A, summary of whole-cell voltage-clamp recordings showing mean (± s.e.m.) percentage change in EPSC amplitude induced by one, four or eight trains of theta-burst stimulation (1, 4 and 8 TBS) (arrow). Increasing the number of TBS trains resulted in LTP of significantly greater magnitude measured at the 2 h post-TBS time point. B, decay characteristics of each form of LTP. The mean time constant of decay (τ, min) is shown for LTP induced by 1, 4 and 8 TBS. Each τ value is significantly different from the others (P < 0.05). Increasing numbers of TBS trains induced LTP with slower decay.

A and B, inhibition of ryanodine receptors (RyRs) selectively inhibits LTP 1. A, mean (± s.e.m.) percentage change in EPSC amplitude induced by 1 TBS (arrow) in control cells and with ruthenium red (Ruth Red; 40 μm) in the pipette; *P < 0.05. B, summary histogram showing mean (± s.e.m.) LTP and τ constants in ruthenium-red-treated cells as percentages of control values for each TBS group. C and D, inhibition of inositol 1,4,5-trisphosphate (IP₃) receptors selectively inhibits LTP 2. C, mean (± s.e.m.) percentage change in EPSCs induced by 4 TBS (arrow) in control cells and cells treated with xestospongin-C (Xest-C; 5 μm, 10 min). D, summary histogram as in B, but showing the effect of Xest-C on the magnitude and decay of LTP 1, 2 and 3. E and F, inhibition of L-type voltage-dependent Ca²⁺ channels (VDCCs) selectively inhibits LTP 3. E, mean (± s.e.m.) percentage change in EPSCs induced by 8 TBS (arrow) in control cells and cells treated with nifedipine (Nifed; 10 μm, 10 min). F, summary histogram as in B, but showing the effect of nifedipine on the magnitude and decay of LTP 1, 2 and 3.

A, section of the apical dendrite of a CA1 pyramidal neurone filled with Oregon Green 488 BAPTA 1 and Alexa-594 showing positioning of line scan through a spine and the underlying dendrite (i); ii, line scan time-series during 1 TBS beginning at arrowheads; iii, current-clamp record during 1 TBS (upper trace; scale bar, 20 mV) aligned with mean ΔF/F (where F is fluorescence) measured simultaneously in the spine (black) and dendrite (purple; scale bars, 50% and 1 s). B, mean ΔF/F traces showing change in [Ca²⁺] in the spine during the first, fourth and eighth TBS trains in controls and drug-treated cells (scale bars, 50%, 1 s). Notice the loss of the Ca²⁺ peaks in the presence of ruthenium red. C–E, examples of Fourier transform (FT) analyses on spine Ca²⁺ transients produced by 1 TBS in controls and drug-treated cells. Notice that the prominent peak at 5 Hz, which corresponds to the burst frequency of TBS, is eliminated by ruthenium red (C), but not by xestospongin-C (D) or nifedpine (E). F–H, summary histograms of the mean (± s.e.m.) magnitude of the 5 Hz peak obtained by Fourier analysis in controls and cells treated with ruthenium red (F), xestospongin-C (G), and nifedipine (H); *P < 0.05. Ruthenium red selectively blocks the 5 Hz peak in the spine Ca²⁺ transients produced by each of 8 TBS trains.

A, voltage-clamp record during paired-pulse stimulation (upper trace; scale bar, 250 pA) aligned with mean ΔF/F measured simultaneously in spines (black) and dendrites (grey; scale bars, 50% and 100 ms). B, mean ΔF/F traces showing the effect of different Ca²⁺ antagonists on paired-pulse evoked Ca²⁺ transients in spines (black) and dendrites (grey; scale bars, 50% and 100 ms). Within-group controls for each antagonist are shown on the left, except for ruthenium red, which is compared with a separate control group. Only d-aminophosphonovalerate (d-AP5; 50 μm) caused any significant change in Ca²⁺ transients evoked by subthreshold paired-pulse stimulation.

A, mean dendritic ΔF/F traces showing change in [Ca²⁺] in the dendrite during the first, fourth and eighth TBS trains, in controls and drug-treated cells (scale bars, 50% and 1 s). Notice the selective inhibition by xestospongin-C during the fourth and eighth trains. B–D, examples of Fourier transform (FT) analyses on dendritic Ca²⁺ transients produced by 1 TBS in controls and cells treated with ruthenium red (B), xestospongin-C (C) and nifedipine (D). The 5 Hz peak in dendritic Ca²⁺ transients is unaffected by any of the antagonists. E–G, summary histograms of the mean (± s.e.m.) area under the curve (AUC; time integral) of dendritic Ca²⁺ transients in controls and cells treated with ruthenium red (E), xestospongin-C (F) and nifedipine (G); *P < 0.05. Xestospongin-C selectively inhibits the dendritic Ca²⁺ transients produced by the second to the eighth TBS.

A, CA1 pyramidal neurone filled with Oregon Green 488 BAPTA 6-F and Alexa-594 showing positioning of line scan through the nucleus (i); ii, current-clamp record during 1 TBS (upper trace; scale bar, 20 mV), aligned with line scan time-series (middle trace) and ΔF/F averaged over the entire nucleus (lower trace; scale bars, 20% and 1 s). B, mean somatic ΔF/F traces showing change in [Ca²⁺] during the first, fourth and eighth TBS trains in controls and drug-treated cells (scale bars, 10% and 1 s). C–E, summary histograms of the mean (± s.e.m.) time integral (AUC) of somatic Ca²⁺ transients produced by each TBS train in controls and cells treated with ruthenium red (C), xestospongin-C (D) and nifedipine (E); *P < 0.05. Note that somatic Ca²⁺ transients are selectively inhibited by nifedipine.

A–C, summary of whole-cell voltage-clamp recordings showing the effect of d-AP5 (50 μm, 10 min) on LTP 1 (A), LTP 2 (B) and LTP 3 (C) over a 2 h post-TBS period. TBS was delivered at the times indicated by the arrows. Inhibition of NMDA receptors inhibits LTP 1 and LTP 2, but only partially affects LTP 3. D–F, mean ΔF/F traces showing the effect of d-AP5 (50 μm) on Ca²⁺ transients produced by the first, fourth and eighth TBS trains in spines (D), dendrites (E) and soma (F). Scale bars, 50% and 1 s (D and E), and 10% and 1 s (F). G–I, summary histograms showing the effect of d-AP5 on the mean (± s.e.m.) time integral (AUC) of Ca²⁺ transients in spines (G), dendrites (H) and soma (I); *P < 0.05. Inhibition of NMDA receptors completely abolishes spine and dendrite Ca²⁺ signals, but has no effect on somatic Ca²⁺.

Schematic diagram describing a proposed mechanism for the differential induction of different forms of LTP by Ca²⁺. Weak conditioning stimulation (e.g. 1 TBS) activates NMDARs resulting in a modest influx of Ca²⁺ into the spine, which in turn activates Ca²⁺-induced Ca²⁺ release (CICR) via ryanodine receptors (RyR). CICR is proposed to activate kinases in the PSD that underlie the expression of LTP 1. Stronger stimulation (e.g. 2–4 TBS) can additionally recruit an IP₃-dependent signalling pathway by activation of mGluRs. Ca²⁺ release from IP₃Rs in the dendrites, possibly combined with other G-protein-coupled mechanisms (e.g. protein kinase C (PKC)), is proposed to activate local dendritic protein synthesis that supports the expression of LTP 2. The NMDAR gates IP₃R activation, possibly via extrasynaptic NMDARs. Repetitive activation of L-VDCCs (e.g. by 5–8 TBS) causes significant Ca²⁺ peaks in the nucleus, which are proposed to trigger the gene transcription necessary for the expression of LTP 3.