Multiple forms of LTP in hippocampal CA3 neurons use a common postsynaptic mechanism

Abstract

We investigated long-term potentiation (LTP) at mossy fiber synapses on CA3 pyramidal neurons in the hippocampus. Using Ca2+ imaging techniques, we show here that when postsynaptic Ca2+ was sufficiently buffered so that [Ca2+]i did not rise during synaptic stimulation, the induction of mossy fiber LTP was prevented. In addition, induction of mossy fiber LTP was suppressed by postsynaptic injection of a peptide inhibitor of cAMP-dependent protein kinase. Finally, when ionotropic glutamate receptors were blocked, LTP depended on the postsynaptic release of Ca2+ from internal stores triggered by activation of metabotropic glutamate receptors. These results support the conclusion that mossy fiber LTP and LTP at other hippocampal synapses share a common induction mechanism involving an initial rise in postsynaptic [Ca2+].

Fig. 1

Contrasting properties of mossy fiber (mf) and commissural/associational (C/A) evoked synaptic responses. (a) Schematic of a hippocampal slice showing stimulating (stim) and recording (record) sites. (b) Synaptically evoked responses could be distinguished based on their different kinetic properties: mossy-fiber-evoked EPSPs and EPSCs have significantly faster rise times than C/A-evoked responses (left; responses evoked with alternate stimulation of s. lucidum and s. radiatum). The group II mGluR agonist DCG-IV (1 μM) in the perfusion medium selectively suppressed mossy-fiber-evoked EPSPs and EPSCs. (Upward arrows designate stimulus onset in all figures.) DCG-IV suppressed responses elicited with s. lucidum stimulation (left, 12.3 ± 3.0% of baseline, n = 6; p < 0.01) but did not affect responses evoked by stimulation in s. radiatum (right, 95.3 ± 7.5%, n = 8; p > 0.1). (c) LTP of mossy fiber and C/A synapses differ in their dependence on NMDA receptor activation. High-frequency trains of stimulation (HFS) were delivered simultaneously to mossy fibers and C/A fibers in the presence of the NMDA antagonists APV (50 μM) and MK-801 (20 μM; n = 5). Left, following HFS, LTP of mossy-fiber-evoked responses (206.5 ± 5.2%) differed significantly from C/A-evoked responses (108.0 ± 6.5%; p < 0.001). Right, cumulative probability plots graphically summarize the data; each point represents the magnitude of change relative to baseline for a given experiment 20–25 min (average) after HFS. The start of the horizontal arrow corresponds to our operational definition for LTP (>20% above baseline). In all figures, responses shown are averages of 3–6 consecutive trials recorded at 0.05–0.1 Hz.

Fig. 2

Mossy fiber LTP induced by three different stimulation protocols. (a) Top, a representative response evoked by one train of B-HFS (15 trains, given every 5 seconds, each consisting of 7 stimuli at 100 Hz with simultaneous postsynaptic depolarization; scale bar, 40 ms). Middle and bottom, responses evoked by L-HFS (100 pulses at 100 Hz); L-HFS-1 and L-HFS-3 differ only by the total number of trains (one and three, respectively, given every 10 seconds for L-HFS-3). These responses were evoked by the first and third stimulation trains using L-HFS-3 (scale bar, 80 ms) (b) Representative responses before (1) and 25 min after HFS (2). (c) Left, time course and magnitude of potentiation evoked by the different stimulation protocols. The magnitude of LTP induced with B-HFS and L-HFS-1 did not differ significantly from each other, but both differed significantly from LTP induced with L-HFS-3 (L-HFS-3, 202.0 ± 16%, n = 15; B-HFS, 140 ± 11%, n = 13; L-HFS-1, 142 ± 11%, n = 9, p < 0.01). Right, cumulative probability graph shows the probability and magnitude of LTP induced for each stimulation protocol 20–25 min after HFS (probability: B-HFS, 54%; L-HFS-1, 67%; L-HFS-3, 87%).

Fig. 3

Effects of postsynaptic BAPTA on the induction of mossy fiber LTP with B-HFS and L-HFS. (a) 1 mM BAPTA blocked the induction of LTP by B-HFS (102 ± 3%, n = 5) but had little effect on LTP induced by L-HFS-3 (187 ± 24%, n = 9, p < 0.01). (b) Higher concentrations of BAPTA (5–10 mM) blocked the induction of mossy fiber LTP induced with B-HFS (99 ± 5%, n = 6) and L-HFS-1 (99 ± 6%, n = 5) and significantly suppressed L-HFS-3-induced LTP (128 ± 11%, n = 18) compared to control (202 ± 16.3%, n = 15, p < 0.001). (c) Cumulative probability plots summarize the effects of 1 mM and 5–10 mM BAPTA on the three different stimulation protocols. Although 5–10 mM BAPTA blocked the induction of LTP with B-HFS and L-HFS-1 in all cases, LTP was induced in at least 30% of the cases when L-HFS-3 was used.

Fig. 4

Ca²⁺ fluorescence imaging during B-HFS and L-HFS. (a) Example fura 2-filled CA3 pyramidal neuron. Colored boxes show where postsynaptic Ca²⁺ transients were measured during high-frequency stimulation. Right, data from individual experiments, including one of the three cells in which BAPTA did not appear to block the Ca²⁺ transient evoked by L-HFS-3. (BAPTA data are from the neuron on the left.) (b) Left, under control conditions, peak [Ca²⁺]_i did not differ significantly for B-HFS and L-HFS-3; however, the half-width was significantly different for the two patterns of stimulation (Kolmogrov-Smirnov, p < 0.001; data not shown). Middle, 1 mM BAPTA significantly suppressed a rise in [Ca²⁺]_i evoked by B-HFS compared to control (Kolmogrov-Smirnov, p < 0.001) but did not significantly affect L-HFS-3-evoked [Ca²⁺]_i. Right, 5–10 mM BAPTA significantly suppressed peak [Ca²⁺]_i for both B-HFS and L HFS-3 (Kolmogrov-Smirnov, p < 0.001). (c) Ca²⁺ transients evoked by L-HFS-3 of C/A. (NMDA antagonists were not present.) [Ca²⁺]_i was evaluated at several dendritic branches and at several locations on an individual dendritic branch. The site with the largest Ca²⁺ response was used for quantitation (control peak, 18.4 ± 1.8% ΔF/F; control half-width, 2.0 ± 0.3 s, n = 7). 5–10 mM BAPTA effectively blocked a rise in [Ca²⁺]_i (3.8 ± 1.2% ΔF/F, n = 6; Kolmogrov-Smirnov, p < 0.05). Ca²⁺ traces and electrical traces are from individual experiments and are averages of three trains of either B-HFS or L-HFS.

Fig. 5

High BAPTA blocks Ca²⁺ signals and LTP. High concentrations of BAPTA (30–50 mM) in the postsynaptic patch pipet blocked Ca²⁺ transients during L-HFS-3 (left, Ca²⁺ responses from the soma and s.lucidum; 2.7 ± 0.3% ΔF/F) and correspondingly prevented LTP induction (101.2 ± 4.3% of baseline, n = 10, p < 0.001 versus control).

Fig. 6

Suppressing synaptic transmission alone does not prevent mossy fiber LTP. (a) Individual experiment showing that the glutamate antagonist kynurenate (10 mM) in the perfusion medium blocked fast synaptic transmission but did not prevent an L-HFS-3-evoked rise in postsynaptic [Ca²⁺]_i (left, average of all experiments, 20.4 ± 5.5% ΔF/F, n = 8), nor did it prevent mossy fiber LTP (right). (b) Combining kynurenate and a low dose of postsynaptic BAPTA (1 mM) prevented a rise in [Ca²⁺]_i (left, average of all experiments, 2.3 ± 1.0% ΔF/F, n = 7) and prevented LTP induction (right). After kynurenate washout, L-HFS-3 evoked a robust Ca²⁺ transient (left), resulting in mossy fiber LTP (right). (c) Right, time course and magnitude of potentiation induced in the presence of 10 mM kynurenate or 1 mM BAPTA and 10 mM kynurenate (p < 0.05). Left, cumulative probability graph showing L-HFS-evoked Ca²⁺ responses when either kynurenate or kynurenate and BAPTA were present. Circled dots represent cases in which LTP was induced in the presence of kynurenate; in all of these cases, a Ca²⁺ transient was observed.

Fig. 7

Ca²⁺ release from internal stores when fast synaptic transmission is blocked. (a) In the presence of kynurenate (8–10 mM), L-HFS-3 elicited a rise in [Ca²⁺]_i. An individual experiment in which the mGluR1 antagonist AIDA (500 μM) blocked the rise in [Ca²⁺]_i during L-HFS. After washout (30 min), a large rise in [Ca²⁺]_i was evoked in the proximal dendrite by L-HFS. In this case, a sustained depolarization was observed both with and without AIDA. (Waveforms are from single stimulation trains.) (b) Ryanodine (ryn; n = 3), thapsigargin (thaps; n = 2), MCPG (n = 3), AIDA (n = 6) or CPCCOEt (n = 3) blocked the rise in postsynaptic [Ca²⁺], consistent with the block of Ca²⁺ release from internal stores. (c) AIDA (n = 3) or CPCCOEt (n = 3) blocked mossy fiber LTP when fast synaptic transmission was blocked (83.1 ± 4.3%, n = 6; data were combined for the two drugs). These results were significantly different from LTP induced without mGluR antagonists present (p < 0.05). Horizontal bar indicates drug application.

Fig. 8

Postsynaptic inhibition of PKA blocked mossy fiber LTP. (a) PKA inhibitor peptide (5–24; 30–100 μM) in the recording pipet significantly reduced potentiation compared to control (133 ± 9%, n = 12 and 221 ± 22%, n = 5, respectively; p < 0.05). (b) Cumulative probability.