Social Recognition Assay in the Rat

Social Recognition Assay in the Rat 1 Joanne R. Mathiasen and Amy DiCamillo 1 UNIT 8.5I 1 Cephalon, West Chester, Pennsylvania ABSTRACT Neuropsychiatric disorders encompass a broad patient population in a variety of disease states across all age groups and are often accompanied by deficits in short-term/working memory. However, most preclinical models that allow for an assessment of cognitive enhancement do not provide robust behavioral readouts with a level of throughput sufficient to support modern drug discovery efforts. The rat social recognition assay presented in this unit is one exception that has been increasingly employed to test new chemical entities for enhancing cognitive activity. The test is simple in design and takes advantage of the spontaneous behavior of rats to investigate conspecifics. The protocol in this unit is designed to evaluate the effects of a test substance on the short-term/working memory of adult male rats employing 30-min or 2-hr pretreatment times. Curr. Protoc. Neurosci. C 2010 by John Wiley & Sons, Inc. 53:8.5I.1-8.5I.15.  Keywords: memory r cognition r social recognition INTRODUCTION Neuropsychiatric disorders encompass a broad patient population in a variety of disease states across all age groups and are often accompanied by deficits in short-term/working memory. The prevalence of cognitive deficit has led to a dramatic increase in the number of drug targets being considered for the enhancement of cognitive function. This presents a particular challenge to preclinical behavioral pharmacology in that most models that allow for an assessment of cognitive enhancement do not provide robust behavioral readouts with a level of throughput sufficient to support modern drug discovery efforts. The rat social recognition assay is one exception that has been increasingly employed to test new chemical entities for cognitive enhancing activity. Social recognition is simple in design and takes advantage of the spontaneous behavior of rats to investigate conspecifics. Since this is a spontaneous behavior, no artificial stimulus, food deprivation, reinforcement, and/or prior training are required. The social recognition assay emerges from the work of Thor and Holloway (1982), who determined that the social memory of adult rats (3 to 4 months old) for juvenile rats (1 month old) decreased as the time interval between presentations of the same juvenile rat was increased. Aged rats (>18 months old) were found to exhibit clear impairment in the social recognition task compared to adult rats. In brief, this protocol is designed to evaluate short-term/working memory of adult male rats following administration of a test substance by various routes of administration (intraperitoneal, oral, or subcutaneous), employing either of two pretreatment times (30 min or 2 hr). NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal, Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals. Behavioral Neuroscience Current Protocols in Neuroscience 8.5I.1-8.5I.15, October 2010 Published online October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142301.ns0805is53 C 2010 John Wiley & Sons, Inc. Copyright  8.5I.1 Supplement 53 BASIC PROTOCOL EVALUATION OF PHARMACOLOGICAL EFFECTS ON SOCIAL RECOGNITION IN ADULT MALE RATS This protocol describes an assay that measures the effect of test compounds on the social recognition memory of an adult male rat for a male juvenile by following a length of time after an initial exposure. Adult rats are exposed two times for 5 min each to the same juvenile rat (familiar juvenile) and these trials are designated trial 1 and trial 2. Social recognition is evaluated behaviorally by timing the adult rat investigation of the juvenile rat with a stopwatch. The investigative behavior includes sniffing, grooming, and/or close following of the juvenile rat. Adult interest not directed at the juvenile per se (e.g., investigating bedding soiled by the juvenile) is not scored. Social recognition (assumed to be short-term/working memory) of the juvenile is lost as the time interval between trial 1 and trial 2 is lengthened from 15 min (memory retained) to 2 hr (memory lost). It is recommended that this time course be established (at least memory at 15 or 30 min and loss of memory at 2 hr) in the laboratory prior to testing compounds (at 2 hr) for the first time. Improvement of social recognition by a potential promnesic compound is indicated by a reduction in the investigation duration at the second encounter with the familiar juvenile 2 hr after the first trial. An important control to allow detection of non-specific effects of compounds on investigative or social behavior is included in all studies where a positive compound effect is observed. In contrast to the experiment in which the adult rat (pretreated with a vehicle or compound) is presented with the same juvenile rat in trial 1 and trial 2, an adult rat treated with an effective dose of compound is subsequently exposed to a novel juvenile rat in trial 2. Assuming that the compound does not have any effects that would disrupt investigation, the investigation time during trial 2 should be comparable to that measured during trial 1. Typically, a compound can be tested in this assay using 84 adult rats and 96 juvenile rats over a 2-week period using 4 working days per week. Usually, five groups are employed, each consisting of 16 to 18 adult rats used once per test group (vehicle control, three doses of test compound, and one dose of positive test compound with novel juvenile control). To reduce the number of juveniles required, juvenile rats can be used repeatedly within the same week, taking care that the same juveniles are used only once within the same day. A power analysis, based on the variability observed once the assay is established and the expected effect size, should be used to estimate the sample size required in each laboratory. Materials Adult male Sprague Dawley rats (350 to 450 g; ∼3 months old) and juvenile male Sprague Dawley rats (75 to 130 g; ∼1 month old) (Charles River Laboratories) Standard rodent diet (LabDiet 5001, PMI Nutrition International) Test compounds (see recipes) Cages (36.8-cm length × 30.5-cm width × 19.1-cm height) Bedding material (Alpha Dri, Shepherd Specialty Papers) Empty cages with filtered (microisolator) hardtop covers Laboratory tape Low-odor permanent marker (Sharpie Magnum, fine-point, blue or black) Metric balance accurate to 1 g Stopwatches with 1-sec precision 3-ml syringes (BD Luer-Lok) and 23-G 3/4 -in. needles (BD PrecisionGlide) Luer gastric needles (18-G × 2-in.; Popper & Sons) Social Recognition Assay in the Rat Prepare animals 1. House and acclimate adult rats (two per cage) to the facility for 2 weeks and juvenile rats (four per cage) for 1 week. Maintain under standard lighting conditions 8.5I.2 Supplement 53 Current Protocols in Neuroscience (12-hr light/dark cycle; lights on at 0700) with food and water provided ad libitum throughout the experiment except during trial 1 and trial 2. The difference in age/weight of the rats is necessary to avoid aggression between male subjects. 2. Transfer all rats to a procedure room at least 30 min prior to the start of the experiment to allow for acclimation to the room. Continue providing free access to food and water, except during testing (trial 1 and trial 2). To avoid olfactory contact separate adult rat cages from juvenile cages using a second rack or shelf if possible. If the cage litter (of either age rat) is very soiled, the strong odor may interfere with the experiment. It is recommended in this case that the home cage litter be changed and a 2-hr acclimation period be allowed before the start of the experiment. This can be avoided by performing routine cage changes 2 days prior to the experiment. To minimize stress to the animal, choose a quiet procedure room with the same or lesser-intensity illumination than that used in the holding area, and with temperature and humidity also similar. It is recognized that illumination levels can significantly affect the level of social interaction. For example, File (1980) noted increased anxiety-related behavior with increasing Lux to 300 in a social interaction test. Therefore, care should be taken to not dramatically change the illumination level from that which is employed in the holding room. The Guide for the Care and Use of Animals (Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council, 1996; http://oacu.od.nih.gov/regs/guide/guide.pdf) recommends light level for a cage to be between 130 and 325 Lux to prevent phototoxic retinopathy in sensitive animals. 3. Set up ten individual test boxes with bedding material and microisolator tops at eye level for viewing by the investigator. Number each box with laboratory tape to match the adult rat number. Test boxes can be relatively close to each other on the laboratory bench but should not be touching. This assay as described can be run reliably testing ten pairs of animals in the same experimental period. Initially, the investigator may wish to start with a reduced number (one to three) of adult/juvenile pairs until comfortable with the assay protocol and timing considerations. 4. After marking the rat tails for identification with a low-odor permanent marker, weigh and record this information for all rats (adults and juveniles). Clean the balance between weighing of the adults and the juveniles so as not to transfer any odors. Marking the adult and juvenile tail with the same number makes the experiment easier to track. Keeping record of the weight of the juveniles is useful for interpreting experiments that may fail and possibly eliminating aggression caused by juveniles that exceed the correct weight range. 5. Assign rats to treatment groups in a blind fashion (coded) with a fixed rotation (e.g., A, B, C, D, etc.) to ensure a distribution of treatments over time. Treatments should be coded by an independent investigator to avoid bias in evaluating the behavior. 6. Remove adult rat number 1 from its home cage and place it into test box number 1 for a 30- min acclimation period prior to trial 1. Place the microisolator top on the test box. Record the time when this acclimation period starts. Test rats 5 min apart; therefore, 6 min later (to allow time for dosing), place adult rat number 2 into test box number 2, and so on until six of the adult rats have been placed into the test boxes. At this time, trial 1 will begin with adult number 1. Acclimation times >60 min should be avoided as this allows rats to enter into deep sleep stages that may lead to decreased arousal when confronted by the juvenile. Behavioral Neuroscience 8.5I.3 Current Protocols in Neuroscience Supplement 53 Initiate trial 1 7. Introduce juvenile 1 into test box 1 with adult 1. Start the 5-min trial timer. Using another stopwatch (starting and stopping as necessary), record the time that the adult spends investigating the juvenile including grooming, sniffing, close following, pawing, and any close contact using the nose and mouth. Do not include time spent in investigations not directed at the juvenile (e.g., investigating urine-soaked bedding, cage surfaces, or fecal boli). If the adult rat is sleeping and does not immediately arouse from sleep upon placement of the juvenile into the test box, gently nudge the adult to wake it so that time is not lost on the 5-min clock and an accurate trial is recorded. Ideally, a separate stopwatch is used for timing investigatory behaviors in each test cage (therefore, requiring 11 stopwatches). 8. At the end of trial 1 (after 5 min), remove the juvenile and return it to its home gang cage. Record the number of seconds for investigation for adult rat 1 in trial 1. If using a 2-hr pretreatment time, gently administer the coded treatment to adult rat 1 (i.p., s.c., or p.o. in a volume of no more than 10 ml/kg i.p. or p.o. or 5 ml/kg s.c.; with 3 ml/kg recommended for s.c.) and return the adult rat to its home cage. Some investigators (Prediger et al., 2005a,b,c) place the juveniles in individual cages between trial 1 and trial 2. This does not appear to be necessary and using gang cages conserves space. Choice of s.c. dose volume should be made based on solubility of compound and a lower volume should be used if possible. 9. Continue placing adult rats no. 7, 8, 9, and 10 at 5-min intervals into their test boxes for acclimation and performing trial 1 with consecutively numbered rats that have been acclimated as described above. Initiate trial 2 10. Place adult rat 1 into the same test box 1 without changing the bedding material 90 min following the start of trial 1 for adult rat 1 to re-acclimatize to the test box (all ten rats will be re-acclimated in the same manner 30 min prior to their trial 2). If using a 30-min pretreatment time, administer coded treatments at this time when placing the adults back into their corresponding test boxes for re-acclimation (again, this will occur for each of the ten rats at 5-min intervals). Rats are dosed during these two handling times (removal from and return to the test box) to minimize the interruption of memory consolidation and to avoid creating retrograde inhibition (Dantzer et al., 1987). Dosing rats and other manipulations can affect memory (see Robbins and Murphy, 2006). Administration of compound immediately after trial 1 permits a pretreatment time of 2 hr. Administration of compound upon replacement of adults into test boxes for re-acclimation before trial 2 allows for a 30-min pretreatment time. The determination of pretreatment time for compounds should be dictated by the compound pharmacokinetics and the planned route of administration. Compound dosing is restricted to the times of animal handling when placing into test boxes for acclimation; it has been demonstrated that there is no effect of the vehicle administration at these times. Caution in the interpretation of positive results is suggested when choosing pretreatment times, keeping in mind that a 2-hr pretreatment time will possibly encompass drug effects on memory consolidation and/or retrieval, and a 30-min pretreatment time prior to trial 2 may be affecting only retrieval and/or late-phase consolidation. An investigator may want to design experiments utilizing both pretreatment times in independent experiments to approach these questions regarding the stages of memory: (1) if the pharmacokinetics of the compound allow, and (2) if this is of interest beyond the simple short-term/working memory design of this protocol. Social Recognition Assay in the Rat 11. After adult rat 1 has been re-acclimated to test box 1 for 30 min (a total of 2 hr since trial 1); re-introduce juvenile 1 to adult rat 1 for trial 2. Start the timer for 5 min. Again, using another stopwatch, record the investigative behavior. At the end 8.5I.4 Supplement 53 Current Protocols in Neuroscience of 5 min, remove the juvenile from the test box. Record the number of seconds of investigation in trial 2 and return adult rat 1 to its home cage. 12. Continue placing adult rats in the corresponding test boxes for re-acclimation and performing trial 2 for the remaining rats. Ensure that each adult rat is matched in trial 2 with the same rat used in trial 1 (the familiar juvenile). 13. Calculate the ratio of investigation duration (RID) of trial 2 (sec)/trial 1 (sec) for each rat. There is considerable independent variability in social investigatory behavior with some rats performing more intense investigatory behaviors than others. The RID provides a normalization of the performance of each individual for comparison across the group. An RID of ∼1.0 indicates that the adult investigated the juvenile in trial 2 for approximately the same duration as trial 1 and suggests a lack of recognition of the juvenile after 2 hr; an RID <1.0 indicates less investigation on the second trial, suggesting some recognition of the juvenile after 2 hr. 14. Compare data from the treated groups with data from the control group RID values after the blind code is broken and all of the data has been collected with proper n values (n ≈ 16/group; determined by power analysis using experimental variance and expected effect size). Perform statistical analysis using a one-way analysis of variance (ANOVA). If supported by the ANOVA, perform a post-hoc test such as the Dunnett’s test with vehicle set as the control group. 15. After establishing the assay, determine from each laboratory an acceptable criterion for minimal average RID values for the vehicle-treated group (e.g., 0.80). Data obtained on test days in which the vehicle-treated RID values fall below this criterion are not included in the final analysis. It is therefore important that a vehicle-treated control group be included on each test day. A criterion for acceptable investigation duration in trial 1 may also be set (e.g., ≥2 min). This insures that the adult rats gain sufficient exposure to the olfactory “signature” of the juvenile. 16. If a significant treatment effect is observed, the highest efficacious dose of the active test agent is tested in a separate group of rats with the introduction of a novel juvenile in trial 2 in the following manner. Using ten adult rats, perform trial 1 according to steps 2 to 14, with one rat receiving vehicle and nine rats receiving the highest efficacious dose of compound. In trial 2, the vehicle-treated adult rat and one adult rat in the treatment group re-investigate their familiar juvenile. The remaining eight adult rats are exposed to novel juveniles. This is repeated in an additional group of rats on a second day, producing an n of 16 in a novel juvenile group, and adding two more rats to the vehicle and high-dose treatment groups. Additional control groups are included to allow detection of non-specific effects of compounds tested on social behavior. In contrast to an experiment in which the adult rat (pretreated with a vehicle or compound) is presented with the same juvenile rat on trial 1 and trial 2, an adult rat that received an effective dose of compound is subsequently exposed to a novel juvenile rat in trial 2. Assuming that the compound does not have any sedative or stimulating effects, the investigation time during trial 2 should be comparable to that of trial 1, resulting in a RID close to 1.0. Novel juveniles should be obtained from different gang cages than those used for familiar juveniles to avoid any gang cage odor-related artifacts. It is acceptable to re-use juvenile rats in the same study. The data analysis for the follow-up test is the same as (included with) the original test, including the novel juvenile exposed group with the other groups, comparing the compound-treated plus novel juvenile group in the post-hoc analysis to vehicle-treated controls with familiar juveniles (see Fig. 8.5I.4). Behavioral Neuroscience 8.5I.5 Current Protocols in Neuroscience Supplement 53 ALTERNATE PROTOCOL EVALUATION OF POSITIVE PHARMACOLOGICAL EFFECTS ON SOCIAL RECOGNITION IN AGED RATS This protocol describes the measurement of the effects of test compounds on age-related deficit seen in the social recognition test. Here, the protocol is as described in the Basic Protocol, but the inter-trial interval is reduced to 30 min. Whereas adult rats retain memory and recognize familiar juveniles after 30 min, as indicated by a significant reduction in the ratio of investigation duration (trial 2 (sec)/trial 1 (sec)), aged rats exhibit an impairment at this time point and will produce a ratio close to 1.0. As with the adult rat protocol, improvement of this age-related deficit in social recognition memory is indicated by a reduction in investigation duration of a familiar juvenile during trial 2. For a standard experiment, the same number of aged rats will be required as used in the Basic Protocol with the addition of a control group of 16 adult rats (3 to 4 months old). Additional Materials (also see Basic Protocol) Male aged, adult, and juvenile rats of the same strain 1. Prepare, treat, and test aged rats using the same procedures described for adult rats in the Basic Protocol using a 30-min inter-trial interval between trial 1 and trial 2. Remove the juvenile immediately after trial 1. Treat one group of aged rats and the adult rats with vehicle only. The definition of “aged” for rodents based on months differs across all strains (described in an editorial by Coleman, 2004). Therefore, a recommendation of an age for an aged rat will depend on the availability of strain and the achievable effect. A reasonable starting point in social recognition studies would be 16.5 to 19.5 months for Long Evans rats (Markham and Juraska, 2007) or 12 to 18 months for Wistar rats (Prediger et al., 2005a), whereas Fox et al. (2005) preferred 24 to 26 month Wistar rats. Aged male rats are commercially available but may be difficult to find sources for purchase. Retired male breeders are typically used and aged in-house. Matched adults and juveniles should be obtained from the same source. The National Institute on Aging (in the U.S.) maintains a colony of aged rats for purchase (http://www.nia.nih.gov/ ResearchInformation/ScientificResources/AgedRodentColoniesHandbook/); however the strain is either Fisher 344 (F344) or Brown Norway (BN). 2. Most often, use a pretreatment time for dosing aged and adult rats in these experiments of 1 hr before trial 1 (equivalent to 90 min before trial 2). Alternatively, administer the test compound following trial 1, producing a 30-min pretreatment time. Unavoidable again are considerations related to the stages of memory with regard to pretreatment times (consolidation, retrieval, and the main effect of short-term/working memory). The most important consideration is to establish a clear deficit in the available aged rats. In the case of aged rats, due to the shortened inter-trial interval (generally 30 min), there will not be much opportunity to investigate the stages of memory with pretreatment times. 3. Do not remove the aged and adult rats from the test box between the trials except to administer the test compound (for a 30-min pretreatment time). 4. Calculate the ratio of investigation duration (RID) of trial 2 (sec)/trial 1 (sec) for each rat. 5. Compare data from the treated groups with data from the control group RID values after all data has been collected using ANOVA and a relevant post-hoc test as described in the Basic Protocol. Social Recognition Assay in the Rat 8.5I.6 Supplement 53 Current Protocols in Neuroscience REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX. Test compounds Soluble compounds: Dissolve compounds in distilled water (for oral administration) or sterile physiological saline (0.9% NaCl; for intraperitoneal, i.p.; or subcutaneous, s.c. injections). Insoluble compounds: Dissolve compounds in 0.5% methylcellulose and 0.2% (w/v) Tween 80 in water (Methocel A15 Premium LV EP, Dow Chemical) for i.p. or s.c. injections (store up to 3 weeks at 4◦ C); pH 2.0 water may solubilize insoluble compounds for oral administration, or 50:40:10 Tween 80/PC/PG (Tween 80/propylene carbonate/propylene glycol) can be used for suspensions. Test compounds should be prepared fresh each day unless stability information is known. Serial dilutions can be made with solutions; however, compounds resulting in suspensions should be separately weighed for each dose level administered. If a new vehicle is to be used, special attention should be paid to vehicle groups to be assured that there is no vehicle effect. ABT-239 (4-(2-benzofuran-5-yl) benzonitrile; Cephalon) was suspended in a vehicle composed of 0.5% methylcellulose and (w/v) 0.2% Tween 80 in sterile distilled water and was administered intraperitoneally (i.p.). A range to include inactive and active doses spans from 0.003 to 1.0 mg/kg i.p. Impaired neurotransmitter release appears to be a common thread through the memory component of various neuropsychiatric disorders. The H3 histamine receptor is considered a novel target for the treatment of cognitive dysfunction, and H3 receptor antagonists modulate the synthesis and/or release of multiple neurotransmitters that may be important for cognitive aspects of multiple neuropsychiatric diseases (Bacciottini et al., 2001; Fernandez-Novoa and Cacabelos, 2001). Recent work with H3 receptor antagonists in social recognition have demonstrated the utility of these agents in rodent models of shortterm/working memory (Fox et al., 2003a,b, 2005; Jia et al., 2006). D-Amphetamine (Sigma-Aldrich) was dissolved in sterile distilled water and also administered i.p. A range to include inactive and active doses spans from 0.1 to 3.0 mg/kg i.p. D-Amphetamine was chosen as a reference compound for two reasons. First, Damphetamine represents a second class of compound (psychostimulants) demonstrating positive effects on short-term/working memory in a variety of models (Aultman and Moghaddam, 2001; Shoblock et al., 2003; Bizarro et al., 2004). Second, D-amphetamine produces a clear demonstration of a false-positive effect with the novel juvenile controls (Perio et al., 1989). COMMENTARY Background Information The original social recognition memory investigation in rats by Thor and Holloway (1982) was expanded by Dantzer and coworkers (Dantzer et al., 1987) to demonstrate that administration of a pharmacological agent (Arg-vasopressin) following the first presentation of a juvenile to an adult rat would restore memory of that juvenile rat when presented at a later trial. The rat social recognition assay has been increasingly employed to test new chem- ical entities for cognitive enhancing activity (Dekeyne et al., 2000; Prediger and Takahashi, 2003, 2005; Van Kampen et al., 2004; Bielsky et al., 2005; Browman et al., 2005; Cowart et al., 2005; Prediger et al., 2005a,b,c, 2006; Mitchell et al., 2006; Bitner et al., 2007; Boess et al., 2007; Depoortere et al., 2007; Di Cara et al., 2007; Hollander et al., 2007; Schreiber et al., 2007). The social recognition model has been pharmacologically validated with a clinically Behavioral Neuroscience 8.5I.7 Current Protocols in Neuroscience Supplement 53 Social Recognition Assay in the Rat active compound, donepezil (Aricept; Jia et al., 2006), suggesting that it may have clinical translational relevance. In addition, a wide variety of putative cognitive enhancers have demonstrated efficacy in this model including other acetylcholinesterase inhibitors (van Kampen et al., 2004; Jia et al., 2006), H3 histamine receptor antagonists (Prast et al., 1996; Fox et al., 2003a, 2005; Cowart et al., 2005; Jia et al., 2006), nicotinic agonists (Perio et al., 1989), stimulants such as caffeine (Prediger et al., 2005), amphetamine (Perio et al., 1989), and a CB1 cannabinoid antagonist (Terranova et al., 1996). There are many behavioral assays that utilize rats to assess multiple domains of memory. The social recognition test is designed to measure short-term/working memory. Short-term memory refers to the capacity for holding a small amount of information in the mind in an active, readily available state for a short period of time. The rat is a communal animal that relies on group and individual odor for the determination of threat, communication of food sources, and sexual information (Galef, 2005). Therefore, a short-term memory for olfactory-based recognition represents a critical cognitive asset for this species. Deterioration of olfactory perception is among the most serious problems of the elderly and a symptom along with the mild cognitive impairment of early Alzheimer’s Disease (Devanand et al., 2000; Wilson et al., 2007; Djordjevic et al., 2008). Decline in the olfactory discrimination ability with age has been attributed to decreases in performance of higher functions in elderly subjects (Kaneda et al., 2000). Age-related olfactory deficits can be seen in rodents (Guan and Dluzen, 1994; Terranova et al., 1994; Roman et al., 1996; Schoenbaum et al., 2002; Prediger et al., 2005b). However, these deficits may be dependent on the task and the stimulus, as olfactory discrimination learning in aged rats has been shown to remain intact under some conditions (Kraemer et al., 2004). The social recognition test in the rat has the advantage of being relatively quick to perform, can be made relatively high throughput, and does not rely on artificial stimulus, food deprivation, reinforcement, or prior training. However, there are some disadvantages to this assay, e.g., the adult rats must be ∼400 g, requiring the use of more test compound than would be needed with other assays that can be performed in younger animals. The weight is important in the social recognition test because a large difference in age/weight be- tween adult and juvenile rats avoids aggressive behavior, which disrupts accurate trial timing. However, this need for additional compound is frequently offset by the finding that many compounds are more potent in the social recognition test than other cognitive assays (e.g., histamine H3 receptor antagonists; Fox et al., 2003a, 2005). This ability of the social recognition test to detect cognitive enhancement at much lower doses than that observed in other memory tests may be an indication of the importance of social recognition memory in rat cognition. While most investigators use male rats, it is important to note that behavioral differences are reported to exist (Markham and Juraska, 2007) between male and female rats in social recognition memory and this effect may rely in part on sexually dimorphic neural pathways, including the extrahypothalmic systems of the neuropeptides arginine-vasopressin and oxytocin (deVries and Miller, 1998; Ferguson et al., 2002). Bluthé et al. (1990) found that female rats (like castrated male rats) investigate juvenile rats less during trial 1 and retain memory for a longer duration than do adult male rats. While it is reported that aged rats investigate juveniles less in trial 1 than adult rats (Prediger et al., 2006), occasionally, protocols will be adjusted to allow a 15-min trial 1 for aged rats (Terranova et al., 1994). This is not practical when testing in a high-throughput environment, due to the shortened inter-trial interval of 30 min between trial 1 and trial 2. An additional consideration in testing aged rats is the pharmacokinetics of the test compound, possibly requiring up to 2 hr for optimal efficacy in some oral formulations. Many investigators pretreat aged and adult rats prior to trial 1 with the test compound. While this should be controlled for in a test with novel juveniles in trial 2, any effects of the test compound that might impair the initial investigation of the juvenile in trial 1 cannot be avoided and may alter interpretation of the results. Critical Parameters and Troubleshooting Experimental environment Acclimation time to the facility for all adult rats should be 2 weeks. Cage litter for the adults should also be changed 2 days prior to experiments. If this is not possible, cages can be changed on the day of the experiment, and a 2-hr re-acclimation time allowed before 8.5I.8 Supplement 53 Current Protocols in Neuroscience the adult rats are moved into the procedure room. During an experiment, in trial 1 or trial 2, attention should be paid to any situation where an adult rat is obsessively distracted by some real or imagined object or spot in the test box that is distracting from the juvenile, and this observation recorded. In rare instances, an RID will be considered an outlier by Grubb’s test or by exceeding 2 standard deviations from the mean, and these recorded observations will be helpful in interpreting these outliers. While low, quiet, constant noise is not a distraction, a loud sudden noise can startle rats and be considered a stress. Due to the short timing in this test, any startle or distraction to the rat during the test trials will cause erratic results and increase variability. In the original work by Thor and Holloway (1982), the social recognition test was performed during the dark phase under a dim red light. Subsequent to this, many investigators performed experiments either during the dark phase or using an inverse light-dark cycle (Perio et al., 1989) still under a red light. Testing during the dark phase under a dim red light requires either light-reversed rats or lightreversed investigators. To keep the procedure simple, it has been found that this precaution is not necessary and the social recognition test can be performed in the light phase under normal laboratory lighting conditions (Hliňák and Krejci, 1995; Prast et al., 1996; Fox et al., 2003a,b; Cowart et al., 2005; Fox et al., 2005; Prediger et al., 2006; Markham and Juraska, 2007). There is no clear agreement in the literature as to an optimal strain of rats to use for the social recognition assay, with previous reports including studies with Long Evans rats introduce adult remove introduce juvenile juvenile (adult) acclimation T1 (Thor and Holloway, 1982; Taylor et al., 1999), Wistar rats (Dantzer et al., 1987; Perio et al., 1989; Terranova et al., 1996; Letty et al., 1997; Millan et al., 2004; van Kampen et al., 2004; Prediger et al., 2005a,b,c), and Sprague Dawley rats (Prast et al., 1996; Fox et al., 2003a, 2005; Cowart et al., 2005; Browman et al., 2005). The lack of an obvious benefit of one strain over another in this assay may be because the behavioral response is mainly an olfactory-based recognition rather than visual, as it is in the novel object discrimination test for which a strain with better vision (e.g., Long Evans) might be more appropriate (Prusky et al., 2002). In optimizing the social recognition assay for drug discovery, it is critical to strike an effective balance between maintaining assay throughput and robust behavior. The timing of the various steps in the social recognition protocol (Fig. 8.5I.1) can significantly alter experimental outcome. The duration of acclimation to the test cage is one such variable since the initial period the adult rat spends investigating new surroundings might detract from interest in investigating a juvenile in the test cage. Likewise, if the adult rat is left to acclimate for longer periods of time, it will tend to fall asleep, especially when the testing is during the light phase. If the time is of a sufficiently long duration to enter into deep sleep, it may be more difficult to arouse the rat when the juvenile is placed in the test box for either trial 1 or trial 2. Some investigators perform the social recognition test in the adult rat home cage, obviating the need for test cage acclimation. This requires individual housing and may not be practical due to space considerations. re-introduce re-introduce end adult juvenile observation ITI T2 (acclimation) Figure 8.5I.1 Social recognition assay timeline. Adult rat is introduced to test cage for acclimation period (30 min) prior to juvenile rat introduction. Trial 1 (T1) proceeds for 5 min. Adult and juvenile rats are removed. Following an inter-trial interval (ITI; 120 min for drug testing), the adult rat is re-acclimated to the test cage (30 min) and the juvenile rat is placed in the test cage for trial 2 (5 min). This ends the assay. Behavioral Neuroscience 8.5I.9 Current Protocols in Neuroscience Supplement 53 A Investigation duration (sec) 225 trial 1 trial 2 200 175 * 150 125 100 75 50 25 0 30-min ITI 120-min ITI B 1.00 RID 0.75 * 0.50 0.25 0.00 30-min ITI 120-min ITI Figure 8.5I.2 Time course for the social recognition model. (A) Mean (± SEM) amount of time (sec) an adult rat spends investigating the same juvenile rat during a 5-min trial 1 (open bars) and trial 2 (dark bars) separated by either a 30- or 120-min inter-trial interval (ITI). In trials separated by a 30-min ITI, but not by a 120-min ITI, adult rats spent significantly less time exploring the familiar juvenile rat relative to trial 1 (*p <0.01 versus trial 1, paired t-test, n = 8/group). (B) Mean (± SEM) ratio of investigation duration during trial 2 and trial 1 (RID; in seconds) was calculated from the data in A. The adult rats retain memory for familiar juvenile rats following a 30-min ITI (RID, 0.70) but not following a 120-min ITI (RID close to unity at 0.94; *p <0.02; 30 min versus 120-min ITI, Student’s t-test). Social Recognition Assay in the Rat The acclimation time may affect subsequent RID points; therefore, keeping all acclimation times consistent is very important. Moreover, it is also recommended that the stopwatch be started only once the adult rat (if sleeping) is aroused (eyes opened, head raised) to avoid errors in timing if the adult is not alert when the juvenile is placed in the test cage. In addition to the aforementioned acclimation time, the amount of time allowed for investigation could theoretically impact on the salience of the juvenile presentation. For example, an adult allowed to investigate until it accumulates 3 min of investigation (flexible trial) time may spend a significantly longer time interacting with the juvenile than an adult limited to a 5-min fixed trial. Previous studies have reported the use of either a fixed or flexible trial duration without clear indica- tions of one method being superior (Thor and Holloway, 1982). Anticipated Results Baseline behavior During acclimation to the test box, adult male rats will spend ∼15 min exploring the test box and then settle down to rest or sleep. For trial 1, when the juvenile is placed in the test box, adult males will then spend ∼2 to 3 min intermittently investigating the juvenile over the 5-min trial. This does not increase if left together in the cage for a longer period of time, and eventually, the adult male and juvenile will go to sleep. In studies where the home cage of the adult male was used as the test box, the investigation times are much shorter, closer to 60 sec. During re-acclimation to the test box 30 min prior to trial 2, the adult rat generally 8.5I.10 Supplement 53 Current Protocols in Neuroscience 1.0 0.9 * RID 0.8 0.7 0.6 0.5 familiar novel Figure 8.5I.3 Ratio of investigation duration (RID; mean ± SEM) for a 30-min inter-trial interval. Adult rats were presented with either a familiar juvenile (open bar) or novel juvenile (dark bar) rat on trial 2. Adult rats remained in the test cage between both trials. Adult rats spend significantly less time investigating the familiar juvenile rat than a novel juvenile rat (*p <0.001, Student t-test; n = 14/group). spends less time exploring the surroundings, however this has not been timed. When the familiar juvenile is presented at trial 2 two hr later for 5 min, the adult does not recognize the juvenile and investigates the juvenile for approximately the same amount of time as in trial 1 producing a ratio of investigation (RID) close to 1.0. Most often, the RID values of vehicle-treated rats will be ≤1.0, but not <0.8. If an adult rat is presented with a familiar juvenile 30 min later in trial 2, the RID value will be much lower than 1.0, closer to 0.6. Figure 8.5I.2 illustrates this inter-trial interval effect (30 and 120 min) on the duration of social investigation by adult male rats. Robust memory retention at the 30-min inter-trial interval and the lack of memory retention at the 120-min inter-trial interval is observed. With additional studies at the 30-min inter-trial interval and a novel juvenile rat presented instead of a familiar rat during trial 2 (Fig. 8.5I.3), it is further demonstrated that the decrease in investigation time of the familiar juvenile rats by the adult rat was in fact due to memory retention and not caused by fatigue or boredom. This approach has been used for interpretation of treatment effects showing that decreased investigation times were specific and indicated enhancement of short-term/working memory (Dantzer et al., 1987). Other approaches to control for false-positive results have also been reported. For example, Engelmann et al. (1995) reported on using two juvenile rats (one familiar and one novel) at trial 2 while Fox et al. (2005) introduced the novel juvenile immediately after the end of trial 2. Although such approaches could produce similar distinction and confirm memory, they also introduced additional experimental variables that need to be specifically addressed. Effects of test compounds An amnesic effect of scopolamine (typical dose of 0.25 to 1.25 mg/kg i.p. 30 min prior to trial 2; and using a 30-min inter-trial interval) is often used to induce a deficit and to demonstrate a pro-cholinergic effect of test compounds (Perio et al., 1989; Terranova et al., 1996; Schreiber et al., 2007; Depoortere et al., 2007; Millan et al., 2007). While this experimental design is not described in detail in this protocol, this can be easily incorporated by establishing a dose-response relationship for scopolamine or any other pharmacological agent (e.g., phencyclidine, MK-801). Promnesic effects of test compounds in the rat social recognition test are evaluated at trial 2, 120 min after trial 1, when the adult rat has no memory for the familiar juvenile rat (see Fig. 8.5I.2). Vehicle-treated rats in trial 2 do not recognize the familiar juveniles, and spend approximately as much investigatory time in trial 2 as they did in trial 1. However, adult rats treated with a cognitively enhancing compound (promnesic agent) do recognize the juvenile rat as familiar and show a significant reduction in investigation duration in trial 2 (RID <1.0). Figure 8.5I.4 shows an example of data obtained with a cognitively enhancing compound, ABT-239, which is a histamine H3 receptor antagonist (Fox et al., 2003a), across a wide dose range that agrees with previously reported data. After establishing that the dose of 1.0 mg/kg was efficacious, a group of adult Behavioral Neuroscience 8.5I.11 Current Protocols in Neuroscience Supplement 53 1.1 1.0 RID 0.9 * 0.8 * * 0.7 * 0.6 0.5 vehicle 0.003 0.01 0.03 0.1 0.3 1.0 1.0+novel ABT-239 (mg/kg 120 min, i.p.) Figure 8.5I.4 Effects of histamine H3 receptor antagonist ABT-239 on short-term memory in the social recognition test at a 120-min ITI. RID refers to ratio of investigation duration [trial 2 (sec)/trial 1 (sec)]; lower ratios indicate improved short-term memory. RID were calculated for each dose (doses on abscissa) of the test compound (see Fig. 8.5I.2 for more details). Lower ratios reflect improved short-term memory. Adult rats were administered the test compounds immediately after the end of a 5-min trial 1, resulting in a 120-min pretreatment time before trial 2. Separate groups of control rats treated with ABT-239 (1.0 mg/kg) and then exposed to a novel juvenile rat in trial 2 (1.0 + novel, respectively) were not significantly different from vehicle-treated rats (*p <0.005 versus vehicle, ANOVA, Dunnett’s post hoc; n = 10–29/group). 1.00 0.75 RID * 0.50 0.25 * 0.00 vehicle 0.001 0.1 familiar 1.0 3.0 * vehicle 1.0 3.0 novel Figure 8.5I.5 D-Amphetamine in the social recognition test at a 120-min ITI. RID refers to ratio of investigation duration [trial 2 (sec)/trial 1 (sec)]; lower ratios indicate improved short-term/working memory. Drug pretreatment time was 30 min prior to trial 2. D-Amphetamine was effective in decreasing RID at 1.0 and 3.0 mg/kg i.p. As a control for any nonspecific effects of the compound, separate groups of control rats received vehicle, 1.0 or 3.0 mg/kg D-amphetamine and then were exposed to a novel juvenile rat in trial 2 (novel). These groups were not significantly different from vehicle-treated rats except with 3.0 mg/kg D-amphetamine. This demonstrates that for Damphetamine, a cognitively enhancing effect can be seen at 1.0 mg/kg; however, the effects at 3.0 mg/kg are not consistent with memory enhancement and illustrate a false-positive effect at this dose. The horizontal line is for orientation purposes relative to previous figures, which have an origin point on the ordinate at 0.5 RID (*p <0.05 versus vehicle ANOVA, Dunnett’s post hoc; n = 12–39/group.) Social Recognition Assay in the Rat 8.5I.12 Supplement 53 Current Protocols in Neuroscience rats given 1.0 mg/kg of test compound (ABT239) was presented with novel juveniles in trial 2 (1.0 + novel) to test for effects of the test compound not related to cognition (such as sedation, stimulation, or undesirable alternative behavioral effects) that would interfere with the assay and produce a false-positive effect. The importance of these control groups is demonstrated in Figure 8.5I.5. DAmphetamine is shown to produce a cognitively enhancing result at a dose of 1.0 mg/kg when the novel juvenile control group is evaluated; yet at 3.0 mg/kg, which also looks to be cognitively enhancing, tests with the novel juvenile reveal that this dose (3.0 mg/kg) is a false-positive result. The novel juveniles tested with the dose of 3.0 mg/kg D-amphetamine were also affected, with RID values significantly different from vehicle-treated controls. Time Considerations ory using a clinically relevant task. Psychopharmacology 153:353-364. Bacciottini, L., Passani, M.B., Mannaioni, P.F., and Blandina, P. 2001. Interactions between histaminergic and cholinergic systems in learning and memory. Behav. Brain Res. 124:183194. Bielsky, I.F., Hu, S.B., Ren, X., Terwilliger, E.F., and Young, L.J. 2005. The V1a vasopressin receptor is necessary and sufficient for normal social recognition: A gene replacement study. Neuron 47:503-513. Bitner, R.S., Bunnelle, W.H., Anderson, D.J., Briggs, C.A., Buccafusco, J., Curzon, P., Decker, M.W., Frost, J.M., Gronlien, J.H., Gubbins, E., Li, J., Malysz, J., Markosyan, S., Marsh, K., Meyer, M.D., Nikkel, A.L., Radek, R.J., Robb, H.M., Timmermann, D., Sullivan, J.P., and Gopalakrishnan, M. 2007. Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways. J. Neurosci. 27:10578-10587. A standard experiment using adult rats and juveniles (five groups of adults with n = 16 and testing ten adult/juvenile pairs per day) would start first with four groups of vehicle and three doses of test compound for 64 adult rats. This would be ∼7 test days. It is possible to test twelve rat pairs per day depending on space constraints and investigator fatigue. An additional 2 working days would be required for testing novel juveniles if a positive effect was seen with a dose of test compound, equaling a total of 9 working days or ∼2 weeks. The adult rats require 2 weeks of acclimation to the facility prior to testing. For each test day, from acclimation time to the procedure room through the last rat in trial 2 and clean up, allow at least 5 hr to test ten adult rats. The test can be broken up into two sets of five rats; however, balance the coded treatment groups such that one group is not tested solely in the morning or afternoon. The trials are timed at 5 min; therefore, set each adult rat trial to be 5 min apart. This does not allow for any time in between rats for dosing, recording trial times, or removing juveniles and adults; therefore, 6 or 7 min between trials should be plenty of time for this, especially with the 120-min inter-trial interval. 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