3 Results

3.1 Effects of brief EE exposure on medial prefrontal cortex protein levels

Western blot analysis of the NMDA receptors in the PSD of the mPFC after EE exposure showed increased expression of the GluN2A subunit (t(10) = 4.86, p < 0.001, +45%) with no changes for the other subunits GluN1 and GluN2B. This result led to an increased GluN2A/GluN2B ratio (t(10) = 2.44, p < 0.05, +20%). The analysis of the expression of the scaffolding proteins related to NMDA receptors revealed an increase of SAP102 after EE exposure (t(10) = 2.39, p < 0.05, +22%) with no changes for PSD95. The analysis of AMPA receptors showed that EE exposure decreased GluA1 subunit expression (t(10) = 2.83, p < 0.05, -22%) while increasing GluA2 levels (t(10) = 3.34, p < 0.01, +25%), a result that led to a significant decrease of GluA1/GluA2 ratio (t(10) = 7.47, p < 0.0001, -38%). Considering the scaffolding proteins related to AMPA receptors, both SAP97 (t(10) = 2.60, p < 0.05, +31%) and GRIP (U = 0, p < 0.01, +18%) protein levels significantly increased after EE exposure. Finally, we found Arc/Arg3.1 significantly increased in the mPFC of the EE group as compared to the NoEE group (t(10) = 3.27, p < 0.01, +30%) (Fig. 1a).
The analysis of the whole homogenate showed a significant increase of vGluT1 (t(10) = 2.49, p < 0.05, +22%) and GLT-1 expression (t(10) = 2.57, p < 0.05, +16%) as compared to NoEE group. In line with the results of the post-synaptic density, only the expression of the GluN2A subunit of NMDA receptors was significantly increased (t(10) = 2.83, p < 0.05, +20%), an effect that was accompanied by SAP102 up-regulation (t(10) = 2.96, p < 0.05, +22%) and, at variance from the PSD, by PSD95 up-regulation (t(10) = 4.82, p < 0.001, +20%) as compared to NoEE group. Analyzing AMPA receptors, the pattern of expression was quite different from the PSD with increased expression of GluA1 subunit (t(10) = 3.53 p < 0.01, +22%) and no effects on GluA2 subunit whereas the related scaffolding proteins showed significantly increased expression [SAP97 (t(10) = 3.90, p < 0.01, +21%) and GRIP (U = 2, p < 0.01, +15%)], in line with data in the PSD. Finally, no changes in Arc/Arg3.1 expression were observed when compared to the NoEE group (Fig. 1b).

3.2 Effects of brief EE exposure on nucleus accumbens protein levels

Western blot analysis in the PSD of the NAc after EE exposure revealed an overall downregulation of NMDA receptor subunits as compared to NoEE group (GluN1: t(10) = 2.77, p < 0.05, -8%; GluN2A: t(10) = 3.44, p < 0.01, -19%; GluN2B: t(10) = 2.68, p < 0.05, -13%); accordingly, the GluN2A/GluN2B ratio was not changed in the EE group. The analysis of the NMDA-related scaffolding proteins revealed a significant decrease of PSD95 after EE exposure (t(10) = 6.48, p < 0.0001, -33%), with no changes in SAP102 expression. Analysis of AMPA receptors shows a significant reduction of GluA2 levels after EE exposure (t(10) = 4.95, p < 0.001, -24%), with no GluA1 alterations. This result led to an increased GluA1/GluA2 ratio (t(6.19) = 2.95, p < 0.05, +22%). The analysis of AMPA-related scaffolding proteins showed a significant increase of SAP97 (t(10) = 3.17, p < 0.01, +10%) and a reduction of GRIP expression (U = 5, p < 0.05, -8%) after EE exposure. Finally, Arc/Arg3.1 expression was significantly decreased in the EE group as compared to the NoEE group (t(6.37) = 3.91, p < 0.01, -33%) (Fig. 2a).
When analyzing the whole homogenate, we found no changes in vGluT1 levels while a significant increase of GLT-1 expression as compared to the NoEE group (t(5.33) = 2.56, p < 0.05, +24%). With respect to NMDA receptors and related scaffolding proteins, no changes were observed except for a significant decrease of PSD95 (t(10) = 2.62, p < 0.05, -13%). Similarly, no changes were observed for AMPARs and related scaffolding proteins. Finally, Arc/Arg3.1 protein levels were significantly decreased as compared to the NoEE group (t(10) = 2.57, p < 0.05, -17%) (Fig. 2b).

3.3 Effects of brief EE exposure on hippocampal protein levels

Western blot analysis in the PSD of the Hipp after EE exposure showed no changes in the main NMDA subunit GluN1 whereas the expression of GluN2A and GluN2B were, respectively, significantly decreased (t(10) = 3.33, p < 0.01, -27%) and increased (t(10) = 2.56, p < 0.05, +13%). This result led to a significant reduction of the GluN2A/GluN2B ratio (t(10) = 4.64, p < 0.001, -36%). The analysis of the NMDA-related scaffolding proteins after EE exposure revealed a significant decrease of the scaffolding protein SAP102 (t(10) = 2.28, p < 0.05, -15%), with no changes for PSD95. At variance from NMDA receptors, no changes in the AMPARs subunits GluA1 and GluA2 expression were observed after EE exposure, with the consequence of an unaltered GluA1/GluA2 ratio. However, we found SAP97 and GRIP levels significantly decreased in the EE group as compared to the NoEE group (t(10) = 2.52, p < 0.05, -17%; t(10) = 3.39, p < 0.01, -11%; respectively). Finally, Arc/Arg3.1 significantly increased after EE exposure (t(10) = 3.57, p < 0.01, +12%) (Fig. 3a).
Examining the whole homogenate, vGluT1 levels were decreased (t(10) = 3.45, p < 0.01, -24%) with no changes for GLT-1 as compared to NoEE group. Notably, the expression of NMDA receptors was not altered after EE exposure, except for a significant decrease for both SAP102 (t(10) = 3.17, p < 0.01, -19%) and PSD95 (t(10) = 3.07, p < 0.05, -11%). Differently from NMDA receptors, the GluA2 subunit of AMPA receptors was significantly increased (t(10) = 2.56, p < 0.05, +27%) while no GluA1 changes were observed. The analysis of AMPA-related scaffolding proteins showed a slight but significant increase of SAP97 as compared to the NoEE group (t(10) = 2.58, p < 0.05, +14%). Finally, Arc/Arg3.1 expression was not altered after EE exposure (Fig. 3b).