Figure 5
From: A critical role for VEGF and VEGFR2 in NMDA receptor synaptic function and fear-related behavior
Hippocampal LTP as well as contextual and cued fear memory are impaired in VEGFR2 conditional knockout mice and upon VEGFR2 inhibition. The three genotypes include control mice or VEGFR2lox/lox, heterozygotes or VEGFR2lox/− and conditional VEGFR2 knockout mice or Nes-cre VEGFR2lox/−. (a) VEGF levels, assessed by Elisa, increased upon chemical LTP induction (tetraethylammonium chloride-artificial cerebrospinal fluid) compared with untreated condition (artificial cerebrospinal fluid) in both heterozygote and knockout adult hippocampal slices (pg VEGF per mg protein from 9.4±0.4 to 10.7±0.7 in VEGFR2lox/− slices, n=7, paired two-tailed t-test, *P<0.05; from 10.3±0.8 to 11.7±0.7 in Nes-cre VEGFR2lox/− slices, n=8, paired two-tailed t-test, *P<0.05). (b) Representative fEPSP traces showing potentiation for the three indicated genotypes (upper trace: averaged first 5 min of baseline, lower trace: averaged last 5 min, stimulus artifacts were removed). Theta burst stimulation (TBS) of Schaffer collaterals was used to induce LTP and fEPSP responses were recorded in stratum radiatum of CA1 region. (Nes-cre VEGFR2lox/− n=11 mice (25 slices), VEGFR2lox/− n=17 mice (36 slices), VEGFR2lox/lox n=8 mice (15 slices). (c) Shortly (5–10 min) after LTP induction with the TBS protocol the fEPSP was significantly reduced in Nestin-cre VEGFR2lox/− knockout mice compared with heterozygotes and controls (169±9% in Nes-cre VEGFR2lox/− versus 201±10% in VEGFR2lox/− and 203±14% in VEGFR2lox/lox, unpaired two-tailed t-test, *P<0.05). (d) An even more pronounced reduction in LTP was observed at 55–60 min after TBS (132±6% in Nes-cre VEGFR2lox/− versus 169±8% in VEGFR2lox/− and 174±9% in VEGFR2lox/lox, unpaired two-tailed t-test, **P<0.01, ***P<0.001). (e) Input–output curves showing fEPSP slopes at different stimulation intensities (fiber volley amplitude). The three groups of mice showed no significant differences. (f) VEGFR2 knockout slices showed normal paired-pulse facilitation at various interstimulus intervals (50, 100, 200 and 400 ms). No significant differences were obtained among the three genotypes. (g–i) VEGFR2 inhibition in hippocampal LTP. (g) Representative fEPSP traces showing potentiation for wild-type slices untreated and treated with 30 μM PTK787 (upper trace: averaged first 5 min of baseline, lower trace: averaged last 5 min, stimulus artifacts were removed). (h) LTP was induced by TBS stimulation of Schaffer collaterals and fEPSP responses were recorded in stratum radiatum of CA1 region. Hippocampal slices were perfused with 30 μM of the VEGFR2 inhibitor PTK787 (PTK) from 10 min before until 10 min after LTP induction (control n=7 mice (11 slices), PTK n=4 mice (7 slices)). (i) LTP magnitude was significantly reduced 55–60 min after TBS in slices treated with PTK (161±10% in control slices vs 130±7% in PTK treated slices, unpaired two-tailed t-test, *P<0.05). (j–l) Fear-conditioning experiment. Animals were placed in a context and presented with an electrical shock (arrow) paired once with an auditory cue (Tone); data were analyzed using a two-way ANOVA (genotype, time) with n=12 per genotype. (j) Cumulative freezing time counted during the 3-min conditioning period. There were no significant differences between genotype (F(2,33)=1.27; P not significant) and all genotypes showed a progressive increase in freezing after the Tone-shock pairing. (k) Cumulative freezing time plotted over time during the 3 min of the context recognition test. There was a significant effect of genotype (F(2,33)=3.30; *P<0.05) and Nes-cre VEGFR2lox/− knockout mice showed reduced freezing time compared with heterozygotes and controls (*P<0.05, Bonferroni post hoc test). (l) Cumulative freezing time during cued testing. There was a significant effect of genotype (F(2,33)=9,59; ***P<0.001) and only controls and heterozygotes showed a major increase in freezing time during the tone presentation as compared with prior to the tone. Nes-cre VEGFR2lox/− knockout mice exhibited strongly reduced level of freezing compared with heterozygotes and controls (***P<0.001, Bonferroni post hoc test). ACSF, artificial cerebrospinal fluid; TEA, tetraethylammonium chloride.
