Figure 5.
Arc/Arg3x.1 mRNA levels were downregulated in Th1-treated primary neuronal cell cultures and in EAE.
a
, Primary neurons obtained from CD1 E16.5 embryos were treated with a Th1 mix or saline [control (C)] for, respectively, 3, 6, and 24 h (n = 5 independent cell cultures). After the removal of the stimulus, neurons did not show any morphological changes with respect to untreated ones (data not shown). RT-PCR analysis showed that CD119 and p55 expression was maintained in Th1-treated neurons.
b
, Then, total extracts from Th1 at 3, 6, and 24 h and vehicle were applied on 10% SDS-PAGE, followed by Western analysis with, respectively, α-GluR1, α-pSer845 GluR1, α-PSD95, and α-TuJ1 as housekeeping gene. Histograms to the right of blots show fold induction (FI) over vehicle (±SD). GluR1 was significantly upregulated during either 6 or 24 h of Th1 treatment: 3 h, 1.6 ± 1.7, NS; 6 h, 1.7 ± 0.4, *p < 0.05; 24 h, 2.6 ± 0.6, *p < 0.05. GluR1 Ser845 was significantly upregulated after either 6 or 24 h of Th1 treatment: 3 h, 0.1 ± 0.01, NS; 6 h, 3.3 ± 0.3, *p < 0.05; 24 h, 3.5 ± 0.2, *p < 0.05. PSD95 was significantly upregulated during either 6 or 24 h of Th1 treatment: 3 h,1.0 ± 0.07, NS; 6 h, 3.1 ± 0.2, *p < 0.05; 24 h, 2.7 ± 0.5, *p < 0.05.
c
, Representative micrograph of vehicle-treated neurons stained for PSD95 (green). Application of Th1 treatment increases the number of PSD95-expressing synaptic boutons in cultured neurons.
d
, Arc/Arg3.1 mRNA levels were measured in neuronal cultures at 3, 6, 12, and 24 h after Th1 treatment by using a real-time RT-PCR assay and the mRNA levels, expressed as percentages ± SEM. *p < 0.05. Histogram in
e
shows bouton fold change over controls (3 h, 2.0 ± 1.1, *p < 0.05; 6 h, 2.5 ± 1.6, *p < 0.05; 24 h, 2.45 ± 1.1, *p < 0.05).
f
, HC and EAE brain coronal sections probed by in situ hybridization for Arc/Arg3.1 mRNA detection display Arc/Arg3.1 mRNA expression within the cortex and within the striatum. Right panels show Arc/Arg3.1 mRNA expression, respectively, in EAE 20 and 30 dpi brains. EAE basal neocortical Arc/Arg3.1 mRNA levels are conserved but were strongly downregulated within EAE striatum. Scale bars:
c
, 10 μm;
f
, 150 μm.