Dynamic changes in seizure state and anxiety-like behaviors during pentylenetetrazole kindling in rats

PTZ


Introduction
Anxiety is characterized by flexible psychological and behavioral states that facilitate coping when potential threats are encountered (Bishop, 2007) [5].However, when it reaches levels that are difficult to control, it is considered a pathological state, "anxiety disorder" (DeMartini et al., 2019) [8].Anxiety disorder is a mental disorder, and its treatment involves the use of benzodiazepines, a class of drugs that enhance the effect of the neurotransmitter gamma-aminobutyric acid (GABA) on GABA A receptors (Baldwin et al., 2005) [4].Anxiety disorders, including generalized anxiety disorder, social anxiety disorder, agoraphobia, and panic disorder, are among the most prevalent mental health issues, with an estimated 10-14% of the population experiencing them within 12 months (Domschke et al., 2024) [10].Elucidating the mechanisms underlying anxiety disorders will improve mental health and quality of life.
The Pentylenetetrazole (PTZ) is a well-characterized prototypical epileptic seizure inducer used in animal studies.In this model, repeated systemic administration of initially sub-convulsant dose of PTZ leads to kindling, that is to the occurrence and then to the progression of secondary generalized seizures.Several studies have reported that PTZkindled animals exhibit anxiety-like behaviors (Godlevsky et al., 2014 [13]; Asadi et al., 2018 [3]; Tariq et al., 2024 [40]).On the other hand, some groups have claimed that PTZ-kindled animals exhibit decreased anxiety-like behaviors (File et al., 1996 [12]; Szyndler et al., 2002 [39]), and the cause of this discrepancy is unknown.We recently reported that PTZ kindling transiently leads to a decrease in epileptic sensitivity in the initial phase of the kindling process, which disappears in the late phase (Kajita et al., 2024) [19].Therefore, it is possible to track anxiety levels during dynamic changes in epileptic susceptibility in individual animals.Elucidating the changes in anxiety levels during the epileptogenesis may lead to the identification of common neurobiological mechanisms between anxiety disorders and epilepsy.
In this study, we hypothesize that anxiety-like behaviors decrease with a decrease in epileptic sensitivity and then increase with the epileptogenesis.Therefore, we compared anxiety-like behaviors in the initial and late phases of PTZ kindling with those in an equal number of saline-injected groups.

Animals
We used 22 male 3-month-old Long-Evans rats (Institute for Animal Reproduction, Kasumigaura, Ibaraki, Japan; https://www.iar.or.jp).The rats were housed in groups in a temperature-and humiditycontrolled room under a 12-h-light/12-h-dark cycle with food and water available ad libitum.Efforts were made to minimize pain and discomfort.A post-hoc power analysis was used to determine whether the number of rats used was appropriate.All the experiments were approved by the Tohoku University Committee for Animal Research, Seiryo Campus (Sendai, Japan).

Experimental schedule
The experimental schedule is shown in Fig. 1 A. After 10 PTZ or saline injections in rats, behavioral tests were performed as a "first trial" to quantify anxiety levels.After completing the behavioral tests, the rats received more 10 injections, and the same behavioral tests were repeated as a "second trial".The interval between the PTZ injection and behavioral tests was one week.Four types of behavioral tests were performed as shown in Fig. 1 B.Although PTZ was not injected from days 20 to 44 in the first trial of the behavioral tests, the sensitivity to convulsant effects induced by PTZ kindling is considered to be maintained for one year (Corda et al., 1991) [7].

Behavioral analysis
Anxiety-like behaviors were analyzed using several behavioral tests that measured spontaneous activity, exploratory behavior, and emotional response in unfamiliar environments or with strangers.The experimental instruments for analyzing rat behavior were crafted by the experimenter.Rat behaviors were recorded using a video camera (GZ-F270-T; JVCKENWOOD Corporation, Kanagawa, Japan).Data analysis was performed manually in blinded manner, except for the open-field test, which was performed using tracking software.Behavioral tests were performed twice: after 10 and 20 PTZ injections.After PTZ kindling, a one-week interval was used before starting the behavioral tests to eliminate short-term memory issues caused by kindling-induced seizures (Hannesson et al., 2001) [16].Behavioral tests were conducted in the specific order described below, starting with the least stressful test for the rats.The rats underwent a maximum of one test per day, and the tests were performed between 1:00 p.m. and 5:00 p.m. Rats were moved to the experimental room 30 min before the test.All equipment was cleaned with 80 % alcohol before each trial.

Open-field test
Spontaneous activity and exploratory behavior in a novel environment were examined using an open-field test.The open field (90 cm × 90 cm × 30 cm: L × W × H, 70 lx) was made of matte black plexiglass divided into a central area that was 54 cm × 54 cm and an outside zone that was 18 cm wide.The rats were placed in the corner of the field, and their movements were tracked for 30 min using a digital camera and tracking software (Smart 3.0; Panlab, Barcelona, Spain).

Light/dark transition test
Exploratory behavior in a novel bright environment was examined using a light/dark transition test.The light/dark transition test involved a light box (white plexiglass: 31 × 31 × 24 cm, 200 lx) and a dark box (black plexiglass: 31 × 20 × 24 cm, 5 lx), and the rats were able to move between the two boxes through a small opening (10 × 10 cm).The rats were placed in the corner of the dark box, and their behavior in and out of the light box was observed for 10 min.Entry into the light box was defined as all four paws in the light box, and exit from the light box was defined as two paws in the dark box.

Elevated plus maze test
Exploratory behavior in a novel open space (also called thigmotaxis) was examined using the elevated plus maze test.The elevated plus maze test comprised four black plexiglass arms (40 × 10 cm, 25 lx) and a junction (10 × 10 cm, 25 lx) at the center and was elevated using stable plastic legs (60 cm).Two opposite arms had walls, the "closed arm," and the other two lacked walls, the "open arm."Rats were placed at the junction with their nose facing the closed arm, and their behaviors were recorded for 10 min.Entry into the open arms was defined as placing all four paws inside the arms, whereas exiting from the open arms was defined as placing two paws at the junction.

Social interaction test
Sociality and emotional responses toward strangers were examined using social interaction tests.The social interaction test was conducted in the same field as the open-field test (90 cm × 90 cm × 30 cm, 70 lx).First, a white acrylic cylindrical cage (23 cm × 30 cm: φ × H) was placed against the wall in the middle of one side of the field, and the rats (subjects) were placed in the opposite corner of the field.The contact time with the empty cage was measured for 10 min.Next, a male rat (stranger) was placed in the cylindrical cage, and the subjects were again placed in the same position.The contact time with the stranger in the cage was measured for 10 min.The grid bars (diameter of 0.5 cm and 2 cm apart) of the cylindrical cage allowed for interaction between the subjects and the stranger.Different male rats were used as strangers in the first (10 injections) and second (20 injections) trials.The area within 15 cm of the cylindrical cage was defined as the interaction zone.Entry into the interaction zone was defined as placing all four paws into the zone.Exits from the interaction zone were defined as placing the two paws outside the zone.

Z score
The analysis of the correlation between anxiety-like behaviors and seizure stages in individual rats involved the transformation of the data to Z scores (Stukalin and Einat, 2019) [37].The Z score is the number of standard deviations a data point is away from the mean.The Z score is computed with the following formula: Z = (x − μ) ∕ σ, where x is the individual value, μ is the population mean and σ is the standard deviation.

Statistical analysis
Behavioral data were collated using Excel (Microsoft, Redmond, WA, USA) for further analyses.R software, version 4.4.0 (R Foundation for Statistical Computing) was used for statistical analysis.The F test was performed to determine the equality of variances of two groups.When equal variances were assumed, the Wilcoxon rank sum test or Wilcoxon matched-pairs signed rank test were performed.When equal variances were not assumed, the Brunner-Munzel test or permuted Brunner-Munzel test (n <10) was performed.For analysis of data correlation, the Spearman correlation test was performed.Values were presented as median and IQRs (25th-75th percentaile), and rounded down to three decimal places.IQRs were calculated using inclusive median.The significance was determined at thresholds of P < 0.05 and P < 0.01.

PTZ kindling induced bimodal changes in the seizure stage
Epileptic seizures induced by PTZ kindling were scored according to the Racine staging system (Fig. 2).The seizure stage first increased to approximately stage 3 (on days 7-13) and then gradually decreased to approximately stage 2 after 10 injections (on day 19).After a 26-day interval (on day 45), the seizure started in approximately stage 1.This stage then increased to approximately stage 3 and persisted.None of the rats reached a fully kindled state after 20 injections (on day 63).The seizure stage showed bimodal changes according to PTZ injection.The seizure stage was transiently suppressed after approximately 10 injections, but this suppression disappeared after approximately 20 injections.Among the 12 rats, two died of PTZ-induced epileptic seizures after six injections (on day 11).

Open-field test
The results of the open-field test are shown in Fig. 3 A. PTZ-injected rats (PTZ group) spent more time in the central area than did saline-Fig.1. Experimental schedule (A) Starting on day 1 of the experiment, the rats were injected intraperitoneally with the PTZ or saline solution every 48 h.One week after the 10 injections, behavioral tests were performed as a "first trial."One week after the first trial, the rats were given 10 additional injections.One week after the 20 injections, the same behavior tests were performed as a "second trial."(B) Four types of behavioral tests were performed in the specific order.PTZ, pentylenetetrazole.
Next, we compared the distances traveled in the open field.There was no significant difference between the two groups after 10 (CTL:

Elevated plus-maze test
The results of the elevated plus-maze test are shown in Fig. 3 C.One rat in the PTZ group fell off the open arm during the trial.This rat was excluded from the data analysis to eliminate fear of falling.The PTZ group spent more time in the open arms than the CTL group after 10 injections (CTL: 1.875, 0.250-3.000;PTZ: 46.690, 3.000-68.000;P = 0.029; n = 10 and 9 rats, respectively).This effect was not detected after 20 injections (CTL: 26.685, 3.500-34.597;PTZ: 12.000, 7.500-39.000;P = 1.000; n = 10 and n = 9, respectively).In both groups, no significant difference in the time spent in the open arms was detected between the 10-and 20-injection groups (CTL: P = 0.058; PTZ: P = 0.203).

Social interaction test
The results of the social interaction test are shown in Fig. 3 D. Some rats climbed to the top of the cage during the trial.These rats were excluded from the data analyses because we could not determine whether this behavior originated from escape or interest in cages or strangers.In both groups, there was no significant difference in contact time with the empty cage after 10

Anxiety-like behaviors correlated with seizure stages after 10 PTZ injections
The correlation between epileptic susceptibility and anxiety levels in individual rats was investigated after 10 PTZ injections.The behavioral data with significant differences detected after 10 PTZ injections (the time spent in the central area in the open-field test, the time spent in the light box in the light/dark transition test, and the time spent in the open arms in the elevated plus maze test) were transformed into Z scores.Higher [− Z scores (− 1 × Z score)] indicate higher anxiety-like behaviors.The − Z scores showed a significant correlation with seizure stage (on day 45) in individual rats, as shown in Fig. 4 (r = 0.395, P = 0.034).These results indicate that anxiety-like behaviors were more suppressed in rats with lower epileptic sensitivity after 10 PTZ injections.

Post-hoc power analysis
A post-hoc power analysis to detect a 5% difference was performed using the parameters for which significant differences were detected in the results after 10 PTZ injections.The post-hoc power analysis showed 33.7% power in time in the open-field test, 90% power in the light/dark transition test, and 86.1% power in the elevated plus-maze test.

Discussion
Bimodal changes in the seizure stage were observed in response to PTZ kindling.The seizure stage was transiently suppressed after 10 injections.In these decreases in epileptic sensitivity, anxiety-like behaviors decreased and correlated with the seizure stage in individual rats.The seizure stages gradually progressed again with continuous injections.Anxiety-like behaviors returned to control levels with the disappearance of the decreases in epileptic sensitivity.

Transient decrease in epileptic sensitivity after repeated stimulation
In this study, transient decrease in epileptic sensitivity was detected after 10 PTZ injections same as our previous report (Kajita et al., 2024) [19].This result is seemingly paradoxical because PTZ kindling is a technique conventionally used to induce epileptic seizures.However, in clinical studies, repetitive transcranial magnetic stimulation therapy reduces focal epileptogenic activity (Graff-Guerrero et al., 2004) [14].In electroconvulsive therapy (ECT), the voltage required to induce a seizure increases over the routine course of ECT (Scott and Boddy, 2000 [33]; Duthie et al., 2015 [11]).In a rat study, transcranial magnetic stimulation decreases the expression of the immediate early gene c-Fos in the cortex (Volz et al., 2013) [43].Based on these studies, we speculate that PTZ-induced stimulation may also enhance the homeostatic increase in epileptic resistance, similar to other types of neuronal stimulation.In addition, the decrease in epileptic sensitivity was transient and disappeared with further continuous stimulation, suggesting that the endogenous antiepileptic system finally collapses with repeated stimulation.This result is consistent with recent clinical reports of patients who experienced prolonged seizures after ECT (Shin et al., 2023 [36]; Saltoglu et al., 2023 [32]).

Transient suppression in anxiety-like behaviors after repeated stimulations
We detected a significant decrease in anxiety-like behaviors in the open-field, light/dark transition, and elevated plus-maze tests after 10 PTZ injections (decrease in epileptic sensitivity).In addition, seizure stage was significantly correlated with anxiety levels in individual rats.These results suggest that decrease in epileptic sensitivity is accompanied by a decrease in anxiety-like behaviors.A decrease in anxiety-like behaviors is detected in fear conditioning and ultrasonic vocalization tests 3-5 days after 12 PTZ injections (Szyndler et al., 2002) [39].In addition, a decrease in anxiety-like behaviors is detected in the elevated plus maze and social interaction tests 24h after 13 PTZ injections (File et al., 1996) [12].These results are consistent with our hypothesis that anxiety-related behaviors decrease during the initial stages of PTZ kindling.

Anxiolytic effects and epileptogenesis
Anxiety-like behaviors were comparable to CTL levels after 20 PTZ injections, suggesting that the anxiolytic effects decrease with the disappearance of decrease in epileptic sensitivity.However, previous studies have shown increased anxiety-like behaviors after PTZ injections of approximately 20.Anxiety-like behaviors increase in the open-field and elevated plus-maze tests after 21 PTZ injections (Godlevsky et al., 2014 [13]; Okwuofu et al., 2021 [29]).In these studies, all animals could acquire fully kindled states, or fully kindled animals were selected for behavioral analysis; on the other hand, the average seizure stage in our rats was approximately stage 3 after 20 injections.None of the rats reached a fully kindled state.Therefore, we speculate that increased anxiety-like behaviors may depend on the epileptogenesis.This hypothesis is supported by a previous study in which fully kindled rats show increased anxiety levels in the elevated plus maze test after 15 PTZ injections (Aghaie et al., 2021) [1].In contrast, mice in the nonfull-kindling state (Racine stage 3.9) do not show significantly increased anxiety levels in locomotor activity assessments and elevated plus-maze tests after 21 PTZ injections (Nieoczym et al., 2021) [28].
In addition, anxiety-like behaviors do not change after a serious seizure induced by a single high dose of PTZ (File et al., 1996) [12], suggesting that excitotoxicity in seizures is not directly responsible for the increase in anxiety.Therefore, neurochemical changes underlying the epileptogenesis may be necessary for increased anxiety-like behaviors.

Possible mechanisms between anxiety disorders and epileptic seizures
Our results showed that anxiety-like behaviors change dynamically during repeated stimulation by PTZ kindling.Anxiety disorders in epilepsy patients are related to the fear of having another seizure (Gray et al., 2023) [15].However, our data rule out the possibility that the anxiety disorders detected in animal models of epilepsy are caused by anxious memories of the seizures themselves, and strongly suggest that anxiety disorders and epileptic seizures share common neurobiological mechanisms.In this study, the findings were limited to behavioral tests, but we discuss the neurochemical changes underlying the decrease in anxiety based on previous studies.
Previous studies have shown an increase in the benzodiazepine receptor and a decrease in PTZ-binding affinity in the mouse forebrain 18h after 11 PTZ injections (Syapin and Rickman, 1981) [38].An increased number of benzodiazepine receptors was also confirmed in the hippocampus, implying a heightened response to benzodiazepines and more powerful recurrent hippocampal inhibition (McNamara et al., 1980) [26].Therefore, we speculate that the dynamic changes in anxiety-like behaviors imply transient potentiation of endogenous inhibitory systems and their dysfunction.
In our previous study, glutamic acid decarboxylase (GAD)65 expression increases in hippocampal somatostatin-positive interneurons (SOM-INs) 24h after 10 injections, but decreases to CTL levels after 20 injections (Kajita et al., 2024) [19].Neither other GABAergic neuron related proteins (GABA receptors, vesicular GABA transporter, and GAD67) change.These data are consistent with other report that repeated electroconvulsive shock for eight consecutive days increases GAD65, but not GAD67 levels in the hippocampus (Jinno and Kosaka, 2009) [18].GAD67 is responsible for homeostatic GABA synthesis, while GAD65 plays a role in activity-dependent rapid GABA synthesis (Tian et al., 1999 [41]; Kanaani et al., 2010 [20]).A lack of GAD65 induces hyperexcitability in the amygdala and hippocampus, leading to increased anxiety-like behaviors in mice (Müller et al., 2015) [27].In addition, GAD65 knockout mice do not exhibit changes in the expression of GABA A receptors but show a diminished response to benzodiazepine (Kash et al., 1997; Kash et al., 1999) [21].According to these studies, GAD65-mediated GABA synthesis would be crucial for suppressing anxiety-like behaviors.
Neural activation is also considered to be regulated by astrocytes.In a rat study, optogenetic neuronal stimulation for eight days induces epileptic resistant states via adenosine release from activated astrocytes (Shimoda et al., 2022) [35].Adenosine release from astrocytes is regulated by the synaptic input of SOM-INs via GABA transporter-3dependent Ca 2+ signaling in the hippocampus (Matos et al., 2018) [25] and cortex (Mariotti et al., 2018) [24].These reports are consistent with our previous finding that GAD65 upregulation after 10 PTZ injections is detected only in SOM-INs of the hippocampus.
From these previous studies, we speculate that GAD65-mediated GABA release from SOM-INs suppresses pyramidal neurons via astrocyte activation in PTZ kindling.Future research should follow how the activity of these pathways changes after 10 and 20 PTZ injections to confirm their relationship with anxiety disorders.

Limitation
One result (increased time spent in the central area in the open-field test after 10 PTZ injections) did not achieve ideal power (80%) in the post-hoc power analysis.Unfortunately, the result of the open-field test is less reproducible.This may be because the number of rats (n = 10) was insufficient.In future studies, a larger number of rats should be prepared if the open-field test is to be used as a tool to detect changes in anxiety-like behavior.

Conclusion
In our study, anxiety-like behaviors decreased after 10 PTZ injections and returned to control levels after 20 PTZ injections, suggesting that a common neurobiological mechanism underlying anxiety disorders and epileptic seizures.The discrepancy in previous studies, in which anxiety levels increased or decreased in PTZ-kindled animals, may be due to differences in the phases of the kindling process.We speculate that these processes are caused by the transient upregulation and dysfunction of the inhibitory system in SOM-IN-activated astrocytes.

Ethics Statement
The animal study was reviewed and approved by the Institute for Animal Experimentation of Tohoku University (Permission number: 2019 IDOU-291-08).

Fig. 2 .
Fig. 2. Seizure stages in PTZ-kindled rats The seizure stages were scored according to the Racine scale (n = 12 rats).The vertical axis indicates the seizure stage.The horizontal axis indicates the number of days from the start of the experiment.The solid black lines indicate the medians and IQRs (25th-75th percentaile), and size of the dots indicate the rat's numbers.

Fig. 3 .
Fig. 3. Anxiety-like behaviors after PTZ kindling Anxiety-like behaviors were compared after 10 or 20 injections using four types of behavior tests.(A) Time spent in the central area (n = 10 rats) and total distance traveled (n = 10 rats) in the open-field test.(B) Time spent in the light box during the light/dark transition test (n = 10 rats).(C) Time spent in the open arms in the elevated plus maze test (n = 10 and 9 rats for CTL and PTZ, respectively).(D) Contact time to empty cage after 10 (n = 10 and 9 rats for the CTL and PTZ groups, respectively) and 20 (n = 8 and 6 rats for the CTL and PTZ groups, respectively) injections.Contact time with strangers after 10 (n = 9 and 8 rats in the CTL and PTZ groups, respectively) and 20 (n = 8 and 6 rats in the CTL and PTZ groups, respectively) injections in the social interaction test.The solid black lines indicate the medians and the IQRs (25th-75th percentile).The Wilcoxon rank sum test, Wilcoxon matched-pairs signed rank test, Brunner-Munzel test, or permuted Brunner-Munzel test were performed to compare the scores in each test between groups (*P<0.05;**P<0.01)and injection times ( # P<0.05; ## P<0.01).

Fig. 4 .
Fig. 4. Correlation between anxiety-like behaviors and seizure stages The correlation between anxiety-like behaviors [− Z score (− 1 × Z score)] and seizure stages.The vertical axis indicates the seizure stage.The horizontal axis indicates the − Z score (the time spent in the central area in the open-field test, the time spent in the light box in the light/dark transition test, and the time spent in the open arms in the elevated plus-maze test).Higher − Z score indicates higher anxiety-like behaviors.Spearman correlation analysis was performed to analyze the correlations among the variables.