Treatment with 1-benzylpiperazine (BZP) during adolescence of male and female hooded rats exposed to environmental enrichment: Subsequent behavioral outcomes

Accepted Manuscript Title: Treatment with 1-benzylpiperazine (BZP) during adolescence of male and female hooded rats exposed to environmental enrichment: subsequent behavioral outcomes Authors: Ellen I. Dixon, Robert N. Hughes PII: DOI: Reference:

S0736-5748(18)30308-3 https://doi.org/10.1016/j.ijdevneu.2018.12.005 DN 2325

To appear in:

Int. J. Devl Neuroscience

Received date: Revised date: Accepted date:

31 October 2018 12 December 2018 18 December 2018

Please cite this article as: Dixon EI, Hughes RN, Treatment with 1-benzylpiperazine (BZP) during adolescence of male and female hooded rats exposed to environmental enrichment: subsequent behavioral outcomes, International Journal of Developmental Neuroscience (2018), https://doi.org/10.1016/j.ijdevneu.2018.12.005 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.

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Treatment with 1-benzylpiperazine (BZP) during adolescence of male and female hooded rats exposed to environmental enrichment: subsequent behavioral outcomes

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Ellen I. Dixon, Robert N. Hughes*

Department of Psychology, University of Canterbury, Christchurch 8140, New Zealand

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Private Bag 4800, Christchurch 4800, New Zealand.

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*corresponding author at: Department of Psychology, University of Canterbury,

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E-mail address: [email protected]

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Highlights:

Adolescent BZP treatment and enrichment affect anxiety at older ages in rats

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Enrichment enhances anxiogenic effects at older ages of adolescent BZP

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treatment

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Adolescent BZP treatment does not seem to affect spatial memory development

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Adolescent BZP effects at older ages can depend on sex and age of testing

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Abstract From 30 days after birth until the completion of the study, male and female rats were caged in same-sexed twos or threes either with (enriched cages, EC) or without

2 several objects for them to explore (standard cages, SC). From 41 to 50 days of age (late adolescence), they received a daily intraperitoneal injection of saline, or 10 or 20 mg/kg of the monoaminergic agonist drug of abuse, 1-benzylpiperazine (BZP). Ten days later (PND60+), their behavior was observed over several days in an open field,

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an elevated plus maze, a light-dark box and (to assess short-term memory) a Y maze in which one of the arms had been changed in brightness between two trials. These

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tests were repeated from 40 days after PND60+, namely PND100+. While open-arm

occupancy at PND100+ in the plus maze was lower following both doses of the drug for SC rats only, other examples of BZP-related heightened anxiety were confined to

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EC rats. This suggested that enrichment had enhanced rather than reduced any

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anxiogenic effects of the drug treatment. There was no plausible evidence of BZP-

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associated impaired spatial memory required to recognize the changed novel Y-

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maze arm. Instead, changes in novelty preferences or neophobia-related anxiety

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were most likely. While there were also some examples of sex and age differences in the later effects of BZP, in most cases these were evident at both ages following

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treatment with both BZP doses. A number of overall BZP, cage, sex and age

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differences, independent of enrichment effects, were also observed. Keywords:

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Benzylpiperazine Adolescence

Environmental enrichment Anxiety

3 Male and female rats 1. Introduction In recent years, there has been a global increase in the abuse of a variety of synthesized substances used recreationally for their psychoactive properties. A

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prominent example is the piperazine derivative, 1-benzylpiperazine (BZP), which is the main active ingredient of so-called “party pills”. The popularity of BZP comes

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from its ability to induce feelings of euphoria, increase energy, decrease fatigue and suppress appetite (Lin et al., 2009). However, such effects can be at the expense of undesirable or even dangerous consequences such as nausea, agitation, acute

Until

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psychosis, rhabdomyolysis and multiple organ failure (Monteiro et al., 2013).

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2008, BZP was legal, freely available to anyone in New Zealand over the age of 18

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and promoted as a “safe” alternative to other illicit psychostimulants, especially methamphetamine (Kerr and Davis, 2011).

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The mechanism of action of BZP for its behavioral effects appears to arise from the elevation of extracellular levels of dopamine and serotonin. This elevation results

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from effects on the release and re-uptake of both transmitters (Monteiro et al., 2013)

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via actions on their respective transporters (DATs and SERTs, Baumann et al., 2004) which are major targets of many psychostimulants (Zahniser and Sorkin, 2009).

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Therefore, contrary to earlier claims, this would make the drug not that different from methamphetamine, a conclusion supported by both behavioral and neurochemical findings (Baumann et al., 2004; Brennan et al., 2007a,b; Herbert and Hughes, 2009; Johnson and Hughes, 2013).

4 For the last two decades there has been increasing attention paid to possible detrimental effects on neurobehavioral development of exposure to drugs of abuse during adolescence when drug use typically begins (Laviola et al., 1999; Patton et al., 2004). Adolescents can be more resistant to the aversive nature of many drugs such

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as their withdrawal effects, while also being more sensitive to their rewarding properties and thus addiction risk (Schramm-Sapyta et al., 2009). Therefore, use at

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this time is regarded as particularly risky not only because of the danger of

developing substance use disorders, but also because adolescence is a period of transition from childhood to adulthood when the still immature brain is highly

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vulnerable to developmental insults and thus possible behavioral impairments

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(Anderson, 2003, 2005; Spear, 2000). Studies with rats and mice have revealed that,

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during adolescence there are changes in limbic and prefrontal areas involving

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overproduction of axons and synapses early on, followed later by extensive synaptic

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pruning particularly in the nucleus accumbens, amygdala and prefrontal cortex (Casey, et al., 2008). Consequently there are structural changes which affect the

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development of vulnerable connections between subcortical and cortical areas in

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transition during adolescence that are important for emotional development (Cunningham et al., 2002) and thus the main focus of the present study, namely anxiety.

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While there have been studies of the longer-term effects in rats of adolescent

exposure to methamphetamine (Riddle et al., 2002; Ye et al., 2014) and other stimulants such as methylene dioxymethamphetamine and methylphenidate (AchatMendes et al., 2003; Fone et al., 2002), there appears so far to be only one such case

5 on record involving BZP (Aitchison and Hughes, 2006). In this preliminary study, male and female PVG/c hooded rats were treated daily with 10 mg/kg BZP from postnatal days 45 (PND45) to PND55 when they were within a similar period of development to late adolescence in humans (Vorhees et al., 2005). Then when they

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reached the equivalent of human adulthood, namely from about 75 days after birth (Anderson, 2003), their behavior was observed (with particular reference to anxiety-

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related behavior) in a Y maze involving a novel brightness change in one arm, an

open field and an emergence apparatus. It was accordingly revealed that, compared with control rats, previously BZP-treated subjects were less responsive to the novel

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Y-maze arm, were less active in the open field (especially its central area) and

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emerged more slowly from a small, darkened chamber into a brightly lit arena.

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These results were interpreted as BZP-related heightened anxiety with some possible

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memory impairment (Aitchison and Hughes, 2006). The responsible mechanism for

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BZP’s pharmacological action is likely to be modification of brain development through effects on relevant processes operated by DA and, to a lesser extent, 5-HT

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(Baumann et al., 2004).

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The present study aimed to determine if any subsequent effects on anxietyrelated behavior of adolescent exposure to two doses of BZP could be modified by concurrent exposure to environmental enrichment in a similar way to what has

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recently been described for methamphetamine (Peterson and Hughes, 2017). As adolescent BZP exposure appeared to lead to higher anxiety in adulthood (Aitchison and Hughes, 2006), and since enrichment can decrease anxiety (Benaroya-Milshtein et al., 2004; Hughes and Collins, 2010), it seemed possible that any longer term

6 anxiogenic effects of the drug might be attenuated by enrichment. This would seem to be the case for effects of acute treatment with scopolamine on anxiety-related behavior in adult rats (Hughes and Otto, 2013). However, enrichment can influence drug effects in different ways. For example, it has been shown to either increase

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voluntary consumption and the reinforcing value of ethanol in rats (Berardo et al., 2016; Rockman et al., 1989) and mice (Rae et al., 2018), or have the opposite effect in

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both species respectively (Deehan et al., 2007; Holgate et al., 2017). It was therefore

conceivable that BZP experienced repeatedly during adolescence in conjunction with enrichment might also differ in its subsequent effects on anxiety-related behavior

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from those following acute administration of other drugs during adulthood.

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Therefore, rats were group-housed in either standard (SC) or enriched cages (EC)

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from weaning at PND30 until after adolescent BZP treatment and the completion of

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all behavioral assessments. Since the earlier study assessed effects of adolescent BZP

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only in adulthood (Aitchison and Hughes, 2006), it was of interest to determine if such effects were also apparent at an earlier stage of development. Therefore,

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behavioral assessments were begun when the rats were 60 days old (PND60+), and

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thus late adolescents/early adults (Anderson, 2003), as well as again during mid adulthood when they were 100 days old (PND100+). Because of some sex differences in effects of adolescent exposure to BZP

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(Aitchison and Hughes, 2006) and enrichment (Peterson and Hughes, 2017), and the need to address the prevalence of male-only animal studies in behavioral pharmacology and neuroscience research (Beery and Zucker, 2011; Hughes, 2007b, 2018), rats of each sex were included in the study.

7 2. Materials and methods 2.1. Animals and cage conditions The subjects comprised 60 male and 60 female PVG/c hooded rats, bred in the Animal Facility of the Department of Psychology, University of Canterbury. Thirty

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days after birth (PND30) they were weaned and 2 males and 2 females from each of 30 litters were housed in 470 x 280 x 230-mm plastic cages in same-sexed groups of 2

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or 3 rats, with free access to food and water. Half of each sex was assigned to an environmental enrichment condition (EC), and the other half was assigned to a standard condition (SC). For rats in the EC condition, their cages contained a

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random selection of objects (such as glass jars, metal lids, marbles, small household

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utensils, tunnels) that were intended to be perceptually enrichening by encouraging

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their active exploration. The objects were replaced each week with a different set.

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The cages of rats in the SC condition were identical to those for EC rats, except that

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objects were not provided. For each cell of the 3 (dose) x 2 (cage) x 2 (sex) design at each testing age there were 10 rats. Social enrichment was provided for rats in both

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the SC and EC conditions as they were all housed with 1 or 2 other cage-mates. The

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cage conditions were continuously provided from the beginning of BZP (or saline) treatment until the end of all behavioral testing at PND100+. The cages were kept in an ambient temperature of 22±2O C on a 12 h light/dark cycle (lights on at 08.00 h),

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and the rats were maintained and treated in accord with requirements of Parts 5 (Codes of Welfare) and 6 (Use of Animals in Research, Teaching and Testing) of the New Zealand Animal Welfare Act, 1999. All experimental procedures had been

8 approved by the Animal Ethics Committee of the University of Canterbury (Approval No. 2016/13R). 2.2. BZP treatment When the rats reached PND41, half of each sex in each cage condition was

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randomly allocated to a BZP treatment group, and the other to a control group. From then on, the treatment groups received an intraperitoneal injection for 10

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successive days of 1-benzylpiperazine (abcr GmbH & Co, Karlsruhe, Germany)

dissolved in 0.9% sterile saline to provide a dose of either 10 or 20 mg/kg. This lower dose had been previously shown to modify behavior in adulthood when

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administered during adolescence (Aitchison and Hughes, 2006), but no information

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is available about effects of a higher dose. The control rats were injected with saline

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vehicle for the same period (0 mg/kg). All injections were in a volume of 1ml/kg.

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Ten days after their last injection on PND50, they began their behavioral testing.

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Because of the numbers of rats involved, this occupied the period from PND60 to PND72 (PND60+). Then when they reached PND100, all rats experienced a second

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round of tests which lasted from PND100 to PND112 (PND100+).

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2.3. Behavioral testing

On PND60+ and again on PND100+, each rat experienced a trial in an open field,

an elevated plus maze, a light-dark box and a Y maze (involving a change of

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brightness in one of the arms between two consecutive exposures) with a two-day interval between each test. The order of testing in the four types of apparatus was randomized for individual rats at each testing age. The former three types of apparatus are commonly used for assessing anxiety-related behavior in rodents

9 (Belzung, 1999; Hascoët et al., 2001; Rodgers and Dalvi, 1997). They each provide slightly different ways of measuring the same underlying process. In other words, anxiety in the open field is measured by location preferences (occupation of the aversive center versus less aversive periphery of the apparatus) as well as amount of

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locomotor activity and frequencies of several anxiety-related responses such as rearing, grooming and defecation. However, anxiety in the plus maze and light-

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dark box involves active choices of aversive areas of the apparatus that provide a conflict between curiosity-related approach and fear-related avoidance, namely,

open versus closed arms of the plus maze, and a light versus dark compartment of

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the light-dark box. Behavior in the Y maze is dependent on ability to recognize the

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arm that has changed in brightness between trials and is thus a measure of short-

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term spatial memory (Hughes and Maginnity, 2007), but can also provide

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information about neophobia-related anxiety or fear (Hughes, 2007a).

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A small CCTV camera was mounted above each type of apparatus with a monitor positioned some distance away to enable the observer to view the rats

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without disturbing them. Illumination was provided by dim, overhead fluorescent

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room lighting. A momentary time-sampling procedure was adopted for recording the frequency of responses. This procedure has proved to be an accurate and reliable method for estimating frequencies and duration of events (Powell et al.,

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1977) which produces test-retest reliability coefficients of between 0.93 and 0.98, and validity coefficients of 0.87 and 0.99 (Detke et al., 1995). In the present study, timesampling involved noting for exactly 3 min in the Y maze, and for 5 min in the other three types of apparatus, the response engaged in by the rat at the onset of

10 successive 3-s auditory signals from a portable timer that were delivered to the observer via an earpiece. At the completion of each rat’s trial, the apparatus was thoroughly cleaned with a 20% Paraquat Blue solution. 2.3.1. Open-field testing

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The black, wooden 60 x 60cm open field comprised 30-cm high walls and a floor divided into 16 numbered squares by means of a grid of intersecting white lines. It

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sat on a 700-mm high table. Each rat was placed in the center of the field and every 3 s it was noted which square the rat was occupying, and if it was engaged in walking, rearing up on hind legs, grooming or remaining immobile. As in previous

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research (e.g., Hughes and Otto, 2013), it was also possible to later obtain a measure

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of ambulation (or distance travelled) by calculating the number of times the rat was

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seen in a different square from that occupied at the onset of the preceding 3-s

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auditory signal. At the end of the trial, the rat was removed and the number of

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eliminated fecal boluses was counted. Heightened anxiety is often regarded as being reflected in lower levels of open-field locomotion, rearing and occupancy of the

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center of the apparatus, as well as higher frequencies of defecation, grooming,

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immobility and occupancy of corners (Archer, 1973; Belzung, 1999). 2.3.2. Elevated plus-maze testing The elevated plus maze consisted of four black 100-cm long, 50-cm wide arms, at

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right angles to each other, that extended from a 15 x 15 cm-long platform. The two closed arms faced each other with end and side metal walls that were 24 cm high and were painted black. The open arms had end and side walls 24 cm high that were constructed from clear Perspex to prevent startled rats from falling or jumping off

11 the maze. This addition to the usual plus-maze design has been shown to not reduce their anxiety-provoking aversiveness (Martínez et al., 2002). Each rat was placed on the center platform of the apparatus facing one of the closed arms. For its 5-min trial, the number of entries (involving all four feet) into each arm was counted as well as

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which arm the rat was occupying at the onset of each 3-s signal. These data later enabled calculation of percentages of arm entries and occupancy that were of the

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open arms. Higher anxiety is accepted as leading to fewer entries into and lower occupation of the open arms (Rodgers and Dalvi, 1997), whereas entries into the closed arms are regarded as a measure of locomotor activity that is relatively

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2.3.3. Light-dark box testing

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uncontaminated by anxiety (Cruz et al., 1994).

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The light-dark box comprised two 30 x 25 x 25 cm (l x w x h) wooden

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compartments that were separated from each other by a wooden partition

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containing a 10 x 10 cm central opening that could be opened and closed with a removable horizontal slide. Each compartment was covered by a hinged lid that

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was made from wood for the dark side and from clear Perspex for the light side. The

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box sat on a 70 cm-high table. Each rat was placed in the dark compartment with the slide separating the two compartments in place. Thirty s later, the slide was withdrawn and the rat’s latency of full emergence (all four feet) into the illuminated

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compartment was recorded with a hand-held stopwatch. It was then noted how often the rat entered this compartment, how often it entered both compartments (transitions), and which compartment it was occupying at the onset of each 3-s signal. Fewer entries into and shorter occupation of the light compartment of the

12 apparatus are generally accepted as indicative of higher anxiety (Hascöet et al., 2001). 2.3.4. Y-maze testing The wooden Y maze was clear-varnished and consisted of two 45 cm-long arms,

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set at an angle of 120O to each other, and a 30 cm-long stem. Both arms and the stem were 10 cm wide and 14 cm high. All the maze (except for the first 15 cm start area

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of the stem) was covered by a clear Perspex hinged lid. Each arm was provided with removable painted metal inserts. These occupied all of the floor and walls of the maze except for the first 5 cm. The maze was positioned on a 70 cm-high table. Each

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rat’s test involved a 5-min acquisition trial, during which it was free to explore all

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parts of the apparatus, followed by a 3-min retention trial. For the acquisition trial

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one of the arms contained a white insert, and the other contained a black insert. At

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the completion of this trial, the rat was removed from the apparatus while both arm

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inserts were replaced by clean black inserts. The rat was then returned to the apparatus where it was faced by two black arms of which one (the novel) had

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changed from white to black. The position of this novel arm was on the left for half

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the rats in each group, and on the right for the other half. For exactly 3 min, the number of entries into each arm (all four feet) was counted, and every 3 s it was noted which arm was being occupied. From the recorded data, it was later possible

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to calculate the percentage of entries into and percentage occupancy of the novel versus familiar arm, as well as total entries of both arms (locomotor activity). Rats’ ability to recognize which maze arm has changed between acquisition and retention trials has been proposed as a measure of curiosity-based short-term spatial

13 memory dependent on the influence of both egocentric and allocentric cues (Hughes and Maginnity, 2007). 2.4. Statistical analysis All data were subjected to separate 4 (BZP dose) x 2 (cage condition) x 2 (sex) x 2

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(testing age) ANOVAs, with repeated measure on the last factor. These were followed by Scheffe post hoc tests (P <0.05) for individual comparisons when BZP

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main effects or interactions were significant. 3. Results

The main effects of BZP, cage condition and sex on all responses recorded in the

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open field, the elevated plus maze, the light-dark box and the Y maze, and results of

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ANOVAs are outlined in Table 1. Scores for each measure at PND60+ and at

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PND100+, and ANOVA results for significance of differences between the two ages

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can be seen in Table 2.

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____________________________ Insert Tables 1 and 2 about here

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____________________________

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3.1. Open-field results

The only responses that were affected by BZP were walking and grooming. For

walking there were significant interactions between BZP dose and cage (F(2, 108) =

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8.10, P < 0.001), and between dose and sex (F(2, 108) = 5.39, P < 0.01) which are outlined in Fig. 1A and B. ____________________________ Insert Fig. 1 about here

14 ____________________________ These interactions revealed that walking occurred more often following treatment with both doses of BZP for EC rats only (Fig. 1A), and more often with 20 mg/kg BZP for males only (Fig. 1B). While there was a significant main effect of BZP dose

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for grooming shown in Table 1, this is more appropriately considered in the light of significant interactions between dose and sex (F(2, 108) = 3.24, P < 0.05), and between

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dose and testing age (F(2, 108) = 3.50, P < 0.05, see Fig. 2A and B). ____________________________ Insert Fig. 2 about here

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For female rats only, grooming occurred less frequently after 10 mg/kg BZP (Fig.

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2A) and then more frequently after 20 mg/kg. For both sexes combined, grooming

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mg/kg PND100+ (Fig. 2B).

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occurred less often after 10 mg/kg BZP at PND60+ but more frequently after 20

As shown in Table 1, overall, EC rats groomed and occupied the center of the

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apparatus less often than SC rats. Female rats engaged in more ambulation and

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walking and less immobility, defecation and occupancy of corners than males. In Table 2 it can be seen that the frequencies of ambulation and grooming were lower at PND100+ than at PND60+.

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3.2. Plus-maze results As shown in Table 1, the only significant effect of BZP on plus-maze behavior

was in the form of a significant dose x cage x testing interaction (F(2, 108) = 6.18, P < 0.01) for occupancy of the open arms, outlined in Fig. 3.

15 ____________________________ Insert Fig. 3 about here ____________________________ This interaction revealed that, while occupancy for SC rats was significantly lower at

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PND100+ following treatment with both BZP doses, no drug effects were apparent at either testing age for EE rats. However, for all rats combined, those from enriched

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cages entered the open arms significantly more often than SC rats, and females

occupied the open arms and entered the closed arms more often than males. As shown in Table 2, both of these responses occurred less often at PND100+ than at

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PND60+.

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3.3. Light-dark box results

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There was a significant dose x cage interaction for entries into the light side of

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the apparatus (F(2, 108) = 5.80, P < 0.01) which can be seen in Fig. 4.

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____________________________ Insert Fig. 4 about here

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____________________________

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This interaction was due to a significantly lower number of entries only by EC rats that had been treated with both doses of BZP. There was also an identical dose x

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cage interaction for light-dark transitions (F(2, 108) = 5.70, P < 0.01, see Fig. 5). ____________________________ Insert Fig. 5 about here ____________________________

16 As with entries into the light, EC (but not SC) rats treated with both doses of BZP made significantly fewer transitions. For all rats combined, the Pearson productmoment correlation between these two responses was highly significant (r(118) = 0.99, P < 0.0001).

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As shown in Table 1, female rats emerged into the light compartment significantly faster than males. Females also entered this side of the apparatus

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significantly more often than males, and made more transitions between both compartments. Emergence latencies were longer and light-side entries and transitions were lower at PND100+ than at PND60+.

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3.4. Y-maze results

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A significant dose x cage x sex interaction for entries of the novel maze arm (F(2,

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108) = 4.92, P < 0.01, see Fig. 6) arose from significantly more entries by SC male rats

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following treatment with 20 mg/kg BZP, but then fewer entries by EC males with

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the same dose. Females from both types of cage were unaffected. Entries into both arms were significantly lower in rats treated with 20 mg/kg BZP (see Table 1).

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As shown in Table 1, while there were no significant main effects of cage for any

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response, female rats made significantly more entries into the novel arm and into both arms than males. However, the correlation between these two responses for both sexes combined was not significant (r(118) = 0.03, ns). For entries into and

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occupancy of the novel arm, significantly higher frequencies of both responses occurred at PND100+ than at PND60+, while there were fewer entries into both arms at the older testing age (see Table 2). 3.5. Summary of BZP effects

17 In view of the complexity of the results of the present study, a summary of the later significant effects of adolescent BZP treatment is presented in Table 3 that outlines these outcomes in relation to the other experimental variables on which they often depended namely, dose, cage, sex and testing age,

Insert Table 3 about here

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____________________________

4. Discussion 4.1. BZP effects

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An unexpected outcome in this study was the relative lack of evidence of

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attenuation of later anxiogenic effects of adolescent exposure to BZP by enrichment.

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In fact, it seemed possible that, as entries into the light compartment of the light-dark

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box following both doses of the drug were lower only for EC rats (Fig. 4),

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enrichment may have actually enhanced anxiogenesis. However, in view of the positive correlation of this response with light-dark transitions and the same pattern

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of BZP effects for both responses, it is likely that what appeared to be active

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avoidance of the light compartment was merely an artifact of the drug’s reduction of locomotor activity. Because of evidence that anxiety or fear can elicit neophobia and thus reduce tendencies for rats to explore novelty (Hughes, 2007a), heightened

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anxiety might also account for the fewer entries of the novel Y-maze arm following 20 mg/kg BZP for EC male rats only (Fig. 6B). But on the other hand, the higher frequencies of open-field walking following both doses for EC (but not SC) rats might suggest the contrary if open-field walking (or locomotor activity) is seen as

18 inversely related to anxiety (Sestakova et al., 2013). However, in some circumstances high levels of locomotor activity can also mean high emotionality or anxiety. For example, it has been shown that with repeated daily tests, high activity in the open field can indicate high emotionality on day 1, but low emotionality on day 2

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(Denenberg, 1969). As there was only one open-field test at each testing age in the present study, it is possible that higher walking frequencies among EC rats following

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treatment with both doses of BZP reflected higher (rather than lower) anxiety in line with what occurred in the light-dark box and possibly Y-maze. Since male rats appear to be more anxious or fearful than females (Archer, 1975; Beatty, 1979; Gray,

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1971) there was some limited support for this view from more open-field walking for

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males, but not females, following 20 mg/kg BZP. But, any anxiety basis for BZP-

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related higher locomotor activity must also be considered in the light of fewer

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entries into both Y-maze arms for all rats treated with 20 mg/kg BZP (Table 1). A

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similar situation prevailed for fewer entries into the light compartment of the lightdark box and fewer transitions between the two compartments for EC rats only

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following both doses of BZP. Clearly, such disparities can only be resolved with

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more empirical evidence.

While the lower occupancy of the plus-maze open arms at PND100+ shown by

SC rats following treatment with both doses of BZP (Fig. 3A) was consistent with

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anxiogenic effects of the drug (as reported earlier for rats which were all kept in standard cages, Aitchison and Hughes, 2006), the lack of any BZP effects for EC rats (Fig. 3B) suggests that, in this case, enrichment may have had a protective function. Although there were no other similar examples of BZP-related higher anxiety just for

19 SC rats alone, more open-field grooming at PND100+ for all rats combined following 20 mg/kg BZP (Fig. 2B) might be interpreted as another case of drug-induced anxiogenesis. This is because grooming can be more evident when rats are in anxiety-provoking situations (Colbern et al., 1978) and less frequent when treated

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with anxiolytic drugs (Dunn et al., 1981). But on the other hand, 10 mg/kg BZP led to the opposite outcome for females only (Fig. 2A) and for all rats at PND60+ (Fig.

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2B) thus suggesting lower anxiety. For the same dose and age, this contrasted with open-arm occupancy in the plus maze for SC rats (Fig. 3A) and entries of the light compartment in the light-dark box for EC rats (Fig. 5). Consequently, rather than

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clarifying any anxiogenic basis for adolescent BZP treatment, these results for

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grooming add to doubts about the validity and reliability of the response as an index

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of anxiety, expressed by other authors (Archer, 1973; Bolles, 1960; Cruz et al., 1994;

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File et al., 1988; Reimer et al., 2015; Spruijt et al., 1992).

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In all cases where EC rats were more affected by adolescent BZP treatment than SC rats, the outcomes suggested that enrichment had enhanced rather than

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attenuated the drug’s subsequent anxiogenic effects. This is contrary to later

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anxiolytic effects of enrichment on adolescent exposure to methamphetamine (Peterson and Hughes, 2017) and acute administration in adulthood of scopolamine (Hughes and Otto, 2013). It is also inconsistent with reductions in the reinforcing

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value and thus addiction potential of other drugs such as heroin (El Rawas et al., 2009), cocaine (Solinas et al., 2008) and ethanol (Holgate et al., 2017). However, there are also examples of enrichment having the opposite effect such as increasing ethanol’s reinforcing value (Berardo et al., 2016) thereby detracting from any

20 perceived beneficial quality of the manipulation. It is accordingly possible that, in the present study, enrichment was similarly detrimental with respect to the action of BZP during adolescence. Because potentially detrimental effects of ethanol are more evident following treatment during adolescence (Berardo et al., 2016), such a

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possibility for BZP is clearly worthy of further investigation. Although the nature of the present study precluded determination of any neurochemical basis for possible

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enhancement of the anxiogenic effects of adolescent BZP, it might be interesting in

future to consider similarities in some central effects of the two manipulations such as, for example, increased dopaminergic and serotonergic activity with both BZP

U

treatment (Baumann et al., 2004; Monteiro et al., 2013) and exposure to enrichment

N

(Brenes et al., 2008; Darna et al., 2015).

A

At first sight, increased entries into the novel Y-maze arm for male (but not

M

female) SC rats treated with 20 mg/kg BZP might suggest some improvement in

ED

their short-term memory (Fig. 6A). However, this seems unlikely in view of the opposite effect of this dose for male EC rats (Fig. 6B) which, as commented on above,

PT

may have been due to enrichment-related enhancement of the drug’s anxiogenic

CC E

action. Therefore, without considerably more research, it is not yet possible to draw any conclusions about effects of adolescent BZP treatment on memory development. Although fewer entries into both arms for all rats following the higher BZP dose was

A

not consistent with the higher frequency of open-field walking following the same dose, the Y-maze response may depend on a different underlying mechanism because it obviously also involved an element of active choice rather than just time spent walking (as in the open-field).

21 4.2. Cage condition effects As shown in Table 1, open-field grooming was lower but occupancy of the center of the field and open-arm entries in the plus maze were higher for EC than for SC rats. Apart from grooming (which, as discussed above, is problematic as an index

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of anxiety), the two other results are consistent with enrichment-related lower anxiety described previously (Benaroya-Milshtein et al., 2004; Hughes and Collins,

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2010; Hughes and Otto, 2013). To some extent, the effects of caging were dependent

on BZP treatment as shown by the significant dose x cage interactions for open-field walking (Fig. 1A) and, in the light-dark box, entries into the light compartment (Fig.

U

4) and transitions between the two compartments (Fig. 5). Whereas EC control rats

N

engaged in less open-field walking than their SC counterparts (consistent with

A

previous findings for locomotor activity, Elliot and Grunberg, 2005), this difference

M

was not significant for BZP-treated rats. However, in the light dark box, EC control

ED

rats entered the light compartment and made more light-dark transitions than SC rats thereby suggesting enrichment-related lower anxiety, but made fewer

PT

transitions when all rats had been treated with 20 mg/kg of BZP. Overall, the

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general pattern of results for rats that had not been treated with BZP was reasonably consistent with enrichment having led to lower locomotor activity and reduced anxiety, as has been described previously (Benaroya-Milshtein et al., 2004; Elliot and

A

Grunberg, 2005; Hughes and Otto, 2013). 4.3. Sex differences There were also a large number of overall sex differences observed in the present study (shown in Table 1) that were generally in agreement with previous reports of

22 female rats being more active than their male counterparts (Archer, 1975). This was evident from their higher levels of open-field ambulation and walking, closed-arm entries in the plus maze, light-dark transitions in the light-dark box, entries into both arms in the Y maze, and lower frequencies of open-field immobility (Table 1).

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However, the significant dose x sex interaction outlined in Fig. 1 also revealed that this sex difference for open-field walking only applied to control rats and those

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treated with 10 mg/kg of BZP. Female rats are known to exhibit less anxiety-related

behavior than males (Archer, 1975; Beatty, 1979; Gray, 1971). There were examples of this sex difference in the present study in the form of females’ lower frequencies of

U

open-field immobility and corner occupancy, and fewer fecal boluses.

N

Although lower female open-field grooming following 10 mg/kg of BZP and

A

then a higher frequency of the response following 20 mg/kg displayed in Fig 2A

M

might appear to reflect anxiety differences, this must again be considered in terms of

ED

the questionable value of grooming as an index of anxiety. More convincing evidence of lower female anxiety was found in their longer occupancy of the plus-

PT

maze open arms, their faster initial emergence into the light compartment of the

CC E

light-dark box and their greater number of entries into this compartment. While the greater number of entries by females into the Y-maze novel arm might suggest better short-term spatial memory than males (Hughes, 2007a), this would be contrary to

A

sex differences in other measures of memory (Jonasson, 2005). Rather, the outcome is more likely to be due to lower neophobia because of their lower anxiety (Hughes, 2007a). The lack of a significant correlation between this response and entries of

23 both maze arms makes it unlikely that the result was merely an artifact of more female locomotor activity. 4.4. Age differences As shown in Table 2, for the majority of responses, there were significant

IP T

differences between PND60+ and PND100+, namely between late adolescence/early adulthood, and mid adulthood (Anderson, 2003). These differences were mainly in

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the form of less locomotor activity and higher levels of anxiety-related behavior at

the older age. The activity differences were evident for open-field ambulation, plusmaze closed-arm, light-dark transitions in the light-dark box, and entries into both

U

Y-maze arms. Higher anxiety at PND100+ was suggested by lower open-arm

N

occupancy in the plus maze (although this only applied to SC rats treated with 10

A

and 20 mg/kg of BZP and EC control rats, Fig. 3) and longer latencies of first

M

entering the light-dark box light compartment followed by fewer entries into this

ED

compartment for all rats. However, while similar age differences in locomotion and anxiety have been shown for rats observed in the open field (Bronstein, 1972;

PT

Stansfield and Kirstein, 2006) and elevated plus maze (Doremus-Fitzwater et al.,

CC E

2009), there have also been reports of the opposite outcomes (Candland and Campbell, 1962; Doremus et al., 2006). Even though the time interval between the rats’ two testing sessions was 60+ days, there is a slight possibility that, rather than

A

age being responsible for the differences between PND60+ and PND100+, repeated measurements might account for the results in a similar fashion to the changes described for repeated measurements of open-field behavior (Walsh and Cummins, 1976).

24 The greater number of entries into and longer occupancy of the Y-maze novel arm shown at PND100+ than at PND60+ suggests either improved short-term memory (Hughes and Maginnity, 2007) or, contrary to observations in the other types of apparatus, lower neophobia-related anxiety (Hughes, 2007a). However, it is

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currently not possible to adequately account for this result especially as reactivity to novelty in rats generally declines rather than increases with age (Barnes, 1990).

SC R

It should be noted that, because the inability to test large numbers of rats in a

single day, the testing period at each age had to be spread over 12 days. This meant that rats tested at the end of each period were up to 12 days older than those tested

U

at the beginning. While this was probably a relatively minor problem at PND100+, it

N

may have had more consequences at the younger age. Consequently, in future

ED

5. Conclusions

M

rats should be ensured.

A

research of this sort, the shortest periods possible between the testing of individual

From the main results of this study, it can be concluded that, while treatment

PT

with BZP during adolescence led to higher anxiety at older ages, this was more

CC E

typical of rats housed in enriched rather than in standard cages thereby suggesting that enrichment had enhanced the anxiogenic action of the drug. So contrary to expectations, enrichment did not appear to have provided protection from the

A

development of BZP-related higher anxiety reported previously (Aitchison and Hughes, 2006) similar to reports of increased voluntary ethanol consumption in rats (Berardo et al., 2016; Rockman et al., 1989). The possibility that enrichment may in some situations be detrimental rather than beneficial is not consistent with most of

25 the literature and, as suggested by van Praag et al., (2000), requires further investigation. The present study also demonstrated that effects of exposure to BZP during adolescence can endure for longer than the assessment period of the previous study (Aitchison and Hughes, 2006) thereby inviting further assessments at even

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older ages. Because of the difficulty in interpreting the effects of adolescent BZP on Y-maze novel-arm preferences, there is a need to disentangle the drug’s possible

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modification of cognitive development from development of anxiety or fear-related neophobia by, for example, adopting more suitable cognitive tests. The lack of

neurobiological assessments in the present study should also be addressed in future

A

CC E

PT

ED

M

A

N

U

related studies.

26 References 1. Achat-Mendes, C., Anderson, K.L., Itzhak, Y., 2003. Methylphenidate and MDMA adolescent exposure in mice: long-lasting consequences on cocaineinduced reward and psychomotor stimulation in adulthood.

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Neuropharmacology 45, 106-115. 2. Aitchison. L.K., Hughes, R.N., 2006.Treatment of adolescent rats with 1-

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benzylpiperazine: a preliminary study of subsequent behavioral effects. Neurotoxicol. Teratol. 28, 453-458.

3. Anderson, S.L., 2003. Trajectories of brain development: point of vulnerability

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4. Archer, J., 1973. Tests for emotionality in rats and mice: a review. Anim.

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36 Figure captions: Fig. 1. Effects observed at PND60+ and PND100+ combined of treatment during adolescence with two doses of BZP on open-field walking frequency for (A) rats housed in standard (SC) and enriched (EC) cages, and (B) for male and female rats.

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*Significantly different from the control group (0mg/kg) P < 0.05; #difference

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between the two cage conditions for 0 mg/kg dose significant, P < 0.05; §sex

A

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PT

ED

M

A

N

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difference for the 0 and 10 mg/kg doses significant, P < 0.05.

37 Fig. 2. Effects observed at PND60+ and PND100+ combined of treatment during adolescence with two doses of BZP on open-field grooming for (A) male and female rats, and (B) for all rats when observed at PND60+, and (B) PND100+. *Significantly different from the control group (0mg/kg) P < 0.05; asignificantly different from 10

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mg/kg, P < 0.05; #sex difference for the 10 and 20 mg/kg doses significant, P < 0.05;

A

CC E

PT

ED

M

A

N

U

SC R

§testing age difference for the 0, 10 and 20 mg/kg doses significant, P < 0.05.

38 Fig. 3. Effects observed at PND60+ and PND100+ separately of treatment during adolescence with two doses of BZP on plus-maze open-arm occupancy in rats housed in (A) standard (SC) and (B) enriched (EC) cages. *Significantly different from the control group (0mg/kg) P < 0.05; #difference between the two testing ages

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for the 10 and 20 mg/kg doses (Fig. 3A) and 0 mg/kg dose (Fig. 3B) significant, P <

A

CC E

PT

ED

M

A

N

U

SC R

0.05.

39 Fig. 4. Effects observed at PND60+ and PND100+ combined of treatment during adolescence with two doses of BZP on entries into the light-dark box light compartment for rats housed in standard (SC) and enriched cages (EC).

*Significantly different from the control group (0mg/kg) P < 0.05; #difference

A

CC E

PT

ED

M

A

N

U

SC R

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between the two cage conditions for the 0 and 20 mg/kg doses significant, P < 0.05.

40 Fig. 5. Effects observed at PND60+ and PND100+ combined of treatment during adolescence with two doses of BZP on transitions between the light-dark box light and dark compartments for rats housed in standard (SC) and enriched cages (EC).

*Significantly different from the control group (0mg/kg) P < 0.05; #difference

A

CC E

PT

ED

M

A

N

U

SC R

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between the two cage conditions for the 0 and 20 mg/kg doses significant, P < 0.05.

41 Fig. 6. Effects observed at PND60+ and PND100+ combined of treatment during adolescence with two doses of BZP on percent entries into the Y-maze novel arm in male and female rats housed in standard (SC) and enriched cages (EC).

*Significantly different from the control group (0mg/kg) P < 0.05; asignificantly

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different from 10 mg/kg, P < 0.05; #sex difference for the 20 mg/kg dose (Fig. 6B)

A

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PT

ED

M

A

N

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significant, P < 0.05.

I N U SC R 42

Table 1. Mean (± S.E.M, in brackets) values of all responses recorded in the open field, the elevated plus maze, the light-dark box and the Y maze, and results of ANOVAs for main effects (at PND60+ and PND100+ combined) of BZP dose, cage and sex. Means (± S.E.Ms) and F ratios for significant main

BZP dose (mg/kg) 10

(n=40)

(n=40)

M

0

ED

Response

A

effects are in bold type.

OPEN FIELD:

58.74

PT

Ambulation

58.25 (1.67

(1.74)

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Walking†#

A

Rearing

34.74

F(2,108)

(n=40)

59.46

0.21

(1.53) 35.47 (1.26)

(1.40) 41.85

20

Cage condition

36.75

0.99

(1.11) 41.64 (1.27)

(1.26)

39.91

0.74

(1.25)

SC

EC

(n=60)

(n=60)

58.12

59.52

(1.32)

(1.36)

35.83

35.47

(1.01)

(1.05)

40.50

41.77

(1.08)

(0.98)

Sex of rats F(1,108)

Males

Females

F(1,108)

(n=60)

(n=60)

0.81

53.03 (1.21)

64.60 (1.00)

55.39

0.09

32.11 (0.94)

39.20 (0.91)

35.97***

0.79

40.02 (1.16)

42.25 (0.86)

2.45

Grooming#Ω

3.35 (0.41)a

2.11 (0.27)a,b

3.28 (0.46)b

3.48*

3.35 (0.38)

2.47 (0.24)

4.16*

2.85 (0.27)

2.97 (0.37)

0.08

Immobility

19.80

20.40 (1.49)

19.74

0.07

20.19

19.77

0.07

24.06 (1.31)

15.90 (0.45)

25.74***

(1.36)

(1.26)

0.85 (0.18)

1.12 (0.33)

0.57

1.50 (0.34)

0.47 (0.15)

8.51**

(1.62) Fecal boluses

1.49 (0.31)

(1.53) 0.59 (0.16)

0.88 (0.44)

2.24

I N U SC R 43

Center occupancy

9.02 (0.60)

9.39 (0.64)

9.57 (0.68)

0.2

10.15

8.51 (0.38)

5.07*

9.38 (0.63)

9.27 (0.38)

0.02

41.87

43.91

1.97

44.98 (1.20)

40.79 (1.79)

8.32**

(1.01)

(1.07)

48.34

51.21

5.86*

48.87 (1.05)

50.59 (0.61)

2.35

(1.06)

(0.57)

48.88

51.54

3.23

47.08 (1.31)

53.34 (0.77)

17.89***

(1.12)

(1.16)

(0.61)

Corner occupancy

42.70

43.24 (1.44)

0.06

(0.98)

M

A

(1.41)

42.72

PLUS MAZE 50.94

49.29 (0.72)

ED

% Open entries

(1.01) 51.86

PT

% Open occupancy§

49.14 (1.15)

(1.32)

49.10

0.97

(1.35) 49.63

1.28

(1.68)

8.65 (0.32)

8.36 (0.30)

8.38 (0.38)

0.32

8.43 (0.22)

8.50 (0.32)

0.05

7.35 (0.23)

9.57 (0.23)

45.27***

Emergence latency

10.61

11.88 (1.43)

13.00

0.35

12.86

10.80

0.77

14.27 (2.15)

9.38 (0.86)

4.37*

(s)

(1.93)

(2.02)

(1.20)

Light-side entries†

8.40 (0.22)

7.96 (0.21)

8.07 (0.22)

0.16

7.51 (0.18)

8.52 (0.22)

13.76**

CC E

Closed-arm entries

A

LIGHT-DARK BOX

(2.61) 7.64 (0.26)

8.00 (0.29)

2.63

I N U SC R 44

occupancy

(1.35)

Light-dark

16.40

transitions†

(0.42)

44.61 (1.61)

44.42

1.06

(1.43) 14.94 (0.53)

15.57

2.48

(0.58)

A

47.09

43.88

46.87

(1.21)

(1.17)

15.57

15.70

(0.42)

(0.43)

56.46

55.96

(0.91)

(1.12)

53.63

53.58

(1.27)

(1.48)

8.22 (0.30)

7.63 (0.33)

3.2

45.04 (1.39)

45.71 (0.98)

0.16

0.05

14.58 (0.35)

16.69 (0.45)

15.39**

0.15

54.83 (1.11)

57.69 (0.89)

4.63*

0.01

55.03 (1.41)

52.18 (1.33)

2.23

2.12

7.20 (0.31)

8.65 (0.29)

13.11**

M

Light-side

ED

Y MAZE % Novel entries

55.94

55.46 (1.01)

PT

(0.90) % Novel occupancy

52.68

54.77 (1.38)

CC E

(1.35) 8.88

arms

(0.35)a

0.68

(1.70) 53.38

0.41

(2.21) 7.89 (0.33)

7.01 (0.41)a

7.22**

A

Entries into both

57.23

Main effect significant *P <0.05, **P <0.01, ***P <0.001; a,bDifference between dose groups with superscript in common significant (P <0.05); †Dose x cage interaction significant (see text); #Dose x sex interaction significant (see text); ¶Dose x cage x sex interaction significant (see text); ΩDose x testing age interaction significant (see Table 2 and text); §Dose x cage x testing age interaction significant (see Table 2 and text).

I N U SC R 45

Table 2. Mean (± S.E.M, in brackets) values of all responses recorded in the open field, the elevated plus maze, the light-dark box and the Y maze at two testing ages (PND60+ and PND100+), and results of ANOVAs for effects of testing age. Means (± S.E.Ms)

A

and F ratios for significant age effects are in bold type.

M

Testing age

PND100+0

F(1,108)

(n=120)

(n=120)

(n=40)

62.47 (1.05)

55.17 (1.25)

30.03***

Walking

35.38 (0.87)

35.93 (0.90)

0.30

Rearing

41.15 (0.86)

41.12 (1.02)

0.01

GroomingΩ

4.30 (0.43)

1.52 (0.13)

42.38***

Immobility

18.68 (1.08)

21.27 (1.33)

2.70

Fecal boluses

1.07 (0.17)

0.90 (0.30)

0.29

Center occupancy

9.68 (0.60)

9.27 (0.38)

0.84

A

CC E

Ambulation

PT

OPEN FIELD:

ED

PND60+

I N U SC R 46

Corner occupancy

42.92 (0.45)

0.02

50.57 (0.66)

48.89 (1.01)

2.16

% Open occupancy§

52.74 (0.75)

47.68 (1.29)

14.53***

9.78 (0.22)

7.14 (0.25)

98.59***

Emergence latency (s)

9.08 (0.73)

14.47 (2.05)

7.46**

Light-side entries

9.57 (0.19)

6.46 (0.18)

20.74***

Light-side occupancy

45.05 (0.85)

45.70 (1.24)

0.25

Light-dark transitions

18.77 (0.37)

12.50 (0.36)

22.02***

PT

Closed-arm entries

ED

% Open entries

M

A

PLUS MAZE

42.86 (1.02)

A

CC E

LIGHT-DARK BOX

Y MAZE

I N U SC R 47

54.26 (1.14)

% Novel occupancy

49.88 (1.58)

Entries into both arms

8.55 (0.31)

58.16 (1.03)

5.57*

57.34 (1.42)

11.48***

7.30 (0.23)

17.23***

M

A

% Novel entries

ED

*P <0.05, **P <0.01, ***P <0.001. ΩDose x testing age interaction significant (see Table 1 and text); §Dose x cage x testing age

A

CC E

PT

interaction significant (see Table 1 and text).

I N U SC R 48

M

BZP effects

ED

Apparatus and response

A

Table 3. Summary of significant effects at two testing days (PND60+, PND100+) of treatment with 10 or 20 mg/kg/day of benzylpiperazine (BZP) during adolescence.

OPEN FIELD

For EC rats only, higher following both doses.

PT

Walking

For males only, higher following the 20 mg/kg/day. For females only, lower following 10 mg/kg/day. For all rats, lower following 10 mg/kg/day at PND60+, and higher following 20 mg/kg/day at PND100+

A

CC E

Grooming

PLUS MAZE

% open-arm occupancy

For SC rats only, lower following both doses at PND100+ only.

I N U SC R 49

LIGHT-DARK BOX

For EC rats only, lower following both doses.

Light-dark transitions

For EC rats only, lower following both doses.

A

Light-side entries

M

Y MAZE

For males only, higher following 20 mg/kg/day.

ED

% novel-arm entries

For EC rats only, lower following 20 mg/kg/day. For all rats, lower following 20 mg/kg/day.

A

CC E

PT

Entries into both arms