1. Volume 95, Issue 4, Pages 928-943.e3 (August 2017)
LTP at Hilar Mossy Cell-Dentate Granule Cell Synapses Modulates Dentate Gyrus Output by Increasing Excitation/Inhibition Balance 
Yuki Hashimotodani, Kaoutsar Nasrallah, Kyle R. Jensen, Andrés E. Chávez, Daniel Carrera, Pablo E. Castillo 
Neuron 
Volume 95, Issue 4, Pages e3 (August 2017)
DOI: /j.neuron
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2. Figure 1
Repetitive Activation of MC Axons Induces NMDAR-Independent LTP at MC-GC Synapses
(A) Top: schematic diagram illustrating the neural circuit in dentate gyrus and the recording configuration. Whole-cell patch-clamp recordings were performed from GCs, and the stimulation electrode was placed in the IML to stimulate MC axons. The LTP induction protocol is depicted on the bottom. OML, outer molecular layer; MML, middle molecular layer; IML, inner molecular layer; GCL, granule cell layer; LPP, lateral perforant path; MPP, medial perforant path; GC, granule cell; IN, interneuron; MC, mossy cell.
(B) MC-GC LTP representative experiment (top) and summary plot (bottom). MC EPSCs were recorded from GCs in whole-cell voltage-clamp mode. After a stable (∼10-min) baseline, the induction protocol was delivered at the time indicated by the vertical arrow. At the end of the experiments, DCG-IV (1 μM) was bath applied in order to verify the identity of the evoked EPSCs. Representative traces, which correspond to the numbers in the time-course plot below (for this and all subsequent figures), are shown on the top.
(C and D) The magnitude of MC-GC LTP depends on the frequency (C) and number of bursts (D) of induction protocol. In (C), the burst number was fixed at 50 times, while the stimulation frequency was changed. In (D), the stimulation frequency was fixed at 100 Hz, while the burst number was changed. Numbers in parentheses, here and in all figures, indicate the number of cells.
(E) MC-GC LTP was normally induced in the presence of 50 μM D-APV during the tetanus.
(F) A pairing-protocol (200 pulses at 2 Hz, paired with 0 mV depolarization) also induced robust MC-GC LTP in the presence of 50 μM D-APV. Presynaptic activity alone (200 pulses, 2 Hz, Vh = −60 mV) also induced robust LTP.
(G) In contrast, pairing-protocol-induced MPP LTP was abolished by D-APV, whereas presynaptic activity alone (200 pulses at 2 Hz) was not sufficient to induce LTP.
(H) Recording configuration (top left), representative traces (bottom left), and summary plot time-course (right), showing that the induction protocol that elicits MC-GC LTP failed to induce LTP at DCG-IV-sensitive MPP inputs.
(I) MC-GC LTP is input specific. Recording configuration (top left) and representative traces (bottom left). Summary plot (right) showing that MC-GC LTP was elicited at the tetanized, but not naive, input.
Data are presented as mean ± SEM.
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3. Figure 2 MC-GC LTP Is Likely Expressed Presynaptically
(A) MC-GC LTP was associated with a reduction of both PPR and CV. Representative traces are shown on left, summary plots (PPR and CV) on right (n = 18).
(B) MC-GC LTP was similarly expressed at the AMPAR- and NMDAR-mediated components of synaptic transmission. Left: compound AMPAR/NMDAR EPSCs were recorded from GCs voltage clamped at −40 mV in the presence of 100 μM picrotoxin. Under control conditions, D-APV (50 μM) removed the slow component but had no effect on the peak amplitude of the compound EPSC. MC-GC LTP was assessed by simultaneously monitoring the AMPAR (peak measurement, red vertical line) and NMDAR component (off-peak measurement, blue vertical line) of MC EPSCs. Right: time-course summary plot showing that the AMPAR and NMDAR components exhibited similar LTP.
(C) Representative experiment using minimal stimulation in IML; time course (bottom) and sample traces (top).
(D) Summary plots demonstrating that MC-GC LTP induced by minimal stimulation is associated with a significant decrease in failure rate and increase in efficacy and potency (n = 8).
(E) BoTx (5–500 nM) included in the intracellular solution did not affect MC-GC LTP.
(F) Control experiments confirming BoTx activity. Intracellulary applied BoTx (5–500 nM) through patch pipette induced rundown of AMPAR-EPSCs recorded from CA1 pyramidal neurons by stimulation of Schaffer collaterals.
Data are presented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p <
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4. Figure 3
MC-GC LTP Requires BDNF/TrkB Signaling, but Not CB1 Receptor Activity
(A) Representative traces (left) and time-course summary plot (right) showing that MC-GC LTP in the presence of the CB1 inverse agonist AM 251 (4 μM) was indistinguishable from MC-GC LTP control in interleaved slices.
(B–D) MC-GC LTP was impaired in the presence of the TrkB inhibitor K252a (15 μM) (B), the TrkB receptor antagonist ANA-12 (15 μM) (C), or the BDNF scavenger TrkB-Fc (D) as compared to control human IgG (TrkB-Fc and IgG were used at 1 μg/mL, with a 20-min preincubation, and were also included in the perfusion).
(E) Single-plane confocal images showing the dentate gyrus of TrkB-floxed mice injected with AAV5.CamKII.eGFP (left) or AAV5.CamKII.GFP-Cre (right). Note the presence of GFP-expressing cells in the granule cell layer of the dorsal blade and the absence of GFP expression in the hilus.
(F) Representative traces (left) and time course (right) showing that MC-GC LTP was abolished in TrkB cKO GCs as compared to control GCs.
Numbers in parentheses represent number of cells. Data are presented as mean ± SEM.
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5. Figure 4
BDNF Application Induces MC-GC LTP via a Presynaptic Mechanism
(A) BDNF induced LTP at MC-GC, but not MPP-GC, synapses. Representative traces (left) and time-course summary plot (right) showing that BDNF bath application (8 nM, 15 min) induced a long-lasting increase in the amplitude of MC-GC EPSCs, but not MPP-GC EPSCs, recorded in the same GC.
(B) BDNF-induced MC-GC LTP was associated with a significant reduction of both PPR and CV.
(C) Representative traces (left) and time-course summary plot (right) showing that brief puffs of BDNF in IML (vertical arrow) induced MC-GC LTP (white circles), and this potentiation was abolished in the presence of the TrkB receptor antagonist ANA-12 (15 μM, gray circles). Puffing BDNF in the MML had no long-lasting effects on MPP-GC synaptic transmission.
(D) BDNF-puff-induced MC-GC LTP was associated with a significant reduction of both PPR and CV.
(E and F) BDNF puffs in IML increased sEPSC frequency but not amplitude. (E) Representative experiment showing traces (left) and sEPSC frequency versus time plot (right). Summary data are presented in (F).
(G and H) No changes were observed by puffing BDNF in the presence of the TrkB antagonist ANA-12 (15 μM). Summary data are presented in (H).
Numbers in parentheses represent number of cells. Data are presented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < n.s., not significant.
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6. Figure 5 cAMP/PKA Signaling Is Involved in MC-GC LTP
(A) The PKA inhibitor H89 (10 μM, 40- to 60-min pre-incubation and also included in the perfusion) blocked MC-GC LTP compared to naive (control) hippocampal slices.
(B) Loading PKI6-22 (2.5 μM) in GCs via recording pipette did not affect MC-GC LTP.
(C) The effect of transient FSK bath application (50 μM) was assessed on MC and MPP-mediated transmission in the same GC. FSK induced much larger potentiation at MC-GC synapses than at MPP synapses. Left: representative traces; middle: time-course summary plot of FSK-induced potentiation at MC- and MPP-GC synapses; right: PPR and CV of MC EPSCs before and after FSK application.
(D) Preapplication of FSK (50 μM bath application for 10 min) occluded MC-GC LTP.
(E) After induction of MC-GC LTP, the stimulation strength was reduced to the original baseline (white arrow) to avoid a ceiling effect, and then, after obtaining a new baseline, FSK (50 μM, 10 min) was applied.
(F) The cell-permeant PKA inhibitor PKI14–22 myristoylated (1 μM bath application) blocked BDNF-induced MC-GC LTP compared to interleaved controls.
(G) FSK-induced LTP (50 μM, 10 min) recorded from cKO and control GCs.
Numbers in parentheses represent number of cells. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗∗p <
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7. Figure 6 Optically Elicited MC EPSCs Show FSK-LTP and MC-GC LTP
(A) Schematic diagram showing the injection of the AAV-hSyn-ChR2(H134R)-EYFP or AAV-hSyn-ChIEF-citrine in the hilus.
(B–E) Infrared/differential interference contrast (IR/DIC) (B and D) and fluorescence images (C and E) showing that ChR2(H134R)-EYFP was selectively expressed in the IML of contralateral dentate gyrus.
(F) Schematic diagram showing optic stimulation of ChR2(H134R)-EYFP-expressing MC axons in contralateral hippocampal slices.
(G) Optically evoked EPSCs (O-EPSCs) recorded from GCs were blocked by TTX (0.5 μM) (top) or sequential application of NBQX (10 μM) and D-APV (50 μM) (bottom).
(H) Representative traces (left) and time-course summary plot (right) showing that, as expected for MC inputs, but not MPP inputs, O-EPSCs were insensitive to bath application of DCG-IV (1 μM).
(I) Top: example of optically induced synaptic responses during the LTP protocol (5 pulses, 30 Hz, repeated 50 times at 2 Hz) in hippocampal slices expressing ChIEF-citrine. Insets: first three and last three burst-induced responses are shown on an expanded timescale. Bottom, representative traces (left) and time-course summary plot (middle) showing robust optically induced LTP, which was accompanied by a significant reduction in PPR and CV (right).
(J) Representative traces (left) and time-course summary plot (middle) showing that transient bath application of FSK (50 μM, 10 min) induced long-lasting potentiation of O-EPSCs, and this potentiation was accompanied by a significant reduction of PPR and CV (right).
Data are presented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p <
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8. Figure 7
Assessing Excitatory and Inhibitory Transmission and Plasticity by Optogenetic Activation of MC Axons
(A) Left: schematic diagram illustrating ChR2(H134R)-EYFP infected MCs project to the contralateral GCs and hilar interneurons. Middle: sample traces showing O-EPSC-IPSC sequence recorded from GCs voltage clamped at −60 mV. Note that ECl− is approximately −89 mV. Biphasic currents were abolished by 10 μM NBQX and 50 μM D-APV (middle, red trace). Summary data are shown on the right.
(B) Sample traces showing O-dIPSC was blocked by 100 μM picrotoxin (blue trace) and then O-EPSC was abolished by subsequent application of 10 μM NBQX and 50 μM D-APV (red trace). Right: summary data showing the amplitude of O-dIPSCs (at time point 30 ms after light illumination) before and after application of picrotoxin (100 μM).
(C) Scatterplot of O-EPSC and O-IPSC amplitudes. Each amplitude was analyzed at peak currents, which underestimates real amplitudes of EPSCs/dIPSCs. Black square shows the mean value. Note that dotted line denotes unity.
(D) Simultaneous recordings of O-EPSCs and O-dIPSCs before and after LTP induction. Representative traces (left) and summary time course (right) showing that optically delivered LTP induction (same as Figure 6I) induced LTP at O-EPSCs, but not at O-dIPSCs. To confirm the disynaptic nature of O-IPSCs, 10 μM NBQX and 50 μM D-APV were applied at the end of every experiment (red trace). The amplitudes of O-EPSCs were measured at peak inward currents and O-dIPSCs were measured at peak outward currents. Dotted line shown in traces indicates time point corresponding to 30 ms after light illumination.
(E) Left top: schematic diagram illustrating ChIEF-citrine-infected MCs project to the contralateral GCs and hilar interneurons. Left bottom: z stack two-photon fluorescence images showing an example of Alexa-Fluor-594-filled interneuron in the molecular layer (ML). Right: time-course summary plot showing that repetitive MC axon photostimulation (5 pulses at 30 Hz, repeated 50 times at 2 Hz) did not induce long-lasting synaptic plasticity. Inset: representative O-EPSCs before and after repetitive stimulation. O-EPSCs were recorded in the continuous presence of 100 μM picrotoxin.
Data are presented as mean ± SEM.
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9. Figure 8 Functional Consequences of MC-GC LTP on the GC Output
(A and B) Representative experiment showing GC firing elicited by burst stimulation in IML (5 pulses, 20 Hz) before and after bath application of picrotoxin (100 μM). (A) Sample traces. (B) Time-course plot of the number of spikes per burst.
(C) Summary data showing the spike probability before and after application of picrotoxin.
(D and E) Representative experiment showing GC firing elicited by burst stimulation in IML (5 pulses, 20 Hz), before and after delivering the LTP induction protocol. (D) Sample traces. (E) Time-course plot of the number of spikes per burst showing the long-lasting enhancement of MC-driven GC firing after LTP induction.
(F and G) Sample traces of two representative experiments showing burst stimulation before and after application of the LTP induction protocol in the presence of 15 μM ANA-12 (F) or 10 μM H89 (G).
(H) Summary plot showing the spike probability under three different conditions. The increase in spike probability after application of the protocol was abolished either in the presence of 10 μM H89 or 15 μM ANA-12.
Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < n.s., not significant.
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