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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2013 Mar 4;110(12):4738–4743. doi: 10.1073/pnas.1222803110

Dysregulation of synaptic plasticity precedes appearance of morphological defects in a Pten conditional knockout mouse model of autism

Koichi Takeuchi a,1, Michael J Gertner a, Jing Zhou b,2, Luis F Parada b, Michael V L Bennett a,3, R Suzanne Zukin a,3
PMCID: PMC3607034  PMID: 23487788

Abstract

The phosphoinositide signaling system is a crucial regulator of neural development, cell survival, and plasticity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulates phosphatidylinositol 3-kinase signaling and downstream targets. Nse-Cre Pten conditional knockout mice, in which Pten is ablated in granule cells of the dentate gyrus and pyramidal neurons of the hippocampal CA3, but not CA1, recapitulate many of the symptoms of humans with inactivating PTEN mutations, including progressive hypertrophy of the dentate gyrus and deficits in hippocampus-based social and cognitive behaviors. However, the impact of Pten loss on activity-dependent synaptic plasticity in this clinically relevant mouse model of Pten inactivation remains unclear. Here, we show that two phosphatidylinositol 3-kinase- and protein synthesis-dependent forms of synaptic plasticity, theta burst-induced long-term potentiation and metabotropic glutamate receptor (mGluR)-dependent long-term depression, are dysregulated at medial perforant path-to-dentate gyrus synapses of young Nse-Cre Pten conditional knockout mice before the onset of visible morphological abnormalities. In contrast, long-term potentiation and mGluR-dependent long-term depression are normal at CA3–CA1 pyramidal cell synapses at this age. Our results reveal that deletion of Pten in dentate granule cells dysregulates synaptic plasticity, a defect that may underlie abnormal social and cognitive behaviors observed in humans with Pten inactivating mutations and potentially other autism spectrum disorders.

The discovery of genes associated with autism spectrum disorders (ASDs) has largely depended on gene linkage analyses within lineages of humans with familial ASDs (1–3). A well-established candidate gene is the tumor-suppressor gene, phosphatase and tensin homolog missing on chromosome 10 (PTEN) (4–7). Pten is a lipid and protein phosphatase best known for its role in suppressing tumor formation by inhibiting cellular survival, proliferation, and cellular architecture (8, 9), but which also plays an important role in brain morphology and synaptic function (10, 11). Pten acts via its lipid phosphatase activity to dephosphorylate phosphatidylinositol (3,4,5)-trisphosphate and negatively regulate the PI3K-mammalian target of rapamycin (mTOR) signaling pathway. Although PTEN mutations are present in 5–10% of people with ASDs (12–14), loss-of-function point mutations in this gene give rise to progressive macrocephaly, a hallmark feature that occurs in nearly 20% of humans with ASDs (5, 6). In addition to anatomical abnormalities, humans with inactivating PTEN mutations exhibit spontaneous seizures and deficits in social and cognitive behaviors (10, 11).

A conditional Pten knockout mouse (cKO) in which both alleles of the Pten gene contain loxP sites and Cre recombinase (Cre) is expressed under the neuron-specific enolase promoter (Nse-Cre) provides a clinically relevant mouse model for humans with inactivating PTEN mutations (15). In Nse-Cre Pten cKO (hereafter Pten cKO) mice, ablation of Pten is primarily focused in the granule cells of the dentate gyrus, pyramidal neurons of the hippocampal CA3, and select populations of postmitotic neurons in the cortex. These mice recapitulate many of the late-onset morphological and behavioral abnormalities observed in humans with inactivating PTEN mutations. Hallmark anatomical features include progressive macrocephaly of the dentate gyrus (DG) and the overlying neocortex and, ultimately, ensuing compression of the CA1 pyramidal cell layer and the deeper layers of entorhinal cortex. At the cellular level, neurons within the granule and polymorphic layers of the DG are enlarged, abnormally oriented, and densely packed, and are intertwined with isolated clusters of atrophic neurons with reduced dendritic arborization (15, 16). Pten cKO mice exhibit autism-relevant behaviors, including seizures and deficits in hippocampus-based social and cognitive behaviors, hypersensitivity to sensory stimuli, anxiety, and spontaneous seizures, evident by 8 wk, an age before the onset of visible morphological abnormalities (15). By 14 wk of age, Pten cKO mice exhibit hypertrophy of dentate granule cells (DGCs) and overlying neocortex, and by 20 wk, severe compression of CA1 pyramidal neurons and select neuronal populations of the entorhinal cortex (15, 16). At the level of intracellular signaling, Pten cKO mice exhibit overactivation of mTOR in DGCs and CA3 pyramidal neurons (15), a defect common to a number of ASDs (11, 17) and thought to underlie neuronal hypertrophy. Moreover, viral-mediated knockdown of Pten in DGCs increases excitatory postsynaptic currents (EPSCs), implicating synaptic deficits in this disorder (18).

Although the prevailing view is that gross changes in morphology are causally related to the social and cognitive deficits in humans with inactivating PTEN mutations, an additional possibility is that synaptic defects precede the morphological abnormalities and contribute to the behavioral deficits associated with PTEN mutations. The present study was undertaken to investigate whether synaptic plasticity deficits precede or are a consequence of anatomical changes. Toward this end, we monitored plasticity at hippocampal synapses at times before, coincident with, and after the appearance of obvious morphological abnormalities. We show that Pten is ablated in DGCs and pyramidal neurons in the hippocampal CA3 in Pten cKO mice. We find that two PI3K- and protein synthesis-dependent forms of synaptic plasticity, theta burst-induced long-term potentiation (TBS-LTP) (19, 20) and metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) (21), are dysregulated at the postsynaptic side of perforant path-to-DGC synapses of Pten cKO mice before the onset of gross morphological defects. Whereas TBS-LTP is enhanced, mGluR-LTD is impaired. In contrast, at this age, synaptic plasticity is normal at CA3–CA1 pyramidal cell synapses, where morphological changes are greatly delayed. These findings reveal a previously unappreciated role for Pten in regulating normal synaptic function and are consistent with a role for Pten in the dysregulation of hippocampal-based synaptic plasticity, cognition, and social interactions associated with the ASD phenotype.

Results

Pten Is Ablated in DGCs and CA3 Pyramidal Neurons in Pten cKO Mice.

To document the neuronal populations and ages at which Pten was deleted in Pten cKO mice, we crossed Nse-Cre mice with Rosa26-stop-lacZ reporter mice (22) and assessed Cre activity by X-Gal staining. By 4 wk of age, X-Gal staining revealed Cre expression in DG, the polymorphic layer, CA3 (but not CA1), and parts of the overlying cortex (Fig. 1A). Nse-Cre; PtenloxP/loxP mice show loss of Pten immunoreactivity in areas where Nse-Cre is expressed (Fig. 1B). By 8 wk, 30–60% of DGCs and CA3 pyramidal cells lacked Pten, whereas CA1 remained relatively unaffected. By 20 wk of age, nearly 100% of Pten cKO mice exhibited enlarged DGCs and hypertrophied mossy fiber projection axons (15). The overlying cortex was also hypertrophied (15), and CA1 was compressed with loss of CA1 pyramidal neurons (Fig. 1 B and C). Thus, in the hippocampus of this mouse with Cre under the Nse promoter, Pten was lost primarily in DGCs and CA3 pyramidal neurons. Neurons of origin of perforant path afferents in layer II of the entorhinal cortex (23) show minimal Cre expression (24) and presumably express normal levels of Pten.

Fig. 1.

Fig. 1.

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Cre and Pten immunostaining are selective to DG and CA3 regions in hippocampus. (A) Cre activity (blue) assessed by X-Gal staining of Nse-Cre; R26-lacZ mouse at 4 wk of age. (Upper) In the hippocampus, Cre activity is present in the dentate gyrus, polymorphic layer, and CA3 region. (Lower) Cre activity is absent in CA1. (B) Pten immunostaining (brown) in wild-type and Pten cKO mice confirms Pten-negative cells (blue) in the granule layer of the dentate gyrus (row 3) at 8 wk (Center) and 20 wk of age (Right) compared with wild-type mice aged at 8 wk (Left). In CA1, Pten-positive cells present at 8 wk, are reduced in number at 20 wk due to compression of CA1. (C) Hematoxylin and eosin staining demonstrates progressive enlargement of the hippocampus of Pten cKO mice. At 8 wk of age, no gross morphological changes are observed (Center). At 20 wk of age, compression of CA1 and overgrowth of dentate gyrus and CA3 are observed (Right). (Scale bars: A–C Upper, 500 μm; A and C Lower, 200 μm; B Lower, 100 μm.)

Basal Synaptic Transmission at DGC Synapses Is Enhanced in Middle-Aged Pten cKO Mice.

To assess basal synaptic transmission in Pten cKO mice, we stimulated afferents in the medial perforant pathway and monitored the input/output relation of field excitatory postsynaptic potentials (fEPSPs) at synapses on DGCs. We focused on the DG because it is a critical checkpoint for the flow of information from the entorhinal cortex to the hippocampus (25) and exhibits progressive neuronal hypertrophy in Pten cKO mice by 14 wk of age (15). The input/output relation (fEPSP slope vs. stimulus intensity) at DGC synapses was unaltered in young (8–12 wk) and old (20–30 wk) Pten cKO mice, but enhanced in middle-aged (14–19 wk) Pten cKO mice relative to that of age-matched WT mice (Fig. 2 A–C). The elevated basal synaptic transmission observed in middle-aged Pten cKO mice may reflect an increase in spine density (15) and number of functional synapses on DGCs at this age (15). In older Pten cKO mice, basal transmission was not detectably altered relative to that of WT mice, consistent with a model whereby the elevated synaptic transmission observed in middle-aged Pten cKO mice has compensated for the reduction in the number of synaptic inputs on DGCs (15).

Fig. 2.

Fig. 2.

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Basal transmission and release probability are elevated in middle-aged Pten cKO mice. (A) Basal synaptic transmission, as assessed by the input/output relation of the fEPSP amplitude as a function of stimulation in nearby stratum moleculare, is normal at DGC synapses of young (8–12 wk) Pten cKO mice. (WT: n = 14 slices, seven mice; Pten cKO: n = 14 slices, seven mice; P > 0.05). (B) The input/output relation is enhanced in middle-aged (14–19 wk) Pten cKO mice (WT: n = 12 slices, six mice; Pten cKO: n = 12 slices, six mice. *P < 0.05, **P < 0.01). (C) The input/output relation is reduced in old (20–30 wk) WT mice to the same values as in old Pten cKO mice (WT: n = 11 slices, six mice: Pten cKO: n = 11 slices, six mice; P > 0.05). (D) PPR is unaltered at DGC synapses of young Pten cKO mice (WT: n = 5 slices, five mice: Pten cKO: n = 13 slices, five mice; P > 0.05. (E) PPR is decreased at DGC synapses of middle-aged Pten cKO mice (WT: n = 6 slices, four mice; Pten cKO: n = 7 slices, four mice. *P < 0.05, **P < 0.01). (F) PPR is unaltered at DGC synapses of old Pten cKO mice (WT: n = 5 slices, five mice; Pten cKO: n = 5 slices, five mice; P > 0.05).

To examine presynaptic function, we monitored the paired-pulse ratio (PPR) at DGC synapses of Pten cKO mice as a function of age. PPR is inversely related to release probability (26). At these synapses, targeted deletion of Pten likely occurs exclusively in postsynaptic DGCs, because levels of Pten appear normal in afferents from the medial perforant pathway (15). PPR was unaltered in young and old Pten cKO mice but decreased in middle-aged Pten cKO mice relative to that of age-matched WT mice (Fig. 2 D–F). The decrease in PPR in middle-aged Pten cKO mice is associated with enhanced release probability and is consistent with enhanced input/output relations observed in Pten cKO mice at these same ages.

Dysregulation of TBS-LTP at DGC Synapses of Pten cKO Mice Is Age Dependent.

Long-lasting forms of synaptic plasticity such as LTP are thought to be neural substrates of learning and memory in WT mice (20). TBS-LTP mimics firing patterns seen in vivo and induces a PI3K-dependent form of LTP (27). To assess TBS-LTP in Pten cKO mice, we stimulated afferents in the medial perforant pathway and monitored fEPSPs at synapses onto DGCs. TBS-LTP was elevated at DGC synapses of young Pten cKO mice, coincident with the onset of behavioral deficits but before the onset of obvious morphological abnormalities (Fig. 3 A and D). In middle-aged Pten cKO mice (Fig. 3 B and D), which exhibit early stages of DGC hypertrophy, and older Pten cKO mice (Fig. 3 C and D), which exhibit more advanced hypertrophy of DGCs and compression of CA1, the magnitude of TBS-LTP was markedly impaired. In contrast, TBS-LTP was unaltered at DGC synapses of WT mice at all ages. Our observation that TBS-LTP is elevated in young Pten cKO mice coincides with the onset of behavioral deficits but precedes the onset of obvious morphological abnormalities (15). Thus, TBS-LTP is impaired at DGC synapses of middle-aged and older Pten cKO mice, despite elevated PI3K signaling (15, 16). A plausible explanation is the marked hypertrophy of DGCs, a morphological change likely to override enhanced PI3K-mTOR signaling and to impair synaptic plasticity.

Fig. 3.

Fig. 3.

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TBS-LTP and mGluR-LTD are dysregulated at DGC synapses of Pten cKO vs. WT mice. (A) TBS-LTP is enhanced at DGC synapses of young Pten cKO mice (WT: to 124.54 ± 3% of baseline; Pten cKO: to 172 ± 4% of baseline; WT: n = 6 slices, four animals; Pten cKO: n = 6 slices, four animals). (B) TBS-LTP is impaired in middle-aged Pten cKO mice (WT: to 140 ± 2% of baseline; Pten cKO: to 110 ± 2% of baseline; WT: n = 6 slices, four animals; Pten cKO n = 6 slices, four animals). (C) TBS-LTP is impaired in old Pten cKO mice (WT: to 140 ± 2% of baseline; Pten cKO: to 111 ± 2% of baseline; WT: n = 6 slices, four animals; Pten cKO: n = 6 slices, four animals). (D) TBS-LTP assessed at 60 min after induction. **P < 0.01. (E–G) mGluR-LTD is impaired at DGC synapses of Pten cKO mice at all ages examined (young: WT: to 85 ± 1% of baseline; Pten cKO: to 96 ± 1% of baseline; n = 6 slices, four mice; middle-aged: WT: to 85 ± 2% of baseline, Pten cKO to 95 ± 1% of baseline; n = 6 slices, four mice; old: WT: to 82 ± 1% of baseline, Pten cKO: to 99 ± 2% of baseline; n = 6 slices, four mice). (H) mGluR-LTD at DGC synapses assessed 60 min after induction. **P < 0.01. In this and all subsequent figures, we pooled data from two sets of controls. In independent experiments, we compared synaptic plasticity in two sets of mice, Nse-Cre Pten+/+ littermates (Pten not floxed) on a C57BL/6 background vs. WT C57BL/6 nonlittermates. The two sets of mice did not exhibit detectable differences in the magnitude of LTP or LTD recorded at DGC synapses (Fig. S1) or CA1 synapses (Fig. S2) at any age examined. We therefore pooled data from the two groups (hereafter denoted as WT mice). (Scale bars: 0.5 mV and 10 ms.)

Deletion of Pten at DGC Synapses Impairs mGluR-LTD at all Ages Examined.

We next examined mGluR-LTD at DGC synapses of Pten cKO mice. mGluR-LTD is an NMDA receptor (NMDAR)-independent, protein synthesis-dependent (28) form of homosynaptic LTD and can be elicited by application of the group mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) at CA1 (29, 30) and DGC synapses (31). mGluR-LTD was significantly impaired in young, middle-aged, and old Pten cKO relative to that of age-matched WT mice (Fig. 3 E–H). Moreover, WT mice did not exhibit an age-dependent change in mGluR-LTD at any age examined. The impaired mGluR-LTD observed in Pten cKO mice suggests a requirement for Pten in mGluR-LTD at DGC synapses.

Basal Synaptic Transmission and Presynaptic Function Are Normal at CA1 Synapses.

To assess basal synaptic transmission at CA3–CA1 synapses of Pten cKO mice, we stimulated Schaffer collateral afferents and monitored the input/output relation of fEPSPs at synapses on CA1 pyramidal neurons. In Pten cKO mice, most CA3 neurons express Cre and are devoid of Pten, whereas postsynaptic CA1 neurons express Pten throughout their lifetime and do not exhibit visible morphological alterations through middle age. (In old Pten cKO mice, compression of CA1 made recordings unstable; therefore we did not perform electrophysiological analysis at CA1 synapses of old Pten cKO mice). The input/output relation (Fig. 4 A and B) and PPR (Fig. 4 C and D) were indistinguishable for young (8–12 wk) and middle-aged (16–20 wk) Pten cKO vs. age matched WT mice. Thus, loss of Pten in DGCs and CA3 pyramidal cells did not alter basal synaptic transmission or PPRs at CA1 synapses.

Fig. 4.

Fig. 4.

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Basal synaptic transmission and paired pulse ratios are not detectably altered at CA1 synapses of Pten cKO vs. WT mice both young and middle-aged. (A and B) Basal synaptic transmission, as assessed by input/output relations, is unaltered at CA1 synapses of Pten cKO mice (WT-young: n = 14 slices, seven mice; Pten-young: n = 14 slices, seven mice; WT-middle-aged; n = 12 slices, six mice; Pten-middle-aged: n = 12 slices, six mice). P > 0.05 at each age. (C) Representative paired-pulse EPSPs at indicated interstimulus intervals in WT (Upper) and Pten cKO (Lower) mice. (D) Mean PPRs at CA1 synapses of Pten cKO vs. WT mice are unaltered at young and middle ages (WT-young: n = 5 slices, five mice; Pten cKO-young, n = 7 slices, five mice; WT-middle-aged; n = 5 slices, five mice; Pten cKO-middle-aged; n = 5 slices, five mice). P > 0.05 at each interval.

Presynaptic Deletion of Pten at CA3–CA1 Synapses Impairs TBS-LTP in Middle-Aged but Not Young Pten cKO Mice.

TBS-LTP was not significantly different in young Pten cKO vs. WT (Fig. 5 A and C). In middle-aged Pten cKO mice, TBS-LTP was significantly impaired (Fig. 5 B and C). We also examined LTP induced by high-frequency stimulation (HFS-LTP) at CA3–CA1 synapses. In young Pten cKO mice, HFS-LTP was unchanged relative to WT (Fig. 5 D and F), but the early posttetanic potentiation (PTP) was enhanced (Fig. S3A). These data suggest that loss of Pten in CA3 could augment the short-term potentiation induced by HFS, but not change LTP at CA3–CA1 synapses. In middle-aged Pten cKO mice, the early potentiation was smaller than in WT mice (Fig. S3B), and HFS-LTP was significantly impaired (Fig. 5 E and F). The defect observed in TBS-LTP and HFS-LTP at CA1 synapses of middle-aged, but not young Pten cKO mice may be due in part to compression of CA1 pyramidal neurons caused by DG and neocortical hypertrophy.

Fig. 5.

Fig. 5.

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TBS-LTP and HFS-LTP, but not mGluR-LTD, are dysregulated at CA1 synapses of middle-aged Pten cKO vs. WT mice. (A) TBS-LTP at CA1 synapses is normal in young Pten cKO mice (WT: to 151.65 ± 2% of baseline; Pten cKO: to 151.12 ± 2% of baseline; n = 4 slices, four mice for each). (B) TBS-LTP at CA1 synapses is impaired in middle aged Pten cKO mice vs. WT mice (WT: to 162.78 ± 3% of baseline; Pten cKO: to 113.12 ± 2% of baseline; n = 4 slices, four mice for each). (C) TBS-LTP assessed at 60 min after induction **P < 0.01. (D) HFS-LTP at CA1 synapses is normal in young Pten cKO mice (WT: to 141 ± 2% of baseline; Pten cKO: to 135 ± 8% of baseline; n = 4 slices, four mice for each). (E) HFS-LTP at CA1 synapses is impaired in middle-aged Pten cKO mice vs. WT mice (WT: to 139 ± 3% of baseline; Pten cKO: to 103 ± 2% of baseline; n = 4 slices, four mice for each). (F) HFS-LTP assessed at 60 min after induction, **P < 0.01. (G) mGluR-LTD at CA1 synapses of young Pten cKO mice is unaltered vs. WT mice. (WT: to 84.5 ± 2% of baseline, n = 5 slices, five mice; Pten cKO: to 79.8 ± 2% of baseline, n = 6 slices, four mice for each). (H) mGluR-LTD at CA1 synapses of middle-aged Pten cKO mice is unaltered vs. WT mice (WT: to 86.6 ± 2% of baseline, Pten cKO to 90.9 ± 1% of baseline, n = 6 slices, four mice). (I) mGluR-LTD assessed 60 min after induction, P > 0.05. (Scale bars: 0.5 mV and 10 ms.)

Presynaptic Deletion of Pten at CA1 Synapses Does Not Alter mGluR-LTD.

We next examined mGluR-LTD at CA3–CA1 pyramidal cell synapses of young and middle-aged Pten cKO mice. At CA1 synapses, mGluR-LTD is protein synthesis- (32) and mTOR-dependent (29, 33) by 3 wk of age. Application of the group I mGluR agonist DHPG induced robust mGluR LTD at CA1 synapses of young and middle-aged Pten cKO mice, comparable to that observed in age-matched WT mice (Fig. 5 G–I). Moreover, mGluR-LTD did not differ significantly with age in either genotype. These results suggest that mGluR-LTD at CA3–CA1 synapses does not require Pten in the presynaptic cells.

Discussion

Pten is a lipid and protein phosphatase best known for its role in suppressing tumor formation by inhibiting cellular survival, proliferation, and cellular architecture (8, 9). At hippocampal synapses, Pten is present pre- (34) and postsynaptically (35), where it is strategically positioned to regulate synaptic plasticity. Humans with inactivating mutations in the PTEN gene exhibit social and cognitive deficits (10, 11) and anatomical abnormalities. A hallmark feature is progressive macrocephaly of the dentate gyrus, which ultimately induces compression of the CA1 pyramidal cell layer. Nse-Cre Pten cKO mice replicate these behavioral and anatomical deficits (15, 16). However, the impact of Pten deletion on activity-dependent synaptic plasticity in this model has been unclear. The present study demonstrates that two PI3K- and protein synthesis-dependent forms of synaptic plasticity, TBS-LTP and mGluR-LTD, are dysregulated at perforant path-to-DGC synapses of Pten cKO mice before the appearance of visible morphological defects. Whereas TBS-LTP is enhanced, mGluR-LTD is impaired. In contrast, synaptic plasticity is unaltered at CA1 synapses at a corresponding age. Moreover, presynaptic function, assessed by PPR, is largely unaltered at all synapses and all ages examined. These findings, which are summarized in Fig. 6, reveal a previously unappreciated role for loss of Pten in the dysregulation of synaptic plasticity, a mechanism relevant to hippocampal-based cognitive and social interaction defects associated with Pten and potentially other ASDs.

Fig. 6.

Fig. 6.

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Summary of synaptic plasticity deficits in Pten cKO mice.

Synaptic Plasticity Deficits at DGC Synapses Precede Morphological Abnormalities.

Although the prevailing view is that gross morphological abnormalities are causally related to the social and cognitive deficits observed in humans with inactivating PTEN mutations, an alternative possibility is that synaptic defects precede morphological abnormalities and potentially contribute to the behavioral deficits associated with humans with inactivating PTEN mutations. An important finding of the present paper is that dysregulation of synaptic plasticity at hippocampal synapses precedes the onset of visible morphological changes in a clinically relevant model of Pten inactivation. This observation argues against the notion that gross morphological alterations are causally related to synaptic defects observed in humans with inactivating PTEN mutations. Moreover, our observation that marked alterations in synaptic function coincide with and/or precede the onset of behavioral deficits is consistent with the concept of “synaptic failure,” postulated to be a causal factor in the dementia associated with Alzheimer’s disease (36). Synaptic plasticity deficits have been reported at CA1, but not DGC, synapses of other mouse models of Pten inactivation, including calcium/calmodulin dependent protein kinase II α (CaMKIIα)-Cre Pten cKO mice (37), GFAP-Cre Pten cKO mice (38), and Pten+/− mice (39, 40). Whereas mice used in the present study exhibit ablation of Pten primarily in DGCs and CA3, but not CA1, pyramidal neurons, in the hippocampus, CaMKIIα-Cre Pten and Pten+/− cKO mice exhibit reduced expression of Pten primarily in CA1 pyramidal neurons. Whereas CaMKIIα-Cre Pten cKO exhibit cognitive, but not morphological, abnormalities before their death at ∼11 wk (37), GFAP-Cre Pten cKO and Pten+/− mice exhibit morphological abnormalities, but not cognitive deficits (38–40). Thus, our findings extend the work of others in that we document deficits in LTP and LTD at DGC synapses in a clinically relevant model of Pten inactivation.

Synaptic Plasticity Is Dysregulated Differentially at DGC vs. CA1 Synapses of Nse-Cre Pten cKO Mice.

Another important finding of the present paper is that synaptic plasticity is dysregulated at perforant path-to-DGC synapses but is unaltered at CA3 to CA1 pyramidal cell synapses of young Nse-Cre Pten cKO mice. Our observation that these postsynaptic forms of LTP and LTD are impaired at perforant path to DGC, but not CA3 to CA1 synapses of young Pten cKO mice is consistent with the cell-specific pattern of Pten deletion. Whereas Pten is ablated in CA3 neurons, it is expressed at normal levels in CA1 neurons (15, 16). Moreover, deficits in synaptic plasticity at DGC synapses precede those at CA1 synapses. Thus, synaptic plasticity deficits in Nse-Cre mice are synapse- and age-specific. Our observation that TBS-LTP is enhanced at DGC synapses of young Pten cKO mice is consistent with the concept that loss of Pten releases a brake imposed on PI3K signaling, enabling PI3K-dependent forms of synaptic plasticity to go unchecked. Our observation that mGluR-LTD is impaired at DGC synapses of young Pten cKO mice is consistent with findings that protein phosphatases are critical for expression of NMDAR-dependent LTD (40, 41) and extends those studies in that we demonstrate that Pten is also essential to mGluR-LTD expression. Insight into the mechanisms underlying a role for Pten in LTD comes from findings by Esteban and coworkers who show that activation of NMDARs triggers association of Pten with the synaptic scaffolding protein PSD-95, which, in turn, recruits Pten to the postsynaptic density and anchors it at spines in an activity-dependent manner (35). Moreover, expression of dominant-negative forms of Pten in organotypic hippocampal slices abolishes LFS- and mGluR-LTD (35). These findings reveal a previously unappreciated role for Pten in activity-dependent synaptic signaling.

Synaptic Plasticity Deficits Are an Early Hallmark of the ASD Phenotype.

Here, we document that synaptic plasticity deficits occur at DGC synapses in a clinically relevant mouse model of Pten inactivation. We further show that synaptic plasticity defects occur before the onset of obvious morphological abnormalities. Moreover, synaptic plasticity defects occur concurrently or before the appearance of behavioral deficits (15), consistent with a possible causal role for synaptic defects in the social and cognitive deficits associated with humans with inactivating PTEN mutations. Accordingly, defects in neuronal excitability and excitatory synaptic transmission occur in other syndromic ASDs that exhibit social and cognitive deficits in the absence of gross macrocephaly, such as Fragile X syndrome (29, 42), tuberous sclerosis complex (TSC1/2) (43, 44), Rett syndrome (45), and Angelman syndrome (46). Although these syndromes arise because of mutations in diverse genes, studies of synaptic defects in syndromic ASDs have uncovered unifying themes and insight into ASD synaptic phenotypes. One such theme is dysregulation of PI3K-mTOR signaling (11, 17). Findings of the present study strengthen this notion in that they link dysregulation of PI3K signaling to aberrant synaptic plasticity at hippocampal synapses in a mouse model of PTEN inactivation. It is hoped that these findings will accelerate the development of novel therapeutic strategies for the amelioration of symptoms associated not only with Pten, but also related ASDs.

Materials and Methods

Details are in SI Materials and Methods.

Drugs.

Agents used were obtained from Tocris Cookson and diluted from stock solutions immediately before use.

Animals.

All procedures involving animals were carried out in accordance with the guidelines of the National Institute of Health for the care and use of laboratory animals and were approved by the Animal Institutes of the Albert Einstein College of Medicine and University of Texas Southwestern Medical Center.

Histology and immunohistochemistry.

Brain sections were prepared by using techniques described (15). Fifty-micrometer vibratome floating sections were prepared for X-Gal staining, and 5-μm paraffin sections were prepared for Pten immunostaining and hematoxylin and eosin (H&E) staining.

Electrophysiology and Data Analysis.

Acute hippocampal slices (400 μm) were prepared from age-matched WT and mutant mice by using conventional techniques (30). fEPSPs were evoked by square pulses (10–100 μA, 100 μs) with a concentric bipolar tungsten stimulating electrode.

Statistics.

Data are expressed as mean ± SE. Student’s t tests and ANOVA were used as appropriate. Significance was taken as P < 0.05.

Supplementary Material

Supporting Information
supp_110_12_4738__index.html (7.2KB, html)

Acknowledgments

We thank Drs. Pablo Castillo and Andres Chavez for reviewing the figures, Dr. Naoki Kaneko for reviewing the manuscript, and all the members of the R.S.Z. laboratory for their help and support. This work was supported by National Institutes of Health Grant MH092877 (to R.S.Z.), a Simons Foundation Explorer award (to R.S.Z.), Simons Foundation Grant SFARI 137078 (to L.F.P.), National Institutes of Health Grant 1P50NS052606 (to L.F.P.), and the generous support of the F. M. Kirby Foundation. L.F.P. is an American Cancer Society Professor, M.V.L.B. is the Sylvia and Robert S. Olnick Professor of Neuroscience, and R.S.Z. is the F. M. Kirby Professor in Neural Repair and Protection.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1222803110/-/DCSupplemental.

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