Altered mGluR5-Homer scaffolds and corticostriatal connectivity in a Shank3 complete knockout model of autism methods
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Δe4-22 -/- mice display core behavioural features of ASDs
To evaluate social preferences in neonates, we examined Δe4-22 juveniles (P15) in a nest-homing test. In a one-way test, the latency to approach a sample of the home nest did not differ among the genotypes ( ). However, when given a choice between the home nest and an unfamiliar nest sample from a developmentally matched litter's nest, the Δe4-22 -/- mice failed to show a preference for home nests, unlike Δe4-22 +/- and Δe4-22 +/+ mice ( ). In a further examination of social behaviour, we monitored the formation of dominance hierarchies over 6 days where three unfamiliar adults of the same genotype were placed into each cage. Since no sex differences were found ( ), the data were collapsed across sex. From the number and direction of agonistic behaviours observed within each triad, a dominance ranking was calculated for eac...
Δe4-22 -/- mice display core behavioural features of ASDs
Mice were next evaluated for sociability. Similar levels of preference for the novel social stimuli in the social affiliation and social preference tests were displayed by the three genotypes ( ); no genotype differences were discerned in the duration or numbers of contacts with the non-social and social stimuli across all test phases ( ). To evaluate social behaviour in a novel environment more critically, we conducted social dyadic tests. In pairings of Δe4-22 males with age-matched C3H males, the time spent in and the numbers of bidirectional interactions did not differ among the genotypes; the mice only displayed mild social investigations characterized by approach behaviours and sniffing of the head, face, anogenital and shoulder areas of the partner mouse ( and ). However, Δe4-22 -/- mice engaged in longer and greater numbers of non-reciprocated...
Δe4-22 -/- mice display core behavioural features of ASDs
Ultrasonic vocalizations (USVs) were assessed in both pups and adults, as communication impairments represent a major feature of SHANK3 -related disorders and ASDs. The P4 Δe4-22 -/- pups emitted significantly fewer USVs that were of shorter duration than Δe4-22 +/- and Δe4-22 +/+ pups ( ). Although the frequencies and amplitudes of calls from Δe4-22 -/- mice were lower than the other two genotypes ( ), all USVs were within a range appropriate for pup distress calls. Upon exposure to oestrous females, Δe4-22 -/- adult males emitted fewer calls, and their calls were significantly shorter in duration and reduced in amplitude relative to the other genotypes ( and ). By comparison, no genotype differences were observed in the peak frequency of adult USVs ( ).
Altered functional connectivity in a frontostriatal circuit
Aside from abnormal network activity, Δe4-22 -/- mice also displayed lower unit firing rates in NAC both before and after introduction of the social stimulus ( ). No genotype differences were observed in cross-frequency phase coupling or phase lock of unit responses (two measures of local connectivity) within the cortical, striatal and thalamic microcircuits ( and ). We quantified directional oscillatory interactions during the social stimulus presentation, as a measure of information transfer across the cortico-striatal-thalamic circuit. In both Δe4-22 -/- and Δe4-22 +/+ mice, oscillatory synchrony was directed from thalamus to cortex and striatum in the 7-11 Hz frequency band; however, the temporal delay between thalamic to striatal oscillatory activity was diminished in the Δe4-22 -/- animal...
Changes in synaptic organization in Δe4-22 -/- mice
To analyze the molecular changes that may contribute to dysfunctional synapses, we examined synaptic proteins in PSD fractions from Δe4-22 striata and hippocampi ( ). Pan-SAPAP and SAPAP3 levels were decreased only in striatum, while GluN2A was reduced only in hippocampus ( ). The most prominent changes in Δe4-22 -/- mice were for the Homer family proteins ( ). Homer1b/c was markedly decreased (18% of +/+) in striatum and mildly reduced in hippocampus (77% of +/+) relative to Δe4-22 +/+ mice. There was no significant change for Homer1a in either brain region. In the Δe4-22 -/- mice, Homer2 was significantly decreased in both brain areas (48% of +/+ in striatum; and 78% of +/+ in hippocampus). Homer3 (the least abundant isoform) was undetectable in striatum, but was reduced in the Δe4-22 -/- hipp...
mGluR5 modulators correct impaired behaviour and LTD
Since Homer1b/c is an important mediator in the mGluR5 signalling pathway, we next examined whether there was any relationship between synaptic Homer1b/c and lever pressing in Δe4-22 -/- mice. After 7 days of lever-press training, we found that those mutants with increased numbers of lever presses had significantly higher levels of Homer1b/c in their striatal PSDs than those with lower lever-press rates ( ). We also observed that 7 days of treatment with CDPPB resulted in a slight increase of synaptic Homer1b/c in Δe4-22 -/- mice ( ), consistent with the partial rescue of lever pressing by this compound. These data suggest that the disruption of mGluR5-Homer scaffolds, due to the reduction of Homer1b/c, may contribute to the impaired instrumental learning and striatal plasticity in the Δe4-22 -/- mice.
Immunocytochemistry and confocal microscopy
Eight-week-old mice were transcardially perfused with 4% paraformaldehyde in 1 × PBS (pH 7.4). The brains were dissected and post-fixed in the same fixative overnight. The brains were then cryoprotected by submerging them in 30% sucrose for 48 h. After embedding into optimal cutting temperature compound (O.C.T.) on dry ice, brains were sectioned on a cryostat at 40 µm thickness. Slices with brain regions of interest were used for further staining with corresponding antibodies. See details in.
Social dyadic test
Male mice were housed individually for >14 days before testing. All testing was performed under red-light illumination (<5 lux) 2-6 h after onset of the dark cycle. Plexiglass test chambers (48 × 26 × 20 cm) were cleaned between each test with LabSan 256CPQ solution (Sanitation Strategies LLC, Williamston, MI) and refilled with 1/8" cob bedding (Andersen Inc., Maumee, OH, USA). Shank3 males were paired to non-familiar C3H/HeJ males (Jackson Labs, Stock No. 000659) of the same age and approximate weight. Each Shank3 mouse and its C3H partner were placed at opposite ends of test chamber and were separated by a solid partition. After 5 min, the barrier was removed and the animals were allowed to interact freely. All tests were filmed and the videos were scored by observers blinded to the genotype of the animals using the Observer XT 9 softwa...
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Evidence quotes (8)
To evaluate social preferences in neonates, we examined Δe4-22 juveniles (P15) in a nest-homing test. In a one-way test, the latency to approach a sample of the home nest did not differ among the genotypes ( ). However, when given a choice between the home nest and an unfamiliar nest sample from a developmentally matched litter's nest, the Δe4-22 -/- mice failed to show a preference for home nests, unlike Δe4-22 +/- and Δe4-22 +/+ mice ( ). In a further examination of social behaviour, we monitored the formation of dominance hierarchies over 6 days where three unfamiliar adults of the same genotype were placed into each cage. Since no sex differences were found ( ), the data were collapsed across sex. From the number and direction of agonistic behaviours observed within each triad, a dominance ranking was calculated for each mouse. All Δe4-22 +/+ mice established a hierarchy rank by day 1 and this remained stable through day 6. In contrast, 75% of hierarchies of Δe4-22 -/- mice were unstable; either dominance was not maintained across days, or no dominant animal could be identified in...
Mice were next evaluated for sociability. Similar levels of preference for the novel social stimuli in the social affiliation and social preference tests were displayed by the three genotypes ( ); no genotype differences were discerned in the duration or numbers of contacts with the non-social and social stimuli across all test phases ( ). To evaluate social behaviour in a novel environment more critically, we conducted social dyadic tests. In pairings of Δe4-22 males with age-matched C3H males, the time spent in and the numbers of bidirectional interactions did not differ among the genotypes; the mice only displayed mild social investigations characterized by approach behaviours and sniffing of the head, face, anogenital and shoulder areas of the partner mouse ( and ). However, Δe4-22 -/- mice engaged in longer and greater numbers of non-reciprocated behaviours (where the Shank3 mouse initiated the social interaction, but the C3H partner did not reciprocate and disengaged its partner by ignoring, leaving, or turning away from the target mouse) than the other genotypes ( and ). Moreover, Δe4-22 -/- animals engaged in more...
Ultrasonic vocalizations (USVs) were assessed in both pups and adults, as communication impairments represent a major feature of SHANK3 -related disorders and ASDs. The P4 Δe4-22 -/- pups emitted significantly fewer USVs that were of shorter duration than Δe4-22 +/- and Δe4-22 +/+ pups ( ). Although the frequencies and amplitudes of calls from Δe4-22 -/- mice were lower than the other two genotypes ( ), all USVs were within a range appropriate for pup distress calls. Upon exposure to oestrous females, Δe4-22 -/- adult males emitted fewer calls, and their calls were significantly shorter in duration and reduced in amplitude relative to the other genotypes ( and ). By comparison, no genotype differences were observed in the peak frequency of adult USVs ( ).
Aside from abnormal network activity, Δe4-22 -/- mice also displayed lower unit firing rates in NAC both before and after introduction of the social stimulus ( ). No genotype differences were observed in cross-frequency phase coupling or phase lock of unit responses (two measures of local connectivity) within the cortical, striatal and thalamic microcircuits ( and ). We quantified directional oscillatory interactions during the social stimulus presentation, as a measure of information transfer across the cortico-striatal-thalamic circuit. In both Δe4-22 -/- and Δe4-22 +/+ mice, oscillatory synchrony was directed from thalamus to cortex and striatum in the 7-11 Hz frequency band; however, the temporal delay between thalamic to striatal oscillatory activity was diminished in the Δe4-22 -/- animals ( ).
To analyze the molecular changes that may contribute to dysfunctional synapses, we examined synaptic proteins in PSD fractions from Δe4-22 striata and hippocampi ( ). Pan-SAPAP and SAPAP3 levels were decreased only in striatum, while GluN2A was reduced only in hippocampus ( ). The most prominent changes in Δe4-22 -/- mice were for the Homer family proteins ( ). Homer1b/c was markedly decreased (18% of +/+) in striatum and mildly reduced in hippocampus (77% of +/+) relative to Δe4-22 +/+ mice. There was no significant change for Homer1a in either brain region. In the Δe4-22 -/- mice, Homer2 was significantly decreased in both brain areas (48% of +/+ in striatum; and 78% of +/+ in hippocampus). Homer3 (the least abundant isoform) was undetectable in striatum, but was reduced in the Δe4-22 -/- hippocampus (20% of +/+). Unexpectedly, we found that mGluR5 was substantially increased in striatum (165% of +/+), but not in Δe4-22 -/- hippocampus. These results indicate region-specific alterations in PSD proteins in Δe4-22 -/- brains, especially in the...
Since Homer1b/c is an important mediator in the mGluR5 signalling pathway, we next examined whether there was any relationship between synaptic Homer1b/c and lever pressing in Δe4-22 -/- mice. After 7 days of lever-press training, we found that those mutants with increased numbers of lever presses had significantly higher levels of Homer1b/c in their striatal PSDs than those with lower lever-press rates ( ). We also observed that 7 days of treatment with CDPPB resulted in a slight increase of synaptic Homer1b/c in Δe4-22 -/- mice ( ), consistent with the partial rescue of lever pressing by this compound. These data suggest that the disruption of mGluR5-Homer scaffolds, due to the reduction of Homer1b/c, may contribute to the impaired instrumental learning and striatal plasticity in the Δe4-22 -/- mice.
Eight-week-old mice were transcardially perfused with 4% paraformaldehyde in 1 × PBS (pH 7.4). The brains were dissected and post-fixed in the same fixative overnight. The brains were then cryoprotected by submerging them in 30% sucrose for 48 h. After embedding into optimal cutting temperature compound (O.C.T.) on dry ice, brains were sectioned on a cryostat at 40 µm thickness. Slices with brain regions of interest were used for further staining with corresponding antibodies. See details in.
Male mice were housed individually for >14 days before testing. All testing was performed under red-light illumination (<5 lux) 2-6 h after onset of the dark cycle. Plexiglass test chambers (48 × 26 × 20 cm) were cleaned between each test with LabSan 256CPQ solution (Sanitation Strategies LLC, Williamston, MI) and refilled with 1/8" cob bedding (Andersen Inc., Maumee, OH, USA). Shank3 males were paired to non-familiar C3H/HeJ males (Jackson Labs, Stock No. 000659) of the same age and approximate weight. Each Shank3 mouse and its C3H partner were placed at opposite ends of test chamber and were separated by a solid partition. After 5 min, the barrier was removed and the animals were allowed to interact freely. All tests were filmed and the videos were scored by observers blinded to the genotype of the animals using the Observer XT 9 software (Noldus Information Technologies, Leesburg, VA). The ethogram for behavioural scoring consisted of 24 behaviours and were collapsed into two categories of behaviour: the total time spent in bidirectional social interaction, consisting of one mouse engaging the other and the other animal reciproca...
Machine-readable layer
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