PreprintPDF Available

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

December 2022

DOI: 10.1101/2022.12.09.519825

License
CC BY 4.0

Authors:

Lucia Carcel

Universidad de Sevilla

L. G. De la Casa

Universidad de Sevilla

Preprints and early-stage research may not have been peer reviewed yet.

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. Experiment 2 revealed that the unconditioned effects of the drug include a deleterious effect on the animals' locomotor activity that probably interferes with drinking behavior. Finally, in a third experiment, we directly tested the potential anxiolytic properties of sodium valproate by injecting the drug before implementing a fear conditioning procedure. These findings are explained in terms of the unconditioned anxiolytic action of the drug and the formation of an association between the context and the effects of the drug that evokes a conditioned response reminiscent of such anxiolytic effect.

Available via license: CC BY 4.0

Content may be subject to copyright.

Unconditioned and Conditioned Anxiolytic Effects of Sodium Valproate on Flavor 7"

Neophobia and Fear Conditioning 8"

María Ángeles Cintado, Gabriel González-Tirado, Lucía Cárcel, Luis Gonzalo De la 10"

Casa* 11"

Laboratory of Animal Behavior & Neuroscience, Dpt. Experimental Psychology. Seville 12"

University (Spain) 13"

14"

* Corresponding author 15"

E-mail: delacasa@us.es (LGD) 16"

17"

All authors contributed equally to this work. 18"

19"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

Abstract 20"

In three experiments with rats, we analyzed the potential anxiolytic effects of 21"

sodium valproate, an anticonvulsant drug that has shown additional pharmacodynamic 22"

effects in animal models, including anxiolytic action. Since previous results have 23"

revealed that injecting valproate before allowing animals to consume a novel flavor 24"

solution resulted in an attenuation of neophobia, we predicted a similar effect when the 25"

novel flavor is presented on a drug-free trial in the presence of a context previously 26"

associated with the drug. In line with this hypothesis, in our first experiment we 27"

observed a reduction in neophobia to a novel flavor for those animals tested in the 28"

presence of the context associated with Sodium Valproate. However, a control group 29"

that received the drug before being allowed access to the novel flavor showed a 30"

significant reduction in consumption. Experiment 2 revealed that the unconditioned 31"

effects of the drug include a deleterious effect on the animals' locomotor activity that 32"

probably interferes with drinking behavior. Finally, in a third experiment, we directly 33"

tested the potential anxiolytic properties of sodium valproate by injecting the drug 34"

before implementing a fear conditioning procedure. These findings are explained in 35"

terms of the unconditioned anxiolytic action of the drug and the formation of an 36"

association between the context and the effects of the drug that evokes a conditioned 37"

response reminiscent of such anxiolytic effect. 38"

39"

40"

41"

42"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

Introduction 43"

Pavlov [1]#described some experiments to show that repeated morphine 44"

administration in the presence of an experimental context resulted in a conditioned 45"

response to that context that mimicked some of the unconditional properties of 46"

morphine, such as nausea, vomiting, excessive salivation, or sleeping. Subsequently, 47"

ample experimental evidence has demonstrated conditioning using the effects of many 48"

different drugs as the Unconditioned Stimulus (US) [2,3,4,5]. These studies have 49"

focused not only on analyzing the learned associations that emerge after pairing the 50"

Conditioned Stimulus (CS) and the US [6,7,8], while also identifying the structures and 51"

neural circuits that constitute the neurobiological bases of learning [9,10,11,12], and the 52"

causes of drug addictions since conditioning has been proposed as a potential trigger of 53"

relapse and tolerance [13,14,15]. 54"

In three experiments, we explored the result of pairing a neutral stimulus (a 55"

distinctive context) with the effects of Sodium Valproate (or"Valproic"Acid,"VPA), one 56"

of the most widely used and effective anticonvulsants for the treatment of epilepsy [16]. 57"

The main action of VPA on the nervous system is related to the levels of gamma-58"

aminobutyric acid (GABA), and its antiepileptic properties are largely based on the 59"

increased levels of this neurotransmitter after its administration [17,18]. It is this agonist 60"

effect on the GABAergic system that underpins its potential anxiolytic properties, 61"

which have already been observed in several experiments with rodents, showing a 62"

reduction in behaviors associated with anxiety after VPA administration [19,20,21,22]. 63"

Thus, for example, Corbett et al. [23] employed various tests to measure anxiety in 64"

rodents treated with VPA (Geller test, cross maze, or social interaction tests) and 65"

observed that, after systematic administration, the animals showed a reduction in 66"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

anxiety-related behaviors. In a similar vein, Kinrys et al. [24] studied the effects of VPA 67"

in people with a social anxiety disorder. After a 12-week treatment with the drug, they 68"

observed a significant reduction in symptoms, suggesting its potential use for treating 69"

this type of disorder. 70"

In a study aimed at exploring the potential anxiolytic properties of VPA, 71"

Shepard [25] analyzed the effects of administering various doses of the drug (100 and 72"

300 mg/kg) on flavor neophobia (using a compound of citric acid and sodium saccharin 73"

dissolved in water). Flavor neophobia is a defensive mechanism that produces a 74"

reduction in consumption of a new flavor [26], and seems to be related, at least to some 75"

degree, to anxiety [27,28]. In terms of survival, this mechanism protects the organism 76"

against potentially dangerous foods since consumption is reduced, thereby limiting any 77"

possible poisonous consequences. Alternatively, if an aversive effect does not follow 78"

consumption, the initial rejection of the flavor disappears, and it becomes a safe or 79"

familiar stimulus due to habituation of neophobia, which results in a progressive 80"

increase in flavor consumption [29]. 81"

The results of the neophobia test in the experiment conducted by Shepard [25] 82"

showed that administering VPA before allowing the animals to consume the novel 83"

flavor resulted in increased consumption (i.e., attenuation of neophobia) compared to a 84"

control group that received a vehicle. Shepard [25] interpreted these results in terms of 85"

the potential anxiolytic action of VPA that would have reduced the anxiety induced by 86"

encountering the novel flavor. 87"

In this paper, we analyzed the potential anxiolytic properties of VPA (300 88"

mg/Kg) and the possibility that such properties could be associated with a neutral 89"

context so that, later on, such a context elicits a conditioned response that mimics the 90"

anxiolytic properties of the drug. More specifically, we wanted to confirm whether the 91"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

context-CS elicited an anxiolytic conditioned response that reduced the intensity of 92"

neophobia reported by Shepard [25]. To this end, in the first experiment we presented a 93"

novel flavor in the presence of a context that had been repeatedly associated with VPA. 94"

The second experiment was designed to replicate the unconditioned effect of VPA on 95"

neophobia and to detect potential alterations in locomotor activity as a side effect of the 96"

drug. Finally, a third experiment was designed to check the anxiolytic properties of 97"

valproate on fear conditioning, a procedure highly dependent on emotional factors 98"

[30,31]. 99"

We anticipated that repeated pairings of a distinctive context with the effects of 100"

the drug would establish the context as a CS capable of evoking an anxiolytic 101"

conditioned response. Consequently, neophobia to a novel flavor would be reduced 102"

when the animal is tested in the presence of the CS-context. Additionally, we expected 103"

that the unconditioned anxiolytic properties of valproate would reduce neophobia and 104"

fear-conditioned intensity. 105"

Experiment 1 106"

The main objective of this experiment was to test whether the repeated pairing 107"

of a context (the CS) with the anxiolytic effects derived from the administration of VPA 108"

(the US) induces a conditioned response to the context that reduces the intensity of 109"

neophobia (and its habituation) in the same way as when the drug is directly 110"

administered prior to consumption of the novel flavor [25]. However, such an effect 111"

would not be observed when VPA is administered after context exposure or in a saline-112"

treated group. Finally, we also expect to replicate the attenuation of neophobia reported 113"

by Shepard [25] when the drug is injected before consumption of the novel flavor. 114"

Method 115"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

Subjects 116"

32 experimentally naïve male Wistar rats (n=8), participated in this experiment. 117"

The mean weight at the start of the experiment was 356 g. At the arrival to the 118"

laboratory, the animals were housed in groups of 2/3 (depending of the animals’ weight) 119"

in type IIIH cages (820 cm2), with wood savings as bedding, and other materials 120"

available in the cages (pieces of fabric, cardboard and wood, stones, etc.), except for the 121"

time they were submitted to the experimental procedure when they were individually 122"

housed. The vivarium was maintained on a 12:12 h light-dark cycle (lights on at 07:00 123"

h), and all behavioral testing was conducted during the light period of the cycle. Four 124"

days before the start of the experimental sessions, each animal was handled daily for 5 125"

min. The day before to initiate the experimental treatment all animals were placed on a 126"

water deprivation schedule (30 min/day access to water) that was maintained across the 127"

entire duration of the experiment. All"experimental"procedures"were"approved"by"the"128"

Ethics"Committee"for"Animal"Research,"University"of"Seville"(Protocol"CEEA-129"

US2020-15/2,"and"were conducted in accordance with the guidelines established by 130"

the EU Directive 2010/63/EU for animal experiments, and the Spanish R.D. 223/1988. 131"

Apparatus and drugs 132"

All experimental sessions were conducted in eight Plexiglas cages, 21 x 18 x 133"

35.5 cm, with paper strips as bedding, located in an experimental room, different to the 134"

vivarium, illuminated by a single 54-W fluorescent white light on the ceiling. All fluids 135"

were provided at room temperature in 150 ml graduated plastic bottles, fitted with 136"

stainless steel spouts. The bottles were attached to the front of each cage during each 137"

trial. The amount of fluid intake was measured by calculating the difference between 138"

the weight of the bottle before and after fluid presentations. Tap water was available in 139"

the bottles during the context habituation stage, and a solution of 0,04% sodium 140"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

saccharine and 0,1% citric acid solution dissolved in tap water was the fluid presented 141"

during neophobia trials. 142"

The drug injected was Sodium Valproate (Merck LifeScience), administered 143"

intraperitoneally at a dose of 300 mg/kg, 30 min before or after starting each 144"

experimental session, depending on the group. A saline solution was used as vehicle. 145"

Procedure 146"

A summary of the experimental treatment appears in Table 1. Each animal was 147"

randomly assigned to one of the following groups: VPA-Sal/Sal, Sal-VPA/Sal, Sal-148"

Sal/VPA, and Sal-Sal/Sal, where the first term is referred to the substance injected 30 m 149"

before context exposure in the conditioning stage, the second term to the substance 150"

injected 30 m after context exposure in the conditioning stage, and the third term to the 151"

substance injected immediately before neophobia trials. 152"

153"

Table 1: Design of Experiment 1. 154"

"155"

GROUP

Context Conditioning

(Days 1-4)

Neophobia Test

(Days 7-9)

Sal – Sal / Sal

Sal – 60 m context (water) - Sal

Sal – 30 min context (flavor)

Val – Sal / Sal

Val – 60 m context (water)- Sal

Sal – 30 min context (flavor)

Sal – Val / Sal

Sal – 60 m context (water)- Val

Sal – 30 min context (flavor)

Sal – Sal / Val

Sal – 60 m context (water)- Sal

Val – 30 min context (flavor)

156"

Val: 300 mg/Kg of Sodium Valproate; Sal: Saline Solution; Flavor: 0,04% sodium 157"

saccharine and 0,1% citric acid dissolved in tap water. See text for additional details. 158"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

159"

The context-conditioning phase took place from day 1 to 4, and it consisted of 160"

one daily session during four consecutive days. On these sessions, each animal was 161"

introduced in the cages located in the experimental room where they remained for 60-162"

min with access to water in the same bottles that were used in the neophobia stage. 163"

Those rats in the Val-Sal/Sal Group received an i.p injection of Sodium Valproate 30 164"

min before being introduced into the experimental context, and the vehicle 30 min after 165"

being returned to their home cages; those rats in the Sal-Val/Sal condition received the 166"

vehicle 30 m before experimental context exposure, and the drug 30 m after they were 167"

removed from the experimental chamber. Finally, the Sal-Sal/Val and the Sal-Sal/Sal 168"

groups received the vehicle injections using the same time schedule described for the 169"

remaining groups. Mean water consumption for each trial was registered after 30 and 60 170"

min. The animals received an additional 30-min period of water access in the home 171"

cages after the context-exposure period. 172"

On Days 5 and 6 the animals remained undisturbed in their home cages where 173"

they received the corresponding 30-min period of access to water. 174"

From Days 7 to 10 those animals in the Val-Sal/Sal, the Sal-Val/Sal, and the Sal-175"

Sal/Sal groups received an i.p. injection of the Vehicle 30 min before to be introduced 176"

in the experimental context, and those rats in the Sal-Sal/Val Group were injected with 177"

sodium valproate. Each one of the three experimental trials consisted in 30-min access 178"

to the citric acid + saccharin solution in the experimental cages to evaluate the intensity 179"

of neophobia and the subsequent habituation of this response. Ml. consumed were 180"

registered as an index of neophobia. An additional 30-min period of access to water was 181"

available in the home cages after each experimental session to ensure an appropriate 182"

level of hydration for the animals. 183"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

Results 184"

Water consumption during context conditioning stage 185"

Fig 1 depicts mean water consumption across the four days of context 186"

conditioning after 30 min (Section A), and 60 min (Section B). As can be seen in the 187"

Fig 1A, when consumption was registered after 30 min, it was lower for the animals in 188"

the VPA-Sal/Sal Group and tended to be reduced across days for the Sal-VPA/Sal 189"

Group. Those groups that did not receive VPA at conditioning stage (Sal-Sal/VPA, and 190"

Sal-Sal/Sal) drank a high and steady amount of water across trials. However, as 191"

depicted in Fig 1B, the differences between groups disappeared during the second 30-192"

min period suggesting that any effect of the drug on consumption was limited to the 193"

first half of each trial. 194"

195"

Fig 1. Mean water consumption across the four days of context conditioning after 196"

30 min (Section A), and 60 min (Section B). 197"

198"

These impressions were confirmed for the statistical analyses. A 4 x 4 mixed 199"

ANOVA (Trials x Groups) conducted on mean amount of water consumed during the 200"

first 30 min on each conditioning trial revealed significant main effects of Trials and 201"

Groups, F(3, 84)=4.49; p<.01, η2=.14, and F(3, 28)=5.95; p<.01, η2=.39, respectively. 202"

The two-way interaction was non-significant, F(9, 84)=1.24; p<.28. The main effect of 203"

Trials was due to a progressive reduction in consumption across trials. Post-hoc 204"

comparisons between groups (Bonferroni, p<.05) revealed that mean water 205"

consumption for group VPA-Sal/Sal (Mean = 7.51 ml, SD = 1.21) was reduced as 206"

compared to Groups Sal-Sal/VPA, and Sal-Sal/Sal (Mean = 9.54 ml, SD = 0.07, and 207"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

10"

Mean = 9.46 ml, SD = 1.15, respectively). No more comparisons between groups were 208"

significant. 209"

A similar 4 x 4 mixed ANOVA (Trials x Groups) conducted on mean amount of 210"

water consumed across the entire 60 min period showed a significant main effect of 211"

Trials, F(3, 84)=11.61; p<.001, η2=.29, confirming the progressive reduction in 212"

consumption across trials that has been observed after 30 min. Neither the main effect 213"

of Groups nor the 2-way interaction was significant, F(3, 28)=2.57; p>.07, η2=.29, and 214"

F(9, 84)<1. 215"

Citric acid + saccharine consumption at neophobia stage 216"

Figure 2 depicts mean citric acid + saccharine consumption across the three 217"

trials of the neophobia stage as a function of Groups. As can be seen in the figure, the 218"

lower consumption corresponded to the animals in the Sal-Sal/VPA Group (that 219"

received an injection of 300 mg/Kg of Valproate 30 min before each neophobia trial). 220"

The higher level of consumption appeared in the VPA-Sal/Sal Group, while the Sal-221"

VPA/Sal, and the Sal-Sal/Sal Groups drank an intermediate amount of the flavored 222"

solution. 223"

224"

Fig 2. Mean citric acid + saccharine consumption across the three trials of the 225"

neophobia stage as a function of Groups 226"

227"

A 3 x 4 mixed ANOVA (Trials x Groups) conducted on mean amount of 228"

flavored solution consumed for neophobia trials revealed that both the main effect of 229"

Trials and Groups were significant, F(2, 56)=54.51; p<.001, η2=.66, and F(3, 230"

28)=49.36; p<.001, η2=.84, respectively. The main effect of Trials reflects an overall 231"

increase in consumption across trials due to neophobia habituation. Post-hoc 232"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

11"

comparisons between groups (Bonferroni, p<.05) on mean consumption collapsed 233"

across test trials revealed that consumption in Group VPA-Sal/Sal was significantly 234"

higher as compared to Sal-VPA/Sal and Sal-Sal/Sal groups. In addition, consumption 235"

for the Sal-Sal/VPA group was reduced as compared to the remaining groups. No more 236"

differences were significant. 237"

The Trials x Group interaction was also significant, F(6, 56)=13.04; p<.001, 238"

η2=.58. In order to identify the source of the interaction, we conducted post-hoc 239"

comparisons between groups (Bonferroni, p<.05) for each Trial that showed higher 240"

consumption in the VPA-Sal/Sal group and reduced consumption in the Sal-Sal/VPA 241"

group as compared to the remaining groups for the first and second test trials. On the 242"

third test trial the Sal-Sal-VPA group drank significantly less than the remaining 243"

groups. No more differences between groups were significant. 244"

Experiment 2 245"

Our hypotheses were partially confirmed in Experiment 1 since neophobia and 246"

its habituation were reduced, i.e., consumption of the novel flavor was increased for the 247"

VPA-Sal/Sal Group for which the flavor was presented in a context previously paired 248"

with the effects of VPA. However, the reduced consumption of the novel flavored 249"

solution for the Sal-Sal/VPA Group at the neophobia stage was unexpected since, using 250"

the same parameters, Shepard [25] observed the opposite result, namely an increase in 251"

consumption of the novel solution when 300 mg/Kg of VPA was i.p. injected 30 m 252"

before access to the solution. Similarly, water consumption was reduced for the group 253"

that received VPA before the conditioning trials, but this reduction was only evident 254"

during the first 30 minutes of each trial. 255"

A possible reason for the reduced consumption observed after VPA 256"

administration could be related to the motor disturbances induced by VPA that have 257"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

12"

been reported in previous studies. These effects include ataxia, hypolocomotion,"258"

reduction"of"grooming, muscle relaxation, or wet"dog"shake"behavior"[32,33,34,35]. "259"

These"disturbances"could"have"disrupted"normal"drinking"behavior,"resulting"in"260"

reduced"consumption.""261"

This"possibility"was"tested"in"the"second"experiment,"which"included"three"262"

groups"exposed"to"the"same"novel"solution"employed"in"the"first"experiment."One"263"

group"received"an"i.p."injection"of"300"mg/kg"30"min"before"flavor"exposure"264"

(Group"VPA-30),"the"second"group"received"VPA"just"before"allowing"the"animal"to"265"

drink"the"novel"flavor"(Group"VPA-0),"while"half"of"the"animals"in"the"third"group"266"

received"saline"30"min"before"flavor"exposure,"and"the"other"half"saline"267"

immediately"before"such"exposure"(Group"Sal) 268"

Neophobia tests were conducted for three consecutive days (30 min access to the 269"

flavor each day) in experimental chambers designed to detect the animals' movements. 270"

Based on the results from Experiment 1, we anticipated a reduction in drinking for those 271"

animals in the VPA groups, which could be even greater for the VPA-0 compared with 272"

the VPA-30 Group. Additionally, we expected a reduction in the global percentage of 273"

locomotor activity for the animals that received VPA compared to those that received 274"

saline. 275"

Method 276"

Subjects 277"

24 experimentally naïve male Wistar rats (n=8), participated in this experiment. 278"

The mean weight at the start of the experiment was 355 g. The animals were housed and 279"

maintained exactly as described for Experiment 1. All procedures were conducted in 280"

accordance with the guidelines established by the EU Directive 2010/63/EU for animal 281"

experiments, and the Spanish R.D. 223/1988. 282"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

13"

Apparatus and drugs 283"

All experimental sessions were conducted in four identical Panlab conditioning 284"

boxes (model LE111, Panlab/Harvard Apparatus, Spain), each measuring 26 x 25 x 25 285"

cm (H x L x W). Each chamber was enclosed in a sound-attenuating cubicle (model 286"

LE116. Panlab/Harvard Apparatus, Spain). The walls of the experimental chambers 287"

were made of white acrylic, and the floor consisted of stainless steel rods, 2 mm in 288"

diameter, spaced 10 mm apart (center to center). Each chamber rested on a platform that 289"

recorded the signal generated by the animal movement through a high sensitivity 290"

Weight Transducer system. Such signal was automatically converted into percent of 291"

general activity, defined as the percentage of the total time that movement was detected 292"

on 2-min periods, by a commercial software (StartFear system software, 293"

Panlab/Harvard Apparatus, Spain). Sampling was performed continuously at a 294"

frequency of 50Hz. All fluids were provided at room temperature in 150 ml graduated 295"

plastic bottles containing a 0,04% sodium saccharine and 0,1% citric acid solution 296"

dissolved in tap water that were attached to the front of each cage during each trial. As 297"

in Experiment 1, the amount of fluid intake was measured by calculating the difference 298"

between the weight of the bottle before and after fluid presentations. 299"

The drug injected was Sodium Valproate (Merck LifeScience), that was i.p. 300"

administered at a dose of 300 mg/kg 30 min before (Group VPA/30) o immediately 301"

before (Group VPA/0) to start each neophobia session. An additional control group 302"

(Group Sal) received only the saline solution. For this control group half of the rats 303"

received the saline injection 30 min before the neophobia trial, and the other half 304"

received the injection immediately before of each trial. 305"

Procedure 306"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

14"

Two days before to start the experimental treatment the standard bottles used in 307"

the vivarium were replaced for the bottles employed during the experimental trials 308"

containing water, in order to habituate the animals to them. 309"

The experimental treatment lasted three days, and each day the animals has 310"

access to 30 min of access to the citric acid + saccharin solution. Those animals in the 311"

VPA-0 and the VPA-30 groups received an i.p. injection of VPA immediately or 30 min 312"

before, respectively, to the start of each experimental trial, that consisted in 30-min 313"

access to the citric acid + saccharine solution. Mean percent of activity and 314"

consumption were registered. An additional 30-min period of access to water was 315"

available at the home cages after each experimental session to ensure an appropriate 316"

level of hydration for the animals. 317"

Results 318"

A preliminary comparison showed no significant differences neither in 319"

consumption nor locomotor activity level between those animals that received the saline 320"

injection 30 or 0 min before novel flavor exposure. Therefore, the data for all the 321"

animals in the Sal Group were unified for the statistical analyses. 322"

Citric acid + saccharine consumption 323"

Fig 3 depicts mean citric acid + saccharine consumption for the three neophobia 324"

trials. As can be seen in the figure, the expected effect of neophobia appeared in the Sal 325"

Group at the first trial, and it was followed by neophobia habituation for second and 326"

third trials. Conversely, the animals in the VPA-30 and VPA-0 groups showed a 327"

reduced amount of drinking across trials that was even more intense for the VPA/0 328"

group. 329"

330"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

15"

Fig 3. Mean citric acid + saccharine consumption for the three neophobia trials as 331"

a function of Groups 332"

333"

A 3 x 3 mixed ANOVA (Trials x Groups) confirmed these impressions. The 334"

analyses revealed significant main effects of Trials and Groups, F(2, 42)=4.3; p<.05, 335"

η2=.17, and F(1, 21)=14.48; p<.001, η2=.58, respectively. The main effect of Trials was 336"

due to an overall increase in consumption across trials. Post hoc comparisons 337"

(Bonferroni, p<.05) revealed that the effect of Groups was due to higher mean 338"

consumption for the Sal (Mean = 7.72 ml, SD = 5.05) as compared to both VPA-30 and 339"

VPA-0 groups (Mean = 2.87 ml, SD = 2.32, and Mean = .61 ml, SD = .43, 340"

respectively). The Trials x Groups interaction was also significant, F(4, 42)=7.63; 341"

p<.001, η2=.42. Post-hoc comparisons between groups (Bonferroni, p<.05) for each trial 342"

showed lower consumption in the VPA-0 group as compared to the VPA-30 for the first 343"

trial and to the Sal-0 for all trials. Consumption in the VPA-30 Groups was also reduced 344"

as compared to Sal Group for the third trial. No more differences between groups were 345"

significant. 346"

Mean Percent activity 347"

Activity percent was computed for each one of the three trials in five periods of 348"

6 minutes. A 3 x 5 x 3 mixed ANOVA, with main factors Trials, 6-min periods, both 349"

within subjects, and Groups revealed a significant main effect of Periods and Groups, 350"

F(4, 168)=64.93; p<.001, η2=.76 y F(2, 21)=41.66; p<.001, η2=.79, respectively. The 351"

main effect of Periods was due to a progressive decrease in activity across time. Post-352"

hoc comparisons between groups (Bonferroni, p<.05) revealed that the main effect of 353"

Groups was due to a higher global level of activity for the Sal (Mean = 69.46%, SD = 354"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

16"

4.14) as compared to the VPA/0 and the VPA/30 groups (Mean = 30.10%, SD = 2.69, 355"

and Mean = 31.41%, SD = 3.41, respectively) 356"

The Periods x Groups interaction was also significant, F(8, 168)=8.68; p<.001, 357"

η2=.45. No more main effects or interactions were significant (all ps>.58). The Periods 358"

x Groups interaction is depicted in Fig 4, that shows mean percent activity for the 6-min 359"

periods collapsed across the three test sessions as a function of Groups. As can be seen 360"

in the figure, and confirmed by post hoc comparisons between groups (Bonferroni, 361"

p<.05), mean activity was lower for the VPA-0 as compared to the VPA-30 Group for 362"

the first 6-min period, and for all periods as compared to the Sal Group. Mean activity 363"

in the VPA-30 Group was significantly reduced as compared to the Sal Group from the 364"

second to the sixth 6 min-Period. 365"

366"

Fig 4. Mean percent activity for the 6-min periods collapsed across the three test 367"

sessions as a function of Groups 368"

369"

Experiment 3 370"

Experiment 2 confirmed disturbances in locomotor activity induced by VPA 371"

administration that could have interfered with drinking behavior. More specifically, a 372"

reduction in movement can impair the animal’s ability to approach the tubes, 373"

consequently reducing the amount of solution consumed. However, we do not have 374"

direct evidence of the potential anxiolytic effect of VPA that might explain the 375"

reduction of neophobia and its habituation observed in Experiment 1 when consumption 376"

took place in the presence of the context associated with the drug. 377"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

17"

The third experiment was designed to obtain a direct behavioral test of the 378"

anxiolytic effect of VPA using a fear-conditioning paradigm, a process that has been 379"

linked to emotional factors [30,31]. To this end, we designed an experiment to 380"

determine whether VPA can reduce fear-conditioning intensity. More specifically, the 381"

experiment comprised three groups: a VPA/Before Group received VPA before 382"

presenting 3 tone-shock pairings; a VPA/After Group received the drug after 383"

conditioning trials; and a Sal/Before Group received a saline injection before 384"

conditioning. Next, the day after conditioning, the tone was repeatedly presented 385"

without the shock in a drug-free session to evaluate the intensity of fear conditioning. 386"

Given the potential anxiolytic effect of VPA, we expected to find reduced fear 387"

conditioning in the group that received VPA before conditioning as compared to the 388"

groups that received saline or VPA after the tone-shock pairings. 389"

Method 390"

Subjects 391"

24 experimentally naïve male Wistar rats (n=8), participated in this experiment. 392"

The mean weight at the start of the experiment was 423 g. The animals were housed and 393"

maintained exactly as described for Experiment 1, except for water that was available 394"

ad libitum. All procedures were conducted in accordance with the guidelines established 395"

by the EU Directive 2010/63/EU for animal experiments, and the Spanish R.D. 396"

223/1988. 397"

Procedure and apparatus 398"

All experimental sessions were conducted in the same conditioning boxes 399"

described in Experiment 2. The conditioning session started with a 180 s period without 400"

any stimulation followed by 3 tone-shock pairings with a 180 s Inter Trial Interval. The 401"

US was a 1-s, 0.5-mA unscrambled AC 50-Hz foot shock from a constant-current 402"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

18"

generator (Model LE100-26) that was delivered to the floor of each chamber. A 403"

loudspeaker was located at the top of each chamber, which produced a 70 dB 2.8-kHz 404"

30 s tone that was used as CS. For conditioning session, an i.p. injection of VPA (300 405"

mg/kg) was injected 30 min before (Group VPA/Before) o immediately after (Group 406"

VPA/After) the start of the experimental treatment. An additional control group (Group 407"

Sal/Before) received a vehicle 30 min before the start of the conditioning stage. One 408"

animal from the VPA/After Group was removed from the experiment due to a failure in 409"

an experimental chamber during the conditioning stage. 410"

The next day, a free-drug extinction session similar for all groups was 411"

conducted. It started with a 180 s period without any stimulation followed by six tone-412"

alone presentations with a 180 s Inter Trial Interval. The chambers’ floor rested on a 413"

platform that registered and recorded the animal’s movements. A percentage score 414"

indicating freezing was computed by the experimental software (PANLAB Startfear) 415"

for the proportion of the total time that immobility was detected. 416"

Results 417"

Fig 5 depicts mean freezing to the Tone-Cs during conditioning (left side) and 418"

extinction (right side) sessions. As can be seen in the figure, activity for the 419"

VPA/Before Group was reduced during the first conditioning trial (i.e., freezing was 420"

higher) as compared to the remaining groups, replicating the deleterious effect of the 421"

drug on locomotor activity observed in Experiment 2. As for the VPA/after and 422"

Sal/Before groups, conditioning was expressed through a high level of freezing in 423"

presence of the tone-CS for the second and third conditioning trials. Regarding the 424"

drug-free extinction stage, fear conditioning was more intense for VPA/After and 425"

Sal/Before groups, as revealed for the higher levels of freezing in presence of the tone 426"

during the first extinction trials for these groups as compared to the VPA/Before Group. 427"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

19"

Fig 5. Mean freezing to the Tone-Cs during conditioning (left side) and extinction 428"

(right side) sessions as a function of Groups 429"

430"

A 3 x 3 mixed ANOVA (Trials x Groups) conducted on mean percent of 431"

freezing during conditioning revealed a significant main effect of Trials and Groups, 432"

F(2, 40)=39.28; p<.001, η2=.66 y F(2, 20)=4.45; p<.05, η2=.31. The main effect of 433"

Trials reflects a progressive increase in freezing across conditioning trials. Regarding 434"

the effect of Groups, post-hoc comparisons (Bonferroni, p<.05) revealed a higher global 435"

level of freezing for the VPA/Before (Mean = 77.09%, SD = 14.68) as compared to the 436"

VPA/After group (Mean = 56.43%, SD = 11.94). No more comparisons were 437"

significant. The Trials x Groups interaction was also significant, F(4, 40)=4.6; p<.01, 438"

η2=.32. In order to explore the source of the interaction we conducted Post-hoc 439"

comparisons (Bonferroni, p<.05) between groups for each trial that that showed 440"

significant differences between the VPA/Before as compared to the Sal/Before and the 441"

VPA/After groups. 442"

A 6 x 3 mixed ANOVA (Trials x Groups) conducted on mean percent of 443"

freezing during extinction revealed a significant main effect of Groups, F(2, 20)=10.70; 444"

p<.01, η2=.52. Post-hoc comparisons between groups (Bonferroni, p<.05) revealed a 445"

lower global level of freezing for the VPA/Before (Mean = 60.54%, SD = 13.99) as 446"

compared to the VPA/After and the Sal/Before groups (Mean = 82.99%, SD = 10.42, 447"

and Mean = 82.96%, SD = 7.89, respectively). The main effect of Trials was non-448"

significant, F(5, 100)=1.33; p>.26, η2=.06. The Trials x Groups interaction was also 449"

significant, F(10, 100)=2.86; p<.01, η2=.22. In order to explore the interaction, we 450"

conducted post-hoc comparisons between groups for each trial (Bonferroni, ps<.05) that 451"

revealed lower levels of freezing, i.e., less fear conditioning, for the VPA/Before Group 452"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

20"

as compared to the Sal/Before and VPA/After groups for the 1st, 2nd, and 4th trials. No 453"

more comparisons were significant 454"

General Discussion 455"

The results of this study have revealed that 300 mg/Kg of VPA resulted in both 456"

an anxiolytic effect and a reduction of the animals’ locomotor activity. Furthermore, the 457"

anxiolytic effect was demonstrated by a reduction in fear conditioning in Experiment 3, 458"

and as a conditioned response that reduced the intensity of neophobia in Experiment 1. 459"

Therefore, the increased consumption of a novel flavor for those rats tested in the 460"

presence of a context previously associated with the effect of VPA can only be 461"

interpreted in terms of an anxiolytic conditioned response induced by the context-CS; 462"

any explanation in terms of residual effects of the drug on the drug-free test day is ruled 463"

out by the fact that the animals that received VPA after context exposure showed no 464"

change in neophobia. Thus, we were able to confirm our main hypothesis that in this 465"

case a Pavlovian association is formed between a distinct context (the CS) and the 466"

effects of the drug (the US). 467"

The reduction in fear conditioning for the group that received VPA before the 468"

tone-shock pairings in Experiment 3 represents a clear demonstration of the 469"

unconditioned anxiolytic effect of VPA. In this case, a possible explanation in terms of 470"

lack of memory consolidation of the tone-shock association can be ruled out for the 471"

absence of an effect on fear conditioning for the group that received VPA immediately 472"

after the conditioning trials. However, we cannot completely rule out an account in 473"

terms of the possible effect of the drug on the perceived intensity of the shock, which 474"

could have weakened the strength of the association with the tone. However, this 475"

possibility is unlikely considering that the level of freezing shown by the VPA-Before 476"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

21"

group at the end of conditioning treatment was at asymptote and similar to the 477"

remaining groups. 478"

The deleterious effect of VPA on locomotor activity was clearly established 479"

when the percentage of activity was recorded after drug administration in Experiment 2. 480"

The greater detrimental effect of the drug on locomotor activity for those animals that 481"

received the drug immediately before the test compared with those that received VPA 482"

30 m before the test, revealed that such an effect was time-dependent and relatively 483"

short-lasting. The reduction in locomotor activity is most likely the result of the lower 484"

water consumption observed when the animals received VPA before the conditioning 485"

stage of Experiment 1 and the reduction in consumption of the novel flavor for the 486"

animals injected with the drug in Experiment 2. This result was largely unexpected 487"

since Shepard [25] reported an increase in the consumption of a novel flavor after 488"

administering both 100 and 300 mg/Kg. of VPA. There are only two apparent 489"

procedural differences between Shepard’s experiments and those reported here: the 490"

context used as the experimental environment and the period for measuring fluid 491"

consumption. Regarding the experimental context, Shepard conducted the entire 492"

procedure in the animal's home cage. However, the present experiments were run in 493"

distinctive cages located outside of the vivarium. This could be a possible factor 494"

responsible for the discrepancy in results since it has been demonstrated that 495"

consumption of a novel flavor is greater when presented in a familiar and safe home 496"

cage than in a novel and potentially dangerous context [36]. A second relevant 497"

difference is related to the periods for which intake was recorded since Shepard 498"

recorded fluid intake after 6, 18, and 30 m, but we only recorded consumption after 30 499"

m. Importantly, Shepard found a significant increase in consumption after 6 m but not 500"

after 18 or 30 m. Therefore, the mentioned procedural discrepancies between 501"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

22"

experiments could, at least in part, explain the different results, although this possibility 502"

merits further investigation. In any case, such differences do not undermine the main 503"

conclusions drawn from our experiments, namely the anxiolytic effect of VPA and the 504"

emergence of an anxiolytic conditioned response through a classical conditioning 505"

process. 506"

Alternative explanations for the changes in neophobia mediated by the 507"

anxiolytic conditioned response observed in the VPA-Sal/Sal Group in the test phase of 508"

Experiment 1 include the effects of context novelty on fluid consumption [37]. More 509"

specifically, considering that VPA administration could have altered context processing 510"

during the conditioning stage, it is possible that such a context could have been rendered 511"

relatively novel at the time of testing for the animals that received the drug. 512"

Furthermore, previous research has revealed that neophobia increases when the flavor is 513"

presented in a novel context, possibly due to exploratory responses interfering with 514"

consumption [38,37]. However, in Experiment 1, we observed an increase in 515"

consumption for the group that received VPA before context conditioning instead of the 516"

reduction that would have been expected if the context had remained functionally novel, 517"

thus ruling out an interpretation of our results in terms of exploratory behavior 518"

interfering with fluid intake. 519"

The role of context in neophobia has received rather limited attention, possibly 520"

because the key factors proposed as determinants of neophobia are mainly related to the 521"

characteristics of the novel flavor [39] or to the deprivation state of the animals at the 522"

time of consumption [40]. However, the experimental context has shown to play a key 523"

role in both neophobia and its habituation [36]. Thus, in a novel context, the 524"

presentation of an unknown flavor produces an increase in neophobia. Consequently, 525"

consumption is reduced compared to when the same flavor is presented for the first time 526"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

23"

in a familiar context [41]. Furthermore, consumption increases when a flavor is 527"

presented in contexts previously associated with appetitive stimuli but decreases when 528"

the flavor appears in a context associated with aversive stimuli [42]. Similarly, other 529"

studies suggest that when a novel taste is presented in a changing environment, the 530"

neophobic response is weaker [43]. In the present paper, we have proposed a new role 531"

for the context in modulating the consumption of novel flavors since neophobia and its 532"

habituation can be reduced when the novel flavor is consumed in the presence of a 533"

context previously associated with the anxiolytic effects of a drug. 534"

Regarding the unconditioned anxiolytic effect of VPA, previous studies have 535"

proposed that this depends on an elevation of GABA levels [44] in addition to blocking 536"

of GABA re-uptake [45]. Moreover, some data indicate that the action of VPA 537"

resembles that of benzodiazepines, with which it also shares anticonvulsant properties 538"

and acts as a muscle relaxant [46,47]. Such anxiolytic effects of VPA have been 539"

demonstrated with a wide variety of procedures, such as the consumption of new flavors 540"

with high palatability [48,49], different types of conflict tasks both with shock [21,50] 541"

and without shock [19], the light-dark aversion test [20], the staircase test [22], the 542"

elevated plus-maze [51], or social interaction [23]. As mentioned above, we have 543"

directly confirmed the anxiolytic effect of VPA using both a fear conditioning 544"

procedure (Experiment 3), and the effects of a conditioning process on neophobia 545"

(Experiment 1). 546"

In sum, we have demonstrated that VPA (300 mg/Kg) induces a reduction in 547"

locomotor activity and has anxiolytic properties. Interestingly, this anxiolytic effect can 548"

be associated with a neutral stimulus (a distinctive context) and expressed as a 549"

conditioned response that reduces the intensity of neophobia and its habituation to a 550"

new flavor. These findings have implications for the potential therapeutic use of VPA, 551"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

24"

our understanding of the mechanisms of classical conditioning using drug stimuli, and 552"

the links between contextual cues and neophobia. 553"

554"

555"

556"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

25"

References 557"

1. Pavlov PI. Conditioned reflexes: An investigation of the physiological activity 558"

of the cerebral cortex. Ann Neurosci. 2010 Jul;17(3):136-41. doi: 559"

10.5214/ans.0972-7531.1017309. PMID: 25205891; PMCID: PMC4116985. 560"

2. Battisti JJ, Uretsky NJ, Wallace LJ. Importance of environmental context in the 561"

development of amphetamine- or apomorphine-induced stereotyped behavior 562"

after single and multiple doses. Pharmacol Biochem Behav. 2000 563"

Aug;66(4):671-7. doi: 10.1016/s0091-3057(00)00214-8. PMID: 10973502. 564"

3. De la Casa LG, Cárcel L, Ruiz-Salas JC, Vicente L, Mena A. Conditioned 565"

increase of locomotor activity induced by haloperidol. PLoS One. 2018 Oct 566"

3;13(10):e0200178. doi: 10.1371/journal.pone.0200178. PMID: 30281607; 567"

PMCID: PMC6169844. 568"

4. Mena A, De la Casa LG. Prepulse inhibition modulation by contextual 569"

conditioning of dopaminergic activity. Behav Brain Res. 2013 Sep 1;252:188-570"

94. doi: 10.1016/j.bbr.2013.06.005. Epub 2013 Jun 10. PMID: 23756135. 571"

5. Siegel"S.."The"role"of"conditioning"in"drug"tolerance"and"addiction."In:."Keehn"JD,"572"

editor."Psychopathology"in"animals:"Research"and"clinical"implications."New"York:"573"

Academic"Press;"1979."Pp."143-168. 574"

6. Ahmed SH, Oberling P, Di Scala G, Sandner G. Amphetamine-induced 575"

conditioned activity does not result from a failure of rats to habituate to novelty. 576"

Psychopharmacology (Berl). 1996 Feb;123(4):325-32. doi: 577"

10.1007/BF02246642. PMID: 8867871. 578"

7. Carey RJ, Carrera MP, Damianopoulos EN. A new proposal for drug 579"

conditioning with implications for drug addiction: the Pavlovian two-step from 580"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

26"

delay to trace conditioning. Behav Brain Res. 2014 Dec 15;275:150-6. doi: 581"

10.1016/j.bbr.2014.08.053. Epub 2014 Sep 9. PMID: 25218870. 582"

8. Cárcel L, De la Casa LG. Temporal Factors Modulate Haloperidol-Induced 583"

Conditioned Catalepsy. Front Behav Neurosci. 2021 Jul 2;15:713512. doi: 584"

10.3389/fnbeh.2021.713512. PMID: 34276319; PMCID: PMC8283013. 585"

9. Eikelboom R, Stewart J. Conditioning of drug-induced physiological responses. 586"

Psychol Rev. 1982 Sep;89(5):507-28. PMID: 7178331. 587"

10. Joyce EM, Koob GF. Amphetamine-, scopolamine- and caffeine-induced 588"

locomotor activity following 6-hydroxydopamine lesions of the mesolimbic 589"

dopamine system. Psychopharmacology (Berl). 1981;73(4):311-3. doi: 590"

10.1007/BF00426456. PMID: 6789349. 591"

11. French ED, Pilapil C, Quirion R. Phencyclidine binding sites in the nucleus 592"

accumbens and phencyclidine-induced hyperactivity are decreased following 593"

lesions of the mesolimbic dopamine system. Eur J Pharmacol. 1985 Oct 594"

8;116(1-2):1-9. doi: 10.1016/0014-2999(85)90178-5. PMID: 2996909. 595"

12. Gold LH, Swerdlow NR, Koob GF. The role of mesolimbic dopamine in 596"

conditioned locomotion produced by amphetamine. Behav Neurosci. 1988 597"

Aug;102(4):544-52. doi: 10.1037//0735-7044.102.4.544. PMID: 3139012. 598"

13. Siegel S. Morphine tolerance acquisition as an associative process. J Exp 599"

Psychol Anim Behav Process. 1977 Jan;3(1):1-13. doi: 10.1037//0097-600"

7403.3.1.1. PMID: 845541. 601"

14. de Wit H, Stewart J. Reinstatement of cocaine-reinforced responding in the rat. 602"

Psychopharmacology (Berl). 1981;75(2):134-43. doi: 10.1007/BF00432175. 603"

PMID: 6798603. 604"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

27"

15. Valyear MD, Villaruel FR, Chaudhri N. Alcohol-seeking and relapse: A focus 605"

on incentive salience and contextual conditioning. Behav Processes. 2017 606"

Aug;141(Pt 1):26-32. doi: 10.1016/j.beproc.2017.04.019. Epub 2017 May 1. 607"

PMID: 28473252. 608"

16. Aldenkamp A, Vigevano F, Arzimanoglou A, Covanis A. Role of valproate 609"

across the ages. Treatment of epilepsy in children. Acta Neurol Scand Suppl. 610"

2006;184:1-13. doi: 10.1111/j.1600-0404.2006.00666.x. PMID: 16776492. 611"

17. Ghodke-Puranik Y, Thorn CF, Lamba JK, Leeder JS, Song W, Birnbaum AK, 612"

Altman RB, Klein TE. Valproic acid pathway: pharmacokinetics and 613"

pharmacodynamics. Pharmacogenet Genomics. 2013 Apr;23(4):236-41. doi: 614"

10.1097/FPC.0b013e32835ea0b2. PMID: 23407051; PMCID: PMC3696515. 615"

18. Szulczyk B, Pasierski M, Nurowska E. Valproic acid potently inhibits interictal-616"

like epileptiform activity in prefrontal cortex pyramidal neurons. Neurosci Lett. 617"

2019 Aug 24;708:134350. doi: 10.1016/j.neulet.2019.134350. Epub 2019 Jun 618"

24. PMID: 31247226. 619"

19. Becker HC, Anton RF. Valproate potentiates and picrotoxin antagonizes the 620"

anxiolytic action of ethanol in a nonshock conflict task. Neuropharmacology. 621"

1990 Sep;29(9):837-43. doi: 10.1016/0028-3908(90)90158-n. PMID: 1981382. 622"

20. de Angelis L. Effects of valproate and lorazepam on experimental anxiety: 623"

tolerance, withdrawal, and role of clonidine. Pharmacol Biochem Behav. 1995 624"

Oct;52(2):329-33. doi: 10.1016/0091-3057(95)00100-b. PMID: 8577798. 625"

21. Lal H, Shearman GT, Fielding S, Dunn R, Kruse H, Theurer K. Evidence that 626"

GABA mechanisms mediate the anxiolytic action of benzodiazepines: a study 627"

with valproic acid. Neuropharmacology. 1980 Aug;19(8):785-9. doi: 628"

10.1016/0028-3908(80)90071-4. PMID: 6106907. 629"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

28"

22. Simiand J, Keane PE, Morre M. The staircase test in mice: a simple and efficient 630"

procedure for primary screening of anxiolytic agents. Psychopharmacology 631"

(Berl). 1984;84(1):48-53. doi: 10.1007/BF00432023. PMID: 6149594. 632"

23. Corbett R, Fielding S, Cornfeldt M, Dunn RW. GABAmimetic agents display 633"

anxiolytic-like effects in the social interaction and elevated plus maze 634"

procedures. Psychopharmacology (Berl). 1991;104(3):312-6. doi: 635"

10.1007/BF02246029. PMID: 1681557. 636"

24. Kinrys G, Pollack MH, Simon NM, Worthington JJ, Nardi AE, Versiani M. 637"

Valproic acid for the treatment of social anxiety disorder. Int Clin 638"

Psychopharmacol. 2003 May;18(3):169-72. doi: 639"

10.1097/01.yic.0000064261.66765.9f. PMID: 12702897. 640"

25. Shephard RA. Valproate enhances fluid consumption suppressed by shock or 641"

neophobia, but not by partial satiation or d-amphetamine, in rats. Life Sci. 642"

1988;43(11):897-904. doi: 10.1016/0024-3205(88)90265-2. PMID: 3137408. 643"

26. Reilly S. Food neophobia: Behavioral and biological influences. Cambridge: 644"

Woodhead Publishing; 2018. https://doi.org/10.1016/B978-0-08-101931-645"

3.00010-0 646"

27. Farrow C, Coulthard H. Multisensory evaluation and the neophobic food 647"

response. In: Reilly S, editor. Food neophobia: Behavioral and biological 648"

influences. Cambridge: Woodhead Publishing; 2018. pp. 219-236. 649"

https://doi.org/10.1016/B978-0-08-101931-3.00011-2 650"

28. Raudenbush B, Capiola A. Physiological responses of food neophobics and food 651"

neophilics to food and non-food stimuli. Appetite. 2012 Jun;58(3):1106-8. doi: 652"

10.1016/j.appet.2012.02.042. Epub 2012 Feb 24. PMID: 22369957. 653"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

29"

29. Domjan M. The essentials of conditioning and learning. 4th ed. American 654"

Psychological Association; 2018. 10.1037/0000057-000. 655"

30. Lang PJ, Davis M, Ohman A. Fear and anxiety: animal models and human 656"

cognitive psychophysiology. J Affect Disord. 2000 Dec;61(3):137-59. doi: 657"

10.1016/s0165-0327(00)00343-8. PMID: 11163418. 658"

31. Tronson NC, Corcoran KA, Jovasevic V, Radulovic J. Fear conditioning and 659"

extinction: emotional states encoded by distinct signaling pathways. Trends 660"

Neurosci. 2012 Mar;35(3):145-55. doi: 10.1016/j.tins.2011.10.003. Epub 2011 661"

Nov 25. PMID: 22118930; PMCID: PMC3290759. 662"

32. Barros HM, Tannhauser SL, Tannhauser MA, Tannhauser M. Effect of sodium 663"

valproate on the open-field behavior of rats. Braz J Med Biol Res. 664"

1992;25(3):281-7. PMID: 1341924. 665"

33. Jose PE, Kullu P, David J, Kulkarni C. Valproate- and aminophylline-induced 666"

'wet dog shakes'--a function of dose and time. Pol J Pharmacol. 1999 Jul-667"

Aug;51(4):357-61. PMID: 10540968. 668"

34. Morag M, Myslobodsky M. Benzodiazepine antagonists abolish 669"

electrophysiological effects of sodium valproate in the rat. Life Sci. 1982 May 670"

10;30(19):1671-7. doi: 10.1016/0024-3205(82)90500-8. PMID: 6285103. 671"

35. Töllner K, Wolf S, Löscher W, Gernert M. The anticonvulsant response to 672"

valproate in kindled rats is correlated with its effect on neuronal firing in the 673"

substantia nigra pars reticulata: a new mechanism of pharmacoresistance. J 674"

Neurosci. 2011 Nov 9;31(45):16423-34. doi: 10.1523/JNEUROSCI.2506-675"

11.2011. PMID: 22072692; PMCID: PMC6633222. 676"

36. De la Casa LG. Context and Taste Neophobia. In: Reilly S, editor. Food 677"

neophobia: Behavioral and biological influences. Cambridge: Woodhead 678"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

30"

Publishing; 2018. pp. 59-75."https://doi.org/10.1016/B978-0-08-101931-679"

3.00004-5 680"

37. Honey RC, Pye C, Lightbown Y, Rey V, Hall G. Contextual factors in 681"

neophobia and its habituation: the role of absolute and relative novelty. Q J Exp 682"

Psychol B. 1992 Nov;45(4):327-47. PMID: 1475403. 683"

38. De la Casa LG, Diaz E, Lubow RE. Effects of post-treatment retention interval 684"

and context on neophobia and conditioned taste aversion. Behav Processes. 2003 685"

Jul 31;63(3):159-170. doi: 10.1016/s0376-6357(03)00080-9. PMID: 12829316. 686"

39. Lin JY, Amodeo LR, Arthurs J, Reilly S. Taste neophobia and palatability: the 687"

pleasure of drinking. Physiol Behav. 2012 Jun 25;106(4):515-9. doi: 688"

10.1016/j.physbeh.2012.03.029. Epub 2012 Mar 29. PMID: 22484563; PMCID: 689"

PMC3348963. 690"

40. Berridge KC. Modulation of taste affect by hunger, caloric satiety, and sensory-691"

specific satiety in the rat. Appetite. 1991 Apr;16(2):103-20. doi: 10.1016/0195-692"

6663(91)90036-r. PMID: 2064389. 693"

41. Archer T, Sjödén, PO. Neophobia in taste-aversion conditioning: Individual 694"

differences and effects of contextual changes. Psychobiology""1979"Dec;7(4):&695"

364–369."https://doi.org/10.3758/BF03326657 696"

42. Vicente L, De la Casa LG. Context Properties Modulate Flavor Neophobia 697"

Habituation. Psicothema. 2021 Nov;33(4):617-622. doi: 698"

10.7334/psicothema2021.164. PMID: 34668477. 699"

43. Modlinska K, Stryjek R. Food Neophobia in Wild Rats (Rattus norvegicus) 700"

Inhabiting a Changeable Environment-A Field Study. PLoS One. 2016 Jun 701"

2;11(6):e0156741. doi: 10.1371/journal.pone.0156741. PMID: 27254150; 702"

PMCID: PMC4890768. 703"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

31"

44. Chapman A, Keane PE, Meldrum BS, Simiand J, Vernieres JC. Mechanism of 704"

anticonvulsant action of valproate. Prog Neurobiol. 1982;19(4):315-59. doi: 705"

10.1016/0301-0082(82)90010-7. PMID: 6134304. 706"

45. Hyden H, Cupello A, Palm A. Inhibition by sodium valproate of the transport of 707"

GABA through the Deiters' neurone plasma membrane. Neuropharmacology. 708"

1984 Mar;23(3):319-21. doi: 10.1016/0028-3908(84)90193-x. PMID: 6427649. 709"

46. Gardner CR, Piper DC. Effects of agents which enhance GABA-mediated 710"

neurotransmission on licking conflict in rats and exploration in mice. Eur J 711"

Pharmacol. 1982 Sep 10;83(1-2):25-33. doi: 10.1016/0014-2999(82)90282-5. 712"

PMID: 6290245. 713"

47. Myslobodsky M, Feldon J, Lerner T. Anticonflict action of sodium valproate. 714"

Interaction with convulsant benzodiazepine (Ro 5-3663) and imidazodiazepine 715"

(Ro 15-1788). Life Sci. 1983 Jul 25;33(4):317-21. doi: 10.1016/s0024-716"

3205(83)80003-4. PMID: 6135965. 717"

48. Cooper SJ, Estall LB. Behavioural pharmacology of food, water and salt intake 718"

in relation to drug actions at benzodiazepine receptors. Neurosci Biobehav Rev. 719"

1985 Spring;9(1):5-19. doi: 10.1016/0149-7634(85)90028-4. PMID: 2579357. 720"

49. Shephard RA, Hamilton MS. Chlordiazepoxide and valproate enhancement of 721"

saline drinking by nondeprived rats: effects of bicuculline, picrotoxin and Ro15-722"

1788. Pharmacol Biochem Behav. 1989 Jun;33(2):285-90. doi: 10.1016/0091-723"

3057(89)90501-7. PMID: 2510184. 724"

50. Vellucci SV, Webster RA. The role of GABA in the anticonflict action of 725"

sodium valproate and chlordiazepoxide. Pharmacol Biochem Behav. 1984 726"

Dec;21(6):845-51. doi: 10.1016/s0091-3057(84)80063-5. PMID: 6151667. 727"

.CC-BY 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.09.519825doi: bioRxiv preprint

32"

51. File SE, Aranko K. Sodium valproate and chlordiazepoxide in the elevated plus-728"

maze test of anxiety in the rat. Neuropsychobiology. 1988;20(2):82-6. doi: 729"

10.1159/000118478. PMID: 3151011. 730"

731"

Supporting information 732"

S1 Fig. Mean water consumption across the four days of context conditioning after 733"

30 min (Section A), and 60 min (Section B). 734"

S2 Fig. Mean citric acid + saccharine consumption across the three trials of the 735"

neophobia stage as a function of Groups. 736"

S3 Fig. Mean citric acid + saccharine consumption for the three neophobia trials as 737"

a function of Groups. 738"

S4 Fig. Mean percent activity for the 6-min periods collapsed across the three test 739"

sessions as a function of Groups. 740"

S5 Fig. Mean freezing to the Tone-Cs during conditioning (left side) and extinction 741"

(right side) sessions as a function of Groups. 742"

S1 File. Data for Experiment 1 743"

S2 File. Data for Experiment 2 744"

S3 File. Data for Experiment 3 745"