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Caffeine facilitates extinction of auditory fear conditioning in rats

Abstract Caffeine is the most widely consumed psychostimulant drug which could affect learning and memory acting through central adenosine receptors. Although caffeine has been suggested to impair the acquisition and the expression of auditory fear conditioning, its effect on the extinction has not been elucidated. To address this issue, in the present study, we investigated whether caffeine affects the extinction of conditioned fear in an auditory fear conditioning paradigm. During conditioning, rats received pairings of auditory cues (conditioned stimulus, CS) and foot shocks (unconditioned stimulus). On the next day, the rats were intraperitoneally administrated saline or caffeine (5 or 10 mg/kg) and then subjected to the extinction training, in which CSs were repeatedly presented without the foot shocks. Twenty‐four hours later, the rats were re‐exposed to the presentations of CSs (retrieval test). We found an acute caffeine administration dose‐dependently decreased freezing rates during the presentations of CS in the extinction training. Furthermore, caffeine‐treated animals showed lower conditioned freezing responses in the retrieval test. These findings suggest that caffeine facilitates the extinction of conditioned fear.

facilitative effects of caffeine on learning and memory have been considered to be mediated by the blockade of Gi/o-coupled adenosine A1 receptors that eventually promote neural plasticity such as long-term potentiation. [7][8][9][10] Associative fear learning and its extinction has been assessed in the auditory or contextual fear conditioning paradigm. During the conditioning, animals learn the association between preceding auditory conditioned stimulus (CS) or surrounding contextual information and subsequent aversive unconditioned stimulus (US) such as mild electric shocks. 11 On the other hand, animals learn CS-No US association during the extinction where only the CSs are repeatedly presented. Understanding the effects of drugs on these learning processes are important to find a potential molecular target for the pharmacological treatment of stress-related mental disorders. Previous studies have shown that caffeine treatments impair both the acquisition and the expression of auditory 12 and contextual fear conditioning. 13,14 However, its effect on the extinction of conditioned fear is yet to be investigated. Given that the extinction of conditioned fear is executed through the distinct neural circuit mechanism from that of acquisition, 15 caffeine could have a different effect on the fear extinction. Furthermore, investigating its effect on the extinction is valuable as a preclinical study aiming at drug therapy for the patient already suffering from stress-related mental disorders. Therefore, in the present study, we examined the effect of acute systemic treatment of caffeine on the fear extinction in the auditory fear conditioning paradigm in rats. The experiment consisted of three phases, fear conditioning, extinction training, and retrieval test ( Figure 1A, see below for details). The fear conditioning was performed in context A while the extinction training and the retrieval test were done in context B.

| ME THODS
In context A, animals were placed in a square transparent Plexiglas chamber with a grid floor (O'Hara & CO., LTD., Tokyo, Japan) which is in a sound-isolating box (Muromachi Kikai CO., LTD., Tokyo, Japan).
The interior was illuminated with 150 lx and background white noise The effect of systemic injection of caffeine on the extinction learning in the auditory fear conditioning. A, Schematic illustration of the experiment. B, Freezing rate during CS periods on each experimental day. BL, baseline period; CAFF-5, caffeine 5 mg/ kg treated group; CAFF-10, caffeine 10 mg/kg treated group; SAL, salinetreated group. Data are presented as the mean ± SEM. *** Indicates a significant difference between SAL and CAFF-10 (P < 0.001). * Indicates a significant difference between SAL/CAFF-5 and CAFF-10 (P < 0.05) (50 dB) was continuously given. In context B, a black-colored square chamber with a smoothed floor was used in the same sound-isolating box. The house light was turned off. The volume of background white noise was changed (60 dB), and the interior was fragranced by putting a small dish filled with peppermint soap (Magic soap, Dr. Bronner's, San Diego, CA, USA) outside of the black chamber.
In the experiment, the dose of 5 or 10 mg/kg was intraperitoneally injected. The dose of 10 mg/kg of caffeine was chosen based on a previous report showing that this dose of caffeine had no anxiolytic and anxiogenic effects in an open-field test and an elevated plus maze test. 16 It has been also reported that this dose of caffeine did not have either rewarding or aversive effects in a conditioned place preference test. 17 During the fear conditioning, animals were placed into the chamber and received pairings of auditory CS (20 seconds, 65 dB, 10 kHz) and electric shock US after a baseline period (120 seconds).
The foot shock US (2-seconds scrambled foot shocks, 0.5 mA) was co-terminated with the CS. The CS-US pairings were repeated four times with variable intertrial intervals (ITIs) on average at 120 seconds. A 120 seconds after the last paring, the rats were removed from the apparatus and returned to the home cage. Twenty-four hours later, the extinction training was performed (day 2). Fifteen minutes before the extinction training, 13 rats received an intraperitoneal injection of SAL (n = 13), 5 mg/kg (CAFF-5, n = 14) or 10 mg/ kg of caffeine (CAFF-10, n = 14). During the extinction training, after a 5-minutes baseline period, CS was repeatedly presented 16 times without the shock US delivery (120-seconds ITI on average).
The rats were returned to the home cage 120 seconds after the last CS presentation. Twenty-four hours later (day 3), we tested whether the drug-induced changes in the fear response observed in the extinction training remained without drug administration. During the retrieval test, after a 5-minutes baseline period, CS was presented twice without the shock US (150-seconds ITI). The rats were returned to the home cage 90 seconds after the last CS presentation.
Rats' behavior was recorded by a head-mount infrared camera, and the freezing behavior was analyzed using ANY-maze software (Stoelting CO., LTD, Wood Dale, IL, USA). Freezing was defined as the cessation of all bodily movements except for breathing.
In this study, the percentages of freezing response during CS were compared using either a one-way ANOVA or a two-way repeated measures ANOVA (drug × trial) followed by Tukey's multiple comparisons (P < 0.05). All statistical analyses were performed using

| RE SULTS
Freezing responses during the fear conditioning, the extinction training, and the retrieval test are shown in Figure 1B and Table S1.
In the conditioning on day 1, animals successively learned auditory fear conditioning. A one-way ANOVA revealed that there was no

| DISCUSS ION
In the present study, we investigated the effect of acute systemic treatment of caffeine on fear extinction in the auditory fear conditioning paradigm. We found that the caffeine treatment generally decreased conditioned fear response during the extinction training.
Importantly, the decreased conditioned response to caffeine was also observed in the next day's retrieval test. Our results suggest the facilitative effect of caffeine on the fear extinction.
The fact that caffeine decreased freezing responses within the extinction training suggests not only its facilitative effects on fear extinction 18 but also its disruptive effects on fear expression. Indeed, previous studies have shown that caffeine treatment impairs the expression of conditioned fear in a retrieval test. 12,13 On the other hand, caffeine has an effect to enhance general locomotor activity in rodents. 19 Given this psychostimulative effect, we cannot rule out the possibility that the reduced conditioned fear response during the extinction training on day 2 could be attributed to increased general activity caused by the caffeine treatment.
The most important result in the present study is that the caffeine treatment immediately before the extinction training reduced conditioned freezing even in the next day's retrieval test, strongly suggesting the facilitating effect of caffeine on the fear extinction. The possibility that the psychostimulative effect of caffeine could remain until the retrieval test could be excluded because the half-life of caffeine in the circulation is normally 3-5 hours, 20 and the facilitative effect of caffeine on the locomotor activity does not last more than 5 hours even at a higher dose