Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities methods
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Reciprocal social interaction
To measure reciprocal social interaction behavior, pairs of juvenile mice were allowed to socially interact at PND25±1 for 5 min after one mouse of the pair being habituated to the test environment for 1 min. Same-sex/same-genotype pairs consisting of non-littermates were used. To enhance the level of social motivation, juvenile mice were socially isolated for 24 h prior to testing. Testing was performed in a clean Makrolon Type III cage with fresh bedding and a metal lid under dim red light. Behavior was recorded using a video camera placed 30 cm away from the cage. Reciprocal social interactions were tested between 0800 and 1800 hours during the light phase of the 12:12 h light/dark cycle.
Reciprocal social interaction
Social interactions were scored by an experienced observer blind to the experimental conditions using the Noldus The Observer XT 10.0 software (Noldus Information Technology, Wageningen, The Netherlands). Parameters of social behaviors included facial sniffing (sniffing the nose and snout region of the partner), anogenital sniffing (sniffing the anogenital region of the partner), following (walking straight behind the partner, keeping pace with the one ahead), push past (squeezing between the wall and the partner), crawling under/over (pushing the head underneath the partner's body or crawling over or under the partner's body), social grooming (grooming the partner) and being socially inactive while having social contact (lying flat or standing still while maintaining close physical contact with the partner; for details see Yang et al. and Terranova and Laviola ). All social behaviors...
Ultrasonic vocalizations
To induce ultrasonic vocalizations (USV) in pups, they were isolated from their mother and littermates on PND8 for 10 min at room temperature (22-24 °C). As described previously,, pups were removed individually from the nest at random and gently placed into an isolation container (10 × 8 × 7 cm; open surface) made of glass, containing clean bedding material. The isolation container was surrounded by a sound-attenuating box (20 × 20 × 20 cm) made of Styrofoam (thickness of walls: 4 cm). USV emission was monitored by an UltraSoundGate Condenser CM 16 Microphone (Avisoft Bioacoustics, Berlin, Germany) placed in the roof of the sound-attenuating box, 20 cm above the floor. After the 10-min isolation period, body weight and body temperature were determined. Body weight was measured using a palm scale (PS6-250; My Weigh Eur...
Interaction-induced USV in juvenile mice
To induce USV in juvenile mice, pairs of juvenile mice were allowed to socially interact at PND25±1 for 5 min after one mouse of the pair being habituated to the test environment for 1 min. Same-sex/same-genotype pairs consisting of non-littermates were used. To enhance the level of social motivation, juvenile mice were socially isolated for 24 h prior to testing. Testing was performed in a Makrolon Type III cage with clean bedding and metal lid under dim red light. USV emission was monitored by an UltraSoundGate Condenser CM 16 Microphone (Avisoft Bioacoustics) placed 15 cm above the cage lid. Reciprocal social interactions were tested between 0800 and 1800 hours during the light phase of the 12:12 h light/dark cycle. Clean cages and lids were used for reciprocal social interactions.
Female-induced USV in adult male mice
To induce USV in adult male mice, male mice at PND~120 were exposed to a wild-type female for 5 min after being habituated to the test environment for 1 min. Testing was performed in a Makrolon Type III cage with clean bedding and a metal lid under dim red light. USV emission was monitored by an UltraSoundGate Condenser CM 16 Microphone (Avisoft Bioacoustics) placed 15 cm above the cage lid. Female exposure occurred between 0800 and 1800 hours during the light phase of the 12:12 h light/dark cycle. Clean cages and lids were used for all female exposures.
Acoustical analysis
For acoustical analysis, recordings were transferred to Avisoft SASLab Pro (version 4.50; Avisoft Bioacoustics) and a fast Fourier transform was conducted (512 fast Fourier transform length, 100% frame, Hamming window and 75% time window overlap). Correspondingly, the spectrograms were produced at 488 Hz of frequency resolution and 0.512 ms of time resolution. Call detection of isolation-induced USV emitted by pups was provided by an automatic amplitude threshold-based algorithm and a hold-time mechanism (hold time: 10 ms). As no USV were detected below 30 kHz, a high-pass filter of 30 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. The accuracy of call detection by the software was verified manually by an experienced user. When necessary, missed calls were marked by hand to be included in the automatic parameter...
Repetitive and stereotyped patterns of behavior
Male PV +/+ ( n =9) and PV -/- ( n =8) mice were tested for restrictive, repetitive behavior using a T-maze reversal paradigm. The mice were first habituated to the maze (entry arm: 35 × 10 cm, T-arms, 40 × 10 cm each, as described previously ) over 5 days immediately prior to the experiment. An appetitive reward (in this case ~200 µl of malted chocolate milk) was placed in a shallow container in one of the short arms of the T-maze and the mouse was trained to retrieve it in 10 consecutive, daily trials until it had reached the criterion, in this case at least 70% of the mice had to enter the correct arm and at least partially consume the reward in at least 8/10 trials on three consecutive days. If a mouse failed to enter either arm, entered the wrong arm or entered the correct arm but failed to consume the reward, this was scored as an inc...
Water-maze reversal learning paradigm
Place navigation testing in the Morris water maze (MWM) was performed as described previously in a round swim tank of 150 cm diameter. The goal platform (14 × 14 cm) was hidden 0.5 cm below the water surface. Animals performed 30 trials (max. duration 120 s), six per day with inter-trial intervals of 30-60 min. During the first 18 trials (acquisition), the hidden platform was kept in the same position and then moved to the opposite quadrant for the remaining 12 trials (reversal). The first trial of the reversal phase served as probe trial to test for spatial retention. Trials were tracked using Noldus EthoVision 2.6 (Noldus Information Technology) and transferred to Wintrack 2.4 software ( http://www.dpwolfer.ch/wintrack ) for analysis.
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Evidence quotes (8)
To measure reciprocal social interaction behavior, pairs of juvenile mice were allowed to socially interact at PND25±1 for 5 min after one mouse of the pair being habituated to the test environment for 1 min. Same-sex/same-genotype pairs consisting of non-littermates were used. To enhance the level of social motivation, juvenile mice were socially isolated for 24 h prior to testing. Testing was performed in a clean Makrolon Type III cage with fresh bedding and a metal lid under dim red light. Behavior was recorded using a video camera placed 30 cm away from the cage. Reciprocal social interactions were tested between 0800 and 1800 hours during the light phase of the 12:12 h light/dark cycle.
Social interactions were scored by an experienced observer blind to the experimental conditions using the Noldus The Observer XT 10.0 software (Noldus Information Technology, Wageningen, The Netherlands). Parameters of social behaviors included facial sniffing (sniffing the nose and snout region of the partner), anogenital sniffing (sniffing the anogenital region of the partner), following (walking straight behind the partner, keeping pace with the one ahead), push past (squeezing between the wall and the partner), crawling under/over (pushing the head underneath the partner's body or crawling over or under the partner's body), social grooming (grooming the partner) and being socially inactive while having social contact (lying flat or standing still while maintaining close physical contact with the partner; for details see Yang et al. and Terranova and Laviola ). All social behaviors were analyzed for frequency of occurrence (that is, number of bouts) and duration in 1-min time bins. In addition to social behaviors, nonsocial behaviors were measured and included rearing (number of times an animal reared on its hind legs), grooming (number of bouts of face, body and genital groo...
To induce ultrasonic vocalizations (USV) in pups, they were isolated from their mother and littermates on PND8 for 10 min at room temperature (22-24 °C). As described previously,, pups were removed individually from the nest at random and gently placed into an isolation container (10 × 8 × 7 cm; open surface) made of glass, containing clean bedding material. The isolation container was surrounded by a sound-attenuating box (20 × 20 × 20 cm) made of Styrofoam (thickness of walls: 4 cm). USV emission was monitored by an UltraSoundGate Condenser CM 16 Microphone (Avisoft Bioacoustics, Berlin, Germany) placed in the roof of the sound-attenuating box, 20 cm above the floor. After the 10-min isolation period, body weight and body temperature were determined. Body weight was measured using a palm scale (PS6-250; My Weigh Europe, Hückelhoven, Germany). For body temperature determination a Testo 110 thermometer (Testo, Lenzkirch, Germany) was used. In addition, the following somatosensory reflexes were determined: surface righting and vertical screen holding. To measure the righting reflex, pups were placed on their...
To induce USV in juvenile mice, pairs of juvenile mice were allowed to socially interact at PND25±1 for 5 min after one mouse of the pair being habituated to the test environment for 1 min. Same-sex/same-genotype pairs consisting of non-littermates were used. To enhance the level of social motivation, juvenile mice were socially isolated for 24 h prior to testing. Testing was performed in a Makrolon Type III cage with clean bedding and metal lid under dim red light. USV emission was monitored by an UltraSoundGate Condenser CM 16 Microphone (Avisoft Bioacoustics) placed 15 cm above the cage lid. Reciprocal social interactions were tested between 0800 and 1800 hours during the light phase of the 12:12 h light/dark cycle. Clean cages and lids were used for reciprocal social interactions.
To induce USV in adult male mice, male mice at PND~120 were exposed to a wild-type female for 5 min after being habituated to the test environment for 1 min. Testing was performed in a Makrolon Type III cage with clean bedding and a metal lid under dim red light. USV emission was monitored by an UltraSoundGate Condenser CM 16 Microphone (Avisoft Bioacoustics) placed 15 cm above the cage lid. Female exposure occurred between 0800 and 1800 hours during the light phase of the 12:12 h light/dark cycle. Clean cages and lids were used for all female exposures.
For acoustical analysis, recordings were transferred to Avisoft SASLab Pro (version 4.50; Avisoft Bioacoustics) and a fast Fourier transform was conducted (512 fast Fourier transform length, 100% frame, Hamming window and 75% time window overlap). Correspondingly, the spectrograms were produced at 488 Hz of frequency resolution and 0.512 ms of time resolution. Call detection of isolation-induced USV emitted by pups was provided by an automatic amplitude threshold-based algorithm and a hold-time mechanism (hold time: 10 ms). As no USV were detected below 30 kHz, a high-pass filter of 30 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. The accuracy of call detection by the software was verified manually by an experienced user. When necessary, missed calls were marked by hand to be included in the automatic parameter analysis. Interaction-induced and female-induced USV were analyzed interactively by an experienced user to avoid false positives due to high levels of background noise resulting from the animals' movements. Total number of USV was calculated for the entire session and in 60-s time bins to visualize...
Male PV +/+ ( n =9) and PV -/- ( n =8) mice were tested for restrictive, repetitive behavior using a T-maze reversal paradigm. The mice were first habituated to the maze (entry arm: 35 × 10 cm, T-arms, 40 × 10 cm each, as described previously ) over 5 days immediately prior to the experiment. An appetitive reward (in this case ~200 µl of malted chocolate milk) was placed in a shallow container in one of the short arms of the T-maze and the mouse was trained to retrieve it in 10 consecutive, daily trials until it had reached the criterion, in this case at least 70% of the mice had to enter the correct arm and at least partially consume the reward in at least 8/10 trials on three consecutive days. If a mouse failed to enter either arm, entered the wrong arm or entered the correct arm but failed to consume the reward, this was scored as an incorrect response. The mouse was allowed 3 min to complete each trial. In order that the mouse was not guided by olfactory clues, a little of the reward was placed outside of each short arm of the maze for the duration of the trials. Once a mouse had reached acquisition, the reward was swapped i...
Place navigation testing in the Morris water maze (MWM) was performed as described previously in a round swim tank of 150 cm diameter. The goal platform (14 × 14 cm) was hidden 0.5 cm below the water surface. Animals performed 30 trials (max. duration 120 s), six per day with inter-trial intervals of 30-60 min. During the first 18 trials (acquisition), the hidden platform was kept in the same position and then moved to the opposite quadrant for the remaining 12 trials (reversal). The first trial of the reversal phase served as probe trial to test for spatial retention. Trials were tracked using Noldus EthoVision 2.6 (Noldus Information Technology) and transferred to Wintrack 2.4 software ( http://www.dpwolfer.ch/wintrack ) for analysis.
Machine-readable layer
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