Conceived and designed the experiments: SAJ JZT. Performed the experiments: SAJ. Analyzed the data: SAJ JZT. Contributed reagents/materials/analysis tools: JZT. Wrote the paper: SAJ JZT.
The authors have declared that no competing interests exist.
The ability to learn and remember conspecifics is essential for the establishment and maintenance of social groups. Many animals, including humans, primates and rodents, depend on stable social relationships for survival. Social learning and social recognition have become emerging areas of interest for neuroscientists but are still not well understood. It has been established that several hormones play a role in the modulation of social recognition including estrogen, oxytocin and arginine vasopression. Relatively few studies have investigated how social recognition might be improved or enhanced. In this study, we investigate the role of the NMDA receptor in social recognition memory, specifically the consequences of altering the ratio of the NR2B∶NR2A subunits in the forebrain regions in social behavior. We produced transgenic mice in which the NR2B subunit of the NMDA receptor was overexpressed postnatally in the excitatory neurons of the forebrain areas including the cortex, amygdala and hippocampus. We investigated the ability of both our transgenic animals and their wild-type littermate to learn and remember juvenile conspecifics using both 1-hr and 24-hr memory tests. Our experiments show that the wild-type animals and NR2B transgenic mice preformed similarly in the 1-hr test. However, transgenic mice showed better performances in 24-hr tests of recognizing animals of a different strain or animals of a different species. We conclude that NR2B overexpression in the forebrain enhances social recognition memory for different strains and animal species.
In all species that live in social groups, conspecific recognition is essential for individual and species survival. The ability to recognize another individual as familiar is the basis on which social relationships are founded. In primates, recognition of a conspecific as familiar relies primarily on visual and auditory cues. In rodents, and many other animals, olfactory or pheromonal cues are the primary cues for the development of social memory
Social memory is an emerging area of interest in memory research. It is not clear as to the cellular mechanisms of controlling the formation of social recognition memory, maximum duration of memory, as well as the brain areas involved in the acquisition and consolidation of social memories. One of the most well-known receptors for the modulation of synaptic plasticity in learning and memory is the
In this study, we set out to examine how changes in the NMDA receptor subunit composition may alter social memory and behavior. Specifically, we have investigated the effects of the genetic overexpression of the NR2B subunit in the forebrain regions, on social recognition between an adult male mouse and a juvenile male of the same strain using several paradigms. Furthermore, we also examine the social memories of the NR2B mice for recognizing different strains. Finally, we also examine NR2B mice on their social recognition of a different animal species (rats). Based on these studies, we find that the NR2B animals have enhanced learning and memory abilities, specifically across different strains and animal species.
Adult male NR2B transgenic animals, and their wild-type littermates, ages 6–9 months, were breed in house from previously generated lines. Animal genotypes were confirmed by PCR analysis of a tail biopsy. Animals were group housed (3–5 per cage) in a standard temperature and humidity controlled animal vivarium with free access to food and water except during social encounters. We used adult male mice aged 6–9 months based on previous studies demonstrating that younger male mice are more aggressive and show more sexually motivated behavior than their older counterparts which masks their recognition abilities
All protocols were approved by the Institutional Animal Care and Use Committee of the Georgia Health Sciences University, and are in strict adherence with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
The first paradigm we used, first described by Thor and Holloway
To assess the effect of acute isolation on social memory we separated group housed animals 24 hours prior to testing into individual cages identical to their original cages, and continued single-housing through the duration of the testing period. During the initial encounter a juvenile male mouse was introduced to the home cage of the adult male for 5 minutes. The amount of time the adult male mouse spent exploring the juvenile was measured.
After the 1-hr or 24-hr interval, either the same juvenile mouse or a novel juvenile mouse was placed back into the home cage of the adult mouse for 5 minutes. The time that the adult mouse spent exploring the juvenile mouse was recorded. A reduction in the amount of time spent exploring the familiar mouse is indicative of the adult mouse remembering the initial encounter with that animal.
The second paradigm that we employed was the habituation/dishabituation paradigm
To assess the habituation-dishabituation paradigm in our animals, we separated group housed transgenic animals and their wild-type littermates 15 minutes prior to testing into individual testing cages identical to their original cages. During the initial encounter a juvenile male mouse was introduced to the home cage of the adult male for 1 minute. The amount of time the adult male mouse spent exploring the white juvenile was measured. Exploration criteria used was the same as previously stated. After one minute the juvenile mouse was removed from the testing cage and placed into a holding cage. After a ten minute delay the juvenile mouse was reintroduced into the testing cage with the adult male mouse for the second trial. This was repeated for four trials. For the fifth trial, a novel juvenile is introduced into the testing cage with the adult male. This trial is used as a control to demonstrate that the reduction in investigation is not due to fatigue or habituation.
To assess the effect of a different color and strain of the stimulus mouse on social memory, we separated group housed transgenic animals and their wild-type littermates (B6× CBA strain) 15 minutes prior to testing into individual testing cages identical to their original cages. During the initial encounter a white juvenile male mouse (BALB/c) was introduced to the home cage of the adult male for 5 minutes. The amount of time the adult male mouse spent exploring the white juvenile was measured. Exploration criteria used was the same as previously stated. At the conclusion of the training the animals used for the 24 hour recall testing were returned to their home cage with their littermates. Animals used for one hour recall training remained in their testing cages for timing reasons.
After 1-hr or 24-hr intervals, either the same juvenile white mouse (BALB/c) or a novel white juvenile mouse (BALB/c) was placed back into the testing cage of the adult mouse for 5 minutes. The time that the adult mouse spent exploring the juvenile mouse was recorded. A reduction in the amount of time spent exploring the familiar mouse is indicative of the adult mouse remembering the initial encounter with that animal.
We also wanted to investigate the social memory between distinct rodent species. We tested the memory of the transgenic NR2B mice and their wild-type littermates for juvenile male rat counterparts (Long Evans). To protect our transgenic animals from previously noted muricide
After the 1-hr or 24-hr interval, either the same rat or a novel rat was placed back into the chamber, and the chamber was placed back into the testing cage with the adult mouse for 5 minutes. The time that the adult mouse spent exploring the chamber was recorded. A reduction in the amount of time spent exploring the rat chamber is indicative of the adult mouse remembering the initial encounter with that animal.
All data are expressed as mean ± SEM. Student T-tests were used to analyze significance of initial exploration versus recall exploration sessions. Differences between groups were analyzed using one-way ANOVA with Tukey-Kramer analysis. Significance level was p<0.05.
To study social memory in mice, we have used two paradigms of social recognition behaviors. In the first paradigm, an exploratory preference was obtained by comparing the amount of time the subject animals spent exploring the stimulus animal in the second trial with that of the first. A reduction in the amount of time spent exploring a familiar animal in the second exposure was interpreted as the recognition of that animal. In the second paradigm, the subject animal was exposed to a stimulus animal several times within a short period of time. The reduction of investigation times in each subsequent encounter was interpreted as a recognition memory of the stimulus animal.
In order to test the social memory in our transgenic animals, we first tested them without any prior isolation from their cage mates. Each animal was separated into a testing cage identical to their home cage and exposed to the stimulus animals for two 5 minute encounter session separated by 1 hour. In the one hour delay test, when a different juvenile was used in the second encounter session both groups had similar initial investigation times (NR2B: n = 5, 144.2±21.60 s; Wt: n = 15, 117.59±13.21 s;
A. Initial encounter and one hour recall using a novel mouse. Here this is no reduction in the investigation time during the second encounter. B. Both the NR2B and the wild-type animals demonstrate a significant reduction in investigation times of the familiar juvenile indicating a memory of that animal. (* p = 0.037, ** p = 0.005) C. There were no significant differences seen between the investigation times in the initial encounter and the second encounter 24 hours later when a novel animal was used at the second encounter. D. Both groups of animals spent significantly less time investigating the familiar animal at the 24 hour recall session. (* p = 0.045, ** p = 0.02).
We then tested the animals using the identical animal in the second encounter, one hour after the first encounter. In this experiment, there were no significant differences in exploration times between the groups in the initial encounter, demonstrating that both groups had similar motivation and interest in the juveniles (NR2B: n = 5, 145.46±24.22 s; Wt: n = 14, 111.52±7.46 s;
We then tested the short-term social memory in our transgenic animals by using a 24 hour isolation protocol. We separated the mice into single cages for a period of 24 hours immediately before testing and continuing throughout the duration of the test to determine if acute isolation would alter their social behavior. For the testing period the subject animal was exposed to the stimulus juvenile animals for two 5 minute encounter sessions separated by 1 hour. In the first session, both groups had similar initial investigation times (NR2B: n = 5, 119.27±11.26 s; Wt: n = 15, 112.45±9.60 s,
A. No reduction in investigation times was seen in the second encounter with a novel animal after one hour. B. Initial encounter and one hour recall using familiar mouse. Here a significant reduction in the investigation times is seen in both the NR2B and the wildtype animals (* p = 0.018, ** p = 0.029) C. There was no reduction in investigation time at the 24 hour recall encounter using a novel mouse. D. A significant reduction was seen in the investigation times of the familiar mouse in both groups after 24 hours. (* p = 0.0136, ** p = 0.001).
We then tested the animals using the identical animal in the second encounter one hour after the first encounter. Again, there were no significant differences in exploration times in the initial encounter, demonstrating that both groups had similar motivation and interest in the juveniles (NR2B: n = 5, 138.71±9.16 s; Wt: n = 15, 127.95±9.26 s,
It has been suggested that socially grouped mice also exhibit long-term social recognition memory. Thus, we examined the long-term social recognition memory of our animals, again using group-housed animals and acute isolation protocols. We first tested long-term social memory of group housed NR2B mice and their wild-type littermates. When we tested the animals using different stimulus juveniles in both encounter sessions, we found no significant differences between the groups in the initial encounter (NR2B: n = 5, 116.62±12.99 s; Wt: n = 15, 114.36±8.62 s,
We then tested our transgenic animals using the identical stimulus juvenile in both encounters. In this experiment, NR2B animals and their wild-type littermates exhibited similar investigation times in the initial encounter (NR2B: n = 5, 110.43±12.53 s; Wt: n = 14, 98.44±6.16 s,
We performed another experiment by using an acute isolation protocol. We separated the mice into single cages for a period of 24 hours prior to a social interaction training session. Again, animals remained isolated through the duration of the retention tests. When the two different stimulus juveniles were used for each encounter, the acute isolation had no effect on either the initial encounter investigation times (NR2B: n = 5, 123.99±17.42 s; Wt: n = 15, 102.86±8.09 s,
When tested using identical animals for both the initial encounter and the second encounter, the NR2B animals and their wild-type littermates had similar initial investigation times (NR2B: n = 5, 103.61±8.82 s; Wt: n = 15, 104.46±10.43 s,
We also wanted to test our animals in another paradigm of social recognition in which a familiar juvenile is presented to the subject mouse multiple times for a small amount of time (1 minute) with a short delay (10 minutes) between exploration sessions. This paradigm is a more ecological approach to examining social behavior by mimicking the animals natural social encounters
During this paradigm the animals were exposed to the stimulus mouse for one minute for four sessions separated by a 10 minute rest period. The NR2B animal showed a significant reduction in exploration times at the second encounter which continued to the fourth encounter. The wild-type animals showed a significant reduction in investigation time from the initial exposure starting at the 3 exposure. During the fifth exposure session, a novel animal was used as a control to account for fatigue and disinterest as a result of repeated exposures. (* = 4.28×10−5, ** = 1.6×10−4, *** = 6.3×10−4; # = 7.7×10−5, # # = 7.1×10−6).
While both transgenic and wild-type mice exhibited the similar social recognition memory within the same strain, we wondered if transgenic overexpression of NR2B may have any effect on the animal's ability to recognize mice from different strains. Our transgenic mice and their littermates are bred on a B6× CBA background (black or agouti), accordingly, we used to BALB/c juvenile mice as stimulus mice. We first examined short-term (1 hour) social memory. We found that when using different white juveniles for the first and second encounter, no significant differences were found between the first session (NR2B: n = 5, 117.13±10.81 s; Wt: n = 15, 123.15±7.03 s,
A. No significant differences were found during the second exposure using a novel mouse after one hour. B. After one hour, both the NR2B and the wild-type animals showed a significant reduction in investigation times of the same white mouse that presented in the first encounter. (* p = 0.044, ** p = 0.005) C. During the 24 recall the NR2B animals and the wild-type animals show no reduction in investigation time when a novel mouse is presented in the second encounter. D. After 24 hours only the NR2B animals show a significant reduction in investigation times of the familiar white mouse indicating that the wild-type animal was unable to form a long-term social recognition memory of the white mouse . (* p = 0.048).
Next we tested the investigation times when using the identical white juvenile mouse for both sessions. Both groups spent similar amounts of time during the initial encounter investigating the white juvenile (NR2B: n = 5, 128.02±12.46 s; Wt: n = 15, 141.2±10.32 s,
We then tested the long-term social recognition memory of our mice using the 24 hour retention test. When different mice were used for each encounter, the investigation times for the first encounter (NR2B: n = 5, 111.73±11.78 s; Wt: n = 14, 131.02±8.59 s,
Interestingly, when using the same BLAB/c juvenile as the stimulus mouse, the NR2B animals showed significant reductions in the investigation times in the second encounter (NR2B: n = 5, 72.66±10.97 s,
In order to test the ability of our transgenic animals to form memories of animal outside of their mouse species, we further examined NR2B mice by using a paradigm in which they were exposed to juvenile Long Evans rats. The rat was maintained in a mesh covered chamber to protect the mice. The chamber was placed inside the mouse's testing cage for five minutes in each session.
When different rats were used in each encounter, there was no significant difference in investigation times between the first encounter (NR2B: n = 9, 155.22±23.37 s; Wt: n = 12, 153.20±13.55 s,
A. In comparison to the initial encounter, neither the NR2B nor the wild-type animals show any reduction in the investigation time of the rats during the second encounter. B. In the one hour recall session with the same rat, both the NR2B and the wildtype animals show a significant reduction in the investigation times indicating that both groups of animals have formed a memory of the familiar rat. (* p = 0.033, ** p = 0.0006) C. There were no significant differences found between the investigation times of the novel rat in the first encounter with the novel rat in the second encounter. D. During the 24 recall session only the NR2B mice showed a reduction in the investigation times of the familiar rat. (* p = 0.001) This indicates that only the NR2B mice were able to form a long-term memory of the rats.
After 24 hours all groups showed similar investigation times in both the first social encounter (NR2B: n = 109, 116.29±9.87 s; Wt: n = 12, 93.77±8.59 s,
In our study, we investigated the cellular mechanisms behind the formation of social memories using Tg-NR2B mice in which NR2B is overexpressed in the forebrain regions. This allowed us to explore the effect of a genetic manipulation of the NR2B∶NR2A subunit ratio of the NMDA receptor on social memory. We have shown previously that our NR2B animals have enhanced learning and memory compared to their wild-type littermates in multiple forms of memory including recognition memory, avoidance memory and emotional fear memory, spatial memory, and spatial working memory,
Because social recognition is crucial for species survival, the maintenance of the established social hierarchy and offspring recognition, it is a good representation of an ethologically relevant task in mice
While the olfactory system has been used for many years to test associative or discriminative memory in rodents, the brain regions involved in social recognition are still being investigated, as some studies give differing results in involved brain regions based on lesion studies and c-Fos activation studies
Our present study is consistent with the notion that NMDA receptor is important for social recognition memory, and furthermore, provides the genetic evidence that NR2B overexpression can lead to improvement in certain aspects of social memory. In our study, the short-term memory of these NR2B animals was unaffected by acute separation. This finding is in agreement with other studies that have explored the effects of isolation on social memory
In the habituation-dishabituation paradigm our animals, we found that investigatory behavior did not return to the first novel exploratory session as in other studies. It has been suggested that other factors may influence such behaviors this type of paradigm
Interestingly, little data in the literature shows the influence of the strain of the stimulus mouse on the ability of the subject mouse to form social recognition memories of the stimulus mouse. Moreover, many studies focus on male-female interactions
Our NR2B animals and their wild-type littermates show comparable short-term social recognition for the rats, but only the NR2B animals showed a long-term social memory of the rat. Our observations are in agreement with some studies which show that mice can form a social recognition memory of other species
In conclusion, we have shown that NR2B transgenic animals have exhibited several interesting social recognition phenotypes. While the NR2B animals show similar short-term and long-term memory in recognizing the same strain stimulus mice in comparison to that of the wild-type animals, the NR2B transgenic animals have enhanced long-term memory capabilities when forming social memories across different strains and species.
The authors would like to thank Brianna Klein for maintenance of the animal colonies and Phillip Wang for valuable assistance with behavioral paradigms.