Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication

Abstract

Background: SHANK3 is a protein in the core of the postsynaptic density (PSD) and has a critical role in recruiting many key functional elements to the PSD and to the synapse, including components of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA), metabotropic glutamate (mGlu) and N-methyl-D-aspartic acid (NMDA) glutamate receptors, as well as cytoskeletal elements. Loss of a functional copy of the SHANK3 gene leads to the neurobehavioral manifestations of 22q13 deletion syndrome and/or to autism spectrum disorders. The goal of this study was to examine the effects of haploinsufficiency of full-length Shank3 in mice, focusing on synaptic development, transmission and plasticity, as well as on social behaviors, as a model for understanding SHANK3 haploinsufficiency in humans.

Methods: We used mice with a targeted disruption of Shank3 in which exons coding for the ankyrin repeat domain were deleted and expression of full-length Shank3 was disrupted. We studied synaptic transmission and plasticity by multiple methods, including patch-clamp whole cell recording, two-photon time-lapse imaging and extracellular recordings of field excitatory postsynaptic potentials. We also studied the density of GluR1-immunoreactive puncta in the CA1 stratum radiatum and carried out assessments of social behaviors.

Results: In Shank3 heterozygous mice, there was reduced amplitude of miniature excitatory postsynaptic currents from hippocampal CA1 pyramidal neurons and the input-output (I/O) relationship at Schaffer collateral-CA1 synapses in acute hippocampal slices was significantly depressed; both of these findings indicate a reduction in basal neurotransmission. Studies with specific inhibitors demonstrated that the decrease in basal transmission reflected reduced AMPA receptor-mediated transmission. This was further supported by the observation of reduced numbers of GluR1-immunoreactive puncta in the stratum radiatum. Long-term potentiation (LTP), induced either with θ-burst pairing (TBP) or high-frequency stimulation, was impaired in Shank3 heterozygous mice, with no significant change in long-term depression (LTD). In concordance with the LTP results, persistent expansion of spines was observed in control mice after TBP-induced LTP; however, only transient spine expansion was observed in Shank3 heterozygous mice. Male Shank3 heterozygotes displayed less social sniffing and emitted fewer ultrasonic vocalizations during interactions with estrus female mice, as compared to wild-type littermate controls.

Conclusions: We documented specific deficits in synaptic function and plasticity, along with reduced reciprocal social interactions in Shank3 heterozygous mice. Our results are consistent with altered synaptic development and function in Shank3 haploinsufficiency, highlighting the importance of Shank3 in synaptic function and supporting a link between deficits in synapse function and neurodevelopmental disorders. The reduced glutamatergic transmission that we observed in the Shank3 heterozygous mice represents an interesting therapeutic target in Shank3-haploinsufficiency syndromes.

Figure 1

Reduced expression of Shank3 in Shank3-deficient mice. (A) Targeting strategy. Top: The genomic structure of Shank3 is shown. The position of the 22t and 32t transcripts are also shown, as is the epitope for antibody N69/46. Bottom: The location of the loxP sites introduced for targeting are shown before and after the activity of the recombinase. The green bar in the wild-type allele indicates the FRT site remaining after deletion of the selection cassette. (B) Expression of Shank3 mRNA. Brain-derived mRNA from wild-type, heterozygous and knockout mice were subjected to quantitative polymerase chain reaction (qPCR) assay for Shank3 mRNA using probes across exons 6 and 7 with normalization against reference genes. (C) Expression of Shank3 in postsynaptic density (PSD) fractions. PSD fractions from wild-type, heterozygous and knockout mice were subjected to immunoblotting with antibody N69/46 to Shank3. The migration of molecular weight markers is shown on the left (in kilodaltons) and an immunoblot for βIII-tubulin as a loading control is shown below. WT, wild-type mice; Het, Shank3 heterozygous mice; KO, homozygous knockout mice. *P < 0.0003.

Figure 2

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 Shank3 heterozygous (red) and wild-type (black) mice, with traces normalized to the first EPSC for comparison. *P < 0.05. WT, wild-type mice; Het, Shank3 heterozygous mice.

Figure 3

Reduced long-term potentiation in Shank3 heterozygous mice. (A) Long-term potentiation (LTP) following high-frequency stimulation. Field recordings of LTP induced with high-frequency stimulation (HFS; 4 times 100 Hz, separated by 5 min) as a function of time in slices from wild-type and Shank3 heterozygous mice. (B) LTP following θ-burst stimulation (TBS). (C) Long-term depression (LTD) following low-frequency stimulation (LFS), an NMDA receptor-dependent form of LTD. (D) LTD following paired-pulse low-frequency stimulation (PP-LFS), a protein synthesis-dependent form of LTD. (E) LTP recorded with whole cell patch-clamp method. LTP was induced with θ-burst pairing (TBP). (E) Normalized EPSP slope is shown as a function of time. (G) Representative EPSP traces before and after (arrow) LTP induction (scale bar: 5 mV, 10 ms). (F) Changes in normalized spine volume following LTP. (H) Representative images showing TBP-induced spine expansion in Shank3 heterozygotes. Images were acquired before and 5 min and 45 min after TBP. Transiently increased and stable spines are indicated by arrowheads and arrow, respectively (scale bar: 1 μm). WT, wild-type mice; Het, Shank3 heterozygous mice.

Figure 4

Decreased density of GluR1-immunoreactive puncta in Shank3 heterozygous mice. (A and B) High-resolution confocal images of puncta from wild-type and heterozygous mice. (C and D) The same images from Figures 4A and 4B are shown after deconvolution. (E and F) The puncta were quantified after thresholding of fluorescence intensity. (G) Quantification of GluR1-immunoreactive puncta. *P < 0.005. WT, wild-type mice; Het, Shank3 heterozygous mice.

Figure 5

Reduced social behaviors in Shank3 heterozygous and wild-type littermate mice. (A) Adult male-female social interactions. Left: Total duration of social interactions, scored as cumulative seconds spent by the male subject in sniffing the nose, anogenital and other body regions of an unfamiliar adult estrus B6 female mouse during a 5-min test session in a clean, empty mouse cage. *P = 0.02. Right: Number of ultrasonic vocalizations emitted during the social interaction test session (*P = 0.003) (n = 12 for wild-type (WT) and n = 14 for heterozygous (Het) mice). (B) Olfactory habitutation and dishabituation to nonsocial and social odors, measured as cumulative time spent sniffing a sequence of identical and novel odors delivered on cotton swabs inserted into a clean cage (n = 8 mice/genotype).