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Burst firing is required for induction of Hebbian LTP at lateral perforant path to hippocampal granule cell synapses


High frequency burst firing is critical in summation of back-propagating action potentials (APs) in dendrites, which may greatly depolarize dendritic membrane potential. The physiological significance of burst firings of hippocampal dentate GCs in synaptic plasticity remains unknown. We found that GCs with low input resistance could be categorized into regular-spiking (RS) and burst-spiking (BS) cells based on their initial firing frequency (Finit) upon somatic rheobase current injection, and investigated how two types of GCs differ in long-term potentiation (LTP) induced by high-frequency lateral perforant pathway (LPP) inputs. Induction of Hebbian LTP at LPP synapses required at least three postsynaptic APs at Finit higher than 100 Hz, which was met in BS but not in RS cells. The synaptically evoked burst firing was critically dependent on persistent Na+ current, which was larger in BS than RS cells. The Ca2+ source for Hebbian LTP at LPP synapses was primarily provided by L-type calcium channels. In contrast, Hebbian LTP at medial PP synapses was mediated by T-type calcium channels, and could be induced regardless of cell types or Finit of postsynaptic APs. These results suggest that intrinsic firing properties affect synaptically driven firing patterns, and that bursting behavior differentially affects Hebbian LTP mechanisms depending on the synaptic input pathway.


The dentate gyrus (DG) is the first layer at which synaptic inputs from the entorhinal cortex arrive among the hippocampal tri-synaptic loop, and has been implicated in pattern separation and conjunctive representations of spatial (context) and non-spatial (items and events) information [1]. The afferent fibers from lateral and medial entorhinal cortex (LEC and MEC) layer II give rise to lateral and medial perforant pathways (LPP and MPP). LPP and MPP carry relatively more non-spatial and spatial information to DG, and innervate distal and intermediate parts of granule cell dendrites in the DG, respectively. Synaptic plasticity-based competitive learning together with the integration of spatial and non-spatial inputs in dentate granule cells (GCs) was recently proposed to underlie the progressive refinement of spatial representation in DG [2].

Heterogeneity in cellular excitability could be one of key mechanisms underlying recruitment of principal cells to a neuronal ensemble or an engram for representation and formation of memories [3]. Recent in vivo recordings of GCs in DG revealed that a majority of GC spikes occurred in bursts, and that active GCs, which comprised only a minor subset of GCs, were morphologically mature and distinct from silent GCs [4,5,6]. While these studies imply heterogeneity among dentate GCs, it remains to be understood how the difference in the excitability among heterogeneous groups of mature GCs is related to the difference in synaptic plasticity. Previously, it was noted that the initial frequency (Finit) of first two action potentials (APs) upon somatic current injection is higher than the rest APs in mature GCs, and T-type voltage dependent Ca2+ channels (T-VDCCs) contribute to the burst firing [7]. Burst firing enhances not only the reliability of pre-synaptic glutamate release [8], but also postsynaptic Ca2+ signaling required for synaptic plasticity [9, 10]. Consistently, burst firing of principal cells plays diverse roles in different cortical regions such as place field formation in CA1 [11], initiation of sharp waves in CA3 [12], and switching thalamic network states for relaying subcortical inputs [13]. Moreover, somatic firings at above a critical frequency has been found to greatly depolarize the dendritic membrane potential through summation of back-propagating APs in neocortical pyramidal cells [14].

In the present study, we found that there are two types of mature GCs displaying low input resistance (< 200 MΩ) in young rats, based on whether the Finit of APs elicited by rheobase current injection is higher than 50 Hz or not, referred to as burst-spiking (BS) and regular-spiking (RS) GCs. Similar heterogeneity has been found in CA3 too [12]. While burst firings of GCs are expected to enhance spike transfer from DG to CA3 due to strong short-term facilitation, it is little understood whether burst firing of GCs has any effect on the input side, that is on the long-term potentiation (LTP) at the LPP or MPP-to-GC synapses. To address this issue, we compared LTP at LPP and MPP synapses between RS and BS cells. We show that Hebbian LTP occurs only at LPP synapses to BS cells but not at those to RS cells, while BS and RS cells do not differ in Hebbian LTP at MPP synapses. The present study on the ionic mechanisms underlying LTP at LPP and MPP synapses to RS and BS-GCs revealed that high and low voltage-activated VDCCs play a key role in LTP induction at LPP and MPP synapses, respectively. These results suggest that activation of L-type VDCCs (L-VDCCs) requires high frequency AP firing, providing an insight into why Hebbian LTP at LPP-GC synapses is induced preferentially in BS cells.


Characteristics and distribution of two types of mature GCs

Burst firing of dentate GCs has been observed both in vivo [5] and ex vivo [7], but its physiological significance in synaptic plasticity is not well understood. We examined firing patterns of mature GCs that have input resistance (Rin) less than 200 MΩ in response to somatic current injection [15,16,17,18,19]. When we applied a step current just above action potential (AP) threshold (rheobase current) for 1 s in whole-cell current clamp mode, a group of cells generated APs in bursts, doublet in majority (82.8%, 18 of 22) and sometimes triplet (18.2%, 4 of 22), while others showed regularly spiking patterns (Fig. 1A). The histogram of initial firing frequency (Finit) showed bimodal distribution (Fig. 1B), so that we nominated cells with Finit under 50 Hz as regular-spiking (RS, black), while cells with Finit over 50 Hz as burst-spiking (BS, red) neurons. The mean value for Finit was 10.6 ± 2.2 Hz (n = 18) in RS-GCs and 147.1 ± 11.2 Hz (n = 22) in BS-GCs. As the injection current increased, Finit increased in RS-GCs, and the difference of Finit between RS- and BS-GCs gradually disappeared (Fig. 1Cc). Despite the remarkable difference in Finit, the number of APs during 1 s depolarization was not significantly different between two groups (Fig. 1Cb). Analyses of AP shapes revealed that the threshold voltage for AP generation was lower, AP duration was longer, and afterhyperpolarization (AHP) was smaller in BS-GCs compared to those in RS-GCs (Fig. 1D). No significant difference was found in passive electrical properties such as input resistance (Rin) and resting membrane potential (RMP) (Fig. 1E). Interestingly, in DG-GCs that have Rin more than 200 MΩ, which are less mature according to the criteria of maturation [20, 21], bursting was very rarely observed (Fig. 1F), suggesting that burst firing is a characteristic feature of fully mature DG-GCs.

Fig. 1
figure 1

Intrinsic properties of regular-spiking (RS) and burst-spiking (BS) mature granule cells. A Representative voltage responses of RS (black) and BS (red) cells to somatic current injection of 250, 300 and 650 pA (1 s duration). Inset, initial firing of BS-GCs at expanded time scale. Initial firing frequency (Finit) was measured as the frequency of first two APs at rheobase current injection. B Bimodal distribution of Finit among mature GCs. Mature GCs were divided into RS and BS with the reference frequency of 50 Hz (RS/BS, n = 18/42). C, a, b Finit (a) and spike numbers (b) of RS and BS cells as a function of injected current amplitude (from 50 to 650 pA). Finit of RS cells increased steeply compared to that of BS-GCs. The general excitability of both GCs was not different throughout all steps (RS/BS, n = 18/22). D Summary bar graphs for analyses of 1st AP waveform evoked by somatic rheobase current injection into RS and BS cells. Mean values for AP threshold were − 34.9 ± 0.5 mV in RS and − 36.7 ± 0.7 mV in BS (*p < 0.05). For afterhyperpolarization (AHP) amplitudes, 17.1 ± 0.5 mV in RS and 12.0 ± 0.5 mV in BS (***p < 0.001). For half-width duration, 0.70 ± 0.01 ms in RS and 0.79 ± 0.01 ms in BS (***p < 0.001; RS/BS, n = 18/22). E Input resistance (Rin; RS, 116.1 ± 9.6 MΩ; BS, 115.3 ± 6.1 MΩ) and resting membrane potential (RMP; RS, − 82.1 ± 1.3 mV; BS, − 81.2 ± 0.9 mV) were not different between RS and BS. F Proportion of RS and BS cells depends on the GC maturity. BS cells were more frequently found in the group of mature GCs (Rin < 200 MΩ) compared to the less mature GC group (Rin ≥ 200 MΩ; RS/BS, n = 16/17). G Distributions (left) and mean values (right) for maximal dendritic branch order in RS (black) and BS (red) cells (RS, 4.5 ± 0.3, n = 8; BS, 5.1 ± 0.1, n = 18, p = 0.10). H Proportion of RS and BS cells along the dorsoventral axis (RS/BS in dorsal, n = 84/148; in ventral, n = 37/8). Error bars indicate S.E.M. *p < 0.05. ***p < 0.001. n.s., not significant (p > 0.05)

To explore whether the bursting behavior is related to morphological properties of GCs, we counted the maximal branch order (MBO) from z-sections of confocal images of biocytin-filled RS- and BS-GCs (Additional file 1: Fig. S1). The MBO of majority (65.4%) of mature GCs was five, while cells with MBO higher than five was only found in BS-GCs and that with lower than 4 is only found in RS-GCs (Fig. 1G). The average MBO of BS-GCs, however, was not different from that of RS-GCs (RS, 4.5 ± 0.3, n = 8; BS, 5.1 ± 0.1, n = 18, p = 0.10, Mann–Whitney test). We then examined whether the relative proportion of BS- and RS-GCs differs along the hippocampal dorso-ventral axis. We found that BS-GCs were dominant in the dorsal DG, and its proportion was opposite in the ventral DG. Among 232 recorded neurons in dorsal DG, 148 (64%) GCs were identified as BS-GCs, while only 8 (18%) out of 45 GCs were BS-GCs in ventral DG, indicating that the dorsal DG harbors more BS-GCs compared to the ventral DG (Fig. 1H).

Subthreshold EPSP summation evoked by a single bout of HFS induces NMDA receptor-dependent LTP at LPP-GC synapses

To investigate whether intrinsic firing patterns have any effects on long-term synaptic plasticity, we recorded excitatory postsynaptic potentials (EPSPs) from RS-GCs or BS-GCs by stimulating lateral perforant pathways (LPP) in the presence of PTX (100 μM, a GABAAR blocker) and CGP52432 (1 μM, a GABABR blocker) (Fig. 2A). After measuring the baseline EPSPs evoked by stimulation of LPP in a 10 s interval for about 5 min, a single bout of high frequency stimulation (HFS, 10 stimuli at 100 Hz) was applied. For the HFS, we tested two different levels of electrical stimulation intensity: low intensity to induce subthreshold response (HFSL) and high intensity to evoke at least 3 APs (HFSH). Compared to MPP-EPSPs, LPP-EPSPs exhibited significantly higher paired pulse ratio and larger normalized area of HFSL-evoked EPSP summation to the 1st EPSP amplitude (Additional file 1: Fig. S3A; Figs. 2B vs. 7B). The average stimulation intensities for HFSL and HFSH of LPP were 15.6 ± 0.9 V (n = 21) and 25.7 ± 1.4 V (n = 18), respectively (Additional file 1: Fig. S3B). The average amplitudes of baseline EPSPs induced by HFSL and HFSH were 5.9 ± 0.3 and 13.8 ± 1.0 mV, respectively (Additional file 1: Fig. S3C). Temporal summations of EPSPs evoked by HFSL reached their peaks between − 60 mV and − 40 mV at the 6th or 7th stimulus. RS- and BS-GCs showed no detectable difference in the temporal summation kinetics (RS, black; BS, red; Fig. 2B). Despite that HFSL of LPP evoked no postsynaptic AP, it induced long-term potentiation (LTP) of EPSP amplitudes in both GC groups, which lasted at least 30 min (Fig. 2C). We denoted this form of LTP as LTPsub, which stands for LTP induced by subthreshold stimulation. The increase in baseline EPSP amplitudes after HFSL was not different between BS-GCs (36.9 ± 8.9%, n = 13) and RS-GCs (35.2 ± 5.3%, n = 12, p = 0.65). The magnitude of LTPsub was correlated with the peak of EPSP summation (r = 0.54, p < 0.001), and significant LTPsub was induced when the peak was higher than − 60 mV (Additional file 1: Fig. S3D). To examine the involvement of NMDA receptor (NMDAR) in LTPsub, we tested the effect of APV (50 μM, a NMDAR blocker) on EPSP responses and LTP expression induced by HFSL. APV profoundly suppressed the baseline EPSPs as well as EPSP summation (Fig. 2E), and abolished LTPsub (Fig. 2F). These results suggest that NMDAR-dependent LTP can be induced at LPP-GC synapses by a single bout of HFS that evokes only a subthreshold voltage response.

Fig. 2
figure 2

NMDAR-dependent LTP at LPP-GC synapses is induced by a single bout of high frequency stimulation (HFS) at subthreshold level. A Schematic diagram illustrating the recording configuration for synaptic stimulation and whole cell recording of mature GC. Lateral perforant pathway (LPP) in outer molecular layer (OML) was electrically stimulated by a bout of HFS (10 stimuli at 100 Hz). Scale bar is 100 μm. B HFSL is defined as low intensity HFS that induce subthreshold response. HFSL-evoked subthreshold responses of RS- (black) and BS-GCs (red) (left), and their cumulative EPSP amplitudes (right). C Time courses of normalized EPSP amplitude before and after HFSL (same as in B). Each point represents averaged value for adjacent 3 EPSP amplitudes (30 s binned). Black dashed line denotes baseline EPSP. Inset, Representative traces for average of 30 EPSP traces before (a) and 26–30 min (b) after HFSL (This holds for inset traces in all subsequent figures except in Fig. 2F and Fig. 7C). D LTP magnitudes before and after HFSL. There was no significant difference between RS and BS (RS/BS, n = 12/13). E Left, Representative traces for EPSP summation in control (black) and after application of APV (brown, 50 μM). Right, Mean values for EPSP area in RS [3.0 ± 0.3 mV s (Con) vs. 1.3 ± 0.2 mV s (APV), n = 5, *p < 0.05] and in BS [2.9 ± 0.3 mV s (Con) vs. 1.9 ± 0.1 mV s (APV), n = 5, *p < 0.05]. Note that the APV effect on subthreshold EPSP summation was examined at synapses which have already underwent LTPsub. F Time course of normalized EPSP before and after HFSL in the presence of APV in both GCs. EPSP amplitude was not potentiated (RS, 1.7 ± 6.0%, n = 3, light brown; BS, − 2.5 ± 4.5%, n = 4, brown). Inset, EPSPs averaged over 1 to 5 min before (a) and after (b) HFSL. Shades and error bars, S.E.M. *p < 0.05. n.s., not significant (p > 0.05)

Postsynaptic burst firing is essential for Hebbian LTP at LPP-GC synapses

We then examined whether AP firings in response to HFSH show any difference between BS and RS (Fig. 3A). The Finit of HFSH-evoked APs was mostly higher than 100 Hz in BS-GCs (128.3 ± 6.9 Hz, n = 21, Fig. 3A). Furthermore, BS-GCs showed a moderate correlation between the Finit of synaptically evoked APs and that of APs evoked by somatic stimulation (r = 0.55, Fig. 3B). In contrast, the Finit of HFSH-evoked APs in RS-GCs was significantly lower than that in BS-GCs (92.0 ± 9.9 Hz, n = 18; p < 0.01; Fig. 3A). These results suggest that mechanisms underlying intrinsic firing pattern contribute to synaptically evoked firing pattern. When the 2nd HFS with higher stimulation intensity (denoted as ‘HFSH-2’) was applied 10 min after HFSL by which LTPsub has been already expressed both in RS and BS, HFSH-2 induced further potentiation of EPSPs in BS-GCs, but not in RS-GCs (Fig. 3C). The time course of this LTP induced by HFSH-2 is shown as the EPSP amplitudes normalized to the EPSP amplitude just before applying HFSH-2 (Fig. 3D). The increase in the EPSP amplitude at 30 min was 44.0 ± 4.8% (n = 7) in BS-GCs, but negligible in RS-GCs (− 4.2 ± 7.0%, n = 12; p < 0.001). These results indicate that BS-GCs express postsynaptic AP-dependent LTP (denoted as LTPAP) distinct from NMDAR-dependent LTPsub. There was a positive correlation between LTPAP magnitudes and Finit of synaptically evoked APs (Fig. 3E). Moreover, when only 1 or 2 APs were elicited by HFS with medium intensity (HFSM), LTP was not induced or not maintained even in BS-GCs (− 10.6 ± 12.8%, n = 6; Additional file 1: Fig. S4A, B), indicating that postsynaptic AP bursts comprised of at least 3 APs at the frequency higher than 100 Hz are essential for the induction of LTPAP.

Fig. 3
figure 3

Hebbian LTP depends on post-synaptic AP bursts, and can be induced only in BS. A Left, Representative voltage responses in RS (black) and BS (red) to HFSH-2 which elicited 3 APs. HFSH-2 is defined as high intensity HFS that was applied following HFSL. Right upper, The boxed traces are superimposed for comparison at expanded time scale. Right lower, Initial AP frequency (Finit) of each group (RS/BS, n = 18/21; **p < 0.01). B Relationship between Finit of APs evoked by somatic current injection and that by synaptic stimulation. Two parameters were significantly correlated in BS (r = 0.55, **p < 0.01) but not in RS (r = 0.11). Black bold line, linear regression in BS. r, Pearson’s correlation coefficient. C Time course of normalized EPSP changes induced by applying two sequential HFS (HFSL and HFSH) in RS (black, n = 5) and BS (red, n = 5). Note that LTPAP was induced on top of LTPsub in BS, not in RS. D Left, Time course before and after HFSH-2. Right, Magnitude of LTPAP in RS- and BS-GCs (RS: − 4.2 ± 7.0%, n = 12; BS, 44.0 ± 4.8%, n = 7, ***p < 0.001). E LTP magnitude as a function of synaptically evoked Finit. Finit was correlated to LTP magnitude (r = 0.50, *p < 0.05). Open circles, individual data; closed circles, averaged value for each group. Black line, linear regression line. F Pairing protocol for LTPAP induction. It consists of subthreshold HFS (HFSL-2) and post-synaptic 3 APs evoked by somatic pulses (2 ms, 3 nA at 100 Hz). G Left, Time course of normalized EPSP before and after a pairing protocol. Right, Pairing protocol-induced LTPAP in RS- and BS-GCs (RS/BS, n = 8/6). H LTP was not induced in the presence of intracellular solution containing BAPTA (10 mM, pink, n = 7). The pairing protocol-induced LTP time courses in RS and BS under control conditions shown in G were merged, and superimposed in gray (n = 14). I Left, Representative voltage traces evoked by HFSH-2 in the presence of APV (brown, 50 μM). No AP burst was elicited in the presence of APV. Right, Time course of normalized EPSP before and after HFSH-2. J Similar as in I, but a pairing protocol was applied instead of HFSH-2. The control trace (gray) was reproduced from panel H for comparison. K Early (LTP5, open circle) and late phase (LTP30, closed circle) LTP induced by a pairing protocol with and without APV (Control, n = 14; APV, n = 11). Shades and error bars, S.E.M. *p < 0.05. **p < 0.01. ***p < 0.001. n.s., not significant (p > 0.05)

To further test the importance of AP frequency for induction of LTPAP, we applied a pairing protocol, in which 10 EPSPs were evoked by HFSL coinciding with 3 APs at 100 Hz evoked by brief current injection to the soma (Fig. 3F, see Methods). The pairing protocol was delivered to the GCs that have already underwent subthreshold LTP. The pairing protocol successfully induced LTP regardless of cell types with no significant difference in the LTP magnitude between RS-GCs and BS-GCs (Fig. 3G), but 3 APs at 50 Hz failed to induce LTP (Additional file 1: Fig. S4C). These findings show that RS-GCs could express LTPAP as if BS-GCs did as long as high frequency APs are paired with synaptic stimulation. Therefore, we did not distinguish BS and RS but pooled the BS and RS data when we analyzed LTP response induced by pairing protocol (gray trace in Fig. 3H). LTP was not induced when intracellular Ca2+ was chelated with a high concentration of BAPTA (10 mM, Fig. 3H). These results confirm that at least 3 APs at the frequency higher than 100 Hz are required to activate LTPAP, and suggest that Ca2+-dependent mechanisms underlie this form of LTP. Finally, we examined the input specificity of LTPAP by monitoring EPSPs evoked by two stimulation electrodes placed at the outer molecular layer. To induce LTPAP using the pairing protocol, HFSL was delivered to one of two stimulation electrodes. LTPAP was induced at the synapse which underwent HFSL paired with 3 APs at 100 Hz, but not at the other synapse (Additional file 1: Fig. S2). Because LTPAP satisfied the conditions for Hebbian LTP, henceforth we will refer to LTPAP as Hebbian LTP.

NMDAR mediates the early phase LTP and facilitates EPSP summation at LPP-GC synapses

To examine whether LTPAP shares the same Ca2+ source with NMDAR-dependent LTPsub, we tested the effect of APV. Because APV profoundly suppressed EPSP summation (Fig. 2E), in the presence of APV it was difficult to generate 3 APs even with high intensity stimulation, and thus LTPAP was not induced (Fig. 3I), indicating that NMDAR current is critical for EPSP summation to elicit high frequency AP generation. However, we could induce LTP by the pairing protocol in the presence of APV (Fig. 3J). Because the time course of LTP development was distinct from that of control pairing-induced LTP (brown vs. gray traces in Fig. 3J), we compared the LTP magnitudes in the APV conditions with the control values for the early and late phases. To this end, we measured normalized EPSP amplitudes averaged over 1 to 5 min and over 26 to 30 min after HFS, and denoted as LTP5 and LTP30, respectively. LTP5 encompasses short-term potentiation (STP) and early phase LTP [22]. LTP5 in the APV conditions was significantly lower, while LTP30 was not different compared to the corresponding control values [LTP5, 8.6 ± 5.5 vs. 25.7 ± 4.6%, p < 0.05; LTP30, 46.4 ± 12.7 vs. 52.62 ± 7.71%, p = 0.57; APV (n = 11) vs. Control (n = 14), Mann–Whitney test, Fig. 3K]. These results suggest that the contribution of NMDAR to Hebbian LTP as Ca2+ source is limited to STP and early phase LTP at LPP-GC synapses, whereas it is essential for EPSP summation and AP burst generation.

T-VDCC contributes to the late phase LTP by facilitating AP bursts at LPP-GC synapses

We showed that burst firing evoked by somatic rheobase current injection (called intrinsic burst firing) has correlation with Finit of synaptically evoked APs which is crucial for LTPAP induction (Fig. 3). We investigated whether ion channel mechanisms underlying intrinsic burst firing also contribute to LTPAP. Since T-VDCC is known to mediate intrinsic bursting in DG-GCs [7], we investigated the role of T-VDCCs in burst firing behavior and LTPAP induction in BS-GCs. Bath application of NiCl2 (50 μM, the blocker of T-VDCC) significantly reduced Finit of intrinsic burst firing (Control, 171.8 ± 13.3 Hz; NiCl2, 38.1 ± 8.89 Hz, n = 9; p < 0.01; Wilcoxon signed-rank test; Fig. 4A). When the bursts were synaptically evoked, NiCl2 partially but significantly reduced the Finit (Control, 128.3 ± 6.9 Hz, n = 21; NiCl2, 91.2 ± 3.5 Hz, n = 13, p < 0.001, Mann–Whitney test; Fig. 4B). Nevertheless, in the presence of 50 μM Ni2+, temporal summation of EPSPs evoked by HFSL was little affected (n = 10, p = 0.11; Fig. 4C), and HFSH was able to induce LTPAP in the BS cells (Fig. 4D). In contrast to APV, the early phase LTP was preserved in the presence of Ni2+ [LTP5 of Ni2+ (n = 7) vs. control (n = 7), 24.9 ± 5.6 vs. 27.5 ± 6.3%, p = 0.90], but no further increase in the EPSP amplitudes was observed (Figs. 4D vs. 3D), and thus LTP30 was lower than the control (20.4 ± 7.7 vs. 44.6 ± 5.7%, p < 0.05, Fig. 4D). Because Ni2+ lowered the Finit of synaptically evoked AP bursts, we tested if suppression of late LTPAP can be rescued by pairing protocol. The mean value for LTP30 measured after the pairing protocol was slightly lower but not significant compared to pairing-induced LTP in control (32.6 ± 13.0 vs. 52.6 ± 7.7%, n = 8, p = 0.19; Fig. 4E), suggesting partial or little contribution of T-VDCC to the LTPAP induction. Similar to HFSH-induced LTP, LTP5 was not different from the control value (25.0 ± 8.2 vs. 25.7 ± 4.6%, p = 0.97). These results suggest that T-VDCC primarily contributes to the late phase LTPAP by enhancing Finit.

Fig. 4
figure 4

T-type Ca2+ channels contributes to the late phase Hebbian LTP by facilitating AP bursts. A Left & Middle, Voltage responses of a BS cell to somatic rheobase current injection before (red, Con) and after bath application of NiCl2 (50 μM, green). Inset of left panel, the boxed trace in an expanded time scale. Right, Mean values for Finit before and after NiCl2 application (n = 9). B Left, Representative EPSP summation in a BS cell evoked by HFSH-2 of LPP in the presence of NiCl2. Right, Finit of AP bursts evoked by HFSH-2 of LPP in BS cells under control and NiCl2 (n = 13) conditions. C EPSP summation evoked by HFSL-2 (left) and the mean area (right) before and after applying NiCl2 (Con, 2.2 ± 0.2 mV s; NiCl2, 2.0 ± 0.2 mV s; n = 10). D Left, Time courses of normalized EPSP in BS cells before and after HFSH-2 with (green) and without (light red) NiCl2. The control time course was reproduced from Fig. 3D for comparison. Right, Magnitude of HFSH-induced LTP in the early (LTP5, open circle) and late (LTP30, closed circle) phases in BS cells (Control, n = 7; NiCl2, n = 7). E Similar as in D, but evoked by a pairing protocol (Control, n = 14, gray; NiCl2, n = 8, green). Shades and error bars, S.E.M. *p < 0.05. **p < 0.01. ***p < 0.001. n.s., not significant (p > 0.05)

Persistent Na+ current amplifies LPP-evoked EPSP summation and is essential for burst firing

Previously, it was shown that T-VDCC in axon initial segment plays a key role in intrinsic burst firing of GCs [7]. Whereas Ni2+ abolished intrinsic bursts (Fig. 4A), it partially reduced Finit of synaptically evoked bursts with little effect on EPSP summation (Fig. 4B, C), implying a possible involvement of dendritic channels in synaptically evoked AP bursts. As a candidate ion channel regulating intrinsic and synaptically evoked bursts, we examined persistent sodium current (INa,P). In CA1 pyramidal cells, INa,P amplifies subthreshold EPSPs leading to spatially tuned firing [23]. We measured Finit of intrinsic bursts in BS-GCs after applying riluzole (10 μM), a typical INa,P blocker [23,24,25]. Riluzole significantly reduced Finit of the intrinsic bursts (Fig. 5A) similar to its effect in CA1 pyramidal neurons [24]. In addition, it markedly suppressed summation of HFSL-evoked EPSPs (Fig. 5B). Due to the substantial inhibition of EPSP summation by riluzole, it was not possible to synaptically evoke AP bursts, even with very high stimulation intensity, and LTP was not induced (Fig. 5C). When 10 EPSP bursts induced by HFSL were paired with 3 APs (pairing protocol), however, the late phase LTP was completely rescued (LTP30, 43.3 ± 14.7 vs. 52.6 ± 7.7%, n = 10, p = 0.34 compared to pairing-induced LTP in control, Mann–Whitney test; Fig. 5E). The rescue of late phase LTP by the pairing protocol suggests that burst APs coincident with synaptic inputs is essential for the late LTP induction. By contrast, early phase LTP was significantly lower than the control value (LTP5, 9.6 ± 6.7 vs. 25.7 ± 4.6%, n = 10, p < 0.05), resulting in the LTP time course similar to that in the APV condition (Fig. 3J, K). This similarity may be explained by assuming that Ca2+ influx through NMDAR mediates the early phase LTP, and that INa,P contributes to NMDAR activation in distal dendrites by amplifying EPSP summation, which cannot be compensated by somatic bursts.

Fig. 5
figure 5

Persistent Na+ current (INa,P) amplifies LPP-evoked EPSP summation and is essential for burst firing. A Left & Middle, AP trains in BS elicited by somatic rheobase current injection in control (red, Con) and after applying riluzole (blue, Ril, 10 μM). Right, Mean Finit before and after application of riluzole (Con, 176.5 ± 12.9 Hz; Ril, 5.7 ± 1.4 Hz; n = 10; **p < 0.01). B Left, EPSP summation evoked by HFSL-2 before and after applying riluzole. Right, Cumulative EPSP amplitudes in control and riluzole conditions (n = 24). C Representative voltage response to HFSH-2 (left) and time course of normalized EPSP before and after HFSH-2 (right, n = 5) in the presence of riluzole. D Similar as in C, but evoked by a pairing protocol. E Early (LTP5, open circle) and late phase (LTP30, closed circle) LTP evoked by a pairing protocol in control and riluzole conditions, showing that late LTPAP was rescued (Con, n = 14; Ril, n = 10). F Cartoon for focal application of riluzole (50 μM) at soma or dendrite during somatic current injection or HFSL of LPP. Scale bar is 100 μm. G Representative traces (upper) and mean Finit (lower) of intrinsic AP bursts with somatic (left) and dendritic (right) puff of riluzole [Soma, 128.8 ± 12.8 Hz (Con) vs. 9.0 ± 3.0 Hz (Ril), n = 12, **p < 0.01; Dend, 137.5 ± 13.3 Hz (Con) vs. 137.6 ± 14.1 Hz, n = 6]. H Similar as in G, but area of subthreshold EPSP summation evoked by HFSL-2. The EPSP summation was not reduced by somatic puff (left) but by dendritic puff (right) [Soma, 2.6 ± 0.3 mV s (Con) vs. 2.5 ± 0.4 mV s (Ril), n = 7; Dend, 2.9 ± 0.5 mV s (Con) vs. 1.1 ± 0.1 mV s, n = 8]. I Left, Procedure for measuring INa,P in GCs. Right, Representative current responses of RS- (black) and BS-GC (red) to a voltage ramp. J Peak INa,P amplitude in RS and BS cells (RS, 144.9 ± 15.6 pA, n = 10; BS, 245.4 ± 16.4 pA, n = 8; ***p < 0.001). Shades and error bars, S.E.M. *p < 0.05. **p < 0.01. ***p < 0.001. n.s., not significant (p > 0.05)

Since riluzole showed profound effects on both intrinsic and synaptically evoked firings, we hypothesized that intrinsic bursting behavior is mainly affected by somatic INa,P, while synaptically evoked AP firings are affected by dendritic INa,P. To test this, we examined the effect of focal puff application of riluzole (Fig. 5F). Peri-somatic puff application of riluzole (50 μM) considerably reduced the Finit of intrinsic burst firings in BS-GCs, whereas dendritic puff had no effect at all (Fig. 5G). On the contrary, the EPSP summation was profoundly diminished by dendritic puff, but not by peri-somatic puff of riluzole (Fig. 5H). These results support our hypothesis for the preferential roles of somatic and dendritic INa,P on the intrinsic and synaptically evoked firing behaviors, respectively. Since BS-GCs have higher Finit both for intrinsic and synaptically evoked APs than RS-GCs, we tested whether difference in INa,P density underlies the different bursting behavior between these two GC types. To measure INaP in the identified GC type, we first examined the AP responses to somatic rheobase current injection using the standard intracellular solution, carefully withdrew the pipette, and then re-patched the same cell again with Cs+- based pipette solution in the presence of Cd2+ (200 μM) and TEA (20 mM) in the bath solution to inhibit Ca2+ and K+ currents (Fig. 5I). We quantified INa,P in each type of neurons using a slowly rising ramp voltage command protocol from a holding potential of − 70 mV to 0 mV for 6 s in voltage-clamp configuration. In consistent with our hypothesis, the peak amplitude of INa,P in BS-GCs was significantly larger than that in RS-GCs (Fig. 5J).

L-VDCC is a major Ca2+ source for LTP induction but little contributes to firing properties

Above results indicate that NMDAR and T-VDCC partially contribute to LTPAP, and that INa,P is essential for intrinsic and synaptically evoked burst firings in BS-GCs. L-VDCC is known as a calcium source for NMDAR-independent slowly developing LTP induced by 200 Hz tetanic stimuli at CA3-CA1 synapses [26, 27]. We studied the role of L-VDCC in burst firing and LTPAP induction. Distinct from drugs tested above, nimodipine (10 μM), an L-VDCC blocker, had no effect on the Finit of APs evoked by somatic current injection (Fig. 6A). Moreover, nimodipine affected neither EPSP summation induced by HFSL nor the Finit of APs evoked by HFSH (Fig. 6B, C). Nevertheless, the late phase of LTPAP was abolished in the presence of nimodipine (LTP30, − 3.1 ± 11.0 vs. 44.6 ± 5.7%, p < 0.01, n = 6, Fig. 6D). Although the early LTP (LTP5) was not different, it was significantly reduced too when one outlier was ignored (LTP5, − 0.9 ± 5.7%, p < 0.05). Furthermore, LTP was not rescued by the pairing protocol (LTP30, − 2.9 ± 10.3 vs. 52.6 ± 7.7%, p < 0.01, n = 7, Fig. 6E), indicating that calcium influx through L-VDCC during AP bursts is essential to induce LTPAP. Although the LTP5 values for HFSH- and pairing-induced LTP were not lowered (HFSH, 7.2 ± 9.3%, n = 6, p = 0.10; Pairing protocol, 4.3 ± 8.2%, n = 7, p = 0.08, Mann–Whitney test), the early increase in normalized EPSP was transient, suggesting that it belongs to short-term potentiation (STP) which decayed within 3 min [22]. These results indicate that L-VDCC mediates both early and late phase LTP but not STP.

Fig. 6
figure 6

Ca2+ influx through L-type Ca2+ channels mediates Hebbian LTP at LPP-GC synapses. A Left & Middle, Representative AP responses to somatic rheobase current injection before (red, Con) and after application of nimodipine (purple, Nimo, 10 μM). Right, Finit of intrinsic bursts was not affected by nimodipine (Con, 198.3 ± 26.8 Hz; Nimo, 206.7 ± 36.0 Hz; n = 5). B Exemplar traces (left) and mean areas (right) of EPSP summation evoked by HFSL-2 of LPP before and after applying nimodipine (Con, 2.1 ± 0.3 mV s; Nimo, 2.3 ± 0.4 mV s; n = 6). C Exemplar voltage response of BS-GCs (left) evoked by HFSH-2 and mean Finit (right) in the presence of nimodipine (Nimo, 154.8 ± 17.5 Hz, n = 5). Black dashed line on the bar graph, control mean Finit in BS cells (128.3 Hz). D Left, Time course of normalized EPSP in BS-GCs before and after HFSH-2. Right, Magnitudes of early (LTP5, open circle) and late phase (LTP30, closed circle) LTP in control and nimodipine conditions (Con, n = 7; Nimo, n = 6). The control LTP time course and magnitudes were reproduced from Fig. 3D (light red). E Similar as in C, D, but applied a pairing protocol instead of HFSH. The control LTP trace and magnitudes were reproduced from Fig. 3H (gray) (Con, n = 14; Nimo, n = 7). F Time course of normalized EPSP in RS- and BS-GCs before and after HFSH-1. G Left, Time course of normalized EPSP in BS-GCs before and after HFSH-1 in the presence of nimodipine. Right, Magnitudes of late phase LTP (LTP30, closed circles). Note little effect of nimodipine on HFSH-1-induced LTP in BS-GCs. Shades and error bars, S.E.M. *p < 0.05. **p < 0.01. n.s., not significant (p > 0.05)

The LTPAP so far was induced by HFSH-2 which was delivered following HFSL-1 in BS-GCs (Fig. 3C). We wondered whether L-VDCC would contribute to LTPAP induced by HFSH-1 (HFSH without preceding HFSL) to a similar extent as HFSH-2-induced LTPAP. The LTP magnitude induced by HFSH-1 was significantly less than the sum of LTP magnitude induced by the HFSL and HFSH-2 sequence (LTPsub + LTPAP) in the BS-GCs (HFSH-1, 63.1 ± 9.6%, n = 8; HFSL-1 + HFSH-2, 108.5 ± 8.2%, n = 5, p < 0.05; Fig. 6F), whereas that in the RS-GCs was not significantly different from the LTPsub + LTPAP sum (HFSH-1, 39.6 ± 10.3%, n = 8; HFSL-1 + HFSH-2, 26.5 ± 12.9%, n = 5, p = 0.72). Importantly, LTP induced by HFSH-1 in BS-GCs was little affected by nimodipine (Nimo, 54.0 ± 10.8%, n = 6, p = 0.66; Fig. 6G). These results suggest that priming of BS-GCs by HFSL-1 is required for the contribution of L-VDCC to LTPAP induced by HFSH-2.

LTP induction at MPP-GC synapses is not affected by firing pattern

Above results indicate that LTP at LPP-GC synapses can be induced by two distinct mechanisms: NMDAR-dependent subthreshold LTP and compound Hebbian LTP, and that the latter heavily depends on activation of L-VDCC resulting from postsynaptic AP bursts. We investigated whether MPP-GC synapses share the same LTP mechanisms with those of LPP-GC synapses. We recorded MPP-evoked baseline EPSPs using an electrode placed in the middle of the molecular layer in a 10 s interval for about 5 min before HFS was applied (Fig. 7A). The stimulation intensity was adjusted so that the peaks of EPSP summation evoked by 100 Hz 10 stimuli remained subthreshold level (around − 60 mV ~ − 40 mV) (denoted as HFSL). Average stimulation intensity of HFSL was 10.9 ± 0.6 V, which is significantly smaller than that used for LTPsub induction at LPP-GC synapses (15.6 ± 0.9 V, Additional file 1: Fig. S3B). The EPSP summation usually reached its peak at 2nd or 3rd stimulation and declined afterwards, consistent with the characteristic short-term depression at MPP-GC synapses [28]. RS- and BS-GCs showed no detectable difference in their subthreshold EPSP responses to HFSL (RS, black; BS, red; Fig. 7B). Unlike NMDAR-dependent LTPsub at LPP-GC synapses, HFSL did not induce LTP either in RS- or BS-GCs [RS, 17.7 ± 8.8% (n = 5); BS, 9.5 ± 7.0% (n = 6); p = 0.66; Fig. 7C]. Moreover, the 1st EPSP amplitude was not affected by APV (MPP, n = 6, p = 0.075; LPP, n = 10, p = 0.047), and the APV effect on the area of EPSP summation was weaker compared to LPP-GC synapses (MPP, p = 0.03; LPP, p < 0.01; Fig. 7D, E). Given that dendritic expression of NMDAR is not different between MPP and LPP synaptic areas of the dentate gyrus [29], lower contribution of NMDAR current may be attributable to lower local depolarization at the MPP synaptic sites.

Fig. 7
figure 7

Postsynaptic AP bursts are not required for LTP induction at MPP-GC synapses. A Similar as in Fig. 2A, but medial perforant pathway (MPP) in medial molecular layer was electrically stimulated with a single bout of train pulses (10 stimuli at 100 Hz). Scale bar is 100 μm. B Left, Subthreshold voltage responses evoked by HFSL of MPP in RS- (black) and BS-GCs (red). Right, Cumulative EPSP amplitudes of EPSP summation. C Left, Time course of normalized EPSP before and after HFSL of MPP. Right, Change in normalized EPSP before and after HFSL (RS, 17.7 ± 8.8%, n = 5; BS, 9.5 ± 7.0%, n = 6). D Left, Representative EPSP summation in control (black) and after application of APV (50 μM, brown). Right, Cumulative EPSP amplitudes in control and APV conditions. E Left, Mean amplitude of 1st EPSP evoked by a bout of HFSL in control and APV conditions at MPP and LPP [MPP, 12.8 ± 1.7 mV (Con) vs. 13.5 ± 1.7 mV (APV), n = 6; LPP, 9.8 ± 0.9 (Con) vs. 7.3 ± 1.1 mV (APV), n = 11, *p < 0.05]. Right, Mean area of HFSL-induced EPSP summation [MPP, 2.3 ± 0.1 mV s (Con) vs. 1.8 ± 0.1 mV s (APV), n = 6, *p < 0.05; LPP, 3.0 ± 0.2 mV s (Con) vs. 1.6 ± 0.1 mV s (APV), n = 10, **p < 0.01]. Both plots show weaker contribution of NMDAR to EPSPs at MPP-GCs than LPP-GCs. F Left, Representative 3 AP bursts evoked by HFSH in RS- (black) and BS-GCs (red). Right, Initial AP frequency (Finit) of each cell type (RS, 101.2 ± 12.7 Hz, n = 11; BS, 152.1 ± 17.3 Hz, n = 7; *p < 0.05). G: Left, Time course of normalized EPSP before and after HFSH. Right, Magnitudes of early (LTP5, open triangle) and late phase (LTP30, closed triangle) LTP, showing that no significant difference in HFSH-induced LTPAP magnitude between RS- and BS-GCs. H LTP magnitudes as a function of Finit of AP bursts evoked by HFSH at MPP-GCs (r = 0.20, p = 0.47). Open symbols, data of individual cells. Closed symbols, averaged value of each group. Black line, linear regression line. r, Pearson’s correlation coefficient. I Left, Voltage responses evoked by a pairing protocol comprised of postsynaptic 3 APs at 100 Hz (upper, black) or 50 Hz (lower, gray) with HFSL of MPP. Middle, Time course of normalized EPSP amplitude before and after the pairing protocol. Right, Magnitudes of early (LTP5, open symbols) and late phase (LTP30, closed symbols). Note that no difference was found in LTPAP magnitude between pairing at 100 Hz and even at 50 Hz. Shades and error bars, S.E.M. *p < 0.05. **p < 0.01. n.s., not significant (p > 0.05)

When the stimulation intensity was increased to evoke 3 APs, Finit was higher in BS-GCs than RS-GCs (Fig. 7F), suggesting that intrinsic bursting mechanisms affect synaptic bursting induced by MPP stimulation, as was shown for LPP-evoked bursts. In spite, LTPAP was induced similarly in both BS and RS [LTP30: BS, 43.8 ± 3.6% (n = 5); RS, 32.1 ± 10.2% (n = 7); p = 0.20; Fig. 7G]. Because of no difference between RS and BS in the LTP magnitudes and time courses, the LTP data from the two cell types were merged for following comparison with LTP under different conditions. At MPP-GC synapses, the LTP magnitude was not correlated with Finit (Fig. 7H, r = 0.20, p = 0.47), suggesting that AP frequency is not critical for the LTPAP induction at MPP-GC synapses. To further test this idea, we tried to induce LTP using a pairing protocol, in which HFSL was paired with 100 Hz or 50 Hz three APs evoked by somatic stimuli to mimic firing of BS- or RS-GCs, respectively. We found that the LTP magnitudes were not significantly different between them (LTP30: 100 Hz (n = 7) vs. 50 Hz (n = 7), 56.1 ± 14.6 vs. 31.2 ± 6.3%, p = 0.26, Fig. 7I).

Hebbian LTP at MPP-GC synapses is mediated by T-VDCC

We characterized the Ca2+ source mediating Hebbian LTP (LTPAP) at MPP-GC synapses. Consistent with the small contribution of NMDAR to EPSP summation at MPP synapses (Fig. 7D), AP bursts were readily evoked by HFSH of MPP in the presence of APV (Fig. 8A). In the presence of APV, the late phase LTP magnitude was not different from the control value [LTP30, 42.7 ± 11.7 vs. 37.0 ± 6.2%, APV (n = 6) vs. control (n = 12), p = 0.75], but the early phase LTP was significantly inhibited [LTP5, 8.6 ± 3.1 (n = 7) vs 31.8 ± 5.8% (n = 14), p < 0.01] (Fig. 8A), suggesting that NMDAR contributes to short-term potentiation and early phase LTP, but not to the late phase LTP at MPP-GC synapses.

Fig. 8
figure 8

Ca2+ influx through T-type is critical to induce Hebbian LTP at MPP-GCs. For comparison, the LTP time courses and LTP magnitudes of RS and BS in Fig. 7G are merged and shown as a control LTP time course and magnitude (gray). A Left, Representative voltage response to HFSH of MPP in the presence of APV (brown, 50 μM). Middle, Time course of normalized EPSP before and after HFSH. Right, Magnitudes of early (LTP5, open triangle) and late phase (LTP30, closed triangle) of LTP, indicating specific reduction of the early phase LTP by APV (Control, n = 12; APV, n = 6). B Similar as in A, but in the presence of nimodipine (Nimo, purple, 10 μM). Nimodipine had no significant effect on both early and late LTPAP. (Control: n = 12; Nimo: n = 6). C Left, Representative voltage response to HFSH of MPP in the presence of NiCl2 (green, 50 μM). Right, Mean Finit in the presence of NiCl2 (NiCl2: 67.0 ± 14.4 Hz, n = 5). Black dashed line denotes the mean value for Finit of both GC types (117.6 Hz, n = 15). D Similar as in A, but in the presence of NiCl2 (green). Late phase of LTP was significantly inhibited. E Similar as in D, but a pairing protocol was applied of HFSH. Both early and late LTP is significantly reduced. Shades and error bars, S.E.M. *p < 0.05. **p < 0.01. n.s., not significant (p > 0.05)

Nimodipine had no significant effect on MPP-evoked AP generation similar to LPP synapses. In stark contrast to LTPAP at LPP-GC synapses (Fig. 6), LTPAP at MPP-GC synapses was not affected by nimodipine (LTP5, 31.3 ± 3.7%, p = 0.66; LTP30, 41.7 ± 13.4%, n = 6, p = 0.96; Fig. 8B), but abolished by NiCl2. NiCl2 significantly reduced the Finit of MPP-evoked APs (Fig. 8C), and abolished the late phase LTP at MPP-GC synapses (LTP30, 6.0 ± 5.3%, n = 5, p < 0.01, Fig. 8D). NiCl2 did not abolish LTP5 (12.8 ± 6.8%, n = 5, p = 0.07), but the early increase in normalized EPSP was not sustained (Fig. 8D), reminiscent of the nimodipine effects at LPP synapses (Fig. 6D). Moreover, the pairing protocol did not rescue the Ni2+ effect on LTPAP at MPP-GC synapses [Ni2+ (n = 7) vs. control (n = 14): LTP5, 7.8 ± 5.1 vs. 31.1 ± 5.9%, p < 0.01; LTP30, 8.0 ± 6.0 vs. 43.7 ± 8.4%, p < 0.01, Fig. 8E], indicating that LTPAP at MPP-GC synapse is mediated by Ca2+ influx through T-VDCC.

HFSH activates mGluR5 signaling pathways at LPP-GC synapses

Pairing presynaptic 10 stimuli at lower frequency (50 Hz) with 3 postsynaptic APs at LPP-GC synapses failed to bring significant potentiation (Additional file 1: Fig. S5A). Moreover, LTPAP was not induced by 1 Hz repeated pairing of a single presynaptic stimulation with postsynaptic AP bursts for 5 min (pre- and post-synaptic sequence, 5 ms interval; Additional file 1: Fig. S5B), indicating that LTPAP at LPP-GC synapses critically depended not only on postsynaptic but also on presynaptic bursts. The requirement of presynaptic bursts is consistent with the condition for spillover of synaptically released glutamate thereby peri-synaptic mGluRs could be activated [30]. For studying downstream signaling of LTPAP at LPP-GC synapses, we adopted the pairing protocol (HFSL of LPP and 3 APs in a GC as shown in Fig. 3F) to avoid possibility that the test drugs may affect postsynaptic AP bursts. In the presence of MPEP (25 μM), the pairing protocol did not induce LTP at LPP-GC synapses (Fig. 9A), suggesting contribution of mGluR5 activation to LTPAP at LPP-GC synapses. Recently, it was shown that two trains of HFS (1 s at 100 Hz, 1 min interval) of LPP induced presynaptic LTP through activation of mGluR5 and endocannabinoid-dependent retrograde signaling [31]. We tested if LTPAP at LPP-GC synapses observed in the present study shares the same mechanism with the LTP form reported in Wang et al. [31]. We could induce LTP at LPP-GC synapses by the pairing protocol even in the presence of AM251, a CB1 inverse agonist (Fig. 9B), arguing against involvement of endocannabinoid signaling in the induction of LTPAP at LPP-GC synapses.

Fig. 9
figure 9

HFSH activates mGluR5 signaling pathways at LPP-GCs, but not at MPP-GCs. A Left, Representative voltage response to a pairing protocol at LPP-GC synapse in the presence of MPEP (dark green, 25 μM). Middle, Time course of normalized EPSP before and after applying a pairing protocol. Right, Magnitudes late phase (LTP30, closed circle) of LTP in the presence of MPEP [6.0 ± 7.7% vs. 52.6 ± 7.7%, MPEP (n = 8) vs. Con (n = 14), **p < 0.01]. B Similar as in A, but in the presence of AM 251 (light blue, 5 μM). Late LTPAP were not affected by AM251 [50.3 ± 9.9% vs. 52.6 ± 7.7%; AM 251 (n = 4) vs. Con (n = 14)]. C Similar as in A, but in the presence of U73122 (orange, 2 μM). Late LTPAP were significantly inhibited [6.7 ± 14.6% vs. 52.6 ± 7.7%, U73122 (n = 5) vs. Con (n = 14), *p < 0.05]. For comparison of LTP at LPP synapses in AC, the time courses and magnitudes of LTP induced by a pairing protocol are reproduced from Fig. 3H (gray). D Similar as in A, but at MPP-GCs. The data for LTP induced by 50 and 100 Hz pairing protocol shown in Fig. 7I were merged and reproduced as control LTP [38.7 ± 18.8% vs. 43.7 ± 8.4%; MPEP (n = 3) vs. Con (n = 14)]. Shades and error bars, S.E.M. *p < 0.05. **p < 0.01. n.s., not significant (p > 0.05)

Because mGluR5 is a Gq-coupled G protein receptor, we tested involvement of phospholipase C (PLC) in the downstream signaling for induction of LTPAP at LPP-GC synapses. After pre-incubation of the slice with U73122 (an inhibitor of PLC, 2 μM) at least for 30 min, LTPAP was abolished (Fig. 9C).

We examined the effect of MPEP at MPP-GC synapses on LTPAP induced by pairing protocol (HFSL of MPP and 3 APs in a GC). In contrast to LTPAP at LPP-GC synapses, that at MPP-GCs was not affected by MPEP (Fig. 9D). This result is consistent with a previous report that more intense stimulation is required for induction of mGluR-dependent LTP at these synapses [32].


Ionic mechanisms underlying Hebbian LTP at LPP and MPP synapses

One of main findings of the present study is that induction of Hebbian LTP (LTPAP) at LPP-GC synapses is critically dependent on high frequency burst firing of pre- and post-synaptic cells. Induction of Hebbian LTP required at least three post-synaptic APs firing at 100 Hz or higher frequency, and thus Hebbian LTP at LPP-GC synapses occurred preferentially at BS-GCs compared to RS-GCs. To scrutinize the mechanisms underlying LTPAP at LPP-BS synapses, we differentiated whether different inward currents contribute to LTPAP by enhancing postsynaptic AP bursts (burst-enhancer) and/or providing Ca2+ influx mediating LTPAP (LTP-mediator). To this end, when an inward current blocker suppressed HFSH-induced LTPAP, we tried to induce LTPAP by applying the pairing protocol in the presence of the blocker. When LTPAP was rescued by the pairing protocol, we regarded it as ‘burst-enhancer’, and otherwise as ‘LTP-mediator’. The other factor to be considered was the time course of LTPAP. As shown in Fig. 3D and G, LTPAP in BS-GCs was comprised of three components: immediate, early and late potentiation. The immediate phase decayed within 3 min, and early and late phases of sustained potentiation lasted more than 30 min [22]. Because the mechanism underlying immediate and early phase potentiation are known to be different from that underlying late phase LTP at CA3-CA1 synapses [26, 27], we measured normalized EPSP amplitudes averaged over early (1–5 min) and late (26–30 min) intervals of the LTP time course, and regarded the former (LTP5) and the latter (LTP30) as magnitudes of STP plus early phase LTP and late phase LTP, respectively. We examined contributions of NMDAR, T-VDCC, INa,P and L-VDCC under this framework. For the late phase LTPAP at LPP-GC synapses, only L-VDCC met the condition for the ‘LTP-mediator’, and other inward currents seem to contribute as a burst-enhancer. For the early phase LTP, the LTP magnitude induced by the pairing protocol was marginally or significantly lower in the presence of blockers of NMDAR and L-VDCC, implying that these two Ca2+-influx channels may mediate the early phase LTP. The differential involvements of NMDAR and L-VDCC in early and late LTP has been shown in CA3-CA1 synapses [27]. Whereas NMDAR-dependent LTP was rapidly expressed in the postsynaptic locus, NMDAR-independent LTP developed more slowly, depended on L-VDCC and expressed in presynaptic locus [26]. These ionic mechanisms of LTP at CA3-CA1 synapses are different from those at LPP-GC synapses, in that L-VDCC contributes to both early and late LTP, while NMDAR does to early LTP. The locus of LTP expression at LPP-GC synapses remains to be investigated. In contrast to LPP-GC synapses, Hebbian LTP at MPP-GC synapses was mediated by T-VDCCs, and BS-GCs had no privilege for induction of LTP at MPP synapses. The requirement of T-VDCC is consistent with [7], which showed that deletion of Cav3.2 gene reduced LTP at MPP-GC synapses.

Ionic mechanisms underlying AP bursts

Most neuronal burst firings are associated with prominent afterdepolarization (ADP), which can be generated by dendritic Ca2+ spikes and/or axo-somatic slow activating inward current. The dendritic contributions to burst firing has been found in hippocampal and neocortical pyramidal neurons [14, 24, 33]. The burst firing of GCs seems to be axo-somatic type, because axo-somatic T-VDCCs played a crucial role [7]. We found that not only T-VDCCs but also INa,P contribute to the burst firings in GCs (Figs. 4 and 5). INa,P is a small fraction of Na+ current that slowly inactivates and exhibits low threshold for activation compared to larger fast and transient fraction of Na+ current. It has been suggested that INa,P not only generates ADP [25] but also amplifies synaptic current [34]. It is very likely that contribution of INa,P to ADP underlies intrinsic burst firing, while INa,P contributes to synaptically evoked AP by amplifying EPSP summation. Consistent with this view, we showed that the Finit of intrinsic bursts in BS-GCs was reduced by local puff of riluzole to the soma, but not by that to the dendrites, suggesting contribution of somatic INa,P. By contrast, dendritic INa,P, but not somatic INa,P, was responsible for enhancing EPSP summation and LPP-evoked AP bursts (Fig. 5G, H). On the other hand, block of T-VDCC using NiCl2 resulted in only partial reduction of Finit of LPP-evoked AP bursts (Fig. 4B), whereas it had stronger effect on MPP-evoked burst firing (Fig. 8C), implying higher density expression of T-VDCC on proximal dendrites compared to distal dendrites. This view is supported by our findings that T-VDCC plays as an LTP-mediator in MPP synapses whereas it plays only a partial role in LTPAP induction at LPP synapses (Figs. 4D, E and 8D, E).

Not only inward current but also outward current may regulate the ADP and temporal summation of EPSPs, which play a critical role in burst firing. Kv4.1 and Kv4.2 are highly expressed in somatic and dendritic regions, respectively [35]. Therefore, downregulation of Kv4.1 and Kv4.2 may contribute to axo-somatic and dendritic burst firing in GCs. Previously, it was shown that mild LTP induction protocol at MPP-GC synapses enhances MPP-induced dendritic Ca2+ transient in 4-aminopyridine sensitive manner, suggesting downregulation of dendritic Kv4.2 [36].

The role of post-synaptic high frequency bursts in Hebbian LTP at LPP synapses

At LPP-GC synapses, LTPAP was induced preferentially in BS cells and the Finit of synaptically evoked burst firing was highly correlated with the LTPAP magnitude (Fig. 3E). Why high frequency bursts are required for LTPAP induction at LPP-GC synapses? L-VDCC was a major Ca2+ source mediating LTPAP (Fig. 6D), although L-VDCC had little influence on both synaptic and AP responses (Fig. 6A, B). In light of these results, it is likely that activation of L-VDCC requires high frequency back-propagating APs (bAPs). A previous study in L5 neocortical pyramidal neurons may provide a hint for addressing the question [14]. Using dual patch recordings at apical dendrite and soma in a single neocortical L5 pyramidal neuron, Larkum et al. discovered nonlinear summation of bAPs at distal apical dendrites: As somatic APs back-propagated along apical dendrites, they were attenuated in amplitude and broadened in width. While low frequency bAPs underwent only such linear attenuation, as the bAP frequency increased above a critical point (100 Hz), bursts of four bAPs summated to readily reach the threshold for activation dendritic Ca2+ channels [14]. We imagine that a similar scenario may be involved in the L-VDCC dependent dendritic Ca2+ signaling evoked by a burst of three somatic APs in GCs. The broadening and attenuation of bAPs at intermediate dendrites has been shown in our previous study [20]. Summation of bAPs at distal dendrites remains to be elucidated in GCs, though it would be a challenging task considering the feasibility of patching on the distal dendrites of GCs.

Hebbian vs. non-Hebbian LTP at LPP-GC synapses

Previously we showed a different form of LTP at LPP-GC synapse, which was critically dependent on dendritic Na+ spikes and activation of NMDA receptors [20]. This form of LTP was induced by theta burst synaptic stimulation (TBS) of LPP, but not by the standard spike time-dependent plasticity (STDP) protocol, which is pairing EPSP with a single somatic AP [20]. The LTP shown in Kim et al. [20] could be induced even without somatic APs as long as TBS elicited dendritic spikes. In contrast, a burst of 100 Hz three APs was required for the induction of LTPAP not only in the pairing protocol but also in the induction protocol of synaptic stimulation alone (HFSH). Therefore, the LTP forms shown in our previous and present studies belong to non-Hebbian and Hebbian LTP, respectively. The strong attenuation of back-propagating somatic APs along the dendrites of mature GCs might be responsible for the no LTP induction by the standard STDP protocol [20]. The results of the present study suggest that postsynaptic AP bursts may overcome the strong dendritic attenuation probably by summation of bAPs in distal dendrites to activate L-VDCCs.

Remarkably, only a single bout of HFS (10 stimuli at 100 Hz) was sufficient for induction of Hebbian LTP. We previously used TBS (4 repeats of 5 Hz 10 bouts of HFS) for induction of dendritic spike-dependent LTP in Kim et al. [20]. The key differences in the LTP induction protocols between these two studies are not only the number of HFS bouts but also the LPP stimulation intensity. The baseline EPSP in our previous and present studies were 7.1 ± 0.5 mV and 13.8 ± 1.0 mV, respectively, indicating that the stimulation intensity required for LTPAP is stronger than that for dendritic spike-dependent LTP.

The subthreshold LTP discovered in the present study has not been described before. It is unique in that ten stimuli which evoked only subthreshold EPSP summation can induce NMDAR-dependent LTP as long as the peak EPSP summation was higher than − 60 mV. Because such weak stimuli have been routinely employed to characterize the baseline properties of synapses, the subthreshold LTP has been ignored in our previous study [20]. Therefore, the dendritic spike-dependent LTP described in Kim et al. [20] has been induced on the top of subthreshold LTP. Given that subthreshold LTP is mediated by NMDAR, the local EPSP summation elicited by high frequency LPP inputs may result in large local depolarization at distal dendrites sufficient for activation of NMDARs, even if it does not elicit somatic APs or dendritic spikes (Note that there was no evidence for dendritic spikes in somatic recordings during the subthreshold LTP induction). Recent in vivo whole-cell recordings in GCs revealed that majority of GCs were under the influence of spatially tuned PP synaptic inputs while only minority of them exhibited spatially tuned firings [6]. It is not clear whether nonspatial LPP inputs are also extensive similar to such spatial inputs. If it is so, subthreshold LTP at LPP-GC synapses might extensively occur over the GC population receiving brief bursts of LPP inputs independent of postsynaptic firings.

Contribution of NMDAR to LTP induction at MPP-GC synapses

In the present study, the HFSH-induced late phase LTPAP was not affected by APV at MPP synapses (Fig. 8A). This result is not consistent with previous field recording studies [28, 37], in which LTP at MPP synapses was abolished by APV. It needs careful investigation to elucidate the reasons underlying the discrepancy, but there are three apparent differences in the experimental conditions. Firstly, previous studies used higher [Ca2+] (4 mM, ref. [28]) or lower [Mg2+] (0.7 mM, ref. [37]). Secondly, the number of pulses used for LTP induction in the previous studies was higher by an order (10 vs. 100 pulses). Thirdly, we might have used stronger stimulation intensity than the previous studies. Lopez-Rojas et al. previously showed a plot of population spike amplitude as a function of initial slope of MPP-fEPSP in the rat DG [36]. In this plot, the population spike activity started to emerge at the initial slope of 2 mV/ms. Judging from the representative fEPSP traces in [28, 37], the baseline fEPSP slope was clearly less than 1 mV/ms. On the other hand, the 1st EPSP amplitude evoked by HFSH of MPP (16.2 ± 1.0 mV, Additional file 1: Fig. S3C) in our study was close to the AP threshold, indicating that the stimulation intensity in the present study might be stronger at least by a factor of two than that in [28, 37]. Therefore, it seems that NMDAR may contribute to LTP at MPP synapses when LTP is induced by a larger number of pulses with weak stimulation intensity, but the necessity of NMDAR may be waived when MPP was stimulated with a smaller number of pulses with high intensity. But this conjecture remains to be tested.

Physiological relevance

Our results cannot be directly extended to in vivo conditions, because LTP in the present study was induced in the presence of GABA receptor blocker. When we tried to induce LTP without GABA receptor blocker, it was not easy for the peak of EPSP summation to reach − 60 mV and to induce LTPsub. Moreover, further increase in the LPP stimulation intensity resulted in a parallel increase in IPSPs, which prevented the peak of EPSP summation from reaching AP threshold. Therefore, we think that both subthreshold and Hebbian LTP cannot be practically induced without reduction of GABAergic inputs. Recently, it was reported that disinhibition is induced at MPP-GC synapses by septal acetylcholine release [38], whereas it is unknown whether similar disinhibition occurs at LPP-GC synapses. It remains to be elucidated whether Hebbian LTP can be induced when feedforward GABAergic inputs are inhibited under an influence of neuromodulator.


Slice preparation and electrophysiology

Acute hippocampal slices (thickness, 350 μm) were prepared from the brains of 17- to 25-day-old Sprague–Dawley rats of either sex. Rats were anesthetized (isoflurane, Forane; Abbott) and decapitated immediately. All the experiments were approved by the University Committee Animal Resource in Seoul National University (Approval #: SNU-210825–6). All brains were obtained coronally for dorsal hippocampus or horizontally for ventral hippocampus (coronal sections located between 4.2 mm and 5.6 mm from the posterior end and transverse sections located 2.8 mm and 4.2 mm from ventral end of the right hemisphere). Slices were prepared in an oxygenated ice-cold sucrose-containing physiological saline using a vibratome (VT1200, Leica), incubated at ~ 36 °C for 30 min, and subsequently maintained in the same solution at room temperature until the recordings. Recordings were performed at near- physiological temperature (33–35 °C) in an oxygenated artificial cerebral spinal fluid (ACSF).

Patch pipettes were obtained from borosilicate glass capillaries (outer diameter = 1.5 mm, inner diameter = 1.05 mm) with a horizontal pipette puller (P-97, Sutter Instruments). The open-tip resistance of patch pipettes was 2.5–4.5 MΩ for somatic recordings. Current- or voltage clamp recordings were performed with an EPC-10 USB Double amplifier (HEKA Elektronik). In current-clamp recordings, series resistance was 8–20 MΩ. Pulse protocols were generated, and signals were low-pass filtered at 3 or 10 kHz (Bessel), digitized (sampling rate: 20 kHz) and stored using Patchmaster software running on a PC under Window 10. Resting membrane potential (RMP) was measured immediately after patch break-in. Input Resistance (Rin) was determined by applying Ohm’s law to the steady-state voltage difference resulting from a hyperpolarizing current step (− 20 pA, 500 ms). Threshold for AP was determined at points at which the derivative of voltage exceeded 40 V/s of somatic stimulations. Pipette capacitance and series resistance (Rs) compensation (bridge balance) were done at the beginning of current-clamp recordings. Recordings were stopped and discarded if RS changed by more than 20% of Rin during the data acquisition.

All experiments were performed on visually identified mature GCs on the basis of the relatively large and round-shaped somata under DIC optics. GCs located at the superficial side of the GC layer in the suprapyramidal blade were purposely targeted. These cells had the average RMP of − 81.6 ± 0.7 mV and Rin of 115.7 ± 5.5 MΩ, that are similar to characteristic intrinsic properties of mature GC population [21]. Cells were filled with a fluorescent dye, Alexa Fluor 488 (50 μM, Invitrogen) at least 5 min and imaged with LED system (Thorlabs) mounted on an upright microscope equipped with a 60 × water immersion objective lens (N.A. 1.0). In order for focal electrical stimulation (100 μs pulses of 5–40 V intensities) of the medial or lateral perforant pathways, a ACSF-filled glass pipette microelectrode (3–4 MΩ) was placed in the vicinity of intermediate or distal part of a visually identified dendrite (typically at < 50 μm distance) of a GC under whole-cell patch. For evaluation of baseline synaptic responses, excitatory postsynaptic potentials (EPSPs) were evoked by applying a pulse every 10 s through a stimulation electrode. All experiments were performed in the presence of the GABA receptor antagonist picrotoxin (PTX, 100 μM) and CGP52432 (1 μM).

Stimulation protocols for the induction of long-term potentiation (LTP)

LTP was induced by either single bout of high-frequency stimulation [39] of afferent fibers or a pairing protocol. HFS consists of 10 stimuli at 100 Hz under current clamp mode. Depending on the stimulation intensity, HFS evoked subthreshold EPSP summation alone or additively post-synaptic APs, which are denoted as HFSL and HFSH, respectively. The pairing protocol is comprised of HFS followed by post-synaptic injection of three suprathreshold current pulses (2 ms, 3 nA) at 100 Hz with a time delay (50 ms, LPP; 10 ms, MPP), similar to a protocol in [40]. The time delay, 50 or 10 ms, was set based on the averaged synaptically evoked firing onset time. For LTP experiments, we monitored baseline EPSPs every 10 s at least for 5 min before applying LTP induction, after which we resumed the EPSP monitoring at least for 30 min. For off-line analysis, EPSP amplitudes were normalized to the mean of baseline values. A time course of normalized EPSP amplitudes was subject to binomial smoothing using a built-in function of IgorPro7 (WaveMetrics). The magnitude of EPSP potentiation was evaluated as a mean of smoothed EPSP amplitudes measured 1 to 5 min or 26 to 30 min after LTP induction (denoted as LTP5 and LTP30, respectively).

Solutions and chemicals

The extracellular solution for dissection and storage of brain slices was sucrose-based solution (87 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 7 mM MgCl2, 0.5 mM CaCl2, 10 mM glucose, and 75 mM sucrose). Physiological saline for experiments was standard ACSF (125 mM NaCl, 25 mM, NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgCl2, 2 mM CaCl2, and 25 mM glucose).

For whole-cell recording, we used K+ rich intracellular solution that contained 115 mM K-gluconate, 20 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 4 mM MgATP, 10 mM Na2-phosphocreatine, and 0.3 mM NaGTP, pH adjusted to 7.2–3 with KOH (~ 300 mOsm). If necessary, 50 μM Alexa 488 were added to the internal solution to detect the dendrites. In subset of experiments for measuring persistent sodium current (INa,P), aCSF containing 20 mM tetraethylammonium chloride (TEA) and 0.2 mM CdCl2 was used, and an internal solution in which K-gluconate and KCl were replaced with Cs-methanesulfonate and CsCl, respectively, at the same concentration. APV (D, L-2-amino-5-phoshonovaleric acid), PTX, riluzole hydrochloride, AM 521 and MPEP hydrochloride were purchased from Tocris Bioscience. CGP52432 was from Abcam. U73122 was from Cayman Chemicals. All other drugs were from Sigma-Aldrich. For all ion channel blockers used in the present study (riluzole, Ni2+, nimodipine), we confirmed that they do not affect the baseline EPSC and its paired pulse ratio. We could safely block INa,P using 10 μM riluzole, because IC50 of riluzole for INa,P is 2.2 μM [41], which is c.a. 20 times lower than IC50 for high voltage-activated Ca2+ channel [42].

Immunohistochemistry and morphological analysis

GCs were filled with 0.2% biocytin (wt/vol) at least 20 min during whole-cell recording. The acute slices (thickness, 350 μm) were fixed overnight at 4 °C in 4% paraformaldehyde (Fujifilm). After fixation, slices were washed for 10 min × 3 times with PBS and then permeabilized with 0.3% Triton X-100 in PBS. Subsequently, slices were treated with 0.3% Triton X-100 and 0.5% BSA in PBS to prevent non-specific staining. Next, they were treated with 0.3% Triton X-100 and streptavidin-Cy3 (1:500) in PBS and were again incubated overnight in 4 °C. After washing steps, slices were finally mounted with DAKO S3023 medium, and coverslips were applied immediately. Confocal images were scanned through a 40 × water-immersed objective (N.A. 0.5) from FV1200 confocal microscope (Olympus Microscopy). Branch orders were manually counted from a series of z-section images (z step: approx. 1 μm, 512 × 512 pixels) displayed using Fluoview software (FV31S).

Statistical tests and data availability

Data obtained from two different conditions were compared using Mann–Whitney U test because the number of samples were less than 30. All data supporting the results presented in the manuscript were included in the figures.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.





Action potential


Back-propagating AP




Cornu ammonis


Dentate gyrus


Excitatory postsynaptic potential

Finit :

Initial firing frequency


Granule cell


High frequency stimulation


High intensity HFS that evokes at least 3 APs


Low intensity HFS that induces subthreshold voltage response


Medium intensity HFS

INa,P :

Persistent sodium current


Lateral entorhinal cortex


Lateral perforant pathway


Long-term potentiation


Hebbian LTP that induced by HFSH

LTPsub :

LTP that induced by HFSL


Maximal branch order


Medial entorhinal cortex


Metabolic glutamate receptor




Medial perforant pathway


N-Methyl-d-aspartate receptor


Phospholipase C



Rin :

Input resistance


Resting membrane potential


Regular spiking


Short-term potentiation


Theta burst synaptic stimulation


Voltage-dependent calcium channel


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This study has been supported by National Research Foundation of Korea (2020R1A2C2006438 to LSH), Seoul National University Hospital and the Health Fellowship Foundation.

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Authors and Affiliations



YSK, WKH and SHL conceived and designed this study. YSK performed the experiments and collected data; YSK, SYK, WKH and SHL analyzed and interpreted data; YSK, WKH and SHL wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Won-Kyung Ho or Suk-Ho Lee.

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Supplementary Information

Additional file 1: Fig. S1.

Representative biocytin-filled RS-and BS-GC. Yellow arrows indicate the maximal dendritic branching points. Scale bar, 100 μm. Fig. S2. Input specificity of postsynaptic AP-dependent LTPat LPP synapses. A: Two stimulation electrodes were placed at outer molecular layer. B: Baseline EPSPs were monitored at the two electrodes. HFSL was delivered to one of two electrodesbut not to the other electrode. Pairing protocolwas applied at the induction pathway. Note that LTPAP was induced only at the synapse that underwent HFSL. Shades and error bars, S.E.M. *p < 0.05. Fig. S3. Stimulation Intensities and baseline EPSP amplitudes to evoke sub- or suprathreshold voltage responses at MPP and LPP synapses. A: Compared to MPP-EPSPs, LPP-EPSPs were significantly higher in paired pulse ratioand in the normalized area of subthreshold EPSP summationto EPSP1 amplitude. B: Mean stimulation intensities used for HFSL and HFSH at MPP and LPP synapses. Both mean intensities for HFSLand HFSHwere significantly stronger at LPP-GCs than MPP-GCs. C: Baseline amplitudes of EPSP evoked by HFSL and HFSH at MPP and LPP synapses. Significantly larger baseline EPSP amplitudes were required at MPP-GCs than LPP-GCs in order to elicit subthreshold responses. But, it was not significant to elicit 3 APs responses. D: Plot of LTPsub magnitude as a function of peak membrane potential of EPSP summation. Error bars, S.E.M. ***p < 0.001. n.s., not significant.. Fig. S4. Conditions for LTPAP induction. A: Time course of normalized EPSP before and after HFSM-2. HFSM-2 is defined by HFS eliciting 1 or 2 APs. Note that LTP was not maintained not only in RSbut also in BS. B: Magnitudes of LTPAP induced by HFSM or HFSH in RS and GS. C: Dependence of LTPAP on the postsynaptic AP frequency. When the post-synaptic AP bursts were elicited at 50 Hz instead of 100 Hz in the pairing protocol, LTPAP was not induced. Shades and error bars, S.E.M. *p < 0.05. Fig. S5. A: Dependence of LTPAP on the synaptic stimulation frequency. When 10 EPSPs were evoked at 50 Hz instead of 100 Hz in the pairing protocol, LTPAP was not induced. B: Single presynaptic stimulation is not sufficient to induce LTP. Left, Representative voltage response to a pairing protocol, in which a single EPSP was coupled to 3 APs at 100 Hz. Middle, Time course of normalized EPSP before and after applying the pairing protocol300 times. Right, LTPAP was not induced by this induction protocol. Black dashed line, mean value for LTP magnitude induced by the conventional pairing protocol comprised of 10 EPSPs and 3 APs at 100 Hz as shown in Fig. 3F. Shades and error bars, S.E.M. *p < 0.05.

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Kim, Y., Kim, S., Ho, WK. et al. Burst firing is required for induction of Hebbian LTP at lateral perforant path to hippocampal granule cell synapses. Mol Brain 16, 45 (2023).

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