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).