All mice were derived from a pure C57BL/6J back ground as fully described before (see Yee et al., 2006
). The experimental subjects were bred by pairing CamKIIαCre:Glyt1tm1.2fl/fl mice with Glyt1tm1.2fl/fl mice to yield litters of an expected 1:1 ratio of CamKIIαCre:Glyt1tm1.2fl/fl (carrying one copy of CamKIIα-driven Cre-expression with homozygously floxed GlyT1 gene) and Glyt1tm1.2fl/fl (with only homozygously floxed GlyT1 gene and no Cre expression) genotypes. The former CamKIIαCre:Glyt1tm1.2fl/fl genotype is referred to as GlyT1ΔFBNeuron
and denoted as the “mutant”, with the latter Glyt1tm1.2fl/fl genotype serving as comparison “control” littermates. Breeding took place in a specific-pathogen free (SPF) breeding facility (Laboratory of Behavioural Neurobiology, ETH Zurich, Schwerzenbach, Switzerland), and litters were weaned and sexed on postnatal day 21. Genotypes were determined by standard PCR on tail biopsies obtained within 10 days after weaning as previously described (Yee et al., 2006
All manipulations and procedures described here had been previously approved by the Swiss Cantonal Veterinary Office as required by the Swiss Act and Ordinance on Animal Protection, which conforms with the ethical standards stipulated in the European Council Directive 86/609/EEC and the NIH publication no. 86-23 (revised 1985) on animal experimentation.
2.2 Housing under different circadian cycle
At the age of 11 weeks, a cohort of 87 mice (cohort A in ) was subdivided into two balanced groups, with respect to genotype and sex, for separate housing between two identical climatized (21±1°C, relative humidity at 55±5%) vivaria differing only in their circadian rhythm. One vivarium was maintained under a “normal” 12/12h light-dark cycle (lights off: 1900–0700h), while the other was under a “reversed” light-dark cycle (lights off: 0700–1900hrs). It thus allowed the direct behavioural comparison between light and dark phases in a between-subjects manner. All behavioural experiments were conducted between 0800 and 1800 h, thus falling within the light phase of animals kept in the “normal” cycle and the dark phase of those kept under the “reversed” cycle. Henceforth, we refer to this between-subject factor as “light-dark phase”. Mice of the same sex were housed in groups of 4 to 6 in Makrolon® Type-III cages (Techniplast, Milan, Italy) with constant provision of water and food (Kliba 3430, Klibamuhlen, Kaiseraugst, Switzerland) unless otherwise specified. The mice were approximately 12-16 weeks old at the time of testing.
Number and distribution of the separate cohorts of animals used in the present study
Four cages (one for each genotype/sex group) each housing four mice of the same sex and genotype, were arranged in each animal vivarium; and a miniature digital camera operating in the visible and infrared spectrum was mounted above the grid cage to allow monitoring of home cage activities. Video outputs of the four cameras in each animal vivarium were fed to a multiplexer (YSQ-430, Sony, Japan) before being transmitted to a PC for digital storage and cage-by-cage analysis. Five-minute 8-bit gray-scale video footages were sampled at 30 min past every o'clock over the 11-day acclimatization period. An image analysis algorithm calculated the number of pixels changed after appropriate thresholding (Open eVision 1.1, Euresys s.a., Belgium) between successive (360 × 288 pixels) binary images 1s apart. The number of pixels changed was expressed as percentage of total pixels per frame, averaged across each hourly 5-min sample. This number was then normalized and expressed as z-scores with respect to each cage's average second-by-second percent pixels changed across the 11-day period). For each cage the 5-h running average time series was calculated and depicted in .
Acclimatization to separate housing conditions with opposite light-dark (12h/12h) cycle
Because our mice were bred under a reversed light-dark cycle, animals allocated to the vivarium with the same circadian rhythm adapted quickly and exhibited the expected diurnal variation in activity, namely, higher in the dark phase and lower in the light phase (). In contrast, animals switched to the normal light-dark cycle experienced a shift of circadian rhythm, and therefore their previous diurnal activity pattern persisted in the first 48h. Thereafter, they underwent a period of adjustment for about two days before the emergence of a new stable diurnal activity rhythm matching the light-dark rhythm (). As shown in the average activity profiles obtained from the two rooms (), their activity cycles were 12-h out-of-phase with each other for the last 7-8 days prior to commencement of experiments. Because all behavioural tests were conducted between 0800 to 1800h, mice kept in the reversed cycle would be tested in the dark phase, and those in the normal cycle in the light phase. The comparison between cycles effectively served as a contrast between light and dark phases while keeping the time and conditions of testing identical.
2.3 Experimental Plan
The cohort of 87 mice, prepared as described above, was first evaluated in an elevated plus maze test of anxiety and an open field test of locomotor activity before being split randomly into two subsets: one destined for the aversive conditioning experiment, and the other for the appetitive conditioning experiment (see ). A separate cohort of naive mice (cohort B), which comprised only female mice kept in reversed light-dark cycle, was prepared especially for test of shock sensitivity.
2.4 Elevated plus maze
The elevated plus maze test of anxiety has been fully described before (Hagenbuch et al., 2006). Briefly, the maze was made of acrylic and consisted of four equally spaced arms radiating from a central square measuring 5 × 5 cm. Each arm was 30 cm long and 5 cm wide. A gray removable plastic floor in-lay was placed in the entire maze surface. One pair of opposing arms was enclosed with opaque walls 14 cm high. The remaining two arms were exposed with a 3-mm-high perimeter border along the outer edges. The maze was elevated 70 cm above floor level, and positioned in the middle of a testing room with diffuse dim lighting (25 lux in the centre of the maze). A digital camera was mounted above the maze and images were captured at a rate of 5 Hz and transmitted to a PC running the Ethovision (Version 3.1, Noldus Technology, The Netherlands) tracking system. A test trial began with the mouse being placed in the central square with its head facing one of the open arms. It was allowed to explore freely and undisturbed for 5 min. The percent time in open arms = [time in open arms/time in all arms] × 100% was used to index anxiety. In addition, the total distance travelled in the entire maze (i.e., arms and central platform) was recorded.
2.5 Open Field Test for spontaneous locomotor activity
This was conducted 48 h after the elevated plus maze experiment. The apparatus consisted of four identical square arenas (40 × 40 cm) surrounded by 25 cm high walls. They were made of wood with a white waterproof plastic surface. The four arenas were arranged in a 2-by-2 configuration, located in a testing room under diffused dim lighting (25 lux). A digital camera was mounted directly above the four arenas, transmitting images to a PC running the Ethovision (Version 3.1, Noldus Technology, The Netherlands) software which tracked the animals with a temporal resolution of 5Hz. Four mice were tested simultaneously. They were placed in the centre of the appropriate arena and allowed to explore undisturbed for 60 min. Afterwards, they were returned to their home cage and the arenas were cleansed with water and dried before the next squad of four mice. Locomotor activity was indexed by distance travelled (in meter) across consecutive 10-min bins.
2.6 Pavlovian aversive conditioning
Five animals from each of the eight experimental groups were selected for this experiment (see ), which commenced 48h after the open field test.
Two sets of chambers were used to provide two distinct contexts. The first set of chambers (context A) comprised four operant chambers (30 × 25 × 29 (high) cm, model E10-10, Coulbourn Instruments, Allentown, PA) individually installed in a ventilated, sound insulated chest. Each chamber was equipped with a grid floor made of stainless steel rods (4 mm in diameter) spaced at an interval of 10 mm centre to centre, and through which scrambled electric shock could be delivered by a shock generator (Model E13-14). The animal was confined to a rectangular region (17.5 × 13 cm) in the centre by a clear Plexiglas enclosure. Illumination inside the chamber was provided by a house light (2.8 W) positioned on the right panel wall, 21 cm above the grid floor. The second set of chambers (context B) comprised four cylindrical (19 cm in diameter) enclosures made of clear Plexiglas, resting on a metal mesh floor, and located in ventilated, sound-insulated, wooden cabinets. They were illuminated by an infrared light source instead of visible light. Each chamber also contained a sonalert tone module (Model SC628, Mallory), which provided an 86 dBA
tone, and a miniature digital camera (sensitive to visible and infrared range) mounted 30 cm directly above the centre of the area of interest. The output of the camera was fed to a multiplexer (YSQ-430, Sony, Japan) before being transmitted to a computer running the Open eVision 1.1 (Euresys, Liège, Belgium) software under the control of a customized Microsoft Visual Basic (version 6) script. The algorithm of the freezing response detection procedure has been validated and fully described before (Richmond et al., 1998
). In brief, successive digitized images (192 × 144, at 8-bit gray scale) obtained at a rate of 1 Hz were compared. The difference in number of pixels between adjacent frames was then computed. If this was less than 0.05% of the total number of pixels in a frame, the animal was considered to be freezing in that 1-s interval. The procedures comprised three distinct phases: (i) conditioning, (ii) test of conditioned context freezing, and (iii) test of conditioned tone freezing across days.
On Day 1, all animals were given three conditioning trials in context A. Each trial consisted of a 30s tone stimulus (conditioned stimulus, CS) followed immediately by a 1-s 0.25 mA foot shock (unconditioned stimulus, US). The first trial was administered 3 minutes after the animals were placed into the chambers. Successive trials were administered every 3 minutes. The conditioning session was concluded with a final 3-min interval.
On Day 2, the animals were returned to context A. They were placed in the test chamber for a period of 8 min.
On Days 3 to 10, conditioned freezing to the tone stimulus was assessed in context B. The tone stimulus was administered 2 min after the animals were placed into the test chamber. The tone remained on for a period of 8 minutes. The expression of freezing or immobility was expressed as percent time freezing.
The three phases were separately analyzed.
2.7 Appetitive Pavlovian conditioning
The animals from cohort A not included in the aversive conditioning experiment (see ) were used in the appetitive conditioning experiment, which commenced 12 days after conclusion of the open field experiment. Five days prior to conditioning, the animals were gradually introduced and acclimatized to a food-restricted diet until they were finally maintained on free feeding 2h per day throughout the experimental period. Their body weight was closely monitored and prevented from falling below 85% of their ad libitum weight.
The apparatus consisted of eight Habitest System operant chambers (29 × 25.5 × 28 high cm, Model E10-10, Coulbourn Instruments Allentown, PA), each located in a ventilated, sound-insulated chamber. Illumination inside the chamber was provided by a house light (2.8 W) positioned on the right panel wall, 21 cm above the grid floor. A partition wall (parallel to the panel wall) was installed to reduce the floor area to 17 × 25.5 cm. A magazine tray was positioned in the middle of the panel wall and 2 cm above floor level. Nose-poke responses to the magazine were detected by an infrared photocell beam (Model H14-01M) placed at the entrance. Liquid reward (0.01 ml of a 20% dilution of a commercial condensed milk, Milch Lait®, Switzerland) was delivered in the magazine by an automated dipper (Model H14-05R) attached to it. Each chamber also contained a sonalert (Model SC628, Mallory), which provided an identical tone (86 dBA) to that used in the Pavlovian aversive conditioning experiment. Each set of four chambers were connected via an interface to a PC running the Graphic State software (Version 1.013) which provided independent control of each chamber and collected all response data.
The animals were first familiarized with consuming the milk reward from the liquid dispenser in a 15-min session, during which the liquid dipper was programmed to be raised (for 5s at a time) at random intervals (mean=30s, range = 5 to 60s). All animals learned to access the magazine and to consume the liquid reward. Conditioning commenced the next day and continued for 14 consecutive days. On each daily conditioning session, the animals were placed in the same test chamber with house light on throughout. Ten discrete conditioning trials, each consisting of a 5-s tone stimulus (the conditioned stimulus, CS) followed immediately by the delivery of the liquid food reward available for 5s (the unconditioned stimulus, US), were presented at random intervals (mean=120s, range=60–180s).
Eight days of extinction immediately followed the last conditioning session. An extinction session was identical to that of the conditioning session except that no reward was available (although the dipper still underwent the motion of delivery).
The approach response was measured by magazine nose pokes. Conditioned approach response was indexed by comparing the frequency of nose pokes during CS presentation with the 5-s pre CS period to control for baseline difference using the ratio: CS nose pokes / (CS nose pokes + Pre-CS nose pokes), calculated daily. A value of 0.5 refers to chance performance such that the nose poking frequency does not differ between the pre-CS and the CS periods. Data collected in the conditioning and extinction phases were separately analysed.
2.8 Shock sensitivity
To evaluate if differences in sensitivity to shock between mutant and controls might contribute to the conditioning phenotypes observed in the aversive Pavlovian conditioning experiment, a separate cohort of naive mice (mutant: n=8, control: n=7) was prepared. Only female mice kept in the reversed cycle (therefore tested in the dark phase) were used here since it was the combination exhibiting the strongest effect size of enhanced conditioned freezing (see Discussion). Four acoustic startle chambers for mice (SR-LAB, San Diego Instruments, San Diego, CA, USA) were used to measure the direct whole body motor response to electric foot shock. Each startle chamber comprised a cylindrical enclosure made of clear Plexiglas attached horizontally on a lightweight mobile platform, which in turn was resting on a solid base inside a sound-attenuated isolation cubicle. A foot shock grid was located along the length of the animal enclosure and was remotely controlled by a programmable shocker. Whole body motion was converted into analogue signals by a piezoelectric unit attached underneath the platform. These signals (in arbitrary units proportional to the force produced) were digitized and stored by a computer. Testing was performed under a constant background noise of 65 dBA produced by a high-frequency loudspeaker mounted directly above the animal enclosure inside each test chamber. The mice were first acclimatized to the apparatus for 2 min after being placed inside the enclosure; 0.5-s foot shocks were then administered in either ascending-descending or descending-ascending sequence (counter-balanced across animals) of the following intensity: 0 (baseline), 0.045, 0.075, 0.105, 0.135, 0.165, 0.195, 0.225 and 0.255 mA. A total of 18 readings were obtained. A 0.5-s response window was defined starting from the shock onset. The force (in arbitrary units) produced by whole body movements was sampled at a rate of 1kHz. The peak response and the latency to peak response were taken to index the reaction to the shock.
2.9 Statistical analysis
All data were analysed by parametric analysis of variance (ANOVA) using the between-subject factors genotype, light-dark phase and sex. Additional within-subject factors were included as determined by the experimental design, such as days, bins, blocks and shock intensity, with polynomial orthogonal contrasts. Statistical significant main effects and interaction terms were further examined by post-hoc pair-wise analysis (based on the pooled error variance) and supplementary restricted analyses to assist interpretation. All statistical analyses were carried out using SPSS for Windows (version 18, SPSS Inc. Chicago IL, USA) implemented on a PC running the Microsoft Windows 7 operating system.