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Disruption of latent inhibition by subchronic phencyclidine pretreatment in rats
Accepted Manuscript Title: Disruption of latent inhibition by subchronic phencyclidine pretreatment in rats Authors: Asmaa M. Al-Ali, Andrew M.J. Young PII: DOI: Article Number:
S0166-4328(19)30338-9 https://doi.org/10.1016/j.bbr.2019.111901 111901
Reference:
BBR 111901
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
4 March 2019 3 April 2019 9 April 2019
Please cite this article as: Al-Ali AM, Young AMJ, Disruption of latent inhibition by subchronic phencyclidine pretreatment in rats, Behavioural Brain Research (2019), https://doi.org/10.1016/j.bbr.2019.111901 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Behavioural Brain Research (short communication)
Disruption of latent inhibition by subchronic phencyclidine pretreatment in rats.
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Asmaa M. Al-Ali1, Andrew M.J. Young
Department of Neuroscience, Psychology and Behaviour, University of Leicester,
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Corresponding author Dr Andrew M.J. Young
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Department of Neuroscience, Psychology and Behaviour
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Lancaster Road, Leicester, LE1 9HN, UK
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University of Leicester
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Lancaster Road,
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Leicester, LE1 9HN
01162297111
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UK
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[email protected]
Current address : Department of Pharmacology and Toxicology, College of Pharmacy,
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University of Basrah, Basrah, Iraq
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Highlights
Subchronic phencyclidine, followed by withdrawal, models schizophrenia in rats Behaviours related to negative and cognitive symptoms are known to be disrupted Latent inhibition, related to positive symptoms, is now also shown to be disrupted Resulting behaviour changes resemble all three symptom domains of schizophrenia
Abstract
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Repeated subchronic treatment with the NMDA-receptor antagonist, phencyclidine, causes
behavioural changes in rats, which resemble cognitive and negative symptoms of schizophrenia.
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However, its effects on behaviours modelling positive symptoms are less clear. This study
investigated whether subchronic phencyclidine pretreatment affected latent inhibition: impaired
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conditioning following repeated preexposure of the to-be-conditioned stimulus.
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Female Lister-hooded rats were pretreated with phencyclidine or saline twice/day for 5 days,
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then remained drug-free for 10 days before latent inhibition testing. Saline pretreated animals
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showed latent inhibition, as expected. However, phencyclidine pretreated animals showed no latent inhibition: the effect of preexposure was attenuated, with no change in basic learning. Thus
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subchronic phencyclidine pretreatment does disrupt latent inhibition, and, importantly, this occurs after withdrawal from the drug, implicating changes in brain function enduring well
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beyond the time that the drug is present in the brain.
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In a separate task, discrimination of a novel object was significantly impaired by phencyclidine pretreatment confirming that five days of subchronic pretreatment was sufficient to invoke
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behavioural impairment previously reported after seven days pretreatment.
Keywords Latent inhibition, Schizophrenia, Phencyclidine
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Abbreviations Conditioned emotional response
DI
Discrimination index
LI
Latent inhibition
NMDA
N-methyl-D-aspartate
NOR
Novel object recognition
NPE
Non preexposed
PCP
Phencyclidine
PE
Preexposed
SI
Suppression index
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CER
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In people, chronic exposure to non-competitive NMDA-type glutamate receptor antagonists (phencyclidine (PCP), ketamine and MK801), causes changes in all three behavioural domains resembling schizophrenia (positive, negative, cognitive), and exacerbates psychosis in schizophrenics [1,2], suggesting a central role for glutamatergic abnormalities in schizophrenia [3]. Given acutely in rats these drugs cause transient behavioural changes, but given chronically
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they bring about long-lasting behavioural deficits, which resemble schizophrenia [2,4,5]. Importantly, after withdrawal from short term chronic (termed sub-chronic) treatment, changes
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endure several months after the end of drug administration [5], indicating long-term
physiological changes, resembling those occurring in schizophrenia [6,7]. In rats, they evoke
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abnormalities in dopaminergic, glutamatergic and GABAergic systems related to schizophrenia
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and behavioural and information processing deficits resembling cognitive and negative
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resembling positive symptoms (review [5]).
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symptoms of schizophrenia (reviews [1,2,5,8]). However, few studies have looked at behaviours
Latent inhibition (LI) is the reduction of associability of a stimulus when it is pre-exposed
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without reinforcement [9] and the effect of preexposure is reduced (disrupted LI) in schizophrenia sufferers [10]. Importantly, several lines of evidence suggest that LI relies on
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attentional process which are primarily associated with positive (as opposed to negative or
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cognitive) symptomatology. First, disruptions of LI are seen only in patients suffering from positive symptoms; second, lesion and local injection studies show that LI is critically dependent on subcortical structures, rather than cortical areas important for negative and cognitive
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symptoms; and third, LI is susceptible to typical antipsychotic drugs (D2 antagonists), which are clinically more effective at treating positive symptoms (see reviews [10,11,12]). Thus LI shows face validity for assessing mechanisms of positive symptomatology [10,11,12]. However, there is debate about the effect of PCP, on LI. In conditioned emotional response (CER) tasks, Weiner and Feldon [13] showed no effect on LI of acute administration of PCP given at preexposure or 4
conditioning, while Traverso and co-workers [14] showed that MK-801 given before preexposure, but not before conditioning, disrupted LI. Similarly, in conditioned taste aversion, acute MK-801 disrupted LI when given at preexposure, but not at conditioning [15], but may potentiate LI when given at conditioning [16]. Importantly, in these experiments, the drug was administered acutely and was present in the brain during at least one stage of testing. While this
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gives insights into effects on ongoing behaviour, relating to transient behavioural changes seen in people taking a drug once, it is unlikely to have much relevance to understanding mechanisms
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of schizophrenia. Conversely, people taking PCP repeatedly exhibit a symptom spectrum and
time course more resembling schizophrenia itself. In rats, subchronic but not acute PCP induces
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sensitisation of mesolimbic dopamine function [17], similar to that reported in schizophrenia
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sufferers [18]. Therefore subchronic drug treatment, followed by withdrawal, where animals are
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drug-free during testing, are more plausible animal models for the disease [2,5]: changes result
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from long-term drug-induced adaptations in brain physiology rather than short term psychopharmacological effects. Although effects of chronic ketamine on LI of conditioned
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avoidance have been reported [19], other studies have shown no effect of chronic treatment of PCP on LI [20]. The present study therefore tested whether LI, would be disrupted after
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withdrawal from subchronic PCP, using an established CER paradigm [21]. Animals were also tested in novel object recognition (NOR) to confirm that the pretreatment was sufficient to
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replicate well-documented deficits in NOR [5,22]. Thirty-two female Lister-hooded rats (225-250g: Charles River, UK), were housed four per cage
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(46 (w) x 40 (d) x 40 (h) cm; double deck, independently ventilated cage: Techniplast, UK), with corn cob and sizzle nest bedding (Datesand, UK), under standard laboratory conditions of temperature (21 ± 2°C), humidity (55 ± 10 %) and light/dark cycle (12 hours light/12 hours dark; lights on 07:00), with food (LabDiet 5LF5, IPS Ltd, UK) and water available ad libitum. Experiments were carried out in the Preclinical Research Facility (designated SPF) and were 5
approved by the University of Leicester Ethical Committee and were covered by personal and project (60/4390) license authority. Once acclimatised to housing conditions (7 days), animals were randomly assigned to drug or vehicle pretreatment groups, and injected (1 ml/kg, i.p.) with PCP (2 mg/kg: n = 16) or vehicle
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(0.9% saline: n = 16) twice daily for five days: drug dose and dosing schedule were based on regimes previously seen to modify behaviour (reviewed in [5,8]) and neurochemical function
[23,24] in our labs and elsewhere. Pretreatment was followed by 1 week without intervention,
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to ensure that PCP was absent during testing [22]: although the plasma half-life of PCP is around 1 hour, due to its lipophilicity, it is believed to be up to 3 days in brain tissue [25]. Animals were
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housed four per cage in mixed groups (two saline-pretreated and two PCP-pretreated animals per
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cage).
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Testing used four identical behavioral chambers housed in sound-attenuating boxes and
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illuminated by a single house light (all Med Associates, USA unless otherwise stated). The
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animals’ licks were counted electronically using a contact lickometer. Mild footshock (see below) was delivered scrambled to the grid floor (0.4 cm diameter bars at 1.5 cm intervals) from
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a shock generator and tone stimuli were delivered through an overhead speaker using a custom built audio-generator. Each animal was tested in the same behavioural chamber throughout the
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experiment, and experimental condition in each chamber was counterbalanced across test groups. All stimulus presentation and lick recording was under computer control (MedPC software).
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Conditioning and LI were measured by conditioned suppression of licking for water in thirsty animals, as previously described [21]. Briefly, after drug washout, animals were handled daily for 5 days and introduced to restricted water access: they received water ad libitum in the home cage for four hours each day (midday to 16:00), for five days per week and free access to water for the two remaining days. This regime was the minimum required to ensure that animals were
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motivated to lick during the testing sessions, between 09:00 and midday. Prior to testing, animals were trained to lick for water in the test chamber, immediately following the period of water restriction. This was repeated daily for 5 days, after which all the animals licked freely in the test chamber. After each lick training session, animals were returned to their home cage for their
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period of ad libitum water availability. Following lick training, animals were randomly allocated to non-preexposed (NPE) and preexposed (PE) groups (n = 8 each for each pretreatment).
For Preexposure animals were
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placed in their respective boxes, with no water available. Animals in the PE groups received 40
presentations of tone (5 sec; 15 dB above background) at 1 min intervals, while NPE animals
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were placed in the box for 40 min without tone presentations. After this, animals were returned
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to their home cage for their period of ad libitum water availability. Next day (Conditioning), all
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animals were placed in their respective boxes without water available. After 5 min they received
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two pairings of tone (as above), followed immediately by mild footshock (1 s train of 6 ms pulses, 25 Hz; 0.3 mA; scrambled to the grid floor), at 5 min intervals. After a further 5 min, they were
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returned to their home cage, for their period of ad libitum water availability. Next day (Testing), animals were placed in their respective boxes with free access to water. Licks were monitored
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continuously and counted electronically. The tone was presented after lick 90 and continued until
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a further 10 licks were completed. The time taken to complete the 10 licks immediately before tone presentation (licks 80 to 90: Time A) and 10 licks during tone presentation (licks 90 to 100: Time B) were measured electronically. Animals were then returned to their home cage for their
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period of ad libitum water availability. Three days later the testing procedure was repeated for each animal over four consecutive days (test days 2 to 5) and Time A and Time B were recorded as above. After each test session, animals were returned to their home cage for their period of ad libitum water availability. On completion of testing they were returned to full water availability in their home cage. 7
For each test session, the suppression index (SI) was, calculated as (Time B - Time A) / (Time A + Time B). SI of 1.0 shows high suppression of licking (good learning), while SI of 0.0 shows no suppression of licking (poor learning). SIs were submitted to 3-way ANOVAs with drug and exposure as between-subject variables,
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and test days as the within-subject variable. Significant interactions were further interrogated with a priori planned comparison using Bonferroni corrected independent samples t-tests.
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Following LI, animals underwent NOR testing as previously described [22,23]. Briefly, four days after extinction testing, animals were habituated to the arena (black plexiglass; 60 cm x 60 cm x 60 cm) on two consecutive days for 30 min. Next day, animals underwent NOR testing.
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Each animal was first placed in the empty arena for 3 min, then removed and placed in a holding
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cage for 1 min, during which time two identical objects (metal cylinders, 12 cm x 8 cm diameter)
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were placed in opposite corners of the arena, 10 cm from the walls. The animal was returned to
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the arena at one of the unoccupied corners and allowed to explore for 3 min, before being
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removed and placed in the holding cage for 1 min. The objects were removed and replaced by one similar (familiar) object and one novel object (glass cylinder; 9 cm x 10 cm diameter), placed
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in the same corners as in acquisition, with the corner occupied by the novel object counterbalanced across animals. The animal was returned to the arena, and allowed to explore
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for 3 min. The session was video recorded, using a webcam (Logitech 510HD), linked to a desktop PC for subsequent scoring.
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Time taken to explore each object during acquisition and test sessions was measured by two independent scorers, blinded to drug conditions. Exploration of an object was defined as rats sniffing, licking or touching the objects with forepaws whilst sniffing at the object [22]. NOR was assessed by comparing the time spent exploring the novel object (TNOVEL) and the familiar
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object (TFAMILIAR) during the test period. Additionally a discrimination index (DI) [22] was calculated as: (TNOVEL - TFAMILIAR) / (TNOVEL + TFAMILIAR) Exploration times were analysed by two-way ANOVA, with significant interactions further interrogated using a priori planned comparisons (differences between TNOVEL and TFAMILIAR)
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using paired samples t-test. The difference in DI between drug conditions was assessed by t-test and the difference in DI from zero (no discrimination) was assessed by one-sample t-test. All
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statistical analysis used SPSS v24 (IBM).
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In LI testing, there was no difference in baseline licking rates (Time A) on test day 1 across the
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four groups. Two-way ANOVA showed no significant main effects of either pretreatment
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(F[1,28] = 1.922; p = 0.177) or preexposure (F[1,28] = 1.63; p = 0.212), and no significant
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Insert table 1 around here
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interaction (F[1,28] = 0.358; p = 0.541: table 1).
On test day 1, NPE animals in both pretreatment conditions exhibited high SI (saline: 0.779 ±
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0.118; PCP, 0.936 ± 0.11), indicating good learning of the tone-shock pairing. In PE animals, the saline-pretreated group showed low SI (0.200 ± 0.178), indicating poor learning to the
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preexposed stimulus, reflecting LI. This effect was substantially reduced in PCP-pretreated animals (SI = 0.677 ± 0.126), showing a disruption of LI by PCP (Figure 1). Extinction of the
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conditioned learning, shown by decreased SI over subsequent test days, occurred in the three groups with high SI (saline NPE; PCP NPE; PCP PE), although mostly they still showed some degree of suppression (Figure 1). Three-way ANOVA revealed a significant main effect of testing day (F[4,112] = 3.308, p = 0.013), and of exposure (F[1,28] = 54.031, p < 0.001) but not of drug treatment (F[1,28] =3.707, p = 0.064). The only significant interaction was the two-way
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drug x exposure interaction (F[1,28] = 6.674, p = 0.015). This interaction was further interrogated using Bonferroni corrected independent t tests of a priori planned comparisons of LI (NPE vs PE) shown in each of the two drug treatment groups for each test day, confirming that salinepretreated animals exhibited LI across all five test days, while PCP-pretreated animals showed
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no significant LI. Insert figure 1 around here
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In NOR testing, during acquisition there was no significant main effect of side (F[1,58] = 0.314; p = 0.577), or of pretreatment (F[1,58] = 2.368; p = 0.129), and no significant interaction between the two (F[1,58] = 0.825; p = 0.368: Figure 2a). At test, there was a significant main effect of
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novelty (F[1,58] = 9.262; p = 0.004), and a significant interaction (F[1,58] = 4.146; p = 0.046),
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but no main effect of pretreatment (F[1,58] = 1.877; p = 0.176). A priori planned comparisons
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of exploration times on familiar and novel objects showed a significant NOR effect in saline-
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pretreated animals but not in PCP-pretreated animals (paired t-test: Figure 2b). There was a
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significant difference in DI between saline-pretreated and PCP-pretreated animals (t[29] = 3.662; p = 0.001; independent samples t-test) and saline-pretreated animals, but not PCP-pretreated
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animals, showed significant discrimination between the objects, since the DI was significantly above zero (saline, t[14] = 6.287; p < 0.001: PCP, t[15] = 0.477; p = 0.640: one-sample t-test:
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Figure 2c).
Insert figure 2 around here
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The results demonstrated that sub-chronic PCP pretreatment disrupted LI. Across repeated extinction testing, the overall suppression in the groups exhibiting conditioning diminished, as would be expected, but LI remained intact in saline-pretreated animals across all 5 days: no LI effect was seen on any day in PCP-pretreated animals. NOR experiments confirmed that the twice daily treatment with PCP for five days was sufficient to produce a robust disruption of 10
NOR in adult rats, previously shown in juvenile rats [23,24], and is similar to that reported in adults treated for seven days [22]. Subchronic PCP pretreatment is well established at producing behavioural deficits mimicking cognitive and negative symptoms of schizophrenia [5], but effects on LI are less certain. The present results show that subchronic PCP disrupts LI, in a CER paradigm, indicating that it does
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indeed affect behaviours resembling deficits in all three behavioural domains. This is in
agreement with results showing ketamine disruption of LI in a conditioned avoidance task [19],
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but at odds with PCP having no effect on LI in both a conditioned avoidance and a taste aversion
task [20]. Notably, the latter study used male rats, whereas the current study used female rats.
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It is known that a sex difference exists in subchronic doses of PCP required to produce enduring
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behavioural deficits. In males a dose of 5 mg/kg is required to produce similar long-term effects
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as a dose of 2 mg/kg in females. Therefore it is possible that the dose used by Tenn and co-
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workers (3 mg/kg in males [20]), was not sufficient to produce long-term deficits in LI. In extinction trials, carried out over four consecutive days, three days after the first test session,
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animals received the tone without footshock, thus extinguishing the tone-footshock association, leading to a reduction in the CER [21], measured as a decrease in SI. At test on Day 1, both
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saline and PCP pretreated NPE groups showed near maximum suppression (SI close to 1.0). Due to this ceiling effect it was impossible to ascertain whether PCP pretreatment affected basic
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learning in NPE animals. However, since stronger conditioned associations extinguish more slowly than weaker ones, we could differentiate the degree of conditioning in these groups by
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examining extinction rates. Although suppression decreased in both groups over four extinction days, there was no significant difference in SI for NPE animals between the two pretreatment groups across extinction days, indicating that PCP pretreatment had not affected basic learning. In summary, subchronic PCP pretreatment disrupted LI, without affecting basic learning, indicating that this pretreatment invokes behavioural abnormalities resembling positive
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symptoms of schizophrenia, as well as cognitive and negative symptoms previously described. In addition, twice daily subchronic PCP pretreatment for five days is sufficient to disrupt behaviour, replicating previous findings using seven days pretreatment.
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Acknowledgements
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AMA-A received a Government of Iraq research studentship.
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5. Neill JC, Barnes S, Cook S, Grayson B, Idris NF, McLean SL, et al. Animal models of cognitive dysfunction and negative symptoms of schizophrenia: Focus on NMDA receptor antagonism. Pharmacology & Therapeutics. 2010;128:419-32. 6. Cochran SM, Kennedy M, McKerchar CE, Steward LJ, Pratt JA, Morris BJ. Induction of metabolic hypofunction and neurochemical deficits after chronic intermittent exposure to phencyclidine: differential modulation by antipsychotic drugs. Neuropsychopharmacology. 2003;28:265-75.
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7. Morris BJ, Cochran SM, Pratt JA. PCP: from pharmacology to modelling schizophrenia. Curr Opin Pharmacol. 2005;5:101-6. 8. Cadinu D, Grayson B, Podda G, Harte MK, Doostdar N, Neill JC. NMDA receptor antagonist rodent models for cognition in schizophrenia and identification of novel drug treatments, an update. Neuropharmacology. 9. Lubow RE, Moore AU. Latent inhibition: The effect of nonreinforced pre-exposure to the conditional stimulus. Journal of Comparative and Physiological Psychology. 1959;52:415-9. 12
10. Gray JA, Joseph MH, Hemsley DR, Young AMJ, Warburton EC, Boulenguez P, et al. The role of mesolimbic dopaminergic and retrohippocampal afferents to the nucleus accumbens in latent inhibition: Implications for schizophrenia. Behavioural Brain Research. 1995;71:19-31. 11. Moser PC, Hitchcock JM, Lister S, Moran PM. The pharmacology of latent inhibition as an animal model of schizophrenia. Brain Research Reviews. 2000;33:275-307. 12. Weiner I. Neural substrates of latent inhibition: the switching model. Psychol Bull. 1990;108:442-61.
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13. Weiner I, Feldon J. Phencyclidine does not disrupt latent inhibition in rats: Implications for animal models of schizophrenia. Pharmacology Biochemistry and Behavior. 1992;42:625-31.
14. Traverso LM, Ruiz G, De la Casa LG. Effect of the NMDA antagonist MK-801 on latent inhibition of fear conditioning. Pharmacology Biochemistry and Behavior. 2012;102:488-94.
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15. Traverso LM, Ruiz G, De la Casa LG. Latent inhibition disruption by MK-801 in a conditioned taste-aversion paradigm. Neurobiology of Learning and Memory. 2003;80:140-6.
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16. Pålsson E, Klamer D, Wass C, Archer T, Engel JA, Svensson L. The effects of phencyclidine on latent inhibition in taste aversion conditioning: differential effects of preexposure and conditioning. Behavioural Brain Research. 2005;157:139-46.
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Figure legends Figure 1: Disruption of LI by subchronic PCP.
Data are mean ± SEM
suppression index (SI) over five testing days; n = 8 per group. *** p < .001; **
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significant LI); Bonferroni corrected independent samples t-test.
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p < .01; * p < .05; significant differences between NPE and PE groups (i.e
Figure 2 : Disruption of NOR by subchronic PCP. (a) Time spent exploring two
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identical objects during acquisition; (b) Time spent exploring the familiar and novel
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objects during test; (c) DI during test. Data are mean ± SEM, n = 15 to 16 per
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group. *** p < 0.001; significant difference in exploration time between familiar
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and novel objects (independent samples t-test); † † p < 0.001; significant difference in DI between saline and PCP pretreated animals (independent samples t-test);
###
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p < 0.01, significant difference from zero (no discrimination: one-sample t-test).
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Fig 1
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Fig 2
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Saline
PCP
NPE
1.96 ± 0.26
2.22 ± 0.57
PE
1.31 ± 0.04
2.00 ± 0.26
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Table 1
Table 1 : Baseline licking rates on Day 1. Mean (± SEM) time to complete 10 licks
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immediately before the onset of the tone (Time A: n = 8 per group).
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S0166-4328(19)30338-9 https://doi.org/10.1016/j.bbr.2019.111901 111901
Reference:
BBR 111901
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
4 March 2019 3 April 2019 9 April 2019
Please cite this article as: Al-Ali AM, Young AMJ, Disruption of latent inhibition by subchronic phencyclidine pretreatment in rats, Behavioural Brain Research (2019), https://doi.org/10.1016/j.bbr.2019.111901 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Behavioural Brain Research (short communication)
Disruption of latent inhibition by subchronic phencyclidine pretreatment in rats.
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Asmaa M. Al-Ali1, Andrew M.J. Young
Department of Neuroscience, Psychology and Behaviour, University of Leicester,
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Corresponding author Dr Andrew M.J. Young
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Department of Neuroscience, Psychology and Behaviour
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Lancaster Road, Leicester, LE1 9HN, UK
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University of Leicester
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Lancaster Road,
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Leicester, LE1 9HN
01162297111
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UK
1
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[email protected]
Current address : Department of Pharmacology and Toxicology, College of Pharmacy,
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University of Basrah, Basrah, Iraq
1
Highlights
Subchronic phencyclidine, followed by withdrawal, models schizophrenia in rats Behaviours related to negative and cognitive symptoms are known to be disrupted Latent inhibition, related to positive symptoms, is now also shown to be disrupted Resulting behaviour changes resemble all three symptom domains of schizophrenia
Abstract
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Repeated subchronic treatment with the NMDA-receptor antagonist, phencyclidine, causes
behavioural changes in rats, which resemble cognitive and negative symptoms of schizophrenia.
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However, its effects on behaviours modelling positive symptoms are less clear. This study
investigated whether subchronic phencyclidine pretreatment affected latent inhibition: impaired
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conditioning following repeated preexposure of the to-be-conditioned stimulus.
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Female Lister-hooded rats were pretreated with phencyclidine or saline twice/day for 5 days,
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then remained drug-free for 10 days before latent inhibition testing. Saline pretreated animals
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showed latent inhibition, as expected. However, phencyclidine pretreated animals showed no latent inhibition: the effect of preexposure was attenuated, with no change in basic learning. Thus
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subchronic phencyclidine pretreatment does disrupt latent inhibition, and, importantly, this occurs after withdrawal from the drug, implicating changes in brain function enduring well
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beyond the time that the drug is present in the brain.
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In a separate task, discrimination of a novel object was significantly impaired by phencyclidine pretreatment confirming that five days of subchronic pretreatment was sufficient to invoke
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behavioural impairment previously reported after seven days pretreatment.
Keywords Latent inhibition, Schizophrenia, Phencyclidine
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Abbreviations Conditioned emotional response
DI
Discrimination index
LI
Latent inhibition
NMDA
N-methyl-D-aspartate
NOR
Novel object recognition
NPE
Non preexposed
PCP
Phencyclidine
PE
Preexposed
SI
Suppression index
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CER
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In people, chronic exposure to non-competitive NMDA-type glutamate receptor antagonists (phencyclidine (PCP), ketamine and MK801), causes changes in all three behavioural domains resembling schizophrenia (positive, negative, cognitive), and exacerbates psychosis in schizophrenics [1,2], suggesting a central role for glutamatergic abnormalities in schizophrenia [3]. Given acutely in rats these drugs cause transient behavioural changes, but given chronically
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they bring about long-lasting behavioural deficits, which resemble schizophrenia [2,4,5]. Importantly, after withdrawal from short term chronic (termed sub-chronic) treatment, changes
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endure several months after the end of drug administration [5], indicating long-term
physiological changes, resembling those occurring in schizophrenia [6,7]. In rats, they evoke
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abnormalities in dopaminergic, glutamatergic and GABAergic systems related to schizophrenia
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and behavioural and information processing deficits resembling cognitive and negative
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resembling positive symptoms (review [5]).
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symptoms of schizophrenia (reviews [1,2,5,8]). However, few studies have looked at behaviours
Latent inhibition (LI) is the reduction of associability of a stimulus when it is pre-exposed
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without reinforcement [9] and the effect of preexposure is reduced (disrupted LI) in schizophrenia sufferers [10]. Importantly, several lines of evidence suggest that LI relies on
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attentional process which are primarily associated with positive (as opposed to negative or
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cognitive) symptomatology. First, disruptions of LI are seen only in patients suffering from positive symptoms; second, lesion and local injection studies show that LI is critically dependent on subcortical structures, rather than cortical areas important for negative and cognitive
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symptoms; and third, LI is susceptible to typical antipsychotic drugs (D2 antagonists), which are clinically more effective at treating positive symptoms (see reviews [10,11,12]). Thus LI shows face validity for assessing mechanisms of positive symptomatology [10,11,12]. However, there is debate about the effect of PCP, on LI. In conditioned emotional response (CER) tasks, Weiner and Feldon [13] showed no effect on LI of acute administration of PCP given at preexposure or 4
conditioning, while Traverso and co-workers [14] showed that MK-801 given before preexposure, but not before conditioning, disrupted LI. Similarly, in conditioned taste aversion, acute MK-801 disrupted LI when given at preexposure, but not at conditioning [15], but may potentiate LI when given at conditioning [16]. Importantly, in these experiments, the drug was administered acutely and was present in the brain during at least one stage of testing. While this
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gives insights into effects on ongoing behaviour, relating to transient behavioural changes seen in people taking a drug once, it is unlikely to have much relevance to understanding mechanisms
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of schizophrenia. Conversely, people taking PCP repeatedly exhibit a symptom spectrum and
time course more resembling schizophrenia itself. In rats, subchronic but not acute PCP induces
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sensitisation of mesolimbic dopamine function [17], similar to that reported in schizophrenia
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sufferers [18]. Therefore subchronic drug treatment, followed by withdrawal, where animals are
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drug-free during testing, are more plausible animal models for the disease [2,5]: changes result
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from long-term drug-induced adaptations in brain physiology rather than short term psychopharmacological effects. Although effects of chronic ketamine on LI of conditioned
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avoidance have been reported [19], other studies have shown no effect of chronic treatment of PCP on LI [20]. The present study therefore tested whether LI, would be disrupted after
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withdrawal from subchronic PCP, using an established CER paradigm [21]. Animals were also tested in novel object recognition (NOR) to confirm that the pretreatment was sufficient to
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replicate well-documented deficits in NOR [5,22]. Thirty-two female Lister-hooded rats (225-250g: Charles River, UK), were housed four per cage
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(46 (w) x 40 (d) x 40 (h) cm; double deck, independently ventilated cage: Techniplast, UK), with corn cob and sizzle nest bedding (Datesand, UK), under standard laboratory conditions of temperature (21 ± 2°C), humidity (55 ± 10 %) and light/dark cycle (12 hours light/12 hours dark; lights on 07:00), with food (LabDiet 5LF5, IPS Ltd, UK) and water available ad libitum. Experiments were carried out in the Preclinical Research Facility (designated SPF) and were 5
approved by the University of Leicester Ethical Committee and were covered by personal and project (60/4390) license authority. Once acclimatised to housing conditions (7 days), animals were randomly assigned to drug or vehicle pretreatment groups, and injected (1 ml/kg, i.p.) with PCP (2 mg/kg: n = 16) or vehicle
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(0.9% saline: n = 16) twice daily for five days: drug dose and dosing schedule were based on regimes previously seen to modify behaviour (reviewed in [5,8]) and neurochemical function
[23,24] in our labs and elsewhere. Pretreatment was followed by 1 week without intervention,
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to ensure that PCP was absent during testing [22]: although the plasma half-life of PCP is around 1 hour, due to its lipophilicity, it is believed to be up to 3 days in brain tissue [25]. Animals were
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housed four per cage in mixed groups (two saline-pretreated and two PCP-pretreated animals per
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cage).
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Testing used four identical behavioral chambers housed in sound-attenuating boxes and
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illuminated by a single house light (all Med Associates, USA unless otherwise stated). The
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animals’ licks were counted electronically using a contact lickometer. Mild footshock (see below) was delivered scrambled to the grid floor (0.4 cm diameter bars at 1.5 cm intervals) from
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a shock generator and tone stimuli were delivered through an overhead speaker using a custom built audio-generator. Each animal was tested in the same behavioural chamber throughout the
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experiment, and experimental condition in each chamber was counterbalanced across test groups. All stimulus presentation and lick recording was under computer control (MedPC software).
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Conditioning and LI were measured by conditioned suppression of licking for water in thirsty animals, as previously described [21]. Briefly, after drug washout, animals were handled daily for 5 days and introduced to restricted water access: they received water ad libitum in the home cage for four hours each day (midday to 16:00), for five days per week and free access to water for the two remaining days. This regime was the minimum required to ensure that animals were
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motivated to lick during the testing sessions, between 09:00 and midday. Prior to testing, animals were trained to lick for water in the test chamber, immediately following the period of water restriction. This was repeated daily for 5 days, after which all the animals licked freely in the test chamber. After each lick training session, animals were returned to their home cage for their
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period of ad libitum water availability. Following lick training, animals were randomly allocated to non-preexposed (NPE) and preexposed (PE) groups (n = 8 each for each pretreatment).
For Preexposure animals were
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placed in their respective boxes, with no water available. Animals in the PE groups received 40
presentations of tone (5 sec; 15 dB above background) at 1 min intervals, while NPE animals
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were placed in the box for 40 min without tone presentations. After this, animals were returned
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to their home cage for their period of ad libitum water availability. Next day (Conditioning), all
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animals were placed in their respective boxes without water available. After 5 min they received
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two pairings of tone (as above), followed immediately by mild footshock (1 s train of 6 ms pulses, 25 Hz; 0.3 mA; scrambled to the grid floor), at 5 min intervals. After a further 5 min, they were
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returned to their home cage, for their period of ad libitum water availability. Next day (Testing), animals were placed in their respective boxes with free access to water. Licks were monitored
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continuously and counted electronically. The tone was presented after lick 90 and continued until
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a further 10 licks were completed. The time taken to complete the 10 licks immediately before tone presentation (licks 80 to 90: Time A) and 10 licks during tone presentation (licks 90 to 100: Time B) were measured electronically. Animals were then returned to their home cage for their
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period of ad libitum water availability. Three days later the testing procedure was repeated for each animal over four consecutive days (test days 2 to 5) and Time A and Time B were recorded as above. After each test session, animals were returned to their home cage for their period of ad libitum water availability. On completion of testing they were returned to full water availability in their home cage. 7
For each test session, the suppression index (SI) was, calculated as (Time B - Time A) / (Time A + Time B). SI of 1.0 shows high suppression of licking (good learning), while SI of 0.0 shows no suppression of licking (poor learning). SIs were submitted to 3-way ANOVAs with drug and exposure as between-subject variables,
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and test days as the within-subject variable. Significant interactions were further interrogated with a priori planned comparison using Bonferroni corrected independent samples t-tests.
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Following LI, animals underwent NOR testing as previously described [22,23]. Briefly, four days after extinction testing, animals were habituated to the arena (black plexiglass; 60 cm x 60 cm x 60 cm) on two consecutive days for 30 min. Next day, animals underwent NOR testing.
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Each animal was first placed in the empty arena for 3 min, then removed and placed in a holding
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cage for 1 min, during which time two identical objects (metal cylinders, 12 cm x 8 cm diameter)
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were placed in opposite corners of the arena, 10 cm from the walls. The animal was returned to
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the arena at one of the unoccupied corners and allowed to explore for 3 min, before being
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removed and placed in the holding cage for 1 min. The objects were removed and replaced by one similar (familiar) object and one novel object (glass cylinder; 9 cm x 10 cm diameter), placed
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in the same corners as in acquisition, with the corner occupied by the novel object counterbalanced across animals. The animal was returned to the arena, and allowed to explore
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for 3 min. The session was video recorded, using a webcam (Logitech 510HD), linked to a desktop PC for subsequent scoring.
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Time taken to explore each object during acquisition and test sessions was measured by two independent scorers, blinded to drug conditions. Exploration of an object was defined as rats sniffing, licking or touching the objects with forepaws whilst sniffing at the object [22]. NOR was assessed by comparing the time spent exploring the novel object (TNOVEL) and the familiar
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object (TFAMILIAR) during the test period. Additionally a discrimination index (DI) [22] was calculated as: (TNOVEL - TFAMILIAR) / (TNOVEL + TFAMILIAR) Exploration times were analysed by two-way ANOVA, with significant interactions further interrogated using a priori planned comparisons (differences between TNOVEL and TFAMILIAR)
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using paired samples t-test. The difference in DI between drug conditions was assessed by t-test and the difference in DI from zero (no discrimination) was assessed by one-sample t-test. All
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statistical analysis used SPSS v24 (IBM).
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In LI testing, there was no difference in baseline licking rates (Time A) on test day 1 across the
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four groups. Two-way ANOVA showed no significant main effects of either pretreatment
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(F[1,28] = 1.922; p = 0.177) or preexposure (F[1,28] = 1.63; p = 0.212), and no significant
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Insert table 1 around here
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interaction (F[1,28] = 0.358; p = 0.541: table 1).
On test day 1, NPE animals in both pretreatment conditions exhibited high SI (saline: 0.779 ±
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0.118; PCP, 0.936 ± 0.11), indicating good learning of the tone-shock pairing. In PE animals, the saline-pretreated group showed low SI (0.200 ± 0.178), indicating poor learning to the
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preexposed stimulus, reflecting LI. This effect was substantially reduced in PCP-pretreated animals (SI = 0.677 ± 0.126), showing a disruption of LI by PCP (Figure 1). Extinction of the
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conditioned learning, shown by decreased SI over subsequent test days, occurred in the three groups with high SI (saline NPE; PCP NPE; PCP PE), although mostly they still showed some degree of suppression (Figure 1). Three-way ANOVA revealed a significant main effect of testing day (F[4,112] = 3.308, p = 0.013), and of exposure (F[1,28] = 54.031, p < 0.001) but not of drug treatment (F[1,28] =3.707, p = 0.064). The only significant interaction was the two-way
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drug x exposure interaction (F[1,28] = 6.674, p = 0.015). This interaction was further interrogated using Bonferroni corrected independent t tests of a priori planned comparisons of LI (NPE vs PE) shown in each of the two drug treatment groups for each test day, confirming that salinepretreated animals exhibited LI across all five test days, while PCP-pretreated animals showed
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no significant LI. Insert figure 1 around here
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In NOR testing, during acquisition there was no significant main effect of side (F[1,58] = 0.314; p = 0.577), or of pretreatment (F[1,58] = 2.368; p = 0.129), and no significant interaction between the two (F[1,58] = 0.825; p = 0.368: Figure 2a). At test, there was a significant main effect of
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novelty (F[1,58] = 9.262; p = 0.004), and a significant interaction (F[1,58] = 4.146; p = 0.046),
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but no main effect of pretreatment (F[1,58] = 1.877; p = 0.176). A priori planned comparisons
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of exploration times on familiar and novel objects showed a significant NOR effect in saline-
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pretreated animals but not in PCP-pretreated animals (paired t-test: Figure 2b). There was a
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significant difference in DI between saline-pretreated and PCP-pretreated animals (t[29] = 3.662; p = 0.001; independent samples t-test) and saline-pretreated animals, but not PCP-pretreated
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animals, showed significant discrimination between the objects, since the DI was significantly above zero (saline, t[14] = 6.287; p < 0.001: PCP, t[15] = 0.477; p = 0.640: one-sample t-test:
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Figure 2c).
Insert figure 2 around here
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The results demonstrated that sub-chronic PCP pretreatment disrupted LI. Across repeated extinction testing, the overall suppression in the groups exhibiting conditioning diminished, as would be expected, but LI remained intact in saline-pretreated animals across all 5 days: no LI effect was seen on any day in PCP-pretreated animals. NOR experiments confirmed that the twice daily treatment with PCP for five days was sufficient to produce a robust disruption of 10
NOR in adult rats, previously shown in juvenile rats [23,24], and is similar to that reported in adults treated for seven days [22]. Subchronic PCP pretreatment is well established at producing behavioural deficits mimicking cognitive and negative symptoms of schizophrenia [5], but effects on LI are less certain. The present results show that subchronic PCP disrupts LI, in a CER paradigm, indicating that it does
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indeed affect behaviours resembling deficits in all three behavioural domains. This is in
agreement with results showing ketamine disruption of LI in a conditioned avoidance task [19],
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but at odds with PCP having no effect on LI in both a conditioned avoidance and a taste aversion
task [20]. Notably, the latter study used male rats, whereas the current study used female rats.
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It is known that a sex difference exists in subchronic doses of PCP required to produce enduring
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behavioural deficits. In males a dose of 5 mg/kg is required to produce similar long-term effects
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as a dose of 2 mg/kg in females. Therefore it is possible that the dose used by Tenn and co-
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workers (3 mg/kg in males [20]), was not sufficient to produce long-term deficits in LI. In extinction trials, carried out over four consecutive days, three days after the first test session,
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animals received the tone without footshock, thus extinguishing the tone-footshock association, leading to a reduction in the CER [21], measured as a decrease in SI. At test on Day 1, both
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saline and PCP pretreated NPE groups showed near maximum suppression (SI close to 1.0). Due to this ceiling effect it was impossible to ascertain whether PCP pretreatment affected basic
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learning in NPE animals. However, since stronger conditioned associations extinguish more slowly than weaker ones, we could differentiate the degree of conditioning in these groups by
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examining extinction rates. Although suppression decreased in both groups over four extinction days, there was no significant difference in SI for NPE animals between the two pretreatment groups across extinction days, indicating that PCP pretreatment had not affected basic learning. In summary, subchronic PCP pretreatment disrupted LI, without affecting basic learning, indicating that this pretreatment invokes behavioural abnormalities resembling positive
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symptoms of schizophrenia, as well as cognitive and negative symptoms previously described. In addition, twice daily subchronic PCP pretreatment for five days is sufficient to disrupt behaviour, replicating previous findings using seven days pretreatment.
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Acknowledgements
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AMA-A received a Government of Iraq research studentship.
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References
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2. Jentsch JD, Roth RH. The Neuropsychopharmacology of Phencyclidine: From NMDA Receptor Hypofunction to the Dopamine Hypothesis of Schizophrenia. Neuropsychopharmacology. 1999;20:201-25.
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13. Weiner I, Feldon J. Phencyclidine does not disrupt latent inhibition in rats: Implications for animal models of schizophrenia. Pharmacology Biochemistry and Behavior. 1992;42:625-31.
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15. Traverso LM, Ruiz G, De la Casa LG. Latent inhibition disruption by MK-801 in a conditioned taste-aversion paradigm. Neurobiology of Learning and Memory. 2003;80:140-6.
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16. Pålsson E, Klamer D, Wass C, Archer T, Engel JA, Svensson L. The effects of phencyclidine on latent inhibition in taste aversion conditioning: differential effects of preexposure and conditioning. Behavioural Brain Research. 2005;157:139-46.
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17. Jentsch JD, Taylor JR, Roth RH. Subchronic Phencyclidine Administration Increases Mesolimbic Dopaminergic System Responsivity and Augments Stress- and Psychostimulant-Induced Hyperlocomotion. Neuropsychopharmacology. 1998;19:105-13.
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18. Breier A, Su TP, Saunders R, Carson RE, Kolachana BS, de Bartolomeis A, et al. Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: evidence from a novel positron emission tomography method. Proc Natl Acad Sci U S A. 1997;94:256974.
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21. Joseph MH, Peters SL, Moran PM, Grigoryan GA, Young AMJ, Gray JA. Modulation of latent inhibition in the rat by altered dopamine transmission in the nucleus accumbens at the time of conditioning. Neuroscience. 2000;101:921-30.
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22. Grayson B, Idris NF, Neill JC. Atypical antipsychotics attenuate a sub-chronic PCP-induced cognitive deficit in the novel object recognition task in the rat. Behavioural Brain Research. 2007;184:31-8. 23. Gupta I, Young AMJ. Metabotropic glutamate receptor modulation of dopamine release in the nucleus accumbens shell is unaffected by phencyclidine pretreatment: In vitro assessment using fast-scan cyclic voltammetry rat brain slices. Brain Research. 2018;1687:155-61. 24. Yavas E, Young AM. N-methyl-D-aspartate modulation of nucleus accumbens dopamine release by metabotropic glutamate receptors: fast cyclic voltammetry studies in rat brain slices in vitro. ACS Chemical Neuroscience. 2017;8:320-8. 13
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25. Bey T, Patel A. Phencyclidine intoxication and adverse effects: a clinical and pharmacological review of an illicit drug. The California Journal of Emergency Medicine. 2007;8:9-14
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Figure legends Figure 1: Disruption of LI by subchronic PCP.
Data are mean ± SEM
suppression index (SI) over five testing days; n = 8 per group. *** p < .001; **
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significant LI); Bonferroni corrected independent samples t-test.
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p < .01; * p < .05; significant differences between NPE and PE groups (i.e
Figure 2 : Disruption of NOR by subchronic PCP. (a) Time spent exploring two
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identical objects during acquisition; (b) Time spent exploring the familiar and novel
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objects during test; (c) DI during test. Data are mean ± SEM, n = 15 to 16 per
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group. *** p < 0.001; significant difference in exploration time between familiar
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and novel objects (independent samples t-test); † † p < 0.001; significant difference in DI between saline and PCP pretreated animals (independent samples t-test);
###
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p < 0.01, significant difference from zero (no discrimination: one-sample t-test).
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Fig 1
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Fig 2
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Saline
PCP
NPE
1.96 ± 0.26
2.22 ± 0.57
PE
1.31 ± 0.04
2.00 ± 0.26
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Table 1
Table 1 : Baseline licking rates on Day 1. Mean (± SEM) time to complete 10 licks
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immediately before the onset of the tone (Time A: n = 8 per group).
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