Abstract: Prolonged consumption of ethanol produces prefrontal cortex (PFC) dysfunction in patients, and this has been demonstrated using structural, physiological and psychological measurements. We therefore wanted to develop an animal model of PFC dysfunction to study whether this state changes sensitivity for ethanol or other behavioural/motivational measures. Adolescent Wistar rats were first screened in the novel object recognition task to establish a pre‐treatment baseline measure of locomotor activity, anxiety‐like behaviour and PFC function. Animals were divided into four treatment groups [saline, 5 mg/kg phencyclidine (PCP), 2.5g/kg ethanol, ethanol + PCP] and injected i.p. for 5 days followed by a 2‐day washout. On the 8th day, animals were allowed to explore a Y‐maze for 10 min. and spontaneous alternations were recorded using the ANY‐maze tracking system. PCP, a classic drug used to induce PFC dysfunction in animals, did not significantly reduce the % correct alternations relative to the 70% level achieved by the saline group. Ethanol and the combination of Ethanol + PCP, however, significantly reduced alternations to approximately 30%. The combined dose was not additive in terms of Y‐maze impairment, and these animals had less total distance travelled and greater time immobile relative to the other groups. We therefore concluded that injection of 2.5 g/kg ethanol for 5 days in Wistar rats produces a more substantial, consistent and valid PFC dysfunction than 5 mg/kg PCP.

It is widely accepted that individuals dependent on alcohol have prefrontal cortex (PFC) dysfunction [1, 2], and considerable structural, physiological and psychological evidence has led to this view. The main structural change after alcohol use is a reduction in frontal cortex volume [3, 4]. Physiological changes were first identified in the 1980s via positron emission tomography (PET) [1]. This decrease in cerebral glucose metabolism in the PFC of alcoholics has been confirmed more recently using [18F]fluorodeoxyglucose as the marker of energy use [1]. A reduction in prefrontal blood flow in alcoholics is consistent across many studies [1], and there is an inverse correlation between alcohol consumption and cerebral blood flow itself [5]. Psychological tests allow for a noninvasive assessment of cognitive abilities in a patient, and the story is the same here. While each psychological test may assess subtly different aspects of PFC function, alcoholics were impaired in the Wisconsin Card Sorting Test, the Porteus Maze test, the Halstead Category Test and the Luria‐Nebraska Neuropsychological Battery [1]. A review of the above evidence suggests increasing PFC damage the more one drinks. Therefore, several have suggested that abstinence from alcohol would reverse this damage [2]. A study from 1975 measured PFC function in alcoholics during the first 6 weeks of abstinence and some recovery was reported [6], but signs of brain atrophy remain long‐term [7]. A more recent hypothesis, however, suggests PFC dysfunction as an obstacle to treatment in the first place. Severe PFC dysfunction and personality characteristics such as neglect or impulsivity would make it difficult to convince the patient to comply with the treatment and take their anti‐alcohol medication. Higher dysfunction at the start of treatment correlates with a poor treatment outcome [8], and a second study found higher relapse rates, more marital problems and employment failure in patients with higher PFC dysfunction [9]. An animal model corresponding to these clinical observations would provide a novel approach in the search for pharmacotherapy to treat alcoholism, particularly at the early stages of sobriety. This model should include repeated exposure, relevant neuro‐adaptation and a measureable behavioural deficit. We became interested in phencyclidine (PCP), which has been used extensively in schizophrenia research. PCP induces a schizophrenia‐like state in patients [10, 11], and repeated injections in animals produce adaptations that are observed in schizophrenic patients [12]. A decrease in the GABAergic marker parvalbumin is consistent across the variety of PCP injection protocols [13-15]. The decrease in prefrontal glucose utilization observed by Cochran et al. [16] also validates the use of PCP. One can question the face validity of using PCP in alcohol research because this assumes the same underlying adaptations for the two drugs. In a previous study, we have found an interaction between PCP and alcohol acutely in terms of nucleus accumbens dopamine release [17] and the two drugs do substitute in discrimination studies [18]. However, we did not observe any signs of PFC dysfunction in terms of RNA expression when we administered 2.58 mg/kg PCP for 5 days [17]. We have recently adopted the 5 mg/kg dose, an age corresponding to adolescence [19] and behavioural measures to assess treatment outcome. Therefore, the goal of this study was to compare PFC dysfunction induced by PCP with a comparable protocol using ethanol injections. Drugs will also be combined to test whether a potentiated PFC dysfunction effect can be induced.

Materials and Methods A total of 44 naïve male Wistar rats weighing 190–210 g upon arrival were used for this study (age of 6 weeks corresponded to adolescence during injection). Animals were kept four per cage in a temperature‐ and humidity‐controlled animal care facility (lights on at 07:00 hr, 12‐hr light/dark cycle) and were allowed to acclimatize to the environment for 1 week before commencement of the study. All experiments throughout the project were approved by the Ethical Committee for Use of Animal Subjects. Animal care procedures followed the guidelines of Swedish legislation on animal experimentation (Animal Welfare Act SFS1998:56) and EU legislation (Convention ETS123 and Directive 86/609/EEC). Three individual groups of animals (n = 12, 16 and 16) were tested, and the treatment groups were balanced across the experiments to ensure replication. The number of animals in each treatment group was saline (n = 8), PCP (n = 12), ethanol (n = 16), ethanol + PCP (n = 8). We chose to include more animals in the ethanol group owing to concerns over the possibility of non‐responders or a highly individual treatment effect. Post‐analysis indicated, however, that we did not actually need 16 animals to obtain our consistent result. The design of the experiment is illustrated in fig. 1. Animals were first tested in the Novel Object Recognition (NOR) task after some brief handling. Performance in this task provided a quick status check on the naïve animal. The Habituation session in the empty box was a test of spontaneous activity, and Centre Time would indicate outliers with respect to anxiety‐like behaviour. Immobility during the Habituation session, perhaps because of neophobia, would also suggest outliers. This is important given the need for mobility to perform spontaneous alternations in the Y‐maze. Figure 1 Open in figure viewer PowerPoint Timeline of the experiment. Each dash on the horizontal line represents 1 day. The Novel Object Recognition task was spread over 2 days in the first week. After at least 2 days of nonhandling, animals were injected once per day (approximately at 13:00 hr) for five consecutive days. After an additional two drug‐ and handling‐free days, animals were tested in the Y‐maze in a drug‐free state. The NOR task was performed in a plastic box of 70 cm by 70 cm by 30 cm high covered in opaque paper (on the outside) to provide a good contrast in colour for behaviour recording. Movement was tracked using the ANY‐maze software and a webcam suspended overhead. Lighting (LED Stixx, Osram, Munich, Germany) was mounted above and outside of the box to ensure even illumination without shadows. Individual testing was performed over 2 days. The first exposure was a Habituation session of 5 min. where animals could explore an empty box. The day after, animals were placed in the box (centre, facing a corner with no objects) which contained two identical soda cans (Loka, silver with diagonal lines and dark writing). Five minutes later, the animal returned to its homecage and the box plus objects were cleaned before the next animal was tested. After 1 hr, the animal was returned for 5 min. to the box which now contained the familiar can (top left) and a novel soda can (Pripps Blå, dark blue with gold writing and a boat) in the bottom right corner. Positioning of the cans and designation of which should be novel was based on a pilot study. Testing was performed in the morning between 08:00 and 12:00 hr. A two‐way repeated‐measures anova was used to compare time in the object zone across the session. A more complete analysis of the population was performed via a histogram of the total times for each individual. The level of significance was p = 0.05 for all tests. After this pre‐test, animals were injected for 5 days (Monday–Friday; approximately at 13:00 hr) on a ‘by cage’ basis. We did not randomize treatments within‐group in case the drug led to a behavioural disadvantage in the homecage environment. Confounders due to drug‐induced changes in dominance roles would be difficult to differentiate from the effect we observed. After the 5 days of injections, animals were left unhandled for the following weekend (Saturday–Sunday) before Y‐maze testing on Monday. Phencyclidine hydrochloride (Sigma‐Aldrich, Stockholm, Sweden) and ethanol (Kemetyl AB, Haninge, Sweden) were dissolved in 0.9% NaCl and injected intraperitoneally (i.p.). For the combination dose, ethanol was followed 60 min. later by PCP injection. The Y‐maze consisted of three arms in grey, nonreflective plastic which fit into a groove in a metal, nonreflective base in the shape of a Y. Arms were 50 cm long, 10 cm wide with walls 20 cm high (Stoelting Europe, Dublin, Ireland). Movement was again tracked using the ANY‐maze software via a camera mounted on the light directly above the maze. No habituation sessions were provided although the same testing room was used. The animal was placed in an arm facing the centre (Arm A) and was allowed to freely explore for 10 min. A correct alternation occurred when the animal moved to the other two arms without retracing its steps (i.e. Arm A to B to C). Movements such as ABA were incorrect. Based on the movement over the entire session, the percentage of correct alternations was calculated. Dysfunction of the PFC was defined as a decrease in % correct alternations relative to saline‐treated controls. Occasionally, an animal would move to another arm and then remain immobile for an extended period of time before moving to the third arm. To prevent misguiding results, if an animal remained immobile for more than 2 min., that particular alternation was excluded. Performance was analysed using the nonparametric one‐way anova (Kruskal–Wallis) followed by Dunn’s multiple comparison test for post hoc investigation. A p < 0.05 was considered significant.

Results Novel Object Recognition results for the entire population of 44 animals are provided in fig. 2. We chose a population‐based analysis because this pre‐test was meant as a screen of performance before drug treatment. Animals with low pre‐test performance may lead to false positives in the test of PFC dysfunction. Animals spent significantly more time exploring the novel object compared to the familiar one (fig. 2A: Mann–Whitney test, p = 0.0022). Analysis of the behaviour over the 5‐min. session confirmed this pattern (fig. 2B: two‐way repeated‐measures anova, F = 8.559, p = 0.0044) with trends to post hoc differences during the first and last minute of the session. A histogram over time in the novel object zone (fig. 2C) indicated a wide variation in actual time spent exploring the objects by the 44 individuals. Of note was that six animals did not actually explore the novel object. After consultation of the videos, three of these were excluded from further analysis while the remaining three received treatment because no objective deviations in terms of activity or immobility could justify their exclusion. However, these were not included in the same treatment groups. Figure 2 Open in figure viewer PowerPoint Population analysis of n = 44 animals after the Novel Object Recognition test prior to any drug treatment. (A) Animals spent significantly more time exploring the novel object compared to the familiar. (B) Time spent exploring the object as a function of session time. Peaks of novel object exploration occurred at the beginning of the session and during the last minute. (C) Histogram over individual novel object exploration time. Note the wide range of time, suggesting performance as a variable trait. **p < 0.01. Spontaneous alternation behaviour in the Y‐maze was affected by drug treatment (fig. 3). Treatment decreased % correct alternations (fig. 3A: Kruskal–Wallis, p = 0.0021) with both 2.5 g/kg ethanol and the combination 2.5 g/kg ethanol + 5 mg/kg PCP significantly less than saline‐treated animals (Dunn’s multiple comparison test: p < 0.001). Therefore, 2.5 g/kg ethanol but not 5 mg/kg PCP alone induced PFC dysfunction. The total number of performed alternations for each group was as follows (median values): saline (26.5), PCP (27.0), ethanol (15.5), ethanol + PCP (8.0). Figure 3 Open in figure viewer PowerPoint Spontaneous alternation behaviour after drug treatment. (A) 2.5 g/kg ethanol and the combination of ethanol + phencyclidine (PCP) significantly decreased percentage of correct alternations while 5 mg/kg PCP was without effect. (B) Ethanol also decreased total distance moved during the 10‐min. session. However, the combination treatment reduced total distance and increased time immobile. (C) Analysis of the time spent in each arm did not identify any group differences with respect to ethanol or PCP. However, the combined treatment did decrease time spent in the Mid Zone and time in Zone A relative to the other groups. *p < 0.05, **p < 0.01, ***p < 0.001 (ethanol relative to Saline). ##p < 0.01 (EtOH + PCP relative to ethanol). The total distance (fig. 3B) moved in the maze was also significantly affected by drug treatment (Kruskal–Wallis, p = 0.0009). Animals treated with 5 mg/kg PCP moved the same amount as controls (p > 0.05), while those treated with 2.5 g/kg ethanol (p < 0.05) or the combination 2.5 g/kg ethanol + 5 mg/kg PCP (p < 0.01) moved significant less. Immobility time (fig. 3C) was also affected by drug treatment (Kruskal–Wallis, p = 0.0028). In this case, only the combined dose of 2.5 g/kg ethanol + 5 mg/kg PCP increased the time spent immobile (p < 0.05). Both of these results led us to conclude that the combined dose of ethanol and PCP may cause a lasting sedative effect which is difficult to interpret. A further analysis of the behaviour in the Y‐maze is provided in fig. 3D. Time spent in Zone A differed across treatment (Kruskal–Wallis: p = 0.0029) and Dunn’s multiple comparison test found a significant difference between 2.5 g/kg ethanol and the combination dose (p < 0.01). However, the ethanol group did not significantly differ from the saline group (p > 0.05). Time spent in Zone B or C did not differ by treatment (Kruskal–Wallis: p = 0.2347 and 0.5602, respectively) while Time spent in Mid Zone did differ (Kruskal–Wallis: p = 0.0072). Again, the combined dose was significantly less than saline‐treated controls (p < 0.05). This analysis again clearly demonstrates a more dramatic effect on behaviour when ethanol and PCP are administered together while treatment with 5 mg/kg PCP alone or 2.5 g/kg ethanol alone do not affect the time spent exploring the maze.

Discussion This study has identified a more reliable method to induce PFC dysfunction in the Wistar rat. Surprisingly, 5 mg/kg PCP did not induce any significant deficits in PFC performance in this study, while a very clear effect was obtained with 2.5 g/kg ethanol and the same injection schedule (5 days of injections and behavioural testing after another 3 days). Ethanol reduced performance to roughly half that of saline‐treated controls and thus is well‐suited to pharmacology studies which attempt to reverse PFC dysfunction. Most importantly, the 2.5 g/kg ethanol method is most valid to study the ethanol‐induced PFC dysfunction observed in patients clinically. The presence of such a robust ethanol effect on spontaneous alternation behaviour is difficult to interpret. Lesions of the hippocampus decrease Y‐maze performance [20-22], but the same is true for lesions of the medial PFC [23, 24] and the more specific prelimbic cortex lesions [25]. This raises the important question of where ethanol is actually having its effect. We chose to use the 2.5 g/kg dose of ethanol because we have characterized this in our microdialysis studies [26]. This is higher than the 1 g/kg typically self‐administered by Wistar rats [27] and is therefore more typical of a ‘heavy‐drinking session’ in human beings but is still well below the ‘binge’ protocols used by several groups [28, 29]. Many studies report hippocampal neuronal death after binge exposure [28, 30] or chronic self‐administration to alcohol [31] while some cite the inhibition of neurogenesis as a key mechanism of action [29, 32]. Therefore, the effect in this study may be a result of deficits in the hippocampus. However, the study by Evrard et al. [30] also noted considerable ethanol‐induced damage in the striatum and PFC. Additionally, spontaneous alternation behaviour in the Y‐maze still involves the PFC [24]. The ethanol‐treated animals often returned to the same arm again (fig. 3A), and this type of perseverance in response is typical in animals with lesions of the medial PFC [33]. The lack of PCP effect on Y‐maze performance can be explained in several ways. Firstly, few studies actually report a spontaneous alternation behaviour deficit after PCP treatment, suggesting this is an inappropriate method. However, four studies do report PCP‐induced deficits in Y‐maze performance in animals [14, 34-36]. A second reason could relate to the variety of dose and treatment protocols for PCP. Previously, we used 2.58 mg/kg for 5 days [16] but did not observe any dysfunction in Wistar rats when measuring Arc or parvalbumin in the medial PFC [17]. By applying drug treatment under adolescence and raising the dose to 5 mg/kg, we could induce deficits in Y‐maze performance (data included in another paper under review). However, the effect was never robust enough to be observed in every experiment without exclusion of animals. A third possibility is some type of behavioural habituation that resulted from the NOR pre‐test. PCP is known to decrease object recognition performance [37, 38] so one would expect that using this task as the post‐test would have demonstrated the PCP effect. We chose not to use NOR as our outcome measure because of the possible need for subjective analysis. The Y‐maze deficit also allows both an increase and a decrease in pharmacological reversal studies, while NOR may have a ceiling effect. Nevertheless, handling of the animals three times during NOR and the general locomotor habituation that this produces could have decreased individual drive to explore the maze. One major line of evidence that refutes a carry‐over effect from NOR concerns the brain areas involved. Lesions of the perirhinal cortex reduce object recognition performance far more than lesions of the hippocampus [39, 40]. Additionally, the spontaneous alternation behaviour task is known to involve a combination of PFC and hippocampus function [24, 41]. An important question for further study is whether PCP and ethanol both have the same mechanism with respect to the deficits they induce in spontaneous alternation behaviour. We chose to inject the combination dose (ethanol then PCP 1hr after) because of the acute potentiation of dopamine release in the nucleus accumbens [17] with hypothesis that this potentiation would manifest in even greater PFC impairment. However, the results of this study do not support that view. The actual percentage of correct alternations was the same for ethanol and the combination group, yet in the remaining measures, the PCP and ethanol effect was more additive than potentiated (figs 2 and 3). Overall, injection of 2.5 g/kg ethanol produced a significant deficit in spontaneous alternation behaviour in the Y‐maze. We would like to conclude that this effect is a result of PFC dysfunction, but to do so, we must differentiate the role of the hippocampus and PFC in this behaviour.

Acknowledgements The studies were supported by the Swedish Research Council – Medicine (2009‐4477 and 2010‐3100), governmental support under the LUA/ALF agreement, Fredrik and Ingrid Thurings Stiftelse, Per‐Erik Lindahl’s stipend fund (Medicine), the Alcohol Research Council of the Swedish Alcohol Retailing Monopoly, Wilhelm and Martina Lundgrens Vetenskapsfond, and Gunnar and Märta Bergendahls Stiftelse.

Conflict of Interest The authors declare that they have no competing or conflicting interests that could influence the experiments or this article.