Generation of Shank1 mutant mice
To generate Shank1 knock-out mice, we constructed a gene-targeting vector to disrupt exons 14 and 15 of the Shank1 gene by homologous recombination (). These exons encode a highly conserved region of Shank1, including the PDZ domain that is required for synaptic targeting of the protein (Sala et al., 2001
). Two independent ES cell lines were identified by Southern blotting and used to generate chimeras that transmitted the mutation into the germ line (). Intercrosses of Shank1 heterozygous mice yielded offspring at the expected Mendelian ratio (representative litter shown in ). Shank1−/−
mice were grossly indistinguishable from wild-type littermates in their home cage and showed similar survival.
Figure 1 Generation and characterization of Shank1 mutant mice. A, Domain structure of Shank1, showing ankyrin repeats (Ank), SH3 and PDZ domains, proline-rich region (Pro), and sterileα motif (SAM) domain. B, Schematic diagram of the Shank1 gene locus, (more ...)
To confirm loss of Shank1 protein expression, we used a Shank1-specific peptide antibody (1356) raised against amino acids 425–440 of Shank1 (Lim et al., 1999
), which lie N-terminal to the targeted deletion. Immunoblotting of wild-type mouse forebrain extracts with antibody 1356 showed a major band of ~240 kDa and several smaller bands that presumably represent alternative splicing and/or degradation products; all these bands decreased in the heterozygote and were eliminated in the homozygous brain (). No bands were seen even in the low molecular weight region of the gel (data not shown). Immunoblotting with pan-Shank antibody 3856, which recognizes Shank2 and Shank3 as well as Shank1 (Lim et al., 1999
), confirmed the loss of the 240 kDa and lower bands corresponding to Shank1 polypeptides. However, an additional set of proteins recognized by 3856 (molecular weight, ~160 −200 kDa) were unchanged in Shank1 knock-outs; these presumably correspond to Shank2 and Shank3 gene products (). These data indicate that the gene targeting resulted in a Shank1 null mutant, with no compensatory increase in Shank2 and Shank3.
We detected no gross abnormalities in the size or histological structure of the brain (including cortex, hippocampus, and cerebellum), based on Nissl-stained sections () (data not shown).
Altered PSD protein composition in Shank1−/− brain
Via its multiple protein–protein interactions, the Shank scaffold has been proposed to play an important role in assembling the PSD (Sheng and Kim, 2000
). We therefore examined the effect of Shank1 disruption on the protein composition of PSD fractions purified from the forebrains of wild-type and Shank1−/−
mice. We used PSD fractions extracted once with Triton X-100 (PSDI), because the twice-extracted (PSDII) or Sarkosyl-extracted (PSDIII) fractions did not yield sufficient protein from individual mice for additional analysis. One-dimensional SDS-PAGE () and two-dimensional gel electrophoresis (data not shown) of purified PSDs revealed no obvious change in the overall protein patterns. By immunoblotting, the Shank1-specific 1356 antibody confirmed the absence of Shank1 protein in the PSD fraction of Shank1 knock-out brains (). The pan-Shank 3856 antibody showed ~40% reduction in total Shank immunoreactivity in PSDs from Shank1−/−
mice (). Interestingly, the ~240 kDa Shank1 protein is more prominent in the PSD preparation compared with crude membrane extracts [compare “Shank (total)” in Figs. , ], suggesting that Shank1 may be more highly enriched in the PSD than Shank2/3.
Figure 2 Altered composition of PSD fractions in Shank1 mutant mice. Analysis of Triton-extracted PSD fractions (PSDI) purified from forebrain of adult wild-type (+/+) and Shank1 knock-out (−/−) mice. A, SDS-PAGE and silver staining of PSD proteins (more ...)
A variety of glutamate receptors, scaffold proteins, and signaling molecules were measured by quantitative immunoblotting (). In Shank1-mutant PSDs, there was a significant reduction of GKAP (~30%). In addition, the level of Homer1b/c showed a modest but significant decrease (~20%). Thus, the levels of two scaffold/adaptor proteins that bind directly to Shank were reduced in Shank1-deficient PSDs. In the same preparations, we detected no significant difference in the abundance of many other proteins that are known to be associated with synapses or the PSD, including NMDA, AMPA, and metabotropic glutamate receptors PSD-95 and GRIP (). Also unchanged were the PSD levels of β
-PIX and cortactin, actin regulatory proteins that can interact directly with Shank but that are not highly enriched in the PSD (Naisbitt et al., 1999
; Park et al., 2003
We then performed immunocytochemistry to examine the distribution of synaptic proteins in dissociated hippocampal neurons cultured from individual wild-type or Shank1−/−
mouse embryos. As expected, staining with the Shank1-specific 1356 antibody was abolished in neurons from mutant animals (). As reported previously (Naisbitt et al., 1999
), staining with pan-Shank antibody 3856 showed Shank to be localized to dendritic clusters that colocalized with the excitatory synapse marker PSD-95 (). In Shank1−/−
neurons, the linear density of Shank puncta (labeled with pan-Shank antibody 3856) along the dendrite was reduced ~50% (). Similar to wild-type neurons (in which 90.3 ± 1.6% of Shank puncta colocalized with PSD-95 clusters), the vast majority of the 3856-immunoreactive Shank puncta in Shank1−/−
neurons (97.1 ± 1.2%; n
= 10 neurons per genotype) also colocalized with PSD-95. These data indicate that the remaining Shank staining (presumably resulting from Shank2 and Shank3) still localizes at excitatory synapses. In line with the biochemical results, there was also a significant decrease in the density of GKAP puncta in Shank1-deficient neurons (). Although we detected no significant change in density of Homer puncta, the immunostaining pattern of Homer was more diffuse in Shank1-deficient neurons (), supporting the idea that Shank is involved in recruiting or stabilizing Homer at synapses (Sala et al., 2001
). There was no difference between cultured wild-type and Shank1 knock-out neurons in the cluster density or staining pattern of PSD-95 or the presynaptic active zone protein Bassoon. Overall, the biochemical and immunostaining data provide consistent evidence that Shank1 is important for synaptic accumulation of GKAP and Homer, supporting previous conclusions based on overexpression and dominant-negative studies of Shank in cultured neurons (Sala et al., 2001
Figure 3 Altered immunostaining of PSD proteins in Shank1-deficient neurons. A, B, Hippocampal neurons in dissociated culture (18–19 DIV) from wild-type (+/+) or Shank1−/− mice were immunostained for the indicated proteins. A, Representative (more ...)
Altered synapse morphology in Shank1 mutant brain
To examine the in vivo role of Shank1 in spine morphology, we performed blinded quantitative analysis of the number and size of dendritic spines in adult wild-type and Shank1−/− mice (). We focused on apical dendrites of CA1 pyramidal neurons of hippocampus, where synapse morphology and plasticity have been studied extensively. Mean spine density showed a slight decrease in Shank1 knock-out mice () ( p < 0.05; t test). Cumulative frequency plots of spine length () and spine head width () revealed a highly significant shift toward smaller spine size (length, p < 0.001, t test, and p < 0.0005, K–S; width, p < 0.001, t test, and p < 0.001, K–S). Although the absolute differences are small, possibly related to the presence of the remaining Shank isoforms, these data affirm that Shank1 is important for spine growth or maintenance in vivo.
Figure 4 Smaller dendritic spines and thinner PSDs in Shank1 mutant mice. A, DiI-labeled dendrites from CA1 pyramidal neurons of adult wild-type (+/+) and Shank1−/− mice (three representative segments shown). Scale bar, 5 μm. B, Quantification (more ...)
We next examined the ultrastructure of synapses, analyzing thin-section electron microscopic (EM) micrographs from wild-type and Shank1−/− mouse brains (). In hippocampal CA1 region, the mean PSD thickness of Shank1−/− synapses was significantly reduced relative to wild type, and cumulative frequency graphs revealed a roughly parallel shift across the range of PSD thickness () [+/+, PSD thickness, 48.4 ± 1.6 nm (mean ± SEM); −/−, 45.8 ± 1.4 nm; p < 0.004, K–S]. The mean length of PSDs decreased in Shank1 mutants but did not reach statistical significance (+/+, 310.5 ± 3.5 nm; −/−, 299.4 ± 8.9 nm; p = 0.295, K–S). However, the cumulative frequency distribution showed that Shank1 mutant synapses suffered primarily a loss of the largest PSDs relative to wild type (). Indeed, PSD length among the largest quartile of CA1 synapses measured per animal was significantly decreased in Shank1 mutants (+/+, 441.8 ± 12.9 nm; −/−, 397.9 ± 15.4 nm; p < 0.003, K–S). These data suggest that although Shank1 is not required for synapse formation, it may be critical for the development and/or maintenance of the largest subset of PSDs and synapses in particular.
Reduced basal synaptic transmission and normal synaptic plasticity in Shank1 mutant mice
What is the effect of Shank1 deficiency on synaptic function? We investigated excitatory synaptic transmission at the Schaffer collateral/commissural-CA1 synapse in acute hippocampal slices from wild-type and Shank1−/− mice (3–5 weeks of age). First, we examined synaptic transmission in Shank1−/− mice by measuring AMPA receptor-mediated field EPSPs in the stratum radiatum using extracellular recording techniques (). The input–output curve for Shank1−/− mice was significantly shifted downward compared with wild type, especially at high stimulus intensities, demonstrating that Shank1 deficiency reduces basal synaptic transmission. PPF of AMPA receptor-mediated EPSCs, measured by whole-cell patch clamping, was similar in wild-type and Shank1−/− slices (+/+, 1.84 ± 0.13, n = 12 cells/6 mice; −/−, 1.74 ± 0.11, n = 9 cells/5 mice; p = 0.588). These results imply that the change in input–output relationship is unlikely to be caused by a change in presynaptic release probability. The ratio of AMPAR and NMDAR EPSCs (AMPA/NMDA ratio) was not significantly different between wild-type mice and Shank1−/− mice (+/+, 1.77 ± 0.35, n = 9 cells/7 mice; −/−, 1.91 ± 0.35, n = 9 cells/5 mice; p = 0.785), suggesting that the Shank1 deficiency does not affect the proportion of synaptic AMPA and NMDA receptors.
Figure 5 Decreased synaptic strength in Shank1 mutant mice. A, Left, Sample traces (average of 10 consecutive responses) represent the responses evoked with seven different stimulus intensities from wild-type (+/+) or Shank1−/− hippocampal slices. (more ...)
Given no change in PPF, the observed difference in synaptic response may be explained by a reduction in the number of functional synapses, in the number of glutamate receptors per synapse, or a combination of the two. To address this question, we examined the amplitudes and frequencies of the AMPAR-mediated mEPSCs (). There was no significant difference in the average mEPSC amplitude between wild-type and Shank1−/− mice. However, the frequency of the mEPSC from Shank1−/− mutants was significantly reduced, suggesting that decreased basal transmission is primarily caused by a reduction in the number of functional synapses. We found no difference in the total length of CA1 apical dendrites in Shank1 knock-out versus wild-type mice (data not shown), implying that the weaker synaptic transmission is not a result of loss of dendrites and total synapses per neuron.
We next examined the effect of Shank1 deficiency on synaptic plasticity at Schaffer collateral/CA1 synapses. LTP was induced in acute hippocampal slices (3- to 5-week-old mice) using tetanic stimulation (100 Hz, 1 s). Similar magnitudes of LTP were obtained in wild-type (1.55 ± 0.06 of baseline at 60 min after tetanus) and mutant (1.58 ± 0.10; p = 0.800) slices (). We also did not detect a difference in LTP in older mice (7- to 9-week-old mice) (data not shown). LTD evoked by low-frequency stimulation (1 Hz, 15 min) was also indistinguishable between wild-type (0.81 ± 0.06 of baseline at 75 min) and Shank1−/− (0.86 ± 0.02; p = 0.464) mice ().
Figure 6 Synaptic plasticity is unchanged in Shank1 mutant mice. A, Top, Sample traces of field EPSPs of wild-type (+/+) and Shank1−/− mice (−/−) recorded at the times indicated in summary graph. Below, Summary graph of the averaged (more ...)
We further tested a stronger stimulus protocol (four tetanic trains separated by 5 min intervals), which has been used widely to induce L-LTP (Kelleher et al., 2004
). This protocol elicited a long-lasting potentiation that was similar in wild-type and Shank1−/−
slices (+/+, 1.29 ± 0.14 of baseline at 180 min; −/−, 1.30 ± 0.08; p
= 0.916) (). Thus, although loss of Shank1 decreases basal synaptic strength, it does not appear to affect standard forms of electrophysiologic plasticity lasting up to a few hours.
Increased anxiety-like behaviors in Shank1 mutant mice
We investigated the behavior of adult Shank1−/− mice in a variety of assays. Although they showed no obvious differences in the home cage, the Shank1−/− mice were significantly less active in a novel open-field environment than their wild-type littermates, as measured by horizontal activity, total distance traveled, and movement time (). The relative reduction in distance traveled and time spent moving (movement time) was similar in the mutant mice; thus, average velocity of movement (calculated as total distance/movement time) was only slightly decreased in mutant mice (+/+, 8.47 ± 0.17 cm/s; −/−, 7.55 ± 0.28 cm/s; p < 0.01). Additionally, mutant mice spent significantly less time in the center zone, a measure of anxiety-like behavior (+/+, 148.6 ± 25.6 s; −/−, 56.1 ± 12.2 s; p = 0.002). The Shank1 mutants demonstrated a mild deficit in motor performance, as revealed by a reduced latency to fall in the accelerating Rotarod test (). Nevertheless, given the small difference in open-field average movement velocity, it is unlikely that motor impairment alone accounts for the large decrease in open-field activity. In the light/ dark exploration test, another anxiety-related task, Shank1−/− mutants similarly had fewer transitions between compartments and showed longer latencies to enter the light side ().
Figure 7 Hypoactivity, rotarod performance, and increased anxiety-related behaviors in Shank1 mutant mice. A, Horizontal activity in open-field test is shown as number of beam breaks (mean ± SEM). Black line, Wild type (n = 24); gray line, Shank1−/− (more ...)
We also observed that Shank1−/− homozygous mice were poor breeders, giving birth only rarely. Moreover, homozygous mutant females did not nurture their pups, and their litters generally died before weaning. Thus, all our studies were performed on offspring of heterozygote intercrosses.
Impaired fear conditioning in Shank1 mutant mice
To examine the role of Shank1 in hippocampus-dependent learning and memory, we turned to a contextual fear conditioning task, in which long-term memory can be established with a single conditioning trial. During the conditioning period, wild-type and Shank1−/− mice were similarly active before the tone-shock pairs and showed similar levels of freezing after footshocks (). However, the Shank1−/− mutants showed significantly less freezing during context testing conducted at 1 h () (+/+, 53.1 ± 5.0%; −/−, 22.2 ± 3.7%; p < 0.0001) and at 24 h after conditioning (+/+, 51.0 ± 4.6%; −/−, 32.8 ± 4.1%; p = 0.006). Given that Shank1 mutants show a decreased freezing response, the data are unlikely to be confounded by the overall hypoactivity of the Shank1 mutants, which could potentially mimic freezing. In contrast, when the conditioned stimulus (tone) was presented in an altered context 48 h after conditioning (cued testing), the Shank1−/− mutants froze to a similar extent as wild type () (+/+, 61.4 ± 5.8%; −/−, 58.4 ± 7.1%; p = 0.757). Thus, Shank1 is required selectively for contextual fear memory, a process believed to depend on intact hippocampus and amygdala function.
Figure 8 Impaired contextual fear memory in Shank1 mutant mice. A, Wild-type mice (black line; n = 20) and Shank1−/− mice (gray line; n = 18) showed similar freezing responses during the conditioning phase. The horizontal bar denotes exposure to (more ...)
Enhanced spatial learning in Shank1−/− mice
We then used the eight-arm radial maze task to assess spatial memory (Olton and Papas, 1979
). This kind of learning accrues over days and weeks and also requires the hippocampus. With successive daily trials, the mice learn the locations of the two arms baited with food, allowing simultaneous evaluation of reference memory (errors counted by entries into arms never containing the reinforcing bait) and working memory (errors counted by entries into arms previously visited during the same trial). Thus, in this task, animals must learn and remember the position of baited arms between trials while rapidly establishing memory of previously visited arms within a trial. The eight-arm radial maze task is less sensitive than the Morris water maze to differences in motor performance, because learning is scored based on the number of errors made rather than the time required to complete the task.
During the acquisition phase, both wild-type and Shank1−/− mice improved their performance with repetitive training () (see Materials and Methods for details of training). Remarkably, the Shank1 mutants showed a steeper learning curve and reached a better performance level with fewer reference memory errors than the wild-type animals () (genotype effect, F(1,27) = 10.98; p < 0.003, repeated measures two-way ANOVA). Even after prolonged training in this protocol, the wild-type mice did not catch up to the mutants' level of performance. Moreover, the Shank1−/− mice made fewer working memory errors than wild type; indeed, the knock-out animals made virtually no mistakes of “revisiting” toward the end of the training period () (genotype effect, F(1,27) = 10.41; p < 0.004).
Figure 9 Enhanced acquisition and impaired retention of spatial memory by Shank1 mutants in the eight-arm radial maze task. Two of eight arms were baited to test simultaneously reference and working memory. A, Total number of reference memory errors during acquisition (more ...)
As another measure of reference memory performance, we also quantified the percentage of trials in which a baited arm was chosen correctly with the first arm selection. Both genotypes selected a baited arm ~25% of the time in initial trials (as expected from random selection of 2 of 8 baited arms), but the Shank1−/− mice improved more quickly and to a higher level () (genotype effect, F(1,27) = 49.72; p < 0.0001). Together, these data indicate that Shank1-deficient mice learn faster and more effectively during repetitive training in the eight-arm radial maze.
Obviously, the enhanced spatial learning of Shank1−/− mice in the radial maze task cannot be accounted for by their relative hypoactivity, which might be expected to prolong the duration of the task. In fact, although the Shank1−/− mice initially took a longer time to complete the task (trials 1–8, genotype effect, F(1,27) = 7.22; p < 0.02, repeated measures two-way ANOVA), the same animals were later able to find the baits in a shorter time than wild-type (trials 21–84, genotype effect, F(1,27) = 4.23; p < 0.05) ().
The surprising results above indicate that Shank1-deficient animals have enhanced spatial learning, seemingly counterintuitive for a gene that encodes a major PSD scaffold protein. Therefore, we examined the long-term stability of spatial memory. To assess memory retention, we retested the mice in the same radial maze task after a period of 28 d in their home cage, during which they had no exposure to the maze. The wild-type animals showed no deterioration in their performance of the radial maze (measured by reference memory errors) after the 4 week “rest” (). In contrast, the Shank1−/− mice performed significantly worse after the 28 d rest, regressing to the wild-type level of reference memory performance. Thus, Shank1 mutants are unable to retain long term the learning enhancement that they gained over the wild-type mice during training.
We further tested the “re-learning” ability of Shank1−/− mice by reversal training, in which the radial maze is rebaited in two different arms (). Although wild-type and Shank1 mutants performed similarly in initial reversal trials (reversal trials 1–16, F(1,27) = 0.30; p > 0.5), the knock-out mice learned the new locations more quickly and made significantly fewer reference memory errors with repetitive training in the newly baited maze (reversal trials 17–36, genotype effect, F(1,27) = 7.81; p < 0.01).
Differences in training protocol can affect learning and memory (Kogan et al., 1997
). We therefore subjected a different cohort of mice to the same radial maze task but applied a more intensive training program in which the number of trials per day was accelerated (). Under this training regimen, the Shank1−/−
mutants performed similarly to the mutant cohort trained under the original protocol, but the wild-type mice improved, making fewer reference memory errors with the more intensive protocol. Nevertheless, despite this improvement, the wild-type mice did not catch up to the performance of Shank1 mutants () (genotype effect, F(1,33)
= 10.86; p
< 0.003). After training, the mice were again tested for long-term retention. Twenty-eight days after the end of intensive training, wild-type mice retained their memory, completing the task with no significant change in reference memory errors. In contrast, Shank1−/−
animals made markedly more reference memory errors after the 4 week break from training (). In summary, Shank1 mutant mice show enhanced acquisition of spatial memory in the radial maze task but impaired long-term retention of this memory after training has finished.