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Advanced Neurology Voluntary running effects in PTEN knockout mouse

2.3. Protein extraction with the ImageJ program (National Institutes of Health,

As described by Vasconcelos et al. , the cerebral cortex USA). For western blotting, food intake, body mass, and
[40]
was homogenized in a Dounce homogenizer filled with behavioral data (open field, elevated plus maze, and social
ice-cold lysis buffer (10 mM HEPES, 1.5 mM MgCl , behavior), statistical analysis was conducted using a two-
way analysis of variance (ANOVA) test, followed by a
2
10 mM KCl, 0.1 mM EDTA) containing protease inhibitors Tukey post-test for multiple comparisons. For behavioral
(0.5 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, data related to the passive inhibitory avoidance task,
and 2 μg/ml antipain) and phosphatase inhibitors non-parametric statistics were employed due to the use
(30 mM sodium fluoride, 3 mM sodium orthovanadate, of a 300-s ceiling in probe sessions. The Kruskal–Wallis
and 20 mM sodium pyrophosphate). The samples analysis of variance was performed, followed by Dunn’s
were then centrifuged at 12,000 ×g for 30 s at 4°C. The multiple comparison test. Social behavior data were
supernatant was removed, and the pellet was re-suspended analyzed using a two-way ANOVA test, and Fisher’s
with the same lysis buffer described above. After 10 min on lysergic acid diethylamide post-tests were conducted .
[43]
ice, 10% v/v NP-40 was added to each sample, vigorously Statistical significance was set at P < 0.05. All graphs were
shaken for 30 s, and then centrifuged at 1,000 ×g for 30 s at plotted as mean values ± scanning electron microscopy
4°C. The supernatant was collected for the western blotting using GraphPad Prism 8.
assay. Protein concentration was measured using the
Bradford colorimetric method . 3. Results
[41]
2.4. Western blotting 3.1. Voluntary running increased food intake and
body mass in both genotypes
Western blotting assay was used to evaluate the expression
of AKT, phospho-AKT, NR1, phospho-NR1, NR2B, PTEN, Females of Pten loxp/+ /Nse-Cre lineage were divided into four
+
synaptophysin, S6, and phospho-S6. The protocol used was groups: WTS, HTS, WTE, and HTE. Food intake and body
based on that described by Laemmli . The amount of protein mass were measured after 10 days of physical exercise.
[42]
in the samples was adjusted to 2.0 μg/μL with the sample buffer The results suggested that mice of both genotypes
(125 mM Tris-HCl, 4% SDS, 20% v/v Glycerol, 200 mM DTT, (WT and HT) presented a higher food intake after
0.02% bromophenol blue, and pH 6.8), incubated for 5 min at 10 days of voluntary running compared to sedentary
95°C, and then immediately placed on ice. Subsequently, each mice (F[1,24] = 41.87, P < 0.0001 for the treatment
sample containing 10 μg total protein was applied to a 10% factor, P < 0.001 for WTS vs. WTE and HTS vs. HTE).
SDS-polyacrylamide (acrylamide/bisacrylamide (37.5:1) gel; No significant difference in food intake was observed
1% SDS) for size-separation of the proteins present. The assay between genotypes (genotype factor, F[1,24] = 0.3964,
was run for 2 h at 90 V. Finally, the proteins were transferred P = 0.5349; interaction factor, F[1,24] = 0.0009911,
to a nitrocellulose membrane (BioRad, Hercules, CA, USA) P = 0.9751) (Figure 1A).
for approximately 90 min at 400 mA.
When analyzing body mass over the same period,
Next, the membranes were stained with Ponceau red the results indicated a difference in body mass between
solution (0.5% Ponceau-S, 5% trichloroacetic acid). The the sedentary and exercise groups (group factor,
excess dye solution was removed by washing with distilled F[1,48] = 10.14, P = 0.0025; time factor, F[1,48] = 0.2000,
water. Subsequently, the membranes were incubated for P = 0.6567; and interaction factor, F[1,48] = 0.07137,
1 h in a solution containing bovine serum albumin in P = 0.7905) (Figure 1B).
TBS-T (100 mM Tris-base, 0.9% NaCl, 0.05% Tween 20) to
+/-
+/+
block non-specific antibody binding. Following this step, 3.2. PTEN and PTEN animals traveled similar
the membranes were incubated with the primary antibody distances over 10 days of running
overnight. The next day, after the washing steps, the To verify whether there was a difference in exercise
membranes were incubated with the secondary antibody motivation between the WT (PTEN ) and HT (PTEN )
+/-
+/+
for 2 h. The chemiluminescence kit (Millipore, Billerica, mice, the distance traveled over 10 days was measured.
MA, USA) was used for detection, and the resulting blots When comparing the daily performance of animals using
were photographed using the G-Box photo documentation the distance traveled on day 1, we observed that both
system (Syngene/Synoptics, Cambridge, England). WT and HT mice covered more distance in a genotype-
independent manner from the 5 and 7 day, respectively
th
th
2.5. Statistical analysis
(time factor, F[9,200] = 13.6, P < 0.0001; genotype
The data obtained from the western blotting assay were factor, F[1,20] = 3.74, P = 0.0544; and interaction factor,
quantitatively analyzed using optical density analysis F[9,200] = 0.65, P = 0.7446] (Figure2A).
Volume 2 Issue 3 (2023) 4 https://doi.org/10.36922/an.0872