Excitatory selective LTP of supramammillary glutamatergic/GABAergic cotransmission potentiates dentate granule cell firing

Abstract

SignificanceIt is now established that many neurons can release multiple transmitters. Recent studies revealed that fast-acting neurotransmitters, glutamate and GABA, are coreleased from the same presynaptic terminals in some adult brain regions. The dentate gyrus (DG) granule cells (GCs) are innervated by the hypothalamic supramammillary nucleus (SuM) afferents that corelease glutamate and GABA. However, how these functionally opposing neurotransmitters contribute to DG information processing remains unclear. We show that glutamatergic, but not GABAergic, cotransmission exhibits long-term potentiation (LTP) at SuM-GC synapses. By the excitatory selective LTP, the excitation/inhibition balance of SuM inputs increases, and GC firing is enhanced. This study provides evidence that glutamatergic/GABAergic cotransmission balance is rapidly changed in an activity-dependent manner, and such plasticity may modulate DG activity.

Keywords: LTP; corelease; dentate gyrus; depolarization; supramammillary nucleus.

Fig. 1.

Depolarization of GCs exhibits LTP of glutamatergic SuM-GC cotransmission. (A, Left) Diagram illustrating injection of AAV-DIO-ChR2(H134R)-eYFP into the SuM of VGluT2-Cre mouse. (Right) Confocal images showing ChR2(H134R)-eYFP-expressing SuM axons in the DG (Upper) and injection site (Lower). (B) A pairing protocol (200 light pulses at 2 Hz, paired with 0-mV postsynaptic depolarization, arrow) induced robust LTP of SuM-GC oEPSCs (white circle). Same pairing protocol still induced LTP in the presence of 50 μM D-AP5 (red circle). Postsynaptic depolarization without presynaptic activity also induced LTP (blue circle), whereas presynaptic activity without postsynaptic depolarization failed to induce LTP (black circle). Representative traces, which correspond to the numbers in the time-course plot below (for this and all subsequent figures), are shown on the top. For this and all subsequent figures, blue bars indicate the time when blue light was delivered to slices. (C) Representative experiment (Upper) and summary plot (Lower) shows repeated depolarizations (2-s duration repeated 10 times every 5 s, arrow) of GCs induced robust LTP of SuM-GC oEPSCs. (D) The magnitude of depol-eLTP depends on the number of depolarizing pulses. Each protocol was applied to the different cells. Summary data at right shows the magnitude of LTP induced by different numbers of depolarizing pulses (once, 132 ± 13% of baseline, n = 11; three times, 167 ± 22% of baseline, n = 11; 10 times, 214 ± 11% of baseline, n = 11). One-way ANOVA, P < 0.001, Tukey’s post hoc test *P < 0.05; ***P < 0.001. (E) Burst APs in GCs at θ frequency (10 bursts of 40-ms current injection, which elicited three to four APs, at 5 Hz, repeated five times every 5 s, arrow) induced LTP. (Inset) Example trace of a single burst APs. (F) Depol-eLTP was normally induced by repeated postsynaptic depolarizations (arrow) in the presence of 50 μM D-AP5. PPR was not changed after induction of depol-eLTP. (Left) Representative traces; (Center) time course summary plot of depol-eLTP (Upper) and normalized PPR (Lower); (Right) summary plot of PPR. Gray bars in (B and C) indicate the time windows for quantification of the magnitude of LTP. Here and in all figures, the magnitude of LTP was measured by comparing baseline responses with the last 10-min responses after LTP induction shown in each experiment. Data are presented as mean ± SEM.

Fig. 2.

Depol-eLTP at SuM-GC synapses is expressed postsynaptically, and GABAergic cotransmission is intact following GC depolarization. (A) The effect of postsynaptic depolarizations on asynchronous synaptic responses in the presence of Sr²⁺. Representative traces (Left) of asynchronous SuM-GC oEPSCs before (10-min baseline) and after induction of depol-eLTP (20 min after GC depolarizations). (Center) Cumulative amplitude and interevent interval distributions of asynchronous events obtained before and after depol-eLTP induction. (Right) Amplitude and frequency summary plots of asynchronous events obtained before and after depol-eLTP induction. For induction of depol-eLTP, extracellular Sr²⁺ solution was replaced by normal artificial cerebrospinal fluid (ACSF) containing Ca²⁺ after 10-min baseline. After confirming the induction of depol-eLTP, extracellular solution was returned to Sr²⁺-containing ACSF. (B) SuM-GC NMDAR-oEPSCs were recorded at −60 mV in the presence of 10 μM NBQX and 100 μM picrotoxin (black trace). NMDAR-oEPSCs were completely blocked by 50 μM D-AP5 (red trace, n = 8, P < 0.001, paired t test). (C) The depol-eLTP induction protocol failed to induce LTP of NMDAR-oEPSCs. (D, Left) Schematic diagram illustrating blockade of glutamatergic transmission by NBQX and D-AP5 leaving GABAergic cotransmission intact at SuM-GC synapses. (Center) Repetitive postsynaptic depolarizations (open circles: 10 pulses; filled circles: 20 pulses) failed to induce LTP of SuM-GC oIPSCs. (Right) PPR of SuM-oIPSCs before and after (0 to 5 min after depolarization) 20 depolarizing pulses. Data are presented as mean ± SEM; ***P < 0.001. n.s., not significant.

Fig. 3.

SuM input-specificity and its target cell-specificity of depol-eLTP. (A) Schematic of recording of electrically evoked MPP-EPSCs and optically evoked SuM-EPSCs from the same GC. Each input was alternately stimulated every 10 s. (B) Repetitive depolarizing pulses of GCs (arrow) elicited LTP of SuM-oEPSCs but not MPP-EPSCs. (C) Experimental diagram. A confocal image of DG obtained from a VGluT2-Cre/VGAT-Venus mouse expressing ChR2(H134R)-mCherry in the SuM axons. Whole-cell recording was performed from a Venus⁺ IN. (D) Repetitive depolarizations of INs failed to induce LTP, while interleaved recordings from GCs exhibited depol-eLTP. (E, Upper Left) ChR2(H134R)-eYFP-expressing SuM axons project to CA2 in addition to the DG. (Right) A confocal image of a biocytin-filled CA2 pyramidal neuron. (Lower Left) Intrinsic electrophysiological properties in responses to 1-s current steps in a CA2 pyramidal neuron. As typical characteristics of CA2 pyramidal neurons, delayed APs and minimal sag were elicited by a positive and negative current injection, respectively. (F) The depol-eLTP induction protocol did not induce LTP at SuM-CA2 pyramidal neuron synapses. Data are presented as mean ± SEM.

Fig. 4.

Molecular mechanisms underlying depol-eLTP. (A) Depol-eLTP required a postsynaptic Ca²⁺ increase through L-VDCCs. Postsynaptic loading with 20 mM BAPTA failed to induce depol-eLTP. Postsynaptic depolarizations (arrow) abolished depol-eLTP in the presence of 30 μM nifedipine. Numbers in parentheses, here and in all figures, indicate the number of cells. (B) Depletion of intracellular Ca²⁺ stores by CPA (30 μM) had no effect on depol-eLTP. (C) Bath application of the PKA inhibitor H89 (10 μM) had no effect on depol-eLTP. (D) Bath application of the PKC inhibitor Gö6983 (1 μM) had no effect on depol-eLTP. (E) Bath application of the CaMKII inhibitor KN-93 (10 μM) abolished depol-eLTP. (F) Postsynaptic loading with the CaMKII inhibitor AIP (10 μM) abolished depol-eLTP. (G) Depol-eLTP was blocked by postsynaptic loading with NEM (500 μM). (H) Postsynaptic loading with BoTx (200 ng/mL) abolished depol-eLTP, while heat-inactivated BoTx (control) normally induced depol-eLTP. Data are presented as mean ± SEM.

Fig. 5.

High NMDAR/AMPAR ratio in GCs and synapse unsilencing induced by depolarization of GCs. (A) Marked difference in the NMDAR/AMPAR ratios at SuM-GC and SuM-IN synapses. (Left) oEPSCs recorded from GCs and INs at −60 mV and +40 mV. oEPSCs at +40 mV were recorded in the presence of 10 μM NBQX. (Center) Quantification of the amplitudes of AMPAR- and NMDAR-mediated currents recorded from GCs and INs. (Right) Summary data showing the NMDAR/AMPAR ratios (GC: 8.5 ± 0.67, n = 17; IN: 1.3 ± 0.16, n = 15, P < 0.001, unpaired t test). (B) Representative experiment of sample traces (Upper: six sweeps overlaid) and time course (Lower). oEPSCs evoked by weak light illumination at −60 mV showed slow rise time and were completely blocked by 50 μM D-AP5. Under this condition (no detectable oEPSCs), repetitive postsynaptic depolarizing pulses (arrow) elicited appearance of oEPSCs. NBQX (10 μM) was applied at the end of experiment to verify the response was mediated by AMPARs. After washout of D-AP5 in the presence of NBQX, NMDAR-oEPSCs were recovered without any potentiation. (C) Summary plots demonstrating that synapse unsilencing was associated with a significant decrease in failure rate, increase in efficacy and potency and no significant change in NMDAR-oEPSCs. Data are presented as mean ± SEM; **P < 0.01, ***P < 0.001. n.s., not significant.

Fig. 6.

TBS of MPP inputs heterosynaptically induces eLTP at SuM-GC synapses. (A) Schematic of recording configuration. MPP inputs were electrically stimulated by a glass electrode and ChR2-eYFP expressing SuM fibers were optically stimulated by a blue light pulse. For induction of LTP, MPP inputs were stimulated by TBS. (B) Representative experiment showing that TBS of MPP inputs (vertical arrow) induced robust LTP of SuM-GC oEPSCs. (Inset) APs during TBS. (C) Summary data showing that TBS-induced LTP was completely blocked by intracellular loading of BAPTA (20 mM). (D) TBS-induced LTP was induced in the presence of D-AP5 (50 μM) but blocked by bath application of KN93 (10 μM). Data are presented as mean ± SEM.

Fig. 7.

SuM inputs trigger spike generation in GCs by increasing excitatory drive associated with the induction of depol-eLTP. (A and B, Upper) Representative traces showing GC firing elicited by burst light illumination (four pulses, 20 Hz) before and after induction of depol-eLTP in the control (A) and in the presence of 100 μM picrotoxin (PTX) (B). (Lower) Time-course plots of the number of spikes per burst (control, n = 7; PTX, n = 11). After 5-min baseline (no spike), the recording was switched to voltage-clamp mode, and GCs were depolarized repetitively to induce depol-eLTP (arrow). Membrane potential was held at −80 mV to −85 mV in current-clamp mode. (C and D) Cumulative number of spikes (C) and frequency (D) in control and PTX (P = 0.054, Kolmogorov–Smirnov test). (E) Time-course plot of the spike probability after induction of depol-eLTP in control (n = 24) and PTX (n = 25). In the presence of PTX, induction of depol-eLTP significantly increased spike probability (P < 0.01, two-way ANOVA). Nonspiking cells were included in the analysis. (F and G) Burst light illumination (four pulses, 20 Hz) was applied while GCs were held at −60 mV to −65 mV in current-clamp mode. Time-courses of the number of spikes per burst were plotted (control, n = 9; PTX, n = 13). (H and I) Cumulative number of spikes (H) and frequency (I) in control and PTX (P < 0.001, Kolmogorov−Smirnov test). (J) Time-course plot of the spike probability after induction of depol-eLTP in control (n = 25) and PTX (n = 24). In the presence of PTX, induction of depol-eLTP significantly increased spike probability (P < 0.001, two-way ANOVA). Nonspiking cells were included in the analysis. Data are presented as mean ± SEM; **P < 0.01, ***P < 0.001.