Using two dimensional (2D) electron microscopic analyses, an increase in the F-actin staining in CA1 hippocampal area of 30 days-old asphyctic rats was observed. Western blot analyses showed an increased in beta actin concentration in sinaptosomal fraction. Moreover, no alterations in MAP-2 dendritic staining was observed in asphyctic animals respect to control group. After four months of PA, the morphological changes of PSDs were studied by three dimensional (3D) electron microscopic analyses. We observed a decrease in the F-actin dendritic spines staining and E-PTA technique showed an increase of PSDs thickness in asphyctic animals. Western blot analyses for beta actin showed no significant difference between groups. These results demonstrate that interruption of the correct brain development by PA induced synaptic dysfunction that might be produced by early changes in the actin organization and long-term misfolding and aggregation of proteins in the PSDs. We hypothesize that the early changes in the PSDs of rat hippocampus might be correlated with important modifications and ubiquitization of the PSDs observed in asphyctic adult rats. Supported by PID 5784, UBACYT M407 and PICT 15001.