Animal Model.
All animal procedures were conducted in accordance within the guidelines of Lehigh University IACUC. Animals were kept on a 12/12 light dark cycle with ad libitum access to food and water. Animals were weaned at 21 days of age and separated by sex into group housing ranging from 2-5 animals per cage. 3-8-month-old naïve, male and female, wildtype (WT) (C57BL/6J), lynx2KO, and double null mutant mice for the alpha7 nAChR and lynx2 (alpha7-lynx2KO mice) were used. Breeding of the lynx2KO mice included null mutant crosses to create knockout mice for experiments, backcrosses the C57BL/6J mice to maintain genetic diversity and avoid genetic drifts, and crosses of heterozygous mice from the backcrosses to other heterozygous mice from different pairs or knockout mice to refresh to the null mutants. The backcross was introduced every three generations. The alpha7- lynx2KO mice were created by crossing lynx2KO null mutant mice with alpha7 null mutant mice. Litters from first generation double heterozygous mice were crossed together and second generation mice were genotyped to find either one allele as a null mutant and the other allele as heterozygous or for double mutants. Combinations of the possible genotypes (such as lynx2KO, alpha7 heterozygous and lynx2 heterozygous and alpha7KO) were crossed until several double mutants were verified. The double mutants were crossed to create mice for experiments. Backcross to C57BL/6J mice were added every 3 generations and added into the breeders as described before to avoid genetic drift. All mice were genotyped using a polymerase chain reaction assay from DNA that was extracted from a tail biopsy. Each tissue sample was genotyped 2-3 times before a confirmation of the genotype was assigned. All mice were involved in one behavioral assay.
Statistics and Graphs.
Power analysis was conducted before data collection. For each experiment, an ANOVA was run to compare output (freezing or latency to dark) to age and sex. When no significant differences were obtained for age or sex, all ages and sexes were combined to analyze by genotype. Statistical analyses between groups were performed using a two-tailed t-test, with a p-value less than 0.05 considered to be statistically significant, by a repeated one-way ANOVA reporting interaction (such as genotype*time) Wilks’s Lambda values followed by either paired T-test or Bonferroni post hoc tests where appropriate, or by a Two-way ANOVA. A Cohen’s d test was used to determine effect size. All behavioral assays were performed with a minimum of 3 replicates or independent cohorts to ensure a biological phenomena as opposed to a one time result. Replicates were temporally separated tests and each included a different set of wildtype and knockout mice. All data are given as 95% confidence intervals (CI) and are represented as AVG ±SEM. *p<0.05, **p<0.01, ***p<0.001.
Fear Conditioning and Extinction
Fear conditioning was carried out in a conditioning hub (Coulbourn Instruments) placed inside an isolation cubicle (Coulbourn Instruments) to prevent ambient light and sound interference. The hub environment was lined with silver metallic walls, washed with isopropyl alcohol between trials, and had a mounted shock floor. On day 1, mice underwent a 2-minute acclimation followed by 2, 30 second sound (80 dB and 8kHz) presentations 2 minutes apart. To induce fear conditioning, during the last 2 seconds of the sound the animal received a mild (0.5 mA) foot shock. Thirty seconds after the last foot shock the animals were returned to their home cage. For the weaker fear extinction paradigm, mice received only 1 tone-shock pairing and a 0.25 mA foot shock. This was run with a different set of mice than the 2 tone-shock pairing. On day 2 and 3 fear extinction took place, with multiple measures taken to control for context. Mice were placed in a different cubicle than the one used for conditioning, the walls of the hub were switched to colorful patterns, a lavender scent was introduced, and between trials, the hub was washed with the novel cleaning agent, ethanol. After 30 seconds of acclimation in the extinction chamber, the animal was presented with 3 sound presentations at the same frequency and intensity as on the training day, each 30 seconds long and 2 minutes apart, but with no foot shocks.
The behavior of the mice was recorded using an in-hub camera and time spent freezing was analyzed using Coulbourn FreezeFrame software. Freezing was considered to be no bout of movement in a 2 second frame. The criteria for an extinguished fear response was a statistically significant decrease in percent freezing to tone (CS) between day 2 and day 3.
Fear Conditioning and Extinction with pharmacological treatment
Fear conditioning was performed as previously described, but with the addition of an IP injection given to lynx2KO mice and WT mice 1 hour prior to fear extinction on day 2 and 3. Mec (a nonspecific nAChR blocker), MLA (an α7 antagonist), and DHβE (a β2* antagonist) can cross the blood brain barrier at several documented doses, with enough potency to produce changes to nAChR function (Turek et al., 1995; Damaj et al., 1995). Doses were chosen based upon such studies. Mec (0.3-2.5 mg/kg, Abcam, Cambridge, MA), DHβE (2 mg/kg, Tocris Bioscience, Bristol, UK), and MLA (5 mg/kg, Sigma – Aldrich, St Louis, MA) were dissolved in 0.9% saline. The control animals were injected with 0.9% saline. Successful fear extinction was defined as a decrease in percent freezing from day 1 to day 2. Day 2 will be analyzed as the final extinction time point.
Light Dark Box
The light dark box assay was conducted in TruScan motion tracking arena (Coulbourn Instruments). A dark walled box was placed inside the back half of the arena with the front half surrounded by clear walls. An arched opening was made between the two areas. lynx2KO or WT mice were initially placed in the light side and their location was monitored for 10 minutes by the TruScan software. Increased latency to enter the dark and increased time spent in the dark indicates heightened anxiety-like behavior. For pharmacological studies, mice were given an. injection, IP, 1 hour prior to the start of testing.
Chronic Social Defeat Stress
CSDS followed a method adapted from the standardized protocol (Golden et. al., 2011). First, CD1 mice were singly housed for 7 days followed by a screen for aggression over 3 days. During each day of screening, an 8-20-week-old WT male was added to the home cage of the CD1 and the latency of the CD1 to attack is recorded. The WT mouse was removed upon attack or after 3 minutes. CD1 aggressors must show aggression in at least 2 subsequent sessions and have an attack latency of less than 60 seconds to be considered as an aggressor.
For non-defeated mice (WT, lynx2KO, a7L2KO) were housed with litter mates after weaning. They were placed in the same room as defeated mice and not disturbed until they were used for social interaction. For defeat, test mice (WT, lynx2KO, α7L2KO) were exposed to a CD1 aggressor mouse for a daily bout of 10 attacks. Compared to C57 (WT), CD1 mice display more aggression than C57 mice (Hsieh et al., 2017). An attack was defined as a physical altercation that included, but was not limited to, biting, shoving, rushing, jumping onto, tail rattling, and kicks. The attacks were limited to 10 to prevent excessive harm to the test mice. Following the bout, the intruder mouse was physically separated from the CD1 aggressor within the CD1 home cage but kept in sensory contact for 24 hours. This was repeated each day for 10 days, after which, the test mice were singly housed for 24 hours to await behavioral testing.
Modified Chronic Social Defeat
We adapted the 10-day CSDS to test the robustness and sensitivity of the post-defeat phenotypes. The same procedure as above was conducted but the defeat sessions occurred over 3 days. After the 3rd day of defeat the test mice were singly housed. We found that the 3 days was sufficient to induce enough of a response in the test mice that conferred with the matching behavioral results of the 10-day protocol.
Social Interaction
The social interaction (SI) test occurred 24 hours after the defeat session ended and mice were singly housed. SI ratios were assessed in Coulbourn TruScan motion tracking arenas. The arena was calibrated with an interaction zone against the back-most wall and a wire containment cage was built to the specifications of the protocol (Golden et al., 2011).
For each phase of social interaction test, post-defeat mice or controls (naïve WT and L2KO mice that did not undergo defeat and were kept in the standard housing of 2-5 littermates), called defeated or non-defeated test mice respectively, were placed in the center arena and tracked for 150 seconds. The initial phase consisted of an empty interaction zone cage. Immediately following the completion of phase 1, the test mouse was removed from the arena and returned to its home cage. During the break, a novel CD1 stranger mouse was added to the wire containment cage within the interaction zone. The same test mouse was again placed in the center of the arena and tracked for 150 seconds. During this second phase, sensory information could be transmitted from the stranger to the test mouse but there was no physical contact.
The location of the test mouse was tracked by the TruScan software. Social interaction ratios were determined based on time in the target zone (24 x 14 x 9 cm box surrounding the interaction zone) with and without the stranger mouse present. The social interaction (SI) ratio was used to assign the test mouse to a group: resilient or susceptible. The SI ratio is calculated by the following formula:
For these studies, an SI ratio above 1.5 was considered ‘resilient’ and below 1.5 ‘susceptible’ rather than the standard 1 as the lynx2KO mice were shifted towards higher SI ratios. We choose an SI ratio of 1.5 as our cutoff to increase the criteria needed to reach the resilient phenotype. Upon separating the groups, the data are presented as the percentage of time spent in the interaction zone with the stranger mouse present.
Test mice first underwent a CD1 social interaction test with a CD1 stranger mouse on day 11. The stranger mice used in the social interaction were either naïve to the test mouse or in some cases for the CD1, the stranger was the most remote CD1 aggressor to that test mouse, from day 1 of CSDS. Only males were used in these studies due to the difficulty of initiating attack behavior directed toward female mice (Takahashi et al., 2017).
Co-Immunoprecipitation (Co-IP)
WT, lynx2KO, and alpha7KO mice were anesthetized with isoflurane and rapidly decapitated. The BLA was bilaterally dissected using visual landmarks and immediately homogenized in the bullet blender tissue homogenizer (NextAdvance, Averill Park, NY, USA), with Co-IP buffer (50 mM Tris, 150 mM NaCl, 0.75% Triton-X 100, Pierce protease inhibitor cocktail). Dynabeads A (ThermoFisher Scientific,Waltham, MA, USA) were pre-incubated with 5µg rabbit anti-lypd1 (lynx2) primary antibodies (ThermoFisher Scientific,Waltham, MA, USA) and thoroughly washed. An input sample of each homogenate was removed and kept at -80 degrees celsius until western blot analysis. Brain homogenates were incubated at a concentration of 13 mg/ml with the Dynabeads-antibody complex for 3 days nutating at RT. After washing, lynx2 (lypd1) protein complexes including interacting proteins were eluted and immediately prepared for western blot analysis. Input (sample prior to the immunoprecipitation) and supernatant (after the immunoprecipitation) lane were to be compared to IP samples in the western blot analysis. Co-immunoprecipitation experiments were performed in three replicates, each with 1 lynx2KO mouse and 2 WT mice for a total of 18 technical replicates with 9 animals. The lynx2KO mice were used as a negative control for the lypd1 antibody and the alpha7KO mice were used as a negative control for the alpha7 antibody.
Western Blot analyses
Samples were denatured in 1x sample buffer (ThermoFisher Scientific,Waltham, MA, USA) at 95°C and run on a 4-20% gradient SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA, USA). Gels were transferred onto activated PVDF membrane via a semi-dry transfer. The membrane was blocked with 5% milk/0.05% Tween-PBS for 1 hour at 4°C followed by an incubation overnight at 4°C in mouse monoclonal anti-nicotinic acetylcholine receptor α7 Subunit (1:1000, Sigma-Aldrich, St Louis, MO) or rabbit polyclonal anti-BDNF (5 μg/mL, Millipore, Burlington, MA, USA) for the BDNF study. After thorough washing, membrane was incubated with conjugated goat anti-mouse (Abcam, Cambridge, MA, USA) at 1:10,000 for 2 hours at 4°C or 1:10,000 or conjugated goat anti-rabbit (Abcam, Cambridge, MA, USA) at 1:10,000 for 2 hours at 4°C for BDNF study. Membranes were incubated in ECL (ThermoFisher Scientific,Waltham, MA, USA) and exposed to film. Loading controls were run using mouse anti-actin primary antibodies (Abcam, Cambridge, MA, USA) at 1:1000 dilution, and goat anti-mouse secondary antibodies (Life Technologies, Carlsbad, CA, USA) at 1:40,000 dilution.