Anxiolytic and sedative effects of sodium valproate with different experimental paradigms in male and female rats

Abstract

Valproic acid or sodium valproate is a widely used drug in the treatment of epilepsy, although it also appears to have anxiolytic and sedative properties derived from its agonistic action on the GABAergic system. To analyze these potential effects of the drug, we conducted three experiments with rats using procedures designed to assess anxiety in rodents. In the first experiment, with a fear conditioning procedure, three groups of male rats were included that received either 100 mg/kg or 300 mg/kg of valproate or an equivalent volume of saline solution. In Experiment 2, recording spontaneous activity in an open field, we compared the effects of valproic acid (300 mg/kg) on male and female rats. In the third experiment, we analyzed the effect of valproic acid using a novelty‐induced hypophagia test and tested again for potential differences as a function of the sex of the animals. The results showed an anxiolytic effect restricted to the 300 mg/kg dose of the drug in Experiment 1. Such an effect was restricted to the female sample in Experiment 2, but in the third experiment affected both sexes. As for the sedative effect, it was observed in all experiments irrespective of the sex of the rats. These findings hold significant implications for the treatment of anxiety disorders since valproate may offer a novel therapeutic approach for anxiety‐related conditions with distinct benefits and fewer side effects. However, clinical studies are needed to validate the translation of these findings from animal models to human patients.

Valproic acid or sodium valproate is a widely used drug in the treatment of epilepsy, although it also appears to have anxiolytic and sedative properties derived from its agonistic action on the GABAergic system. To analyze these potential effects of the drug, we conducted three experiments with rats using procedures designed to assess anxiety in rodents. Our findings hold significant implications for the treatment of anxiety disorders since valproate may offer a novel therapeutic approach for anxiety‐related conditions with distinct benefits and fewer side effects.

1. INTRODUCTION

Valproic acid or sodium valproate (VPA) is one of the most used anticonvulsant drugs for the treatment of epilepsy, ever since its identification as a drug that prevents seizures induced by the administration of pentylenetetrazol. ¹ Recent studies suggest that VPA could be useful for treating a wide range of conditions, such as bipolar disorder, ² , ³ , ⁴ migraines, ⁵ , ⁶ , ⁷ motor‐related disorders, ⁸ , ⁹ and even in cancer treatment. ¹⁰ , ¹¹ , ¹² Among these novel and promising applications of VPA, of special relevance is its potential use as an anxiolytic agent ¹³ , ¹⁴ , ¹⁵ due to its agonistic action on the GABAergic system, similar to the mechanisms of action of benzodiazepines. ⁹ , ¹⁶ , ¹⁷ , ¹⁸

Numerous studies have demonstrated the utility of VPA in various anxiety‐related pathologies due to its impact on GABA activity. ¹⁹ , ²⁰ Specifically, the use of VPA has proven effective in treating panic disorder, ²¹ , ²² , ²³ , ²⁴ bruxism, ²⁵ alcoholism, ²⁶ generalized anxiety disorder, ²⁷ , ²⁸ , ²⁹ bipolar depression, ³⁰ posttraumatic stress disorder, ³¹ and the reduction of anxiety in individuals without pathologies. ³²

The main action of VPA as a GABAergic agonist is an increase in the levels of this neurotransmitter, which exerts an inhibitory action on the central nervous system. ³³ , ³⁴ This action occurs through different pathways by inhibiting certain proteins involved in its degradation pathway, such as ABAT (4‐Aminobutyrate Aminotransferase) and ALDH5A1 (Aldehyde Dehydrogenase 5 Family Member A1), ³⁵ , ³⁶ increasing the levels of this neurotransmitter by enhancing the activity of glutamate decarboxylase, which converts Glutamate into GABA, ³⁷ , ³⁸ or by increasing the availability of the GABA precursor, α‐ketoglutarate, by inhibiting α‐ketoglutarate dehydrogenase (α‐KDHC). ³⁹ , ⁴⁰

This action of VPA on the nervous system appears to be closely related to anxiety. In fact, there is currently abundant scientific evidence demonstrating the existence of a GABAergic deficit in anxiety disorders ²⁰ and a notable presence of GABA neurons in circuits crucial for the acquisition, modulation, and extinction of fear, ⁴¹ implicating structures such as the amygdala ⁴² , ⁴³ and the hippocampus (Möhler & Rudolph, 2002). ⁴⁴ It is important to note that the mechanism of action of VPA is very complex and encompasses different neurochemical pathways and, although the main and most studied pathway of action is related to the GABAergic system, it also involves other neurotransmitters, including glutamate, ⁴⁵ , ⁴⁶ dopamine, and acetylcholine. ⁴⁷

A noteworthy advantage of using VPA is its lack of potential for tolerance. ⁴⁸ In contrast to the addiction problems associated with benzodiazepine treatment, valproate acts through a different pathway to induce anxiolytic effects, suggesting its suitability for long‐term use. ³² Therefore, considering all the data mentioned, it appears that this drug could be valuable in the treatment of anxiety disorders.

In addition to its anxiolytic properties, VPA appears to have sedative properties and has been proposed as an alternative treatment to antipsychotic drugs for managing agitation and delirium. ⁴⁹ Its mechanism of action for controlling agitation involves increasing the synthesis of GABA, inhibiting its degradation, and enhancing its activity on receptors. ⁵⁰

Rodent‐based behavioral tests and models are extensively used to explore how stress induces anxiety‐related behaviors and to develop new treatments for anxiety disorders such as generalized anxiety disorder, panic disorder, or posttraumatic stress disorder. ⁵¹ , ⁵² Specifically, animal models that exhibit increased anxiety in open‐field tests and reduced latency to feed in novel environments can mimic the hypervigilance and avoidance behaviors characteristic of generalized anxiety disorder and posttraumatic stress disorder, making these tests valuable in translational research aimed at understanding and treating anxiety disorders.

Therefore, to assess the potential anxiolytic and sedative effects of VPA, we conducted three experiments with Wistar rats that received either VPA or an equivalent volume of saline (SAL) solution. In each experiment, the animals were subjected to a task designed to evaluate anxiety. Specifically, in Experiment 1, we tested the potential anxiolytic effect of two doses of VPA (100 and 300 mg/kg) with a fear conditioning procedure, ⁵³ , ⁵⁴ in which the freezing response to a conditioned stimulus predicting the occurrence of an electric shock was analyzed as an index of anxiety. In Experiment 2, we tested the potential anxiolytic effect of VPA (300 mg/kg) on locomotor activity in an open field ⁵⁵ and compared the effects of VPA on male and female rats. This task is also suitable for evaluating the sedative properties of VPA, as it allows for the identification of differences in the locomotor activity of the animals. ⁵⁶ Finally, in Experiment 3, we assessed the effects of VPA on the suppression of feeding induced by an unfamiliar context, a procedure that has also been used to evaluate anxiety. ⁵⁷ , ⁵⁸ Again, in this experiment, we evaluated potential sex differences in VPA administration. In all cases, we predicted a reduction in anxiety‐associated responses for the animals that received VPA compared to their respective controls. Regarding sex differences, we did not have specific hypotheses since, although some sex differences have been observed in rodents when VPA has been administered prenatally, ⁵⁹ there is no evidence in the literature following acute drug administration.

2. EXPERIMENT 1

A procedure commonly associated with anxiety is fear conditioning. ⁶⁰ The main objective of this experiment was to determine whether the administration of two doses of VPA (100 and 300 mg/kg) prior to the pairing of a tone (CS) and a shock (US) resulted in a reduction in fear conditioning. In addition, a control group was injected with a saline solution before the conditioning procedure. To assess potential differences between the groups, a drug‐free test was conducted after conditioning in which the tone was presented alone, and the animals' freezing levels were recorded as an index of conditioning.

The estimation of the sample size for each group in this and the next experiments, based on our previous experience, the expected variability, and the effort to reduce the number of animals used, was 8 animals per group (for a significance level of α = 0.05 and a power of 95%).

2.1. Methods

2.1.1. Subjects

Twenty‐four experimentally naive male Wistar rats (n = 8) participated in this experiment. The mean weight at the start of the experiment was 390 g (ranging from 328 to 434 g). Upon arrival at the laboratory, the animals were housed in groups of 2 or 3, depending on their weight, in type IIIH cages (820 cm²) with wood shavings as bedding. Various enrichment materials, such as pieces of fabric, cardboard, wood, stones, etc., were provided in the cages, except during the experimental procedures when the rats were individually housed. The vivarium was maintained on a 12:12‐h light–dark cycle with lights on at 07:00 h. All behavioral testing took place during the light phase of the cycle. Two days before the start of the experimental sessions, each animal was handled daily for 5 min. The animals had unrestricted access to water and food. All experimental procedures were conducted following ethical guidelines and were approved by the Ethics Committee for Animal Research at the University of Seville (code number CEEA‐US2015‐28/4). These procedures were carried out in accordance with the guidelines established by the EU Directive 2010/63/EU for animal experiments and Spanish R.D. 53/2013.

2.1.2. Apparatus

All experimental sessions took place in four identical Panlab conditioning boxes (model LE111, Panlab/Harvard Apparatus, Spain), each measuring 26 × 25 × 25 cm (height × length × width). These chambers were enclosed within sound‐attenuating cubicles (model LE116, Panlab/Harvard Apparatus, Spain). The walls of the experimental chambers were made from white acrylic, and the floor consisted of stainless‐steel rods, 2 mm in diameter, spaced 10 mm apart (center to center). Each chamber was situated on a platform that recorded signals generated by the animals' movements through a highly sensitive weight transducer system. This signal was automatically converted into a percentage of freezing, defined as the proportion of time the animal spent without movement, using commercial software (StartFear system software, Panlab/Harvard Apparatus, Spain). The unconditioned stimulus (US) was a 1‐s, 0.5‐mA unscrambled AC 50‐Hz foot shock administered via a constant‐current generator (Model LE100‐26) to the floor of each chamber. A loudspeaker positioned at the top of each chamber emitted a 70 dB 2.8‐kHz 30‐s tone, which served as the conditioned stimulus (CS).

2.1.3. Drugs and solutions

VPA (Merck Life Science S.L.U.) was dissolved in sterile saline solution (0.9%) and administered via intraperitoneal injection (IP) at doses of 100 and 300 mg/kg. The doses were selected based on previous studies, both of which are among the most used in the literature. ⁵⁶ , ⁶¹ , ⁶² Control animals received injections of sterile saline at a volume equivalent to that administered to the VPA‐treated animals. The drugs were administered 20 min before initiating the experimental procedure. This time corresponds to the approximate time at which the drug reaches its peak concentrations, and its effects are most noticeable. ⁶³

2.1.4. Procedure

The animals were randomly divided into three groups: SAL, VPA‐100, and VPA‐300. The experimental protocol for all animals began with a single 17‐min baseline session, designed to measure each animal's general activity without the influence of the drug and to acclimatize the rats to the new environment. The following day, the context conditioning session commenced. Twenty minutes prior to this session, one‐third of the animals received an IP injection of 100 mg/kg of VPA (VPA‐100 Group), the next third received 300 mg/kg of VPA (VPA‐300 Group), and the remaining third received an equivalent dose of saline (SAL group). Twenty minutes after the drug administration, the conditioning session began, consisting of a 180‐s period without any stimulation, followed by three tone‐shock pairings with a 180‐s Inter‐Trial Interval.

After a 48‐h interval without additional manipulations to allow the animals to metabolize the drug, a free‐drug extinction session similar for all groups was conducted. This session began with a 180‐s period without any stimulation, followed by a presentation of the tone alone (30 s). A percentage score representing freezing behavior was calculated as an index of conditioning.

2.2. Results

Figure 1 depicts the mean freezing to the Tone‐CS at baseline and conditioning (Section A), and for the extinction trial (Section B). As can be seen in Section A, there were no differences in freezing between groups at baseline. Regarding conditioning, the mean percent of freezing varied across groups, but reached a high level at the third conditioning trial for all groups, indicating that conditioning treatment was effective. Finally, for the drug‐free extinction trial depicted in Section B, the expression of fear conditioning was more intense for the VPA‐100 and SAL groups compared to the VPA‐300 Group.

FIGURE 1.

Mean percent of freezing to the Tone‐CS at baseline and conditioning (Section A) and for the extinction trial (Section B) as a function of groups. SAL: Saline; VPA‐100: 100 mg/kg of Valproate; VPA‐300: 300 mg/kg of Valproate. The arrows indicate significant differences between groups (*p < 0.05). Error bars represent SEMs.

The statistical analyses confirmed these impressions. Thus, the mean percent of freezing during the baseline trial was 21.61% (range 13.63%–43.04%). A one‐way ANOVA with Groups as the main factor revealed that there were no significant differences between groups, F(2, 21) < 1.

A 3 × 3 mixed ANOVA (Trials × Groups) conducted on mean percent of freezing during conditioning revealed significant main effects of Trials and Groups, F(2, 42) = 19.20; p = 0.000, η ² = 0.48, and F(2, 21) = 9.74; p = 0.001, η ² = 0.48, respectively. The main effect of Trials reflects an overall increase in freezing across trials. Post hoc comparisons between groups (HSD Tukey, p < 0.05) revealed a higher global level of freezing for the VPA‐100 and VPA‐300 (mean = 75.11%, SD = 9.22, and mean = 77.63%, SD = 11.32, respectively) compared to SAL Group (mean = 56.83%, SD = 10.22). The Trials × Group interaction was also significant, F(4, 42) = 3.20; p = 0.022, η ² = 0.23. An analysis of simple effects (p < 0.05) revealed that the interaction was due to a higher level of freezing for the VPA‐300 Group compared to the SAL Group in the first conditioning trial, and a higher level of freezing for the VPA‐100 compared to the SAL Group in the second conditioning trial.

A one‐way ANOVA with Groups as the main factor conducted on the mean percent of freezing for the extinction trial revealed that the differences between groups were significant, F(2, 21) = 20.07; p < 0.000, η ² = 0.66. Post hoc comparisons between groups (HSD Tukey, p < 0.05) indicated a significantly lower mean level of freezing for the VPA‐300 Group compared to the VPA‐100 and SAL groups. The differences between SAL and VPA‐100 groups were nonsignificant.

3. EXPERIMENT 2

In Experiment 1, fear conditioning was less intense after the administration of 300 mg/kg of VPA prior to its establishment. Since the 100 mg/kg dose was not effective in reducing fear conditioning—in fact, conditioning was even higher in this group compared to the control group—we administered the 300 mg/kg dose in subsequent experiments to analyze the anxiolytic effect of VPA.

A potential limitation of Experiment 1 is associated with a possible analgesic effect of VPA, ⁶⁴ which could have reduced the perception of the intensity of the shock used as the US. Consequently, the observed reduction in fear during the extinction phase of Experiment 1 could be interpreted as due to reduced perceived intensity of the US in the VPA‐300 Group compared to the SAL Group.

To avoid potential confounding factors, Experiments 2 and 3 utilized procedures to evaluate anxiety that does not involve the administration of external aversive stimuli, thus eliminating the influence of VPA's potential analgesic action. Specifically, in Experiment 2 the rats' spontaneous activity in an open field was recorded, a technique commonly used to analyze anxiety in rodents. ⁵¹ One group received 300 mg/kg of VPA before being introduced to the open field, while another group received an equivalent volume of SAL. Additionally, in this experiment, we evaluated potential differences in the effects of VPA based on the sex of the animals.

Based on previous results indicating the potential anxiolytic ¹³ , ¹⁴ , ¹⁵ and sedative ⁶⁵ effects of VPA, we anticipated an increase in the time spent on the central platform of the open field, indicative of the anxiolytic properties of the drug, and a reduction in general activity due to its sedative effects. Due to the absence of previous results in this field with VPA in male vs. female rodents, we do not have a specific hypothesis regarding sex differences.

3.1. Methods

3.1.1. Subjects

Thirty‐two experimentally naive Wistar rats, 16 males and 16 females (n = 8), participated in this experiment. At the start of the experiment, the mean weight for males was 415 g (ranging from 369 to 467 g) and 254 g (ranging from 222 to 290 g) for females. The animals were housed and maintained as described in Experiment 1.

3.1.2. Apparatus and procedure

To register motor activity, each animal was placed for 5 min. in a transparent 45 × 45 cm cage surrounded by16 infrared lights (spaced 2.5 cm) forming a double grid of cells (Actitrack, Panlab, Barcelona, Spain). The 5‐min duration of the test was selected based on our laboratory's previous experiments and aligns with Gould et al. ⁶⁶ suggestion indicating that a brief test period more effectively highlights exploratory behavior and novelty responses rather than baseline activity. The following indices of locomotion were recorded by a computerized control unit: Mean time spent in the central area (a 23 × 23 cm area in the middle of the field) and mean resting time (defined as the absence of significant locomotor movements other than respiration) during the 5 min that the animals remained in the open field.

3.1.3. Drugs and solutions

VPA (Merck Life Science S.L.U.) was dissolved in sterile saline solution (0.9%) and administered via IP injection at a dose of 300 mg/kg. Control animals received injections of sterile saline at a volume equivalent to that administered to the drug‐treated animals. The drugs were administered 20 min before initiating the experimental procedure.

3.2. Results

Figure 2 depicts the mean time spent in the central area of the open field (Section A) and mean resting time (Section B), in both cases expressed in seconds, as a function of the drug injected (VPA vs. SAL) and the sex of the rats. As can be seen in Section A of Figure 2, VPA‐treated females spent more time in the central area of the open field, an index of the anxiolytic effect of the drug, than those in the SAL Group. However, such a difference did not appear in males. As for resting time, a variable indicative of the potential sedative properties of the drug, both VPA‐treated males and females showed an increase in resting time compared to the animals that received the vehicle.

FIGURE 2.

Mean time spent in the central area of the open field (Section A) and mean resting time (Section B) for the groups injected with Saline (SAL) or Valproate (VPA) as a function of rats' sex. The arrows indicate significant differences between groups (*p < 0.05). Error bars represent the standard error of the mean.

A 2 × 2 ANOVA with Drug (VPA vs. SAL) and Sex (Males vs. Females) as the main factor was conducted on mean time spent in the central area of the open field and mean resting time. The analyses for time in the central area revealed significant main effects of Drug and Sex, F(1,28) = 30.51, p = 0.012, η ² = 0.21, and F(1,28) = 30.51, p = 0.000, η ² = 0.52, respectively. The main effect of Drug reflects that those animals in the VPA Group spent more time in the central zone than those in the SAL Group (mean = 51.07 s, SD = 38.95, and mean = 31.50 s, SD = 18.35, respectively). The main effect of sex was due to female rats spending more time in the central zone as compared to the males (Mean = 61.44 s, SD = 33.51, and Mean = 21.14 s, SD = 8.31, respectively). The Drug × Sex interaction was also significant, F(1,28) = 6.78, p = 0.015, η ² = 0.19. Post hoc comparisons (HSD Tukey, p < 0.05) indicated that the interaction, which is depicted in Figure 2A, was due to longer time in the central zone spent for the VPA‐treated as compared to the SAL‐treated, but only in the females.

The ANOVA on mean resting time revealed significant main effects of Drug and Sex, F(1,28) = 16.26, p = 0.000, η ² = 0.37, and F(1,28) = 6.78, p = 0.014, η ² = 0.20, respectively. The main effect of the Drug reflects more resting time in the VPA than in the SAL Groups (mean = 98.76 s, SD = 46.72, and mean = 53.21 s, SD = 17.27, respectively). The main effect of sex was due to more resting time for males as compared to females (mean = 90.71 s, SD = 42.82, and mean = 61.26 s, SD = 35.80, respectively). Finally, the Drug × Sex interaction was nonsignificant, F(1,28) = 1.52, p = 0.228, η ² = 0.05. Although the interaction was not significant, we conducted post hoc comparisons (HSD Tukey, p < 0.05) to identify possible differences between groups. The analyses revealed that males who received VPA increased resting time compared to both males and females in the SAL condition. No more differences between groups were significant.

4. EXPERIMENT 3

Experiment 2 revealed an anxiolytic effect of VPA that was restricted to the females, and a sedative effect both in males and females. In Experiment 3, we again analyzed the potential anxiolytic effect of VPA using the novelty‐induced hypophagia test, a procedure that typically results in an increase in the latency to initiate food consumption, ⁵⁷ , ⁶⁷ as well as a reduction in total consumption ⁵⁸ , ⁶⁸ in the presence of a new context. These results are considered a measure of anxiety in rodents. Additionally, in this experiment, we evaluated again potential differences in the effects of VPA based on the sex of the animals.

We anticipated a reduction in time to start feeding, and an increase in the amount of food consumed due to a reduction of context‐novelty induced anxiety for those animals in the VPA Group. Considering the results of Experiment 2, although with caution due to the very distinctive nature of the procedure employed in this experiment, we expect that these results will be restricted to females.

4.1. Methods

4.1.1. Subjects

Thirty‐two experimentally naive Wistar rats, 16 males and 16 females (n = 8) participated in this experiment. Mean weight at the start of the experiment was 424 g (ranging from 349 to 496 g) for males, and 257 g (ranging from 227 to 298 g) for females. The animals were housed and maintained as described for Experiments 1 and 2, except that a food restriction regime (1‐h of food access daily) was implemented three days before the start of the experiment.

4.1.2. Apparatus and procedure

The novelty‐induced hypophagia test was conducted in eight Plexiglas cages measuring 378 mm x 217 mm, devoid of any bedding. The cages were in an experimental room separate from the vivarium. The same standard food that the animals received daily was utilized in this test. Each animal was individually placed in a cage, and 100 g of food was provided on the cage feeder. The food test lasted 15 m, and the behavior of each animal was videotaped and categorized by two observers. The dependent variables were time to start feeding (sec.) and total amount consumed (calculated by subtracting the food weight at the end of the session from the initial weight).

4.1.3. Drugs and solutions

VPA (Merck Life Science S.L.U.) was dissolved in sterile saline solution (0.9%) and administered via IP injection (300 mg/kg). Control animals received injections of sterile saline at a volume equivalent to that administered to the VPA‐treated animals. The injections were administered 20 min before initiating the experimental procedure.

4.2. Results

Figure 3 depicts the mean time to start feeding (Section A) and the amount of food consumed (Section B) as a function of the drug injected (VPA vs. SAL) and the sex of the rats. As can be seen in Section A of Figure 3, both VPA‐treated males and females start feeding sooner than those animals in the SAL Group, revealing the reduced impact of the novel context on feeding due to the anxiolytic effect of the drug. As for the mean amount of food consumed, that is depicted in Section B of the figure, both VPA‐treated males and females consumed less food compared to the animals that received the vehicle, probably due to movement restrictions that interfered with eating behavior because of the sedative effect of the drug.

FIGURE 3.

Mean time to start feeding (s) (Section A) and mean amount of food consumed (g) (Section B) for the groups injected with Saline (SAL) or Valproate (VPA) as a function of rats' sex. The arrows indicate significant differences between groups (*p < 0.05). Error bars represent the standard error of the mean.

A 2 × 2 ANOVA with main factors Drug (VPA vs. SAL) and Sex (Males vs. Females) was conducted on mean time to start feeding. Rat's weight was introduced as a covariate in the analyses. The analysis revealed a main effect of Drug, F(1,27) = 6.92; p = 0.014, η ² = 0.20, due to a reduced time for those animals in the VPA Group compared to the SAL Group (mean = 48.44 s, SD = 21.83, and mean = 79.88 s, SD = 43.31, respectively), reflecting the anxiolytic effect of VPA. Neither the main effect of Sex nor the Drug x Sex interaction was significant (ps > 0.22). Although the interaction was not significant, we conducted post hoc comparisons (HSD Tukey, p < 0.05) to identify possible differences between groups. The analyses revealed no significant differences between groups.

To identify differences in total amount consumed, a 2 × 2 ANOVA with main factors Sex (Males vs. Females) and Drug (VPA vs. SAL) was conducted on mean consumption (rat's weight was introduced as a covariate). The analysis revealed a significant main effect of Sex and Drug, F(1,28) = 23.89; p = 0.000, η ² = 0.46, and F(1,28) = 12.06; p = 0.002, η ² = 0.30, respectively. The main effect of Sex reflects a higher amount of food consumed by males compared to females (Mean = 3.01 g, SD = 1.03, and Mean = 1.67 g, SD = 0.81, respectively). The main effect of drug was due to a higher amount of food consumed for those animals in the SAL Group compared to the VPA Group (mean = 2.81 g, SD = 0.94, and mean = 1.87 g, SD = 1.14, respectively). This unexpected reduction in food consumption in the VPA‐treated animals may result from the sedative effect of the drug, which could interfere with eating behavior. Finally, the Drug × Sex interaction was nonsignificant, F(1,28) = 3.09; p = 0.090, η ² = 0.01. Although the interaction was not significant, we conducted post hoc comparisons (HSD Tukey, p < 0.05) to identify possible differences between groups. The analyses revealed that VPA‐treated females consumed less food than the remaining groups. No more differences between groups were significant.

5. GENERAL DISCUSSION

The results indicate that VPA (300 mg/kg) exerted an anxiolytic effect when administered before pairing a CS (auditory tone) with an aversive US (electric shock), as the conditioning intensity appears reduced in a drug‐free trial compared to a group of animals that had received a lower dose of VPA (100 mg/kg) or SAL (Exp. 1). Since this result could be affected by potential analgesic effect of VPA, in subsequent experiments, we used procedures that did not involve aversive stimuli. The results of Experiment 2 indicate that VPA (300 mg/kg) exerted an anxiolytic effect that was restricted to the female rats when evaluated in an open field, since the VPA‐treated female rats spent more time in the central area than the female rats injected with the vehicle. However, in Experiment 3, the significant main effect of VPA revealed a reduction of latency to initiate food consumption in a novel context for both male and female rats compared to the control groups.

As for the sedative effect of VPA, it was observed in the first conditioning trial of Experiment 1, before the animals had received the CS and the US, with male rats injected with 300 mg/kg of VPA showing more freezing than those injected with 100 mg/kg or an equivalent amount of saline. Consistent with this result, VPA‐treated males in Experiment 2 exhibited significantly more resting time in the open field compared to the control groups.

Although the results are consistent, suggesting an anxiolytic and sedative effect following the administration of 300 mg/kg of VPA, there are some aspects that should be further analyzed in future research. Specifically, the fact that the anxiolytic effect of VPA did not appear in male rats when measuring the time spent in the center of the open field was unexpected, because it contrasts with previous results where the administration of 300 mg/kg of VPA in male rats did lead to an increase in the percentage of time spent in the central zone of the open field. ⁶⁹ However, the discrepancy in the results may be related to the different treatments the animals received: in our experiment, Valproate was administered only once 20 min before conducting the open field test, whereas in the experiment by Kandeda et al., ⁶⁹ the animals had been previously injected with kainate and received the corresponding dose of VPA twice a day for a total of 14 days.

A possible explanation for the differences observed between males and females in our Experiment 2, with females spending more time in the central area of the open field but males not showing this effect, could be a result of the greater sedative effect of the drug observed in males in this same experiment. This increased sedation might have more significantly affected locomotor activity in males and, therefore, contributed to the reduction of time spent in the central zone.

The potential anxiolytic effect of VPA could be related to its action on the central nervous system. Although it is still not possible to precisely define all the effects of VPA on the organism, ⁷⁰ there is abundant evidence of its role as a GABAergic agonist. ³⁴ , ⁷¹ Such effect is similar to that of benzodiazepines, with which VPA seems to share not only an anxiolytic effect but also anticonvulsant properties and a muscle relaxant action. ¹⁸ , ⁷² , ⁷³ In fact, different authors have compared both types of drugs and reached similar conclusions about the effects of VPA and various types of benzodiazepines on anxiety‐related disorders and behaviors. ⁴⁸ , ⁷⁴ , ⁷⁵ , ⁷⁶ Other studies suggest that the effects of VPA could also be related to the blockade of voltage‐dependent sodium, potassium, and calcium channels, although the possible mechanisms of this pathway are largely unknown. ⁷⁷ , ⁷⁸ , ⁷⁹

Another fact that reflects the similarity between VPA and benzodiazepines is the sedative properties found in our experiments. Previous research has already shown various motor impairments as a consequence of VPA administration. ⁷⁶ , ⁸⁰ , ⁸¹ , ⁸² In fact, some recent studies suggest a potential use of VPA to treat agitation and delirium states. ⁸³ , ⁸⁴

Regarding sex‐related differences associated with VPA, despite the limited experimental evidence available, there appears to be some differentiation in pharmacokinetics based on gender. ⁸⁵ , ⁸⁶ Thus, in the treatment of epilepsy with VPA, a significantly higher remission of seizures has been found in males than in females. ⁸⁷ However, to our knowledge, there is no research on possible sex differences on the anxiolytic or sedative effects of VPA with which to compare our findings, although considering the significant variability found with benzodiazepines, it might be interesting to continue this line of research using different doses and animal models of anxiety, as well as controlling the estrous cycle in females.

Although further research is needed to determine the clinical application of VPA in individuals who respond partially or do not respond to conventional therapy, ¹⁷ , ²⁷ the existing experimental evidence supports its potential as an anxiolytic agent. Nonetheless, caution must be exercised when drawing conclusions from animal models, as the transition to clinical use may not always be straightforward. ⁸⁸ Given the increasing prevalence and severity of anxiety disorders, ⁸⁹ it is crucial to revisit the assessment mechanisms of such medications. However, human evaluation of VPA and its efficacy appears to be more advanced, with a substantial body of empirical evidence regarding its effectiveness in anxiety tests with healthy subjects ³² and individuals with various disorders. ¹⁷ , ²⁷ , ³¹

Of particular interest for the use of VPA in anxiety treatment are the benefits it presents compared to other drugs. One of its advantages over benzodiazepines is that VPA appears to not generate tolerance, making it an ideal drug for prolonged or chronic treatments. ³² , ⁴⁸ Furthermore, the prolonged use of benzodiazepines leads to significant cognitive impairment, ⁹⁰ , ⁹¹ , ⁹² whereas VPA appears to have promising neuroprotective effects proven in various neurodegenerative diseases ⁹³ and central nervous system disorders. ⁹⁴ When reviewing the effectiveness of different treatments for generalized anxiety disorder, VPA has proven to be one of the most effective drugs, even surpassing many benzodiazepines. ⁷⁴ , ⁹⁵ This fact, combined with the absence of tolerance, makes it an ideal option for treating this type of disorder, especially considering the serious issues of abuse and dependence on benzodiazepines that have emerged in recent decades. ⁹⁶ Additionally, some studies have considered the possibility that VPA prescription could alleviate the effects of discontinuing prolonged benzodiazepine treatments. ⁹⁷ The results in this domain are still somewhat contradictory, as some studies report a reduction in the severity of withdrawal syndrome when VPA is administered, ⁹⁸ while others do not find such an effect, but a consistent improvement in the absence of benzodiazepine consumption related to VPA treatment was observed. ⁹⁹

Also, in the analysis of any drug, it is important to consider its side effects. The most frequent adverse effects of VPA are gastrointestinal disturbances (such as dyspepsia, nausea, and vomiting), weight loss, and a paradoxical reaction to the drug that can include symptoms like sedation, tremors, ataxia, and cognitive impairment. ⁸¹ , ⁸² , ¹⁰⁰ Therefore, it is essential not only to consider the potential positive effects of VPA on anxiety reduction but also to continuously monitor the occurrence of side effects.

Finally, we consider it necessary to develop additional research involving different doses of VPA, other experimental paradigms designed to assess anxiety, or the inclusion of positive controls in the experiments to compare the effects of VPA with other well‐established anxiolytic drugs, such as diazepam or lorazepam. Therefore, despite the experimental evidence on the anxiolytic and sedative properties of VPA, further research is needed to determine under which circumstances it should be used in individuals who respond partially or do not respond to conventional therapy, ¹⁷ , ²⁷ as the clinical use of valproate has not yet been standardized for anxiety disorders. ¹⁰¹

AUTHOR CONTRIBUTIONS

All authors contributed equally to the work presented in this paper. Each author was involved in the study design, data collection, analysis and manuscript preparation.

FUNDING INFORMATION

This research was funded by Agencia Estatal de Investigación (AEI) of Spain (grant no. PID2019‐107530GB‐I00/AEI/10.13039/501100011033).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Reviewer Board: All experimental procedures were approved by the Ethics Committee for Animal Research at the University of Seville (code number CEEA‐US2015‐28/4). These procedures were carried out in accordance with the guidelines established by the EU Directive 2010/63/EU for animal experiments and Spanish R.D. 53/2013.

Informed Consent: Not applicable.

Registry and the Registration No. of the study/trial: The conducted research was not preregistered.

Animal Studies, ethic Statement: All experimental procedures were carried out in accordance with the guidelines established by the EU Directive 2010/63/EU for animal experiments and Spanish R.D. 53/2013.

Supporting information

Data S1.

Cintado MA, De la Casa LG, González G. Anxiolytic and sedative effects of sodium valproate with different experimental paradigms in male and female rats. Neuropsychopharmacol Rep. 2024;44:737–748. 10.1002/npr2.12483

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available in the Data S1 of this article and in idUS (Depósito de Investigación de la Universidad de Sevilla) at https://doi.org/10.12795/11441/159039. ¹⁰²