Regional differences in hippocampal calcium handling provide a cellular mechanism for limiting plasticity

Abstract

Although much is known about the mechanisms underlying synaptic plasticity, the cellular mechanisms that negatively regulate plasticity in some brain regions are considerably less studied. One region where neurons do not reliably express long-term potentiation (LTP) is the CA2 subfield of the hippocampus. Given the connection between synaptic plasticity and increases in postsynaptic [Ca(2+)], and that CA2 neurons express a large number of calcium-regulating proteins, we tested the hypothesis that the relative lack of LTP in CA2 results from differences in the calcium dynamics of these neurons. By measuring calcium-dependent fluorescence transients in dendritic spines, we show that CA2 neurons have smaller action potential-evoked intracellular Ca(2+) transients because of a higher endogenous Ca(2+)-buffering capacity and significantly higher rates of Ca(2+) extrusion when compared with CA1 and CA3 neurons. Perfusion with higher external [Ca(2+)] during induction restores LTP to CA2 neurons, suggesting that they possess the cellular machinery required for plasticity, but that the restriction of postsynaptic [Ca(2+)] limits its expression. Camstatin, an analogue of the calcium-modulating protein Pep-19 strongly expressed in CA2 neurons, blocked LTP and increased Ca(2+) extrusion in CA1 neurons, suggesting a role for extrusion in the regulation of plasticity in CA2. In agreement with this idea, we found that intracellular introduction of a PMCA pump inhibitor (carboxyeosin) allows for the induction of LTP in CA2 neurons. Our results indicate that regulation of postsynaptic [Ca(2+)] through modulation of extrusion and/or buffering regulates expression of LTP in CA2 and potentially other brain regions.

Fig. 1.

Ca²⁺ buffering in dendritic spines of hippocampal neurons. (A) CA1, CA2 and CA3 spines; linescans (dashed line) were performed on spines located <150 μM from the soma on secondary or tertiary dendrites. (B) Ca²⁺-dependent fluorescence traces evoked by single and high frequency (40 AP at 83 Hz; 30 AP at 63 Hz) trains of action potentials, bars under high frequency traces indicate time of AP firing (∼500 ms). Traces represent averages of 15 and 5 trials for single and high frequency conditions respectively. Single AP traces are fit with an exponential decay function (dashed line) used in calculation of τ_decay. (C) Plots of Δ[Ca²⁺]_AP⁻¹ as a function of indicator concentration (K_b). Linear fits extrapolated back to 0 K_b (dotted lines) provided estimates of Δ[Ca²⁺]_AP^[dye]=0 and the endogenous buffering capacity (K_E = -K_b0). Endogenous τ_decay was computed using the same method. Error bars, SEM.

Fig. 2.

High bath Ca²⁺ restores LTP and significantly increases free calcium in CA2 neurons. (A) Ca²⁺ handling in CA2 dendrites and spines with 10 mM bath calcium. Ca²⁺ dependent fluorescence traces evoked by single and high frequency (40 AP at 83 Hz; 30 AP at 63 Hz) trains of action potentials. (B) Plots of Δ[Ca²⁺]_AP⁻¹ as a function of indicator concentration (K_b). Linear fits extrapolated back to 0 K_b (dotted lines) provided estimates of Δ[Ca²⁺]_AP^[dye]=0 and the endogenous buffering capacity (K_E = -K_b0). Error bars indicate SEM. (C) Response of CA2 neurons to high frequency stimulation (HFS) using 4 mM (gray circles, n = 10), 10 mM (black circles, n = 12), and 10 mM Ca²⁺ ACSF with 50 μM APV (open circles, n = 6) during LTP induction. Similar experiments in CA1 neurons using 4 mM Ca²⁺ ACSF are shown for comparison (gray triangles, n = 8). Time of perfusion (0–3 min) with high Ca²⁺ ACSF is indicated by solid gray bar. Arrow indicates onset of HFS. Error bars, SEM.

Fig. 3.

Camstatin blocks LTP and dramatically increases extrusion in CA1 neurons. (A) Ca²⁺ dependent fluorescence traces evoked by single and high frequency (40 AP at 83 Hz; 30 AP at 63 Hz) trains of action potentials. Exponential decay fits to traces of single AP evoked fluorescence demonstrate a significant shortening in τ_decay with camstatin (50 ms) vs. controls (140 ms). (B) Plots of Δ[Ca²⁺]_AP⁻¹ as a function of indicator concentration (K_b). Linear fits extrapolated back to 0 K_b (dotted lines) provided estimates of Δ[Ca²⁺]_AP^[dye]=0 and the endogenous buffering capacity (K_E = -K_b0). Error bars, SEM. (C) The response of CA1 neurons to HFS was measured using normal internal solution (gray triangles, n = 9), and internal solution containing 10 μM camstatin (black circles, n = 11). Heat inactivated camstatin failed to block LTP (open circles, n = 5). The response to continuous 0.05-Hz stimulation was also measured with 10 μM camstatin (gray circles, n = 6).

Fig. 4.

Ten micromolar intracellular carboxyeosin (CE) reduces rates of Ca²⁺ extrusion and rescues LTP in CA2 neurons. (A) Single AP responses measured with the calcium indicator X-Rhod 5F (300 μM) in CA2 neurons with and without 10 μM CE. Average τ_decay (n = 8) are indicated for the single dye concentration shown and are significantly longer with CE. (B) Synaptic responses from CA2 neurons loaded with the PMCA pump inhibitor CE (black circles, n = 10) after HFS. Arrow indicates timing of HFS (100 Hz). The impact of CE on baseline synaptic responses (gray circles, n = 8) and the response to 0.01% DMSO vehicle controls (n = 6) over the same time period is also shown. Error bars, SEM.