The T-type calcium channel antagonist, Z944, alters social behavior in Genetic Absence Epilepsy Rats from Strasbourg

Accepted Manuscript Title: The T-type calcium channel antagonist, Z944, alters social behavior in Genetic Absence Epilepsy Rats from Strasbourg Authors: Wendie N. Marks, Nadine K. Zabder, Stuart M. Cain, Terrance P. Snutch, John G. Howland PII: DOI: Reference:

S0166-4328(18)31215-4 https://doi.org/10.1016/j.bbr.2018.12.021 BBR 11693

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

23 August 2018 30 November 2018 10 December 2018

Please cite this article as: Marks WN, Zabder NK, Cain SM, Snutch TP, Howland JG, The T-type calcium channel antagonist, Z944, alters social behavior in Genetic Absence Epilepsy Rats from Strasbourg, Behavioural Brain Research (2018), https://doi.org/10.1016/j.bbr.2018.12.021 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.

The T-type calcium channel antagonist, Z944, alters social behavior in Genetic Absence Epilepsy Rats from Strasbourg Wendie N. Marks (PhD)a *, Nadine K. Zabder (BSc)a, Stuart M. Cain (PhD)b, Terrance P. Snutch (PhD)b, and John G. Howland (PhD)a a

University of Saskatchewan, Department of Physiology, Saskatoon, SK, Canada Michael Smith Laboratories and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada

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*Corresponding

author at: University of Saskatchewan, Department of Physiology, Health Sciences Building, 107 Wiggins Rd, Saskatoon, SK, Canada; Tel: +1-306-966-2032; Fax +1306-966-4298; Email address: [email protected]

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Highlights  Male and female GAERS show social interaction deficits  The T-type calcium channel antagonist Z944 reverses some social deficits in GAERS  Z944 decreases introductory and aggressive behaviors in GAERS and control rats  Z944 increases anxiety behaviors in GAERS in an open field and elevated plus maze

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Abstract Abnormalities in social behavior are a co-morbid symptom of idiopathic generalized epilepsies such as childhood absence epilepsy. The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model is a spontaneously occurring absence epilepsy phenotype closely correlated to that of human absence epilepsies. Similar to the human conditions, GAERS display social abnormalities. Previous studies have only demonstrated social abnormalities in female GAERS, whereas social problems are observed in male and female patients. Seizures in GAERS result in part due to a gain-of-function missense mutation in the Cav3.2 T-type calcium channel gene. This study examined the effects of the pan-T-type calcium channel antagonist, Z944, on social interaction behaviors in male and female GAERS using an open field social interaction test. A second objective of this study was to examine the effects of Z944 on anxiety-like behavior in an open field locomotion test and elevated plus maze. Results showed a decrease in social activity in GAERS relative to non-epileptic control (NEC) rats. Acute, systemic Z944 (5 mg/kg; i.p.) consistently reduced introductory and aggressive behaviors in both GAERS and NECs, whereas, strain effects were observed for over-and-under crawl behaviors. In the open field locomotion test and elevated plus maze, Z944 increased anxiety-like behaviors in GAERS, whereas, Z944 produced inconsistent effects on anxiety-like behaviors in NECs. The results of this study suggest that the regulation of T-type calcium channel activity may be a useful strategy for the development of new therapeutic approaches for the treatment of social and affective abnormalities observed in absence epilepsy disorders.

Key words: GAERS, social interaction, T-type calcium channel, anxiety, Z944

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1. Introduction The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model is a spontaneously occurring absence epilepsy phenotype closely correlated to that of human absence seizures [1]. GAERS were derived from the selective breeding of an outbred Wistar colony of rats in Strasbourg that displayed bilateral and spontaneous spike-wave discharges (SWDs) throughout the cortex [2], a pathological phenotype characteristic of absence seizures [3,4]. Rats from the same Strasbourg colony free of any spontaneous SWD were also selectively bred to create the non-epileptic control (NEC) strain [2]. Seizures in GAERS are produced by abnormalities in thalamocortical circuitry in part due to a gain-of-function missense mutation in the Cav3.2 Ttype calcium channel gene [5–7]. Strong evidence also implicates a role for T-type calcium channels in the pathophysiology of seizures in children with absence epilepsy [8,9]. Absence epilepsy patients display interictal behavioral impairments in social and cognitive functioning, as well as comorbid psychiatric disorders [10]. Consistent with absence epilepsy patients, GAERS display an anxiety-like phenotype [11–18], and cognitive deficits are observed in associative, and recognition memory tasks [13,19]. Recently, we demonstrated that GAERS also display social motivation abnormalities as demonstrated through a sociability task [20]. Importantly, acute systemic treatment with the pan-T-type calcium channel antagonist, Z944, not only decreases the severity of seizures in GAERS [21], but also improves both the cognitive and social abnormalities observed in this strain [19,20]. Importantly, Z944 has also been shown to alter object recognition memory and social motivation in the NEC strain suggesting that T-type calcium channel regulation is not only involved in the modulation of seizure activity, but also cognition [19,20]. Interestingly, sociability deficits were only observed in female GAERS [20], whereas in disorders such as childhood absence epilepsy, social problems are observed in both male and female patients [10]. As social interaction in the sociability task is measured by the preference of a rat to explore a cage containing a stranger rat as opposed to an empty cage, it is possible that the sociability task does not measure alterations in qualitatively rich social behaviors, such as aggression. Researchers have previously quantified offensive and defensive behaviors in GAERS directed towards a stranger conspecific in a resident versus intruder home cage scenario [22]; however, only male GAERS were used. In this previous study, similar social behaviors in male GAERS relative to controls were observed [22]. The objectives of the present study were to: 1) determine whether the sex differences in sociability performance previously observed in GAERS are also observed in an open field social interaction test that enables a detailed and ethologically valid analysis of social interaction behavior, and 2) determine whether Z944 reverses potential behavioral alterations in an open field social interaction test. We have previously observed decreases in locomotor activity with Z944 treatment [13,19,20]. Considering decreased locomotor activity can be an indication of an increase in anxiety-like behavior in rodents [23], and that increased anxiety in rats could affect social interaction behaviors, a third objective of this study was to examine the effects of Z944 on anxiety-like behavior in both open field and elevated plus maze (EPM) tests. In the open field social interaction test, two rat conspecifics freely interact within an open field for 10 minutes. Given the deficits in sociability previously observed in female GAERS [20], we expected to observe a decrease in stranger proximity and introductory behaviors in female GAERS as these behaviors constitute similar exploratory behaviors that rats make in the sociability task. We also anticipated that altered stranger proximity and introductory behaviors in 2

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female GAERS would be restored by acute systemic Z944 treatment. Alternatively, we expected to observe comparable stranger proximity and introductory behaviors in male GAERS relative to NEC rats. We also expected to observe increased anxiety-like behaviors in GAERS in the open field locomotor and EPM tests. The effects of rat strain, sex, and drug treatment on the nonintroductory behaviors of the social interaction test, and the effects of drug treatment on the anxiety-like behaviors in the open field locomotion test and EPM are exploratory and therefore specific hypotheses were not made. 2. Materials and methods

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2.1 Animals All experiments were performed using GAERS and NEC rats bred at the University of Saskatchewan Lab Animal Services Unit (Saskatoon, Canada) [13] that ranged in age from 11-14 weeks. We have confirmed the presence of spontaneous SWDs in GAERS that are consistent in morphology with absence seizure activity typically observed in this strain [14] in the littermates of a subset of the GAERS used for this study [13,19]. Rats were maintained on a 12 hr day-night cycle (lights on at 7am) in a temperature-controlled room (21°C). All experiments were carried out during the light cycle. Standard rat chow and water were available ad libitum. Rats were group housed (2 or 3 per cage) in standard, ventilated polypropylene cages throughout the entire experiment. Rats were separated from their cage mates only during the testing sessions. Separate groups of animals were used for each behavioral task. Experimental procedures were conducted in accordance with the Canadian Council on Animal Care guidelines for humane animal use and were approved by the University of Saskatchewan Animal Research Ethics Board, project protocol #20130118.

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2.2 Drug preparation A detailed description of the characterization and synthesis of Z944 is provided in Tringham et al., 2012. On each testing day, Z944 was freshly prepared in a solution of 10% dimethyl sulfoxide (DMSO; Sigma Aldrich, St. Louis, MO) and 90% sodium carboxymethyl cellulose (0.5% in saline, Sigma Aldrich). All injections were administered intraperitoneally at a dose of 5 mg/kg in a volume of 5 ml/kg. The dose of Z944 used for the current study is based on findings from previous research which examined the dose-dependent effects of Z944 on sociability performance in female GAERS [20]. It was shown that a 5 mg/kg dose of Z944 improved sociability performance in female GAERS, whereas, a 10 mg/kg dose significantly affected locomotor performance in the task [20]. Injection of Z944 or vehicle was performed 15 min prior to task performance.

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2.3 Open field social interaction test The testing apparatus consisted of a circular arena (150 cm in diameter, 45 cm-high walls) made of white industrial plastic painted black (Fig. 1). Both the test and stranger rats freely explored the circular arena separately for a 10 min habituation period 24 hr prior to testing. During testing, the test rat and a stranger rat of the same strain, sex, and treatment freely explored the arena for 10 min. On test day, all test rats were marked on their back with black marker for identification while the stranger rats were left unmarked. The number of subjects per group in the open field social interaction test consisted of the following: male NEC (11 vehicle, 11 Z944), female NEC (13 vehicle, 11 Z944), male GAERS (10 vehicle, 10 Z944), female 3

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GAERS (14 vehicle, 12 Z944). All behavior was recorded with a video camera mounted above the circular arena. Behavior was scored using JWatcher (http://www.jwatcher.ucla.edu/) by a researcher blind to the strain, treatment, and sex of the test rats. Introductory, 20 cm proximity, over crawl, under crawl, and aggressive behaviors were scored based on previously described manual categorization and analysis of social interaction behaviors [24–26]. Stranger proximity consisted of the test and stranger rat being within a 20 cm proximity of each other, which may include active or non-active exploration. Introductory behaviors consisted of: nosing – rats touched or sniffed at each other’s noses, anogenital sniff – rat sniffed the hind end of the other rat, sniff – sniffed each other anywhere on the body except for the nose or anogenital region, follow – rat followed closely behind the other rat with purpose. Over crawl behaviors occurred when the test rat stepped on top of, stepped over, or pinned the stranger rat. Under crawl behaviors occurred when the test rat crawled under or was pinned by the other rat. Aggressive behaviors consisted of: threat or thrust – test rat aggressively approached the stranger rat with quick convulsive movements or was the aggressor in a fight, boxing – both rats stood up on their hind legs while facing each other and usually bat their forelegs together.

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2.4 Open field locomotion test The testing apparatus is identical to that used for the open field social interaction test. Rats freely explored the testing arena for a 10 min habituation period 24 hr prior to testing. On test day, rats freely explored the arena for 10 min. The number of subjects per group in the open field locomotion test consisted of the following: male NEC (5 vehicle, 4 Z944), female NEC (8 vehicle, 9 Z944), male GAERS (4 vehicle, 5 Z944), female GAERS (4 vehicle, 5 Z944). Locomotor behavior was recorded and scored using Noldus Ethovision EX 11.5. A circle of 100 cm in diameter in the middle of the testing arena was considered the inner circle. The distance travelled, and time spent in the inner and outer circles of the arena were analyzed. A reduced distance travelled and time spent in the inner circle was considered indicative of anxiety-like behaviors [27].

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2.5 EPM The maze was constructed from a plywood base (45 cm from the ground) painted white with white corrugated plastic floor and walls. The floor of the maze consisted of perpendicular interlocking arms (110 cm in length and 10 cm in width), with two pairs of arms containing either 45-cm-high walls (closed arms), or no walls (open arms) with a 0.5 cm edge. A trial began by placing a rat in the center of the maze, facing an open arm. The rat then freely explored the maze for 5 min. The number of subjects per group in the EPM consisted of the following: male NEC (8 vehicle, 8 Z944), female NEC (8 vehicle, 8 Z944), male GAERS (8 vehicle, 8 Z944), female GAERS (8 vehicle, 8 Z944). Locomotor activity and time spent in the closed and open arms was tracked using Noldus Ethovision EX 11.5. Less time spent in the open arms or more time spent in the closed arms was interpreted as anxiety-like behaviors [28]. 2.6 Data analysis All figures are representative of the means with the error bars showing the standard error of the mean (SEM). Statistical analyses of the social interaction and open field locomotion tests were conducted using SPSS Version 20 for Windows (IBM). A correlation matrix of the social interaction subcategories scored for the open field social interaction test was initially examined to determine the appropriateness of running a MANOVA. As the subcategories were not 4

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correlated, all statistical analyses were performed with separate four-way mixed-factor ANOVAs (Time Bin as the repeated-measures factor; Strain, Sex, and Treatment as the between-subjects factors). Each time bin represents the cumulative sum of behavior over a five min segment. The EPM was analyzed with separate univariate ANOVAs (Strain, Sex, and Treatment as the between-subjects factors). Significant interactions were followed up with independent-samples post hoc t-tests with Bonferroni corrections. Effect size was calculated with partial η2 which represents the total variability in each dependent variable that can be attributed to the independent variables. Small, medium, and large effect sizes are considered partial η2 values of 0.01, 0.06, and 0.014 respectively. Statistical significance was set at p ≤ 0.05 unless otherwise stated. The statistical results for significant main effects or two-way interactions that were qualified by significant three-way interactions are not reported in the results section. Nonsignificant post hoc results are also not reported in the results section. 3. Results

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3.1 Strange proximity The 10 min social interaction test was divided into two, five min time bins to assess changes in social behaviors over time. The amount of time rats spent within a 20 cm proximity of each other was initially analyzed to examine the duration of all passive and active social activity (Fig. 3A). Analysis revealed significant within-subjects Time Bin by Sex (F(1,84) = 6.67, p = 0.012, partial η2 = 0.074) and Time Bin by Treatment (F(1,84) = 27.92, p < 0.001, partial η2 = 0.249) interactions. Significant between-subjects main effects of Sex (F(1,84) = 29.80, p < 0.001, partial η2 = 0.262) and Strain (F(1,84) = 9.06, p = 0.003, partial η2 = 0.097) were also observed. Post hoc analysis of the interaction terms revealed that overall, vehicle treated rats significantly decreased in their duration of proximity from time bin one to two, whereas Z944 treated rats significantly increased in proximity (both p ≤ 0.001). A significant decrease in proximity was observed in Z944 treated rats relative to vehicle treatment only in the first time bin (t(90) = 6.52, p < 0.001). Examination of the group means to determine the nature of the main effects showed that overall, female rats had significantly decreased proximity relative to male rats. Also, GAERS showed significantly decreased proximity relative to NECs.

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3.1.1 Introductory behaviors The introductory behaviors nosing, sniffing, ano-genital sniffing, and following were combined for the analysis of frequency (Fig. 2A), and duration (Fig. 3B). A significant withinsubjects main effect of Time (F(1,84) = 61.19, p < 0.001, partial η2 = 0.421) and a significant Time Bin by Sex (F(1,84) = 4.26, p = 0.042, partial η2 = 0.048) interaction was found for the frequency of introductory behaviors. The between-subjects analysis revealed a significant main effect of Strain (F(1,84) = 6.49, p = 0.013, partial η2 = 0.072) and Treatment (F(1,84) = 57.48, p < 0.001, partial η2 = 0.406). Comparisons of the group means for the significant main effect terms revealed that all rats decreased in the frequency of introductory behaviors from time bin one to two. Further, GAERS showed a significantly decreased frequency of introductory behaviors relative to NEC rats, and all rats treated with Z944 showed a significantly decreased frequency of introductory behaviours relative to vehicle treated rats. Post hoc analysis of the Time Bin by Sex interaction revealed a significant sex difference in the frequency of introductory behaviors only for the second time bin (t(90) = -3.42, p = 0.001) where female rats showed less introductory behaviors relative to males. 5

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Analysis of the duration of introductory behaviors also revealed a significant withinsubjects main effect of Time (F(1,84) = 59.49, p < 0.001, partial η2 = 0.415) and a significant Time Bin by Sex (F(1,84) = 5.87, p = 0.018, partial η2 = 0.065) interaction. A significant between-subjects main effect of Treatment (F(1,84) = 45.29, p < 0.001, partial η2 = 0.350) was also observed. Similar to the frequency of introductory behaviors, all rats decreased in the duration of introductory behaviors from time bin one to two. Also, Z944 significantly decreased the duration of introductory behaviors relative to vehicle treated rats. Post hoc analysis of the Time Bin by Sex interaction revealed a significant sex difference in the duration of introductory behaviors only for the second time bin (t(90) = -3.38, p = 0.001), where female rats showed less introductory behaviors relative to males.

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3.1.2 Over crawl behaviors Significant between-subjects main effects of Strain (F(1,84) = 4.49, p = 0.037, partial η2 = 0.051) and Treatment (F(1,84) = 9.26, p = 0.003, partial η2 = 0.099) were found for the frequency of over crawl behaviors (Fig. 2B). Comparisons of the means for strain and treatment groups revealed that overall, GAERS showed a reduced frequency of over crawl behaviors relative to NECs, whereas rats treated with Z944 showed an increased frequency of over crawl behaviors relative to vehicle treated rats. Analysis of the duration of dominance behavior (Fig. 3C) revealed a significant withinsubjects Time Bin by Strain interaction (F(1,84) = 6.02, p < 0.016, partial η2 = 0.067). Post hoc analysis of this interaction showed GAERS had a decreased duration of dominance behavior relative to NECs during bin two (t(90) = 2.44, p = 0.016). The between-subjects analysis of duration showed a significant Strain by Treatment by Sex (F(1,84) = 6.59, p = 0.012, partial η2 = 0.073) interaction. Post hoc analysis revealed a significant decrease in the duration of dominance behavior in vehicle treated male GAERS relative to vehicle treated male NECs (t(19) = 2.8, p = 0.011). However, Z944 treatment in male GAERS significantly increased the duration of dominance behavior relative to vehicle treated male GAERS (t(18) = -3.13, p = 0.006).

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3.1.3 Under crawl behaviors Exploration of the frequency of under crawl behaviors (Fig. 2C) revealed a significant between-subjects Sex by Strain by Treatment interaction (F(1,84) = 4.31, p = 0.041, partial η2 = 0.049). Post hoc analysis showed a significant decrease in under crawl behaviors in vehicle treated male GAERS relative to vehicle treated male NECs (t(19) = 3.25, p = 0.004). A treatment effect was also observed only in male GAERS with Z944 treatment significantly increasing the frequency of under crawl behaviors relative to vehicle treatment (t(18) = -2.97, p = 0.008). Inspection of the three-way interaction also revealed an effect of sex within vehicle treated GAERS as females had a higher frequency of under crawl behaviors relative to males (t(22) = 2.85, p = 0.009). Analysis of the duration of under crawl behaviors (Fig. 3D) showed a significant between-subjects main effect of Treatment (F(1,84) = 6.25, p = 0.014, partial η2 = 0.069) and a significant Sex by Strain (F(1,84) = 9.65, p = 0.003, partial η2 = 0.103) interaction. Examination of the group means determined that overall, rats treated with Z944 showed significantly increased duration of under crawl behaviors relative to vehicle treated rats. Post hoc analysis of the interaction term revealed a significant decrease in the duration of under crawl behaviors in male GAERS relative to male NECs (t(40) = 3.31, p = 0.002). Analysis of the interaction term

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also showed that, within NEC rats, females had a significantly decreased duration of under crawl behaviors relative to males (t(44) = -3.24, p = 0.002).

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3.1.4 Aggressive behaviors The behaviors threat-thrust and boxing were combined to examine the frequency (Fig. 2D) and duration (Fig. 3E) of aggressive behavior. Analysis of the frequency of aggressive behavior over time revealed a significant within-subjects Time Bin by Strain by Treatment interaction (F(1,84) = 4.81, p = 0.031, partial η2 = 0.054). Between-subjects analysis of the frequency of aggressive behavior revealed a significant Strain by Treatment interaction (F(1,84) = 5.57, p = 0.021, partial η2 = 0.062). Post hoc analysis showed a reduction in aggressive behavior in vehicle treated GAERS relative to vehicle treated NECs in the second time bin (t(46) = 2.76, p = 0.008). Post hoc analysis also showed that Z944 significantly reduced aggressive behavior in NECs in the first and second time bin (both p ≤ 0.011), and in GAERS in the first time bin (t(44) = 3.26, p = 0.002). When the duration of aggressive behavior was considered, a significant within-subjects Time Bin by Treatment interaction (F(1,84) = 6.39, p = 0.013, partial η2 = 0.071) and a significant between-subjects main effect of Treatment (F(1,84) = 18.80, p < 0.001, partial η2 = 0.183) was found. Post hoc analysis showed that the duration of aggressive behavior significantly increased from time bin one to two in vehicle treated rats (t(47) = -2.66, p = 0.011). However, Z944 significantly reduced the duration of aggressive behaviors overall for both time bins.

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3.2 The effects of rat strain and Z944 on open field locomotion The 10 min locomotion test was divided into two, five min time bins to assess potential changes in locomotor behavior over time. Distance travelled within the entire circular arena was analyzed to examine general locomotor activity (Fig. 4A). Within-subjects analysis revealed significant Time Bin by Treatment (F(1,36) = 25.65, p < 0.001, partial η2 = 0.416) and Time Bin by Treatment by Sex (F(1,36) = 5.39, p = 0.026, partial η2 = 0.130) interactions. A significant between-subjects main effect of Strain (F(1,36) = 110.78, p < 0.001, partial η2 = 0.755) was also found. Post hoc analysis of the Time Bin by Sex by Treatment interaction revealed a significant decrease in locomotor behavior from time bin one to two in vehicle and Z944 treated female rats, as well as Z944 treated male rats (all p < 0.001). Analysis of the Time Bin by Treatment interaction revealed a significant decrease in locomotor behaviour in Z944 treated rats occurred only for the second time bin (t(42) = 3.03, p = 0.004). Overall, a significant decrease in locomotor behavior in GAERS relative to NECs was observed.

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3.2.1 Inner circle The distance travelled (Fig. 4B) and duration of time spent (Fig. 4C) in the inner circle of the open field was analyzed to assess anxiety-like behavior. A significant Time Bin by Strain interaction (F(1,36) = 7.25, p = 0.011, partial η2 = 0.168) was found for the distance travelled in the inner circle. A significant between-subjects Strain by Treatment interaction (F(1,36) = 4.89, p = 0.033, partial η2 = 0.120) was also found. Post hoc analysis revealed a significant decrease in distance travelled within the inner circle from time bin one to two only in NECs (t(25) = 4.36, p < 0.001). Post hoc analysis also showed strain differences in distance travelled within the inner circle were only observed in vehicle treated rats with GAERS traveling less distance than NECs

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(t(19) = -4.85, p < 0.001). Analyses further revealed that Z944 significantly reduced the distance travelled in the inner circle only in NEC rats (t(24) = 4.61, p < 0.001). Analysis of the duration of time spent in the inner circle of the open field revealed a significant within-subjects Time Bin by Treatment interaction (F(1,36) = 9.06, p = 0.005, partial η2 = 0.201). The between-subjects analysis showed a significant Strain by Treatment interaction (F(1,36) = 7.06, p = 0.012, partial η2 = 0.164). Post hoc analysis showed a significant reduction in the duration of time spent in the inner circle in vehicle treated GAERS relative to vehicle treated NECs (t(19) = -3.67, p = 0.002). Further, in the NEC strain, Z944 significantly reduced the duration of time spent in the inner circle relative to vehicle treatment (t(24) = 6.47, p < 0.001) for both time bins one and two (both p < 0.001).

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3.2.2 Outer circle Analysis of the distance travelled in the outer circle of the open field (Fig. 4D) revealed significant within-subjects main effects of Time (F(1,36) = 158.80, p < 0.001, partial η2 = 0.815) and Strain (F(1,36) = 84.77, p < 0.001, partial η2 = 0.702). Inspection of the group means revealed that all rats decreased in distance travelled in the outer circle from time bin one to two. Overall, GAERS travelled significantly less distance in the outer circle relative to NECs.

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3.3 The effects of rat strain and Z944 on EPM The total distance travelled (Fig. 5A), percent time spent in the closed arms (Fig. 5B), and percent time spent in the open arms (Fig. 5C) of the EPM was analyzed. For total distance travelled, a significant main effect of Strain (F(1,56) = 30.74, p < 0.001, partial η2 = 0.354) showed that GAERS travelled less distance overall relative to NECs. Analysis of the percentage of time spent in the closed arms revealed significant Strain by Sex (F(1,56) = 5.65, p = 0.021, partial η2 = 0.092), and Strain by Treatment (F(1,56) = 14.70, p < 0.001, partial η2 = 0.208) interactions. Post hoc analysis of the Strain by Sex interaction showed in the GAERS strain, female rats spent a significantly greater proportion of time in the closed arms relative to males (t(30) = -2.76, p = 0.010). Further, female GAERS spent a significantly greater proportion of time in the closed arms relative to female NECs (t(30) = -4.21, p < 0.001). Post hoc analysis of the Strain by Treatment interaction showed Z944 significantly reduced time spent in the closed arms relative to vehicle rats in the NEC strain (t(30) = 2.72, p = 0.011). However, Z944 treated GAERS spent significantly more time in the closed arms relative to Z944 treated NECs and vehicle treated GAERS (both p ≤ 0.021). Analysis of the effects of Strain, Sex, and Treatment on the percentage of time spent in the open arms revealed non-significant effects (all p ≥ 0.073).

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4. Discussion In these experiments, we characterized social interaction behaviors in male and female epileptic (GAERS) and non-epileptic (NEC) rats treated with the T-type calcium channel blocker, Z944. We further examined the effects of Z944 in GAERS and NEC on open field locomotor activity and activity in the EPM. The most notable findings are the following: an overall decrease in social activity in drug-naïve GAERS relative to NECs was observed. When examining the effects of Z944 on social interaction behaviors, however, the effects were dependent on both the type of social behavior as well as strain. Although Z944 reduced introductory and aggressive behaviors in both GAERS and NECs, Z944 increased over crawl and under crawl behaviors in GAERS. When the effects of sex are considered, females displayed a quicker reduction in social activity over time relative to male rats. In the open field locomotion 8

test and the EPM, GAERS showed significant reductions in locomotor activity relative to NECs, and increased anxiety-like behavior. Evidence from the open field locomotion test suggests Z944 increases anxiety-like behavior in the GAERS and NEC strains, a result that is not consistent with reduced anxiety-like behavior in the NEC strain in the EPM.

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4.1 Social interaction behavior in drug naïve GAERS and NEC rats A reduction in introductory behavior and stranger proximity was anticipated in female GAERS with the effects of strain and sex on over-and-under crawl and aggressive behaviors being exploratory. However, both male and female GAERS demonstrated significantly decreased frequency or duration of all the social interaction subtypes, an effect we did not expect to observe in male GAERS. We have previously examined sociability performance in male and female GAERS relative to NECs. In the sociability task, we observed decreased performance only in female GAERS [20]. Other researchers have investigated social interaction behavior in a home versus intruder cage context in male GAERS [22]. The duration of partner investigation and allogrooming activity, offensive, defensive, and self-grooming behaviors were examined. No alterations in behavior were found in male GAERS in this study [22]. However, alterations in social behavior are noted in both male and female rodents in other models of epilepsy [29–33]. The reduction in social interaction behaviors observed in GAERS could be attributed to the occurrence of absence seizures during task performance which would result in a general decrease in locomotor activity. However, we have previously shown that GAERS do not display SWDs during an object recognition memory task, despite demonstrating a decrease in locomotor activity [19]. Seizures in GAERS typically occur during periods of quiet wakefulness and are interrupted by periods of sensory stimulation and heightened attention [2]. However, as we did not measure seizure activity during the social interaction task, we cannot correlate the decreased locomotor activity observed during task performance with the occurrence of seizures in this study. The nature of the inconsistent findings in male GAERS may relate to the differences in how social behaviors were measured between studies. First, the context of the social tasks varied between the current study and previous measures of social interaction behavior in GAERS making direct comparisons difficult. Second, sociability performance in GAERS was examined in non-treatment matched conspecifics previously [20]; whereas, the current study examined social behavior in treatment-matched rats. Differences in social activity have been observed in non-treatment matched relative to treatment matched groups in a study examining the effects of early-life lipopolysaccharide exposure on social behavior in adolescent Sprague Dawley rats [34]. Future research should consider examining social behavior to both treatment and nontreatment matched conspecifics. Third, we chose to also examine the frequency of social interaction behaviors as frequency may be a more accurate measure of behaviors that occur only for brief instances, as we found for the over-and-under crawl, and aggressive behaviors. Indeed, when only duration is considered, we observed a non-significant effect of strain on introductory behaviors, an effect consistent with the findings of previous researchers on measures of partner investigative and allogrooming in male GAERS [22]. Lastly, although the categorization of behaviors used for the current study are typical for an open field social interaction test [24–26], the qualitative difference in the categorization of social interaction behaviors in the home versus intruder cage test used by previous researchers may also explain the lack of consistency between previous findings in GAERS and the current study [22].

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Consistent across studies that investigated the interaction of sex and the GAERS strain is a decrease in social activity in female GAERS. One potential explanation for this finding is that female GAERS have a greater seizure severity which may have resulted in greater behavioral impairment. Although SWDs in GAERS have been shown to be comparable across males and females [35], to the best of our knowledge, it has not been determined whether fluctuations in the estrus cycle affects SWDs in female GAERS. In the WAG/Rij model of absence epilepsy, an increase in SWDs have been observed during the proestrus phase suggesting that higher levels of progesterone enhance SWDs in this strain [36]. However, it is yet to be directly examined in animal models of absence epilepsy whether seizure severity correlates with alterations in social behaviors. Clinical evidence suggests that seizure severity does not mediate the severity of social abnormalities in patients with juvenile onset generalized epilepsies as social deficits are observed regardless of seizure control [37,38]. Rather, psychosocial abnormalities in patients may be attributed to abnormalities in brain functioning which are present prior to seizure onset and have a genetic or developmental origin [37].

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4.2 The effects of Z944 on social interaction behaviors in GAERS and NEC rats We hypothesized that Z944 would decrease introductory behaviors and stranger proximity in NEC rats, with an anticipated reversal of these behavioral impairments in female GAERS, a hypothesis made based on previous observations in a sociability task [20]. However, GAERS of both sexes showed an unaltered duration of introductory behaviors with a decrease observed only for the frequency of these behaviors and the duration of stranger proximity in the first time bin. Given introductory behaviors comprise most of the social interaction behaviors, a reduction in the frequency but not the duration of these behaviors likely reflects a qualitative difference in the way these two strains explore stranger rats rather than an overall reduction in introductory behavior in GAERS. Further, Z944 treated rats showed a non-significant difference in stranger proximity by the second time bin. As such, Z944 may generally decrease introductory behaviors in rats that show normal functioning of these specific social behaviors. Accumulative evidence suggests that Z944 can impair normal cognitive performance; however, Z944 improves alterations in cognition observed in the GAERS disease-state phenotype [19,20]. Examination of over-and-under crawl behaviors showed that GAERS displayed very little activity in these specific social subtypes. However, Z944 significantly increased over-and-under crawl behaviors in GAERS. Importantly, this demonstrates that Z944 did not globally decrease social interaction behaviors, which also suggests that Z944 did not produce a general decrease in locomotor activity. Further support for a non-generalized effect of Z944 on locomotor activity is demonstrated by total locomotor activity in the EPM (discussed in detail in section 4.3). As Z944 produced a general decrease in stranger proximity in both rat strains during the first time bin, it is important to note the possibility that Z944 may have increased anxiety during task performance. By the second time bin, however, the stranger proximity in Z944 treated animals was similar to those of vehicle treated rats and actually increased in duration rather than decrease possibly indicating decreased anxiety by the second time bin. Aggressive social behaviors in rodents are commonly measured as an indication of responsiveness to psychotropic drugs [39]. Unlike introductory behaviors, the occurrence of aggressive social behavior increases rather than decrease over time when two rodents are introduced to a novel environment and territories are established [40,41]. Indeed, in this experiment, total social activity decreased over the testing session, whereas aggressive behaviors increased over time in vehicle treated rats. However, we observed an overall decrease in 10

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aggressive behaviors in Z944 treated rats. Both typical and atypical antipsychotics are well documented to have anti-aggressive properties [39,42–44]. Therefore, the data from the current study suggests that Z944 may have antipsychotic properties. Indeed, both Z944 and the pan Ttype calcium channel antagonist, TTA-A2, have previously been demonstrated to reduce amphetamine or MK-801 induced hyperlocomotion, an assay predictive of antipsychotic efficacy [45,46]. Further, many neuroleptic agents block T-type calcium channels suggesting that their therapeutic efficacy may be associated with their T-type calcium channel activity [47,48]. Further research into the potential antipsychotic efficacy of Z944 is warranted. One potential explanation for the reduction in introductory and aggressive behaviors observed is that Z944 is having a sedative effect that is resulting in a decrease in locomotor activity. Indeed, other researchers have reported sedative effects of Z944 at doses of 30 and 100 mg/kg [49]. Although we have observed a decrease in locomotor activity at doses of 10 and 5 mg/kg [19,20], measures of sedation are not observed with Z944 treatment at these lower doses [21,49]. Further, in the current study, not all social behaviors were reduced with Z944 treatment as over-and-under crawl behaviors were increased. Although, previous research and the current findings suggest that the reduction in social interaction behaviors observed in this study are not the result of a sedative effect of Z944, we did not directly measure sedation in our testing cohorts. Therefore, we cannot definitely conclude that the rats did not experience a sedative effect from Z944. A detailed understanding of the mechanisms underlying the mediating effects of T-type calcium channels on social interaction behavior are yet to be determined. However, it is known that T-type calcium channels are involved in the regulation of neuronal burst firing [50]. Patterns of burst firing correlate with cognitive performance in rats [51–53]. In particular, burst firing in the thalamo-cortical network is implicated in regulating states of arousal, perception, and cognition (Jones, 2007). As thalamo-cortical areas are shown to be involved in mediating social interaction behaviors in rodents [54,55], and burst firing in the thalamo-cortical network is altered in GAERS [6], it is possible that the regulation of T-type calcium channel dependent burst firing in the thalamo-cortical network contributes to the altered social interaction behaviors observed in this study. However, further research is needed to directly examine these speculations.

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4.3 The effects of Z944 on anxiety-like behavior in GAERS and NEC rats Anxiety-like behavior in GAERS in the EPM and open field test have been widely reported [11–16,56], although to our knowledge the factor of Sex has only been controlled for by our laboratory [13]. In the current study, we observed an increase in anxiety-like behavior in the open field in both male and female GAERS; whereas, in the EPM, female GAERS spent more time in the closed arms of the maze relative to female NEC or their male conspecifics. In a previous study, we observed a female-specific increase in anxiety-like behavior in GAERS in the EPM [13]. Although surmounting evidence demonstrates an increased anxiety-like phenotype in GAERS overall, the current study and previous results from our laboratory suggest that female GAERS are particularly prone to display elevations in anxiety-like behaviors. T-type calcium channels have been implicated to play a role in the modulation of anxiety as Cav3.2 deficient mice display an elevated anxiety-like phenotype in a light/dark conflict test, EPM, and open field test [57]. The current study is the first to directly examine the effects of a Ttype calcium channel antagonist on anxiety-like behavior in rats. The effects of Z944 on anxietylike behaviors in the open field test and EPM are complex and task dependent. Z944 enhanced 11

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anxiety in both the GAERS and NEC strain in the open field test as demonstrated through a decrease in locomotor activity in general or in the inner circle. However, in the EPM, we observed a significant decrease in the amount of time spent in the closed arms in Z944 treated NEC rats relative to vehicle treated NECs, behavior indicative of a decrease in anxiety. Whereas, we observed an increase in time spent in the closed arms in Z944 treated GAERS. Importantly, in the EPM, the dose of Z944 used (5 mg/kg) did not decrease overall locomotor activity as observed following Z944 treatment at higher doses [19,20]. As a decrease in locomotor activity was not consistently observed in both the open field and EPM tasks following Z944 treatment, it suggests that alterations in locomotor activity are not the result of a general sedative-like effect of Z944. Therefore we believe that Z944 did not attenuate the anxiety-like phenotype observed in GAERS, but instead increased it. However, our findings should be interpreted with caution as sedation was not directly measured in our testing cohorts and thus, we cannot conclude with certainty that a decrease in locomotor activity during the open field task was not the result of a sedative effect of Z944 treatment. The findings of this study agree with previous research which demonstrated that Cav3.2 deficient mice show increased anxiety in a light/dark conflict test, EPM, and open field test [57]. This suggests that the therapeutic properties of Z944 in GAERS on cognition [19,20] do not generalize to affective disturbances. However, further tests should be conducted to determine whether Z944 ameliorates other affective disturbances observed in GAERS such as increases in depressive-like behavior [12]. As well, further research on the effects of Z944 on anxiety-like behavior is needed to resolve the inconsistent findings observed in the open field test and EPM in NECs.

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4.4 Conclusions In summary, alterations in social activity in both male and female GAERS were observed using an open field social interaction test, an effect not observed for sociability or home cage intruder tasks with this strain. The effects of Z944 on social interaction behavior depended not only on rat strain, but also the behavior subtype. Z944 consistently reduced the introductory and aggressive behaviors in both GAERS and NECs, whereas, strain effects were observed for the over-and-under crawl behaviors. This pattern of results suggests that distinct neural circuits that mediate social behaviors may be more susceptible to alterations in T-type calcium channel activity, an effect that this is further mediated by the T-type calcium channel mutation observed in GAERS. Several interesting and unanticipated effects of Z944 on behavior warrant further investigation. Of note, a Z944-induced reduction in aggressive behavior is consistent with the effects of neuroleptic agents on social behaviors. Future research that examines the effects of Z944 in animal models of schizophrenia may be particularly fruitful. As the current study investigated the acute effects of Z944 treatment, a characterization of the effects of chronic Z944 administration are justified and particularly relevant to the therapeutic potential of the drug. Overall, the results from the current study suggest that investigation into the regulation of T-type calcium channel activity may not only advance the understanding of the biological mechanisms of the social and affective abnormalities observed in disorders such as absence epilepsy, but may also be a useful strategy for the development of new therapeutic approaches.

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Funding This research was supported by a Brain Canada Multi-Investigator Research Initiative Grant with matching support from Genome British Columbia, the Michael Smith Foundation for Health Research, and the Koerner Foundation (PI: TS). Work in the laboratory of TS is supported by an operating grant from the Canadian Institutes for Health Research (CIHR; #10677) and a Canada Research Chair in Biotechnology and Genomics-Neurobiology. Work in the laboratory of JH is supported by an operating grant from CIHR (#125984) and matching funds for these experiments from the Saskatchewan Health Research Foundation and the University of Saskatchewan. Data Availability The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

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Declarations of interest: none

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Figure legends Figure 1. Schematic of the open field social interaction test. See methods for details.

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Figure 2. Frequency of introductory behaviors, over crawl behaviors, under crawl behaviors, and aggressive behaviors in the open field social interaction test. (A) GAERS showed significantly decreased introductory behaviors relative to NECs (*p ≤ 0.05). Z944 significantly decreased introductory behaviors relative to vehicle treatment (#p ≤ 0.05). In the second time bin, all female rats had decreased introductory behaviors relative to male rats (&p ≤ 0.05). (B) GAERS demonstrated significantly decreased over crawl behaviors relative to NECs (*p ≤ 0.05). Z944 significantly decreased over crawl behaviors relative to vehicle treatment (#p ≤ 0.05). (C) Vehicle treated male GAERS demonstrated significantly decreased frequency of under crawl behaviors relative to vehicle treated male NECs (*p ≤ 0.05). Z944 treated male GAERS showed significantly increased under crawl behaviors relative to vehicle treated male GAERS (#p ≤ 0.05). Vehicle treated female GAERS demonstrated a significantly increased frequency of under crawl behaviors relative to vehicle treated male GAERS (**p ≤ 0.05). (D) NEC rats treated with Z944 showed significantly decreased aggressive behaviors relative to vehicle treated NEC rats (*p ≤ 0.05). In time bin 1, Z944 treated GAERS showed significantly decreased aggressive behaviors relative to vehicle treated GAERS (**p ≤ 0.05). In time bin 2, vehicle treated GAERS showed significantly decreased aggressive behavior relative to vehicle treated NECs (#p ≤ 0.05).

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Figure 3. Duration of stranger proximity, introductory behaviors, over crawl behaviors, under crawl behaviors, and aggressive behaviors in the open field social interaction test. (A) GAERS showed significantly decreased stranger proximity relative to NECs (*p ≤ 0.05). Z944 significantly decreased stranger proximity relative to vehicle treatment in the first time bin (**p ≤ 0.05). All female rats had decreased stranger proximity relative to male rats (#p ≤ 0.05). (B) Z944 significantly decreased introductory behaviors relative to vehicle treatment (#p ≤ 0.05). In the second time bin, all female rats had decreased introductory behaviors relative to male rats (&p ≤ 0.05). (C) In male rats, vehicle treated GAERS showed significantly decreased over crawl behaviors relative to vehicle treated NECs across both time bins (*p ≤ 0.05). However, Z944 treated male GAERS showed significantly increased over crawl behaviors relative to vehicle treated male GAERS across both time bins (#p ≤ 0.05). In time bin 2, GAERS showed significantly decreased over crawl behaviors compared to NEC rats (**p ≤ 0.05). (D) Male GAERS demonstrated a significantly decreased duration of under crawl behaviors relative to male NECs (*p ≤ 0.05). In NEC rats, females showed significantly decreased under crawl behaviours relative to males (**p ≤ 0.05). Overall, Z944 treatment significantly increased under crawl behavior relative to vehicle treatment (#p ≤ 0.05). (E) Z944 significantly reduced aggressive behaviors relative to vehicle treatment (#p ≤ 0.05).

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Figure 4. Locomotor activity in the open field locomotion test. (A) GAERS showed significantly decreased total distance travelled compared to NECs (*p ≤ 0.05). In the second time bin, rats treated with Z944 showed significantly decreased locomotor activity relative to rats treated with vehicle (#p ≤ 0.05). (B) Vehicle treated GAERS travelled significantly less distance in the inner circle relative to vehicle treated NECs (#p ≤ 0.05). NEC rats treated with Z944 travelled significantly less distance in the inner circle relative to NEC rats treated with vehicle (*p ≤ 0.05). (C) Vehicle treated GAERS spent significantly less time in the inner circle relative to vehicle treated NECs (#p ≤ 0.05). Within NEC rats, Z944 significantly reduced the amount of time spent in the inner circle relative to vehicle treatment (*p ≤ 0.05). (D) Overall, GAERS showed significantly less distance travelled in the outer circle compared to NECs (*p ≤ 0.05).

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Figure 5. Exploratory activity in the elevated plus maze. (A) GAERS travelled significantly less distance throughout the entire elevated plus maze compared to NECs (*p ≤ 0.05). (B) In female rats, GAERS spent a significantly greater percentage of time in the closed arms compared to NECs (#p ≤ 0.05). In GAERS, females spent a significantly greater percentage of time in the closed arms relative to males (&p ≤ 0.05). NECs treated with Z944 spent a significantly decreased amount of time in the closed arms relative to vehicle treated NECs (**p ≤ 0.05). GAERS treated with Z944 spent a significantly increased amount of time in the closed arms relative to GAERS treated with vehicle (*p ≤ 0.05). (C) No significant group differences were noted for the percentage of time spent in the open arms of the elevated plus maze.

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