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Unconditioned and Conditioned Anxiolytic Effects of Sodium Valproate on Flavor Neophobia and Fear Conditioning

María Ángeles Cintado, Gabriel González, Lucía Cárcel, Luis Gonzalo De la Casa  


In three experiments with rats, we analyzed the potential anxiolytic effects of sodium valproate, an anticonvulsant drug that has shown additional pharmacodynamic effects in animal models, including anxiolytic action. Since previous results have revealed that injecting valproate before allowing animals to consume a novel flavor solution resulted in an attenuation of neophobia, we predicted a similar effect when the novel flavor is presented on a drug-free trial in the presence of a context previously associated with the drug. In line with this hypothesis, in our first experiment we observed a reduction in neophobia to a novel flavor for those animals tested in the presence of the context associated with Sodium Valproate. However, a control group that received the drug before being allowed access to the novel flavor showed a significant reduction in consumption.


Pavlov [1] described some experiments to show that repeated morphine administration in the presence of an experimental context resulted in a conditioned response to that context that mimicked some of the unconditional properties of morphine, such as nausea, vomiting, excessive salivation, or sleeping. Subsequently, ample experimental evidence has demonstrated conditioning using the effects of many different drugs as the Unconditioned Stimulus (US) [2–5]. These studies have focused not only on analyzing the learned associations that emerge after pairing the Conditioned Stimulus (CS) and the US [6–8], while also identifying the structures and neural circuits that constitute the neurobiological bases of learning [9–12], and the causes of drug addictions since conditioning has been proposed as a potential trigger of relapse and tolerance [13–15].


Thirty-two experimentally naïve male Wistar rats (group size n = 8), participated in this experiment. The mean weight at the start of the experiment was 356 g. At the arrival to the laboratory, the animals were housed in groups of 2/3 (depending of the animals’ weight) in type IIIH cages (820 cm2), with wood savings as bedding, and other materials available in the cages (pieces of fabric, cardboard and wood, stones, etc.), except for the time they were submitted to the experimental procedure when they were individually housed. The vivarium was maintained on a 12:12 h light-dark cycle (lights on at 07:00 h), and all behavioral testing was conducted during the light period of the cycle. Four days before the start of the experimental sessions, each animal was handled daily for 5 min. The day before to initiate the experimental treatment all animals were placed on a water deprivation schedule (30 min/day access to water) that was maintained across the entire duration of the experiment. All experimental procedures were approved by the Ethics Committee for Animal Research, University of Seville (Protocol CEEA-US2020-15/2), and were conducted in accordance with the guidelines established by the EU Directive 2010/63/EU for animal experiments, and the Spanish R.D. 223/1988.


We conducted preliminary one-way ANOVAs on mean percent freezing during the 180 s period prior to the onset of the first CS-tone for conditioning and testing stages with Groups as main factor. The analyses for the conditioning stage revealed a significant effect of Groups, F(2, 20) = 47.63; p < .001, η2 = .82. Post-hoc comparisons between groups (Bonferroni, p < .05) revealed a higher level of freezing for the VPA/Before (Mean = 87.42%, SD = 27.03) as compared to the VPA/After and Sal/Before groups (Mean = 13.88%, SD = 3.84, and Mean = 19.37%, SD = 6.34). This result confirms the reduction in locomotor activity induced by VPA.


The results of this study have revealed that 300 mg/Kg of VPA resulted in both an anxiolytic effect and a reduction of the animals’ locomotor activity. Furthermore, the anxiolytic effect was demonstrated by a reduction in fear conditioning in Experiment 3, and as a conditioned response that reduced the intensity of neophobia in Experiment 1. Therefore, the increased consumption of a novel flavor for those rats tested in the presence of a context previously associated with the effect of VPA can only be interpreted in terms of an anxiolytic conditioned response induced by the context-CS; any explanation in terms of residual effects of the drug on the drug-free test day is ruled out by the fact that the animals that received VPA after context exposure showed no change in neophobia. Thus, we were able to confirm our main hypothesis that in this case a Pavlovian association is formed between a distinct context (the CS) and the effects of the drug (the US).

Citation: Cintado MÁ, González G, Cárcel L, De la Casa LG (2023) Unconditioned and conditioned anxiolytic effects of Sodium Valproate on flavor neophobia and fear conditioning. PLoS ONE 18(7): e0279511.

Editor: Dragan Hrncic, Belgrade University Faculty of Medicine, SERBIA

Received: December 7, 2022; Accepted: June 16, 2023; Published: July 7, 2023

Copyright: © 2023 Cintado et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting information files.

Funding: This research was funded by Agencia Estatal de Investigación (AEI) of Spain (grant no.: PID2019-107530GB-I00/AEI/10.13039/501100011033). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


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