SAP102 null mice are viable and have normal brain structure
We generated SAP102 mutant mice by deleting N-terminal exons and introducing a frame shift into the SAP102 gene on the mouse X chromosome (). Southern blotting and PCR confirmed the structure of the altered locus in targeted ES cells and mice ( and data not shown). We analyzed SAP102 expression in wt (+/Y, wt) and hemizygous (−/Y, mutant, null) male mice using antibodies raised against the N terminus of the protein. No full-length or truncated SAP102 protein could be detected in adult forebrain extracts from SAP102 mutant mice ( and data not shown). Immunohistochemical staining showed robust expression of SAP102 in the hippocampus, cortex, and olfactory bulb of wt mice but complete absence in mutant mice, indicating that the mutation produces a null allele ().
Figure 1 Generation of SAP102-targeted mice. a, SAP102 has multiple protein–protein interaction domains, including three tandem PDZ (PSD-95/Discs large/zona occludens-1), an Src homology 3 (SH3), and a guanylate kinase (GK) domain (top). We replaced SAP102 (more ...)
Consistent with the human condition (Tarpey et al., 2004
), SAP102 mutant mice are viable and fertile. Crosses between heterozygous female and wt male mice produced heterozygous (+/−) female, hemizygous (−/Y) male and wt mice of both sexes in the expected Mendelian ratios (χ
= 5.95; p
= 0.11). Crosses between hemizygous males and heterozygous females also produced Mendelian ratios of the expected offspring genotypes, including hemizygous males and homozygous (−/−) females (χ
= 0.37; p
= 0.95). Several crosses between hemizygous male and homozygous female mice were also fertile, producing all-null litters of approximately normal size (data not shown). Brain sections stained histochemically with Nissl or immunohistochemically against PSD-95, PSD-93, NR1, NR2A, NR2B, or MAP2B showed no alterations in the morphology of the SAP102 mutant brain generally or the hippocampus in particular (data not shown) (supplemental Fig. 1, available at www.jneurosci.org
as supplemental material). We found no change in neuronal cell density in hippocampal area CA1 or dentate gyrus (supplemental Fig. 2, available at www.jneurosci.org
as supplemental material). Adult mutant mice displayed no gross physical or neurological abnormalities or overt seizures, and there was no difference between wt and hemizygous males in weaning body weight (data not shown).
SAP102 was required for normal spatial learning
Because humans with SAP102 mutations show mental retardation, we assessed spatial learning and reference memory in the SAP102 mutant mice using a standard water maze protocol. Initial training used a visible platform to ensure that the mice can use visual cues to navigate to the plat-form. This was followed by hidden plat-form training in which the ability of the mice to use memory-guided navigation was tested (). During the visible platform training, the platform was elevated above the water and curtains were drawn around the tank to eliminate extramaze spatial cues. We observed an interaction of day and genotype (F(2,48) = 3.413; p < 0.0411), and post hoc analysis showed that the mutant mice were faster on days 2 and 3 (with no difference between the two) compared with day 1, whereas the wt mice did not improve over the 3 d (, left). The mice were then trained for 5 d on a hidden platform task with extramaze cues present and a sub-merged platform (, middle). The wt mice showed improvement over the 5 d, but the mutants failed to improve (F(4,100) = 3.059; p < 0.0201). Probe tests, in which the escape platform was removed from the pool, followed 10 min (H1) and 24 h (H2) after training. Performance did not differ between the two probe tests, so they were combined for additional analyses. Although no difference between the wt and knock-out mice was determined for combined probe trial (H1+H2, F(1,26) = 2.915; p < 0.10), the wt mice spent more time in the training quadrant than predicted by chance (t(13) > 3.386; p < 0.005), whereas the mutants did not (t(12) > 1.1799; p < 0.095).
Figure 2 Impaired spatial learning in SAP102 mutant mice. a, Watermaze timeline. Dark triangles indicate probe trials conducted at the end of the training trials; vertical lines indicate training trials. b, Results of training sessions for each day of the visible (more ...)
We next examined flexibility of spatial learning using a reversal platform protocol, in which the hidden platform was switched to the opposite side of the pool (). The path length decreased over the 5 d of reversal platform training (, right) (F(4,100) = 5.597; p < 0.0004). There was no difference between the wt and mutant mice overall and no interaction of group and day, indicating that all mice were able to find the platform equally well. Memory for the platform was assessed throughout training using probe tests at the end of each day of reversal training (R1–R5) as well as 24 h and 1, 2, and 8 weeks after the last day of training (R6–R9).
The wt mice rapidly acquired the new platform position (, R2–R9), performing above chance level from the first block of training (R1+R2, t(13) > 2.272; p < 0.041). In contrast, mutant mice performed no better than chance in the initial stages of training. They showed gradual improvement in their performance with training, eventually spending more time than chance in the target quadrant after completion of the 6 d training (R7, t(12) > 2.533; p < 0.025). They then retained this information for an additional 2 weeks (better than chance, R8) but, by 8 weeks after training, were back to chance levels (R9). Direct comparison of the groups confirms that the mutant mice were impaired relative to the wt mice in the early stages of training (R1+R2, F(1,25) = 4.534; p < 0.043) but that, with additional training, this deficit was alleviated (no group difference from R3+R4 onward). These results demonstrate that the SAP102 mutant mice are impaired in the initial stages of learning (R1+R2) and that this impairment can be overcome by continued training (H1+H2, R3+R4, or R5+R6). Together, these data indicate that, in the absence of SAP102, mice show a spatial learning impairment as tested in early learning, and this impairment can be overcome with continued training.
Loss of SAP102 results in changes in spatial strategy usage
Humans and animals, when confronted with problems, including spatial tasks, can choose among multiple strategies to achieve their goal (Wolfer et al., 1997
; Graziano et al., 2003
; Bohbot et al., 2004
). Mice have been shown to use seven strategies for locating the platform in the water maze, and these can be correlated with escape latency and ranked in order of increasing efficiency: thig-motaxis, circling, random search, scanning, self-orienting, approaching target, and direct finding (Graziano et al., 2003
) (examples in ). To evaluate strategy usage, each individual swim trace for the visible and reversal probe trials was assigned to the seven categories. Although both mutants and wt used all of the strategies, their preferred (modal) strategies were different (, M). In the nonspatial, visible version of the task (), the wt mice preferred the self-orienting strategy, and, surprisingly, the mutants used the more efficient approaching strategy. When the mice were placed in the hippocampus-dependent submerged platform tasks (hidden and reversal) (), the wt mice used the approaching strategy, in contrast to the SAP102 mutants that used the less efficient circling strategy. With extended training, the control mice switched the most efficient, “direct finding” strategy (R5+R6), whereas the mutant mice stayed fixated on the circling strategy ().
Figure 3 Strategy choices in the water maze. Shown are the percentages of wt and SAP102−/Y mice that choose a particular search strategy. a, Sample traces of the paths for each of the seven search strategy categories.DF,Direct finding;A,approaching target;O,orienting;S, (more ...)
SAP102 and wt mice both use the approaching strategy but in the different tasks, indicating that both groups of animals are capable of using this strategy but deploy it under distinct conditions. Although approaching was optimal for the mutants in the visible platform, they did not use it to find the hidden platform. This could arise if the mutants were unable to engage the approaching strategy when using spatial cues, either caused by an impairment in the cognitive processing of spatial information by NMDA receptor signaling or perhaps as a result of confounding factors attributable to sensorimotor, perceptual, or motivational defects.
To eliminate the possibility of confounds, we tested the mice in a secondary battery that included the assessment of motor coordination, muscle strength, and anxiety with the rotorod, grip strength, open-field exploration, and the elevated plus maze (data not shown) (supplemental Fig. 3, available at www.jneurosci.org
as supplemental material). In the rotorod, no differences between the wt and mutant mice were detected at low or medium speeds. At the highest speed, the wt mice did not learn over days, whereas the mutant mice improved. There was no difference in grip strength detected. In the open field, there were no differences detected for time in the inner area, latency to reach the inner square, or stretch attend postures, but the mutant mice moved less as measured by line crosses, rearing, and the duration of time spent immobile. On the elevated plus maze, using the criterion defined by Weiss et al. (2000)
, differences need to be detected in four measures before an altered fear profile can be claimed (head dips, distance traveled, percentage time in open arms, and stretch attend postures). With these animals, there were no differences detected in the time spent in the open, stretch attend postures, or head dips, whereas the total distance traveled was lower for the mutants.
Overall, there are limited differences observed in this secondary battery, most of which show that the mutants had an altered locomotor phenotype. These differences, however, were not straightforward because, when motivation for a task was low (e.g., elevated plus maze or open-field exploration), the mutants’ performance was worse than that of the wt mice, but when the motivation for the task was high, (e.g., rotorod or swimming in the water maze), the mutants performed as well or better than the wt mice. The essence of the secondary battery is twofold. First, there is no overall difference in the “anxiety/fear profile,” and, second, there is no consistent deficit with motor coordination or strength. As a result, it is safe to conclude that the poorer performance observed by the mutants in the water maze was not attributable to increased fear or motoric phenotypes in the SAP102 mice. These data indicate that impaired learning performance in SAP102 mutants, although overcome by extended training, is ultimately achieved despite the use of suboptimal strategies.
Normal basal synaptic transmission in SAP102 mutant mice
Before assessing synaptic plasticity, we examined basic features of synaptic transmission. Because previous studies of PSD-95 and NMDA receptor knock-outs showed no major changes in basal synaptic transmission, we did not expect to find significant changes (Forrest et al., 1994
; Sakimura et al., 1995
; Kutsuwada et al., 1996
; Tsien et al., 1996
; Migaud et al., 1998
). We first determined input–output functions for excitatory synaptic transmission at Schaffer collateral fiber inputs onto pyramidal cells in the hippocampal CA1 region. AMPA receptor-mediated synaptic transmission in slices from SAP102 mutant mice was normal (), as was paired-pulse facilitation (). The NMDA receptor-mediated component of EPSCs recorded using whole-cell voltage-clamp techniques was not different in SAP102 mutant mice (). SAP102 may have an important role in the trafficking of NR2B-containing NMDA receptors (Sans et al., 2000
), and changes in the subunit composition of synaptic NMDA receptors could alter the time course of NMDA receptor-mediated synaptic currents in SAP102 mutants. However, single-exponential curve fits to the decaying phase of EPSCs recorded at membrane potentials of +40 mV were also the same in CA1 pyramidal cells from SAP102 and wt mice (). This suggests that there is not a decrease in NR2B-containing NMDA receptors at synapses in adult SAP102 mutant mice but does not rule out a role for SAP102 at earlier time points in development in which SAP102 is expressed at higher levels (Sans et al., 2000
). Together, these results indicate that AMPA and NMDA receptor-mediated synaptic transmission is normal at Schaffer collateral fiber inputs to the hippocampal CA1 region of adult SAP102 mutant mice.
Figure 4 Basal synaptic transmission and postsynaptic receptor function are normal in SAP102 mutant mice. a, The input–output curves for wt (open symbols; n = 5 mice, 10 slices) and SAP102 mutant mice (filled symbols; n = 5 mice, 8 slices) are shown. Presynaptic (more ...)
Altered LTP and spike-timing-dependent plasticity in SAP102 mutants
We next examined the issue that different patterns of synaptic activity, which lead to changes in synaptic strength, might use MAGUK proteins. Because PSD-95 mutants showed abnormal LTP over a wide range of stimulation conditions, we were particularly interested to ask whether SAP102 was required over the same wide range or, more interestingly, might have a requirement in more specific ranges of synaptic activity. Thus, we examined the ability of several different stimulation protocols to induce LTP in the CA1 region of hippocampal slices from SAP102 mutant mice. As shown in , the induction of LTP by a conventional high-frequency stimulation protocol (two trains of 100 Hz stimulation) was normal in SAP102 mutant mice. In contrast, a long, low-frequency (5 Hz) train of synaptic stimulation that induced only modest LTP in wt slices induced a twofold increase in synaptic strength in slices obtained from SAP102 mutants ( p < 0.005 compared with wt) (). This is in stark contrast to PSD-95 mutants, which showed significant increases in LTP in both stimulation paradigms.
Figure 5 Loss of SAP102 results in specific enhancement of hippocampal synaptic plasticity induced by theta frequency and spike timing-dependent stimulation. a, High-frequency stimulation-inducedLTP is normal in SAP102 mutants. Sixty minutes after 100 Hz stimulation (more ...)
We then examined the response of excitatory synapses in the hippocampal CA1 region to spike timing-dependent plasticity protocols, a more physiologically relevant paradigm (Thomas et al., 1998
; Meredith et al., 2003
). In wt cells, pairing of single pulses of presynaptic fiber stimulation with a burst of postsynaptic APs induced robust LTP, whereas pairing with single postsynaptic APs did not (). This is consistent with previously published data and demonstrates that AP bursts are the essential trigger for LTP induction in these cells. Remarkably, however, in SAP102 mutant cells, pairing with single postsynaptic APs induced robust LTP (). This indicates that SAP102 plays an instrumental role in determining the rules governing how changes in synaptic strength are induced by coincident presynaptic and postsynaptic activity. Such inappropriate induction of LTP could result in degradation of information storage, producing the learning deficits observed in the SAP102 mutant mice. Moreover, in whole-cell current-clamp experiments, we found that pairing EPSPs with bursts of postsynaptic action potentials (100 pairings at 10 Hz) induced similar levels of LTP in cells from wt and SAP102 mutant mice (). We conclude that SAP102 was not required for normal synaptic transmission but was necessary for frequency-specific forms of LTP and in particular that form of LTP induced by low-frequency (5 Hz) patterns of synaptic activity.
SAP102 couples NMDA receptors to the MAPK/ERK pathway
The electrophysiological specificity of the LTP phenotype provides an important clue toward the identification of the down-stream signaling pathway that must be altered in SAP102 mutants, namely, that the enzyme(s) involved must be selective for 5 Hz and not 100 Hz LTP. Previous studies show that MAP kinase kinase (MEK)/ERK signaling has precisely this property because MEK inhibitors preferentially block 5 Hz but not 100 Hz LTP in mouse CA3–CA1 slices (Winder et al., 1999
). Although this suspicion was subsequently confirmed, we initially took a less biased approach using proteomic assays that measure the function of many different kinases. This differential proteomics strategy examined the levels and/or phosphorylation of 48 different postsynaptic proteins from the hippocampus of mutant mice ( and data not shown) (for a full list of phosphorylation sites, see supplemental Materials and Methods, available at www.jneurosci.org
as supplemental material). Strikingly, these studies showed a change in only one protein: the phosphorylated form of ERK2 (phospho-ERK). Elevation of phospho-ERK in SAP102 mutants was confirmed using Western blotting () and ELISA assays (), in which a modest (~25%) but consistently reproducible increase in basal phospho-ERK was seen in the mutant mice. Consistent with the idea that these changes in basal ERK activity contribute to the enhancement of LTP in SAP102 mutants, the MEK inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o
-aminophenylmercapto)butadiene] blocked the enhancement of 5 Hz stimulation-induced LTP in SAP102−/y
Figure 6 Altered postsynaptic signaling in SAP102 mutant mice. a, No change in total hippocampal levels of postsynaptic proteins in mutant mice. i, MAGUKs; ii, postsynaptic receptors; iii, postsynaptic signaling proteins. b, Elevated basal ERK phosphorylation (more ...)
We next examined NMDA receptor activation of ERK and other members of the ERK pathway. Hippocampal slices from wt and mutant mice were stimulated by application of NMDA (20 μM for 3 min), and protein phosphorylation and levels were analyzed. In electrophysiological experiments using the same NMDA treatment, we found that NMDA had no lasting effects on synaptic transmission in wt slices and induced a significant but transient potentiation of synaptic transmission in slices from SAP102−/y mice (data not shown). shows that ERK phosphorylation, normalized between wt and SAP102−/Y levels at the time of stimulation, was attenuated in response to NMDA stimulation in the absence of SAP102. The response to phospho-MEK1/2, the upstream activator of ERK in the MAPK pathway, was also attenuated (), although basal phospho-MEK1/2 levels were unaltered. To confirm that the attenuated phospho-ERK response was not an artifact resulting from normalization of the SAP102−/Y levels to that of wt, we performed additional independent experiments without normalization. We found an ~30% reduction in phospho-ERK levels in mutant slices compared with wt after NMDA application despite elevated basal phospho-ERK (). These results show that SAP102 regulates MAPK-mediated NMDA receptor signaling.
Although MEK and ERK have been extensively characterized in the context of the induction of LTP, the physiologically relevant substrates remain less well understood. We attempted to discover the downstream effects of the altered ERK activity in SAP102 mutants by ERK substrates for which reagents were available. We found no change in phosphorylation of Kv4.2, Elk-1, pS383, p70, ribosomal S6 kinase-4 (RSK-4), or Menkes protein MNK-1 (data not shown). It is thus likely that the downstream alteration is mediated by some other, as yet undescribed, ERK substrate.
SAP102 and PSD-95 perform distinct but overlapping functions
SAP102 and PSD-95 have similar protein domains, binding partners, subcellular localization, and regional expression patterns (Cho et al., 1992
; Muller et al., 1996
; Fukaya and Watabe, 2000
), suggesting they may have overlapping and specific functions. The availability of knock-outs of both genes allows a genetic approach to this issue. We first asked whether SAP102 expression was altered in PSD-95 mutant mice. Western blots of hippocampal extracts from 10 wt and 10 mutant animals showed a robust and reproducible elevation of SAP102, suggesting some compensation for loss of PSD-95 in these mice (). We had already found no change in PSD-95 protein levels in hippocampus extracts of SAP102 mice (), but a change in PSD-95 localization to the NMDA receptor complex was another possible compensatory mechanism. We used coimmunoprecipitation to examine the levels of PSD-95 associated with NMDARs in SAP102 mutants. We found that more PSD-95 coimmunoprecipitated with NR1 in forebrain extracts from 10 SAP102 mutant mice than from 10 of their wt littermates (). These results suggest that PSD-95 and SAP102 can be recruited to the same NMDA receptor complexes and may compete for interaction. Moreover, these results suggest that SAP102 and PSD-95 have partial functional overlap and that the phenotypes observed in the two mutant mouse strains may be tempered by compensation by the other MAGUK. If this was the case, mice with mutations in both proteins should display a more severe phenotype than that of the individual mutations. To examine this prediction, we crossed the SAP102 and PSD-95 strains. Of 88 weaned pups from these crosses, none were double null, and only two females were heterozygous for SAP102 and homozygous for PSD-95, reflecting a severely skewed distribution of genotypes among the offspring (). Additional analysis indicated that the double-mutant mice die perinatally, with 4% of pups being double knock-out at birth (compared with an expected 12.5%) and none surviving past postnatal day 3. These data show that SAP102 and PSD-95 have partial function overlap and that the presence of at least one is necessary for postnatal survival.
Figure7 Overlapping but distinct functional roles of SAP102 and PSD-95. a, SAP102 protein expression is elevated in the hippocampus of PSD-95 mutant mice. Shown are Western blots on hippocampal extracts from wt and PSD-95 homozygous animals. b, Increased PSD-95 (more ...)