Altered Striatal Synaptic Function and Abnormal Behaviour in Shank3 Exon4-9 Deletion Mouse Model of Autism

Abstract

Shank3 is a multi-domain, synaptic scaffolding protein that organizes proteins in the postsynaptic density of excitatory synapses. Clinical studies suggest that ∼ 0.5% of autism spectrum disorder (ASD) cases may involve SHANK3 mutation/deletion. Patients with SHANK3 mutations exhibit deficits in cognition along with delayed/impaired speech/language and repetitive and obsessive/compulsive-like (OCD-like) behaviors. To examine how mutation/deletion of SHANK3 might alter brain function leading to ASD, we have independently created mice with deletion of Shank3 exons 4-9, a region implicated in ASD patients. We find that homozygous deletion of exons 4-9 (Shank3(e4-9) KO) results in loss of the two highest molecular weight isoforms of Shank3 and a significant reduction in other isoforms. Behaviorally, both Shank3(e4-9) heterozygous (HET) and Shank3(e4-9) KO mice display increased repetitive grooming, deficits in novel and spatial object recognition learning and memory, and abnormal ultrasonic vocalizations. Shank3(e4-9) KO mice also display abnormal social interaction when paired with one another. Analysis of synaptosome fractions from striata of Shank3(e4-9) KO mice reveals decreased Homer1b/c, GluA2, and GluA3 expression. Both Shank3(e4-9) HET and KO demonstrated a significant reduction in NMDA/AMPA ratio at excitatory synapses onto striatal medium spiny neurons. Furthermore, Shank3(e4-9) KO mice displayed reduced hippocampal LTP despite normal baseline synaptic transmission. Collectively these behavioral, biochemical and physiological changes suggest Shank3 isoforms have region-specific roles in regulation of AMPAR subunit localization and NMDAR function in the Shank3(e4-9) mutant mouse model of autism.

Keywords: Phelan-McDermid syndrome; Shank3; autism spectrum disorder; grooming; mouse model.

Figure 1

Genetic deletion of Shank3 exons 4–9. (A) Schematic of the Shank3 gene displaying exons and their respective domains (ANK—Ankyrin repeat domain; SH3—Src Homology 3 domain; PDZ—PSD95/Dlg1/Zo1 domain; SAM—Sterile alpha motif) (top). Schematic of the targeted portion exons 4–9 of Shank3 (middle) and the insertion of the targeting construct following recombination (bottom). (B) Southern blot of ScaI and KpnI-digested DNA from control (lane 1) and neo-resistant ES cells (lanes 2–5) reveals 13.5 kb and 11.1 kb ScaI and KpnI fragments reflecting proper targeting in clones that were selected for blastocyst injections (asterisks).

Figure 2

Altered Shank3 isoform expression from whole striatum lysates in Shank3^e4–9 mutant mice. (A) Quantification and representative western blots of striatum whole tissue lysates with C-terminal Shank3 antibody (top) and N-terminal Shank3 antibody (bottom) showing decrease (HET) or complete loss (KO) of the C-1, C-2, N-1, and N-2 bands of Shank3 using Shank3 C and N antibodies in Shank3e4–9 mutants compared to WT. Additionally, there was a significant decrease in C-3, C-7, and N-3 bands in both HET and KO mice. (B) Quantification of other synaptic proteins from striatal lysates shows no significant differences. For each analysis, data were normalized to β-actin levels and then to the average of WT (*P < 0.05, **P < 0.01, ***P < 0.001, n = 8 WT, 7 HET, 8 KO).

Figure 3

Striatal synaptosome analysis in Shank3^e4–9 mutant mice. (A) Quantification and representative Western blots of striatal synaptosomes with C-terminal Shank3 antibody (top) and N-terminal Shank3 antibody (bottom). There is complete loss of the C-1, C-2, N-1, and N-2 bands of Shank3 using Shank3 C and N antibodies in Shank3e4–9 KO mice and significant decrease in same bands in HET mice compared to WT. Significant decreases are also observed in HET and KO mice for C-5, C-6, C-7, and N-3 and in KO mice only for C-3, C-5, N-4, and N-5 (*P < 0.05, **P < 0.01, ***P < 0.001 as indicated, n = 6) (B) Quantification of other synaptic proteins from striatal synaptosomes shows significant decreases in GluA2, GluA3, Homer1 b/c, and PSD95 in Shank3e4–9 KO mice. For each analysis, data were normalized to β-actin levels and then to the average of WT. Representative blots are shown inset for proteins showing significant differences. (*P < 0.05, **P < 0.01, ***P < 0.001, n = 11 WT, 11 HET, 12 KO).

Figure 4

Motor and anxiety tests in Shank3^e4–9 mutant mice. (A) All genotypes showed similar locomotor habituation over 2 hr in the locomotor box. (B) In the rotarod test all genotypes showed similar motor learning and coordination over 2 days and 8 trials. In the open field test all genotypes spent a similar amount of time in the center (C) and traveled similar distances (D). (E) In the dark/light test all three genotypes showed similar latencies to enter the light chamber from the dark chamber; additionally all genotypes spent equivalent times in either the dark or light chamber (F). In the elevated plus maze all three genotypes spent a similar percentage of time in open arms and closed arms respectively (G) and traveled similar distances in this task (H). All genotypes performed similarly in the marble burying test (I) and nest building test (J–K); (n = 20 WT, 16 HET, 20 KO).

Figure 5

Startle, Prepulse inhibition and Fear Conditioning in Shank3^e4–9 mutant mice. (A) All three genotypes displayed similar startle amplitude following a range of dB stimuli. (B) Prepulse inhibition of acoustic startle is unchanged among the genotypes. All three genotypes were tested in a one trial cue-dependent (C) and context-dependent (D) fear conditioning paradigm. There was no significant difference among genotypes in in level of freezing (n = 20 WT, 16 HET, 20 KO).

Figure 6

Vocalization and grooming abnormalities in Shank3^e4–9 mutant mice. (A) Shank3e4–9 KO mice displayed increased time spent grooming during the observation period. (B) All genotypes display similar number of grooming bouts during the 10-min observation period. (C) Both KO and HET mice spend more time grooming per bout than WT mice. (*P < 0.05; **P < 0.01, n = 20 WT, 16 HET, 20 KO). (D) Both HET and KO mice display abnormalities in the number of ultrasonic calls following separation from their mother early in life. At age P4 and P6 KO mice display an increase in number of calls compared to WT mice, while HET mice displayed increased calls at ages P4 and P12 (*P < 0.05; **P < 0.01, P4: n = 8 WT, 20 HET, 6 KO; P6: n = 21 WT, 10 HET, 14 KO; P8: n = 26 WT, 32 HET, 16 KO, P10: n = 17 WT, 26 HET, 7 KO; P12: n = 29 WT, 25 HET, 15 KO).

Figure 7

Social interaction in Shank3^e4–9 mutant mice. Direct social interaction between age/sex-matched adult pairs of mice of the same genotype scored as (A) number of interaction bouts and (B) time spent interacting. (C) Interaction with a juvenile target mouse. All genotypes displayed similar time interacting during the initial and recognition periods of the juvenile social interaction test. (D) Time spent interacting with an empty cage in an open arena (inanimate) followed by time spent interacting with a social target in that cage. No difference was observed in time spent interacting among genotypes. (*P < 0.05, ***P < 0.001, ****P < 0.0001; n = 20 WT, 16 HET, 20 KO).

Figure 8

Shank3^e4–9 mutant mice exhibit significantly impaired spatial and novel object recognition. (A) Schematic representation of the spatial learning and object recognition test. For four consecutive days mice were habituated to the arena (44 × 44 × 44 cm) for 5 min (not shown in schematic). Following habituation all mice received 7 trials each with inter-trial interval as depicted in schematic and described in Methods. (B) The mean time spent interacting with objects A, B, and C (baseline; trial 5). (C) Mean interaction time during trial 6 (spatial test) with objects A, B, and C after A has been moved to a novel location. (D) The mean interaction time with novel object B and familiar objects A and C (trial 7, novel object recognition test). Following 7 days of Morris water maze training we analyzed (E) latency to platform, (F) total distance traveled to reach platform, and (G) % thigmotaxis (*P < 0.05; **P < 0.01, ***P < 0.001, n = 20 WT, 16 HET, 20 KO). Probe trials conducted one day after training on day 8 (H) and one day following reversal training, day 13 (I) showed no difference in spatial preference among groups. (J) Latency to reach the platform in the visible platform version of the water maze conducted at the end of all testing.

Figure 9

Synaptic plasticity and basal synaptic transmission at hippocampal CA3-CA1 synapses in Shank3^e4–9 HET and KO mice. (A) LTP is decreased in Shank3e4–9 KO mice, but not in Shank3e4–9 HET mice. Arrow indicates 100 Hz conditioning stimulus. Inset: Average of 10 consecutive traces immediately preceding 100 Hz stimulation for 1 s (black) and at 60 min post-tetanus (gray) in WT (left), HET (middle), and KO (right) mice. Scale bar: 0.2 mV; 5 ms. (B) Summary data of mean fEPSP slope for final 10 min of recording normalized to pre-tetanus baseline (n = 8 WT, 8 HET, and 7 KO slices). (C) mGluR-LTD is normal in Shank3e4–9 HET and KO mice. Bar indicates 10 min bath application of 100 μM DHPG. Inset: Average of ten consecutive traces immediately preceding DHPG wash-in (black) and at 60 min after the start of DHPG washout (gray) in WT (left), HET (middle), and KO (right) mice. Scale bar: 0.2 mV; 5 ms. (D) Summary data of mean fEPSP slope for final 10 min of recording normalized to pre-DHPG baseline (n = 12 WT, 9 HET, 8 KO slices). *P < 0.05. (E) NMDA/AMPA ratio is unchanged in Shank3e4–9 HET and KO mice. Inset: ten consecutive traces (gray) and average trace (black) from WT (left), HET (middle), and KO mice (right) at −70 mV (bottom) and +40 mV (top) (n = 30 WT, 35 HET, and 24 KO cells). Scale bar: 200 pA, 50 ms. (F) Cumulative frequency plot of mEPSC amplitude, (G) mean mEPSC amplitude, and (H) mean frequency of events are unaffected in Shank3e4–9 HET and KO mice. Inset: 1 min raw traces from WT (top), HET (middle), KO (bottom) mice. Scale bar: 15 pA; 1.5 s (n = 21 WT, 19 HET, 19 KO cells). (I) Paired-pulse ratio is not affected in Shank3e4–9 HET or KO mice at interstimulus intervals 30–500 ms (n = 8 WT, 9 HET, 10 KO slices). (J) Input-output relationship of stimulus intensity to fEPSP slope is unchanged in HET and KO mice compared to WT controls. Inset: fEPSP slope plotted against fiber volley amplitude (n = 15 WT, 10 HET, 7 KO slices).

Figure 10

Striatal excitatory transmission is impaired following Shank3 exon 4–9 deletion. (A) Image capture of electrode placement in dorsal striatum using IR-DIC microscopy at 10× resolution. Stimulating electrode (stim) was placed just inside of the corpus collosum (cc) and patch clamp electrodes (rec) were used to record whole-cell EPSCs from MSNs 150–200 μm away. Inset: 10 consecutive traces (gray) and average trace (black) from WT (left), HET (middle), and KO mice (right) at −70 mV (bottom) and +40 mV (top). Scale bar: 200 pA (WT), 400 pA (HET), 170 pA (KO), 25 ms. (B) NMDA/AMPA ratio is decreased in Shank3e4–9 HET and KO mice (n = 18 WT, 17 HET, and 18 KO cells). (C) Mean mEPSC amplitude (Inset: 1-min raw traces from WT (top), HET (middle), KO (bottom) mice. Scale bar: 15 pA; 1.5 s) and (D) mean frequency of events are unaffected in HET and KO mice (n = 25 WT, 29 HET, and 20 KO cells). **P < 0.01, ***P <0.001.