), heterozygous (Dlx1+/−
) and wild-type (Dlx1+/+
) mice [22
], all age-matched at the time of testing, were maintained on a C57BL/6 J × CD1 background [18
]. Wild-type C57BL/6 J mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were used as an additional comparison group for spontaneous alternation and spatial working memory tasks. Mouse colonies were maintained at the Massachusetts Institute of Technology (MIT) or at the University of California, San Francisco (UCSF), in accordance with National Institutes of Health, MIT, and UCSF guidelines for care and use of lab animals.
General behavioral assays were performed on one set of mice (aged 3–6 months), which were all males from three genotype groups (Dlx1−/−
), in the following testing order: zero maze, open field, locomotor activity, food intake, rotarod, and finally tail suspension. We did not perform fear conditioning, prepulse inhibition, or working memory tasks on this set of mice. Mice were group-housed (4–6 mice per cage) in standard polycarbonate mouse cages (29
13 cm). They had free access to food and water, and were maintained in a 12 hour light/dark cycle. Between subjects, the testing apparatus was cleaned with a 0.25% bleach solution, wiped down with water, and dried. With the exception of tests requiring the monitoring of photobeam activity, all animals were tested during the light cycle. Experimental conditions were counterbalanced by genotype.
Multiple sets of the mice (age
2 months) were tested for assays in the following order: visual behavioral assays and then fear conditioning (context only, or context and trace). Some sets of these mice were finally tested with prepulse inhibition (aged
A different set of mice (all males) were tested with the spontaneous alteration task and spatial working memory task. Dlx1 mice (Dlx1+/+ and Dlx1−/−) tested on these tasks were 11–15 weeks old and group-housed; C57BL/6 J mice used as age matched controls were 12–15 weeks old and group-housed. All mice were tested on the spontaneous alteration task first and then on the spatial working memory task 1–2 weeks later. These mice were not tested on any other behavioral assays reported in this manuscript.
For all behavioral tests, investigators were blind to the genotypes. Approximately 3 min before each assay, the animal was removed from its home cage and placed in a clean holding cage for transfer; an exception to this was the tasks requiring photobeam activity monitoring, where animals were placed directly into the test chambers.
A zero maze (34 cm inner diameter, 46 cm outer diameter, placed 40 cm off the ground on 4 braced legs) was used as previously described [23
]. Mice were removed and placed in the center of one of the closed quadrants. The test lasted 6 min. Latency in entering (all four paws) an open quadrant, time spent in the open quadrants, and number of closed-to-open quadrant transitions was live-scored in a blinded fashion by a remote observer.
A 4-unit open field consisting of a white Kydex box divided into four separate 50
38 cm chambers was used, allowing four animals to be tested concurrently. A video camera was mounted directly above the chambers to monitor the animals’ activity and movements. Four mice were simultaneously placed into the open field chambers. A video tracking system (Poly-track, San Diego Instruments, San Diego, CA, USA) was used to measure distance traveled in 5 min monitoring sessions. Assignments to the four chambers were counterbalanced by genotype. Mouse activity within 7 cm of the chamber walls was defined as occurring within a peripheral zone; activity further away from the walls was considered to occur within a center zone.
mice were not tested due to limited availability of activity cages) were housed individually in larger cages (48
13 cm) with bedding, food and water, under a 12-hour light/dark cycle (lights on 7 AM). To assess activity, we used a photobeam system (FlexField, San Diego Instruments, San Diego, CA, USA), which records horizontal locomotor activity (as monitored by a 4
8 array of infra-red photobeams). Data were collected for three days, 24 h each day, with animals placed in the system at noon on day 1.
During the locomotor activity run described above, daily food (standard UCSF laboratory chow pellets) intake measures were made by weighing the food daily.
Motor coordination was assessed with an Accurotor rotarod machine (Accuscan Instruments, Columbus, Ohio, USA). The rotation rate was accelerated from zero to 50 rpm over 5 min. Four animals were tested concurrently in separate 11 cm-wide compartments, on a rod that was approximately 3 cm in diameter and was elevated 35 cm. Each animal was assessed over nine trials with 20 min inter-trial intervals (ITIs). For each trial, the latency to fall from the rod was recorded.
Using adhesive tape, animals were suspended by the tail from a 1.2 cm diameter metal bar elevated 30–35 cm. When suspended, rodents either make “escape attempt” movements, or adopt a characteristic immobile posture. The total time for which the mouse was immobile was measured during a 6 min test period.
All mice were allowed to rest undisturbed in their transportable home cage at least seven days prior to the fear conditioning. To minimize occurrence of handling-induced seizures in the mutant mice [18
], we habituated all animals for 3–5 sessions (~10 min in each session, once a day) prior to the day of the first experiment. In these habituation sessions, mice were acclimated to the room and handling of the experimenter. The experimenter wore gloves, habituated the mice to handling, allowing them to walk or run from hand to hand, until the mice showed no apparent signs of stress or fear.
An automated fear-conditioning system (TSE Systems, Bad Homburg, Germany) was used for fear conditioning. Some mice were tested with contextual fear conditioning only (without exposure to a light or tone); the others were tested with both contextual and trace fear conditioning. For each mouse from the Dlx1−/−
groups, training (Day 1) consisted of a 3 min exposure to the conditioning box (context). In the case of contextual fear conditioning only, this was followed by a foot shock (2 sec, 1.0 mA, constant current). In the trace conditioning sessions, the 3-minute exposure to the context was followed by a visual or auditory cue. In the case of visually cued fear conditioning, the cue was a 30 sec flickering light at 1 Hz, followed by a 15 sec trace interval, and then a foot shock (2 sec, 1.0 mA, constant current). Because a visual cue tends to be less effective in eliciting a defensive response than an auditory cue and often requires more conditioned stimulus (CS)—unconditioned stimulus (US) pairings to the light alone [24
], the light-pulse-shock sequence was repeated twice before removing the mouse from the box (15 sec ITI). In the case of an auditory cue, after a 3 min exposure to the box, the mouse was subjected to the tone (30 sec, 10 kHz, 75 dB sound pressure level (SPL), 200 ms interval), a 15 sec trace interval, a foot shock (2 sec, 1.0 mA, constant current), and finally a 15 sec interval before removing it from the context. Given a positive correlation of shock intensity (ranging from 0.25 mA to 1.0 mA) with percent of freezing in mice [25
], we used a shock intensity of 1.0 mA, which is at the higher end of this range, to ensure that both the wild-type and mutant mice would be conditioned with the foot shock.
The contextual memory test (Day 2) was performed 24 h after training by re-exposing the mice for 3 min to the conditioning context. Freezing, defined as a lack of movement except for heart rate and respiration, associated with a crouching posture, was assessed every 10 sec, for 180 sec, by a trained observer who was unaware of the genotypes. The number of observations indicating freezing was expressed as a percentage of the total number of observations. Baseline freezing for the contextual test refers to freezing (if any) on day 1 during the 3 min exposure to the context, before the tone and foot shock in the training session. We performed the contextual memory test alone, or combined it with the trace conditioning. The results did not differ in the two cases. We report the results on contextual fear conditioning that was carried out alone, i.e. without a visual or auditory cue paired to the shock.
The trace test was performed on day 3, 24 h after the contextual memory test. Mice were placed in a novel context. After a 30 s habituation period, a 180 s flickering light (1 Hz) or a 180 s tone (10 kHz, 75 dB SPL, 200 ms interval) was presented in the absence of a foot shock; freezing behavior was recorded every 10 sec, over 18 intervals (see above). Baseline freezing for the trace test refers to freezing (if any) on day 3 in the second context, before presenting the light or tone.
Mice were tested using an ASR-PRO1 acoustic startle reflex test apparatus (Med Associates, St. Albans, VT, USA).
Mice were acclimated to the testing room for 1 h prior to testing. For 2 min before start of trials, mice were acclimated to the apparatus. Trials were given at an interval of 3–8 s (randomized). Trials consisted of either a 40 ms startle stimulus alone (110 dB white noise) or a startle stimulus preceded 100 ms earlier by a 20 ms white noise prepulse at 8 dB, 12 dB, or 16 dB above background (60 dB white noise). Five trials for each stimulus configuration were recorded using Startle Reflex Software (Med Associates, St. Albans, VT, USA).
Visual cliff test
Mice were placed in a 60
60 cm box with a clear Perspex base. A high-contrast grating was attached to the underside of one half of the box; the clear half of the box was placed such that it protruded from the table, revealing a drop to the floor of approximately 90 cm. Mice were placed in the center of the box and their behavior was monitored for 5 min, via a digital video camera mounted above the box. The amount of time spent in each half of the box was recorded. To confirm the role of the visual system in this task, the test was also performed under dark (red light) conditions.
Visual water maze test
Mice were tested in a circular tank (diameter 1.2 m) filled with opaque water. In the center of the target quadrant, a platform (11
11 cm) was submerged below the water surface, but with a visual cue (a cylinder with high-contrast gratings on the surface) above the water to guide the mouse. The swimming paths of the mice were recorded by a video camera, and analyzed off line by the Videomot 2 software (TSE Systems, Bad Homburg, Germany). Each mouse was placed into the maze from four random points of the tank, and allowed to search for the platform. The amount of time that each mouse took to find the platform was recorded and averaged for comparison.
Spontaneous alternation task (Y-maze)
Three open arms of an automated radial maze were used to form a Y-maze. Proximal visual cues were attached to the arms and to the hub of the maze to orient the mice in space. C57BL/6 J, Dlx1+/+
mice were placed in the hub and allowed to traverse the three arms for 8 min. Based on the natural levels of activity in the maze, which peaked at about 2 min, the data were analyzed for the first 2 min, as well as for the entire 8 min of maze exploration. Alternation was considered to be ABC, BCA or CAB, etc, in any order or overlapping occurrence; but not ABA or BBC. The number of entries into each arm, the percent of alternation ((number of alternations / (total number of arm entries-2))
100), and the percent of repeat entries into the same arm were calculated.
Spatial working memory task (novel arm in a Y-maze)
For this task, the animal (a C57BL/6 J, Dlx1+/+ or Dlx1−/− mouse) first explored two randomly selected arms of the Y-maze for 5 min, followed by a 2 min ITI in which the mouse was confined to the center hub of the maze, with all maze arms closed. Next, all three Y-maze arms were opened, and the mouse was free to explore the entire maze for 5 min. The data from the retrieval phase were analyzed to determine if mice recognized the novel arm, expressed by enhanced exploration.
Two measures of novelty exploration/working memory were used to analyze the data from the retrieval phase of this assay: the number of entries into the Y-maze arms and the total amount of time spent in each of the arms.
All p values reported in the results were obtained either from ANOVA for overall comparisons of three or more groups of mice (using genotype as the independent variable) except the repeated measures ANOVA described below, or from t-tests for comparisons between two groups of mice. Locomotor activity of Dlx1 mice was analyzed using repeated measures ANOVA with genotype as the between factor, and activity counts over time as the within factor.
Analysis of the prepulse inhibition assay was carried out using repeated measures ANOVA with genotype as the between group variable and prepulse intensity as the within group variable. Where a significant effect was seen between groups of mice, a post-hoc comparison was performed using the Tukey HSD test.
Statistical analysis of the spontaneous alternation task was performed using one-way ANOVA with genotype as the independent variable and number of entries, percent alternation, and percent repeated entries as dependent variables. Data from spatial working memory task were analyzed using repeated measures ANOVA, with genotype as the between variable, number of entries, and time spent in arms as within variables (separate ANOVA for each).