Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication methods
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Basal glutamatergic synaptic transmission is reduced in Shank3 heterozygous mice
Altered basal synaptic properties in Shank3 heterozygous mice. (A) Reduced α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptor responses in Shank3 heterozygous mice. Slices were incubated in the presence of 2-amino-5-phosphonopentanoic acid (APV) and mean field excitatory postsynaptic potential (field EPSP) slope as a function of fiber volley is shown for slices derived from wild-type and heterozygous mice. The inset shows representative traces for a given stimulus intensity (0.5 mA) in the input/output (I/O) graph (arrow indicates the trace from wild-type; scale: 10 ms, 0.5 mV). (B) Normal N -methyl-D-aspartic acid (NMDA) receptor responses in Shank3 heterozygous mice. Slices were incubated in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and mean field EPSP slope as a function of fiber volley is shown. (C) Miniature excitatory postsynaptic cur...
Immunoblotting
PSD fractions were prepared as follows. Hemibrains of wild-type, heterozygous and homozygous Shank3 mice were homogenized in 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-A containing 4 mM HEPES, pH 7.4, 0.32 M sucrose, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail (both from Roche). Nuclear fractions were precipitated by centrifuging twice at 700 g for 15 min, and the resulting supernatants were further centrifuged at 21,000 g for 15 min. The precipitates were resuspended in HEPES-B containing 4 mM HEPES, pH 7.4, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail, homogenized and rotated at 4°C for 1 hour. The lysates were centrifuged at 32,000 g for 20 min and washed twice with HEPES-C containing 50 mM HEPES, pH 7.4, 0.5% Triton X-100, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail. Finally,...
Hippocampal slice electrophysiology
Methods of recording, imaging and analysis were carried out according to our previously published protocols [, ]. All experiments were conducted on CA1 pyramidal cells at 32°C in acute slices taken from Shank3 heterozygous mice and wild-type littermates. Spines were visualized using calcein contained in the patch pipette, making use of a two-photon laser scanning system modified from Olympus Fluoview FV 300 driven by a Chameleon two-photon laser (Coherent, Santa Clara, CA, USA). Baseline synaptic responses were evoked using a glass pipette positioned ~20 µm away from the imaged spines and recorded at the soma. Long-term potentiation (LTP) was induced with a θ-burst pairing (TBP) protocol in which two trains of θ-burst stimuli (each train, separated by 20 s, consisted of five bursts at 5 Hz, and each burst contained five pulses at 100 Hz) were paired with brief, sm...
Extracellular recordings
Hippocampal slices (350 µm thick) were prepared from 4- to 6-week-old heterozygous mice and their wild-type littermate controls. Slices were perfused with Ringer's solution containing (in mM): NaCl, 125.0; KCl, 2.5; MgSO 4, 1.3; NaH 2 PO 4, 1.0; NaHCO 3, 26.2; CaCl 2, 2.5; and glucose, 11.0. The Ringer's solution was bubbled with 95% O 2 and 5% CO 2 at 32°C during extracellular recordings (electrode solution: 3 M NaCl). Slices were maintained for 1 hour prior to establishment of a baseline of field excitatory postsynaptic potentials (fEPSPs) recorded from stratum radiatum in area CA1, evoked by stimulation of the Schaffer collateral-commissural afferents (100-µs pulses every 30 s) with bipolar tungsten electrodes placed into area CA3 [ ]. Test stimulus intensity was adjusted to obtain fEPSPs with amplitudes that were one-half of the maximal response. The EPSP initia...
Extracellular recordings
Paired-pulse responses were measured with an interstimulus interval (ISI) of 50 ms and are expressed as the ratio of the average responses to the second stimulation pulse (FP2) to the first stimulation pulse (FP1). LTP was induced by either a high-frequency stimulus (four trains of 100-Hz, 1-s stimulations separated by 5 min) or TBS (15 bursts of four pulses at 100 Hz separated by 200 ms). To induce long-term depression (LTD), Schaffer collaterals were stimulated by low-frequency stimulation (LFS; 900 pulses at 1 Hz, 15 min) or by a paired-pulse low-frequency stimulation (PP-LFS; 1 Hz for 20 min, 50-ms interstimulus interval [ ]) to induce mGlu receptor-dependent LTD. Data were expressed as means ± SD, and statistical analyses were performed using analysis of variance (ANOVA) or Student's t -test, with significance set at an α level of 0.05.
Measurement of GluR1-immunoreactive puncta
Three-month-old animals were anesthetized with 250 µl of 15% chloral hydrate and perfused transcardially with 1% paraformaldehyde for 1 min followed by 4% paraformaldehyde for a total of 13 min. The brains were then removed, hemisected, cut in 50-µm-thick sections using a Leica VT1000S Vibratome (Vibratome, Bannockburn, IL, USA) and subsequently stored in phosphate-buffered saline (PBS) until use. Sections were incubated at 37°C for 5 min, followed by incubation in acidified pepsin (1 ml in a 0.2 N HCl solution) for 6.5 min. The tissue was then washed at room temperature in PBS-B (3 × 20 min) and incubated in a 0.3% Triton X-100, 0.5% bovine serum albumin (BSA), 5% normal goat serum for 1 h on an orbital shaker. The blocking step was followed by overnight incubation in the primary antibody (rabbit polyclonal antiglutamate receptor 1 AB1504; Millipore, Billerica, MA...
Behavioral analyses
Shank3 wild-type and heterozygote breeding pairs were imported from Mount Sinai School of Medicine to the National Institute of Mental Health. Mice were maintained by breeding C57BL/6 wild-type mice with Shank3 heterozygotes and housed in a conventional temperature- and humidity-controlled vivarium. Littermates were housed by sex in mixed genotype groups of two to four per cage on a 12:12-h circadian cycle with lights on at 0600. Behavioral experiments were conducted between 1000 and 1600 in dedicated testing rooms.
Behavioral analyses
Male-female social interactions were evaluated in a 5-min test session as previously described [, ], with the exception that subject males were group-housed and individually tested in clean cages with clean litter. Each of the 12 wild-type and 14 heterozygous male subject mice, ages 2.5-4 months, was paired with a different unfamiliar estrus C57BL/6J female. A digital closed-circuit television camera (Panasonic, Secaucus, NJ, USA) was positioned horizontally 30 cm from the cage. An ultrasonic microphone (Avisoft UltraSoundGate condenser microphone capsule CM15; Avisoft Bioacoustics, Berlin, Germany) was mounted 20 cm above the cage. Sampling frequency for the microphone was 250 kHz, and the resolution was 16 bits. The entire apparatus was contained in a sound-attenuating environmental chamber (ENV-018V; Med Associates, St. Albans, VT, USA) illuminated by a single 25-Watt red light. V...
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Evidence quotes (8)
Altered basal synaptic properties in Shank3 heterozygous mice. (A) Reduced α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptor responses in Shank3 heterozygous mice. Slices were incubated in the presence of 2-amino-5-phosphonopentanoic acid (APV) and mean field excitatory postsynaptic potential (field EPSP) slope as a function of fiber volley is shown for slices derived from wild-type and heterozygous mice. The inset shows representative traces for a given stimulus intensity (0.5 mA) in the input/output (I/O) graph (arrow indicates the trace from wild-type; scale: 10 ms, 0.5 mV). (B) Normal N -methyl-D-aspartic acid (NMDA) receptor responses in Shank3 heterozygous mice. Slices were incubated in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and mean field EPSP slope as a function of fiber volley is shown. (C) Miniature excitatory postsynaptic currents (mEPSCs) from wild-type and Shank3 heterozygous mice. Left: Amplitude of mEPSCs. * P < 0.01. Right: Frequency of mEPSCs. * P < 0.03. (D) Sample traces of mEPSCs. Scale: 1 s, 40 pA. (E) Cumulative probability of mEPSC amplitude. (F) Paired-pulse ratio. Left: Representative EPSC traces from S...
PSD fractions were prepared as follows. Hemibrains of wild-type, heterozygous and homozygous Shank3 mice were homogenized in 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-A containing 4 mM HEPES, pH 7.4, 0.32 M sucrose, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail (both from Roche). Nuclear fractions were precipitated by centrifuging twice at 700 g for 15 min, and the resulting supernatants were further centrifuged at 21,000 g for 15 min. The precipitates were resuspended in HEPES-B containing 4 mM HEPES, pH 7.4, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail, homogenized and rotated at 4°C for 1 hour. The lysates were centrifuged at 32,000 g for 20 min and washed twice with HEPES-C containing 50 mM HEPES, pH 7.4, 0.5% Triton X-100, Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail. Finally, postsynaptic density fractions were resuspended in HEPES-C containing 1.8% sodium dodecyl sulfate (SDS) and 2.5 M urea. Fifty-two µg of each PSD fraction were loaded to 4-12% SDS-polyacrylamide gel electrophoresis (PAGE gel (Invitrogen, Carlsbad, CA, USA), transferred to polyvinylidene fluoride...
Methods of recording, imaging and analysis were carried out according to our previously published protocols [, ]. All experiments were conducted on CA1 pyramidal cells at 32°C in acute slices taken from Shank3 heterozygous mice and wild-type littermates. Spines were visualized using calcein contained in the patch pipette, making use of a two-photon laser scanning system modified from Olympus Fluoview FV 300 driven by a Chameleon two-photon laser (Coherent, Santa Clara, CA, USA). Baseline synaptic responses were evoked using a glass pipette positioned ~20 µm away from the imaged spines and recorded at the soma. Long-term potentiation (LTP) was induced with a θ-burst pairing (TBP) protocol in which two trains of θ-burst stimuli (each train, separated by 20 s, consisted of five bursts at 5 Hz, and each burst contained five pulses at 100 Hz) were paired with brief, small postsynaptic depolarization. Volume analysis of individual spines was performed as detailed previously [ ]. Briefly, the integrated fluorescence intensity inside a spine head was measured for individual spines at different time points and normalized to the fluorescence intensity of the dendrites...
Hippocampal slices (350 µm thick) were prepared from 4- to 6-week-old heterozygous mice and their wild-type littermate controls. Slices were perfused with Ringer's solution containing (in mM): NaCl, 125.0; KCl, 2.5; MgSO 4, 1.3; NaH 2 PO 4, 1.0; NaHCO 3, 26.2; CaCl 2, 2.5; and glucose, 11.0. The Ringer's solution was bubbled with 95% O 2 and 5% CO 2 at 32°C during extracellular recordings (electrode solution: 3 M NaCl). Slices were maintained for 1 hour prior to establishment of a baseline of field excitatory postsynaptic potentials (fEPSPs) recorded from stratum radiatum in area CA1, evoked by stimulation of the Schaffer collateral-commissural afferents (100-µs pulses every 30 s) with bipolar tungsten electrodes placed into area CA3 [ ]. Test stimulus intensity was adjusted to obtain fEPSPs with amplitudes that were one-half of the maximal response. The EPSP initial slope (mV/ms) was determined from the average waveform of four consecutive responses. Input-output (I/O) curves were generated by plotting the fEPSP slope versus fiber volley amplitude in low-Mg 2+ (0.1 mM) solution. AMPA receptor-mediated and NMDA receptor-mediated I/O relationships were measured...
Paired-pulse responses were measured with an interstimulus interval (ISI) of 50 ms and are expressed as the ratio of the average responses to the second stimulation pulse (FP2) to the first stimulation pulse (FP1). LTP was induced by either a high-frequency stimulus (four trains of 100-Hz, 1-s stimulations separated by 5 min) or TBS (15 bursts of four pulses at 100 Hz separated by 200 ms). To induce long-term depression (LTD), Schaffer collaterals were stimulated by low-frequency stimulation (LFS; 900 pulses at 1 Hz, 15 min) or by a paired-pulse low-frequency stimulation (PP-LFS; 1 Hz for 20 min, 50-ms interstimulus interval [ ]) to induce mGlu receptor-dependent LTD. Data were expressed as means ± SD, and statistical analyses were performed using analysis of variance (ANOVA) or Student's t -test, with significance set at an α level of 0.05.
Three-month-old animals were anesthetized with 250 µl of 15% chloral hydrate and perfused transcardially with 1% paraformaldehyde for 1 min followed by 4% paraformaldehyde for a total of 13 min. The brains were then removed, hemisected, cut in 50-µm-thick sections using a Leica VT1000S Vibratome (Vibratome, Bannockburn, IL, USA) and subsequently stored in phosphate-buffered saline (PBS) until use. Sections were incubated at 37°C for 5 min, followed by incubation in acidified pepsin (1 ml in a 0.2 N HCl solution) for 6.5 min. The tissue was then washed at room temperature in PBS-B (3 × 20 min) and incubated in a 0.3% Triton X-100, 0.5% bovine serum albumin (BSA), 5% normal goat serum for 1 h on an orbital shaker. The blocking step was followed by overnight incubation in the primary antibody (rabbit polyclonal antiglutamate receptor 1 AB1504; Millipore, Billerica, MA, USA), which was made in blocking solution at the appropriate dilution (1 µg/ml). The tissue sections were then washed in PBS-B (5 × 5 min) and incubated in secondary antibody (goat antirabbit Alexa Fluor 488, Invitrogen) in a 2% BSA and 0.3% Triton PBS-B solution at the appropriate dilut...
Shank3 wild-type and heterozygote breeding pairs were imported from Mount Sinai School of Medicine to the National Institute of Mental Health. Mice were maintained by breeding C57BL/6 wild-type mice with Shank3 heterozygotes and housed in a conventional temperature- and humidity-controlled vivarium. Littermates were housed by sex in mixed genotype groups of two to four per cage on a 12:12-h circadian cycle with lights on at 0600. Behavioral experiments were conducted between 1000 and 1600 in dedicated testing rooms.
Male-female social interactions were evaluated in a 5-min test session as previously described [, ], with the exception that subject males were group-housed and individually tested in clean cages with clean litter. Each of the 12 wild-type and 14 heterozygous male subject mice, ages 2.5-4 months, was paired with a different unfamiliar estrus C57BL/6J female. A digital closed-circuit television camera (Panasonic, Secaucus, NJ, USA) was positioned horizontally 30 cm from the cage. An ultrasonic microphone (Avisoft UltraSoundGate condenser microphone capsule CM15; Avisoft Bioacoustics, Berlin, Germany) was mounted 20 cm above the cage. Sampling frequency for the microphone was 250 kHz, and the resolution was 16 bits. The entire apparatus was contained in a sound-attenuating environmental chamber (ENV-018V; Med Associates, St. Albans, VT, USA) illuminated by a single 25-Watt red light. Videos from the male subjects were subsequently scored by an investigator uninformed of the subject's genotype on measures of nose-to-nose sniffing, nose-to-anogenital sniffing and sniffing of other body regions, using Noldus Observer software (Noldus Information Technology, Leesburg, VA, USA) as pre...
Machine-readable layer
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