Behavioral Neuroscience 2015, Vol. 129, No. 1, 74 – 85

© 2014 American Psychological Association 0735-7044/15/$12.00 http://dx.doi.org/10.1037/bne0000024

Phenotypic Characterization of Nonsocial Behavioral Impairment in Neurexin 1␣ Knockout Rats Frederic Esclassan, Jennifer Francois, Keith G. Phillips, Sally Loomis, and Gary Gilmour

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Eli Lilly & Co. Ltd, Windlesham, United Kingdom Neurexins are neuronal presynaptic proteins that play a key role in mediation of synapse formation. Heterozygous partial deletions in the neurexin-1 gene (NRXN1, 2p16.3) have been observed in autism spectrum disorder (ASD) patients. NRXN1-␣ knockout (KO) mice present behavioral impairments that resemble some of the core ASD symptoms of social impairment and inflexibility/stereotypy. At present, a thorough assessment of cognitive function has yet to be completed. Rats, containing a biallelic deletion of the NRNX1-␣ gene on a Sprague Dawley background were compared to littermate wild types across a range of tasks designed to test functional domains disrupted in ASD and other neurodevelopmental disorders, including sensory perception (prepulse inhibition), attention (latent inhibition), associative learning (instrumental and Pavlovian conditioning), and memory (rewarded alternation T maze and spatial discrimination). NRXN1␣ KO rats were found to present with large and persistent nonsocial deficits, including hyperactivity, deficits in simple instrumental learning, latent inhibition, and spatialdependent learning. No deficit in sensorimotor gating was observed, despite the presence of an exaggerated startle response. Although KO animals were also able to learn a simple Pavlovian conditioning discrimination, they did display impaired latent inhibition. The presence of pronounced impairments in several domains in NRXN1␣ KO rats clearly suggests that nonsocial cognitive deficits can also be measured in an animal model of ASD. Further exploration of those deficits, both clinically and preclinically, as planned in the Innovative Medicines Initiative’s European Autism Interventions: A Multicenter Study for Developing New Medications program, may help to better understand the brain circuitry involved in ASD and therefore open new avenues to advance novel therapies. Keywords: autism, cognition, neurexin1, phenotype

number of candidate genes have been associated with increased disease susceptibility, many of which have known synaptic and axodendritic functions. Among these, heterozygous partial deletions and other disruptions in the gene coding for neurexin-1 (NRXN1, 2p16.3) have been repeatedly observed in ASD patients (Autism Genome Project Consortium et al., 2007; Bucan et al., 2009; Ching et al., 2010; Dabell et al., 2013; Gauthier et al., 2011; Glessner et al., 2009; Kim et al., 2008; Yan et al., 2008) and also other neurodevelopmental disorders including schizophrenia (Duong et al., 2012; Shah et al., 2010; Stewart, Hall, Kang, Shaw, & Beaudet, 2011). The incidence of total ASD cases that may be attributable to NRXN1 gene deletion has been estimated at 0.5% (Etherton, Blaiss, Powell, & Sudhof, 2009), compared to an estimated incidence of exonic NRXN1 deletion in the normal population of 0.02% (Ching et al., 2010; Kirov et al., 2009). Most of this work to date has shown that deletions or disruptions specifically of the alpha isoform of NRXN1 (Ching et al., 2010) are particularly implicated, thereby providing a candidate molecular target to manipulate in animal models. In this regard, studies using NRXN1-␣ knockout (KO) mice have already been conducted and potentially recapitulate aspects of ASD with regard to the behavioral phenotype presented. NRXN1-␣ KO mice exhibit deficits in social behavior, including reduced social approach and investigation behaviors (Grayton, Missler, Collier, & Fernandes, 2013). They also show impairments in synaptic transmission, sensory processing, and nest building; increased levels of repetitive grooming; and improvements in motor learning (Etherton et al., 2009).

Autism spectrum disorder (ASD) is a neurodevelopmental disorder clinically characterized by cardinal features of impaired social interaction and communication deficits, as well as pathological expression of restricted and repetitive behaviors (Won et al., 2013 for review). ASD can be highly heritable and a large

This article was published Online First November 24, 2014. Frederic Esclassan and Jennifer Francois, In Vivo Pharmacology, Lilly Research Centre, Lilly Research Laboratories, Eli Lilly & Co. Ltd, Windlesham, England, United Kingdom; Keith G. Phillips, Neurosymptomatics, Lilly Research Centre, Eli Lilly & Co. Ltd., Windlesham, United Kingdom; Sally Loomis and Gary Gilmour, In Vivo Pharmacology, Lilly Research Centre, Lilly Research Laboratories, Eli Lilly & Co. Ltd, Windlesham, England, United Kingdom. Frederic Esclassan and Jennifer Francois contributed equally to the preparation of this article. At the time of preparation, all authors were employees of Eli Lilly & Co. Ltd. The authors participate in the European Autism Interventions- A Multicenter Study for Developing New Medications Project that receives support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115300, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013), from the EFPIA companies in kind contribution and from Autism Speaks. Correspondence concerning this article should be addressed to Gary Gilmour, Lilly Research Laboratories, Eli Lilly & Co. Ltd, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, UK. E-mail: [email protected] 74

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NRXN1 KO RAT BEHAVIORAL PHENOTYPE

Further, sex-dependent differences in response to novelty and habituation processes have also been observed (Laarakker, Reinders, Bruining, Ophoff, & Kas, 2012). Although cognitive deficits are not always viewed as a core component of ASD symptomatology, impairments in cognitive domains of perception, attention, and executive function are commonly observed (Kas et al., 2014) and their relationship to social and communication differences remains significantly underresearched. As such, phenotyping animals lacking NRXN1-␣ across several behavioral and cognitive domains might help to understand the link between NRXN1 biology and cognitive dysfunction observed in ASD patients, perhaps ultimately offering new approaches for the development of novel therapies. This work was conducted in the context of the European Unionfunded Innovative Medicines Initiative, European Autism Interventions: A Multicenter Study for Developing New Medications (EU-AIMS). The major intentions of EU-AIMS are to facilitate development and validation of translational models of ASD, to hopefully advance novel therapies for treatment (Murphy & Spooren, 2012). Compared to mice, the use of rats in this context affords a broader repertoire of cognitive tests with which animals can be assessed and also somewhat simplifies the ability to combine behavioral work with in vivo measurement of other neurophysiological parameters. In the present study, Sprague-Dawley rats with a homozygous NRXN1-␣ KO received initial cognitive assessment in behavioral tasks designed to test functional domains disrupted in ASD, including perception, attention, associative learning, and executive function mechanisms.

Methods Subjects All experiments were conducted in accordance with the Council Directive, 2010/63EU of the European Parliament and the Council of September 22, 2010 on the protection of animals used for scientific purposes (www.eur-lex.europa.eu/LexUriServ/LexUriServ .do?uri⫽OJ:L:2010:276:0033:0079:EN:PDF), with approval of the Lilly Research Laboratories Institutional Animal Care and Use Committee. Adult male and female NRXN1-␣ KO rats were generated by SAGE® Labs (Saint-Louis, MO) in collaboration with Autism Speaks (www.autismspeaks.org). Rats were homozygous KOs, containing a biallelic deletion of the NRNX1-␣ gene on a Sprague Dawley background strain. All animals used in this study were generated from a Heterozygous ⫻ Heterozygous breeding strategy. A total of 21 NRXN1-␣⫺/⫺ rats (12 female, 9 male) were compared with 23 littermate NRXN1-␣⫹/⫹ wild type (WT) controls (12 female, 11 males), derived from 11 different litters. Upon arrival, rats were housed in standard housing conditions (07:00 hr to 19:00 hr light phase, controlled temperature and humidity, ad libitum water) for a period of 7 days before behavioral training started. During this time, they were acclimated to the food restriction regime (i.e., maintained at no less than 85% of their free-feeding weight) and were handled regularly.

Experimental Overview Animals were assessed in behavioral tests in the following order: (1) instrumental responding under a variable interval (VI)

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15-s schedule; (2) open field; (3) appetitive latent inhibition (LI); (4) spatial reversal; (5) prepulse inhibition (PPI); and (6) T maze alternation. For all assays, apart from LI and T maze, both gender and genotype effects were assessed. For LI, only the genotype effect was assessed, and for T maze only male rats were assessed. Instrumental responding under a VI 15-s schedule. Testing was conducted in standard operant chambers housed in sound and light attenuation chambers (Med Associates, St. Albans, VT). Two retractable levers were located either side of a recessed magazine where food pellets (Noyes, 45 mg, Formula P) were delivered from an automatic pellet dispenser. Experimental sessions were controlled and data recorded using programs written in-house using MedPC IV software (Med Associates, St. Albans, VT). Rats first underwent a 30-min session of magazine approach training, where a nose poke was rewarded with delivery of a pellet under a VI 60-s schedule of reinforcement (range ⫽ 15–105 s). Thereafter, both levers were presented and animals were rewarded for pressing under a VI 15-s schedule of reinforcement. Each daily test session was 30 min long and the animals were trained for 10 consecutive days. The total number of head entries and lever presses were recorded. Open field. Three identical circular arenas (50 cm high ⫻ 80 cm diameter) made of opaque Perspex were used. Experiments were run in the dark with only a strip of LED lights fixed to the inside of the top edge to deliver even lighting (5 lux) intensity inside each arena. Arenas were based on infrared light tables (100 cm long ⫻ 100 cm wide, 80 cm high) and monitored using overhead infrared cameras. The cameras fed into a Quad videoswitch, which in turn fed into a computer fitted with a video capture card. Testing consisted of two 10-min sessions spaced by 60 min, with the arena being cleaned between sessions. Image analysis was carried out with the video tracking software Ethovision XT V8 (Noldus, Wageningen, The Netherlands). LI. Testing was conducted in standard operant chambers as described for the VI task above. On the wall opposite the food receptacle, a single 100 mA house light was situated directly next to an auditory speaker, which allowed for the delivery of auditory stimuli via a programmable generator (ANL-926, Med Associates, St. Albans, VT). Animals were randomly allocated to either a preexposure or nonpreexposure group. For the preexposure condition, rats were exposed to 40 presentations of a 20-s tone (2000Hz, 90dB) that acted as a conditioned stimulus (CS), each separated by an average interval of 110 s. The CS had no consequence during this phase. For the nonpreexposure condition, animals were only exposed to the context (i.e., the operant chambers) with no CS presentation. Animals were trained on either of these protocols for 10 consecutive days. After the last preexposure session all rats underwent an appetitive Pavlovian conditioning procedure. These sessions consisted of a randomized presentation of 15 Pavlovian trials and 15 blank trials. For Pavlovian trials, each CS (20-s tone) was immediately followed by the delivery of two sucrose pellets. Blank trials were virtual trials where no CS or pellets were delivered and were used to assess the specificity of the response to the Pavlovian trials. Animals were trained on this procedure for 4 consecutive days. During CS presentation and virtual CS presentation during a blank trial, the number of head entries were recorded and compared to a 20-s pre-CS and a 20-s post-CS period. An index of acquisition was then calculated from the ratio of head entries as follows: Ratio ⫽ (CShead entry – PreCShead entry)/ (CShead entry ⫹ PreCShead entry). This ratio was calculated for each

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Pavlovian and Blank trial and then the averaged across a session for each trial type. Spatial reversal. The same arena as described for the open field test was used for this experiment, except that the gray Perspex external walls were removed to uncover an extramaze white cue panel oriented “North” and four equally spaced food magazines positioned at “North-East,” “South-East,” “South-West,” and “North-West” within the maze (see Figure 1). All cardinal orientations are relative to the position of overhead cameras used to track behavior in each arena. Each food magazine was connected to a pellet dispenser fixed on the outside of the arena. An LED indicator light placed 2 cm above each magazine was used to cue when food reward was available from that magazine. Arenas were monitored using overhead infrared cameras and Ethovision XT V8 video tracking software (Noldus, Wageningen, The Netherlands) was used for image analysis. Rats were initially habituated to the arena in a single 15-min session where each magazine light cued availability of single food pellet rewards (Test Diet, St. Louis, MO) from all four magazines under a fixed interval 15-s schedule. Subsequently, rats were trained to associate object approach behavior in a trigger zone (16 cm diameter, 20 cm from the wall, Object 1, Figure 1) with delivery of reward. All entries in trigger zones were measured relative to the detected nose point of animals. During this phase of training, the correct trigger zone was the same across all arenas and was a zone that was not subsequently used in discrimination and reversal stages of the study. To trigger delivery of reward, animals had to learn to pause by the object in the correct trigger zone. A pause was defined by Ethovision as less than 2% change in the overhead profile of the rat between successive video samples,

when the nose point of the animal was in the correct trigger zone. Upon successful pausing in the trigger zone, reward delivery was randomly cued by the magazine light at one of the four magazines. Two seconds after an animal entered the active reward zone, the indicator light in this zone was turned off and a new trial could start. Animals were allowed to complete as many trials as possible in a 30-min session and were trained in this manner for 4 consecutive days. For every session the floor and objects were cleaned with alcohol wipes between animals. Object training was followed by spatial discrimination and spatial reversal (see Figure 1) stages of testing. Animals were trained to discriminate between two identical objects placed in two opposite trigger zones. The two magazines equidistant from the objects were used to deliver rewards. Animals were rewarded for pausing only on the object located in the correct trigger zone (S⫹) while a pause in the incorrect trigger zone (S-) had no consequence. Trigger zones were counterbalanced across arenas and reward location was randomized for each correct trial. Animals received 30 min sessions of spatial discrimination testing over 3 consecutive days. Following this, spatial reversal testing used 40-min test sessions, where the first 10-min used similar locations of S⫹ and S- to that of the spatial discrimination sessions. During the next 30 min of the session the rule was reversed, where the previously nonrewarded spatial location (S-) was now associated with reward while no consequence followed a pause in the previously rewarded S⫹ zone. The total number of correct pauses and the percentage of correct pauses (number of correct pause/total number of pauses) were calculated for each animal for all test sessions.

Figure 1. Overview of the spatial reversal apparatus and protocol. Habituation (15 s), object training, spatial discrimination phase, and reversal steps of the task. The rewarded trigger zone (S⫹) used for each step of the task is shown in gray, the nonrewarded zone (S-) is shown by the dashed circle. Stars represents the location of the magazine were reward are delivered.

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NRXN1 KO RAT BEHAVIORAL PHENOTYPE

Prepulse Inhibition (PPI). Testing was conducted in eight ventilated sound-attenuated startle chambers controlled by SDI software (SR-LAB, San Diego Instruments, San Diego, CA). Each chamber contained a transparent acrylic tube (length: 20.3 cm, diameter: 8.9 cm) attached to a Plexiglas frame within a ventilated enclosure. Acoustic pulses and prepulses were delivered via a speaker mounted 17.5 cm above the tube. Movement inside the tube was detected by a piezoelectric accelerometer below the frame that transduced motion from within the cylinder. The amplitude of the whole body startle to an acoustic pulse was defined as the maximum of 100 one-ms accelerometer readings collected from pulse onset. PPI testing comprised of an habituation session, test session and pulse intensity, each separated by 24 hours. On Day 1, habituation to the startle chamber and stimuli was achieved by exposing rats to 15 min of background white noise at a level of 65 dB, followed by eight startle trials (40 ms, 120 dB); the intertrial interval averaged 15s (range ⫽ 10 –25 s). The total session lasted 17 min. The mean amplitude of the startle trials was calculated for each animal and used to create matched treatment groups for testing. For the test session, PPI was measured for four prepulse intensities: ⫹4, ⫹8, ⫹12 and ⫹ 16 dB above background noise (65 dB). The interstimulus interval was 120 ms and was defined as the time between onset of the prepulse and onset of the pulse. Animals were given a 5-min acclimation period (white noise only) followed by a sequence of six startle trials. This was followed by a pseudorandomized sequence of: eight prepulse-pulse ⫹ 4 dB trials (40-ms 69dB prepulse, 40-ms 120dB pulse); eight prepulse-pulse ⫹ 8 dB trials (40-ms 73dB prepulse, 40-ms 120dB pulse); eight prepulse-pulse ⫹ 12 dB trials (40-ms 77dB prepulse, 40-ms 120dB pulse); eight prepulse-pulse ⫹ 16 dB trials (40-ms 81dB prepulse, 40-ms 120dB pulse); eight no stimulus trials (recordings taken with no stimulus present), and eight pulse alone trials (40 ms, 120 dB). Finally, a sequence of six startle trials ended the test session. The intertrial interval averaged 15 s (range ⫽ 10 –25). The session lasted 25 min. Basal startle amplitude was calculated as the average of the eight pulse alone trials. PPI was calculated, for each animal and at each prepulse intensity, using the following formula: 100 ⫻ ([pulse alone – prepulse-pulse]/pulse alone). Startle amplitude was calculated as the average of the eight pulse trials. T maze rewarded alternation. The T maze (Apogee Engineering Analysis Solutions, Norwich, U.K.) track was constructed of matt black, 80-mm wide Perspex with 200-mm high transparent Perspex walls. The external lengths of maze edges were 860 mm (choice end), 1050 mm (return arm), and 225 mm (delay end) in length. The center-arm was 830 mm in length, and a door was located 630 mm from the choice point forming a holding area used during the delay phase of the task. Entry of the rats into specific areas of the maze was detected using infrared beam breaks and passed to a microcontroller (Arduino Mega 2560). Custom-made Matlab programs automatically controlled the maze protocol, allowing it to run without human intervention. Rewards were delivered by two pellet dispensers located at the end of each reward arm. Each trial on the maze comprised of two stages. During the sample (forced) phase, the rat was released from the holding area at the base of the T maze and allowed to run along the center-arm and forced to turn toward one of the reward areas to receive a sugar pellet reward. The animal then returned to the holding area. At the start of the test phase, the animal was allowed a free choice between the two arms of the T maze and was rewarded for visiting

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the arm not entered on the sample phase. At the end of the test phase, the animal was able to return to the start area, where it was confined for a 2-s intertrial interval. Left/right allocations for the sample and choice runs were pseudorandomized with no more than three consecutive sample runs to the same side. Animals were allowed to run 20 trials in a 1-hr period and had to complete a minimum of eight trials to be included in any measurement. The total number of trials performed, the accuracy (number of correct choice/number of trials) and the bias (maximum number of trial on one side [left or right]/total number of trial) was calculated for each animal. Statistics. All data were analyzed using Statistica v9.0 (Statsoft Ltd, Bedford, U.K.). Analysis of variance (ANOVA) was used to analyze all datasets, with sex (male or female) and genotype (KO or WT) as main factors for all assays, except LI and T-Maze. These studies did not include a main factor of sex as a result of a lack of power to determine this effect in each case. PPI and LI studies had additional main factors of preppulse (4 levels: ⫹4, ⫹8, ⫹12, ⫹16) and preexposure (preexposed or nonpreexposed), respectively. VI, open field, and spatial reversal studies also had a repeated measure of session, and spatial reversal studies had an additional repeated measure of day. Significant main effects or interactions were subsequently explored with Fisher’s least significant difference post hoc analyses. Several datasets required logarithmic transformation (open field, number of head entries during LI, and startle response during PPI) before being appropriate to conduct ANOVA.

Results Age and Body Weight Animals ages were from 18 to 24 weeks (average 21 weeks) at the time of testing and no differences in age were observed between sex or genotype (genotype, F(1,39) ⫽ 0.42, ns; sex, F(1,39) ⫽ 0.091, ns, Figure 2A). Before behavioral testing began, NRXN1 KO animals were found to be significantly lighter than WT littermate controls (genotype, F(1,39) ⫽ 50.78, p ⬍ .001; Sex ⫻ Genotype, F(1,39) ⫽ 14.05, p ⬍ .001, Figure 2B). This was apparent for both males and females despite there being a strong main effect of sex, F(1,39) ⫽ 359.24, p ⬍ .001, where female animals were generally much lighter than males.

Open-Field As presented in Figure 2C, both male and female NRXN1 KO rats were significantly hyperactive during both open-field sessions (genotype, F(1,39) ⫽ 0.6, p ⬍ .001). Although WTs displayed a significant level of habituation across sessions as activity levels decreased from Session 1 to Session 2 (Session ⫻ Genotype, F(1,39) ⫽ 12.02, p ⬍ .01, WT Session 1 vs. WT Session 2, p ⬍ .01), NRXN1 KO failed to habituate as their activity in Session 2 was not significantly different from that observed in Session 1 (Session ⫻ Genotype, F(1,39) ⫽ 12.02, p ⬍ .01, KO Session 1 vs. KO Session 2, p ⫽ .69).

PPI NRXN1 KO animals displayed a significantly greater startle response compared to WT animals (genotype, F(1,38) ⫽ 4.76, p ⬍

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LI

Figure 2. Age, weight, and locomotor activity in an open-field. All data are presented as Mean ⫾ SEM. A: Age in weeks at the beginning of behavioral testing. No significant difference of either sex (male, M, white bars and female, F, gray bars) or genotype (wild type [WT]⫹/⫹ and neurexin1 knockout [KO]⫺/⫺, open and striped bars) was observed. B: All groups were weighed before the beginning of behavioral testing. For both sexes (male, M, white bars and female, F, gray bars) a significant effect of genotype (WT ⫹/⫹ and NRXN1 KO⫺/⫺, open and striped bars, respectively) was observed with the NRNX1 KO animals were lighter than WT. C: Both sexes displayed hyperactivity in an open-field that did not habituate with repeated exposure. ⴱ p ⬍ .05, ⴱⴱⴱ p ⬍ .001 compared with WT.

.05), irrespective of sex (Figure 3, insets). Average PPI was calculated for both male and female and the statistical analysis revealed a significant main effect of sex (F(1,152) ⫽ 5.61, p ⬍ .05), with males presenting a globally greater PPI compared to females. No significant effects of genotype, Sex ⫻ Genotype, or Sex ⫻ Genotype ⫻ Intensity were found. The globally increased startle response observed in NRXN1 KO rats therefore did not lead to a measurable disruption of PPI (see Figure 3).

Instrumental Responding (VI 15) During magazine approach training, both male and female NRXN1⫺/⫺ animals exhibited significantly greater levels of head entry behavior than WT animals (genotype, F(1,39) ⫽ 25.43, p ⬍ .001, Figure 4A). During VI 15 training (Figure 4B), no difference was observed in instrumental learning capacity between female WT and KO animals. However, a deficit was observed for the male NRXN1 KO rats from Day 5 of acquisition onward, where they performed significantly fewer lever presses than male WT littermate controls (Sex ⫻ Day ⫻ Genotype, F(9,351) ⫽ 4.97, p ⬍ .001).

The statistical analysis reveal no significant main effect of sex (head entries, F(1,34) ⫽ 0.13, p ⫽ .7; blank, F(1,34) ⫽ 1.47, p ⫽ .2; or Pavlovian trial, F(1,34) ⫽ 0.96, p ⫽ .42) or Sex ⫻ Group (head entries, F(1,34) ⫽ 2.3, p ⫽ .1; blank, F(1,34) ⫽ 0.009, p ⫽ .92; or Pavlovian trial, F(1,34) ⫽ 1.82, p ⫽ .15) or Sex ⫻ Genotype (head entries, F(1,34) ⫽ 0.48, p ⫽ .49; blank, F(1,34) ⫽ 0.12, p ⫽ .72; or Pavlovian trial, F(1,34) ⫽ 0.12, p ⫽ .97) interaction for any of the parameters tested, therefore male and female were pooled for the final analysis in order to improve the power of the study. The total number of head entries during each Pavlovian conditioning session was recorded and no significant difference could be observed between genotype (genotype, F(1,38) ⫽ 0.223, ns; Genotype ⫻ Exposure, F(1,38) ⫽ 0.267, ns, Figure 5A). As presented in Figure 5B, WT and NRXN1 KO animals that were not preexposed to the CS both acquired the Pavlovian conditioning task after 4 consecutive days at a similar rate of acquisition (genotype, F(1,38) ⫽ 3.16, ns; WT nonpreexposed vs. KO nonpreexposed, p ⫽ .16). Head entry ratios were raised from 0.08 ⫾ 0.09 to 0.64 ⫾ 0.08 for WT animals, and from 0.25 ⫾ 0.06 to 0.62 ⫾ 0.08 for KO animals during this period. Preexposing WT animals to the CS for 10 days before the start of the conditioning procedure resulted in a significant delay in learning during Pavlovian trials from Day 2 onward (Session ⫻ Group, F(3,60) ⫽ 4.1, p ⬍ .05). This typical LI effect, observable in WT/preexposed animals, was not found in KO/ preexposed animals. The KO/preexposed animals showed a decreased head entry ratio compared to the nonpreexposed group on Day 2 of conditioning, whereas no significant difference could be observed on Days 1, 3, and 4 (Session ⫻ Group, F(3,54) ⫽ 0.92, ns; Day 2 nonpreexposed vs. preexposed, p ⬍ .05). No significant differences were observed between any groups for blank trials (Figure 5C), confirming the behavioral specificity of the LI effect.

Spatial Reversal Statistical analysis of the number of pauses performed by animals revealed a significant effect of Sex ⫻ Genotype, F(1,38) ⫽ 14.63, p ⬍ .001 (Figure 6A), highlighting that male NRXN1 KO animals performed fewer pauses then their littermate controls over test sessions (planned comparison M/KO vs. M/WT, p ⬍ .01). This effect was not observed in females (planned comparison F/KO vs. F/WT, p ⫽ .3). Analysis of acquisition and reversal of spatial discrimination revealed significant main effects of sex (F(1,38) ⫽ 10.94, p ⬍ .01), genotype (F(1,38) ⫽ 32.362, p ⬍ .001), and session (F(4,152) ⫽ 13.08, p ⬍ .001), and further a significant Sex ⫻ Genotype interaction (F(1,38) ⫽ 8.77, p ⬍ .01). On Day 1 of acquisition, female NRXN1 KO animals presented impairment in task acquisition with a significantly lower percentage of pauses in the correct location. However, these animals displayed equivalent performance levels to the WT controls during subsequent acquisition sessions (Figure 6B). During the 30-min period on Day 4 when the discrimination rule was reversed, no significant differences were observed between female WT and KO animals, either in terms of the 30-min averaged performance (Figure 6B, planned comparison F/KO vs. F/WT, p ⫽ .86) or the time-course of learning rate (Figure 6C, Sex ⫻ Genotype, F(1,37) ⫽ 10.67, p ⬍ .01, Sex x Genotype ⫻ Time, F(7,259) ⫽ 1.79, ns; planned comparison F/KO versus F/WT, p ⫽ .95). In contrast to females, male

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Figure 3. Startle response and pre-pulse inhibition. Data are presented as mean ⫾ SEM. Average prepulse inhibition measured in male (M) and female (F) NRXN1 knockout (KO) (⫺/⫺striped bars) and wild type (WT) (⫹/⫹ plain bars) animals. Prepulse inhibition was calculated at each prepulse intensity (69, 73, 77, and 81 dB) using the following formula: 100 ⫻ ([pulse alone {120dB} – prepulse-pulse]/pulse alone). No significant difference in prepulse inhibition was observed between either sex or genotype. The average startle response is presented in the insets and was measured on 8 120dB pulse trials. A greater startle response was observed for KO animals irrespective of sex. ⴱ p ⬍ .05 compared with WT.

NRNX1 KO rats displayed a significantly lower percentage of pauses in the correct location across all 3 days of acquisition compared to WT controls (planned comparison M/WT vs. M/KO, p ⬍ .001). Averaged across the reversal session, correct pauses were also significantly lower in male KO animals (Figure 6B, planned comparison p ⫽ .86), and remained significantly lower than those reached at the end of acquisition (Figure 6B, planned comparison Day 3 vs. Day 4 11– 40 min, p ⬍ .001). This effect was confirmed by the time-course data that showed NRXN1 KO animals had a significantly lower performance at every time-point measured after the first 5 min (Figure 6C). T maze rewarded alternation. After excluding animals that performed less than 8 trials over the 30 min session (one male KO and

two male WT), the total number of trials, accuracy and bias calculated for each animal were averaged. After applying this exclusion criterion, a total of seven animals were left per group and no significant differences were found in the number of trials completed by NRXN1 KO animals compared to WT controls (Figure 7A). With regard to accuracy, a significant main effect of the genotype (F(1,12) ⫽ 14.03, p ⬍ .01) was observed but no Day ⫻ Genotype interaction (F(2,24) ⫽ 0.230, ns), where NRXN1 KO animals displayed significantly lower levels of performance compared to WT littermate controls across all three testing sessions (Figure 7B). Similarly, for the bias measure a significant main effect of the genotype (F(1,12) ⫽ 5.95, p ⬍ .05) was observed but no Day ⫻ Genotype interaction

Figure 4. Acquisition of instrumental responding under a VI 15 schedule. Data are presented as mean ⫾ SEM. A: Total number of head entries performed by wild type (WT) (⫹/⫹) and NRXN1 knockout (KO) (⫺/⫺) rats during magazine approach training. Irrespective of sex, NRXN1 KO animals performed significantly more head entries than WT controls. ⴱⴱ p ⬍ .01, ⴱⴱⴱ p ⬍ .001 compared with WT. B: Total number of lever presses performed by WT (⫹/⫹, circle) and NRXN1 KO (⫺/⫺, semifilled circle) animals during acquisition of VI 15 responding. No difference was observed in instrumental learning in the female (F, gray circle) animals, while a significant deficit was observed for male (M, white circle) NRXN1 KO rats compared with respective controls. ⴱ p ⬍ .05 compared to M ⫹/⫹.

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Figure 5. Appetitive latent inhibition procedure. Data are presented as mean ⫾ SEM. A: Total number of head entries performed in each session. No significant effect of preexposure or genotype was observed. B: Pavlovian trials. C: Blank trials. Head entry ratios were calculated for preexposed (gray triangles) and nonpreexposed groups (white circles), for wild type (⫹/⫹) and NRXN1 knockout (⫺/⫺) animals. ⴱ p ⬍ .05, ⴱⴱ p ⬍ .01, ⴱⴱⴱ p ⬍ .001, compared with nonpreexposed group.

(F(2,24) ⫽ 1.40, ns), thereby showing that male KO animals showed a consistent increase in bias across all three test session compared to WTs.

Discussion These results are, to the best knowledge of the authors, the first profiling of a transgenic rat with an ASD-relevant genetic modulation across a broad repertoire of behavioral assays. Homozygous NRXN1␣ KO rats were found to present with large and persistent nonsocial deficits, including hyperactivity, deficits in simple instrumental learning, LI and spatialdependent learning. These deficits may be relevant in the context of ASD and other neurodevelopmental psychiatric disorders. Some of these deficits also appeared to vary significantly by sex, where in the broadest sense male KO rats were found to be more greatly impaired than females, which might be consistent with the human situation where the incidence of ASD in males is approximately four times that of females (Kirkovski, Enticott, & Fitzgerald, 2013). The most noticeable behavioral phenotype of NRXN1␣ KO rats was that of pronounced hyperactivity in both sexes, as measured initially in this study in an open-field environment. This hyperactivity could potentially relate to decreases in body weight in KO rats that were also apparent. However, a more thorough assessment of circadian activity, feeding behavior, and metabolism would be required to further explore those relationships. If hyperactivity can be considered to be a form of increased behavioral output, this was also observed in both male and female KO animals during the magazine approach training phase of the VI 15 task, where all KO animals showed an increase in head entry behavior. Also, during acquisition of a spatial reversal task, male NRXN1␣ KOs made significantly fewer pauses in total than WT animals did. Overall, these data suggest that a pronounced hyperactivity is found in NRXN1␣ KO animals that may be somewhat independent of test environment or behavioral endpoint. Interestingly, ASD patients often show comorbidity with several ADHD symptoms, including hyperactivity (Murray, 2010; Reiersen & Todd, 2008), and

the presence of significant overlap of shared biological processes disrupted in children with these two neurodevelopmental conditions has already been demonstrated (Martin et al., 2014). However, surprisingly, a previous study has reported an opposite phenotype in mice compared to rats, where homozygous NRXN1␣ mutant mice were less active in a novel environment (Grayton et al., 2013). Also in contrast, NRXN1␣ KO rats failed to show habituation to an open field upon reexposure to that environment, yet heterozygous KO mice were hyperactive and presented an enhanced habituation that was especially evident in males (Laarakker et al., 2012). This difference is difficult to explain at present, although several other cofactors such as anxiety and arousal may be relevant here. Indeed, previous studies have shown increased anxiety in NRXN1␣ KO mice (Grayton et al., 2013). The exaggerated responses to sensory stimuli, evident as a greater acoustic startle response, as well as observations of unpredictable home cage behavior (personal observations) reported in NRXN1␣ KO rats in the present study also tend to suggest a profile of higher anxiety in these animals although further studies would be required to definitively conclude this. Variable expression or reduced penetrance of the NRXN1␣ knockout manipulation combined with other genetic and/or environmental factors could influence the ultimate phenotype between species, with the rat homozygous NRXN1␣ KO profile being at the more pronounced end of the phenotypic scale. Although NRXN1␣ KO rats displayed signs of auditory hypersensitivity, these exaggerated acoustic startle responses did not lead to significant differences in sensorimotor gating as measured by the PPI paradigm. This result may seem surprising given reports of gating deficits in different sensory modalities across the ASD spectrum (Fournier, Hass, Naik, Lodha, & Cauraugh, 2010). Although PPI deficits have been observed in adult ASD (Perry, Minassian, Lopez, Maron, & Lincoln, 2007), fragile X (Yuhas et al., 2011) and Asperger’s syndrome (McAlonan et al., 2002), other studies have failed to see these effects (Kohl et al., 2014; Oranje, Lahuis, van Engeland, Jan van der Gaag, & Kemner, 2013; Ornitz,

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Figure 6. Acquisition and reversal of a spatial discrimination task. Data are presented as mean ⫾ SEM. A: Total number of pauses performed by male and female wild type (WT) (⫹/⫹, open circle) and NRXN1 knockout (KO) (⫺/⫺, semifilled circle) animals. B: Average group performance for male (white circle) and female (gray circle) rats across acquisition (d1-d3) and reversal (REV) stages. The % correct pause (number of pauses in the correct location/number of trials) was calculated for each session as an index of learning. The REV test session has been plotted as the first 10 min (REV1), where the rule was as for d1– d3 acquisition, vs. the final 30 min where the rule was reversed (REV2). ⴱ p ⬍ .05, ⴱⴱ p ⬍ .01, ⴱⴱⴱ p ⬍ .001 compared to WT; ## p ⬍ .01 compared with d4 0 –10 session. C: Timecourse of reversal learning performance for male (white circle) and female (gray circle) rats during the reversal stage. Data are presented in 5-min time bins. Time 5–10 corresponds to baseline (prereversal, REV1) performance, whereas Time 15– 40 corresponds to the period during which the rule was reversed (REV2). ⴱ p ⬍ .05, ⴱⴱ p ⬍ .01, ⴱⴱⴱ p ⬍ .001 compared with WT.

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Figure 7. T maze rewarded alternation task. Data are presented as mean ⫾ SEM. Animals that performed less than 8 trials were excluded from the analysis. A: Average total number of trials performed by wild type (WT) (⫹/⫹, open bars) and NRXN1 knockout (KO) (⫺/⫺, striped bars) males during three consecutive test sessions. B: Average accuracy and relative average bias of WT (⫹/⫹ open circle) and NRXN1 KO (⫺/⫺, semifilled circle) males during three consecutive test sessions. ⴱ p ⬍ .05, ⴱⴱ p ⬍ .01, main significant effect compared with WT controls.

Lane, Sugiyama, & de Traversay, 1993; Yuhas et al., 2011). Such inconsistent results may possibly suggest that sensorimotor gating deficits might only occur in subgroups of ASD patients or at different stages during the course of the illness. As a limitation however, the PPI measure obtained in the present study could be confounded by the exaggerated baseline startle response in KO animals, potentially masking the ability to observe any deficit in the first place. Yet as auditory hypersensitivity is consistently described in the ASD literature (Baron-Cohen, Ashwin, Ashwin, Tavassoli, & Chakrabarti, 2009; Gomes, Pedroso, & Wagner, 2008; Kohl et al., 2014; Tomchek & Dunn, 2007), this should be investigated further in NRXN1␣ KO animals as a core feature of the autism phenotype. NRXN1␣ KO animals displayed a number of learning difficulties, where their learning ability differed depending on the process under question. For instance, simple stimulus-response contingencies were preserved, as demonstrated by a similar rate of learning of all nonpreexposed animals (i.e., the control condition) during an appetitive Pavlovian LI task. In contrast to this, a striking sexdependent impairment in action-outcome associations was found during acquisition of an instrumental response task. Although female KO animals acquired a simple VI 15 instrumental schedule for food reward in a normal manner, male KOs were greatly impaired, showing little evidence of learning even after 10 days of training. It would seem unlikely that an alternative explanation for

this effect lay in differing levels of motivation to obtain food reward, as KO animals demonstrated apparently equivalent levels of motivation to perform the Pavlovian task mentioned above. Despite aspects of learning theory being used to a degree in educational strategies in autistic children, there has been surprisingly little clinical investigation on the nature of reward-based learning in ASD. One study reported that ASD patients performed as well as their mental age-matched controls in an instrumental conditioning task (Reed, Staytom, Stott, & Truzoli, 2011), suggesting a discrepancy with the data obtained from the NRXN1␣ KO animal model. However, studies that directly assess instrumental learning in patients are rare and this topic certainly requires further exploration, both clinically and preclinically, before conclusions on the translational validity of this aspect of the model can be determined. In parallel to the effects on simple nonspatial instrumental learning, NRXN1␣ KO rats also presented impairments when learning two tasks involving simple spatial discrimination; the rewarded alternation T maze and location discrimination in an open field environment. In rodents, spatial learning is well known to engage hippocampal circuitry, which may suggest some relationship of these deficits to impairments in episodic memory reported in ASD patients, also potentially attributable to alteration in hippocampal function (Bigham, Boucher, Mayes, & Anns, 2010). Note, however, that both the high levels of expressed hyperactivity and relative failure to acquire an instrumental re-

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sponse may still be potentially confounding regarding interpretation of the spatial discrimination deficits observed. NRXN1␣ KO rats showed significant side bias when performing the T maze, which although possibly reflective of learning and memory impairments, may also reflect a form of stereotyped behavior or behavioral rigidity. With such a complex phenotype being presented by the KO rats, it may ultimately be very difficult to unequivocally ascribe impairment in any single assay to dysfunction of a single psychological domain. Though reservations regarding alternative explanations of the origins of cognitive impairments must be held, the LI deficit observed in KO animals is much less likely to have been confounded by concomitant changes in activity, stereotypy, or behavioral inflexibility. Compared to WT animals, NRXN1␣ KO rats show no delay in learning a Pavlovian cue– outcome association after extensive exposure to the same cue in a noncontingent setting. To the best knowledge of the authors, LI has not yet been studied in ASD patients. Such measurements might be very informative however, notwithstanding the translational potential of the LI measure itself across species. Study of LI in the context of these constructs may offer a greater resolution with regard to the nature of the psychological impairment in ASD. Of particular interest might be the reported deficits in divided attention in ASD (Ciesielski, Knight, Prince, Harris, & Handmaker, 1995; Sinzig, Morsch, Bruning, Schmidt, & Lehmkuhl, 2008; Yerys, Wallace, Jankowski, Bollich, & Kenworthy, 2011) and what this might mean for LI processes in these patients. Another reason why the LI effect in NRXN1␣ KO rats might be important is the bridge it may offer to schizophrenic phenotypes. LI deficits are a common finding in schizophrenia (Lubow, 2005), and NRXN1 gene alterations have also been linked to a high risk of developing schizophrenia (Rujescu et al., 2009). Thus, whereas the LI deficit observed in NRXN1␣ KO rats might not specifically or exclusively relate to the human autistic phenotype, it may point to deeper construct validity of this aspect of the model. A final point should be made regarding expectations of translational validity of the NRXN1␣ KO rat behavioral phenotype. On the basis of the work done so far, it is difficult to consider there to be clear and simple phenotypic homology from mouse to rat to human, with there being as many differences in behavior as there are commonalities across species, where even the commonalities might potentially arise from disruption to different psychological processes. Sex differences, which were readily apparent in the rat, and the phenotypic difference between mono- and biallelic deletions must also be considered properly in an evaluation of translational validity. Multiple human studies have shown a significantly higher frequency of NRXN1 defects in patient populations in comparison to control populations, but NRXN1 alterations have also been identified in normal parents and healthy controls (Bucan et al., 2009; Ching et al., 2010; Feng et al., 2006; Hedges et al., 2012; Kirov et al., 2009; Rujescu et al., 2009; Sanders et al., 2011; Schaaf et al., 2012; Zweier et al., 2009). Therefore, there are clearly other genetic and/or environmental factors influencing the ultimate phenotype and degree of neurocognitive disabilities caused by defects in NRXN1 expression. What is important is that the availability of NRXN1␣ transgenic rats should technically facilitate some of the integrated studies that are now required to probe regional and circuit activity (e.g., via electrophysiology or neuroimaging methodologies) in the context of impairment to

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more completely understand the construct validity of aspects of the behavioral phenotype. Only by completing this level of work will a robust understanding of translational validity emerge. Altogether, the presence of pronounced nonsocial cognitive deficits in NRXN1␣ KO rats is clearly evident and is especially interesting in the context of recent hypotheses suggesting a link between cognition and social differences observed in ASD (Rajendran & Mitchell, 2007). Further exploration of these deficits, as planned in the Innovative Medicines Initiative EU-AIMS program, will help to determine the true translational validity of the models, to better understand the brain circuitry involved in ASD and hopefully open new approaches to novel therapies.

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Received July 28, 2014 Revision received September 24, 2014 Accepted October 2, 2014 䡲