CA1 areas were stimulated by stimulating Schaffer collaterals with 12TBS or 4TBS, dissected out quickly at different time points after TBS, and processed for Western blotting. For each individual time point, the ratio of pTrkB signal to total TrkB signal was normalized against the control condition from the same experiment. Three mice were used for each experiment with multiple time points, and each time point contains CA1 from 4 hippocampal slices. The same experiment was repeated at least 6 times (n = 6) in (A) and (B), each with new batch of animals.
(A) Examples of TrkB activation in hippocampal slices induced by 12TBS and 4TBS. No obvious difference was found in the pattern of TrkB activation.
(B) Quantification of time courses of TrkB activation induced by 4TBS with and without the protein synthesis inhibitor anisomycin (40 µM). The pTrkB time course induced by 12TBS is included for comparison.
(C) Protein synthesis independence of TrkB activation measured 1 hour after 12TBS. Both example (top) and quantification (bottom, n = 4) are shown. Inhibition of protein synthesis by anisomycin (40 µM) does not block the TBS-induced increase in pTrkB (*, P <0.05, one-way ANOVA; no difference was found between “12TBS” and “12TBS+anisomycin” groups).

(A) Double staining of pTrkBY816 (green) and DAPI (blue) of hippocampus slice. The proximal and distal areas of hippocampal CA1 neurons are highlighted by the yellow and red boxes, respectively. The position of the stimulating electrode is indicated by the white circle.
(B–C) Double staining of pTrkBY816 (green) and DAPI (blue) of proximal region from cell body with or without 12TBS. Note the CA1 neuronal cell body stained by DAPI located in the upper part of the images.
(D–E) Double staining of pTrkBY816 and DAPI of distal region from cell body with or without 12TBS. Note the marked increase in pTrkB staining after 12TBS.
(F–G) Quantification of the number of pTrkB positive puncta in the proximal and distal regions from cell body of CA1 neurons, respectively (* P<0.001, t-test).

(A) Two-pathway recording scheme. Two independent Schafer collateral pathways that project to the same CA1 neurons were stimulated by two stimulating electrodes positioned on either side of the recording electrode. S1 was stimulated by strong TBS (12TBS) whereas S2 was stimulated by weak TBS (4TBS).
(B) Lack of facilitation between the two Schafer collateral pathways. Upper: the superimposed red and green fEPSP traces induced by S2 were same, regardless of whether S1 was stimulated 30 msec earlier. Lower: S1-induced fEPSPs were the same regardless of S2 stimulation.
(C) Failure to induce L-LTP by weak TBS (4TBS). Application of 4TBS alone induced only E-LTP (last <2 hours), but not L-LTP.
(D) Weak TBS in S2 induced L-LTP when preceded by strong TBS in S1. In a typical “two pathway” experiment using slices from TrkB^F616A mice, a 12TBS to S1 was followed by a 4TBS to S2. Application of 12TBS resulted in a robust L-LTP in S1. Application of 4TBS to S2, which normally induces only E-LTP, now elicited L-LTP. Treatment with vehicle (indicated by the black bar) around the time of 4TBS had no effect on L-LTP in TrkB^F616A slices.
(E) Inhibition of TrkB activation blocked E-LTP → L-LTP conversion in S2. The “two-pathway” experiment was done exactly the same way as in (D), except 1NMPP1 (5 µM) was applied instead of vehicle (DMSO). Treatment with 1NMPP1 blocked L-LTP in S2, without affecting L-LTP in S1 in TrkB^F616A slices.
(F) Lack of effect of 1NMPP1 on L-LTP in wild type hippocampal slices. 1NMPP1 was applied to hippocampal slices from C57BL/6 mice around the time of 4TBS (black bar). 12TBS induced a 166±10% potentiation in S1, whereas 4TBS induced a 138±5% potentiation in S2.
(G) Lack of effect of vehicle on L-LTP in TrkB^F616A slices. L-LTP was induced in both S1 (158±9%) and S2 (135±10%) when vehicle was applied at the time of 12TBS.
(H) 1NMPP1 inhibited L-LTP in S1 (104±5%) but left L-LTP intact in S2 (135±5%) when it was applied at the time of 12TBS.
The number of slices tested is indicated in brackets. At least 3 animals were tested for each experiment.

Mice were trained in an inhibitory avoidance (IA) task with 2 weak foot shocks (0.1 mA, 100 ms) at 1 sec interval. Latency to step through the door from illuminated chamber to dark chamber was recorded as measurement of memory. Data was plotted as mean ± s.e.m. and Newman-Keuls analysis was performed after one-way ANOVA. The number of tested mice is indicated in each column.
(A) and (C) STM (60min) and LTM (24h) were assessed with different groups of wild type mice or TrkB^F616A mice, respectively. The weak IA training only induced STM but not LTM.
(B) Exploration in novel environment (open filed with four objects, OF) consolidated weak IA induced STM into LTM. 1NMPP1 did not block the consolidation in wild-type mice.
(D) Novelty-promoted LTM in TrkB^F616A mice was blocked by IP injection of 1NMPP1.

Hippocampal neurons were treated with BDNF-beads (green) for 30 min, and stained with an antibody against phosphorylated TrkB (pTrkB, red). Local pTrkB immunoreactivity is dramatically increased in dendrites touched by BDNF-beads (arrow).
(A) Preferential activation of TrkB on dendrites in contact with BDNF-beads. The area highlighted by the blue box is enlarged and shown in the lower right. Note the strong pTrkB signals (red) associated with the BDNF-beads (green), indicated by arrows.
(B) Blockade of BDNF-beads induced TrkB activation by k252a. Neurons treated with k252a and BDNF-beads (green) show little pTrkB immunoreactivity even when dendrites were touched by BDNF-beads (arrow).
(C) Touching by beads alone does not activate TrkB. Neurons were treated with uncoupled beads (green) without BDNF. Little pTrkB immunoreactivity was detected along the dendrites even when dendrites were touched by beads (arrow).
(D) Low levels of basal TrkB activation in cultured hippocampal neurons. Little pTrkB immunoreactivity was detected in dendrites in absence of BDNF beads (asterisk).
(E) Abundant TrkB immunoreactivity (red) along the dendrites of cultured hippocampal neurons. Arrows show the place where BDNF-bead touched dendrite.
(F) Quantification of pTrkB immunoreactivity under different conditions. The intensity of an individual pTrkB immunofluorescent spot along the dendrite was quantified by Image J. The intensities of all spots from the point touched by BDNF beads (0–5 µm, 5–10 µm, 20–50 µm) were summed and presented as arbitrary units (AU). pTrkB signal gradually decreases along the dendrites from the point of BDNF-bead contact. Note that there is little pTrkB signal on the dendritic branch not contacted by BDNF-bead, despite the fact that other branches are contacted by BDNF-beads. The measured regions of interest are indicated as “n” for each condition. ***: P<0.001; Newman-Keuls analysis following one-way ANOVA.
(G–I) Confocal images of total TrkB immunoreactivity (red) along the dendrites of cultured hippocampal neurons treated with (H) or without (G) BDNF-conjugated beads, and the quantifications (I). Note that total TrkB level with BDNF-bead treatment was comparable to control without BDNF-conjugated beads.

(A) 1NMPP1 blocked 12TBS-induced TrkB activation in TrkB^F616A mice. The CA1 regions of hippocampal slices from TrkB^F616A mice were microdissected 1 hour after 12TBS. For each experiment, 3 mice were used, and for each condition, 4 CA1 slices were used. The same experiment was repeated 5 times (n = 5), each with new batch of animals. 1NMPP1 (5 µM), but not vehicle control (DMSO), blocked the 12TBS-induced increase of pTrkB in TrkB^F616A mice (*, P <0.003; one-way ANOVA).
(B) Blockade of 12TBS induced L-LTP by inhibiting TrkB activation with 1NMPP1 in adult hippocampal slices derived from TrkB^F616A mice. 1NMPP1 (5 µM), applied to slices throughout the experiment, completely blocked L-LTP (blue diamonds), while vehicle control had no effect (black circles). 1NMPP1 did not affect L-LTP in the control mice (C57BL/6) (pink diamonds).
(C) 1NMPP1 applied immediately after 12TBS also blocked L-LTP in TrkB^F616A slices.
(D) Inhibition of L-LTP by 1NMPP1 applied between 0 and 40 min after 12TBS (red) but not 45 min after 12TBS in TrkB^F616A slices (black).
(E) Stable baseline recording were obtained for over 4 hours without any treatment.
(F) Indistinguishable STPs in 1NMPP1- and vehicle-treated TrkB^F616A slices. STPs were induced by 2TBS. In both cases, fEPSPs returned to baseline at 60 min after 2TBS.

The “two pathway” experiment was done exactly the same way as in Figure 5, except that 4TBS was applied to S2 40 min before (instead of 60 min after) 12TBS applied to S1. Application of 1NMPP1 or vehicle (DMSO) is indicated by the black bar.
(A) Treatment with 1NMPP1 (5 µM) in TrkB^F616A slices blocked L-LTP in S2, without affecting L-LTP in S1.
(B) L-LTP was induced by 12TBS in S1 and by 4TBS in S2 in TrkB^F616A slices treated with vehicle around the time of 4TBS.
(C) L-LTP was induced by 12TBS in S1 and by 4TBS in S2 in wild type (C57BL/6) slices treated with 1NMPP1 around the time of 4TBS.
(D) Synaptic tagging model for two pathway recording. Short-lived synaptic tag can be generated by strong (12TBS) and weak (4TBS) stimulation, while the PRPs can only be induced by strong stimulation. Overlap of the tag wave and PRP wave indicates the capture of PRPs, leading to L-LTP in that specific pathway.

(A) Normal short-term memory after TrkB inhibition by 1NMPP1. At 10 min after IP-injection with 1NMPP1 or vehicle, mice were trained in an inhibitory avoidance (IA) task with 2 weak foot shocks (0.1 mA, 100 ms) at 1 sec interval. Latency to step through the door from illuminated chamber to dark chamber was recorded as measurement of memory and plotted as mean ± s.e.m.
(B) Normal pain sensory after TrkB inhibition by 1NMPP1. Hot plate test was performed at 55°C. Latency to lick fore paw and hind paw was plotted as mean ± s.e.m.
(C–E) Normal emotional state and locomotion after TrkB inhibition by 1NMPP1. Elevated zero maze tests were performed using the ring-shaped platform consisting of two walled (white Plexiglas) sections separated by open sections of equal length. Activity of each mouse was recorded for five minutes. Mice injected with 1NMPP1 spent similar amount of time in open arms as compared with those injected with vehicle (C). In addition, 1NMPP1 did not affect the number of times that mice entered the open arms (D) and the total distance that mice traveled in the maze (E).