Adult forebrain NMDA receptors gate social motivation and social memory

Accepted Manuscript Adult forebrain NMDA receptors gate social motivation and social memory Stephanie Jacobs, Joe Z. Tsien PII: DOI: Reference:

S1074-7427(16)30162-9 http://dx.doi.org/10.1016/j.nlm.2016.08.019 YNLME 6528

To appear in:

Neurobiology of Learning and Memory

Received Date: Revised Date: Accepted Date:

20 June 2016 12 August 2016 25 August 2016

Please cite this article as: Jacobs, S., Tsien, J.Z., Adult forebrain NMDA receptors gate social motivation and social memory, Neurobiology of Learning and Memory (2016), doi: http://dx.doi.org/10.1016/j.nlm.2016.08.019

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Adult forebrain NMDA receptors gate social motivation and social memory Stephanie Jacobs1, and Joe Z. Tsien1*

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Brain and Behavior Discovery Institute and Department of Neurology, Medical College of Georgia at Augusta University Augusta, GA 30907, USA

ABSTRACT: Motivation to engage in social interaction is critical to ensure normal social behaviors, whereas dysregulation in social motivation can contribute to psychiatric diseases such as schizophrenia, autism, social anxiety disorders and post-traumatic stress disorder (PTSD). While dopamine is well known to regulate motivation, its downstream targets are poorly understood. Given the fact that the dopamine 1 (D1) receptors are often physically coupled with the NMDA receptors, we hypothesize that the NMDA receptor activity in the adult forebrain principal neurons are crucial not only for learning and memory, but also for the proper gating of social motivation. Here, we tested this hypothesis by examining sociability and social memory in inducible forebrain-specific NR1 knockout mice. These mice are ideal for exploring the role of the NR1 subunit in social behavior because the NR1 subunit can be selectively knocked out after the critical developmental period, in which NR1 is required for normal development. We found that the inducible deletion of the NMDA receptors prior to behavioral assays impaired, not only object and social recognition memory tests, but also resulted in profound deficits in social motivation. Mice with ablated NR1 subunits in the forebrain demonstrated significant decreases in sociability compared to their wild type counterparts. These results suggest that in addition to its crucial role in learning and memory, the NMDA receptors in the adult forebrain principal neurons gate social motivation, independent of neuronal development.

KEY WORDS: social motivation, sociability, social memory, NR1 knockout, NMDA receptor

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1. Introduction Social interactions are complex behaviors requiring multiple cognitive processes and interpretation of multifaceted social stimuli. A key aspect of a social interaction is the motivation of each organism to initiate and respond to the other. Social interaction is thought to produce an internal reward state which will, in turn, reinforce and promote approach behaviors (Ikemoto et al. 2015). Impaired or abnormal social behaviors have been known to exacerbate many psychiatric conditions including schizophrenia (Lai et al. 2014; Witten et al. 2014; Vaskinn et al. 2015), autism ( Lin et al., 2012; Wang et al. 2013; Enter et al. 2012; Bambini-Junior et al. 2014; Devine 2014), depression (Iniguez et al. 2014; Zanier-Gomes et al. 2015), and post-traumatic stress disorder (Eagle et al. 2013; Sripada et al. 2015; Sripada et al. 2016). Many of these conditions have also been found to be affected by dopamine activity in the brain. Dopamine has been widely studied for its roles in reward and motivational behaviors. For example, DA neurons showed a marked increase in calcium transients during social interactions (Gunaydin et al., 2014) , whereas decreased dopamine activity in the prefrontal cortex has been indicated in the altered social behaviors following social defeat stress (Watt et al. 2014; Jin et al. 2015; Novick et al. 2015). Further, when the DA neurons in the VTA were optogenetically stimulated, the mice significantly increased the investigation of a novel conspecific while the investigation of a novel object remained unchanged (Gunaydin and Deisseroth 2014). Largely missing thus far from the field of social cognition, is the analysis of downstream molecules that participate in regulation of social motivation during adulthood. It has been shown that the dopamine receptors interact closely with the N-methyl-d-aspartate (NMDA) receptor in the forebrain neurons in the cortex, hippocampus, striatum, etc. (Lee et al. 2002; Fiorentini et al. 2003; Sarantis et al. 2009; Varela et al. 2009; Hu et al. 2010; Vastagh et al. 2012). Consistent with this rationale, several studies showed that a genetic knockdown of the NMDA receptors throughout development in whole body resulted in altered social behavior (Mohn et al. 1999). 3

Deletion of the NMDA receptors in the GABAergic interneurons during development also caused deficits in social memories (Belforte et al. 2010). Similarly, social behaviors were impaired when NR1 was knocked out in parvalbumin (PV)-positive interneurons during development. These PV NR1 knockout mice displayed lower levels of approach to a novel mouse indicating reduced social motivation, as well as lower levels of exploration of the novel mouse, relative to the wild-type mice (Saunders et al. 2013). These observations added to the notion that impaired neural development may be the main cause for social impairment. However, the NMDA receptor is known for its role in regulating cognitive functions that is independent of neural development. The question is whether the NMDA receptors in adult principal excitatory neurons gate cognitive computational signals for social motivation and social interactions. Here we seek to determine, and differentiate, if the NMDA receptor in the forebrain neurons regulates social motivation vs. social memory in the adult brain. We examined this question by analyzing the inducible and forebrain-specific NMDAR1 knockout mice (iFB-KO) (Tsien et al. 1996a; Cui et al. 2004) in a sociability test, social recognition memory tests and non-social recognition test.

Results 1.1.

Novel Object Recognition

1.1.1. Short-term novel object recognition The novel object paradigm was utilized to determine the non-social object recognition memory in the iFB-KO mice.

In all our experiments, we temporally inactivated the NMDA

receptors in the forebrain principal neurons of adult mice five days prior to behavioral tests by feeding these mice tetracycline (Shimizu et al. 2000; Cui et al. 2004). First, the iFB-KO mice were tested in their ability to learn and remember an object for a short period of time - namely, a one-hour novel recognition test. These mutant mice, as well as littermate control mice, were first

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allowed to investigate two identical objects in the training phase and, after the delay time, were allowed to investigate one of the familiar objects from the training phase and one novel object (Figure 1a). A preference for the novel object over the familiar object indicated a memory for the familiar object as rodents are more likely to spend more time investigating an unfamiliar object. Both the iFB-KO mice and their wild-type littermates spent approximately equal amounts of time investigating the objects in the training phase (Wt: 32.02 ± 4.14 s; iFB-KO: 36.19 ± 4.47 s; p < 0.05; Figure 1b), spending an approximately equal percentage of time with both objects (Wt: 50.55 ± 1.73%; iFB-KO: 49.41 ± 2.20%; Figure 1c). When the distance that the animals travelled while investigating the two objects was measured, it was found that the iFB-KO mice travelled significantly less than the wild-type mice during the training phase (Wt: 2128.28 ± 176.20 cm; iFB-KO: 1577.59 ± 155.67 cm; p < 0.05; Figure 1d). After one hour the iFB-KO mice showed only a slight preference for the novel object (39.70 ± 4.39s), spending significantly more time with the familiar object (35.26 ± 4.27s) than their wild-type littermates (22.16 ± 3.65s; p < 0.05). Conversely, the wild-type mice spent a significantly greater percentage of time with the novel object (37.92 ± 4.98s; 62.15 ± 4.43%, p < 0.05; Figure 1c) than the iFB-KO mice (54.17 ± 2.74%). The wild-type animals spent significantly more time investigating the novel object in the recall session, than the familiar object in the training session (p < 0.05) indicating the wild-type mouse’s memory of the object from the training phase. Interestingly, there were no significant differences between the distanced travelled between the two groups while exploring the objects (Wt: 1588.46 ± 225.46 cm; iFB-KO: 1285.4 ± 110.30 cm). Long-term novel object recognition Similarly, we tested the iFB-KO mice in a 24-hour novel object recognition paradigm, where the delay between the training session and the recall session was 24 hours (Figure 2a). In the training session, the iFB-KO mice investigated a novel object as much as their wild-type 5

littermates (Wt: 38.83 ± 4.19 s; iFB-KO: 37.75 ± 37.75 s; Figure 2b). Over the course of the training round, the iFB-KO animals spent nearly the same amount of time investigating each object, as did their wild-type littermates (Wt: 50.90 ± 3.17%; iFB-KO: 51.59 ± 5.11%; Figure 2c). Both groups of animals also ambulated similar distances while exploring both objects (Wt: 1908.20 ± 205.16 cm; iFB-KO: 1515.49 ± 177.76 cm; Figure 2d). This demonstrates that the iFB-KO mice show similar motivation to explore a novel object as their wild-type littermates. After a 24-hour delay, each mouse was placed into the novel object arena with one of the, now familiar, objects from the training session, and a novel object (Figure 2a). The mice were allowed to explore for five minutes. The iFB-KO mice spent equal amounts of time investigating each object (familiar: 35.63 ± 4.58s; novel: 35.82 ± 4.50s). Their wild-type littermates, however, spent significantly more time investigating the novel object than the familiar object (familiar: 23.64 ± 3.43s; novel: 38.64 ± 5.33s; p < 0.05). The wild-type mice spend a significantly greater percentage of time investigating the novel object in the recall session than the object in the training session (61.67 ± 3.04%; p < 0.01) indicating their memory of the familiar object. Interestingly, the wild-type mice spent a significantly larger percentage of time investigating the novel object in the recall session than the iFB-KO mice (p < 0.05). These data indicate that the iFB-KO mice are unable to form a long-term memory of the object. Interestingly, when the distance travelled by the animals was plotted, the iFB-KO mice were found to travel significantly shorter distances than the wild-type animals (Wt: 1830.09 ± 182.02 cm; iFB-KO: 1264.06 ± 195.91 cm; p < 0.05). Taken together, the above experiments suggest that the mutant mice exhibited normal motivation to explore non-social objects, yet they were significantly impaired in the formation of long-term novel object recognition memories. 1.2.

Social Discrimination To investigate the social recognition memory of the iFB-KO mice, we utilized a social

discrimination paradigm consisting of a training session in which the subject mouse is presented 6

with a novel juvenile mouse, and a recall session in which the subject animal is allowed to investigate the familiar animal from the training session and a novel juvenile (Figure 3a). A preference for the novel stimulus animal over the familiar animal indicates a memory for the familiar conspecific. The social discrimination paradigm is advantageous due to the ability for each animal to act as its own control. For these tasks, we enclosed the stimulus juvenile males in wire mesh enclosures so that the interaction time is that of the subject animals and not influenced by the interest of the stimulus mouse. 1.2.1. Short-term social discrimination To determine the short-term social recognition memory of the iFB-KO mice, we tested them in a one-hour social discrimination paradigm. In the training session, the iFB-KO mice spent significant less time exploring the novel juvenile conspecific than their wild-type littermate (Wt: 80.43 ± 4.92s; iFB-KO: 57.13 ± 7.66s; p < 0.05; Figure 3b). These data indicate a reduced motivation for exploring a conspecific. When the distance that the mice travelled in the experimental arena was measured no significant differences were found (Wt: 1511.95 ± 184.55 cm; iFB-KO: 1187.98 ± 116.26 cm; Figure 3d). In the recall session, the iFB-KO mice did spend more time investigating the novel conspecific over the familiar conspecific (familiar: 43.39 ± 5.75s; novel: 56.19 ± 7.41s), but this difference was not significant. The investigation times resulted in a slightly greater percentage of time with the novel animal over the familiar animal (55.15 ± 5.39%; Figure 3c) but not significantly greater than chance, indicating that the iFB-KO mice did not form a memory for the conspecific. In the recall session, the wild-type littermates did spend significantly more time investigating the novel conspecific than the familiar conspecific (familiar: 38.69 ± 6.24s; novel: 63.03 ± 5.18s). The wild-type mice spend a significantly greater percentage of time investigating the novel conspecific than the familiar conspecific (63.16 ± 3.79%, p < 0.01). When the distance that the mice travelled while exploring the conspecifics

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was measured the wild-type mice did travel more than the iFB-KO mice but the difference was not found to be significant (Wt: 1475.52 ± 206.45 cm; iFB-KO: 1062.32 ± 120.01 cm; p = 0.09). 1.2.2. Long-term social discrimination To determine the long-term social recognition memory of the iFB-KO mice, we first allowed the mice to explore a novel mouse in the training session followed by a 24-hour recall session with the now-familiar mouse from the training session and a novel mouse (Figure 4a). Interestingly, the iFB-KO mice spent significantly less time investigating the stimulus conspecific than the wild-type mice (Wt: 96.95 ± 8.80s; iFB-KO: 58.57 ± 4.05s; p < 0.001; Figure 4b). When the distance travelled by the mice during the training session was measured, there were no significant differences found between the two groups (Wt: 1402.78 ± 159.41 cm; iFB-KO: 1178.59 ± 114.13 cm; Figure 4d). This indicated that the mutant mice had similar locomotor activity. After a 24-hour delay, the subject mice were allowed to explore the familiar mouse from the training session or a novel mouse.

When allowed to freely explore both stimulus

conspecifics, the iFB-KO mice did not show a preference for either animal (familiar: 53.01 ± 6.64s; novel: 64.82 ± 7.66s; Figure 4b) spending approximately equal time with each animal (53.84 ± 3.82%; Figure 4c). Conversely, the wild-type animals spend more time exploring the novel conspecific than the familiar conspecific (familiar: 32.35 ± 5.07s, novel: 70.89 ± 4.05s). The wild-type mice spent a significantly greater percentage of time investigating the novel mouse (69.95 ± 2.99%) than the iFB-KO mice (p < 0.01). Additionally, the wild-type mice ambulated significantly more during this task than the iFB-KO mice (Wt: 1609.90 ± 223.75 cm; iFB-KO: 1064.81 ± 122.52 cm; p < 0.05). These data indicate that the iFB-KO mice showed reduced social interaction during training, as well as impairment in social discrimination memory. 1.3.

Habituation-Dishabituation To further verify such social deficits, we also investigated the social memory of the iFB-

KO mice using the habituation-dishabituation social recognition paradigm. In this paradigm, the 8

subject mouse is exposed to the stimulus mouse for four one-minute sessions, separated by 10 minutes (Figure 5a). In a fifth one-minute session, a novel conspecific is used as a control to confirm that any reduction in exploration times is a result of prior exposure to the conspecific, not fatiguing effects (Dantzer et al. 1987; Winslow and Camacho 1995). In this paradigm, the wild-type mice showed significant decreases in the amount of time spent investigating the conspecific from the first session to the second session (1: 22.64 ± 2.15s; 2: 16.87 ± 1.65s; p < 0.05; Figure 5b), indicating a memory of the conspecific. The wild-type mice continued to decrease the amount of time spent investigating the conspecific in the third and fourth sessions (3: 12.27 ± 1.02s; 4: 6.51 ± 0.81s). The wild-type mice significantly decreased their exploration of the familiar conspecific from the first session to the second session (p < 0.05), from the second session to the third session (p < 0.05), and from the third session to the fourth session (p < 0.001), indicating their memory of the familiar mouse. When a novel conspecific was presented in the fifth session, the wild-type mice investigated it to a similar degree as the conspecific in the first trial (25.35 ± 4.07s), and significantly more than the stimulus mouse in the fourth session (p < 0.001). The iFB-KO mice show similar exploration in the initial exploration session as the wildtype animals (17.75 ± 1.58s). Interestingly, the iFB-KO mice show only modest decreases in investigation times from one trial to the next in the second, third and fourth sessions (2: 14.55 ± 1.97s; 3: 16.44 ± 2.04s; 4: 12.93 ± 1.08s). However, by the fourth session, the iFB-KO mice investigated the familiar conspecific significantly less than in the first exploration session (p <0.05). This effect is confirmed with the fifth session in which the iFB-KO mice spend significantly more time exploring the novel conspecific in that session than the familiar conspecific in the fourth session (5: 21.45 ± 1.85s; p < 0.001).

1.4.

Sociability test

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Finally, we tested the motivational preference for the iFB-KO mice for an object or a stimulus mouse when both are presented simultaneously. To measure the social approach behavior in the iFB-KO mice, we placed the conspecific male in the wire enclosure to allow the subject to approach and investigate either the object or the other mouse, without interference from the stimulus mouse (Figure 6a). Interestingly, when given a choice, the iFB-KO mice spend significantly more time investigating the novel object than the wild-type mice (Wt: 16.86 ± 2.28 s; iFB-KO: 40.43 s; p < 0.05; Figure 6b). Remarkably, the iFB-KO mice spent significantly less time investigating the novel conspecific mouse than the wild type mice (Wt: 96.52 ± 11.49 s, iFB-KO: 62.07 ± 5.84 s; p = 0.01). The iFB-KO mice spent a significantly smaller percentage of their time investigating the conspecific mouse than the novel object (Wt: 84.03 ± 1.89%; iFB-KO: 64.98 ± 5.98%; p = 0.01; Figure 6c). These results suggest that unlike the wild-type mice, which preferred to interact with conspecifics over objects, the loss of the NMDA receptor in the forebrain significantly reduced sociability.

2. Discussion NMDA hypofunction, or impaired NMDA function, has been implicated in several neuropsychological disorders including schizophrenia, autism, PTSD and mood disorders (Akbarian et al. 1996; Humphries et al. 1996; Gao et al. 2000; Tsai and Coyle 2002). It has been previously reported that the developmental knockdown of the NR1, expressing only 5 – 10% of the normal levels of the NR1 subunit impaired social behavior. These knockdown mice exhibited social withdrawal and social avoidance behaviors (Mohn et al. 1999) and reduced social interaction (Duncan et al. 2004; Halene et al. 2009). Similarly, when the NR1 subunit was knocked out in GABAergic neurons in the cortex and hippocampus during postnatal development, the mice were highly susceptible to social isolation-induced anxiety, and were impaired in nest building and short-term social recognition memory (Belforte et al. 2010). Interestingly, when the ablation of the NR1 subunits occurs in adulthood, the schizophrenia 10

phenotype in these mice was not observed. The adult knockout mice showed no deficit in social recognition, acoustic startle, spontaneous alteration or anxiety-related behaviors (Belforte et al. 2010), demonstrating the developmental dysfunction as a necessary precursor to schizophrenia. Thus, both these knockout and knockdown studies have led to the notion that these social deficits were due to abnormal development of neural circuits. However, given the role of the NMDA receptor in regulating cognitive behavior during the adulthood (Tsien et al. 1996b; McHugh et al. 1996; Tang et al., 1999; Kuang et al., 2010; Zhang et al., 2013), we reasoned that the NMDA receptors may regulate social motivation as a function of cognitive computation in the adult brain and is independent of neural development. By using an inducible knockout approach, we have demonstrated that the NR1 subunit regulates sociability, impairs social recognition and non-social object recognition. The iFB-KO mice showed significantly less social motivation and sociability. This is in line with other aforementioned observations in which reduced NR1 expression reduces sociability and social investigation. Interestingly, this study also provides evidence for impaired social memory in a conditional NR1 knock-out, and demonstrates that iFB-KO mice are impaired in object recognition memory, even at short time durations. These data are in agreement with other similar knock-out studies which describe the role of the NR1 subunit and the NMDA receptor in many forms of learning and memory, including object recognition memory (Rampon et al. 2000). Interestingly, Rampon et al also described impairments in social transmission of food preference and decreased fear response in a hippocampal-dependent fear conditioning paradigm (Rampon et al. 2000). It is important to point that inducible knockout of the NMDA receptors in the adult forebrain did not seem to alter motivation to explore non-social items such as novel objects. In light of this specific motivational effect, we conclude that the NMDA receptor in the adult forebrain plays a unique role in regulating social motivation, which is independent of neural development.

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Our current finding is also consistent with our previous evidence showing that postnatal forebrain overexpression of the NR2B subunit of the NMDA receptor enhanced social learning (Jacobs and Tsien 2012; Jacobs et al. 2015), whereas NR2A overexpression reduced social memory (Jacobs and Tsien 2014). These observations collectively point to the notion that certain aspects of impaired social motivation and social behavior seen in schizophrenia, depression, and autism patients may be readily alleviated with therapeutic treatment in adulthood. Given the fact that the NR2B subunit of the NMDA receptor plays a crucial role in enhancing memory, faster fear extinction, and social behaviors (Tang et al. 1999; Jacobs and Tsien 2012; Jacobs et al. 2015), one approach is to boost NMDA receptor functions by enhancing NR2B expression by its up-regulator, magnesium threonate (MMFS-01), or using NR2B-containing NMDA agonists such as rapastinel (GLYX-13) (Slutsky et al. 2010; Burgdorf et al. 2011; Moskal et al. 2014; Burgdorf et al. 2015; Liu et al. 2015). Preliminary testing with these compounds has been shown to enhance cognitive functioning in humans, by increasing the NR2B available in the brain. MMFS-01 was found to enhance executive function in older adults with cognitive impairments (Liu et al. 2015). Rapastinel was found to enhance cognition, and positively affect emotional learning in humans (Burgdorf et al. 2011; Moskal et al. 2014; Burgdorf et al. 2015). Such clinical successes highlight the importance of the basic understanding of the NR2B mechanisms for the development of new strategies to treat neurological diseases (Cyranoski 2012; Wang et al. 2014). It might be of great interest to examine whether enhancing NR2B-containing NMDA receptor can reduce social impairment in autism and schizophrenic patients. Further investigation of physiological signature in the forebrain regions of the iFB-KO mice will also lead to better understanding how the NMDA receptor actually gates social motivation.

3. Methods 12

3.1.

Animals and Genotyping For all experiments, adult male mice (8 – 12 months old) were used as subject mice.

Inducible NR1 forebrain-specific knockout mice (iFB-KO mice) were generated as previously described (Shimizu et al. 2000; Cui et al. 2004). The iFB-KO mice express the heterozygous CaMKII-Cre transgene (specific to the excitatory neurons in the forebrain regions), a heterozygous NR1-GFP transgene under the control of the tet-O promoter, heterozygous tetracycline transactivator (tTA) transgene, and the homozygous floxed – NR1 gene. Littermates lacking the Cre transgene were used as wild-type littermates. To knockout the NR1 gene in the forebrain, the mice were treated with doxycycline (mg/ml) for five days prior to testing. Genotyping was performed via tail biopsy samples using Cre primers, NR1-GFP primers, tTA primers and floxed NR1 primers. To reduce animal numbers, the same cohort of mice was used for each test (iFB-KO: n = 13; Wt: 11), at least one day of rest was allowed between paradigms. Stimulus animals were juvenile (one month old) males from the same genetic background as the iFB-KO mice. Stimulus animals were used for only one paradigm. Mice were maintained in a temperature and humidity controlled vivarium with a 12:12 light-dark cycle. All testing was done during the light phase. Mice were allowed free access to food and water, except during experimental procedures. Mice were extensively handled prior to any testing paradigm. To reduce the number of animals used, a single cohort of animals was utilized for the experiments with at least 24 hours of rest between trials. Subject animals were not reused for multiple experiments. Separate objects were used in the sociability task, the onehour novel object recognition task and the 24-hour recognition task to avoid conflicting results. All objects were tested prior to experimental procedures for balance and were emotionally neutral and novel to the cohort of animals used. All testing procedures were conducted in specially designed, sound- dampened, dimly lit behavioral rooms. Experimenters were blind to the genotype of the animals. This study was carried out in strict accordance with the 13

recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee of Georgia Regents University. Animals were habituated to the testing environment and the wire enclosures used for the social memory paradigms for three days, 10 minutes per day before testing. For these experiments a long plastic rectangular arena (55 cm x 32 cm x 20 cm) was used. The stimulus objects or animals were placed 7 cm from the edge of the arena, centered laterally (Figure 7). Stimulus animals were habituated to the wire enclosures prior to the beginning of the experiment. Stimulus animals were placed inside wire enclosures so that encounters would be initiated by the subject animals. The subject animal was said to be exploring the stimulus if the animal’s nose was directed toward the stimulus within 2 cm of the object or wire enclosure, touching, sniffing or licking the enclosure or object. 3.2.

Novel Object Recognition The novel object recognition paradigm was used to test the short-term (one hour) and

long-term (24 hours) memory in the same environment (Tang et al. 1999) and under similar conditions to the social recognition paradigm (Jacobs and Tsien 2012; Jacobs and Tsien 2014; Jacobs et al. 2015). To test the novel object memory of the iFB-KO mice, two identical objects were placed 7 cm from the edge of the arena in training phase in the appropriate location, as denoted by the shaded circles (Figure 1a). The subject mouse was placed into the arena for five minutes to freely explore. At the conclusion of the five minutes, the subject mouse is removed from the arena and returned to the home cage. The arena and objects were thoroughly cleaned with 70% ethanol. After the described time (one hour for short-term memory (Figure 1a), 24 hours for long-term memory (Figure 2a)), the recall phase began. For the recall phase, one of the identical objects was removed and replaced with a novel object. The subject mouse was placed into the arena and allowed five minutes to freely explore both the novel object and the 14

now-familiar object from the training phase. After the five minutes, the subject mouse was removed from the arena, and both the arena and the objects were thoroughly cleaned with 70% ethanol. The amount of time the mouse spent investigating the objects was determined, according to the criteria above, and recorded. Exploration of the object was defined as the mouse facing the object within 3 cm of the object, sniffing, touching or licking the object. The distance traveled during each phase was determined using BIOBSERVE software. 3.3.

Social Discrimination The social discrimination paradigm was used to test the short-term (one hour) and long-

term (24 hours) social recognition memory of the iFB-KO. This paradigm was employed because each mouse could act as its own control by having both the novel mouse and the familiar mouse presented simultaneously (Engelmann et al. 1995). After being habituated to the wire enclosure, a stimulus mouse was placed into the wire enclosure and then placed into the arena 7 cm from the edge. The subject mouse was placed into the arena for five minutes to freely explore the stimulus mouse. At the conclusion of the five minutes, the subject mouse and the stimulus mouse were removed from the arena and returned to their respective home cages. The arena and wire enclosure were thoroughly cleaned with 70% ethanol. The amount of time the subject mouse spent investigating the stimulus mouse in the wire enclosure was determined, according to the criteria above, and recorded. After the described time (one hour for short-term memory (Figure 3a), 24 hours for long-term memory (Figure 4a)) the recall phase was initiated. For the recall phase, the now-familiar stimulus mouse from the training phase is placed into the wire enclosure and placed into the arena; this is repeated with a second novel stimulus mouse. The subject mouse was then placed into the arena and allowed five minutes to freely explore both stimulus mice in their wire enclosures. After the five minutes, the subject mouse and the stimulus mice were removed from the arena and returned to their respective home cages. The arena and the wire enclosures were thoroughly cleaned with 70% ethanol. The amount of time 15

the mouse spent investigating the stimulus animals were determined, according to the criteria above, and recorded. The distance traveled during each phase was determined using BIOBSERVE software. 3.4.

Habituation-Dishabituation Finally, we utilized the habituation-dishabituation paradigm (Dantzer et al. 1987; Winslow

and Camacho 1995) in which the subject animal was allowed to investigate a single stimulus juvenile male for a short duration several times in succession. In the habituation phase, the subject mouse reduces the exploration of the stimulus mouse at each successive trial. The reduced investigation times in subsequent exposures are a result of a short-term memory of the familiar mouse. The dishabituation phase consists of a fifth exposure session in which the subject mouse is exposed to a novel juvenile male. This trial is used as a control for fatigue or disinterest of the subject mouse. To investigate the habituation-dishabituation paradigm in the iFB-KO mice, a juvenile male mouse was placed into the wire enclosure and placed into the arena (Figure 5a). The subject mouse was allowed to explore the stimulus mouse in the wire enclosure for one minute, after which both animals were returned to their respective home cages. After a 10-minute delay, the stimulus mouse was placed back into the wire enclosure, and then into the arena and the testing paradigm was repeated. This stimulus mouse was used for a total of four habituation sessions. For the fifth trial, a novel juvenile male was placed into the wire enclosure and then into the arena. The subject mouse is then given one minute to investigate the novel stimulus mouse. The amount of time the subject mouse spent exploring the mice in each session was measured using the exploration criteria stated above. 3.5.

Sociability

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To test the sociability in the iFB-KO mice, a novel object was placed at one end of the testing arena and a juvenile male stimulus mouse was placed in the wire enclosure at the opposite end (Figure 6a). This task investigates the preference of the subject mouse to explore either an object or a conspecific (Moy et al. 2004). An increase in the investigation of the object over the animal demonstrates that the subject animal is less social (Moy et al. 2004). The subject mouse was placed into the enclosure and allowed five minutes to freely investigate the object and the stimulus animal. At the conclusion of the five minutes, the subject mouse and the stimulus mouse were placed back into their respective cages. The object, wire enclosure, and the testing arena were thoroughly cleaned with 70% ethanol. The amount of time that the animal spent exploring each object was determined, according to the criteria listed above. The preference index was determined as the amount of time spent exploring the mouse divided by the total amount of time that the animal explored either the object or the conspecific mouse. 3.6.

Data Analysis Significance between genotypes was determined using a Student’s T-test or ANOVA

where appropriate. The difference was judged to be significant if p < 0.05.

4. Acknowledgements The authors would like to thank Fengying Huang for her assistance in the maintenance of the animal colony.

This work was supported by the National Institutes of Health

(R01NS079774).

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6. Figure Legends Figure 1: The iFB-KO mice have impaired short-term object recognition memory. a. Schematic illustrating experimental setup of the novel object paradigm in which the subject animal was allowed to freely explore two identical novel objects in the training round, followed by one familiar object and one novel object.. b. The iFB-KO mice were unable to distinguish the familiar object from the novel object and spent nearly equal time exploring each object. The wild-type mice spent significantly more time exploring the novel object than the familiar one (*p < 0.05). c. The wild-type mice spent a significantly greater percentage of their time with the novel conspecific than the iFB-KO mice in the recall session. The wildtype mice also spent significantly more time with the novel object that the familiar object in the training session indicating a memory for the familiar object. d. The iFB-KO mice did travel significantly less while exploring the novel objects in the training session (*p < 0.05), but this did not result in significant differences in the exploration times. This difference was not observed in the recall session.

Figure 2: The iFB-KO mice have impaired long-term object recognition memory. a. Schematic illustrating experimental setup of the novel object. b. The iFB-KO mice spent nearly equal time exploring each object in the recall session. The wild-type mice spent significantly more time with the novel object than the familiar object in the recall (*p < 0.05). c. The wild-type mice spent a significantly greater percentage of their time with the novel

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conspecific than the iFB-KO mice in the recall session. The wild-type mice also spent significantly more time with the novel object that the familiar object in the training (**p < 0.01). d. The iFB-KO mice did travel significantly less than the wild-type mice in the recall session during the exploration of the two objects (*p < 0.05).

Figure 3: Short-term social recognition ability is impaired in the iFB-KO mice. a. Schematic illustrating experimental setup of the social recognition paradigm. b. The iFB-KO mice spent significantly less time exploring the conspecific in the recall session than the wild-type animals (*p < 0.05). The wild-type mice spent significantly more time with the novel conspecific than the familiar conspecific in the recall session (**p < 0.01), while the iFB-KO mice did not demonstrate a significant preference for either animal. c. The wild-type mice spent a significantly greater percentage of their time with the novel conspecific, while the iFB-KO mice showed little preference for the novel conspecific. d. There were no significant differences in the distance travelled during either session in this social discrimination task.

Figure 4: The iFB-KO mice are impaired in long-term social recognition. a. Schematic illustrating experimental setup of the social recognition paradigm. b. In the 24-hour social recognition training session the iFB-KO mice spent significantly less time exploring the novel conspecific than the wild-type animals (***p < 0.001). The wild-type mice spent significantly more time with the novel conspecific than the familiar conspecific in the recall session, whereas the iFB-KO mice did not demonstrate a preference for either animal. The wild-type mice spent significantly less time exploring the familiar conspecific than the iFB-KO mice (*p < 0.05). c. The wild-type mice spent a significantly greater percentage of their time with the novel conspecific, while the iFB-KO mice showed little preference for the novel conspecific in the 24-hour recall session (**p < 0.01). d. The iFB-KO mice ambulated significantly less during the recall phase than the wild-type mice (*p < 0.05). 22

Figure 5: The iFB-KO mice are unable to learn a familiar conspecific as quickly as the wildtype animals in the habituation-dishabituation paradigm. a. Schematic illustrating experimental setup of the social habituation-dishabituation paradigm. b. In this paradigm, the iFB-KO mice showed only modest decreases in the exploration of the familiar conspecific, whereas the wild-type animals significantly decrease their exploration of the familiar conspecific over the first four trials (*p < 0.05, **p <0.001). Both groups did significantly increase their exploration of the novel conspecific in the fifth trial.

Figure 6: Decreased sociability in the iFB-KO mice. a. Schematic illustrating experimental setup of the sociability paradigm. b. The iFB-KO mice spent significantly more time exploring the novel object than the wild-type mice (*p < 0.05). Interestingly, the iFB-KO spent significantly less time with the novel conspecific that the wild-type mice (**p = 0.01). c. The wild-type mice spent a significantly greater percentage of their time with the novel conspecific than the iFB-KO mice (**p = 0.01).

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Highlights    

NR1 subunits are ablated in forebrain regions of the iFB-KO mice. iFB-KO mice are impaired in recognition memory. iFB-KO mice have significantly reduced social interaction. Social motivation is regulated by the NMDA receptor in the forebrain regions.

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