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. 2008 Sep 24;28(39):9857-69.
doi: 10.1523/JNEUROSCI.3145-08.2008.

The Neurotrophin-Inducible Gene Vgf Regulates Hippocampal Function and Behavior Through a Brain-Derived Neurotrophic Factor-Dependent Mechanism

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The Neurotrophin-Inducible Gene Vgf Regulates Hippocampal Function and Behavior Through a Brain-Derived Neurotrophic Factor-Dependent Mechanism

Ozlem Bozdagi et al. J Neurosci. .
Free PMC article

Abstract

VGF is a neurotrophin-inducible, activity-regulated gene product that is expressed in CNS and PNS neurons, in which it is processed into peptides and secreted. VGF synthesis is stimulated by BDNF, a critical regulator of hippocampal development and function, and two VGF C-terminal peptides increase synaptic activity in cultured hippocampal neurons. To assess VGF function in the hippocampus, we tested heterozygous and homozygous VGF knock-out mice in two different learning tasks, assessed long-term potentiation (LTP) and depression (LTD) in hippocampal slices from VGF mutant mice, and investigated how VGF C-terminal peptides modulate synaptic plasticity. Treatment of rat hippocampal slices with the VGF-derived peptide TLQP62 resulted in transient potentiation through a mechanism that was selectively blocked by the BDNF scavenger TrkB-Fc, the Trk tyrosine kinase inhibitor K252a (100 nm), and tPA STOP, an inhibitor of tissue plasminogen activator (tPA), an enzyme involved in pro-BDNF cleavage to BDNF, but was not blocked by the NMDA receptor antagonist APV, anti-p75(NTR) function-blocking antiserum, or previous tetanic stimulation. Although LTP was normal in slices from VGF knock-out mice, LTD could not be induced, and VGF mutant mice were impaired in hippocampal-dependent spatial learning and contextual fear conditioning tasks. Our studies indicate that the VGF C-terminal peptide TLQP62 modulates hippocampal synaptic transmission through a BDNF-dependent mechanism and that VGF deficiency in mice impacts synaptic plasticity and memory in addition to depressive behavior.

Figures

Figure 1.
Figure 1.
Homozygous and heterozygous VGF knock-out mice have impaired contextual fear conditioning compared with wild-type mice. Homozygous Vgf/Vgf (n = 5), heterozygous Vgf+/Vgf (n = 8), and wild-type Vgf+/Vgf+ (n = 7) male mice were trained and tested for contextual fear conditioning as described in Materials and Methods. Data are expressed as the mean ± SEM percentage of time spent freezing during the 3 min period of testing, 24 h after training (***p < 0.001).
Figure 2.
Figure 2.
VGF homozygous mutant mice have impaired performance in the Morris water maze. Mice were tested in the Morris water maze as described in Materials and Methods. Performance in the four daily cued and uncued trials was evaluated by video camera, and parameters were averaged for each day (days 1–4). In A and B, sample computer-generated tracings of swim paths are shown for wild-type (A) and VGF knock-out (B) mice in uncued (left) and cued (right) trials. C, D, Distance traveled (centimeters) (C, cued; D, uncued) was quantified for each genotype on each day (ANOVA, mean ± SEM; n = 5 mice of each genotype per group; *p ≤ 0.05). On day 4, the target platform was removed, and a 60 s probe trial was performed. E, Search paths were tracked, and time spent in the target region of the maze (a circular region of 20 cm diameter centered on the location of the target platform) and the nontarget region (a circular region of 20 cm diameter placed at the center of the maze) was quantified. F, Probe trial performance was measured by calculating a spatial learning index: (timeover target − timeover nontarget)/(timeover target + timeover nontarget). ANOVA demonstrated a significant effect of genotype on learning index (F(2,12) = 5.615; p = 0.019), which was confirmed with a nonparametric test (Kruskal–Wallis rank sum, p = 0.049). Bonferroni's correct pairwise comparisons revealed a significant difference between knock-out and wild-type learning indices (p = 0.025), but differences between wild-type and heterozygote (p = 1.000) and knock-out and heterozygote (p = 0.076) indices were not significant.
Figure 3.
Figure 3.
LTP in VGF mutant mice is indistinguishable from wild-type mice, but LTD is impaired in homozygous VGF knock-out mice. In A, to determine whether VGF modulates the generation of LTP at Schaffer collateral–CA1 hippocampal synapses, LTP was induced by tetanic stimulation (4 × 1 s trains, 100 Hz, separated by 5 min) to the Schaffer collaterals in hippocampal slices taken from homozygous Vgf/Vgf knock-out mice (filled circles), heterozygous Vgf/Vgf+ mice (open triangles), and wild-type mice (open circles) (n = 4 mice per group, 2–3 slices per animal), and field EPSP slope in CA1 was determined during the 120 min recording period after tetanus. The inset shows representative EPSP traces that were obtained for each genotype, recorded before (1) and 120 min after (2) tetanic stimulation (calibration: 10 ms, 0.5 mV). In B, LTP induced by theta-burst stimulation (10 bursts of four pulses at 100 Hz separated by 200 ms) was found to be comparable in hippocampal slices from wild-type, heterozygous, and homozygous VGF knock-out mice [inset, representative traces for each genotype, recorded before (1) and 90 min after (2) theta-burst stimulation]. In C, no significant differences among the three genotypes were detected when early-phase LTP was stimulated in hippocampal slices by a single 100 Hz pulse of 1 s duration [inset, representative traces for each genotype, recorded before (1) and 90 min after (2) stimulation]. In contrast, in D, LTD induced by low-frequency stimulation was significantly reduced in hippocampal slices from homozygous VGF knock-out mice compared with wild-type or heterozygous VGF mutant mice (n = 2–4 mice per group, 2–3 slices per animal; p < 0.01) [inset, representative traces for each genotype, recorded before (1) and 60 min after (2) LFS stimulation]. All field EPSP measurements shown are mean ± SD (calibration: 10 ms, 0.5 mV). KO, Knock-out; WT, wild type; Het, heterozygous.
Figure 4.
Figure 4.
VGF-derived C-terminal peptide TLQP62 potentiates CA1 field EPSPs. In A, hippocampal slices were treated with varying doses of TLQP62 (0.1–10 μm), and fEPSP slope in the CA1 region was determined. The bar indicates the duration of TLQP62 treatment (n = 6 rats for each TLQP62 concentration tested). The inset in A shows representative EPSP traces before (1), 30 min after (2), or 150 min after (3) initial exposure to 10 μm TLQP62. In B, input–output curves plotting stimulus intensity (milliamperes) against field EPSP slope (millivolts per millisecond) are shown in control slices (filled circles), slices treated with 10 μm TLQP62 (open triangles), and 60 min after TLQP62 washout (open circles) (n = 6 rats). In C, VGF-derived peptides AQEE30 (1–100 μm) and LEGS25amide (100 μm; open circles) and a scrambled control TLQP62 peptide (10 μm; filled squares) were applied to hippocampal slices, and field EPSP slope in the CA1 region was determined (n = 4 rats). In D, to investigate whether the truncated peptide TLQP21 blocked TLQP62-mediated potentiation, hippocampal slices were pretreated with TLQP21 (10 μm; filled triangles) before administration of TLQP62 or were treated with TLQP21 after TLQP62-mediated potentiation had been initiated (10 μm; filled circles) (n = 3 rats). In E, TLQP62 (10 μm) was applied to hippocampal slices from wild type (WT), heterozygous Vgf+/Vgf (Het), and homozygous Vgf/Vgf (KO), and field EPSP slope in the CA1 region was determined. The means of the fEPSP slope at 60 min of TLQP62 treatment were as follows: 170 ± 35% (WT), 160 ± 27% (Het), and 167 ± 27% (KO) (n = 2–4 mice per genotype, 3–4 slices per animal; p > 0.05, ANOVA). All field EPSP measurements shown are mean ± SD.
Figure 5.
Figure 5.
VGF-derived peptide TLQP62 enhances excitatory transmission in the CA1 region via a mechanism that is BDNF dependent and is blocked by inhibitors of Trk tyrosine kinase activity or tissue plasminogen activator (tPA), but not by function-blocking anti-p75NTR antiserum. In A, hippocampal slices were incubated for 2 h in the BDNF scavenger TrkB–Fc (5 μg/ml; diamonds), the NT-3 scavenger TrkC–Fc (5 μg/ml; triangles), the NGF-scavenger TrkA–Fc (5 μg/ml; squares), or Ringer's solution (circles). After the 2 h incubation, slices were transferred to the recording chamber and were perfused with 10 μm TLQP62 for 1 h. In control experiments, treatment with Fc alone (5 μg/ml) had no effect on TLQP62-induced potentiation (supplemental Fig. 2A, available at www.jneurosci.org as supplemental material). The means of the fEPSP slope at 60 min of TLQP62 treatment were 260 ± 11% (TLQP62 alone), 264 ± 11% (TLQP62 + TrkC–Fc), 211 ± 9% (TLQP62 + TrkA–Fc), and 106 ± 8% (TLQP62 + TrkB–Fc) (n = 4; TLQP62 vs TLQP62 + TrkB–Fc, p < 0.01; TLQP62 vs TLQP62 + TrkA–Fc and TLQP62 vs TLQP62 + TrkC–Fc, p > 0.05; ANOVA). In B, treatment with the Trk tyrosine kinase inhibitor K252a (100 nm, filled triangles; 200 nm, filled diamonds) blocked TLQP62-induced potentiation, whereas treatment with its analog K252b had significantly less effect (100 nm, open triangles; 200 nm, open diamonds). The means of the fEPSP slope at 60 min of TLQP62 treatment were 260 ± 11% (TLQP62 alone), 224 ± 16% (TLQP62 + 100 nm K252b), 194 ± 12% (TLQP62 + 200 nm K252b), and 101 ± 2% (TLQP62 + 100 nm K252a) (n = 4; TLQP62 vs TLQP62 + 100 nm K252a, p < 0.01; TLQP62 vs TLQP62 + 100 nm K252b, p > 0.05; TLQP62 vs TLQP62 + 200 nm K252b, p = 0.02; t test). In C, preincubation with function blocking anti-p75NTR antiserum did not decrease the magnitude of TLQP62 potentiation but slowed the decline in potentiation after peptide treatment was stopped, whereas normal rabbit serum had no effect (supplemental Fig. 4B, available at www.jneurosci.org as supplemental material). The means of the fEPSP slope at 60 and 120 min of TLQP62 treatment were, respectively, 260 ± 11 and 101 ± 10% (TLQP62 alone) and 264 ± 10 and 181 ± 13% (TLQP62 + anti-p75NTR) (n = 4; p < 0.05 at 120 min, t test). In D, inhibition of tPA activity using tPA STOP (open circles) blocked TLQP62-induced potentiation. The means of the fEPSP slope at 60 min of TLQP62 treatment were 260 ± 11% (TLQP62 alone) and 99.5 ± 12% (TLQP62 + tPA STOP) (n = 4; p < 0.01, t test). All field EPSP measurements shown are mean ± SD (n = 4 rats per group, 1 slice per rat).
Figure 6.
Figure 6.
TLQP62-induced potentiation does not involve activation of NMDA-type glutamate receptors, and neither occludes nor is occluded by tetanic stimulation. In A, the effect of previous saturating LTP induced by tetanic stimulation (arrows) on subsequent TLQP62-induced facilitation of hippocampal synaptic transmission was measured. Application of TLQP62 (10 μm) was found to increase fEPSP slope after the induction of stable LTP. The bars indicate duration of TLQP62 treatment. In B, after TLQP62-induced potentiation reached a plateau, stimulus intensity was reduced to match the original baseline, and tetanic stimulation (4 trains of 100 Hz; arrow) caused potentiation that was indistinguishable from potentiation in the control condition. In C, hippocampal slices were incubated for 15 min with the NMDA glutamate receptor blocker AP-5 (50 μm) and were transferred to the recording chamber and perfused with TLQP62 (10 μm) in the presence of AP-5 for 1 h. There was no effect of AP-5 on TLQP62-induced facilitation; the means of the fEPSP slope at 60 min of TLQP62 treatment were 260 ± 11% (TLQP62 alone) and 243 ± 11% (TLQP62 + AP-5) (n = 4; p > 0.05, t test). Insets, Representative EPSP traces were recorded at times shown by the numbers on the graphs. Calibration: 10 ms, 0.5 mV. All field EPSP measurements shown are mean ± SD (n = 4 rats per group, 1 slice per rat).

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