Hippocampal neuronal and astrocytic responses to noradrenaline and natural arousal

  1. Sian N. Duss
  2. Maria Wilhelm
  3. Alina-Mariuca Marinescu
  4. Runzhong Zhang
  5. Fritjof Helmchen
  6. Johannes Bohacek
  7. Peter Rupprecht

  • Curated by eLife

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    eLife Assessment

    This important work uses a sophisticated combination of neuromodulator imaging, optogenetics, and two-photon calcium imaging to examine how locus coeruleus-mediated norepinephrine signaling influences distinct hippocampal cell types. The evidence is solid and provides novel insights into cell type-specific responses to norepinephrine release. However, the conclusions would be strengthened by a more thorough analysis of the differences between locomotion-associated activity and optogenetic stimulation of the locus coeruleus.

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Abstract

The locus coeruleus (LC)-noradrenaline (NA) system is a central component of the brain’s response to arousal and stress. Yet how LC activity contributes to the cellular response profiles observed during natural arousal remains unclear. Here, we directly compared natural arousal with selective LC activation in mouse CA1, using physiologically titrated optogenetics, fiber photometry of NA and calcium signals, chronic two-photon imaging, and behavioral monitoring. While natural arousal robustly activated astrocytes, pyramidal cells, and interneurons, direct LC stimulation revealed a striking divergence in cellular response polarity and sensitivity. At levels of LC activation that correspond to arousal and moderate stress, we observed strong and reliable calcium responses in astrocytes, whereas pyramidal neurons and interneurons remained largely unaffected. Only at high-intensity LC stimulation did neurons exhibit a response, characterized by broad population-level inhibition of both pyramidal cells and interneurons, alongside the transient activation of an interneuron subpopulation that occupied distinct laminar positions in CA1. Thus, LC-driven NA release produces cell-specific effects in hippocampal CA1 that are distinct from and, at the population level for neurons even opposite to, cellular dynamics during natural arousal. Together, our results reveal a divergence in how astrocytes and neurons respond to LC-driven NA release and suggest that noradrenergic effects in the hippocampus at moderate levels of arousal and stress are predominantly mediated by astrocytes.

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  1. eLife

    eLife Assessment

    This important work uses a sophisticated combination of neuromodulator imaging, optogenetics, and two-photon calcium imaging to examine how locus coeruleus-mediated norepinephrine signaling influences distinct hippocampal cell types. The evidence is solid and provides novel insights into cell type-specific responses to norepinephrine release. However, the conclusions would be strengthened by a more thorough analysis of the differences between locomotion-associated activity and optogenetic stimulation of the locus coeruleus.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Duss et al. use several complementary and state-of-the-art strategies to characterize the effects of norepinephrine release from LC axons on post-synaptic cell types in the hippocampus. While a large body of research supports an important role for NE signaling in hippocampal function, the precise role by which NE promotes these effects remains poorly elucidated, in large part due to the complexity that adrenergic subtypes can be expressed in a variety of cell types and promote a variety of responses. Towards assessing this, the authors first establish an optogenetic strategy by which their delivery stimuli mimic endogenous activation of LC in 'moderate' and 'high' acute stress events, using NE sensors to titer stimulation patterns to similar levels of NE release. They then conduct a series of 2P imaging experiments in mice and compare response properties of various cell types in the hippocampus (excitatory and inhibitory neurons, and astrocytes) when the animal is 'naturally' or optogenetically aroused (via activation of the LC). The results are surprising. Whereas natural arousal causes activation of astrocytes, pyramidal cells, and interneurons, optogenetic activation of the LC does almost the opposite, with only astrocytes responding positively. Another important finding from the study is that astrocytes seem to be the most responsive cell type in the hippocampus to NE release, suggesting they could be key components for downstream functional effects of NE release in this brain region.

    Strengths:

    (1) The study was methodically done with respect to the characterization of how optogenetic parameters related to levels of NE release. Also, the analysis of their calcium imaging of various cell types in the hippocampus was very comprehensive.

    (2) Related, their discovery that cell types in the hippocampus respond differently to NE release, while not a completely unexpected finding, is something that has not been addressed experimentally in such a direct way before (to my knowledge).

    (3) Their finding that optogenetic stimulation of the LC produces opposing results to when these cells are naturally activated has wide implications for the LC field and potentially beyond.

    Weaknesses:

    I was surprised that no efforts were made to further assess what might be causing this discrepancy in hippocampal responses to optogenetic vs. natural activation of the LC. Some experiments that I felt were missing:

    (1) The authors go to great lengths to measure NE release in a variety of arousing conditions (tail lift, foot shock, 5Hz LC opto, 20Hz LC opto), but then in their 2P imaging, they're comparing the opto results to a 'natural' arousal state defined as when the mice were in motion. Maybe I missed it, but I wasn't sure that they ever checked the level of hippocampal NE release in this running state, similar to what they did in the other arousal conditions. Thus, it wasn't clear to me how comparable this state was to the optogenetic stimulation.

    (2) The authors do a nice experiment to show that increases in the hippocampal NE sensors are dependent on LC activity via optogenetic inhibition of the LC (Figure 1, Supplement 3). It seems like a missed opportunity to include a similar strategy in their 2P testing, to assess whether the differing responses of pyramidal cells, interneurons, and astrocytes are truly due to NE release. I could imagine it might be difficult to precisely time LC inhibition with periods of movement, but I imagine that mice would still run even if the LC is inhibited.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    The manuscript aims to determine the extent to which LC-mediated NA release in the CA1 region of the hippocampus (at both population and cellular levels) contributes to physiological arousal responses associated with innate behaviors (stress, locomotion). The manuscript is divided into two parts in which the authors compare time-locked responses in astrocytes, interneurons (pan-targeting), and pyramidal (CaMKIIa-driven targeting) cells.

    In the first part of the manuscript, the authors perform bulk recordings of either NA release or calcium activity locked onto either 'natural arousal' events (tail lift, foot shock, force swim) or direct optogenetic activation of LC somas. A first aim is to identify an optogenetic stimulation frequency that would mimic NE release in the target area by low- and high-intensity stressors. In the second aim, they compared evoked responses across cell types and concluded that stressors and direct LC activation trigger similar responses in astrocytes but not in interneurons or pyramidal cells.

    In the second - and most extended - part of the manuscript, the authors performed 2-photon cellular recordings of these different cell populations and compared responses evoked by the onset of locomotion vs. direct activation of the LC. Doing so, they observed a great degree of heterogeneity across these two conditions and across cell types. They conclude that NA effects on the hippocampus are primarily mediated by astrocytes and that LC-NA neuromodulation alone does not recapitulate the full breadth of 'natural arousal' modulations. They conclude that other neuromodulators likely contribute to how the hippocampus responds to high arousal levels.

    Strengths:

    Overall, the manuscript is well written and the figures are particularly clear.

    Optogenetics is a very successful technique in contemporary neuroscience, yet one important identified limitation is that it operates largely in a non-physiological regime, driving spike rates in regions rarely visited under normal physiological operations. This has raised valid concerns about the physiological relevance of findings obtained from studies using this technique. Here, the authors aimed at calibrating optogenetic manipulations of the LC so as to match the physiological release of NA observed in specific behavioral contexts. This is a valuable endeavor that could bring the field towards more reproducible and broadly valid findings.

    Another important open question is how different cell types coordinate to support global network activity and adaptive behavior. By recording distinct cell populations from the same region (CA1) and in response to the same category of endogenous versus exogenous events (locomotion or LC activation), it becomes possible to unravel important and specific operation modes, here also linked to a specific category of neuromodulation signaling.

    Weaknesses:

    This manuscript was difficult to review. There is clearly a lot of work and effort that went into it, and the multiple techniques seem well implemented, often with appropriate controls. Yet, the general framing, the links between experiments and interpretations, unfortunately, look questionable in my opinion. Below, I unpack what I think are the 4 main weakness points.

    (1) Incomplete calibration of optogenetic manipulations to physiological regimes

    While mapping optogenetic stimulation protocols to physiological variations is valuable, the proposed approach suffers from major limitations. First, the only parameter that is calibrated is the peak of NE release (as estimated from GRAB-NE fluorescence). Thus, it excludes other important aspects of the response, including trial-to-trial variability and the temporal dynamics of the response. Furthermore, stressor and LC activation conditions are simply non-comparable in terms of the duration of the stimulation (e.g., 3 min swim test versus 10s optogenetic stimulation), likely involving neuromodulation at different timescales (phasic vs. tonic). Albeit not explicitly mentioned, the number of trials and inter-trial interval between successive stimulations are also likely unmatched. On another note, the identification of the best stimulation frequency seems based on a grid of predefined values, while a more precise, continuous assessment could have easily been used. Finally, even though phasic NE release is known to depend on baseline tonic NE levels (especially with a sensor that reports a sublinear function of NE concentration), this dimension is ignored.

    (2) Weak links between imposed stressors and spontaneous locomotion

    The general approach is surprising: authors calibrated the optogenetic stimulation protocol on a range of stress-related behaviors and applied this to locomotion behavior. Indeed, while the first part of the manuscript uses different stressors in freely moving contexts to 'naturally' elevate arousal, the second part uses spontaneous locomotion bouts in a head-fixed situation as proxies for heightened 'natural' arousal. These two parts are very difficult to relate, and it is entirely unclear how NE regimes observed in the first context generalize to the second. Yet, on several occasions, the authors directly relate the first (fiber photometry, Fig.1) and second (2-photon, Fig. 2-6) parts of the manuscript. For instance, they conclude in favor of a "weak alignment between astrocytic responses to arousal and to LC stimulation on a cellular basis, despite the similarity of the bulk response." It remains unclear why closer preparations weren't used in the two parts, such as time-locked change in GRAB-NE2m fluorescence according to either locomotion onset or in a fear conditioning assay, both using fiber photometry in a head-fixed setting.

    (3) LC optogenetics and spontaneous locomotion differ by more than the origin of the arousal drive

    By directly comparing spontaneous locomotion and LC activation, the authors imply that the only difference between these two conditions is the origin of arousal: endogenous vs. exogenous, respectively. Furthermore, they interpret LC activation as triggering a pure NA effect while locomotion would reflect the conglomerate modulation from multiple neuromodulatory systems. On the one hand, LC activation likely results in the recruitment of other arousal centers (the raphe serotonin system, for instance, see 10.1101/2025.03.26.644382). On the other hand, differences between these conditions span well beyond specific arousal centers (see the massive motor-related activity in cortical dynamics: 10.1038/s41593-019-0502-4). Another, more methodological concern is the larger instability of the field of view during locomotion by comparison to optogenetic activation. While I am sure the authors corrected for movement-related translation in x and y directions, there might still be residual motion artefacts in the z direction that could account for some of the differences between the two conditions.

    (4) Loose equivalence between locomotion and natural arousal

    On many occasions, the authors draw a direct equivalence between spontaneous locomotion and 'natural arousal'. Arousal is a multifaceted concept that relates to far more behavioral readouts and network states than just locomotion. For instance, imagine a freezing mouse in response to a threat: locomotion would be absent, but the animal would still be quite aroused. It is ok to leave aside a particular readout and focus on other one(s) (especially thus in the case of arousal, which has many aspects). However, in that case, a single readout cannot be equated with 'natural arousal' as a whole. Instead, terms like 'locomotion' or 'locomotion-linked arousal' should be preferred. Indeed, in the particular case of locomotion, what is being readout is the upper part of the arousal continuum, whereas pupil size or whisker pad movements can also provide a more complete readout, including the lower and intermediate parts of that same continuum. While it is not necessary to include other arousal readouts (once claims are appropriately modified), the motivation for leaving out available readouts (lines 187-201) feels like a post-hoc rationalization.

    In sum, these 4 points call in my opinion for a profound change in how results are presented and interpreted. If agreed, a solution could be to leave aside the first part of the manuscript, to provide a more accurate picture of the differences between optogenetic activation and spontaneous locomotion, and to better flag the limitations of the approach (a part that I believe is entirely missing in the current version).

  4. eLife

    Reviewer #3 (Public review):

    Summary:

    In this study, the authors focused on the CA1 region of the hippocampus to compare Ca2+ dynamics in astrocytes, pyramidal neurons, and interneurons in response to optogenetic stimulation of locus coeruleus-triggered noradrenaline (NA) release, or movement (natural arousal)-triggered NA release. The most striking finding is that all studied cell types responded differently to LC stimulation compared to natural arousal. The description of these findings is important as a resource for further mechanistic studies on how multiple neuromodulator systems may interact or for predicting the consequences of the selective impairment of the noradrenergic system.

    Strengths:

    The technical design and conduct of the experiments, analysis including statistics, as well as the presentation of the results, are timely and very solid.

    Weaknesses:

    The identity and localization of NA receptors responsible for effects on neurons are less clear, and therefore, the difference between LC stimulation and natural arousal is less surprising. However, the presented data are consistent with the established finding that astrocytes directly sense NA mainly through α1 adrenergic receptors, yet in this study, astrocytes that responded strongest to LC stimulation did not respond strongest to natural arousal, and vice versa for other astrocytes.

    The authors seem to favor diversity of astrocyte responsiveness as an explanation, but also mention differences in LC activation pattern and distance of individual astrocytes to NAergic nerve terminals. Therefore, this warrants a careful consideration of a critical aspect of the experimental design. The authors delivered Ca2+/NA sensors as well as the optogenetic tools via AAV. While Figure 1 Supplement 3 suggests that most LC neurons were transduced, AAV transduction will almost certainly lead to a diversity in copy numbers per cell. On the receptor side, this can lead to an artificial diversity in Ca2+ response detection sensitivity among individual cells, but more importantly, for the LC, this could account for a different pattern of activation by optogenetic stimulation compared to activation by natural arousal. Such a problem would remain unnoticed with the currently presented matching of optogenetic and natural arousal stimulations of LC using population NA sensor signals (Figure 1, fiber photometry).

    Major suggestion:

    A critical experiment to test for this caveat would be to ideally express the NA sensor in astrocytes (due to their space-filling process arborizations and direct response to NA; but expression in neurons, as present, would work as well) and study the spatial pattern of NA release using two-photon microscopy, comparing multiple days and LC stimulation by optogenetics versus natural arousal. In case these experiments revealed nonuniform NA signal patterns, stable over days, but different when caused by optogenetic stimulation versus natural arousal, it would possibly shift the interpretation of the astrocyte response patterns towards depending mainly on NA release rather than diversity in NA responsiveness. Such a finding would be consistent with studies that compared arousal-mediated Ca2+ dynamics in NAergic terminals and Bergmann glia in the cerebellum (PMID: 36790089). On the other hand, in case these added experiments revealed similar NA release patterns in response to optogenetic stimulation versus natural arousal, then the presented findings would convincingly represent a biological phenomenon.

    Minor suggestion:

    Using "movement" as a proxy for arousal is very appropriate. To avoid the misunderstanding that different phenomena have been studied, it may be useful to acknowledge that early studies of noradrenergic signaling to astrocytes have found that speed of locomotion does not correlate well with astrocyte Ca2+ responses, and electromyographic signals have been used as a "proxy for movement" (PMID: 24945771).

  5. eLife

    Author response:

    We thank the reviewers for their positive and constructive feedback and for the careful reading of our manuscript.

    We plan to address the reviewers’ comments and, specifically, to more thoroughly compare movement-associated activity with optogenetic stimulation of the locus coeruleus (LC), with new experiments, clarifications, and additional analyses.

    (1) We plan to perform new experiments using two-photon imaging of noradrenaline (NA) sensors in head-fixed mice during both optogenetic LC stimulation and spontaneous movement. This will, if successful, allow us to directly compare the spatial and temporal structure of NA release across conditions, and to quantify NA amplitude during locomotion versus LC stimulation.

    (2) We will analyze existing NA fiber photometry data for movement-related NA release and compare it to release evoked by LC stimulation.

    (3) In general, we plan to more prominently highlight the limitations of our study that were brought up by the reviewers. In particular, we will expand our discussion of other neuromodulatory systems and their interactions with the LC-NA system, and will tone down conclusions of our study if they cannot be supported by the additional planned experiments and analyses.

    Finally, a reviewer suggested the additional experiment to inhibit LC while performing two-photon imaging in head-fixed animals. These experiments have, due to their technical complexity, a low likelihood of success. In addition, recent work from the lab of Emily Macé already performs LC inhibition during functional recordings (doi: 10.64898/2026.03.06.710089). This work supports our interpretation that the contribution of LC-evoked NA release does not dominate movement-related signals. We will discuss these recent findings in the revised version of our manuscript.

    Together, we believe that these planned experiments, analyses, and revisions will address all main concerns raised by the reviewers.

  6. Version published to 10.64898/2026.01.16.699885 on bioRxiv