KCNH2-3.1 expression impairs neuronal function
Supplementary Information
Online Methods
Quantification of dendritic spines
Confocal microscopic images were taken from the largest dendrites in each neuron. Using an LSM 510 Image Examiner (Carl Zeiss MicroImaging Inc., Thornwood, NY), the GFP-filled spines were classified into three groups (mushroom, filapodia/ thin, stubby). Mushroom spines are mature spines exhibiting a large head and a thin neck. Stubby spines are unstable synaptic protrusions that have a thick neck and no head. Filapodia are immature spines that have a long thin neck(1). Quantification was performed blind to source of tissue.
General health
General health, motoric reflexes, and empty cage behavior were assessed as previously described(2). The test procedures used the following protocol:
Response to Handling
The mice were picked up and responses to handling were noted. These included vocalization, biting, excessive struggling, and jumping.
Empty cage behavior
The mouse was placed in a new empty cage with bedding and observed for threeminutes. Transfer freezing, wild running, stereotypy, grooming, rearing, digging, and exploration were noted. The presence of exploration was rated on a scale from 1-3. 1 represented minimal movement, 2 was fast movement and more exploration, but with brief pauses in between, and 3 was continuous movement and exploration with no pauses. Grooming was tallied and timed with a stopwatch. Rearing events and digging events were also tallied.
Physical Characteristics
The mice were picked up and placed in the palm of the hand. Fur condition, bald patches, piloerection, missing whiskers, body tone, skin color, limb tone were noted and any abnormalities were recorded.
Reflexes
The following neurological reflexes were tested:
Righting reflex: The mice were lifted by the tail using one hand and the other hand was held horizontally and parallel to the ground and placed against the back of the mice. Mice with normal righting reflexes flipped over onto the hand.
Whisker twitch: A cotton-tipped dowel was used to brush against the whiskers. Care was taken to insure that the dowel was moved into place from behind and below eye level so the mice did not see the dowel coming. Mice with normal whisker twitch turned to face the dowel.
Ear twitch: A cotton-tipped dowel was used to touch the mouse’s ear. The touch should produce an ear twitch.
Corneal reflex: A cotton-tipped dowel formed was used to make contact with the mouse’scornea. An intact reflex will produce an eye blink.
Physical Abilities
Trunk curl: The mice were held upside down by the tail. Mice exhibiting a normal trunk curl would curl upward.
Forepaw reaching:The mice were held by the tail over a flat surface and slowly lowered and any forepaw reaching toward the table was noted.
Positional passivity:The forepaws of the mice were placed on a slightly elevated platform and whether the mice moved or remained stationary was noted.
Petting escape: The mice were placed on a flat surface while being petted on the back. Any struggling or vocalizations were noted.
Wire hang:The mice were placed on the flat part of the wire cagetop. The wire cage top was turned over and the latency for the mice to fall off was recorded. The cutoff time was 60 s.
Behavioral testing
Locomotor activity
Locomotor activity was conducted as previouslydescribed with a few modifications(3). The testing arena was a Plexiglas® open field (42 x 42 x 30 cm) equipped with infrared photobeam sensors arranged to detect horizontal and vertical movement (Accuscan; Columbus, OH, USA). The testing arena was illuminated with red light (9 ± 2 lux). Naïve mice were allowed to freely explore the arena for 60 minutes. Distance travelled (cm) was recorded during the test period.
Acoustic startle and prepulse inhibition
Acoustic startle response and PPI were measured using four SR-Lab Systems (San Diego Instruments, San Diego, CA, USA) as described previously(4). Test sessions began with a 5-min acclimation period. Over the next 10.5 min, mice were presented with each of seven trial types in six blocks of trials for a total of 42 trials. The order in which trial types were presented was randomized within each block. The interval between trials was 10-20 seconds. One trial type measured the response to no stimulus (baseline movement), and another presented the startle stimulus alone. The startle stimulus was a 40 ms 120 dB sound burst. The other five were acoustic prepulse plus acoustic startle stimulus trials. Prepulse tones were 20 ms in length and 74, 78, 82, 86, and 90 dB, presented 100 ms before the startle stimulus. A background level of 70 dB white noise was maintained over the duration of the test session.
Cued and contextual fear conditioning
Mice were placed in a fear conditioning chamber (Med Associates, Inc.; St. Albans, VT, USA) and allowed to habituate to the chamber for 120 s. Freezing time was recorded during the habituation phase. Mice were then given three tone-shock pairings 120 s apart. For the pairing, the conclusion of the tone (conditioned stimulus, 30s, 2.8 kH, 80 dB), was followed by a footshock (unconditioned stimulus, 2s, 0.5mA) delivered through the grid floor. As the measure of conditioning, freezing behavior was measured for 120 s after the final tone-shock pairing. Twenty-four hours later, the mice were returned to the testing chamber and freezing behavior was measured for 300 s as a measure of contextual fear conditioning.
Discrete paired-trial variable-delay T-maze task
The procedure for this T-maze task was similar to one previously used in our laboratory(3). Mice were habituated to single housing for one week and were then food restricted to a level of 85% of their free-feeding weight. The mice were given 8 days for their weight to stabilize and received access to 10 reward pellets (5TUL 14 mg pellets, TestDiet, Richmond, IN) during the last 3 days of this period. Following habituation to single housing and stabilization of body weight, mice were habituated to the T-maze apparatus over the course of 2 sessions. The T-maze apparatus was made of clear acrylic (dimensions of arms: 40 X 10.2 X 17.5 cm). A recessed food cup was located at the end of each arm. During habituation sessions mice were allowed to retrieve reward pellets from the food cups. At the beginning of the session, each cup was baited with two reward pellets. The cups were re-baited continuously. Mice were allowed to retrieve 16 reward pellets during Session 1 and 20 reward pellets during Session 2. Each session automatically ended after 10 minutes if the mouse did not retrieve the maximum number of reward pellets. On the day following habituation, mice were given one session of 10 forced-alternation runs. For this session, one goal arm was blocked and the mouse had 2 minutes to consume the reward pellet located in the open arm. After an intertrial interval of at least 15 minutes, the mouse was returned to the maze for another forced run with the open/closed arms switched. Training for the discrete paired-trial delayed alternation task began on the following day. Training consisted of 10 paired trials each day. A paired trial consisted of a pseudo-randomly chosen forced run where one arm was blocked and the other arm was baited with a single reward pellet. The mouse was given 4 minutes to consume the pellet. Following consumption, the mouse was returned to the home cage for a 4-second intratrial delay. After the intratrial delay, the mouse was returned to the maze with access to both arms. The arm blocked on the forced run was now baited with 2 reward pellets. Again, the mouse was given 4 minutes to consume the reward pellets. After an intertrial interval of at least 15 minutes, mice were returned for another trial. If the mouse traveled down the unbaited arm, this was recorded as an error and the mouse was removed from the maze. The normal intertrial interval followed incorrect trials as well. Each testing session utilized a pseudo-randomly chosen pattern of 10 forced runs. Each day, the same pattern was used for each mouse. Mice were trained using these parameters for 20 days or until they reached 80% accuracy for three consecutive days. Mice that failed to reach 80% accuracy for three consecutive days within the 20-day training period were excluded from the study. Two mice each from both genotype groups failed to reach criterion. Mice were then tested using variable intratrial intervals (4, 30, 60, and 240 seconds) and a 20-second intertrial interval. Mice were given four trials of each intertrial interval on four consecutive days.
Novel object recognition
The novel object recognition test was conducted in a Plexiglas® open field arena (42 x 42 x 30 cm). On day 1 of testing, mice were allowed to freely explore the empty arena for 60 minutes. On day 2, mice were placed back into the open field for a 10 min period where they were allowed to explore two identical copies of an object. The objects were either rectangular boxes (4 x 4 x 7 cm) or Erlenmeyer flasks (4 x 7 cm). Both types of objects could be either white or black. One hour later, mice were placed back into the open field with a copy of the object encountered previously and a copy of a novel object. The novel object differed from the familiar object in both color and shape. The testing sessions were videotaped and later scored by an observer blind to genotype. Time spent exploring each object was recorded and a discrimination ratio to determine recognition memory was obtained. The discrimination ratio was calculated by subtracting the amount of time spent exploring the familiar object from the amount of time spent exploring the novel object divided by the total exploration time.
Temporal order object recognition
The temporal order object recognition test was conducted in the same open field arena as the novel object recognition test and the object location test and utilized the same 60-min exploration procedure during day 1. On day 2, mice were exposed to two sample phases. During the first sample phase, mice were allowed to explore two identical copies of an object for five minutes. The second sample phase was also five minutes long and mice were allowed to explore two copies of a different object. In the test phase, mice were presented with one copy of the object from the first sample and one copy from the second sample. The discrimination ratio is calculated using the same formula as for the novel object recognition and object location tests.
Barnes maze
The Barnes maze testing procedure was adapted from a previously published study(5).The Barnes maze consisted of a white acrylic circular table (1 m in diameter) placed on an elevated platform (1.5 m from the floor). The table had twelve, identical holes (4 cm in diameter) equidistant along the perimeter of the table located 1.8 cm from the edge. A black escape box was placed under one of the holes. All extra-maze cues were kept constant during testing.
Habituation Phase
Day 1 of Barnes Maze testing was the habituation phase. All mice were habituated to the maze and the black escape box by placing the mouse in an upside down wire pencil holder on the table. The mouse had one minute to enter the goal box. Upon entering the goal box, the wire cage was then placed over the hole and the mouse was kept in the box for one minute. After one minute the mouse was removed from the maze and placed in its home cage.
Testing Phase
Testing began on the day immediately following habituation. The mice were placed in the middle of the cleaned maze always facing the same direction and were given four minutes to discover the goal box. Upon entering the goal box, the hole was then covered and the mice were kept inside for an additional minute. The mice were then removed and placed back into the home cage. Mice were tested twice a day for ten consecutive days.
Probe trial
Probe trials occurred on Day 12 and Day 18. The mice had four minutes to find the goal box. Upon entering the goal box, the mice were immediately removed from the maze and placed back into its cage. Mice who did find the goal box in the allotted time were immediately removed from the maze and placed back in the home cage. Each mouse was only given one probe trial each day.
Trace fear conditioning
Mice were placed in a fear conditioning chamber (Med Associates, Inc.; St. Albans, VT, USA) and received five unpaired tones followed by five tone-shock pairings. For the pairing, the conclusion of the tone (conditioned stimulus, 30s, 2.8 kH, 80 dB), was followed by a footshock (unconditioned stimulus, 2s, 0.5mA) delivered through the grid floor. After each tone, freezing behavior was recorded for 20 seconds. Data were analyzed using VideoFreeze software (Med Associates, Inc). Freezing behavior was analyzed during the period following the unpaired tones and after the final tone-shock pairing.
Statistical analyses
T-tests were used to analyze the data for the dendritic morphology, electrophysiology, object location test, acoustic startle, fear conditioning experiments, and the days to criterion measure of the discrete paired-trial variable-delay t-maze task. Repeated measures ANOVAs were used to analyze the data from the variable-delay portion of the T-maze task, locomotor activity, prepulse inhibition, Barnes maze training, and electrophysiology experiments, where appropriate. We also conducted a planned comparison of the control and KCNH2-3.1 transgenic groups at the 4-sec interval, the testing interval well-within the accepted limits of PFC-dependent working memory(6, 7), using a t-test. The data met the assumptions of the chosen statistical test and variance was similar between experimental groups. No specific statistical methods were used to estimate sample size, but the samples sizes were similar to previously published studies(3, 8). No specific randomization strategy was used for these experiments. All data are represented as the mean ± SEM.Post-hoc analyses were conducted using Bonferroni-corrected pairwise comparisions. Significance was set at p < 0.05.
SupplementaryTable and Figures
Table 1
Supplementary Figure 1
Baseline locomotor activity
(a) Mice were allowed to freely explore a novel open field for 60 minutes under low, red-light conditions (8 ± 2 lux). Baseline locomotor activity was unchanged by KCNH2-3.1 expression [F(1, 31) = 1.28, p = 0.27]. Both KCNH2-3.1 transgenic mice and their littermate controls significantly decreased locomotor activity over time [F(11, 341) = 18.81, p < 0.001].There was also a nonsignificant genotype X timepoint interaction [F(11, 341) = 1.78, p = 0.056.
Supplementary Figure 2
Startle amplitude and prepulse inhibition (PPI)
(a) Startle amplitude was measured in response to the presentation of a 40 ms, 120 dB sound burst. There were no differences between the groups in response to no stimulus [t(23) = 0.12, p = 0.90] or startle amplitude [t(23) = 0.54, p = 0.60]. (b) PPI, a measure of sensiromotor gating, was also normal in KCNH2-3.1 transgenic mice. There was no main effect of genotype [F(1, 23) = 0.93, p = 0.35] or genotype X prepulse sound level interaction [F(4, 92) = 0.40, p = 0.81]. Both KCNH2-3.1 transgenic mice and their littermate controls exhibited the normal increase in PPI as the prepulse intensity increased [F(4, 92) = 12.44, p < 0.001].
Supplementary Figure 3
Cued and contextual fear conditioning
KCNH2-3.1 transgenic mice showed no difference inbaseline freezing [t(18) = 1.99, p = 0.06] or conditioning to either the cue [t(18) = 0.63, p = 0.95] or context [t(18) = 0.16, p = 0.87] compared to the control mice.
Supplementary Figure 4
Dendritic spine morphology in the hippocampus
(a) Confocal microscopic images of representative dendrites from control (top) andKCNH2-3.1 transgenic mice (bottom). Prototypical mushroom (red arrows), thin/filapodia (white arrows), and stubby (yellow arrows) are identified in each image. (b) KCNH2-3.1 transgenic mice have fewer mushroom [t(28) = 2.41, p = 0.02] and stubby [t(28) = 4.56, p < 0.001] spines. There were no differences in the number of thin spines/filopodia [t(28) = 0.41, p = 0.68]. n = 18/control and n = 12/KCNH2-3.1. Data are represented as the mean ± SEM. *p < 0.05; ***p < 0.001
Supplementary Figure 5