Cholinergic modulation shifts the response of CA1 pyramidal cells to depolarizing ramps via TRPM4 channels with potential implications for place field firing

  1. Crescent L Combe
  2. Carol M Upchurch
  3. Carmen C Canavier
  4. Sonia Gasparini

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    This manuscript by Combe et al. presents the role of cholinergic modulation in the spike rate adaptation in pyramidal place cells. Using combined electrophysiology, pharmacological, and multi-compartment computational modeling, the authors identify the downstream pathway (e.g. activation of TRPM4 channel) that shapes the firing pattern under the triangular-shaped ramps. The study demonstrates solid evidence, and the findings are important for bridging pyramidal neurons' molecular/channel properties to behavior-level implications (place field firing).

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Abstract

A synergistic combination of in vitro electrophysiology and multicompartmental modeling of rat CA1 pyramidal neurons identified TRPM4 channels as major drivers of cholinergic modulation of the firing rate during a triangular current ramp, which emulates the bump in synaptic input received while traversing the place field. In control, fewer spikes at lower frequencies are elicited on the down-ramp compared to the up-ramp due to long-term inactivation of the Na V channel. The cholinergic agonist carbachol (CCh) removes or even reverses this spike rate adaptation, causing more spikes to be elicited on the down-ramp than the up-ramp. CCh application during Schaffer collateral stimulation designed to simulate a ramp produces similar shifts in the center of mass of firing to later in the ramp. The non-specific TRP antagonist flufenamic acid and the TRPM4-specific blockers CBA and 9-phenanthrol, but not the TRPC-specific antagonist SKF96365, reverse the effect of CCh; this implicates the Ca 2+ -activated nonspecific cation current, I CAN , carried by TRPM4 channels. The cholinergic shift of the center of mass of firing is prevented by strong intracellular Ca 2+ buffering but not by antagonists for IP 3 and ryanodine receptors, ruling out a role for known mechanisms of release from intracellular Ca 2+ stores. Pharmacology combined with modeling suggest that [Ca 2+ ] in a nanodomain near the TRPM4 channel is elevated through an unknown source that requires both muscarinic receptor activation and depolarization-induced Ca 2+ influx during the ramp. Activation of the regenerative inward TRPM4 current in the model qualitatively replicates and provides putative underlying mechanisms for the experimental observations.

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  1. Version published to 10.7554/elife.84387 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This study combines in vitro somatic and dendritic recordings and computational modeling to study how cholinergic agonists modulate the response of CA1 pyramidal neurons to triangular current injections. The authors have previously used a similar approach (Upchurch, 2022, JNeuroscience) to show that CA1 neurons exhibit asymmetric AP firing (more firing on the upward ramp) in response to such current injections and that this effect is due to Na channel inactivation. The present work builds on these results by showing that cholinergic modulation changes this response, i.e., there is more firing on the downward part of the ramp. This change appears to require an intracellular Ca2+ concentration increase (mediated via IP3 and voltage-gated Ca2+ channels), which activates TRPM4 channels. In this scheme, cholinergic activity increases IP3, and the depolarizing current injection opens voltage-gated Ca2+ channels. This study will be of some interest to cellular neurophysiology experts working on the hippocampus.

    1. This study claims that the triangular current injections recapitulate hippocampal place cell activity. However, it has been shown recently that the asymmetric firing of CA1 place cells is due to synaptic weight changes resulting from synaptic plasticity (e.g., Bittner et al., 2017). This suggests that the asymmetric firing of place cells is primarily the result of asymmetric synaptic input. Therefore, the authors should test whether carbachol similarly affects a synaptically driven membrane potential ramp. If this is not the case, the strong claim that this work has implications for place cell firing is not justified, in my opinion.

    We have added the results showing the effects of cholinergic modulation on a synaptically-driven membrane potential ramp, obtained by electrically stimulating the Schaffer collaterals with a stimulation frequency that was adjusted according to a linear, symmetric ramp (see also Hsu et al, Neuron 99,147-162, 2018). These results have been added to the manuscript in the Results section for new Figure 2 (lines 169-197) and in the Methods section (lines 716-726).

    1. Along the same lines, it has been shown before that the precision of spike timing depends on the stimulation pattern in vitro (Mainen and Sejnowski, 1995). Constant stimuli led to imprecise AP firing trains, whereas current injections that included fluctuations resembling synaptic input generated spike trains that were more reliable and reproducible in terms of timing. This study concluded that a low intrinsic noise level in spike generation was essential in generating informative spike sequences. Following this pivotal work, the authors could add noise to their current stimulus and observe the effect on the AP firing patterns. If this is not possible, the authors should at least report the sweep-to-sweep variability for the data shown, e.g., in panels 1A2, 1B2, 1D2, and 1E2.

    We thank the reviewer for this suggestion to acknowledge the variability in the data across trials and we have added the Mainen and Sejnowski, 1995 citation to the manuscript (see Results lines 128-134). We addressed sweep-to-sweep variability among the various trials.

    1. In most of the data presented in this manuscript, Carbachol appears to induce a 3 mV hyperpolarization and increase input resistance. As a result, the amount of current injected during Carbachol is drastically lower than during the controls. This should be emphasized more, and the input resistance should be quantified for each experimental condition. It should also be discussed whether this change in input resistance can account for the changes in the firing pattern observed. Finally, it should be clearly stated how the amount of the current injected was chosen for each cell, and data from a range of injected current ramps should be shown for each cell.

    We thank the reviewers for this comment, which made us realize that our initial presentation was not clear, in particular with regard to the traces that were chosen as examples in the initial submission of the paper. We now clarify on page 5 (lines 113-125) of the manuscript as follows:

    “In some trials, under control conditions, we applied a baseline depolarization prior to the ramp, in order to capture the variability observed in vivo (Harvey et al Nature 461:941–946, 2009; Epsztein et al. Neuron 70:109–120, 2011). Application of the cholinergic agonist carbachol (CCh, 2 µM) caused a depolarization of 2-6 mV. We compensated for this depolarization by injecting tonic hyperpolarizing current to reestablish the original membrane potential (see also Losonczy, et al., Nature 452, 436-442, 2008), as indicated by an offset from the 0 pA current level in the traces of the injected current ramps. The amplitude of background fluctuations in the resting membrane potential increased from a few tenths of a mV in control to 2-4 mV in CCh. Moreover, the threshold for action potential generation became more hyperpolarized. For all these reasons, we were not able to consistently vary the membrane potential using baseline depolarizations in the presence of CCh, because baseline depolarization alone frequently evoked spiking.”

    For this reason, many of the carbachol example traces in the initial submission had more hyperpolarized Vm than their control counterparts. Acetylcholine also caused a depolarization in a dose-dependent manner, that was compensated for in the same way. In this new version of the manuscript, we systematically report the effects of cholinergic agonists on membrane potential and neuronal excitability. Further, we show example traces with resting membrane potentials within 1 mV for each pharmacological comparison, therefore removing this variable and hopefully making results clearer. We also now state how the amount of injected current was chosen for each condition, and that the amount of injected current was generally lower in the presence of cholinergic agonists. Both the tonic hyperpolarizing current and the amplitude of the injected ramp for each example can now be appreciated in each figure.

    Finally, the reviewers’ comment also made us realize that, in principle, the center of mass of firing could be systematically skewed by the initial membrane potential, the amplitude of the current ramp injection and/or the input resistance. For this reason, we added a supplementary figure (1-2) where the adaptation index was plotted as a function of each these variables. In all cases, it is apparent that the main factor determining whether the center of mass of firing is shifted earlier or later in the ramp is the presence or absence of carbachol rather than initial membrane potential, current injection amplitude, or input resistance.

    1. It remains unclear how the current result that TRPM4 channels can mediate the firing pattern change relates to the previous finding that the current injection evoked CA1 neuronal firing pattern is due to long-term Na channel inactivation.

    We thank the reviewers for this suggestion, which helps to clarify our initial results. New Figure 8 addresses the connection between long-term inactivation of Na+ channels and the activation of TRPM4 channels, as characterized by the model (see Results lines 375-391). Furthermore, the model was instrumental in assessing how the Ca2+ and voltage-dependence of TRPM4 channels synergize to contribute to the shift in the center of mass of firing (Figure 9). Figure 9 illustrates the positive feedback loop between Ca2+ entry and the additional depolarization produced by Ca2+ activation of TRPM4 channels that can potentially accelerate firing (see Results lines 392-427).

    1. Figure 8: Panel C is supposed to confirm the prediction from the model that the carbachol-mediated change of firing activity is related to intracellular Ca2+ domains. However, the example cell shown is depolarized to -52 mV, and there is no hyperpolarization following Carbachol. Is this an effect of the high concentration of BAPTA? Again, what was the current injected under this experimental condition?

    Again, we thank the reviewer for pointing out the lack of clarity in the presentation of our results. We have now rewritten the results section for former Figure 8 (now Figure 10) to more clearly present these findings. The reviewer is correct that with the combination of 30 mM BAPTA + 10 nM free Ca2+ added to the intracellular solution (panel C of current Figure 10) the addition of carbachol did not change the membrane potential, as there were no changes in the holding current. Also, the amplitude of the ramp is comparable in control conditions and in the presence of carbachol under these conditions.

    We have now added all these details in the Results section for figure 10C.

    Reviewer #2 (Public Review):

    The manuscript focuses on the cholinergic modulation of TRPM4 channels in the CA1 pyramidal neurons. The authors presented solid convincing evidence that TRPM4 but not TRPC channels are the Ca2+-activated nonselective cation channel in CA1 pyramidal neurons being modulated by activation of muscarinic receptors. Using bi-directional ramp protocol, the authors revealed that ACh modulation could lead to forward shifts in place field center of mass, whereas decreased ACh modulation could contribute to backward shifts. This represents a significant molecular/cellular finding that links neuromodulation of intrinsic properties to place field shifts, a phenomenon seen in vivo. The authors used a computational approach to model this CA1 neuron spiking to further reveal the mechanism.

    To further improve the manuscript, I have the following suggestions/questions:

    1. The triangular ramp stimulation (introduced by the same group; Upchurch et al., 2022) makes it possible to emulate the hill-shaped depolarization during place field firing. However, one concern is the time scale/duration of the ramp (2 sec) compared to the physiological pattern (100ms~200ms in the in vivo recording in freely moving rat, Epsztein et al., 2011). Using a longer ramp to generate more spikes for calculating the adaptation index is understandable. However, considering the Ca entry/accumulation during prolonged depolarization, repeating one set of experiments with a shorter ramp is crucial to verify the major findings.

    When determining the duration of the current injections for our ramps, we relied on the data recorded in vivo in freely moving rats (Epsztein et al. Neuron 70:109–120, 2011) or in head-fixed mice running on spherical a treadmill immersed in virtual reality (Harvey et al Nature 461:941–946, 2009). In those papers, the voltage deflections are shown as a function of time, and gray bars or boxes represent the time the animals spend traversing the place field. We interpret those figures as showing that the hill-shaped depolarizations have variable durations, on the order of 1-20 s; we therefore think that our experiments with 2 and 10 second-long ramps cover a fair range of these durations. The place fields in Epsztein et al., 2011 were 4 cm long, and the authors give an example in Figure 3, in which the 2 meter track is traversed 1.5 times in 3 minutes. At that rate, the rat spent on average 2.4 seconds in each place field. We interpret the numerous shorter epochs of firing on the order of 100-200 ms shown Figure 2 in Epsztein et al. as the result of ongoing theta modulation within one overall depolarization during a single place field traversal. The following quote from that paper supports our interpretation “Some (Figure 2E, trace 1), but not all (trace 2), passes revealed spiking associated with a series of large (to ~-25 mV), long-lasting (~100 ms) depolarizations (Kandel and Spencer, 1961; Wong and Prince, 1978; Traub and Llinás, 1979; Takahashi and Magee, 2009) occurring rhythmically at ~4–5 Hz (theta frequency).” We thank the reviewer for pointing out these traces; our results are more directly applicable to the traces without theta modulation. Adding theta modulation is beyond the scope of this study but will be considered in future studies. Our average results in Figure 1 show that carbachol similarly affects 2 s and 10 s ramps, therefore we decided to present only the data on 2 second ramps for all the subsequent figures (see Results lines 156-157).

    1. Strictly speaking, the term "Ca2+-induced Ca2+ release (CICR)" is only used in ER Ca2+ release via ryanodine receptors (RyR) rather than IP3Rs. The author should be careful since it is used in the abstract (Line 36). In addition, pharmacology inhibition experiments should be incorporated to further dissect the role of RyR-induced CICR.

    We thank the reviewer for pointing out the possible confusion regarding the use of the term Ca2+-induced Ca2+ release (CICR) and we removed it from the text. Further, for this resubmission, we have pharmacologically dissected the role of IP3 vs ryanodine receptors in the cholinergic shift in the center of mass of firing due to the activation of TRPM4 channels, as suggested by the reviewer (see new Figure 6). To our surprise, neither the IP3R antagonist, Xestospongin C (1-2 µM), nor the RyR antagonist ryanodine (40 µM) were effective in preventing the cholinergic shift of the center of mass of firing when added to the intracellular solution (see Results lines 310-340).

    1. Applying strong buffering BAPTA not only removed the IP3R-TRPM nanodomain but also hindered Ca entry via VGCC. To validate the role of ER Ca2+ release in regulating TRPM, depletion of ER Ca2+ pool with SERCA inhibitor (e.g. thapsigargin) would be a more direct way to test the model (also make sure to add TRPC inhibitor to avoid the store-operated Ca2+ entry).

    We agree with the reviewer that 30 mM BAPTA also disrupts intracellular Ca2+ elevation via voltage-dependent Ca2+ channels on the neuronal membrane. Given that our experiments excluded a role of Ca2+ release from the intracellular stores (see below), our new model includes a nanodomain where, during cholinergic activation, the Ca2+ entry through VGCC is amplified to reach micromolar concentrations, through a currently unknown mechanism. As pointed out by the reviewer, the experimental results with 30 mM BAPTA support the existence of a nanodomain for the activation of TRPM4 channels, regardless of the nature of the calcium source.

    We have also addressed the role of ER Ca2+ release in our experiments.

    1. How does the TRPM current overcome the long-term inactivation of Nav? A channel state model should be added to the manuscript to make it easier to understand.

    Figure 11C now shows the Markov model of the NaV channel and new Figure 8 is devoted to explaining the mechanism by which current through the TRPM4 channels overcomes the long-term inactivation of the NaV channel.

    Reviewer #3 (Public Review):

    Combining slice physiology and simulation, Combe and colleagues discovered that TRPM4 channels activated by Ca2+ in nanodomains mediate ICAN currents in CA1 pyramidal neurons that drive the cholinergic modulation of firing rate. The finding is novel and interesting.

    Strengths:

    1. Identification of TRPM4 channels as the carrier of ICAN currents with independent pharmacological inhibitors and other supporting evidence.
    1. Physiological and simulational verification of physically closely located Ca2+ source and TRPM4 channels required for ICAN activation.

    Weaknesses:

    1. The conclusion of the cholinergic role in down-ramp or backward firing shifts is not convincing.

    We agree with the reviewer that our interpretation is somewhat speculative, and we have now included disclaimers throughout the manuscript as well as placed most of these interpretations in a portion of the discussion titled “Ideas and speculations: Implications of our results for place fields in intact rodents”. In addition, we added the word “potential” in the title.

  3. eLife

    eLife assessment

    This manuscript by Combe et al. presents the role of cholinergic modulation in the spike rate adaptation in pyramidal place cells. Using combined electrophysiology, pharmacological, and multi-compartment computational modeling, the authors identify the downstream pathway (e.g. activation of TRPM4 channel) that shapes the firing pattern under the triangular-shaped ramps. The study demonstrates solid evidence, and the findings are important for bridging pyramidal neurons' molecular/channel properties to behavior-level implications (place field firing).

  4. eLife

    Reviewer #1 (Public Review):

    This study combines in vitro somatic and dendritic recordings and computational modeling to study how cholinergic agonists modulate the response of CA1 pyramidal neurons to triangular current injections. The authors have previously used a similar approach (Upchurch, 2022, JNeuroscience) to show that CA1 neurons exhibit asymmetric AP firing (more firing on the upward ramp) in response to such current injections and that this effect is due to Na channel inactivation. The present work builds on these results by showing that cholinergic modulation changes this response, i.e., there is more firing on the downward part of the ramp. This change appears to require an intracellular Ca2+ concentration increase (mediated via IP3 and voltage-gated Ca2+ channels), which activates TRPM4 channels. In this scheme, cholinergic activity increases IP3, and the depolarizing current injection opens voltage-gated Ca2+ channels. This study will be of some interest to cellular neurophysiology experts working on the hippocampus.

    1. This study claims that the triangular current injections recapitulate hippocampal place cell activity. However, it has been shown recently that the asymmetric firing of CA1 place cells is due to synaptic weight changes resulting from synaptic plasticity (e.g., Bittner et al., 2017). This suggests that the asymmetric firing of place cells is primarily the result of asymmetric synaptic input. Therefore, the authors should test whether carbachol similarly affects a synaptically driven membrane potential ramp. If this is not the case, the strong claim that this work has implications for place cell firing is not justified, in my opinion.

    2. Along the same lines, it has been shown before that the precision of spike timing depends on the stimulation pattern in vitro (Mainen and Sejnowski, 1995). Constant stimuli led to imprecise AP firing trains, whereas current injections that included fluctuations resembling synaptic input generated spike trains that were more reliable and reproducible in terms of timing. This study concluded that a low intrinsic noise level in spike generation was essential in generating informative spike sequences. Following this pivotal work, the authors could add noise to their current stimulus and observe the effect on the AP firing patterns. If this is not possible, the authors should at least report the sweep-to-sweep variability for the data shown, e.g., in panels 1A2, 1B2, 1D2, and 1E2.

    3. In most of the data presented in this manuscript, Carbachol appears to induce a 3 mV hyperpolarization and increase input resistance. As a result, the amount of current injected during Carbachol is drastically lower than during the controls. This should be emphasized more, and the input resistance should be quantified for each experimental condition. It should also be discussed whether this change in input resistance can account for the changes in the firing pattern observed. Finally, it should be clearly stated how the amount of the current injected was chosen for each cell, and data from a range of injected current ramps should be shown for each cell.

    4. It remains unclear how the current result that TRPM4 channels can mediate the firing pattern change relates to the previous finding that the current injection evoked CA1 neuronal firing pattern is due to long-term Na channel inactivation.

    5. Figure 8: Panel C is supposed to confirm the prediction from the model that the carbachol-mediated change of firing activity is related to intracellular Ca2+ domains. However, the example cell shown is depolarized to -52 mV, and there is no hyperpolarization following Carbachol. Is this an effect of the high concentration of BAPTA? Again, what was the current injected under this experimental condition?

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript focuses on the cholinergic modulation of TRPM4 channels in the CA1 pyramidal neurons. The authors presented solid convincing evidence that TRPM4 but not TRPC channels are the Ca2+-activated nonselective cation channel in CA1 pyramidal neurons being modulated by activation of muscarinic receptors. Using bi-directional ramp protocol, the authors revealed that ACh modulation could lead to forward shifts in place field center of mass, whereas decreased ACh modulation could contribute to backward shifts. This represents a significant molecular/cellular finding that links neuromodulation of intrinsic properties to place field shifts, a phenomenon seen in vivo. The authors used a computational approach to model this CA1 neuron spiking to further reveal the mechanism.

    To further improve the manuscript, I have the following suggestions/questions:
    1. The triangular ramp stimulation (introduced by the same group; Upchurch et al., 2022) makes it possible to emulate the hill-shaped depolarization during place field firing. However, one concern is the time scale/duration of the ramp (2 sec) compared to the physiological pattern (100ms~200ms in the in vivo recording in freely moving rat, Epsztein et al., 2011). Using a longer ramp to generate more spikes for calculating the adaptation index is understandable. However, considering the Ca entry/accumulation during prolonged depolarization, repeating one set of experiments with a shorter ramp is crucial to verify the major findings.

    2. Strictly speaking, the term "Ca2+-induced Ca2+ release (CICR)" is only used in ER Ca2+ release via ryanodine receptors (RyR) rather than IP3Rs. The author should be careful since it is used in the abstract (Line 36). In addition, pharmacology inhibition experiments should be incorporated to further dissect the role of RyR-induced CICR.

    3. Applying strong buffering BAPTA not only removed the IP3R-TRPM nanodomain but also hindered Ca entry via VGCC. To validate the role of ER Ca2+ release in regulating TRPM, depletion of ER Ca2+ pool with SERCA inhibitor (e.g. thapsigargin) would be a more direct way to test the model (also make sure to add TRPC inhibitor to avoid the store-operated Ca2+ entry).

    4. How does the TRPM current overcome the long-term inactivation of Nav? A channel state model should be added to the manuscript to make it easier to understand.

  6. eLife

    Reviewer #3 (Public Review):

    Combining slice physiology and simulation, Combe and colleagues discovered that TRPM4 channels activated by Ca2+ in nanodomains mediate ICAN currents in CA1 pyramidal neurons that drive the cholinergic modulation of firing rate. The finding is novel and interesting.

    Strengths:

    1. Identification of TRPM4 channels as the carrier of ICAN currents with independent pharmacological inhibitors and other supporting evidence.
    2. Physiological and simulational verification of physically closely located Ca2+ source and TRPM4 channels required for ICAN activation.

    Weaknesses:

    1. The conclusion of the cholinergic role in down-ramp or backward firing shifts is not convincing.
  7. Version published to 10.1101/2022.10.24.513511v3 on bioRxiv
  8. Version published to 10.1101/2022.10.24.513511v2 on bioRxiv
  9. Version published to 10.1101/2022.10.24.513511v1 on bioRxiv