Fig 1.
1μM ZnCl2 enhances the magnitude of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in hippocampus.
(A) Time course of TBS-induced LTP of SCH-CA1 fEPSP slopes in control (dark circles, n = 15), compared to 1μM ZnCl2 (lighter circles, n = 10). Bath-applied 1μM ZnCl2 (solid bar) significantly and persistently enhanced magnitude of LTP compared to untreated control slices. LTP was significantly enhanced 50 minutes post TBS (*, P<0.05; Student’s t-test). Sample traces next to graph compare baseline (solid wave) to post TBS (dashed wave) for all conditions. (B) The theta burst stimulus (TBS) stimulating protocol used for all LTP experiments (2 trains 3 minutes apart, each train consisting of 10x 100Hz/5pulse bursts at 200ms interburst intervals).(C) Summary of all control slice LTP vs ZnCl2 treated slices, demonstrating that ZnCl2 significantly enhances SCH-CA1 LTP at 1μM (*, P<0.05; Student’s t-test). Statistical data are presented as mean ± SEM.
Fig 2.
Chelation of endogenous Zn2+ inhibits CA1-LTP.
(A) Bath application (solid bar) of the Zn2+ chelater 1mM CaEDTA (open triangles, n = 7) significantly reduced the magnitude of SCH-CA1 LTP compared to untreated control slices (dark circles, n = 7). Bath application of the Zn2+ chelator 50μM TPEN (solid bar) converted control LTP to LTD (light circles, n = 6; Comparison of three groups P<0.05; 1-way ANOVA). Sample traces next to graph compare baseline (solid wave) to post TBS (dashed wave) for all conditions. (B) Summary of mean ± SEM fEPSP slopes 50 minutes post-TBS for untreated control slices versus slices pre-treated with 1mM CaEDTA (*, P<0.05; Dunnett’s post hoc test) and slices pre-treated with 50μM TPEN, where LTP converted to LTD (*, P<0.05; Dunnett’s post hoc test).
Fig 3.
Zn2+ enhancement of LTP requires activation of postsynaptic NMDA receptors.
(A) ZnCl2 (1μM, light circles, n = 9) does not alter basal synaptic transmission after 1 hour of exposure compared to control slices (dark circles, n = 5; P>0.05, Student’s t-test). (B) Summary of baseline responses showing that ZnCl2 did not alter baseline synaptic transmission compared to untreated control slices. (C) TBS did not induce LTP in the presence of either 50μM D-AP5 (filled circles, n = 6), or 1μM ZnCl2 + 50μM D-AP5 (light circles, n = 7). (D) Summary of mean fEPSP slopes 50 minutes post TBS. There was no significant difference between D-AP5 alone and 1μM ZnCl2 LTP + D-AP5 at 50 minutes post TBS. (P>0.05, Student’s t-test) (E) Sample of control paired-pulse evoked SCH-CA1 EPSCs (dark trace, average of 6 responses) versus EPSCs in 1μM [ZnCl2] (light trace, average of 6 responses) during a paired-pulse stimulus at an interval of 50ms. (F) Paired-pulse profiles of PPF ratios at interpulse intervals from 10–500 msec in control (n = 6, dark circles) vs. 1μM [ZnCl2] (n = 6, gray circles), showing no significant differences (P>0.05, Student’s t-test). Statistical data are presented as mean ± SEM.
Fig 4.
ZnCl2 enhances NMDA receptor-dependent fEPSP and NMDA evoked currents.
(A) Effect of bath application of ZnCl2 on pharmacologically-isolated NMDAR fEPSPs. ZnCl2 (black bar) produced a slow, persistent enhancement of NMDAR fEPSPs (light circles, n = 7) compared to untreated control slices (dark circles, n = 8). D-AP5 (50μM) was bath applied after one hour (n = 2 for control and ZnCl2), which eliminated NMDAR fEPSP responses in both groups. (B) Summary of mean ± SEM NMDAR fEPSP amplitude % increase over baseline after 1 hour bath application of ZnCl2 in control vs 1μM ZnCl2 (*, P<0.05; Student’s t-test). (C) Effect of bath application of 1μM ZnCl2 (light circles, n = 5) on pharmacologically-isolated SCH NMDA evoked currents in CA1 pyramidal neurons by pressure injection of NMDA compared to untreated control neurons (dark circles, n = 6). 1μM ZnCl2 significantly and persistently increased NMDA evoked currents amplitudes in CA1 pyramidal neurons. (D) Mean ± SEM NMDA evoked currents amplitude (*; P<0.05, Student’s t-test) and total area (*; P<0.05, Student’s t-test), calculated as the % increase over baseline after 40 minutes ZnCl2 application, compared to its control baseline.
Fig 5.
Zn2+ is required for the enhancement of NMDAR fEPSPS which requires NR2B-containing NMDARs and activation of Src family kinases (SFK).
(A) Pharmacologically-isolated NMDA fEPSP amplitudes in the presence of the NR2B-selective NMDAR antagonist Ro25-6981 (1 μM) in control slices (squares, n = 6), and after co-application of 1μM ZnCl2 (light circles, n = 6). Ro25-6981 completely blocked the enhancement of NMDAR fEPSPs by 1μM ZnCl2. Sample traces for each treatment shown to right, before (dark traces) and 50 minutes after (light traces) drug application. (B) Summary of NMDAR fEPSP decrease versus baseline (100%) after 70 minutes drug application in slices treated with 1μM Ro25-6981 and those treated with Ro25-6981 plus 1μM ZnCl2 (P>0.05; Student’s t-test). (C) Time course of NMDA fEPSP amplitudes in slices pre-treated for 5 minutes with the SFK inhibitor PP2 (top bar, 10μM, dark squares, n = 8) prior to bath application of 1μM ZnCl2 (lower bar) plus PP2, versus application of the two drugs simultaneously (light circles, n = 6). (D) Summary of NMDA fEPSP amplitudes after 1 hour recording. Simultaneous treatment with 1μM ZnCl2 + PP2 significantly enhanced the magnitude of NMDA fEPSPs compared to control ACSF (*, P<0.05; 1-way ANOVA, Dunnett’s multiple comparison test), and this potentiation was completely blocked by pretreatment with 10μM PP2 (Control n = 8, vs pretreatment, P>0.05; Dunnett’s multiple comparison test). Mean NMDA fEPSPs in PP2 alone was not statistically different from control (Control vs PP2, P>0.05; Dunnett’s multiple comparison test). Statistical data are presented as mean ± SEM.
Fig 6.
NR2B blockade and Src kinase inhibition block Zn2+ enhancement of Schaffer collateral-CA1 LTP.
(A)Time course of LTP of SCH-evoked fEPSPs in 1μM ZnCl2 + Ro 25–6981 slices (circles, n = 8), compared to LTP in slices treated with 1μM Ro 25–6981 (black bar, squares, n = 7). There was no significant difference in the magnitude of LTP between groups. Sample traces for each treatment shown to right, before (dark traces) and 50 minutes after (light traces) TBS. (B) Summary of SCH-CA1 LTP 50 minutes post-TBS in slices treated with Ro25-6981 and Ro25-6981 + ZnCl2, compared to drug-free control slices (n = 7; P>0.05; 1-way ANOVA). (C) Time course of LTP elicited by TBS (arrow) in the presence of 10μM PP2-treated slices (squares, n = 6), compared to LTP in PP2 + 1μM ZnCl2 treated slices (circles, n = 5). The magnitude of LTP in the two groups were not significantly different (P>0.05; Student’s t-test). Sample traces for each treatment shown to right, before (dark traces) and 50 minutes after (light traces) drug application. (D) Summary of normalized fEPSP slope 50 minutes after TBS in slices treated with 1μM ZnCl2 + PP2 versus PP2. Statistical data are presented as mean ± SEM. (E) Western blot analysis showing the Mean ± SEM of phosphorylation at Y1472 on NMDA NR2B receptor subunits in the untreated control slices, compared to slices treated with 1μM ZnCl2 or with 1μM ZnCl2 and the PP2 inhibitor. Zn2+ treatment did not significantly alter the state of phosphorylation of the NR2B subunits at Y1472 (P>0.05; Dunnett’s multiple comparison test).