NSG2 mutant mouse model
Constitutive “NSG2 KO em1” (C57BL/6N-Nsg2em1(IMPC)KMPC/KMPC; RRID:IMSR_MOP1805013) mice were purchased from and generated by the Korean Mouse Phenotyping Center (KMPC), and details of the CRISPR-mediated gene targeting strategy can be found at https://mousephenotype.kr/. Heterozygote (NSG2+/-) animals were bred to produce a viable colony from which the cohort reported here was generated. Offspring were weaned at between 21-23 days of age, ear tagged, and housed with same sex littermates at 22°C on a 12-h reverse light/dark cycle (dark period: 0800-2000). The mice had free access to standard chow and water at all times except where noted below (D-R learning paradigm). Behavior tests were performed on a cohort of 12 wild-type (wt; NSG2+/+), and 13 KO (NSG2-/-) male mice (total n=25) and all animals were between the ages of 2 and 6 months during testing. NSG2 genotyping was performed via Polymerase Chain Reaction (PCR). Tail snips (~0.5 cm) were digested in 200 μl of DirectPCR Lysis Reagent (Viagen Biotech, Los Angeles, CA) and 2μl of Proteinase K (Zymo Research, Irvine, CA) at 55°C for 24h followed by incubation at 95°C for 10min to inactivate Proteinase K. PCR amplification of NSG2 was carried out using Q5 DNA polymerase according to the manufacturer's recommendations (New England Biolabs, Ipswich, MA). The following primers were used to determine specific allelic expression: 5’-AGGGGGCTTGTCATCTCTGA-3’ (NSG2 forward), 5’-GTCCCCTTCCATTCCATCCC-3’ (NSG2 reverse). 1 μl of digested genomic DNA was used in a 20 μl PCR reaction containing both primers (5 μM each). The following cycling parameters were used: 1 cycle of 95°C for 3 min, 30 cycles of 95°C (30s), 68°C (30s), 72°C (30s) and 1 cycle of 72°C for 5 min (C1000 Touch Thermal Cycler, Bio-Rad, Hercules, CA). 2–5 μl of the PCR reactions were subjected to gel electrophoresis using a 3% agarose gel in tris-borate-ethylenediamine tetraacetic acid buffer. Amplicons were visualized to identify mice with wt (227 bp), KO (220 bp), and heterozygote (227 and 220 bp) genotypes.
Western Blot Analysis
Brains from wt and NSG2 KO mice were homogenized in 1X RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), 1x proteinase inhibitor (Thermo, #A32955) with a micro-homogenizer while kept on ice. Detergent-insoluble material was removed by centrifugation at 13,000x g for 10 min. Protein concentration was determined using the Pierce BCA Protein Assay Kit (#23225). Twenty micrograms of protein were mixed with 1X NuPAGE LDS Sample Buffer (Invitrogen, #NP0007), 1X NuPAGE Sample Reducing Agent (Invitrogen, #NP0009) and incubated at 95oC for 20 minutes. The samples were separated on a 15% Tris-Glycine gel with a 4% stacker using a Tris/Glycine/SDS buffer and transferred overnight onto an Immobilon-FL Transfer Membrane using a Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked for one hour (0.5% Non-Fat Dry Milk in PBS) and then probed with the primary antibody: rabbit anti-NSG2 (Abcam; ab189513, 1:1000) for 2h at room temp (0.5% Non-Fat Dry Milk/0.1% Tween-20 in PBS). The blots were washed 3x5min with PBS and incubated one hour with secondary antibody [Goat anti-Rabbit horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch Laboratories, Inc., 111-035-003, 1:10,000)]. After another 3x5min wash with PBS, signal was developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo, #34580), and imaged on a Bio-Rad ChemiDoc. The unstripped membrane was then probed with the primary antibody: mouse anti-NSG1 (Santa Cruz; sc-390654, 1:1000) and secondary: Goat anti-Mouse horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch Laboratories, Inc., 115-035-003, 1:10,000).
Immunohistochemistry
WT and NSG2 KO mice were anesthetized and perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were removed and immersed in 4% PFA, 20% sucrose, and 30% sucrose, each for 12–24 h. Sagittal sections were sliced on a sliding knife microtome (American Optical). Free-floating sections were permeabilized and blocked simultaneously in 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and 10% donkey serum (MilliporeSigma) for 1 hr in PBS. Sections were stained with goat anti-NSG1 (Thermofisher; PA5-37939, 1:1000) and rabbit anti-NSG2 (Abcam; ab189513, 1:500) in 0.25% Triton and 5% donkey serum in PBS overnight at 4°C. Sections were washed three times in PBS followed by secondary antibody labeling (donkey anti-goat and donkey anti-rabbit [1:1000]; Thermofisher) in the same buffer as primary antibodies. Sections were then mounted to microscopy slides (Superfrost plus, Fisher Scientific), immersed in Fluoromount-G, and imaged on a slide-scanning microscope (Zeiss Axion Scan.Z1) with Colibri 7 LED light source. Standard fluorescence calibration was performed on wild-type control brain sections to ensure proper dynamic range of signals and imaging conditions were maintained on NSG2 KO brains
Behavioral Analyses
All behavioral assays were performed essentially as described previously [12] with the exception of the Morris Water Maze and touchscreen discrimination-reversal tasks. Where appropriate, mice were acclimated to testing rooms for at least 30 min. prior to testing and all apparati were cleaned using 70% ethanol and dried between individual trials to eliminate olfactory distractions. All of the experiments were performed during the active phase between the hours of 0900–1200, except the Morris Water Maze, which was separated into two session (0930-1100 and 1200-1430). Tests were performed in a room illuminated by red lights, except for homecage behavioral monitoring, which occurred during both light and dark phases. Behavioral assays were performed in the following order: discrimination-reversal operant learning, open field, rotarod, Catwalk XT, elevated zero maze (EZM), trace fear conditioning (TFC), homecage monitoring. All tests were separated by a 24–48 interval.
Anxiety-related tasks
The open-field test was used to assess exploratory behavior, anxiety, and gross locomotion. Mice were randomly placed in one of the corners of a white Plexiglas open field chamber (29 X 29 X 29cm) and allowed to freely explore for 30 minutes. An overhead camera (Med Associates Basler acA1300-60) and Ethovision tracking software XT (Noldus Information Technology, Wageningen, Netherlands) were used to track velocity, distance traveled, and duration in the center or the border areas. The elevated zero maze (EZM) consisted of an elevated circular platform (64 cm high; 50.8 cm min diameter and 60.96 cm max diameter) with two opposing enclosed sections enclosed by a wall (20 cm high) and two opposing open sections, each with a platform 5 cm wide. Each mouse was randomly placed in one quadrant within the maze and allowed to explore for 5 min while tracked via the same overhead camera and video-tracking software as noted above to monitor the position of the central point of the mouse body. The software measured latency and cumulative time spent in the open and closed areas of the maze.
Motor-related tasks
The rotarod task was used to assess motor performance and fatigue resistance in rodents using the Panlab Rota Rod model LE8205 (Harvard Apparatus, Holliston, MA). The rod was initiated to rotate at a constant initial speed of 4 RPM. Once the mouse was positioned, the rod accelerated to 40 RPM in 300 s. The time spent on the rod and the RPM reached at the time of falling was captured for each of five trials. A 30-s inter-trial interval (ITI) was used throughout the experiment. The CatwalkXT system (Noldus, Leesburg, VA) was used to analyze gait and fine motor coordination. Mice were allowed to walk freely across the glass walkway to reach a dark goal box and footprint detection occurred as animals passed through a black tunnel illuminated from one side by a reflected green fluorescent light. Three trials capturing at least three stride lengths each were captured for each animal during one testing session. Trials were included in the analysis (were “compliant”) if the mouse crossed the recording area in under 5 seconds and did not show a maximum speed variation greater than 60%. Compliant trials were analyzed via automatic detection but were also reviewed manually; trials that did not represent continuous forward movement were excluded.
The extraction of 174 traits was performed in an automated fashion using Catwalk XT 8.1 Software. These data were then exported and evaluated statistically as described previously [66]. Briefly, data for each trait was z-score transformed and normalized, and then evaluated statistically for group differences using FDR-corrected unpaired T-tests.
Circadian regulation
Spontaneous homecage activity was collected in wild-type and NSG2 KO mice to assess diurnal cycles and locomotor behavior in a non-aversive environment. All mice were individually housed in a standard home cage with corncob bedding with ad libitum food and water and left undisturbed for a 72-h period under their normal illumination conditions (lights on at 2000h and off at 0800h). Horizontal activity was automatically measured by photocell beam break for 72 h using the PAS-Homecage system (San Diego Instruments, San Diego, CA). The first 9h were considered an acclimation period and excluded from analysis, while the middle 48h (two cycles) were used for data analysis (starting at 0800h, light offset). Repeated measures ANOVA was performed across the entire circadian period and two-way ANOVA was performed on cumulative light and dark activity across the 48h period to assess light (sleep) and dark (wake) phases separately.
Learning and memory
Pavlovian trace fear conditioning (TFC) was used to measure associative learning. Animals were first placed into a Habitest® System (Coulbourn Instruments, Allentown, PA) for 180 s to habituate to apparatus. After the no stimulus period, a 90 dB, 5000 Hz white noise auditory tone was presented for 30 s (conditioned stimulus [CS]) followed by a 20-s interval that terminated with a 2 second 0.7 mA footshock (unconditioned stimulus [US]). After the first CS/US pairing there was a 90-s interval followed by another CS/US pairing. The CS/US pairings were repeated 5 times on day 1 (acquisition) with a randomized interval averaging 120 s. Each session was ended 60 s after the final pairing. Memory for the CS was tested absent the shock in the same chamber 24 h (day 2) after acquisition. Morris Water Maze (MWM):Equal numbers of wt and KO animals were separated into two sessions (mid-morning/mid-afternoon) to minimize inter-trial interval between animals. All animals were trained for 6 days (4x1min trials/day, 15 min inter-trial interval) to find a hidden platform submerged beneath opaque (white, non-toxic paint) water (26°C) using spatial cues on the wall, as previously described [27]. Platform location by quadrant as well as animal placement were randomized to eliminate bias. Animals were singly housed and kept warm in cages between trials. On the seventh day, a probe trial was performed via removal of the platform and the percentage of time spent in the target quadrant vs. the other quadrants was recorded as a measurement of spatial working memory. On day 8, animals were tested on a single cued trial where a tall, visible cue was placed on the platform in the target quadrant. Time to reach the platform was recorded as a measure of general motor function. All behavioral data were collected using Ethovision XT8 software (Noldus, Netherlands), and then processed in Prism (GraphPad, V9).
Touch screen discrimination reversal (D-R): Operant behavior was conducted as previously described [67] in a chamber measuring 21.6 x 17.8 x 12.7 cm (model # ENV-307W, Med Associates, St. Albans, VT) housed within a sound- and light-attenuating box (Med Associates, St. Albans, VT). A solid acrylic plate was used to cover the grid floor of the chamber to facilitate ambulation. A peristaltic pump delivered 10µl of liquid strawberry milkshake (strawberry Nesquik mixed with skim milk) into a magazine. A house-light, tone generator and an ultra-sensitive lever was located on one end of the chamber, while a touch-sensitive screen (Conclusive Solutions, Sawbridgeworth, U.K.) was on the opposite side of the chamber covered by a black acrylic aperture plate, which creates two 7.5 x 7.5 cm touch areas separated by 1cm and located at a height of 0.8cm from the floor of the chamber. KLimbic Software Package v1.20.2 (Conclusive Solutions) controlled and recorded stimulus presentation and touches in the response windows. Eight week-old mice were food restricted and maintained at 85% free-feeding body weight and subjected to 3 days of acclimation to the behavior room and liquid reward. Mice were habituated to the operant chamber and retrieving reward from the magazine by being placed in the chamber for ≤30 minutes with liquid available in the magazine. Once a mouse retrieved at least 10 liquid reward retrievals, during a habituation session, it began the pre-training regimen. First, mice were trained to obtain reward by pressing a lever within the chamber on an FR1 schedule. Once a mouse showed willingness to press the lever and collect 30 rewards in a <30 minute-session, it was moved to touch training. During this stage, a lever press led to the presentation of a white (variously-shaped) stimulus in 1 of the 2 response windows (spatially pseudorandomized). The stimulus remained on the screen until a response was made. Touches in the blank response window had no effect, while a touch to the white stimulus window resulted in reward delivery, immediately cued by a tone and illumination of the magazine. Once a mouse was able to initiate, touch and retrieve 30 rewards in a <30 minute-session, it was moved to the final stage of pre-training. This stage was identical to touch-training except that responses at a blank window during stimulus presentation now produced a 10-second timeout, immediately signaled by illumination of the house light, to discourage indiscriminate screen responding. Errors made on this pre-training stage (as well as on discrimination and reversal, see below), were followed by correction trials in which the same stimulus and left/right position was presented until a correct response was made. Once a mouse was able to make ≥75% (excluding correction trials) of responses at a stimulus-containing window in a 30-trial session, it was moved onto discrimination testing.
Pairwise discrimination and reversal was tested as previously described [23]. Mice were first trained to discriminate two novel, approximately equally-luminescent stimuli, presented in a spatially pseudorandomized manner, over 30-trial sessions (5-second inter-trial interval). The stimulus designated as correct was counterbalanced across mice and genotypes. Responses at the correct stimulus window resulted in a single liquid food reward, cued by a 1-second tone and illumination of the magazine. Responses at the incorrect stimulus window resulted in a forced timeout, signaled by illumination of the house-light. Correction trials following errors were presented, with the same stimuli, in the same spatial orientation, until a correct response was made. Discrimination criterion was ≥85% correct responding out of 30 trials, excluding correction trials, over two consecutive sessions. Reversal training began on the session after discrimination criterion was attained. Here, the designation of correct verses incorrect stimuli was reversed for each mouse. As for discrimination, there were 30-trial daily sessions until the mice reached a criterion of ≥85% correct responding (excluding correction trials) over two consecutive sessions. For discrimination and reversal, the dependent variables were correct and incorrect trials, correction errors, reaction time (time from lever press initiation to screen touch) and magazine latency (time from screen touch to reward retrieval). In order to examine distinct phases of reversal (early perseverative and late learning), we separately analyzed errors and correction errors for sessions where performance was below 50% correct and performance above 50% correct as previously described [26]. Further analyses of trial types were performed for trials >50% correct to assess trial-type biases. These trials were separated according to consecutive trial pairs as: perseverative (incorrect-incorrect), regressive (correct-incorrect), lose-shift (incorrect-correct), win-stay (correct-correct.)
Statistical analysis
For Homecage, TFC and rotarod analyses, we used repeated measures ANOVA with post-hoc tests unless otherwise stated. For open field and EZM, unpaired t-tests were used with statistical significance set a priori at p = 0.05 following validation of normally distributed data. Mann-Whitney U test was used for non-parametric data.