Synaptic underpinnings of altered hippocampal function in glutaminase-deficient mice during maturation

Abstract

Glutaminase-deficient mice (GLS1 hets), with reduced glutamate recycling, have a focal reduction in hippocampal activity, mainly in CA1, and manifest behavioral and neurochemical phenotypes suggestive of schizophrenia resilience. To address the basis for the hippocampal hypoactivity, we examined synaptic plastic mechanisms and glutamate receptor expression. Although baseline synaptic strength was unaffected in Schaffer collateral inputs to CA1, we found that long-term potentiation was attenuated. In wild-type (WT) mice, GLS1 gene expression was highest in the hippocampus and cortex, where it was reduced by about 50% in GLS1 hets. In other brain regions with lower WT GLS1 gene expression, there were no genotypic reductions. In adult GLS1 hets, NMDA receptor NR1 subunit gene expression was reduced, but not AMPA receptor GluR1 subunit gene expression. In contrast, juvenile GLS1 hets showed no reductions in NR1 gene expression. In concert with this, adult GLS1 hets showed a deficit in hippocampal-dependent contextual fear conditioning, whereas juvenile GLS1 hets did not. These alterations in glutamatergic synaptic function may partly explain the hippocampal hypoactivity seen in the GLS1 hets. The maturity-onset reduction in NR1 gene expression and in contextual learning supports the premise that glutaminase inhibition in adulthood should prove therapeutic in schizophrenia.

Fig 1. Evoked synaptic responses in hippocampus slices

(A) Schaeffer collateral input to CA1 pyramidal neurons recorded with whole cell voltage clamp. AMPA and NMDA responses were measured at +40 mV from 5–105 ms post-stimulus as charge transfer (integrated EPSC). (A1) Sample control traces (gray) and after D-APV 100 μM application (black) are shown from WT (left) and GLS1 het (right) cells. Traces shown are the averages of 10 responses. (A2) The AMPA receptor response (left) was measured from traces after D-APV application. The NMDA receptor response (middle) was calculated by subtracting the response after D-APV from control. The ratio of AMPA to NMDA charge transfer is shown on the right. There were no significant genotypic differences. (B) LTP of Schaeffer collateral-CA1 fEPSP. fEPSP slope data are shown as per cent of baseline (determined as the average of the preceding 15 min). Each point indicates a 5 min average. The dashed line indicates the control baseline (100%). The tetanus was delivered at the arrow. * p < 0.05 between genotypes. There was a significant genotypic reduction in LTP in GLS1 hets.

Fig 2. GLS1 expression

(A) WT GLS1 expression is reported as fold change relative to adult HIPP levels. Highest expression levels were observed in hippocampus (Hipp) and frontal cortex (FC). Differences between adults and juveniles were observed in FC. (B) GLS1 expression in GLS1 het samples is reported as fold change relative to matched WT samples. GLS1 expression was down regulated in the Hipp and FC of both juveniles and adults by 40–60%. n’s shown are for GLS1 hets; WT n’s are in panel A. Data represent fold change (2^−ΔΔCt) ± s.e.m. compared to control, following GAPDH normalization. * p < 0.05 between age groups. ^ p < 0.05 relative to adult HIPP. # p < 0.05 relative to juvenile HIPP. ◇ t-test, p < 0.05 relative to WT values for the same region.

Fig 3. NR1 and GluR1 gene expression

(A) NR1 expression. (A1) WT NR1 expression is reported as fold change relative to adult Hipp. No differences were found between Hipp and FC in adults or juveniles. (A2) NR1 expression in GLS1 het samples are reported as fold change relative to matched WT samples. NR1 expression was down regulated in Hipp (at trend level) and FC of adults, but not juveniles. (B) GluR1 expression. (B1) WT GluR1 expression is reported as fold change relative to adult Hipp levels. GluR1 expression was significantly lower in the FC than in the Hipp of both adult and juvenile samples. (B2), GluR1 expression levels in GLS1 hets are reported as fold change relative to matched WT mice. No genotypic differences were found. See Fig 2 legend for definition of significance symbols.

Fig 4. NR1 protein levels

NR1 protein was reduced in the Hipp of adult GLS1 het mice. Representative examples of NR1 (~120 kDa) and GAPDH at (~38 kDa) bands for total protein samples (H), synaptosomal membrane fractions (LP1) and cytosolic fractions (S3) were resolved by SDS-PAGE (shown above). The histograms depict relative mean levels of NR1 in WT and GLS1 het mice. Data were combined from two independent experiments. Significant genotypic differences were found in LP1. ◇ t-test, p < 0.05.

Fig 5. Fear conditioning in adult and juvenile mice

(A) Fear conditioning in adult WT and GLS1 het mice. (A1) On Day 1 (Training), all animals showed increasing levels of freezing during tone presentation, indicating the acquisition of a fear response. (A2), On Day 2 (Tone Test), all animals showed enhanced freezing during the tone period compared to the pre-tone period. (A3) On Day 3 (Context Test), GLS1 hets showed significantly reduced contextual learning. (B) Fear conditioning in juvenile WT and GLS1 het mice. (B1) On Day 1, animals showed increasing levels of freezing during tone presentation, indicating the acquisition of a fear response. (B2) On Day 2, both genotypes showed increased freezing when presented with the tone in a different context. (B3) On day 3, no genotypic differences in contextual learning were seen. ◇ t-test, p < 0.05.