HFS-induced mGluR activation causes increases in AP firing and its temporal precision
We recently reported that high-frequency stimulations (HFS; 100 Hz for 0.5 s) to Schaffer Collateral (SC) synapses trigger mGluR-dependent Ca2+ release in apical dendrites of CA1-PN, which in turn induce calmodulin-dependent increase in persistent Na current (INa,P) that leads to E-S potentiation [32]. To investigate the effects of dendritic INa,P increases on intrinsic excitability of CA1-PNs, we tested whether action potential (AP) firing in CA1-PNs evoked by depolarizing current injection is affected by HFS. To rule out any NMDA receptor-dependent changes or the recruitment of GABAergic inhibition, we performed this experiment in the presence of APV (50 μM) and PTX (100 μM) in the bath. We adjusted the current amplitude so that the AP number during 1-s depolarization was between 6 and 8 under control conditions and repeated this stimulation at 10 s interval (Fig. 1a and b). We then applied HFS through a stimulation electrode which was positioned at 80~ 100 μm away from the soma. After HFS to the SC pathway, there was no change in resting membrane potential (RMP; control: − 64.31 ± 0.55 mV, after HFS: − 64.25 ± 0.61 mV, n = 8), but AP number increased significantly (Fig. 1a and b). To examine the time course of the HFS effect on intrinsic excitability, we plotted the number of APs as a function of time (Fig. 1b). The increase in AP numbers following HFS did not last long and the increased AP numbers decreased gradually to the basal level within 2 min (Fig. 1b), which is consistent with the effect of HFS on E-S potentiation reported in our previous paper [32].
As a mechanism for HFS-induced increases in intrinsic excitability, we examined the involvement of INa,P using INa,P blocker riluzole [30]. In the presence of riluzole (10 μM), HFS-induced increases in AP number were abolished (Fig. 1a and b). These results support the idea that HFS-induced enhancement of dendritic INa,P facilitates AP firing. We previously showed that HFS-induced enhancement of dendritic INa,P is mediated by mGluR5 activation [32], and confirmed here that HFS-induced increases in AP number were indeed abolished in the presence of mGluR5 blocker MPEP (Fig. 1d). However, afterhyperpolarization (AHP) following repetitive APs was affected neither by HFS nor by riluzole (Fig. 1a and c).
To further investigate the physiological relevance of INa,P enhancement on neuronal excitability, we examined the effect of HFS on firing activity of APs evoked by a slow current ramp (250 pA/350 ms; Fig. 2). Voltage responses obtained by applying the same ramp for 10 times were superimposed (Fig. 2a), and the effects of HFS on the spike onset time and jitters were analyzed (Fig. 2b). The spike onset time was measured by the duration from the initiation point of the ramp to the first AP. After HFS, spike onset time was advanced by 6.5 ± 2.0 ms (n = 3), but this advancement did not occur in the presence of riluzole (Fig. 2a and b). Spike timing jitter, which represents spike timing precision, was quantified as the standard deviation of the spike onset latencies which were measured while the same ramp was applied for 10 times. The spike timing jitter in control was 2.0 ± 0.3 ms (n = 3), while it decreased to 1.2 ± 0.4 ms after HFS, and this decrease was also reversed by riluzole (Fig. 2a and b). These results suggest that HFS-induced enhancement of INa,P not only advances the spike onset but also increases the temporal precision. To investigate how increased INa,P affect both spike onset and temporal precisions, we further analyzed the depolarizing phase during a slow current ramp, and found that the rate of membrane depolarization exhibited prominent changes on the later phase of slow depolarization (Fig. 2c), which might account for the facilitation of AP generation and a decrease in spike jitter. Somatic spike threshold was slightly hyperpolarized (control: − 41.5 ± 1.0 mV; after HFS: − 42.0 ± 1.2 mV, n = 3, Fig. 2d), but there was no statistical significance.
To mimic more physiological voltage responses occurring in vivo [10, 11], we injected an oscillating sine wave current with theta frequency (5 Hz) via somatic pipette, while current amplitudes were adjusted to evoke APs at 5th or 6th sine wave. Representative voltage traces obtained in responses to the injection of 8 sine waves were superimposed (Fig. 2e). After HFS, AP firing was advanced so that 1st AP was evoked at 3rd or 4th sine wave. To examine the effect of HFS on AP onset time, we measured AP onset at 7th sine wave (indicated by a grey box, Fig. 2e). The onset time was reduced by 12.1 ± 1.0 ms and spike timing jitter was also significantly reduced (n = 3, Fig. 2f). These results suggest that activity-dependent advancement of AP firing and increased temporal precision may play a role during the realistic network oscillation.
Group I mGluR-dependent changes in the intrinsic excitability of CA1-PNs
It is well known that group I mGluR activation affects intrinsic excitability in a variety of neuronal types in the brain [6, 24], including hippocampus [26]. To compare the effects of HFS-induced mGluR activation with those of global mGluR activation on intrinsic excitability, we examined the effect of bath application of an agonist for group I mGluRs, DHPG, on intrinsic excitability. Intrinsic excitability was assessed by applying 1-s depolarizing current steps with varying magnitude in the presence of synaptic blockers (50 μM APV and PTX 100 μM PTX). CA1-PNs in control conditions showed tonic firing followed by AHP in response to a depolarizing current injection (Fig. 3a), and firing frequency and AHP increased as the magnitude of current increased (Fig. 3b and c). We next applied DHPG (50 μM) into the bath. Application of DHPG gradually depolarized the RMP from − 62.9 ± 1.6 mV (n = 7) to − 56.9 ± 1.7 mV (n = 7; Fig. 3d). To minimize the effect of RMP changes on spike frequency, we applied depolarizing step pulses after injecting currents to adjust the RMP to match control values (Fig. 3a). DHPG caused a significant increase in the number of APs at all applied current amplitudes (Fig. 3a and b). We also observed generation of afterdepolarization (ADP) following repetitive firing in the presence of DHPG (Fig. 3a and c). We next determined whether the DHPG effects on intrinsic excitability is mediated by mGluR5 activation. DHPG-induced changes in intrinsic excitability were reversed by mGluR5 antagonist MPEP (Fig. 3e–g), but not by mGluR1 antagonist LY367385 (Fig. 3f and g).
The difference between the effects of HFS and DHPG bath application on intrinsic excitability suggests that target ion channels underlying the effects of mGluRs are not homogenously distributed. To detect the location of mGluR5-induced changes in neuronal excitability, DHPG was applied locally by pressure application from a glass pipette into the dendritic or perisomatic region (Fig. 3h). Interestingly, only the dendritic puff application of DHPG cause the instantaneous increase in firing rates whereas the somatic puff did not trigger changes in spiking frequency (Fig. 3i). RMP depolarization was induced by DHPG application to perisomatic region, but the magnitude was variable and much smaller than that induced by DHPG bath application (Fig. 3a). Furthermore, no significant change in afterpotential was observed during DHPG local application to either perisomatic or dendritic region (Fig. 3h). Taken together, it can be suggested that significant RMP depolarization and ADP generation require global activation of mGluR5, while local activation of mGluR5 at dendritic region can sufficiently modulate AP firing by selectively targeting dendritic INa,P.
We further examined the role of INa,P in DHPG effects using riluzole. Among DHPG effects, RMP depolarization (− 55.5 ± 1.3 mV, n = 4) and ADP generation were not affected by riluzole, while increased AP firing was completely reversed by riluzole (Fig. 4a, b and c). To determine the cellular location where INa,P modulation leads to increased AP firing, we employed the local puff applications of riluzole (50 μM) to the dendritic or perisomatic area in the presence of DHPG. DHPG-induced increases in AP firing was substantially inhibited by a block of dendritic INa,P (Fig. 4d), but not by block of INa,P in perisomatic regions (Fig. 4e). These results together with the effects of HFS support the idea that enhancement of dendritic INa,P mediated by group I mGluR activation is the underlying ionic mechanism of the increased AP firing.
mGluR5-dependent Ca2+ release mediated by cADPR-RyR pathway underlies increased intrinsic excitability
We have recently reported that mGluR5-dependent INa,P increase is mediated by Ca2+/calmodulin signaling that is activated by intracellular Ca2+ release via cADPR/ryanodine receptor (RyR) pathway [32]. However, Park et al. [20] showed that mGluR-dependent increase in R-type Ca2+ currents mediates ADP generation by DHPG. It is possible that different Ca2+ sources are involved in regulating different ion channels. To examine this possibility, we analyzed changes in intrinsic excitability under experimental conditions where cADPR/ RyR pathway was blocked. Under these conditions, DHPG-induced RMP depolarization were not significantly affected, but DHPG-induced increase in AP firing as well as DHPG-induced ADP generation were almost completely abolished (Fig. 5a to e), suggesting that these effects are dependent on cADPR/RyR-mediated Ca2+ release.
HFS to Shaffer collateral pathway induces Ca2+ release which is confined to proximal apical dendrites [32], and the effect of HFS on intrinsic excitability is selective for modulating the timing and precision of AP firing, which is mediated by INa,P enhancement (Figs. 1 and 2). By contrast, global activation of mGluRs by DHPG induces complex effects on intrinsic excitability (Fig. 3), suggesting that mGluRs target multiple ion channels. We tested the hypothesis that interaction between mGluR5 and target ion channels occurs locally in a restricted region of the neuron. To test this idea, we used Ca2+ buffer with different kinetics, BAPTA and EGTA. In the presence of 8 mM BAPTA, a fast Ca2+ buffer in the intracellular solution, DHPG effects on both AP firing and ADP were completely abolished (Fig. 5f and h), while these effects were diminished but not completely abolished in the presence of EGTA, a slow Ca2+ buffer (Fig. 5g and h). More powerful effects of BAPTA on inhibiting DHPG-mediated changes compared with EGTA effects suggest that the distance between the Ca2+ source and the target ion channels is very close within a nanometer scale so that a slow Ca2+ buffer is not sufficient for blocking Ca2+ signaling. However, DHPG-induced depolarization still occurred in the presence of BAPTA or EGTA, suggesting that the ion channel mechanisms responsible for DHPG-induced depolarization, possibly activation of TRPC channels [7] or inhibition of leak K+ channels [8] are not Ca2+-dependent.