Brain Research 1814 (2023) 148429

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Brain Research
journal homepage: www.elsevier.com/locate/brainres

Investigation of the protective effect of long-term exercise on molecular

pathways and behaviours in scopolamine induced alzheimer’s
disease-like condition
Seda Kose a, *, Meltem Donmez Kutlu a, Samet Kara b, Sait Polat b, Kubra Akillioglu a
a
Cukurova University Medical Faculty, Department of Physiology, Division of Neurophysiology, Adana 01330, Turkey
b
Cukurova University Medical Faculty, Department of Histology and Embryology, Adana 01330, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Despite research, the role of exercise in treatment and prevention of neurodegenerative diseases remains unclear.
Alzheimer’s disease Our study, investigated that protective effect of treadmill exercise on molecular pathways and cognitive be­
Amyloid beta haviours in a scopolamine-induced model of Alzheimer’s disease.
BDNF
For that purpose, male Balb/c mice subjected to exercise for 12 weeks. During the last 4 weeks of exercise,
Exercise
p-GSK3ßSer389
mice were given an injection of scopolamine (2 mg/kg). Following injection, open field test and Morris water
Scopolamine maze test were used to assess emotional-cognitive behaviour. Hippocampus and prefrontal cortex of mice were
isolated, and levels of BDNF, TrkB, and p-GSK3ßSer389 were assessed by western blotting, and levels of APP and
Aß-40 were analysed by immunohistochemistry.
In our study, scopolamine administration increased anxiety-like behaviour in open field test, while negatively
affecting spatial learning and memory in Morris water maze test. We found that exercise had a protective effect
against cognitive and emotional decline. Scopolamine decreased levels of p-GSK3ßSer389, BDNF in hippocampus
and prefrontal cortex.Whereas TrkB decreased in hippocampus and increased in prefrontal cortex. There was an
increase in p-GSK3ßSer389, BDNF, TrkB in the hippocampus, and p-GSK3ßSer389, BDNF in the prefrontal cortex in
the exercise + scopolamine group. Immunohistochemical analysis showed that scopolamine administration
increased APP and Aß-40 in hippocampus and prefrontal cortex in neuronal and perineuronal areas whereas Aß-
40 and APP were reduced in exercise + scopolamine groups.
In conclusion, long-term exercise may have a protective effect against scopolamine-induced impairments in
cognitive-emotional behaviour. It can be suggested that this protective effect is mediated by increased BDNF
levels and GSK3ßSer389 phosphorylation.

1. Introduction formed by amyloid beta (Aß) deposition. Aß is formed by two consecu­

Alzheimer’s disease (AD) is one of the most common progressive
neurodegenerative diseases in old age and accounts for 60–70% of de­
mentia cases (Lane et al., 2018). An estimated 40 million people
worldwide are living with dementia. Most of them are over the age of 60
(Scheltens et al., 2016). Today, the elderly population is growing as life
expectancy increases around the world due to widespread access to
healthcare and technological advances. It is estimated that the number
of Alzheimer’s cases will triple by 2050 due to the growing elderly
population and the risk of developing dementia (Alzheimer’s, 2019).
The most common pathological finding in AD is senile plaques
tive cleavages of amyloid precursor protein (APP) by secretases (Chen
et al., 2017). Aß plaques accumulate in the neocortex, posterior cingu­
late, precuneus, and association areas of the limbic cortex (Miller-
Thomas et al., 2016). Neurotoxic Aß plaques that accumulate outside the
cell cause mitochondrial and synaptic damage and disrupt cell functions
by leading to hyperphosphorylation of the tau protein (Özkay et al.,
2011). In addition, various cellular and molecular signalling pathways
triggered by inflammation lead to Aß accumulation (Akiyama and
Barger, 2000). It has been reported that the synthesis and/or release of
several inhibitory or excitatory neurotransmitters, particularly acetyl­
choline (ACh), are altered in AD. ACh is known to play an important role

* Corresponding author at: Medical Faculty, University of Cukurova, 01330, Turkey.

E-mail address: kakillioglu@cu.edu.tr (S. Kose).

https://doi.org/10.1016/j.brainres.2023.148429
Received 16 February 2023; Received in revised form 17 May 2023; Accepted 27 May 2023
Available online 1 June 2023
0006-8993/© 2023 Elsevier B.V. All rights reserved.
S. Kose et al.
in learning, memory, and neuroplasticity (Kandimalla and Reddy,
2017). Slotkin et al. reported that decrease in choline acetyltransferase
(ChAT) enzyme activity and a decrease in ACh synthesis in AD (Slotkin
et al., 1990). In summary, although the factors that cause AD are still
under debate, cholinergic dysfunction, Aß accumulation, tau hyper­
phosphorylation, inflammation, DNA damage, and mitochondrial
damage all contribute to the development of the disease (Lane et al.,
2018).
Acetylcholine is abundant in the central nervous system. It is the
main neurotransmitter active in the cerebral cortex, basal ganglia, and
basal forebrain (Hampel et al., 2018). Acetylcholine is involved in many
important physiological processes, such as learning memory, attention,
stress response, and sleep-wakefulness (Ferreira-Vieira et al. (2016)).
Cholinergic loss in AD is due to the degeneration of cholinergic neurons
in the nucleus basalis Meynert (NBM) and their axons that project to the
cerebral cortex. Nicotinic and muscarinic receptors in the cerebral cor­
tex are also altered in AD (Hampel et al., 2018). The acquisition, storage,
and use of new information depend on adequate cholinergic trans­
mission (Lim et al., 2015). Therefore, cognitive and emotional behav­
iour can be greatly affected by disruptions in cholinergic transmission.
The non-selective muscarinic receptor antagonist scopolamine has
been used experimentally in neuropsychopharmacology to induce
cognitive deficits. As M1 and M5 receptors are more abundant in the
CNS, it is thought that the effects of scopolamine are mediated through
these receptors. (Klinkenberg and Blokland, 2010). Cholinergic trans­
mission indicates its effects on emotional and cognitive functions mainly
through the M1 receptor. Several studies have shown that scopolamine
affects locomotor activity and impairs emotional and cognitive func­
tions. A study has shown that infusing scopolamine into the amygdala
increases locomotor activity. (Nomura et al., 1994). In another study,
infusion of scopolamine into the hippocampus did not affect locomotor
activity but increased anxious behaviour (Smythe et al., 1996). It has
been reported that rats given scopolamine showed hyperactivity in the
open field test and memory impairment in the MWM test (Jafarian et al.,
2019). In our study, the AD-like condition was induced by scopolamine
(a non-selective muscarinic receptor antagonist). As mentioned in the
literature, this model is often used as a good model for learning and
memory impairment and causes Alzheimer’s-like physiopathological
changes in the brain. In addition, the fact that scopolamine is easy to use,
has no serious side effects, and is cheaper than other methods is one of
the reasons why it was preferred in our study.
Signaling pathways involving the activation of glycogen synthase
kinase-3ß (GSK-3ß) are one of the mechanisms proposed to explain the
physiopathological processes involved in the development of AD.
Glycogen synthase kinase-3 (GSK-3) is a structurally active serine-
threonine kinase found in all tissues. GSK-3ß is active in cells that are
not normally stimulated. GSK-3ß is rendered inactive by the phosphor­
ylation of its specific sites. This enzyme is rendered inactive by phos­
phorylation of Ser9, Ser21, and Ser389. GSK-3ß is involved in
remodelling cell morphology by phosphorylating cytoskeletal proteins
that make up the cytoskeleton (microtubule-related proteins, tau, and
kinesin light chain) in the central nervous system, affecting functions
such as synaptic plasticity, neurofilament stabilisation, and new synapse
formation (Hur and Zhou, 2010). At the same time, GSK-3ß can also
trigger processes that lead to the accumulation of Aß. GSK-3ß plays a role
in the pathogenesis of AD by upregulating Aß production and sup­
pressing acetylcholine synthesis (Tang, 2019). M1 receptors, which are
abundant in the hippocampus and prefrontal cortex, have been shown to
influence the cleavage process of APP. M1 receptor activation through
the PKC pathway leads to an increase in sAPPα and a decrease in Aß by
inhibiting BACE, while it reduces Aß-induced apoptosis and p-tau by
inhibiting GSK-3ß (Fisher, 2012). GSK-3ß has also been shown to be
involved in the conversion of short-term memory into long-term mem­
ory and the formation of new neurons (Llorens-Martin et al., 2014).
Studies in mouse models of AD have reported that abnormal activation
of GSK-3ß leads to the loss of dendritic spines. Although studies have
2
Brain Research 1814 (2023) 148429
found evidence that GSK-3ß plays a role in the physiopathology of late-
onset AD, its mechanisms of action have not been elucidated (Llorens-
Martin et al., 2014, Souder and Anderson, 2019). It is believed that the
discovery of GSK-3ß’s mechanisms of action on the pathways that lead to
AD will contribute to both a better understanding of the causes of the
disease and the development of new treatments (Souder and Anderson,
2019) Fig. 1a and b.
Today, the fact that the benefits of pharmacological therapy in Alz­
heimer’s patients are very limited is leading researchers to investigate
non-pharmacological methods (Zucchella et al., 2018). In parallel, it is
believed that exercise, which is a non-pharmacological method, may be
an effective way to treat age-related neurodegenerative diseases, and
research is underway in this area. For example, several studies have
shown that exercise can protect against neuronal damage through
BDNF, increase plasticity in the brain, and thus have a positive effect on
cognitive disorders, (Koo et al., 2013, Vaynman et al., 2004, Xu et al.,
2021). In a study using scopolamine to induce an amnesia model in
mice, it was shown that the spatial learning ability of scopolamine-
treated animals was impaired and that this impairment was improved
by treadmill exercise. The same study reported that treadmill exercise
also improved spatial learning in healthy mice (Heo et al., 2014). The
aim of our study was to investigate the protective effect of long-term
exercise on emotional-cognitive behaviour and changes at the cellular
level induced by scopolamine in the AD model. For this purpose, the
effect of scopolamine, administered for 28 days to Balb/c mice that had
undergone 12 weeks of treadmill exercise, was assessed on cognitive
behaviour using the Morris water maze (MWM) test and on emotional
behaviour and locomotor activity using an open field test. At the cellular
level in the hippocampus and prefrontal cortex, BDNF, tyrosine receptor
kinase-B (TrkB), and phosphorylated GSK-3ß Ser389 (p-GSK3ßSer389)
levels were assessed by Western blotting, and APP and Aß-40 levels were
analysed by immunohistochemistry. We chose the Balb/c mouse strain
for this study because there are some emotional behaviours that may be
specific to the Balb/c strain. For example, Balb/c mice show more
anxiety-like behaviour and are susceptible to social defeat stress (Groves
et al., 2013, Mehta and Schmauss, 2011).
2. Results
2.1. Body weight
In body weight measurements ANOVA found a significant difference
between weeks in the S-Saline [χ2 (11) = 152.3p < 0.001], E-Saline [χ2
(11) = 162.9p < 0.001], S-SCOP [χ2 (11) = 103.4p < 0.001], and E-
SCOP [χ2 (11) = 129.6p < 0.001] groups. There was a significant in­
crease in body weight in all groups compared to the first week in all
weeks (p < 0.01).
In body weight comparisons between groups, the second week [H
(3,53) = 8.32p = 0.04], third week [H (3,53) = 26.14p < 0.001], fourth
week [H (3,53) = 19.24p < 0.001], sixth week [H (3,53) = 9.24p =
0.02], and seventh week [H (3,53) = 13.17p = 0.004] between groups
significant difference was detected. A significant decrease was detected
in the E-Saline group compared to the S-Saline group on the third (U =
30.0p < 0.001 z = − 3.49) and, fourth (U = 59.0p = 0.02 z = − 2.28)
weeks. A significant decrease was found in the E-SCOP group on the
third (U = 10.5 p < 0.001 z = − 3.49), fourth (U = 9.5 p < 0.001 z =
− 3.54), sixth (U = 28.5 p = 0.01 z = − 2.39) and, seventh (U = 14.5 p =
0.001 z = − 3.27) weeks compared to the S-SCOP group. There was no
significant difference between the groups in the first week [H (3,53) =
1.58p = 0.66], fifth week [H (3,53) = 6.19p = 0.10], eighth week [H
(3,53) = 1.03p = 0.79], ninth week [H (3,53) = 3.43p = 0.33], tenth
week [H (3,53) = 3.94p = 0.26], eleventh week [H (3,53) = 3.09p =
0.37], and twelfth week [H (3,53) = 1.70p = 0.63] Fig. 2.
S. Kose et al. Brain Research 1814 (2023) 148429

Fig. 1. a) Muscarinic receptors of acetylcholine, b) possible effect of scopolamine on beta amyloid deposition.

3
S. Kose et al.
Fig. 2. Body weight for twelve weeks. The data are presented as the mean ±
standard error. N = 11–15. For statistical analysis, see inside the text.
2.2. Open field test
In the open field test, there was a significant drug [F (1,49) = 39.17p
< 0.001] and exercise [F (1,49) = 7.18p = 0.01] effect in time spent in
the center, but no drug × exercise [F (1,49) = 1.35p = 0.25] interaction.
The time in the center was significantly reduced in the S-SCOP group
compared to the S-Saline group (p < 0.01). It increased significantly in
the E-Saline group compared to the S-Saline (p = 0.02) and E-SCOP (p <
0.001) groups. No significant difference was found between the E-SCOP
group and the S-SCOP group (p = 0.74) Fig. 3a.
In the open field test, significant drug effect [F (1,49) = 30.25p <
0.001], exercise effect [F (1,49) = 31.26p < 0.001] and drug × exercise
interaction [F (1,49) = 4.48p = 0.039] were found. Distance traveled
increased significantly in the E-Saline group compared to the S-Saline
group (p = 0.049). It increased significantly in the E-SCOP group
compared to the E-Saline (p < 0.001) and S-SCOP (p < 0.001) groups.
There was a tendency to increase in distance traveled in the S-SCOP
group compared to the S-Saline group (p = 0.09) Fig. 3b.
There was a significant drug [F (1,49) = 4.06p < 0.05] and exercise
[F (1,49) = 43.79p < 0.001] effect on the rearing, but no drug × exercise
[F (1,49) = 0.10p = 0.75] interaction. There was a significant increase in
the frequency of rearing in the E-Saline group compared to the S-Saline
(p < 0.001) group, and in the E-SCOP group compared to the S-SCOP (p
= 0.001) group. No significant difference was found in the S-SCOP group
compared to the S-Saline group (p = 0.63) and in the E-SCOP group
compared to the E-Saline group (p = 0.34) Fig. 3c.
While there was a significant exercise effect [F (1,49) = 15.60p <
0.001] and drug × exercise interaction [F (1,49) = 4.85p < 0.05] on the
frequency of entry to the center, no drug effect [F (1,49) = 0.36p = 0.54]
was found. A significant increase was found in the frequency of entry to
the center, in the E-SCOP group compared to the S-SCOP group (p =
0.001). No significant difference was found in the E-Saline and S-SCOP
groups compared to the S-Saline group (p = 0.54, p = 0.68, respec­
tively), and in the E-SCOP group compared to the E Saline group (p =
0.20) Fig. 3d.
In the open field test, entry time to the center found a significant
exercise [F (1,49) = 33.70p < 0.001] effect, but no drug [F (1,49) =
3.35p = 0.073] effect and drug × exercise [F (1,49) = 3.49p = 0.68]
interaction. Entry time to the center decreased in the E-Saline group
compared to the S-Saline group, and in the E-SCOP group compared to
the S-SCOP group (p < 0.05, p < 0.001, respectively). A trend to increase
in entry time to the center was found in the S-SCOP group compared to
the S-Saline group (p = 0.06). No significant difference was found in the
E-SCOP group compared to the E-Saline group (p = 1.00) Fig. 3e.
4
Brain Research 1814 (2023) 148429
2.3. Morris water maze test
2.3.1. Training trials
In the Morris water maze test, ANOVA found a significant difference
between days in the S-Saline group escape latency for five days in the
training trials [χ2 (4) = 26.2p < 0.001]. Compared to the first day, the
escape latency was significantly reduced on the second (Z = − 2.3 p =
0.02), third (Z = − 2.02p = 0.04), fourth (Z = − 2.09p = 0.03), and fifth
(Z = − 3.58p < 0.001) days. In the E-Saline group, ANOVA detected a
significant difference between days in the escape latency [χ2 (4) = 25.7
p < 0.001]. A significant decrease was found on the fourth (Z = − 2.5 p
= 0.011) and fifth (Z = − 4.3 p < 0.001) days compared to the first day.
In the training trials, there was a significant difference between days in
the S-SCOP group in the escape latency [χ2 (4) = 17.5 p = 0.002]. In the
escape latency, a significant decrease was found in the time to find the
platform on the second (Z = − 2.7 p = 0.006), third (Z = − 4.0 p <
0,001), fourth (Z = − 2.7 p = 0.006), and fifth (Z = − 2.7 p = 0.007) days
compared to the first day. ANOVA detected a significant difference be­
tween days in the escape latency in the E-SCOP group [χ2 (4) = 10.8 p =
0.028]. Compared to the first day, a significant decrease was found in
the escape latency on the second (Z = − 2.6 p = 0.008), third (Z = − 3.3 p
= 0.001), fourth (Z = − 2.8 p = 0.004), and fifth (Z = − 2.6 p = 0.007)
days.
There was a significant difference between the groups in the escape
latency on the first [H (3,212) = 17.04p = 0.001], fourth [H (3,212) =
10.04p = 0.01], and fifth [H (3,212) = 19.16p < 0.001] days of the
training trials. When the E-Saline group was compared with the S-Saline
group, no significant difference was found in the first (U = 1618.0p =
0.3 z = − 1.0), second (U = 1760.5p = 0.8 z = − 0.2), third (U = 1701.0p
= 0.5 z = − 0.5), fourth (U = 1556.0p = 0.1 z = − 0.1) and fifth (U =
1553.0p = 0.1 z = − 0.1) days. When the S-SCOP group was compared
with the S-Saline group, there was a significant increase in the escape
latency on the first (U = 980.0p = 0.006 z = − 2.7), fourth (U = 992.0p
= 0.02 z = − 2.2) and fifth (U = 840.0p = 0.001 z = − 3.2) days, but no
difference was found on the second (U = 1098.0p = 0.1 z = − 1.5) and
third (U = 1263.5p = 0.6 z = − 0.3) days. There was a significant in­
crease in the escape latency on the first (U = 1058.5p = 0.008 z = − 2.6)
and fifth (U = 1015.5p = 0.008 z = − 2.6) days when the E-SCOP group
was compared with the E- Saline group, but there was no significant
difference on the second (U = 1436.5p = 0.9 z = − 0.0), third (U =
1434.0p = 0.9 z = − 0.0) and fourth (U = 1244.0p = 0.2 z = − 1.2) days.
There was no significant difference in the escape latency on the first (U
= 948.0p = 0.2 z = − 1.1), second (U = 910.5p = 0.2 z = − 1.2), third (U
= 1042.0p = 0.9 z = − 0.1), fourth (U = 876.5p = 0.1 z = − 1.5) and fifth
(U = 941.5p = 0.3 z = − 0.9) days when the E-SCOP group was
compared with the S-SCOP group Fig. 4a.
In the Morris water maze test, ANOVA found a significant difference
between days in the S-Saline group in swimming speed for five days in
the training trials [χ2 (4) = 17.14p < 0.01]. Swimming speed decreased
significantly on the third (Z = − 2.10p = 0.03), fourth (Z = − 3.22p =
0.001) and fifth (Z = − 3.34p = 0.001) days compared to the first day.
ANOVA detected a significant difference between days in swimming
speed and duration in the E-Saline group [χ2 (4) = 32.22p < 0.001].
Swimming speed decreased significantly on the third (Z = − 2.27p =
0.02), fourth (Z = − 3.90p < 0.001), and fifth (Z = − 4.63p < 0.001) days
compared to the first day. In the training trials, there was a significant
difference between the days in the S-SCOP group in swimming speed [χ2
(4) = 9.8p = 0.043]. A decrease in swimming speed was detected on the
fourth day compared to the first day (Z = − 1.9p = 0.04). ANOVA found
no significant difference in swimming speed between days in the E-SCOP
group [χ2 (4) = 8.6p = 0.071].
In the Morris water maze test, a significant difference was found
between the groups on the first [H (3,212) = 16.50p = 0.001], third [H
(3,212) = 15.20p = 0.002], fourth [H (3,212) = 9.32p = 0.025] and fifth
[H (3,212) = 21.43p < 0.001] days in swimming speed in the training
trials. When the E-Saline group was compared with the S-Saline group,
S. Kose et al. Brain Research 1814 (2023) 148429

a)
100

Time spent in the center (s)

80

60 ###

**
40

20 S-Saline
S-SCOP
E-Saline
E-SCOP
0

b) 6000 c)
###,^^^ 70

5000
60
***
Distance traveled (cm)

^^

Frequency of rearing
4000 50

*
40
3000

30
2000
20
S-Saline S-Saline
1000 S-SCOP S-SCOP
E-Saline 10 E-Saline
E-SCOP E-SCOP
0 0

d) 60
e) 40
^^
Frequency of entry to the center

50
Entry time to the center (s)

30

40

30 20

20
* ^^^

10
S-Saline S-Saline
10 S-SCOP S-SCOP
E-Saline E-Saline
E-SCOP E-SCOP

0 0

Fig. 3. a)time spent in the center (s). *p < 0,05, **p < 0,001 compared to S-Saline group, ###p < 0,001 compared to E-Saline group. b) The distance traveled (cm).
*p < 0,05 compared to S-Saline group, ###p < 0,001 compared to E-Saline group, ^^^p < 0,001 compared to S-SCOP group. c) Frequency of rearing. ***p < 0,001
compared to S-Saline group, ^^p < 0,01 compared to S-SCOP group. d) Frequency of entry to the center. ^^p < 0,01 compared to S-SCOP group. e) Entry time to the
center (s). *p < 0,05 compared to S-Saline group, ^^p < 0,01 compared to S-SCOP group. ANOVA was performed followed by Tukey HSD test. The data are presented
as the mean ± standard error. N = 11–15.

there was an increase in swimming speed on the first (U = 1309.0 p =
0.01 z = − 2.5) day but no significant difference was found on the other
days. When the S-SCOP group was compared with the S-Saline group, a
significant increase in swimming speed was detected on the first (U =
935.0 p = 0.011 z = − 2.5), third (U = 975.0 p = 0.023 z = − 2.2), fourth
(U = 946.0 p = 0.014 z = − 2.4) and fifth (U = 729.0 p < 0.001 z = − 3.8)
days. Compared to the E-Saline group, there was a significant increase in
swimming speed on the third (U = 1049.0 p = 0.016 z = − 2.4) and fifth
(U = 1041.0 p = 0.014 z = − 2.4) days in the E-SCOP group but no
significant difference was found on the other days. No significant dif­
ference was found between the groups when the E-SCOP group was
compared with the S-SCOP group Fig. 4b.
2.3.2. Test
Mice, whose training trials were completed for five days in the Morris
water maze, were taken to the experiment from the opposite quadrant of
the quadrant with the platform in by removing the platform was on the
6th day. On the 6th day, a significant difference was found between the
groups when looking at the time spent on the target quadrant where the
platform is located [H (3,53) = 21.52p < 0.001]. The time spent in the
target quadrant was significantly reduced in the S-SCOP group
compared to the S-Saline group (U = 7.0p < 0.001 z = − 3.9). There was
no significant difference in the time spent in the target quadrant in the E-
Saline group compared to the S-Saline group (U = 108.0p = 0.852 z =
− 0.1). A decreasing tendency was found in the E-SCOP group compared
to the E-Saline group (U = 51.5p = 0.06 z = − 1.8). There was a sig­
nificant increase in the time spent in the target quadrant in the E-SCOP
group compared to the S-SCOP group (U = 19.5p = 0.004 z = − 2.8)
Fig. 5a.
There was no significant difference in swimming speed between the
groups on the test day [H (3,53) = 1.038p = 0.79] Fig. 5b.

5
S. Kose et al. Brain Research 1814 (2023) 148429

Fig. 4. a) escape latency in the morris water maze (s). b) swimming speed (cm/s). *p < 0,05 **p < 0,01 compared to S-Saline group, #p < 0,05, ##p < 0,01
compared to E-Saline group. Friedman’s ANOVA test was performed follewed by Mann-Whitney U test. The data are presented as the mean ± standard error. N
= 11–15.

Fig. 5. a) time in target quadrant in the morris water maze (s). b) swimming speed (cm/s). ***p < 0,001 compared to S-Saline group, ^^p < 0,01 compared to S-SCOP
group. Kruskal Wallis was performed folewed by Mann Whitney U test. The data are presented as the mean ± standard error. N = 11–15.

2.4. Immunohistochemical analysis
The hippocampus and prefrontal cortex tissues were taken to
determine the expression of Aß-40 and APP was analysed by immuno­
histochemical method. No immune markers were observed in the hip­
pocampus and prefrontal cortex sections where no primary antibody
was administered as a negative control Fig. 6a, 6b.
When the hippocampus tissue sections obtained from the S-Saline
group were evaluated for Aß-40 immunoreactivity, no immunostaining
was observed in the cytoplasm of neurons and glial cells. In addition, no
staining was observed in nerve fibers and perineuronal areas Fig. 7a.
In the evaluation of hippocampus tissue sections obtained from the S-
SCOP group for Aß-40 expression, the presence of pronounced Aß-40
expression in the cytoplasm of neurons and perineuronal areas was
noted. This expression was particularly evident in the cytoplasm of
neurons. In addition, it was observed that there was no immunoreac­
tivity in glial cells Fig. 7b.
When hippocampus tissue sections obtained from the E-Saline group,
were examined for Aß-40 immunoreactivity, it was observed that there
was no expression in the cytoplasm of neurons and glial cells. In addi­
tion, no immunoreactivity was observed in the perineuronal area Fig. 7c.
In the evaluation of hippocampus tissue sections obtained from the
E-SCOP group in terms of Aß-40 expression, the presence of rather
minimal Aß-40 expression in the cytoplasm and perineuronal areas of
neurons located in the CA1 region was remarkable. In addition, while
immunoreactivity was not observed in many neuron cytoplasms in this
region, Aß-40 expression was observed in a small number of neuron
cytoplasms. However, no expression was observed in the cytoplasm of

Fig. 6. Immunohistochemical analysis of the prefrontal cortex and hippocampus. a) No immune markers were observed in hippocampus sections where no primary
antibody was administered as a negative control. b) No immune markers were observed in prefrontal cortex sections where no primary antibody was administered as
a negative control. Bar = 50 μm.

6
S. Kose et al. Brain Research 1814 (2023) 148429

Fig. 7. Immunohistochemical analysis of the hippocampus. a) Aß-40 immunoreactivity of the S-Saline group, No staining is observed in neurons (arrows), glial cells
(arrow heads) and perineuronal areas (*) in hippocampus b) Aß-40 immunoreactivity of the S-SCOP group, Strong expression of Aß-40 immunoreactivity in neuron
cytoplasm (ok) is observed. In addition to this, it appears that Aß-40 immunoreactivity is not expressed in glia cells (arrowhead), c) Aß-40 immunoreactivity of the E-
Saline Group, It is observed that Aß-40 immunoreactivity is not expressed in neuron cytoplasms (arrow) and glia cells (arrow head), d) Aß-40 immunoreactivity of the
E-SCOP group, Aß-40 immunoreactivity is observed in neurons, neuron cytoplasm and perineuronal areas (ok). It is observed that Aß-40 is not expressed in glia cells
(arrowhead), e) APP immunoreactivity of the S-Saline group, high expression of APP immunoreactivity is observed in the cytoplasm of neurons (arrow). In glia cells,
APP expression (arrow head) is not observed, f) APP immunoreactivity of the S-SCOP group, Strong expression of APP immunoreactivity in the cytoplasm of neurons
(arrow) is observed. However, APP is not expressed in glia cells (arrow head) or its weak expression is observed in some glia cells, g) APP immunoreactivity of the E-
Saline group, Strong expression of APP immunoreactivity is observed in the cytoplasm of neurons (arrow). Whereas in glia cells, there is no expression of APP or its
weak expression (arrowhead) is observed in some glial cells, h) APP immunoreactivity of the E-SCOP group, high expression of APP immunoreactivity in the
cytoplasm of neurons (arrow) is observed. However, APP is not expressed in glia cells (arrow head) or its weak expression is observed in some glia cells. Bar = 50 μm,
k) Aß-40, l) APP, **p < 0.01, ***p < 0.001 compared to S-Saline group, ^^^p < 0.001 compared to S-SCOP group ###p < 0.001 compared to E-Saline group. Mann
Whitney U test.

7
S. Kose et al.
Fig. 7. (continued).
glial cells Fig. 7d.
When the APP immunoreactivity of the hippocampus sections ob­
tained from the S-Saline group were evaluated, significant APP immu­
noreactivity was observed in the cytoplasm of the neurons and the
perineuronal areas. In contrast, APP immunoreactivity was not observed
in glial cells Fig. 7e.
In the examination of hippocampus tissue sections obtained from the
S-SCOP group for APP expression, significant immunoreactivity was
observed in the cytoplasm of neurons and perineuronal areas. However,
no APP immunoreactivity was observed in glial cells Fig. 7f.
In the evaluation of hippocampus tissue sections obtained from the
E-Saline group in terms of APP immunoreactivity, the presence of
expression in the cytoplasm of neurons and perineuronal areas was
noted. However, no APP expression was observed in glial cells Fig. 7g.
In the evaluation of hippocampus tissue sections obtained from the
E-SCOP group in terms of APP immunoreactivity, the presence of sig­
nificant APP expression in the cytoplasm of neurons and perineuronal
areas was noted. However, no APP immunoreactivity was observed in
glial cells Fig. 7h.
Prefrontal cortex tissue sections taken from the S-Saline group were
also evaluated for Aß-40 immunoreactivity. Aß-40 expression was not
observed in the neuron cytoplasm and perineuronal area. In addition, no
Aß-40 immunoreactivity was observed in glial cells Fig. 8a.
The evaluation of prefrontal cortex tissue sections obtained from the
S-SCOP group for Aß-40 revealed the presence of Aß-40 expression in the
cytoplasm of neurons and perineuronal areas. While expression was not
observed in the majority of glial cells, the presence of minimal immu­
noreactivity in a few glial cells was noted Fig. 8b.
Aß-40 immunoreactivity was evaluated in prefrontal cortex tissue
sections taken from mice belonging to the E-Saline group. In the eval­
uation for Aß-40 immunoreactivity, there was no immunoreactivity in
the cytoplasm of neurons and glial cells, and perineuronal areas Fig. 8c.
Immunohistochemical evaluation was performed for Aß-40 anti­
bodies in prefrontal cortex tissues taken from mice belonging to the E-
SCOP group. In the examination of Aß-40 in the tissue sections taken, the
presence of minimal expression of Aß-40 was observed in a small
number of neurons. In addition, no expression was observed in glial cells
Fig. 8d.
In the examination of prefrontal cortex tissue sections obtained from
the S-Saline group for APP, pronounced immunoreactivity of APP was
observed in the cytoplasm of neurons and the perineuronal space. In
addition, no APP immunoreactivity was observed in glial cells in the
prefrontal cortex Fig. 8e.
In the examination of prefrontal cortex tissue sections obtained from
the S-SCOP group for APP, significant APP expression was observed in
the cytoplasm of neurons and perineuronal areas. However, no APP
immunoreactivity was observed in glial cells Fig. 8f.
In contrast, the evaluation of prefrontal cortex tissue sections ob­
tained from the E-Saline group for APP, showed significant APP
expression in the cytoplasm of neurons and the perineuronal area.
However, no immunoreactivity was observed in glial cells Fig. 8g.
However, in the evaluation of prefrontal cortex tissue sections
8
Brain Research 1814 (2023) 148429
obtained from the E-SCOP group for APP, the presence of pronounced
APP immunoreactivity was noted both in the cytoplasm of neurons and
in the perineuronal areas. No immunoreactivity was observed in the
cytoplasm of glial cells Fig. 8h.
2.5. Western blot analysis
A significant difference was found between the groups in the
expression of TrkB in the hippocampus [H (3, 16) = 13.29p = 0.004].
TrkB expression was significantly decreased in the S-SCOP group
compared to the S-Saline group (U = 0.0p = 0.01 z = − 2.4). TrkB
expression increased significantly in the E-SCOP group compared to the
S-SCOP group (U = 0.0p = 0.02 z = − 2.3). TrkB expression increased
significantly in the E-SCOP group compared to the E-Saline group (U =
0.0p = 0.01 z = − 2.3). There was no significant difference in TrkB
expression in the E-Saline group compared to the S-Saline group (U =
4.0p = 0.21 z = − 1.2).
There was a significant difference between the groups in the
expression of BNDF in the hippocampus [H(3, 16) = 11.13p = 0.011]. A
significant increase in BDNF expression was found in the E-Saline group
compared to the S-Saline group (U = 0.0p = 0.01 z = − 2.4). BDNF
expression was significantly decreased in the S-SCOP group compared to
the S-Saline group (U = 0.0p = 0.014 z = − 2.4). BDNF expression
increased significantly in the E-SCOP group compared to the S-SCOP
group (U = 1.0p = 0.01 z = − 2.3). There was no significant difference in
BDNF expression in the E-SCOP group compared to the E-Saline group
(U = 4.0p = 0.24 z = − 1.1).
Significant difference was found between groups in hippocampus p-
GSK3ßSer389 expression [H (3, 16) = 12.98p = 0.005]. p-GSK3ßSer389
expression was significantly decreased in the S-SCOP group compared to
the S-Saline group (U = 0.0p = 0.014 z = − 2.4). p-GSK3ßSer389
expression increased significantly in the E-SCOP group compared to the
S-SCOP group (U = 0.0p = 0.02 z = − 2.3). There was no significant
difference in p-GSK3ßSer389 expression in the E-SCOP group compared to
the E-Saline group (U = 6.0p = 0.56 z = − 0.5) Fig. 9a – d.
Significant difference was found between groups in prefrontal cortex
TrkB expression [H(3, 16) = 7.8p = 0.04]. TrkB expression increased
significantly in the S-SCOP group compared to the S-Saline group (U =
0.0p = 0.013 z = − 2.4). TrkB expression increased significantly in the E-
SCOP group compared to the E-Saline group (U = 0.0p = 0.01 z = − 2.3).
There was no significant difference in TrkB expression in the E-Saline
group compared to the S-Saline group (U = 4.0p = 0.12 z = 0.0) and E-
SCOP group compared to S-SCOP (U = 8.0p = 1.00 z = 0.0).
There was also a significant difference between the groups in pre­
frontal cortex BDNF expression [H(3, 16) = 10.07p = 0.018]. A signif­
icant increase in BDNF expression was found in the E-Saline group
compared to the S-Saline group (U = 0.0p = 0.01 z = − 2.4). BDNF
expression was significantly decreased in the S-SCOP group compared to
the S-Saline group (U = 0.0p = 0.014 z = − 2.4). BDNF expression
increased significantly in the E-SCOP group compared to the S-SCOP
group (U = 0.0p = 0.02 z = − 2.3). There was no significant difference in
BDNF expression in the E-SCOP group compared to the E-Saline group
S. Kose et al. Brain Research 1814 (2023) 148429

Fig. 8. Immunohistochemical analysis of the prefrontal cortex. a) Aß-40 immunoreactivity of the S-Saline group, It is observed that Aß-40 immunoreactivity is not
expressed in neuron cytoplasms (arrow) and glia cells (arrow head), b) Aß-40 immunoreactivity of the S-SCOP group, high expression of Aß-40 immunoreactivity in
neuron cytoplasm (ok) is observed. However, it is observed that Aß-40 immunoreactivity is not expressed in glia cells (arrow head), c) Aß-40 immunoreactivity of the
E-Saline group, It is observed that Aß-40 immunoreactivity is not expressed in neuron cytoplasms (arrow) and glia cells (arrow head), d) Aß-40 immunoreactivity of
the E-SCOP group, Weak expression of Aß-40 immunoreactivity in the cytoplasm and perineuronal area of some neurons is observed (arrow). Expression of this
antibody (arrow head) is not observed in glia cells, e) APP immunoreactivity of the S-Saline group, high expression of APP immunoreactivity in the cytoplasm of
neurons (arrow) is observed. However, it is observed that APP is not expressed in glia cells (arrowhead), f) APP immunoreactivity of the S-SCOP group, high
expression of APP immunoreactivity in the cytoplasm of neurons (arrow) is observed. However, it is observed that APP is not expressed in glia cells (arrow head), g)
APP immunoreactivity of the E-Saline group, Strong expression of APP immunoreactivity in the cytoplasm of neurons (arrow) is observed. However, APP is not
expressed in glia cells (arrow head) or its weak expression is observed in some glia cells, h) APP immunoreactivity of the E-SCOP group, Strong expression of APP
immunoreactivity in the cytoplasm of neurons (arrow) is observed. However, APP is not expressed in glia cells (arrow head) or poor expression of this antibody is
observed in some glia cells, Bar = 50 μm. k) Aß-40, l) APP, *p < 0.05, ***p < 0.001 compared to S-Saline group, ^^^p < 0.001 compared to S-SCOP group, ###p <
0.001 compared to E-Saline group. Mann Whitney U test.

9
S. Kose et al. Brain Research 1814 (2023) 148429

Fig. 8. (continued).

a) b) 1,8

1,6 ^,#
1,4

1,2

TrkB (fold change)

1,0

0,8
*
0,6

0,4 S-Saline
S-SCOP
0,2 E-Saline
E-SCOP
0,0

c) 1,2
d) 1,6
* ^ ^
1,4
1,0
(fold change)

1,2
BDNF (fold change)

0,8
* 1,0

0,6
0,8
Ser389

*
0,4 0,6
p-GSK3

S-Saline 0,4
S-Saline
0,2 S-SCOP
S-SCOP
E-Saline
E-SCOP 0,2 E-Saline
E-SCOP
0,0
0,0

Fig. 9. a)western blot image of hippocampus. b) TrkB fold change, c) BDNF fold change, d) p-GSK3ßSer389 fold change. *p < 0,05 compared to S-Saline group, ^p <
0,05 compared to S-SCOP group, #p < 0,01 compared to E-Saline group. Kruskal Wallis was performed followed by Mann Whitney U test. N = 4.

Fig. 10. a)western blot image of prefrontal cortex. b) TrkB fold change, c) BDNF fold change, d) p-GSK3ßSer389fold change, *p < 0,05 compared to S-Saline group, ^p
< 0,05 compared to S-SCOP group. ##p < 0.001 compared to E-Saline group. Kruskal Wallis was performed followed by Mann Whitney U test. N = 4.

10
S. Kose et al.
(U = 8.0p = 1.00 z = 0.0).
There was a significant difference between the groups in the
expression of p-GSK3ßSer389 in the prefrontal cortex [H (3, 16) = 14.32p
= 0.002]. p-GSK3ßSer389 expression was significantly decreased in the S-
SCOP group compared to the S-Saline group (U = 0.0p = 0.014 z =
− 2.4). p-GSK3ßSer389 expression increased significantly in the E-SCOP
group compared to the S-SCOP group (U = 0.0p = 0.021 z = − 2.3). p-
GSK3ßSer389 expression was significantly decreased in the E-SCOP group
compared to the E-Saline group (U = 0.0p = 0.021 z = − 2.3) Fig. 10a –
d.
3. Discussion
The present study showed that long-term physical exercise may
protect against the decrease in memory performance and anxiety-like
behaviour observed with scopolamine administration in Balb/c mice.
Our main findings were as follows: (1) scopolamine administration
increased anxiety-like behaviour in the open field test, while negatively
affecting spatial learning and memory performance in Morris water
maze test; (2) long-term physical exercise had a protective effect against
cognitive and emotional behaviors impaired by scopolamine adminis­
tration; (3) scopolamine decreased levels of p-GSK3ßSer389, BDNF in
hippocampus and prefrontal cortex. Whereas TrkB decreased in hippo­
campus and increased in prefrontal cortex; (4) there was an increase in
p-GSK3ßSer389, BDNF, TrkB in the hippocampus, and p-GSK3ßSer389,
BDNF in the prefrontal cortex in the exercise + scopolamine group; (5)
immunohistochemical analysis showed that scopolamine administration
increased APP and Aß-40 in hippocampus and prefrontal cortex in
neuronal and perineuronal areas whereas Aß-40 and APP were reduced
in exercise + scopolamine group.
Many studies in the literature have shown that the use of scopol­
amine has a negative effect on spatial learning and memory performance
(Jafarian et al., 2019). The cholinergic system, which is highly
concentrated in the thalamus, striatum, limbic system, and cortex, is
involved in learning and memory, attention, and other higher brain
functions. Scopolamine, a non-selective blocker of muscarinic acetyl­
choline receptors, can cause atrophy and neurodegeneration in the rat
brain. Scopolamine is known to cause deterioration in learning-memory,
and motor activity, mainly by blocking muscarinic receptors in the
hippocampus and cerebral cortex and increasing acetylcholinesterase
activity (Klinkenberg and Blokland, 2010). In particular, the degenera­
tion of neurons from the Mynert nucleus in the basal forebrain to the
hippocampus and cortex leads to a decline in cognitive function. Studies
show that scopolamine administration has opposite effects on anxiety-
like behaviour. In addition to studies showing that it reduces anxiety-
like behaviour, some studies suggest that it increases anxiety-like
behaviour which is in line with our findings (Furey and Drevets, 2006,
Jafarian et al., 2019).
The administration of scopolamine to mice caused a decrease in
BDNF levels in the hippocampus and the prefrontal cortex, accompanied
by a deterioration in cognitive and emotional function. Many studies in
the literature show that scopolamine reduces BDNF levels (Safar et al.,
2016, Tang, 2019). BDNF mRNA levels have been shown to decrease in
Alzheimer’s patients in post-mortem examinations (Garzon et al., 2002).
A post-mortem study of Alzheimer’s patients with clinical mild cognitive
impairment reported a correlation between BDNF levels in the parietal
cortex and decline in cognitive function (Peng et al., 2005). BDNF plays
an important role in neuronal development, growth, differentiation, and
synaptic function in areas such as the cortex, hippocampus, and
cholinergic basal forebrain neurons during early development and in
adulthood (Marini et al., 2004). All of these regions are severely
damaged in Alzheimer’s patients (Giannakopoulos et al., 1998). Studies
have reported that BDNF levels decrease when Aß proteins are applied to
normal nerve tissue (Rosa et al., 2021). Activity-dependent synaptic
plasticity is important in the cellular mechanisms of learning and
memory. BDNF is known to be important for learning and memory,
11
Brain Research 1814 (2023) 148429
NMDA receptor-mediated long-term potentiation (LTP), and its main­
tenance (Kocahan and Doğan, 2017, Navakkode and Korte, 2012). In
addition, cholinergic neurons reflected from the basal forebrain to the
hippocampus play an important role in the formation of learning and
memory. Acetylcholine is known to act by facilitating NMDA receptor-
mediated transmission in the hippocampus through muscarinic and
nicotinic receptors. Processes such as inhibition of GABA release,
blockade of potassium permeability, and activation of intracellular sig­
nalling pathways are reported to lower the membrane threshold for LTP
formation (Krnjevic et al., 1988). Gil-Bea et al. reported in their study
that increasing BDNF expression via muscarinic receptors is important
for memory formation in the hippocampus (Gil-Bea et al., 2011). They
suggest that BDNF activates the intracellular signalling pathways
necessary for synaptic plasticity in neurons, such as the formation of new
synapses through TrkB receptors with an autocrine effect. Although
there are many studies in the literature reporting a decrease in BDNF
levels with scopolamine administration, the intracellular signalling
pathways involved in the reduction of BDNF levels due to a decrease in
muscarinic transmission have not been fully elucidated.
BDNF acts through the tyrosine kinase receptor (TrkB) and the pan-
neurotrophin receptor (p75) in the central nervous system. The effect of
BDNF on cognitive function is usually mediated by the TrkB receptor,
while it binds with low affinity to the p75 receptor. It is generally
thought that it exerts effects such as apoptosis and inhibition of neuronal
growth through this receptor. It is also known that this receptor localises
together with TrkB receptors (Bibel et al., 1999). Our study also looked
at the levels of TrkB receptors, which mediate the effect of BDNF. It was
found that as the level of BDNF in the hippocampus decreased, the level
of TrkB also decreased. TrkB levels have been reported to decrease in
patients with Alzheimer’s disease (Ginsberg et al., 2006). It has been
reported that scopolamine administration reduces hippocampal BDNF
and TrkB levels in mice and rats (Kim et al., 2021, Saikia et al., 2018). In
the AD model induced by Aß25-35 in rats, it has been shown that there is a
decrease in BDNF and TrkB levels throughout the brain (Du et al., 2020).
TrkB triggers signalling pathways associated with functions such as
intracellular growth, differentiation, and plasticity through three
distinct signalling pathways. These signaling pathways are Ras-mitogen-
activated protein kinase (MAPK, MEK), phosphatidylinositol 3 kinase
(PI3K), and phospholipase C-gamma (PLC-) (Minichiello, 2009). Akt,
also known as protein kinase B, plays an important role in cellular
functions such as cell proliferation, apoptosis, and glucose metabolism
(Song et al., 2005). While many of the studies have reported that Akt
activation increases in AD, there are also studies that report that it de­
creases (Griffin et al., 2005, Lee et al., 2009). Aß induces Akt by acti­
vating caspase-3, thereby activating GSK-3ß (Jo et al., 2011). Apoptotic
processes are triggered by the activation of GSK-3ß. In our study, we
found that TrkB expression increased despite a decrease in BDNF by a
mechanism we cannot explain in the prefrontal cortex. This may be
related to the upregulation of TrkB due to the decrease in BDNF.
The Thr390 (human)/Ser389 (mouse) phosphorylation of GSK-3ß
occurs in a limited number of tissues compared to the phosphorylation
of Ser9 and Ser21. Ser9 and Ser21 phosphorylation are found in many
tissues, while Ser389 phosphorylation is found in the brain, thymus, and
spleen. The target for Ser9 and Ser21 phosphorylation is cytoplasmic
GSK-3ß, whereas the primary target for Ser389 phosphorylation is nu­
clear GSK-3ß. In addition, Ser9 phosphorylation of GSK-3ß is common to
all brain regions, whereas Ser389 phosphorylation has been shown to be
particularly prevalent in the hippocampus and cortex (Thornton et al.,
2018). Thr390 (human)/Ser389 (mouse) phosphorylation of GSK-3ß by
p38 MAPK has also been reported as an alternative pathway for GSK-3ß
inactivation. It has been reported that p-GSK3ßSer389 is excessive in the
brain and thymocytes, where p38 MAPK activity is high (Thornton et al.,
2008). GSK-3ß has been reported to cause the excessive phosphorylation
of the tau protein observed in AD and the accumulation of Aß plaques
(Wang et al., 2008). While GSK-3ß Ser9 phosphorylation hasbeen re­
ported to decrease in AD, Ser389 phosphorylation has not been studied
S. Kose et al.
in the literature. In our study, we found that GSK-3ßSer389 phosphory­
lation was decreased in the hippocampus and prefrontal cortex in the
scopolamine-induced AD model. Thornton et al. reported in their study
that learning-memory, locomotor activity, and anxiety-fear behaviours
were not affected in Ser389 knockout mice, but unexpected fear be­
haviours were observed (Thornton et al., 2018). In our study, a decrease
in p-GSK3ßSer389 levels in the hippocampus and prefrontal cortex was
observed, along with a decrease in spatial learning memory performance
with scopolamine administration.
The p38 MAPK signalling pathway plays an important role in phys­
iological processes such as neuronal plasticity and neurodegenerative
diseases. p38 MAPK is activated when it is phosphorylated by MKK6 and
MAPK3(MKK3) in the presence of cytokines, growth factors, and the
effects of cellular stress. Highly homologous isoforms of p38 MAPK
encoded by four different genes were expressed (p38a, p38ß, p38y, and
p38δ). The fact that they have different isoforms in different tissues al­
lows the p38 MAPK signalling pathway to have important functions in a
wide range of physiological processes. These functions include apoptotic
processes, inhibition of proliferation, and control of cell differentiation
(Zarubin and Han, 2005). The MAPK pathway has been shown to be
altered in AD. Phosphorylation of the ERK, JNK, and p38 forms of the
MAPK pathway is reported to be associated with cognitive decline in AD
(Ghasemi et al., 2014). Studies have shown that scopolamine adminis­
tration increases the phosphorylation of p38, JNK, and ERK in the MAPK
signalling pathway (Moosavi et al., 2018, Pak et al., 2021). Our current
study did not assess p-p38 MAPK protein levels, but previous studies
have reported different results, such as that p-p38 MAPK activity in­
creases, decreases, or does not change with scopolamine administration
(Ban et al., 2020, Jahanmahin et al., 2019, Moosavi et al., 2018). It has
been suggested that these differences may depend on the duration of
scopolamine administration and the strain of animal used. In our study,
the decrease in p-GSK3ßSer389 levels may be due to decreased activation
of the p38 MAPK pathway. Wang et al. showed in their study that BDNF
increases the levels of p-p38 and phosphorylated AKT (Wang et al.,
2019). They proposed that BDNF phosphorylates p38 via the PI3K
pathway using TrkB receptors. In our study, we found that BDNF and
TrkB levels decreased. This may have resulted in a decrease in phos­
phorylation of the p38 MAPK pathway and thus a decrease in p-
GSK3ßSer389 levels.
In our study, it cannot be excluded that scopolamine administration
has an effect on changes in cognitive and emotional functions, a
decrease in BDNF-TrkB and p-GSK3ßSer389 levels, and various signalling
pathways due to muscarinic receptor blockade. As expressed by
Thornton and colleagues in their study, GSK-3ßSer389 phosphorylation
may mediate the effects of scopolamine on anxiety and fear behaviour,
while other scopolamine-activated signalling pathways may also
mediate its effects on cognitive function (Thornton et al., 2018).
In our study, we observed that scopolamine administration increased
APP and Aß-40 expression in the hippocampus and prefrontal cortex in
neuronal and perineuronal areas. It has been reported that, physiologi­
cally, APP has a trophic effect on the central nervous system (Dawkins
and Small, 2014). It has been reported that APP knockout mice have
smaller brains and that this affects neurogenesis (Wang et al., 2016).
APP has also been reported to play a role in LTP by regulating calcium
release (Kim et al., 2000). At physiological levels (picomolar), Aß has
been reported to reduce potassium channel activity and apoptosis (Yu
et al., 2006). It has also been reported to increase acetylcholine release,
protect neurons during aging, and be important for learning memory by
increasing synaptic plasticity (Morley et al., 2010). It is well known that
in Alzheimer’s disease, there is an uncontrolled and excessive produc­
tion and accumulation of Aß. In rats, scopolamine (2 mg/kg) once a day
for 6 weeks has been shown to increase Aß plaques and decrease BDNF
levels in the hippocampus (Safar et al., 2016). The same study reported
that scopolamine increased GSK-3ß mRNA and protein expression in the
hippocampus. The M1, M3, and M5 receptors trigger intracellular sig­
nalling pathways by activating PKC, while M1 and M4 activate the
12
Brain Research 1814 (2023) 148429
enzyme adenyl cyclase. In some cells, M2 and M4 receptors trigger
intracellular signalling pathways involving phospholipase C. It has been
shown that activation of M1 and M3 receptors by acetylcholine increases
sAPPα secretion through the PKC pathway and also reduces Aß plaques
(Welt et al., 2015).
In AD, the degeneration of cholinergic neurons in the brain and a
decrease in the synthesis and release of ACh lead to a disruption of
cholinergic transmission. ACh is responsible for regulating learning and
cognitive functions that depend on the hippocampus. It acts on the
hippocampus and cerebral cortex in the brain, particularly through M1
receptors (Klinkenberg and Blokland, 2010). Studies in transgenic ani­
mals suggest that a cholinergic deficit plays a role in the accumulation of
Aß and the formation of phosphorylated tau (Ramos-Rodriguez et al.,
2013). It was first suggested by Nitsch et al. that stimulating M1 and M3
receptors can increase sAPPα and thus prevent the formation of Aß
(Nitsch et al., 1992). M1 receptor activation increases sAPP via ADAM17
(tumor necrosis factor-converting enzyme) and decreases Aß via PKC
inhibition of BACE-1. It also reduces Aß-induced apoptosis and p-tau by
phosphorylating GSK-3ß (Fisher, 2012). Administration of a mAChR
antagonist such as scopolamine leads to an increase in Aß and hyper­
phosphorylation of tau caused by GSK-3ß. It has been reported that M2
and M4 are ineffective in activating α-secretase and may even have an
inhibitory effect on the release of these receptors sAPPα (Nitsch et al.,
1992).
In studies, increasing the inactive (phosphorylated) form of GSK-3ß
in BDNF-TrkB mediated signalling keeps the conversion of APP to Aß
plaques under control. It has been reported that Aß plaques increase
with an excessive increase in the active form of GSK-3ß due to a decrease
in BDNF transmission. The active form of GSK-3ß has also been reported
to increase tau phosphorylation by activating tau kinases (Llorens-
Martin et al., 2014, Safar et al., 2016). In our study, increased Aß plaques
could also be explained by a decrease in phosphorylated GSK-3ß.
In accordance with the literature, scopolamine did not affect body
weight. In mice that exercised, there was a significant decrease in body
weight in the third and fourth weeks compared to sedentary subjects,
while a relative decrease was seen in other weeks (Baek et al., 2020,
Zhao et al., 2020).
In Balb/c mice that were exercised for 12 weeks, it was found that
their anxiety-like behaviour decreased with an increase in center la­
tency, time spent in the center, frequency of entry to the center, and the
number of rearing in the open field test. There are many studies in the
literature that show the positive effects of physical exercise on emotional
and cognitive function, which is consistent with our findings (Pietrelli
et al., 2012). Physical exercise is known to have a positive effect on
learning and memory performance through cellular events such as
neurogenesis, increased LTP, and increased dendritic and synaptic
branching (van Praag et al., 1999). Studies report that the beneficial
effects of exercise on the central nervous system are related to the
intracellular effects of BDNF (Russo-Neustadt et al., 1999). In our study,
we also found that treadmill exercise increased BDNF levels in the
hippocampus and prefrontal cortex. It has been reported that BDNF
levels, which are increased by physical activity, cause neuronal plas­
ticity by activating intracellular MAPK/ERK and phospholipase C sig­
nalling pathways through TrkB receptors (Minichiello, 2009, Ying et al.,
2002). The reasons why BDNF levels increase in the brain during exer­
cise are not fully explained in the literature. The fact that BDNF crosses
the blood-brain barrier and is synthesised outside the brain, for example
by T and B lymphocytes, monocytes, and skeletal muscles, makes it
difficult to identify the actual source of increased BDNF levels. The re­
searchers suggest that in addition to BDNF released from peripheral
tissues, several myokines (iris, cathepsin, etc.), glycolysis products
(lactate), ketone bodies (β-hydroxybutyrate) and cytokines (TNFα, IL-
1β, IL-18) cross the blood-brain barrier and regulate BDNF release in the
central nervous system (Delezie and Handschin, 2018, Tong et al.,
2012). Physical exercise has also been shown to increase choline uptake,
muscarinic receptor density, and the number of neurons expressing
S. Kose et al.
acetylcholine transferase in the hippocampus (Fordyce and Farrar,
1991).
In our study, it was found that p-GSKßSer389 levels did not change in
the exercise group. Bayod et al. showed an increase in the level of p-
GSK3ßSer9 in the hippocampus of rats that were exercised on a treadmill
5 times a week (for 30 min at a speed of 12 m/min) for 36 weeks (Bayod
et al., 2011). In a study investigating the effects of an enriched envi­
ronment, such as physical exercise, it is reported that an increase in
BDNF in the hippocampus causes an increase in the levels of p-GSK3ßSer9
(Hu et al., 2013).
The fact that the p-GSK3ßSer389 levels did not change with long-term
physical exercise in our present study may be interpreted as an adap­
tation to long-term exercise. As mentioned above, although there are
studies on p-GSKßSer9 in the literature, p-GSKßSer389 has not been studied
in physical exercise.
In our study, we found that long term physical exercise may protect
against the decrease in memory performance and anxiety-like behaviour
observed with scopolamine administration in Balb/c mice. Heo Y-M
et al. report that exerciseprotects against cognitive decline and induces
neurogenesis by increasing hippocampal BDNF levels in mice adminis­
tered 1 mg/kg scopolamine before treadmill exercise for 14 days (Heo
et al., 2014). Rosa et al. reported that the anxiety-like behaviour
observed with cerebroventricular Aß-40 administration in mice was
reduced by short-term treadmill exercise (Rosa et al., 2021). Although
the beneficial effects of short-term exercise in AD have been shown in
the literature, there are less studies on the effects of long-term exercise.
It has also been reported to increase cortical BDNF levels in trans­
genic mice that were given treadmill exercise for 12 weeks (5 days a
week, 12 m/min) (Koo et al., 2013). In our study, we found that BNDF,
TrkB, and p-GSK3ßSer389 levels decreased in the scopolamine treated
groups and increased in the exercise + scopolamine groups. Immuno­
histochemical examination also showed that the increased expression of
Aß and APP in the prefrontal cortex and hippocampus decreased with
scopolamine administration in the exercise groups. Physical activity has
been shown to have a beneficial effect on AD in studies for many years.
Exercise has been reported to increase brain blood flow and hippo­
campal volume, reduce inflammation, oxidative stress, Aß plaques, and
reduce neuroinflammation by preventing the occurrence of obesity and
diabetes (Ribarič, 2022). As a result of these, it is suggested that exercise
improves neurogenesis, synaptic plasticity, angiogenesis, and cognitive
performance. Unfortunately, the cellular mechanisms and molecular
pathways underlying all these events are not fully understood.
Throughout the studies, it has been reported that conditions such as
decreased BDNF, oxidative stress, and inflammation cause the accu­
mulation of Aß plaques, while physical exercise reverses these negative
effects by increasing BDNF. In our study, physical exercise may have
increased p-GSK3ßSer389 through the p38 MAPK pathway by increasing
scopolamine-reduced BDNF levels, thereby reducing Aß plaque accu­
mulation. In addition, as mentioned in the literature, the accumulation
of Aß plaques may be prevented by the effect of exercise on reducing
neuroinflammation. Moreover, since Ser389 phosphorylation uses nu­
clear GSK3ß as a substrate compared to Ser9 phosphorylation, is
expressed only in limited tissues, and the activation stimuli are different
compared to Ser9 phosphorylation, elucidation of this signaling
pathway will contribute to the understanding of the protective role of
physical exercise against the effects of scopolamine.
While p-GSK3ßSer389 was evaluated in our study, total GSK3ß could
not be evaluated. In addition, the p-p38 MAPK signalling pathway could
not be evaluated. These are accepted as the limitations of our study. The
small sample size of mice used in molecular studies is another limitation.
In the literature, the effects of long-term exercise on AD have
generally been investigated in transgenic animal models and non-
transgenic models (Aβ or tau directly into the brain via intra­
cerebroventricular (i.c.v.) or intrahippocampal injections) (García-Mesa
et al., 2011, Souza et al., 2013, Yang et al., 2022). The results of our
study are important because it is one of the limited number of studies
13
Brain Research 1814 (2023) 148429
investigating the molecular and behavioural effects of long-term exer­
cise, particularly in AD with cholinergic impairment. In addition, in our
study, not only the benefits of long-term exercise alone, but also the
benefits of this exercise in the adult period as a result of the changes in
the developing brain were revealed. In short-term exercise models
applied in the literature, mostly adult or old mice are used. Our study, in
which mice that voluntarily received treadmill exercise as soon as they
were separated from their mothers on day 21 were evaluated at 15
weeks of age, differs in this respect. However, although short-term ex­
ercise has been reported to increase BDNF and TrkB levels, the mecha­
nisms related to this have not been elucidated. In addition to BDNF and
TrkB, we also analysed pGSK3ßSer389 levels, which have not been pre­
viously studied in the literature in mice subjected to long-term exercise,
and tried to elucidate the mechanisms by which the positive effects of
exercise occur. Although short- and long-term exercise has been shown
to increase BDNF expression in the hippocampus in various animal
models, it is emphasised that this effect of exercise is less in old animals
than in young animals (Adlard et al., 2005, Müller et al., 2020). Despite
the unclear mechanisms of how exercise modifies the processes of AD,
the long preclinical period of the disease suggests that symptoms may be
significantly delayed by interventions such as exercise that are effective,
sustainable, patient-adaptable and have few side effects (Ribarič, 2022).
Studies with rodent models of AD have shown that exercise can alleviate
the symptoms of neurodegeneration (Belarbi et al., 2011, Nay et al.,
2021). There are studies suggesting that exercise performed before the
onset of AD-like neuropathology may slow the progression of the dis­
ease. Results showing that exercise interventions after the development
of Aß pathology do not have a positive effect on cognitive and neuro­
pathological parameters are also reported (Richter et al., 2008, Zhao
et al., 2015). Ultimately, it was found that while the administration of
scopolamine caused a deterioration in cognitive and emotional behav­
iour, prolonged physical exercise had a protective effect against these
negative scopolamine-induced behaviours. Scopolamine may have
shown its effects on cognitive and emotional function by reducing the
levels of BDNF, TrkB, and p-GSK3ßSer389 in the hippocampus, BDNF and
p-GSK3ßSer389 in the prefrontal cortex and increasing the levels of Aß-40
and APP in both areas. Long-term physical exercise may have a pro­
tective effect by increasing levels of BDNF, TrkB, and p-GSK3ßSer389 in
the prefrontal cortex and hippocampus and reducing levels of Aß-40 and
APP.
4. Experimental procedure
4.1. Ethic
The experimental protocols were approved by the Ethics Committee
of Cukurova University Medical Sciences Experimental Application and
Research Center. Procedures in the study were performed in accordance
with the NIH Guide for the Care and Use of Laboratory Animals (date:
August 12, 2020, and decision number: 2/5).
4.2. Animals
In our study, male Balb/c mice were used at the age of 3 weeks (15
±2 g). The experimental animals were provided by Cukurova University
Medical Sciences Experimental Application and Research Center, and
were housed in the animal care room in our department during the
experiment. The room temperature (22±1 ◦ C) and humidity (40–60%)
were kept under constant control during the 12-hour light/dark cycle of
the animal care room. Water and food were provided ad libitum to the
mice.
4.3. Exercise protocol
Voluntary treadmill exercise was performed in mice in accordance
with the exercise protocol previously described by Zhao et. al. Mice in
S. Kose et al.
the exercise group underwent a 6-day acclimatisation period before
starting the 12-week exercise programme to get them used to the
treadmill. The treadmill was set at a walking speed of 5 m/min for the
first 2 days (days 1–2), then increased to 8 m/min on days 3–4 and 12 m/
min on days 5–6. The mice were then subjected to a 12-week exercise
protocol that included 45 min of exercise per day, 5 days per week, from
6 to 8p.m. Each day, the mice were exercised at a series of speeds in the
following order: 5 m/min for 5 min, 8 m/min for 5 min, 12 m/min for
30 min, and 5 m/min for 5 min. Mice in sedentary control groups were
placed on the static treadmill for the same amount of time (Zhao et al.,
2020) Fig. 11.
4.4. Drug
In our study, the muscarinic acetylcholine receptor antagonist
scopolamine (scopolamine hydrobromide trihydrate, 99%) was admin­
istered during the last 4 weeks of a 12-week exercise programme to
induce an AD model. The administration dose of scopolamine was pre­
pared by dilution with saline solution to 2 mg/kg. The prepared stock
solution was kept at − 20 ◦ C in 1 ml Eppendorf tubes. The stock solution
was diluted with saline before injection. On the days of injection, it was
kept at +4 ◦ C.
In the scopolamine groups, 2 mg/kg (0.1 ml/10 g body weight vol­
ume) of scopolamine was administered intraperitoneally once a day
after exercise for 28 days (Hou et al., 2014, Safar et al., 2016, Satou
et al., 2018, Sharma and Gill, 2022). The control groups received the
same volume of saline intraperitoneally once a day. The body weights of
the animals were measured on the days of injection, and their weight
was also monitored weekly to monitor their somatic growth.
Four different experimental groups (N = 69) were formed: sedentary-
saline (S-Saline, n = 15), exercise-saline (E-Saline, n = 15), sedentary-
scopolamine (S-SCOP, n = 11), and exercise-scopolamine (E-SCOP, n
= 12). The mice were randomly divided into four groups. The
randomization was performed according to a computer-generated
randomization list. After exercise and scopolamine administration, the
mice were subjected to an open field test and Morris water maze test to
assess emotional and cognitive function, respectively. At the end of the
injections, the prefrontal cortex and hippocampus of four mice from
each group that were not involved in the behavioural experiments were
isolated. BDNF (Cat No.: 28205–1-AP), TrkB (Cat No.: STJ96109), p-
GSK3ßSer389 (Cat No.: 14850–1-AP), and beta actin (Cat No.: 20536–1-
AP) levels were assessed by western blot analysis; APP and Aß-40 levels
were assessed by immunohistochemical analysis.
Fig. 11. Mice during treadmill exercise.
14
Brain Research 1814 (2023) 148429
4.5. Behavioural experiments
Mice were taken to the experiments on the last 8 days of the 28-day
injection period, 30 min after the injection. Before starting the behav­
ioural tests, the mice underwent hand adaptation for 3 days. The mice
were first subjected to the open field test and, after a one-day rest, to the
Morris water maze test.
4.5.1. Open field test
The open field setup used in the test was made of black plexiglas,
60x60x24, open at the top and surrounded by a 1 cm thick wall at the
sides. The area of the assembly adjacent to the wall is called the pe­
riphery, and the remaining area is called the central area. The mice were
brought into the experimental room 3 min before the experiment to
acclimatise to the environment in which the experiment would be
conducted, and then brought into the experiment. The room tempera­
ture was set at 22±1 ◦ C and the illuminance at 100 lx. Mice were left in
the open field from any corner, and their behaviour was recorded by a
video-camera for 5 min. In the open field test, after the mice were left in
the open field, entry time to the center (sec), time spent in the center
(sec), frequency of entry to the center, distance travelled (cm), and the
number of rearing were recorded. In the open field test, the locomotor
activities of the mice were evaluated with the distance traveled variable,
and the anxiety-like behavior and exploratory behaviors of the mice
were evaluated with the frequency of rearing, time spent in the center,
frequency of entry to the center, and entry time to the center variables.
4.5.2. Morris water maze test
The Morris water maze used in the test consists of a 95-cm-diameter,
45-cm-deep cylinder made of galvanised sheet metal with a black inte­
rior. There is an escape platform (27 cm high and 10 cm in diameter)
that can be moved inside the pool. During the experiments, the inside of
the pool was filled with tap water to 1 cm above the platform level. The
water temperature was adjusted to 21±1 ◦ C. Black geometric shapes
were hung on the walls of the laboratory, in contrast to the light-colored
wall, within the field of view of the mice, to provide a spatial cue to the
mice swimming in the pool. The light in the pool is set at 50 lx.
Mice were subjected to consecutive training trials 4 times a day for 5
days in a Morris water maze. In each trial, mice left in the pool in one of
the four imaginatively divided quadrants were given 60 s to find the
escape platform. Animals were removed from the pool after remaining
on the platform for 10 s. During the training trials, if the mice could not
find the platform for 60 s, they were guided by hand to spatially learn
the location of the platform and were removed from the pool by keeping
them on the platform for 10 s. The position of the platform was kept
fixed in the same quadrant for all training trials. Between training trials,
mice were kept in their cages at room temperature for 30 s.
On day 6, the test day of the experiment, the platform was removed
from the pool, and the experimental animals were left in the pool in the
quadrant opposite the quadrant where the platform had been before its
removal. For the test, mice were floated in the pool for 60 s, and the time
spent in the target quadrant was recorded by a camera. Spatial learning
and memory of mice were assessed using ETHOVISION XT (NOLDUS,
version 4.1) (Akıllıoğlu, 2010.). In the Morris water maze test, for the
first 5 days, the escape latency (sec) and the average swimming speed to
the platform (cm/sec) were recorded during the mice’s 60-second
training trials. On the 6th day of the experiment, the time spent in the
target quadrant for 60 s and the average swimming speed (cm/sec) were
recorded. On the first five days of the MWM test, mice were evaluated
for their spatial learning, and on the sixth day, for their memory.
4.6. Western blot analysis
4.6.1. Obtaining total protein samples
The prefrontal cortex and hippocampus of mice were isolated before
western blotting to obtain proteins from the tissue samples to be used.
S. Kose et al.
The isolated tissues were kept at − 80 ◦ C. The prefrontal cortex and
hippocampus tissues were weighed on a sensitive scales. 3 ml of RIPA
(for each 1 ml of RIPA, 10 μl PMSF, 10 μl sodium orthovanadate, and
10–20 μl protease inhibitor were added) was added per 1 g of tissue and
homogenised at 70–80 RPM for at least 5 min. The protein sample and
the RIPA mixture were homogenised until creamy and transferred to 1
ml Eppendorf tubes. Eppendorf tubes were centrifuged at 14,000 RPM
at +4 ◦ C for 10 min. The supernatant obtained by centrifugation was
stored at − 20 ◦ C. The total protein level of the samples was determined
by the BCA (bicinchoninic acid) protein assay using albumin standards
of known protein levels.
3 μl of the gel electrophoresis standard was loaded into the first well
of the SDS-PAGE gel, and 30 μl of protein samples were loaded into each
of the other wells. The proteins were run at 80 V for the first 20 min and
then at 100 V for about 1–1.5 h until the proteins reached the end of the
membrane. At the end of the run, the gel was transferred to the PVDF
membrane. After transfer to the membrane, the gel was gently shaken
for 1 h at +4 ◦ C in 20 ml of 5% blocking solution. After 1 h, the mem­
brane was washed 3 times for 5 min with a washing solution at +4 ◦ C in
a shaker. Primary antibodies (ß-actin 1:1000, BDNF 1:500, TrkB 1:500,
and p-GSK3ßSer389 1:500) were applied immediately after washing. The
membrane was incubated with the primary antibody overnight at +4 ◦ C
on a shaker. After the primary antibody, the secondary antibody
(1:5000) was added to the membrane and incubated for 1 h at +4 ◦ C on a
shaker. Enhanced Chemi Luminescence (ECL) solution was then used for
imaging. After exposure to the ECL solution, the radiation generated on
the membrane was transferred to the 18x24 cm x-ray film, making the
protein bands visible. Quantitative analyses of band protein densities
were performed using the Image Lab program. The analysis was nor­
malised by dividing the specific protein density amounts by the band
intensity amount of ß-actin protein. The amount of band intensity %
change in comparison to the control group was expressed as: [(Prt C.
sample/Prt C.ß-actin)/Prt C.control mean. X 100]. While the protein
density value of the control group was taken as 100 %, the protein fold
increase of the control group was taken as 1.
4.7. Immunohistochemical method
In our study, 4 µm thick sections were taken from the hippocampus
and prefrontal cortex tissues of 4 mice from each group for histological
analyses. Histological sections of tissues were made on polylysine-
coated slides to determine APP and amyloid beta-40 (Aß-40) activity.
The sections kept in the refrigerator were dried before the marking
process was started, then put back in the refrigerator and left for 15 min.
The sections, which were dried again after removal from the refriger­
ator, were deparaffinized in xylol in an oven at 60 ◦ C for 15 min. Tissue
sections were then passed through the xylol series at room temperature.
It was hydrated in a series of gradually decreasing degrees of alcohol and
taken in distilled water. The sections were then placed in citrate solution
(pH: 6) and left in a water bath at 95 ◦ C for 45 min to reveal the antigens.
Sections cooled for 45 min at room temperature were washed in distilled
water and immersed in 3 % hydrogen peroxide (H2O2) for about 10 min
to prevent endogenous peroxides. Sections were washed in distilled
water and phosphate buffer (PBS, pH: 7.2–7.4). After carefully wiping
and drying each section, the blocking solution (ab93705, Abcam, USA)
was added to prevent non-specific binding. After 15 min at room tem­
perature, the sections were passed through distilled water and phos­
phate buffer, and the surrounding tissue was dried. The primary
antibodies anti-APP (27320–1-AP, Proteintech, USA) at 1:200 dilution
and anti-Aß-40 (NBP1 44047, Novus Biologicals, USA) at 1:200 dilution
were then applied. All primary antibody coated sections were stored
overnight at +4 ◦ C in a humid chamber. Instead of primary antibodies,
dilution solution was dripped onto sections taken for the negative con­
trol. Sections treated with primary antibodies and kept at +4 ◦ C over­
night and then at room temperature for 1 h were then washed with
distilled water and PBS. Biotin (ab93705, Abcam, USA.) was applied and
15
Brain Research 1814 (2023) 148429
left at room temperature for 15 min. After 15 min, the sections were
washed in distilled water and PBS, dried, and Avidin (ab93705, Abcam,
USA) was added and left for 20 min at room temperature. Amino­
ethylcarbozol (AEC, ab93705, Abcam, USA) solution was dripped onto
the sections that had been passed through distilled water and PBS, left
for 10 min, and then washed in tap water. Tissue sections were stained
with hematoxylin for 30 s before counterstaining. After staining, the
sections were washed in tap water and allowed to dry. Tissues were
covered with a coverslip by dripping water-based sealant. Photographs
were taken using an Olympus BX53 light microscope (Japan).
The immunohistochemical scoring method was used to investigate
the expression of APP and Aß-40. For immunohistochemical scoring, the
H-scoring system was used in 10 randomly selected areas of a 40-mm
lens. The H-score was calculated by summing the products of the 4
different staining intensities (3+++ (strong staining), 2++ (medium
staining), 1+ (weak staining), 0 (no staining)) together with the per­
centage of positive cells. Differences in expression between groups were
evaluated using the Mann-Whitney U test. p < 0.05 was considered
significant Fig. 12.
4.8. Statistical analysis
Data were expressed as mean ± standard error (SE). p < 0.05 was
considered significant. Two-way ANOVA was used for normally
distributed and parametric data, followed by Tukey’s test for pairwise
group comparisons. Non-normally distributed and non-parametric data
were analysed using the Kruskal-Wallis test, followed by the Mann-
Whitney U test for pairwise group comparisons. As body weight and
the Morris water maze test did not follow a normal distribution, Fried­
man’s ANOVA was used, followed by the Wilcoxon test for within-group
comparisons and the Mann-Whitney U test for between-group compar­
isons. SPSS version 20.0 was used for statistical analysis.
5. Conclusion
In our study, we found that physical exercise may protect against the
memory impairment and anxiety-like behaviour observed with scopol­
amine induced Alzheimer’s Disease-like condition in Balb/c mice. There
are many studies in the literature aimed at understanding the physio­
pathology of AD. In the scope of these studies, it is aimed to investigate
the effects related to the decrease of BDNF and the accompanying
disruption of intracellular signalling pathways. It is mainly reported that
increased GSK-3ß mediate Aß plaque formation and excessive tau
phosphorylation, whereas Ser9 phosphorylation reduces these adverse
effects. According to the results of our study, it can be suggested that the
decrease in GSK3ßSer389 phosphorylation is important in the physiopa­
thology of AD, and the effects of physical exercise are mediated by
GSK3ßSer389 phosphorylation. In particular, the investigation of the p38
MAPK/GSK3ßSer389 pathway, which is known to be active in specific
tissues such as the brain and spleen, will be important in further studies
to understand the role of this pathway in exercise and AD.
6. Limitations
Our study evaluated p-GSK3ßSer389, but due to budget limitations, we
were unable to evaluate total GSK3ß or the p-p38MAPK signalling
pathway. Furthermore, the small sample size of mice used in molecular
studies was another limitation of our study. Despite these limitations, we
still believe that our research provides valuable insights into the area,
and that further research should be conducted to gain a better under­
standing of the mechanisms involved.
CRediT authorship contribution statement
Seda Kose: Validation, Investigation, Writing – original draft,
Writing – review & editing. Meltem Donmez Kutlu: Validation,
S. Kose et al.
Fig. 12. Timeline of the experiment.
Investigation, Writing – review & editing. Samet Kara: Validation,
Investigation, Visualization, Resources. Sait Polat: Validation, Investi­
gation, Visualization, Resources. Kubra Akillioglu: Conceptualization,
Methodology, Validation, Formal analysis, Resources, Writing – original
draft, Writing – review & editing, Visualization, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This study is a part of the MSc thesis of Seda Köse. This study was
supported by a research grant (TYL-2020-13222) from the Scientific
Research Projects Unit of Cukurova University.
Funding
This work was supported by the Scientific Research Office of
Cukurova University (I.U BAP, Project number: TYL-2020-13222).
Our study was presented partly at of the Turkish Society of Physio­
logical Sciences, 46th National Physiology Congress, 8–10 October
2021, Virtual Meeting. The abstract of the presentation was printed in
“Acta Physiologica 2022; Volume 234, Supplement 724: PC05.” It also
presented partly at the Turkish Society of Physiological Sciences, 47th
Turkish Physiology Congress, 1-4 November 2022, Antalya, Turkey. The
abstract of the presentation was printed in “Acta Physiologica 2023;
Volume 237, Supplement 727: OC57.”
This study was supported by a research grant (TYL-2020-13222)
from the Scientific Research Projects Unit of Cukurova University.
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