Experimental procedures were performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee.
Kv4.2 KO mice were generated in a 129S6 genetic background as described previously . For the current experiments, mice were backcrossed to the C57BL/6J strain for nine generations. To control for potential effects of maternal genotype , KO mice and WT controls were littermates bred from heterozygous parents. Males and females were used. Mice were housed in same-sex groupings in a temperature- and humidity-controlled vivarium under a 12-hour light/dark cycle (lights on at 06.00 hours) and testing conducted during the light phase after 1 hour of acclimation to the test room. Three separate, naïve cohorts were tested. One cohort of mice was screened on a battery of neurological measures, including prepulse inhibition and home-cage activity, and basal home-cage corticosterone levels. A second cohort was tested on a battery of assays for anxiety-, fear- and stress-related phenotypes, with putatively more stressful tests conducted later in the sequence and at least 6 days between each test: novel open field, light/dark exploration, elevated plus-maze, pavlovian fear conditioning and extinction, and forced-swim test/swim-induced corticosterone response. A third cohort was tested for behavioral responses to repeated inescapable forced swims. The experimenter remained blinded to genotype during testing. The number of mice tested in each assay is given in the corresponding figure legends.
Initial phenotypic screen
Neurological test battery
Functional observational battery
Gross physical and neurological abnormalities were examined using a simple functional observation battery, as described previously [16, 23, 24]. Basic physical health was evaluated by examining for missing whiskers, bald patches, piloerection, exophthalmus, straub tail, kinked tail, kyphosis, lordosis, core body temperature, reactivity to handling, and body weight. Simple sensory reflexes were measured via orienting responses to an approaching hand and to physical touch via palpebral closure on touch of the eye, twitch of pinna on touch, and an orienting response to tail pinch. Mice were observed for splayed limbs, and forepaw and hind limb clutch when suspended upside-down by the tail, and wild running, freezing, trembling, sniffing, licking, rearing, jumping, seizures, defecation, urination, head bobbing, circling, abnormal gait, retropulsion, and prancing forelimbs when placed in a novel, bare, standard holding cage (130 × 135 × 350 mm) for 1 minute.
Pain perception was tested using the hot-plate test. a flat plate (Columbus Instruments, Columbus, OH, USA) was heated to 55°C, and the mouse placed upon it. The latency to first hind-paw lick was manually timed, with a maximum response latency of 30 seconds. and genotypes statistically compared via Student's t-test.
Acoustic startle and prepulse inhibition of startle
Acoustic startle and prepulse inhibition of startle (PPI) was tested as described previously . The mouse was placed in a clear Plexiglas holding cylinder (San Diego Instruments SR-LAB System; San Diego, CA, USA) for an acclimation period of 5 minutes. A 65-dB broadband background noise was presented during acclimation and throughout the test session. Five pulse-alone trials (120 dB broadband sound pulse for 40 ms) began and ended the session, and were not included in the analysis. Testing consisted of presentation of 120 dB startle trials and prepulse + startle trials (noise prepulse sound for 20 ms, followed 100 ms later by the 40 ms 120-dB broadband sound pulse). There were three different prepulse intensities (3, 6 and 12 dB above background), each presented 10 times with a variable interval (range 12-30 seconds) between each presentation. There were vie pulse-alone trials before and after each prepulse + startle trial. Basal activity in the startle chambers was measured during the no-stimulus trials. Startle amplitude was measured starting from the onset of the startle stimulus via whole-body vibrations (sampled every 1 ms over 65 ms), transduced into analog signals by a piezoelectric unit attached to the platform on which the cylinders rested. This was used to derive an average startle amplitude over the 65 ms recording period. PPI at each prepulse intensity was calculated as:
The effects of genotype and PPI intensity were analyzed using two-way analysis of variance (ANOVA), with repeated measures for PPI intensity.
The mouse was individually housed in a standard holding cage as before, and left undisturbed for a 48-hour acclimation period under normal vivarium conditions. Activity was measured over 24 hours using a photocell-based activity monitor (Opto M3; Columbus Instruments, Columbus, OH, USA) and expressed as the 12-hour average activity during the light and dark phase, as described previously . The effects of genotype and circadian phase were analyzed using two-way ANOVA, with repeated measures for cycle.
Battery of anxiety-, fear-, and stress-related behaviors
Novel open field
The novel open-field test  was conducted as described previously  in a 400 × 400 × 350 mm square arena (60 lux) constructed of white Plexiglas. Testing was conducted under 65 dB white noise to minimize external noise disturbances (Sound Screen, Marpac Corporation, Rocky Point, NC, USA). The mouse was placed in the perimeter and allowed to explore the apparatus for 30 minutes. Total distance traveled and time spent in the center square (200 × 200 mm) was measured by a video-tracking system (Ethovision; Noldus Information Technology Inc., Leesburg, VA, USA).
Light-dark exploration test
The light-dark exploration test  was conducted as described previously . The mouse began the test in an opaque black Plexiglas shelter (390 × 130 × 160 mm) with a 130 × 80 mm opening at floor level that opened onto a large white Plexiglas square arena (390 × 390 × 350 mm) illuminated to approximately 90 lux. Percentage time spent in the lighted compartment, frequency of entering the lighted compartment, and total distance traveled in the entire apparatus over a 15 min session were measured by a video-tracking system (Ethovision Noldus Information Technology Inc). Mice that did not enter the lighted compartment were recorded as having a latency to exit time of 900 seconds. Genotypes were compared using Student's t-test.
The elevated plus-maze test  was conducted as described previously . The apparatus consisted of two open arms (300 × 50 mm; 90 lux) and two closed arms (300 × 50 × 150 mm; 20 lux), extending from a 50 × 50 mm central area and elevated 200 mm from the ground (San Diego Instruments, San Diego, CA, USA). The walls were made from black acrylonitrile butadiene styrene (ABS) plastic and the floor from white ABS plastic. A 50 mm high raised lip around the perimeter of the open arms prevented the mice from falling off the maze. Testing was conducted under 65 dB white noise (Sound Screen, Marpac Corporation, Rocky Point, NC, USA) to minimize external noise disturbance. The mouse was placed in the center of the maze facing an open arm, and allowed to explore the apparatus for 6 minutes. Time spent in the open arms and number of entries into the open and closed arms were measured by a video-tracking system (Ethovision Noldus Information Technology Inc). Genotypes were compared using Student's t-test.
Pavlovian fear conditioning and extinction
Pavlovian fear conditioning and extinction was assessed as described previously [32, 33]. The mouse was placed in context A: a 270 × 270 × 110 mm chamber with transparent walls and a metal rod floor. To provide a distinctive olfactory environment, the chamber was cleaned between subjects with a 79% ethanol/20% water/1% vanilla extract solution. After acclimation period of 180 seconds, the mouse received three pairings (interval of 60-120 seconds after each pairing) of an auditory tone (30 seconds, 80 dB, white noise) and footshock (2 seconds, 0.6 mA scrambled footshock), in which the shock was presented during the last 2 seconds of the tone. The presentation of stimuli was controlled by a freeze monitoring system (Med Associates Inc, Georgia, VT, USA).
Twenty-four hours later, expression of fear to the tone and subsequent within-session extinction was tested. Mice were placed in context B: a novel context (Plexiglas cylinder with black/white-checkered walls and a solid floor, cleaned with a 1% acetic acid/99% water solution) housed in a novel room. After an initial acclimation period of 180 seconds, the mouse received 500 presentations of the tone alone (each tone for 30 seconds, with a no-stimulus interval of 5 seconds). Twenty-four hours later, extinction retrieval was probed with three tone presentations in context A. Four hours later, mice were presented with three tones in context B to assess fear renewal.
Freezing (no visible movement except that required for respiration) in response to the tone was manually scored every 5 seconds and converted to a percentage:
Freezing during extinction trials were averaged into 100 five-trial blocks for analysis, and the first and last trial blocks compared. The effect of genotype and trial/trial block during conditioning and extinction were analyzed using two-factor ANOVA, with repeated measures for trial/trial block, followed by Bonferroni post hoc tests. The effect of genotype on extinction retrieval and fear renewal were analyzed using Student's t-test.
Responses to swim stressors
Forced-swim test behavior and corticosterone response
The forced-swim test  was conducted as described previously . The mouse was gently lowered into a 200 mm-diameter cylinder filled with water 24 ± 1.0°C for test lasting 6 minutes. Immobility (cessation of limb movements except for minor movement necessary to keep the mouse afloat) was manually scored every 5 seconds during minute 3 to 6. Genotypes were compared using Student's t-test.
Mice were returned to the home cage for 30 minutes after forced swim, and then killed via cervical dislocation and decapitated. The trunk blood was collected, left to coagulate at room temperature for 1-2 hours, and then separated by centrifugation at 4°C for 30 seconds at 13,000 rpm (20000 x g). Serum was collected and analyzed for corticosterone (bound and free) using a commercial radioimmunoassay (MP Biomedicals, Orangeburg, NY) as described previously . Genotypes were compared using Student's t-test.
Repeated inescapable forced swim
The repeated inescapable forced-swim (riFS) test was conducted as described previously . The mouse was gently lowered into a cylinder filled with water at 24 ± 1.0°C. The cylinder was of a larger diameter (300 mm) than that used for the forced-swim test used above (200 mm) and had a 40 × 40 mm escape hole located 40 mm above the waterline and out of reach for the mouse. A platform (plastic wiffle ball) was magnetically held 100 mm below the water, also out of reach. After 1 minute, the platform was remotely released (by turning off the magnet) and quickly (within < 2 seconds) floated vertically upwards along a pole to sit on top of the water, directly under the escape hole. If the mouse attempted to climb onto or hold the platform, it sank below the water. The test terminated 20 seconds after the platform was presented, regardless of the number of attempts by the mouse to use it to reach the escape hole. There was one trial per day (10.00 to 12.00 hours) for 10 consecutive days. Immobility was manually scored every 5 seconds during the 1 minute before platform presentation. The effect of genotype and trial were analyzed using two-way ANOVA, with repeated measures for trial.
Knockout and wildtype mice were compared using Student's t-test with the exception of the prepulse inhibition of startle, home cage activity, fear conditioning and extinction, and riFS testing, which was analyzed using a 2-factor analysis of variance (ANOVA) with Bonferroni posttest. The threshold for statistical significance was P < .05. GraphPad Prism 5 statistical software (La Jolla, CA) was used for all statistical analysis.