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Citation: Ilbay, G.; Dogan, Z.I.;
Balıkcı, A.; Erdogan, S.; Karaoglan
Kahilogulları, A. Effects of Postnatal
Caffeine Exposure on Absence
Epilepsy and Comorbid Depression:
Results of a Study in WAG/Rij Rats.
Brain Sci. 2022,12, 361. https://
doi.org/10.3390/brainsci12030361
Academic Editor: Maria Nobile
Received: 22 January 2022
Accepted: 6 March 2022
Published: 8 March 2022
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brain
sciences
Article
Effects of Postnatal Caffeine Exposure on Absence Epilepsy and
Comorbid Depression: Results of a Study in WAG/Rij Rats
Gul Ilbay 1, *,† , Zeynep Ikbal Dogan 1 ,† , Aymen Balıkcı 1, Seyda Erdogan 2and Akfer Karaoglan Kahilogulları 3
1Department of Physiology, Faculty of Medicine, Kocaeli University, Kocaeli 41100, Turkey;
zynp_ikbal@hotmail.com (Z.I.D.); pt_eymen@hotmail.com (A.B.)
2Department of Neurology, Faculty of Medicine, Ankara University, Ankara 06560, Turkey;
dr_seyda@yahoo.com
3Department of Psychiatry, Health Sciences University Dı¸skapı Yıldırım Beyazıt Training and Research
Hospital, Ankara 06500, Turkey; akferkaraoglan@hotmail.com
*Correspondence: gulilbay@yahoo.com
† These authors contributed equally to this work.
Abstract:
The present study aims to investigate effect of early caffeine exposure on epileptogenesis
and occurrence of absence seizures and comorbid depression in adulthood. For this purpose, Wistar
Albino Glaxo from Rijswijk (WAG/Rij) rats were enrolled in a control and two experimental groups
on the 7th day after the delivery. The rats in experimental groups received either 10 or 20 mg/kg
caffeine subcutaneously while animals in control group had subcutaneous injections of 0.9% saline.
The injections started at postnatal day 7 (PND7) and were continued each day for 5 days. At
6–7 months
of age, electroencephalogram (EEG) recordings and behavioral recordings in the forced
swimming test, sucrose consumption/preference test and locomotor activity test were carried out.
At 6 months of age, 10 mg/kg and 20 mg/kg caffeine-treated WAG/Rij rats showed increased
immobility latency and active swimming duration in forced swimming test when compared with
the untreated controls. In addition, 20 mg/kg caffeine treatment decreased immobility time. In
sucrose preference/consumption tests, WAG/Rij rats in 10 mg/kg caffeine group demonstrated
higher sucrose consumption and preference compared to untreated controls. The rats treated with
20 mg/kg caffeine showed higher sucrose preference compared to control rats. The exploratory
activity of rats in the 10 mg/kg caffeine-treated group was found to be higher than in the 20 mg/kg
caffeine-treated and control groups in the locomotor activity test. At 7 months of age, caffeine-treated
animals showed a decreased spike-wave discharge (SWD) number compared to the control animals.
These results indicate that postnatal caffeine treatment may decrease the number of seizure and
depression-like behaviors in WAG/Rij rats in later life. Caffeine blockade of adenosine receptors
during the early developmental period may have beneficial effects in reducing seizure frequency and
depression-like behaviors in WAG/Rij rat model.
Keywords: chronic absence seizure; caffeine; depression; WAG/Rij rats
1. Introduction
Depression is a common mental disorder affecting 280 million people worldwide [
1
].
Despite the availability of effective treatments for mental disorders including depres-
sion, people who experience depression are often not diagnosed correctly or receive no
treatment [2]
. Absence seizure incidence varies from 0.7 to 4.6 per 100,000 in the general
population [
3
]. Similar to depression, many people with epilepsy do not receive appropriate
and adequate treatment for their condition. This is defined as a phenomenon called the
treatment gap.
Many developing countries with limited resources struggle to develop and implement
policies to prevent an increase in the treatment gap. Prevention or early diagnosis and
intervention programs have been developed to reduce the burden of these two disorders.
Brain Sci. 2022,12, 361. https://doi.org/10.3390/brainsci12030361 https://www.mdpi.com/journal/brainsci
Brain Sci. 2022,12, 361 2 of 15
Effective community-based programs have been shown to reduce depression [
1
]. However,
development of psychosocial interventions is not enough to prevent all depression cases
due to the multifactorial nature of the disorder including biological, environmental and
personal factors. Moreover, there is still a group of patients who do not respond to current
treatments even when they access quality health care in a timely fashion.
Epileptogenesis is defined as the development and extension of tissue which is able to
generate spontaneous seizures and refers to the process by which the previously normal
brain is functionally altered towards the generation of abnormal electrical
activity [4,5]
.
Epileptogenesis is considered to occur in three phases: occurrence of a precipitating injury
or event, a latent period and chronic, established epilepsy. The latent period of epilepto-
genesis is a clinically silent period characterized by ongoing remodeling processes that
may last from days to years after the injury. Several mechanisms may be responsible for
epileptogenesis, including plasticity and trafficking of GABA-A receptors, network re-
organization, increased inflammatory processes, changes in gene and receptor expression
and seizure or status epilepticus-induced alterations in ion channels. Recent data indicate
that epileptogenesis is not restricted over a limited time—contrary to what was previously
thought—and continues to progress after the expression of spontaneous seizures making
the process a moving target [
5
,
6
]. Therefore, interventions during this period might be
used both to prevent the subsequent development of epilepsy and disease modification.
From a therapeutic perspective, disease modification consists of antiepileptogenesis and
comorbidity modification. Antiepileptogenic treatment can be given before or after the
epilepsy onset and may result in complete prevention or delay of epilepsy development
or reduce the severity of the disease. The other step of treatment is preventing the burden
of comorbid diseases such as anxiety, depression, somato-motor impairment or cogni-
tive decline. Despite the large number of anti-seizure medications, they fail to prevent
epilepsy development and provide disease modification. Even seizure control cannot be
achieved in up to 30% of epilepsy patients with current anti-seizure drugs. Thus, the
development of novel therapeutic approaches to prevent and treat epileptogenesis and its
comorbid disorders is crucial. There have been an increasing number of studies investi-
gating the antiepileptogenic effects of different medications in animal models of structural
and genetic epilepsies.
A number of clinical observations show a relationship between epilepsy and depres-
sion. Both depressive disorders and epilepsy have a genetic basis. In 1986, researchers
described one of the most well-known rat models as a valid genetic animal model of absence
epilepsy with comorbidity of depression absence epilepsy [
7
]. Rats of Wistar Albino Glaxo
from Rijswijk (WAG/Rij) strain, around 2 months of age, showed spontaneous, generalized
spike-wave discharges (SWDs) in electroencephalograms (EEG) along with decrease of
consciousness and an interruption of continuous activity, as in human absence epilepsy.
After the age of six months, around 16–20 SWDs/h appeared in the EEG of WAG/Rij rats
and continued in a lifelong manner, therefore serving as the chronic animal model for
absence epilepsy [
8
]. WAG/Rij rats are also confirmed as an animal model for chronic
depression since they exhibit depression-like symptoms [
9
]. Depression-like behavioral
symptoms emerge at 3–4 months of age and tend to be aggravated parallel to the increase
of SWDs [10].
The molecules investigated in animal models of genetic epilepsies include anti-seizure
drugs such as levetiracetam, ethosuximide, zonisamide, vigabatrin and perampanel; an-
tidepressants such as clomipramine, fluoxetine and duloxetine; statins such as atorvastatin,
simvastatin and pravastatin, immunosuppressants or immunomodulator drugs such as
rapamycin, etericobix and fingolimod [
5
,
6
,
11
]. Early long-term fingolimod treatment was
shown to have antiepileptogenic and antidepressant effects in WAG/Rij rats, however the
antiepileptogenic effects were transitory given that the absence seizures returned to control
levels 5 months after treatment discontinuation [
11
]. The effect of
liraglutide—a novel
glucagon-like peptide-1 analogue—on epileptogenesis and comorbid behavioral alter-
ations was investigated in the mouse intrahippocampal kainic acid model of temporal
Brain Sci. 2022,12, 361 3 of 15
lobe epilepsy and the WAG/Rij rat model of absence epilepsy [
12
]. It was proposed that
liraglutide had neuroprotective effects with several mechanisms including reduced neuroin-
flammation and increased pro-survival cell signaling. Liraglutide showed antiepileptogenic
effects and prevented comorbid memory impairment and anxiety-like behavior in kainite
induced epilepsy. However, there was no modifying effect on epileptogenic process in the
WAG/Rij rat model of absence epilepsy. Recently, the disease modifying effect of two his-
tone deacetylase inhibitors, sodium butyrate and valproic acid and their co-administration,
was studied in WAG/Rij rats, based on the evidence pointing to the link between epigenetic
mechanisms such as alterations in histone acetylation and the epileptogenic process [
13
].
The results of the study demonstrated the antiepileptogenic effects of each drug with
enhancement by co-administration. Additionally, one month after treatment withdrawal,
depressive-like behavior and cognitive performance showed improvement with early-
chronic treatment. Therefore, the investigators proposed histone deacetylase inhibitors as
a potential candidate in management of epileptogenesis and psychiatric comorbidity.
The evidence of environmental factors leading to epigenetic modifications in the
development of epilepsy and comorbid depression have also been shown in the WAG/Rij
rat model [
14
]. The onset of absence epilepsy was delayed, and depression symptoms were
reduced in the WAG/Rij pups that received intensive early maternal care by their foster
Wistar mothers [
15
]. Our previous study showed that early neonatal tactile stimulations
may decrease seizure activity and comorbid depression-like behaviors at adult ages in
WAG/Rij rats [
16
]. These examples demonstrate the effect of early environmental factors
on genetic absence epilepsy and comorbid depression.
Environmental factors, modulating adenosine, have recently been reported as other
factors that can control the development of epilepsy [
17
]. Adenosine, a neuromodulator that
terminates seizures in the brain through activation of adenosine A
1
receptors and decreases
neuronal excitability also regulates cognitive and psychiatric behavior [
18
,
19
]. Gomes et al.
reported that major depression significantly affects the expression and functioning of the
adenosinergic system, with data on A1R and A2AR [20].
Due to having molecular structural similarities to adenosine, caffeine can bind to the
A
1
and A
2A
adenosine receptor subtypes and block adenosine from binding thereby acting
as an adenosine antagonist [21].
There are numerous studies investigating chronic low-dose caffeine effects on seizures
and epilepsy in adult rats and they demonstrate different outcomes. Some of these studies
did not find a relation between exposure to chronic low-dose caffeine or withdrawal
from it and seizures [
22
]. However, there are studies reporting decrease in the duration
of convulsions in animals that received caffeine for several days in comparison to ones
that received saline, in addition to studies reporting seizures triggered by a single-high
caffeine dose [
23
]. In adult rats with genetic absence epilepsy, seizure activity decreased
after an acute dose of caffeine, while chronic doses had no effect [
24
]. Moreover, seizure
susceptibility was demonstrated to decrease in young animals by chronic, low-dose caffeine
exposure. Rat pups exposed to chronic low doses of caffeine in the first weeks after
birth showed an increase in seizure threshold for generalized tonic-clonic seizures in
adulthood [
13
]. Tchekalarova et al. demonstrated that neonatal caffeine treatment caused
suppression of rhythmic metrazol activity evoked by PTZ in adult rats in another study [
25
].
Overall, the impact of caffeine on seizure seems to be dependent on age, seizure model,
caffeine dose, and administration method [23].
Studies on caffeine and its relationship to depression are not as numerous as the ones
on epilepsy but there are promising results about the impact of caffeine as an adenosine
receptor antagonist on symptoms of depression. Epidemiological studies report beneficial
effects of intermediate levels of caffeine consumption (300–550 mg/day) on depressive
symptoms in non-clinical populations while higher doses have negative effects [
26
]. These
effects are also shown in animal models [27].
Brain Sci. 2022,12, 361 4 of 15
Considering the burden of the disorders and high comorbidity, we have investigated
the effects of early intervention of caffeine on comorbid epilepsy and depression in the
chronic animal model of absence epilepsy and comorbid depression.
2. Materials and Methods
2.1. Ethical Approval
Data from the experimental study that was approved by the Ethics Committee of
Kocaeli University (KOU-7/1-2016) is revisited for this paper.
2.2. Animals and Caffeine Treatment
Progeny of pregnant WAG/Rij rats kept in separate cages in an environment at
22–23 ◦C
and on a 12–12 h light–dark cycle with free access to water and food were enrolled
in the study. The births were monitored and on the 7th day following birth (PND7), pups
were randomly assigned to either a control group or to one of the two experimental groups.
Rat pups in experimental groups were given subcutaneous injections of 10 or 20 mg/kg
caffeine. The control group were injected with 0.9% saline. The injections started at PND7
were continued for 5 days. We chose the period of 7–11 days for the intervention since
adenosine receptors are present but not fully mature in the various brain areas at this
developmental stage [
19
]. Rat pups stayed with their mother and were separated just for
a short amount of time during injection. Litters were weaned on postnatal day 21. After
weaning, rats were housed in different cages according to the experimental groups and
were maintained under normal conditions until 180 days of age.
2.3. Behavioral Tests
All WAG/Rij rats (n= 9 for saline, n= 10 for 10 mg/kg caffeine and n= 9 for 20 mg/kg
caffeine treatment) were individually exposed to a forced swimming test (FST), sucrose
consumption test (SCT) and locomotor activity test at the age of 6 months between
10:00 am
and 3:00 pm each day of the tests.
2.3.1. Force Swimming Test
Depressive-like behaviors are assessed with FST, which has been used in many studies
with some minor modifications [
14
,
28
]. The experiment was conducted in a transparent
cylinder (47
×
38 cm) filled with water 22
±
1
◦
C. Initially, rats (PND180) were forced to
swim for 15 min as part of the pretest session and were dried before being put back into
their cages. Twenty-four hours after the pretest session, rats were resubmitted to the forced
swimming test for 5 min and their swimming behavior was recorded using video cameras.
Immobility (duration of passive swimming), immobility latency and swimming time (total
duration of active swimming) were assessed by a blinded rater for each rat. Immobility is
defined as having no additional activity other than the required act of keeping the head
above water.
2.3.2. Sucrose Consumption/Preference Test
Anhedonia and motivation were assessed with the sucrose consumption/preference
test. The drinking of sucrose solution (20%) and the number of approaches to the bottle
were calculated during fifteen minutes for each rat. The bottles were weighed at the
beginning and at the end of the test in order to measure the sucrose intake. Animals were
not subjected to any of food and water deprivation. Values of sucrose consumption on the
4th day were used to compare the differences between rat groups after a 3-day adaptation
procedure as defined in previous studies [29].
2.3.3. Locomotor Activity Test
A locomotor activity was recorded by the rat activity monitoring system (Commat
Ltd., Ankara, Turkey) consisting of a Plexiglas test chamber, computer and activity software.
Rats were taken to the test room for 1 h and were then placed into plexiglass chamber
Brain Sci. 2022,12, 361 5 of 15
(42 ×42 ×30 cm
) of the system. The chamber had infrared photocells, with pairs of 15 in-
frared photobeams and detectors located every 2.5 cm in the horizontal plane (bottom) and
every 4.5 cm in the vertical plane (upper). Locomotor activities measured with interruption
of photocell beams were recorded by computer for 10 min. The repeated beam breaks
at the same photobeams are used as a measure of stereotypic activity. The breaks in the
upper set of photobeams were used as a measure of vertical activity. Breaking more than
1 consecutive
photobeam in the bottom set of photobeams was used as a measure of ambu-
latory activity. Total distance travelled (in cm) and numbers of stereotypic, ambulatory and
vertical movements were analyzed [16].
2.4. Surgery, EEG Recordings and Assessment of Absence Seizures
At 7 months of age, 7 animals in each group were equipped with electrodes. Tripo-
lar electrodes (MS3333/2A; Plastic One, the United States) were put over the frontal
(
AP 2.0 mm,
L 3.5 mm), the parietal (AP
−
6.0 mm L 4.0 mm) and the cerebellar cortex
(reference electrode) with stereotaxic surgery under Xylazin (5 mg/kg ip) and Ketamine
(60 mg/kg ip) anesthesia. After 2 weeks of recovery, animals were connected to the MP150
EEG recording system. After the rats that were free to move were habituated to record-
ing conditions for 1 h, EEG recordings from each subject were taken for 4 h between
10:00 am–2:00 pm
. Numbers and durations of spike wave discharges (SWD) which met the
following criteria defined by van Luijtelaar et al. were analyzed: duration 1–10 s, having
spike and wave with frequencies between 7–10 Hz and doubled amplitude to background
activity [30].
2.5. Statistical Analyses
Data are as means
±
S.E.M. The analyses were run in GraphPad Prism 7.03 (
San Diego
,
CA, USA), using a one-way ANOVA followed by post hoc tests. Figure legends demonstrate
statistical tests and sample sizes. The level of 0.05 was made use of as a threshold for
statistical significance.
3. Results
3.1. Behavioral Measures in the Forced Swimming Test
In rat pups treated with caffeine, latency to immobility was significantly longer than
the rats in the control group (10 mg/kg vs. control p= 0.023; 20 mg/kg vs. control
p< 0.001)
(Figure 1A). The immobility durations of the rats in the group treated with
20 mg/kg
caf-
feine injection were significantly less than for rats in the control group (
p< 0.05
) (Figure 1B).
The 10 mg/kg caffeine group also showed shorter immobility duration compared with the
control group (Table 1) but the difference was not statistically significant.
When swimming time was evaluated, it was found that the rats in the 10 mg/kg and
20 mg/kg caffeine treatment groups were significantly more active than the rats in the
control group (10 mg/kg vs. control p< 0.05; 20 mg/kg vs. control p< 0.01) (Figure 1C).
3.2. Sucrose Consumption/Preference Test
Sucrose consumption in the 10 mg/kg caffeine treatment group was significantly
higher compared to the control group (p= 0.01) (Figure 2A). Caffeine treatment groups
had a higher sucrose preference (%) compared to the control group (10 mg/kg vs. control
p< 0.001; 20 mg/kg vs. control p< 0.05) (Figure 2B).
3.3. Locomotor Activity
In the locomotor activity test, significant differences amongst the 10 mg/kg caffeine
treatment group, the 20 mg/kg caffeine treatment group and the control group were found.
The locomotor activity distance of the 10 mg/kg caffeine-treated group was significantly
higher in comparison to the control group (p< 0.01) (Figure 3A). Mean number of ambula-
tory activities in the 10 mg/kg caffeine treatment group was significantly higher than the
other two groups (10 mg/kg vs. control p= 0.001; 10 mg/kg vs. 20 mg/kg caffeine p< 0.05)
Brain Sci. 2022,12, 361 6 of 15
(Figure 3B). Rats treated with 20 mg/kg caffeine showed significantly higher stereotypic
movements than those of the 10 mg/kg caffeine treatment and the control group (p= 0.01)
(Figure 3C). No change was observed amongst the groups in vertical movements.
Brain Sci. 2022, 12, x FOR PEER REVIEW 6 of 15
224
217
169
139
When swimming time was evaluated, it was found that the rats in the 10 mg/kg and
20 mg/kg caffeine treatment groups were significantly more active than the rats in the
control group (10 mg/kg vs. control p < 0.05; 20 mg/kg vs. control p < 0.01) (Figure 1C).
Figure 1. Behavioral measures in the forced swimming test control (n = 9), 10 mg/kg (n = 10) and 20
mg/kg (n = 9) caffeine-treated WAG/Rij rats. Data are shown as mean ± SEM. Changes in (A) Latency
Figure 1.
Behavioral measures in the forced swimming test control (n= 9), 10 mg/kg (n= 10) and
20 mg/kg (n= 9) caffeine-treated WAG/Rij rats. Data are shown as mean
±
SEM. Changes in
(A) Latency
until first immobility period, (
B
) Time spent in immobility, (
C
) Swimming time. Data
marked with asterisks are significantly different. * p< 0.05, ** p< 0,01 *** p
≤
0.001 versus the control
group (one-way ANOVA with post hoc Tukey test).
Brain Sci. 2022,12, 361 7 of 15
Table 1. Immobility durations of the rats according to the study groups.
Control Group
Seconds
10 mg/kg Caffeine Group
Seconds
20 mg/kg Caffeine Group
Seconds
278 214 168
268 208 207
212 215 200
264 29 207
201 217 156
179 192 156
218 189 130
169 160 157
224 217 169
139
Brain Sci. 2022, 12, x FOR PEER REVIEW 7 of 15
until first immobility period, (B) Time spent in immobility, (C) Swimming time. Data marked with
asterisks are significantly different. * p < 0.05, ** p < 0,01 *** p ≤ 0.001 versus the control group (one-
way ANOVA with post hoc Tukey test).
3.2. Sucrose Consumption/Preference Test
Sucrose consumption in the 10 mg/kg caffeine treatment group was significantly
higher compared to the control group (p = 0.01) (Figure 2A). Caffeine treatment groups
had a higher sucrose preference (%) compared to the control group (10 mg/kg vs. control
p < 0.001; 20 mg/kg vs. control p < 0.05) (Figure 2B).
Figure 2. Effects of postnatal caffeine treatment on sucrose consumption (A) and preference (B) in
control (n = 9), 10 mg/kg caffeine-treated (n = 10) and 20 mg/kg caffeine treated (n = 9) WAG/Rij rats.
Data are shown as mean ± SEM. Data marked with asterisks are significantly different. * p < 0.05, **
p ≤ 0.01 *** p < 0.001 versus the control group (one-way ANOVA with post hoc Tukey test).
3.3. Locomotor Activity
In the locomotor activity test, significant differences amongst the 10 mg/kg caffeine
treatment group, the 20 mg/kg caffeine treatment group and the control group were
found. The locomotor activity distance of the 10 mg/kg caffeine-treated group was signif-
icantly higher in comparison to the control group (p < 0.01) (Figure 3A). Mean number of
ambulatory activities in the 10 mg/kg caffeine treatment group was significantly higher
Figure 2.
Effects of postnatal caffeine treatment on sucrose consumption (
A
) and preference (
B
) in
control (n= 9), 10 mg/kg caffeine-treated (n= 10) and 20 mg/kg caffeine treated (n= 9) WAG/Rij rats.
Data are shown as mean
±
SEM. Data marked with asterisks are significantly different.
*p< 0.05,
** p≤0.01, *** p< 0.001 versus the control group (one-way ANOVA with post hoc Tukey test).
Brain Sci. 2022,12, 361 8 of 15
Brain Sci. 2022, 12, x FOR PEER REVIEW 8 of 15
than the other two groups (10 mg/kg vs. control p = 0.001; 10 mg/kg vs. 20 mg/kg caffeine
p < 0.05) (Figure 3B). Rats treated with 20 mg/kg caffeine showed significantly higher ste-
reotypic movements than those of the 10 mg/kg caffeine treatment and the control group
(p = 0.01) (Figure 3C). No change was observed amongst the groups in vertical movements.
Figure 3. The different components of locomotor activity in control (n = 9), 10 mg/kg (n = 10) and 20
mg/kg (n = 9) caffeine-treated WAG/Rij rats. Data are shown as mean ± SEM. (A) ** Significantly
Figure 3.
The different components of locomotor activity in control (n= 9), 10 mg/kg (n= 10) and
20 mg/kg (n= 9)
caffeine-treated WAG/Rij rats. Data are shown as mean
±
SEM. (
A
) ** Significantly
longer distance traveled (cm) compared with control rats (p< 0.01), (
B
) Higher numbers of ambulatory
activity treated with 10 mg/kg caffeine compared with control (*** p= 0.001) and 20 mg/kg caffeine-
treated WAG/Rij (* p< 0.05) rats, (
C
) Higher numbers of stereotypic activity treated with 20 mg/kg
caffeine compared with control 10 mg/kg caffeine treatment group and control group (** p= 0.01);
(one-way ANOVA with post hoc Tukey’s test).
Brain Sci. 2022,12, 361 9 of 15
3.4. Effects of Postnatal Caffeine Treatment on SWDs in Adult WAG/Rij Rats
SWDs were detected on the EEG background, which are repetitive complexes with
a frequency
of 7–10 Hz (Figure 4). SWD number and total duration were summed for 4 h
in caffeine-exposed and control WAG/Rij rats.
Brain Sci. 2022, 12, x FOR PEER REVIEW 9 of 15
longer distance traveled (cm) compared with control rats (p < 0.01), (B) Higher numbers of ambula-
tory activity treated with 10 mg/kg caffeine compared with control (*** p = 0.001) and 20 mg/kg
caffeine-treated WAG/Rij (* p < 0.05) rats, (C) Higher numbers of stereotypic activity treated with 20
mg/kg caffeine compared with control 10 mg/kg caffeine treatment group and control group (** p =
0.01); (one-way ANOVA with post hoc Tukey’s test).
3.4. Effects of Postnatal Caffeine Treatment on SWDs in Adult WAG/Rij Rats
SWDs were detected on the EEG background, which are repetitive complexes with a
frequency of 7–10 Hz (Figure 4). SWD number and total duration were summed for 4 h in
caffeine-exposed and control WAG/Rij rats
Figure 4. Figure shows examples of SWDs and the power spectra of EEG during the SWD in
WAG/Rij rats.
Figure 5 depicts the results for number and total duration of SWDs in three groups.
Rats in groups treated with caffeine showed less SWD number; however, a statistically
significant decrease was found only in the 20 mg/kg caffeine treatment group (p < 0.05)
(Figure 5). When total SWD duration was compared, no statistically significant difference
was found between the groups but total durations of SWD of the 10 and 20 mg/kg caffeine-
treated group were lower than control group. Total SWD duration was 850 ± 150 in the
Figure 4.
Figure shows examples of SWDs and the power spectra of EEG during the SWD in
WAG/Rij rats.
Figure 5depicts the results for number and total duration of SWDs in three groups.
Rats in groups treated with caffeine showed less SWD number; however, a statistically
significant decrease was found only in the 20 mg/kg caffeine treatment group (p< 0.05)
(Figure 5). When total SWD duration was compared, no statistically significant difference
was found between the groups but total durations of SWD of the 10 and 20 mg/kg caffeine-
treated group were lower than control group. Total SWD duration was 850
±
150 in the
control group, 489
±
114 in the 10 mg/kg caffeine-treated group and 656
±
154 in the
20 mg/kg caffeine-treated group.
Brain Sci. 2022,12, 361 10 of 15
Brain Sci. 2022, 12, x FOR PEER REVIEW 10 of 15
control group, 489 ± 114 in the 10 mg/kg caffeine-treated group and 656 ± 154 in the 20
mg/kg caffeine-treated group.
Figure 5. Number (A) and total duration (B) of SWDs in control (n = 7) and 10 mg/kg (n = 7) and 20
mg/kg (n = 7) caffeine-treated WAG/Rij rats. * p < 0.05 in comparison with control group; (one-way
ANOVA with post hoc Tukey’s test). Data are means ± S.E.M.
4. Discussion
Caffeine is a central nervous system stimulant, and several interactions may contrib-
ute to its stimulative effects. For instance, it modulates the GABA-A receptors and changes
the response to inhibitory gamma-aminobutyric acid. Additionally, the molecular struc-
ture of caffeine (1,3,7-trimethylxanthine) is similar to adenosine [31]. Adenosine is a pu-
rine and two different types of purinergic receptors have been described: selective for
adenosine (P1) and selective for ATP/ADP (P2) [32]. The adenosine selective P1 receptors,
have four subtypes with A1 and A2A subtypes showing high affinity for adenosine. Ac-
tion of A1 receptors results in inhibition of adenylate cyclase followed by inhibition of
glutamatergic transmission. The effect of seizure suppression of adenosine, as an inhibi-
tory neuromodulator is supposed to be mainly based on A1 receptors [33]. Caffeine is a
competitive non-selective P1 receptor antagonist binding to the A1 receptor as well as the
Figure 5.
Number (
A
) and total duration (
B
) of SWDs in control (n= 7) and 10 mg/kg (n= 7)
and
20 mg/kg
(n= 7) caffeine-treated WAG/Rij rats. * p< 0.05 in comparison with control group;
(one-way ANOVA with post hoc Tukey’s test). Data are means ±S.E.M.
4. Discussion
Caffeine is a central nervous system stimulant, and several interactions may contribute
to its stimulative effects. For instance, it modulates the GABA-A receptors and changes the
response to inhibitory gamma-aminobutyric acid. Additionally, the molecular structure of
caffeine (1,3,7-trimethylxanthine) is similar to adenosine [
31
]. Adenosine is a purine and
two different types of purinergic receptors have been described: selective for adenosine
(P1) and selective for ATP/ADP (P2) [
32
]. The adenosine selective P1 receptors, have four
subtypes with A1 and A2A subtypes showing high affinity for adenosine. Action of A1
receptors results in inhibition of adenylate cyclase followed by inhibition of glutamatergic
transmission. The effect of seizure suppression of adenosine, as
an inhibitory
neuromodu-
lator is supposed to be mainly based on A1 receptors [
33
]. Caffeine is
a competitive
non-
selective P1 receptor antagonist binding to the A1 receptor as well as the A2A
receptor [23]
.
Contrary to A1 receptors, the action of A2A receptors results in activation of adenylate
cyclase further causing an increase in glutamatergic transmission [
34
]. Accordingly, the
Brain Sci. 2022,12, 361 11 of 15
answer to the question of the effects of caffeine on the development of seizure activity is
not simple.
Previous research showed that caffeine may cause neuronal hyperexcitability by
increasing Ca
2
influx, changing potassium currents and antagonism of inhibitory A1
adenosine receptors [
23
,
34
,
35
]. In fact, caffeine was previously proposed as an animal model
of seizures [
36
]. However, the results of animal studies have suggested the effect may be
towards either increasing seizure susceptibility or protecting against seizures; depending on
age, the animal epilepsy model, administration type (a single dose or long-term exposure)
and caffeine dose [
23
]. The results of studies investigating the effects of caffeine exposure in
adult animals are conflicting. Interestingly, a recent study investigating the effect of agonists
and antagonists of adenosinergic activity on seizures and inflammation in WAG/Rij rats
found that caffeine administered rats show a decreased SWD number on EEG linked
to
an increase
of cytokine levels in the thalamus [
37
]. The results of this study may be
important for pointing to the potential interactions between the adenosinergic system,
inflammation and seizures. Indeed, adenosine A2A receptors are found in immune cells and
involved in tissue protection by reducing inflammation [
38
]. The role of adenosine A2A and
P2 receptors via inflammatory processes attracts attention in other neurological disorders
such as Parkinson’s disease, memory impairment, multiple sclerosis or amyotrophic lateral
sclerosis [34].
Animal studies on the effect of maternal caffeine exposure reported an increased
seizure susceptibility by hyperthermia in the offspring of rat dams receiving caffeine
during pregnancy [
39
]. Additionally, a delayed migration of GABAergic neurons into
the hippocampus was reported. In humans a few studies pointed to this issue. A study
analyzing 35,596 children in Denmark found no association between maternal caffeine
intake and the risk of febrile seizures in the first three months of life [
40
]. So far, in humans
there is no clear evidence that maternal caffeine intake increases the risk of seizures.
The protective effects of caffeine stand out especially when administered to young
animals having an immature brain, as in our study [
23
]. This may be most likely due to
changes in the density and sensitivity of adenosine A1 and A2A receptors during brain de-
velopment at young ages [
41
–
43
]. The present study showed that repeated treatment with
caffeine from postnatal day 7 to 11 has an impact on EEG parameters of absence seizures
and depression-like behaviors in adult WAG/Rij rats that have a genetic predisposition to
absence epilepsy and comorbid depression. Caffeine treatment groups have
a decrease
in
both number and total duration of SWDs. Caffeine treatment groups showed increased im-
mobility latency, swimming time and reduced immobility duration in the forced swimming
test. Sucrose preference and sucrose consumption were also increased with caffeine treat-
ment. Caffeine-treated rats were more active and showed more stereotypic and exploratory
activity in the locomotor activity test in comparison to the control rats.
There are experimental studies which suggest that seizure susceptibility could be
modified by early caffeine treatment during development. The findings of these studies
indicated that early caffeine exposure exerts predominantly anticonvulsant effects, but
the effects may change by age, dose and model [
44
]. For example, caffeine treatment
during the second postnatal week (P7–P11) resulted in decreased seizure threshold to
PTZ and picrotoxin while they showed full resistance to bicuculline at 25-day-old Wistar
rats [
45
]. Data on the effect of early caffeine treatment on seizure activity in adulthood
is limited. Postnatal caffeine treatment was shown to decrease the seizure susceptibility
to different convulsive agents such as PTZ, picrotoxin and kainic acid in adult rats [
46
].
Tchekalarova et al.
showed that a chronic low dose of caffeine 7–11 days after birth caused
suppression of seizures in a PTZ model absence seizure [
13
]. Similar to these convulsive
model results, postnatal caffeine treatment induced suppression on seizure expression in
adulthood in our nonconvulsive model of genetic absence epilepsy. The results of our
study support the idea that caffeine exposure during the developmental period can have
a permanent effect on seizure activity in adulthood. Common suggested mechanisms
explaining the effect of caffeine on seizure activity are its inhibition on adenosine receptors
Brain Sci. 2022,12, 361 12 of 15
and permanent changes of adenosine receptor subtypes in different brain areas [
47
]. Effects
of neonatal caffeine on number of SWDs could be associated with blockade of the A
1
and
A
2
receptor subtypes in our study; Adenosine receptors exist but are immature in the
second neonatal week (P7–P11) of development that we administered caffeine [44].
It was previously pointed out that the adenosinergic system might have been involved
in modulation of absence epilepsy and depression mechanisms.
A firm, well-documented relationship is defined between SWDs and vigilance level.
Absence seizures occur more during drowsiness and light slow-wave sleep and less often
in high arousal states [
48
]. Considering this relation with occurrence of SWDs and vigilance
level, the reduced seizure activity could be associated with increased locomotor activity in
animals treated with caffeine. Enhanced locomotor activity was also previously reported in
rats treated with caffeine at postnatal days 7–11 [49].
It can also be suggested that caffeine exposure may induce epigenetic modifications,
and these epigenetic changes can lead to long-lasting effects in brain excitability by affecting
the adenosinergic system. Pathological changes in DNA methylation homeostasis, which
underlie epileptogenesis, were previously demonstrated [
50
]. It is known that a number
of genes are involved in the development of absence epilepsy in WAG/Rij rats. The
epileptic phenotype (SWDs) in WAG/Rij rats is determined by one gene with a dominant
mode of inheritance while other genes determine the number of seizures. The number
and duration of SWDs are determined by different genes [
14
]. Treatment with caffeine
reduced the number of SWDs significantly while the decrease in the total duration of SWDs
was not statistically significant. It seems like early-life caffeine exposure has influence on
mechanisms of seizure generation rather than seizure termination. It should be emphasized
that SWD occurrence is triggered in the somatosensory cortex while reticular thalamic
nucleus is known to be associated with SWD termination [51].
In this study, we found that postnatal caffeine treatment has a positive impact on
depression as measured with forced swimming and sucrose preference tests. These are the
first data on the antidepressant-like effects of postnatal caffeine exposure. It has been known
that caffeine leads to alteration in mood and anxiety in humans and animals [
27
]. Caffeine
in low doses induces antidepressant effects without any major negative consequences
on health [
52
]. Experimental studies also suggested that the adenosinergic system and
its nonselective receptor antagonist caffeine have an important role in the regulation of
depressive-like behavior [
53
]. Caffeine and selective A
2A
receptor antagonists reduce
immobility in the forced swimming test and the tail suspension test in mice which shows
their potential role as antidepressants [
54
,
55
]. Chronic caffeine exposure was also found to
reduce anxiety and depression in adult animals in chronic unpredictable stress models [
52
].
On the other hand, the relationship between depression and presence of SWDs was
shown in genetic absence epilepsy models [
9
]. Depression-like behaviors are triggered by
occurrence and repetition of seizure activity. Therefore, reduced depression-like symptoms
in the caffeine treated rats might be the consequence of reduced seizure activity.
We used forced swimming and sucrose preference tests to measure depression-like
symptoms in this study. A forced swimming test is generally used in evaluating antide-
pressant effect. The test in general has numerous advantages but it may be influenced by
factors such as sleep abnormalities, or it may reflect an adaptive behavior to survive. We
did not evaluate sleep abnormalities, which is a limitation for the study. Considering the
methodology used, we do not think the differences in the forced swimming test are linked
to development of an adaptive behavior. In this study, results of the forced swimming test
and sucrose preference test are not consistent for different doses of caffeine administration.
The inconsistency between two tests may be related with the symptoms that the tests aim to
measure; it has been assumed that the sucrose preference test measures “anhedonia” while
the forced swimming test measures “despair”. Unfortunately, we cannot explain the reason
behind the lack of dose-dependency for the sucrose preference test shown in this study.
In summary, the present study results show that postnatal intervention of caffeine
reduces the frequency of absence epileptic seizure and comorbid depressive symptoms
Brain Sci. 2022,12, 361 13 of 15
and leads to permanent locomotor hyperactivity in adulthood. It can be suggested that the
blockade of adenosine receptors by caffeine during the early developmental period medi-
ates the effects of caffeine on absence epilepsy, depressive-like behaviors and locomotor
activity in adulthood in WAG/Rij rats. Further research is required to explore the role of
caffeine as an epigenetic regulator on the prevention of epileptogenesis and depressive-like
symptoms in absence epilepsy.
Author Contributions:
Conceptualization, G.I.; Methodology, Z.I.D. and A.B.; Formal Analysis,
A.B.; Investigation, Z.I.D. and A.B.; Data Curation, Z.I.D.; Writing—Original Draft Preparation, G.I.;
Writing—Review & Editing, A.K.K. and S.E.; Visualization, Z.I.D. and A.B.; Supervision, G.I.; Project
Administration, G.I.; Funding Acquisition, G.I. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by grant 2017/026 from the Kocaeli University Research Fund.
Institutional Review Board Statement:
Our study was approved by the Kocaeli University Animal
Care and Use Committee in accordance with the Declaration of Helsinki and the guidelines of the
care and use of laboratory animals for scientific purposes (KOUHADYEK-7/1-2016).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available on request from the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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