Calcium-Dependent Protein Kinase C Is Not Required for Post-Tetanic Potentiation at the Hippocampal CA3 to CA1 Synapse

Abstract

Post-tetanic potentiation (PTP) is a widespread form of short-term synaptic plasticity in which a period of elevated presynaptic activation leads to synaptic enhancement that lasts tens of seconds to minutes. A leading hypothesis for the mechanism of PTP is that tetanic stimulation elevates presynaptic calcium that in turn activates calcium-dependent protein kinase C (PKC) isoforms to phosphorylate targets and enhance neurotransmitter release. Previous pharmacological studies have implicated this mechanism in PTP at hippocampal synapses, but the results are controversial. Here we combine genetic and pharmacological approaches to determine the role of classic PKC isoforms in PTP. We find that PTP is unchanged in PKC triple knock-out (TKO) mice in which all calcium-dependent PKC isoforms have been eliminated (PKCα, PKCβ, and PKCγ). We confirm previous studies and find that in wild-type mice 10 μm of the PKC inhibitor GF109203 eliminates PTP and the PKC activator PDBu enhances neurotransmitter release and occludes PTP. However, we find that the same concentrations of GF109203 and PDBu have similar effects in TKO animals. We also show that 2 μm GF109203 does not abolish PTP even though it inhibits the PDBu-dependent phosphorylation of PKC substrates. We conclude that at the CA3 to CA1 synapse Ca(2+)-dependent PKC isoforms do not serve as calcium sensors to mediate PTP.

Significance statement: Neurons dynamically regulate neurotransmitter release through many processes known collectively as synaptic plasticity. Post-tetanic potentiation (PTP) is a widespread form of synaptic plasticity that lasts for tens of seconds that may have important computational roles and contribute to short-term memory. According to a leading mechanism, presynaptic calcium activates protein kinase C (PKC) to increase neurotransmitter release. Pharmacological studies have also implicated this mechanism at hippocampal CA3 to CA1 synapses, but there are concerns about the specificity of PKC activators and inhibitors. We therefore used a molecular genetic approach and found that PTP was unaffected when all calcium-dependent PKC isozymes were eliminated. We conclude that PKC isozymes are not the calcium sensors that mediate PTP at the CA3 to CA1 synapse.

Keywords: post-tetanic potentiation; protein kinase C; synaptic plasticity.

Figure 1.

Effects of PKC inhibitor on post-tetanic potentiation (PTP) at Hippocampal CA3→CA1 synapses. A, Domain structures of Ca²⁺-dependent PKC isoforms (classical PKCs). B, CA3 to CA1 synapses were stimulated at 0.4 Hz and synaptic responses were measured with an extracellular electrode. As indicated, different protocols were used to induce PTP with tetanic stimulation time t = 0. Left, Average normalized field EPSPs (fEPSPs). Right, representative traces of the averages of baseline responses (black) and the first three responses after tetanic stimulation (gray). These five protocols were used in the same slice, and three to five trials per protocol were recorded for the average (n = 12, 4; 12 slices from 4 animals, denoted similarly in other figures). Scale bar: 0.2 mV, 10 ms. C, Similar experiments were conducted as in A using the tetanic protocol 50 stim at 50 Hz to induce PTP, but with paired stimulation (Δt = 50 ms) to monitor the paired-pulse ratio (PPR). Inset, Scaled representative traces of the averages of baseline responses (black) and the first three responses after tetanic stimulation (gray; n = 47, 16). D, Similar experiments as in B, but with whole-cell voltage-clamp recordings from CA1 neurons (n = 29, 10). E, PTP induced at CA3→CA1 synapses was monitored with or without the presence of broad spectrum PKC inhibitor GF (2 or 10 μm; 1 h preincubation). Left, Average normalized fEPSPs. Right, representative traces of the averages of baseline responses (black) and the first three responses after tetanic stimulation (gray). (Control: n = 42, 15; 2 μm GF: n = 10, 2; 10 μm GF: n = 8, 2). Scale bar: 0.2 mV, 10 ms. F, Similar recordings as in E, but PTP was induced with 400 stimuli at 100 Hz (Control: n = 17, 7; 2 μm GF: n = 10, 2; 10 μm GF: n = 8, 2). Scale bar: 0.2 mV, 10 ms.

Figure 2.

Expression of PKC isoforms in the hippocampal CA1 region. A, Western blot of classical PKC isoforms from WT and PKCαβγ TKO brain lysates. B, Confocal images of Ca²⁺-dependent PKC isoforms α, β, and γ in the hippocampal CA1 region from the indicated genotypes. SP, Stratum pyramidale; SR, stratum radiatum. Scale bar, 25 μm.

Figure 3.

PTP at CA3→CA1 synapses is unaltered in PKCαβγ TKO animals. A, Averaged fEPSP amplitude plotted against fiber volley amplitude, for stimuli between 10–80 μA. Inset, Example extracellular recordings evoked by a range of stimulus intensities in WT and PKC TKO slices. Stimulus intensities from top: 10, 30, 50, and 70 μA, respectively. Scale bar: 0.2 mV, 5 ms. B, Representative traces of paired stimulation (Δt = 50 ms) evoked in WT and TKO slices. WT PPR: 1.68 ± 0.04, n = 22; TKO PPR: 1.66 ± 0.03, n = 34. p = 0.7. Scale bar: 0.2 mV, 20 ms. C, Normalized fEPSPs as a function of time for the indicated induction protocols in WT (open symbols; n = 12, 4) and TKO (filled symbols; n = 9, 3). Right, representative traces of the averages of baseline responses (black) and the first three responses after tetanic stimulation (gray). D, Amplitude of PTP induced by a train consisting of 5–50 stimuli at 50 Hz and for 400 stimuli at 100 Hz. E, Comparison of PTP time course induced using different protocols in WT and TKO slices. F, Similar experiments as in A using the tetanic protocol 50 stimuli at 50 Hz to induce PTP, but with paired stimulation (Δt = 50 ms) to allow for the monitoring of the PPR. Left, Average normalized fEPSP. Right, representative traces of the averages of baseline responses (black) and the first three responses after tetanic stimulation (gray; n = 36, 14).

Figure 4.

The phorbol ester PDBu enhances synaptic responses and occludes PTP in both WT and TKO animals. A, Representative experiment showing normalized fEPSPs as a function of time in a WT slice. fEPSPs were recorded in 1.5 mm external Ca²⁺ and PTP (50 stimuli at 50 Hz induction) was monitored before and after the application of the PKC activator PDBu (1 μm). Induction of PTP is indicated by arrowheads. B, Average normalized fEPSPs during PDBu application in WT animals (n = 17, 7). fEPSPs were recorded every 5 s to monitor the effects of PDBu but displayed in the plot every 25 s for clarity. C, Comparison of PTP before and after the application of PDBu in WT slices. D–F, Similar to A–C, but in TKO animals (n = 13, 4). G, PTP (50 stimuli at 50 Hz induction) in the presence of the indicated concentrations of the PKC inhibitor GF in WT (open symbols) and TKO (filled symbols) animals. (WT control: n = 42, 15; WT + 2 μm GF: n = 10, 2; WT + 10 μm GF: n = 8, 2; TKO control: n = 29, 11; TKO + 2 μm GF: n = 7, 2; TKO + 10 μm GF: n = 8, 2). H, Protein analysis of phosphorylated levels of PKC substrates in the presence of PKC activator PDBu (1 μm) with and without the preincubation in the indicated concentrations of GF for 1 h. I, Summary of physiology experiments. Empty bars: WT; filled bars: TKO. J, Quantification of phosphorylated levels of PKC substrates in H. Intensity is normalized to WT control conditions (n = 5 for both genotypes).