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

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.

Fig. 1.

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.)

Fig. 2.

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).

Fig. 3.

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.)

Fig. 4.

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.

Fig. 5.

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.)

Fig. 6.

Summary of synaptic plasticity deficits in Pten cKO mice.