A novel role for the lateral habenula in fear learning
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Summary: The manuscript examined the role of the lateral habenula (LHb) on contextual and cued fear conditioning with tests occurring at different time points since acquisition. The investigation provided important controls and systematic examination of testing and training conditions in places. The findings are interesting and likely of broad significance. However, the reviewers felt that the investigation lacked focus; that is, a more hypothesis-driven examination of the potential role of the LHb in the differential disruption of contextual and cued fear was viewed as necessary to make a major impact on a broad range of readers. Currently, there is not a clear and strong interpretation of the data, and more studies are necessary to further explore some of the options put forward by the authors.
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Reviewer #3:
In this manuscript, Sachella et al examine the contributions of the lateral habenula (LHb) to fear conditioning. They use 3 different paradigms: (1) a contextual fear conditioning paradigm, (2) a cued fear conditioning paradigm, (3) a combination paradigm where both context and cues can predict shocks. They also manipulate the LHb in several ways: (1) using muscimol, (2) using inhibitory optogenetics, (3) using excitatory optogenetics. The results are thought-provoking and would represent a novel contribution to the field, but I am left confused about some of the major points. My suggestions for improvement/clarification of the manuscript are as follows:
Major Comments:
Some important points need to be brought up in the introduction in order to frame the problem the authors are addressing and motivate the study. First, the …
Reviewer #3:
In this manuscript, Sachella et al examine the contributions of the lateral habenula (LHb) to fear conditioning. They use 3 different paradigms: (1) a contextual fear conditioning paradigm, (2) a cued fear conditioning paradigm, (3) a combination paradigm where both context and cues can predict shocks. They also manipulate the LHb in several ways: (1) using muscimol, (2) using inhibitory optogenetics, (3) using excitatory optogenetics. The results are thought-provoking and would represent a novel contribution to the field, but I am left confused about some of the major points. My suggestions for improvement/clarification of the manuscript are as follows:
Major Comments:
Some important points need to be brought up in the introduction in order to frame the problem the authors are addressing and motivate the study. First, the introduction needs more background on separate circuits controlling cued vs contextual fear conditioning (hippocampus, amygdala). This only comes up in the discussion. Readers also need more background on connections between known structures for fear conditioning and the LHb. There should also be explicit discussion of the well characterized connections between LHb and dopamine neurons, including how LHb inputs help generate reward prediction errors that may be important for fear conditioning. The idea that prediction errors contribute to the authors' observations could be foreshadowed here.
In general, the muscimol experiments are nicely done. However, muscimol is always administered during training. I am left wondering whether LHb activity is required during the initial learning of the association or for consolidation later. It would be ideal to also include a test of muscimol infusion immediately following the FC training, during a memory consolidation period. This is important because the authors at times seem to argue that the LHb is important specifically for memory consolidation, but later in the discussion claim that activity during the training (related to prediction errors) is an explanation for their results.
I'm struggling with the interpretation of the experiments in Figures 3 + 4 using the cue + context FC paradigm and talking about "reconsolidation." These are really key to the paper so making sure the experiments are clear is a must. From the cue + context test, it seems that having both cues + contexts available for memory provides a much stronger memory. I am uncertain about why the authors think this is so and whether the effect is independent of the LHb? For the "reconsolidation" experiment, I can't figure out what's new. The no-reconsolidation group should look like Figure 2 muscimol group, and it mostly does. The reconsolidation group should look like the Figure 3 muscimol group, and it mostly does. So this looks to me more like a replication of Figures 2+3 (with no vehicle control) than anything else. What did we learn that could not be learned from the experiments in Figures 1-3? The suggestion is that "FC training under inactivation of the LHb creates a cued memory whose retrieval depends on contextual information." (lines 154-155). I don't disagree with this interpretation necessarily but it seems vague, and there is no circuit-level insight as to the mechanism.
The ArchT experiments, as the authors already recognize, are problematic because of potential heating and other artifacts. 25s of continuous 10mW green light is a lot. I am not left with much confidence in interpreting these experiments and therefore I am not sure why they are included in the paper. There are other methods of optogenetic inhibition that would be better suited perhaps, or the results could be replicated with chemogenetics, where the authors could ensure DREADD viruses did not spread into the medial habenula.
The oChIEF experiments are interesting, but again very difficult to interpret. There is no data showing what the stimulation does to LHb firing, which is a concern given the very long light stimulation (through the whole experiment). Therefore, it is unclear whether the authors' hypothesis that the light stimulation interferes with normal function is correct. The design here also does not take advantage of the temporal precision of optogenetics.
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Reviewer #2:
In this work by Sachella and colleagues, the role of the lateral habenula (LHb) is investigated for its role in fear conditioning during initial encoding and subsequent retrieval in a later setting. This diencephalic nucleus has received a significant amount of attention in the preceding decade after its connectivity and regulation of neuromodulatory systems during learning and motivation was discovered. However, much less is known about its function in fear learning and memory. Building on the findings the authors report in an earlier avoidance setting, the present study deftly employs a series of pharmacological and optogenetic tools to identify the potential time-limited role of LHb in fear memory. Overall, the findings fit well with their previous work, and builds upon these observations by adding in more contemporary …
Reviewer #2:
In this work by Sachella and colleagues, the role of the lateral habenula (LHb) is investigated for its role in fear conditioning during initial encoding and subsequent retrieval in a later setting. This diencephalic nucleus has received a significant amount of attention in the preceding decade after its connectivity and regulation of neuromodulatory systems during learning and motivation was discovered. However, much less is known about its function in fear learning and memory. Building on the findings the authors report in an earlier avoidance setting, the present study deftly employs a series of pharmacological and optogenetic tools to identify the potential time-limited role of LHb in fear memory. Overall, the findings fit well with their previous work, and builds upon these observations by adding in more contemporary genetic tools to parse these aspects of the task. In particular, I was very enthusiastic about the further exploration of LHb in an associatively-learned fear approach; the strategies that have been highly successful in our understanding of amygdalo-hippocampal fear systems here are compelling applied to the LHb which traditionally has been better understood in stress and motivational settings. However, while the studies themselves were carefully conducted, it was not clear that these observations provided a conceptually transformative approach to the understanding of these neurobehavioral processes. Furthermore, some potential limitations in controls and isolation of important circuit function limits the impact of these findings. Specific concerns are numbered below:
First, while the optogenetic inhibition of LHb via ArchT selectively during the cue confirms the pharmacological observations in the preceding experiments, the use of that approach did not significantly extend those observations. Other controls such as a neural stimulus (CS-) or equivalently-applied optical inhibition during the inter-trial interval may have provided insights into the selectivity of the manipulation on the stability of the fear memory beyond that observed in the pharmacological approach. Adding to this, it would also have been of value (particularly with the optogenetic approaches where this would be quite straightforward) to explore some of the encoding vs retrieval vs expression distinctions that the LHb may contribute by providing stimulation/inhibition selectively during memory retrieval/expression in the 24h/Day7 test days.
The authors comment on the potential circuit-related contributions of LHb to portions of the amygdalo-hippocampal fear system, which would be of tremendous interest, yet without some isolation of these pathways in their approach, the authors are correct that these predictions would be largely speculative.
The use of optogenetics in the final study was quite unorthodox and I am not sure I found it entirely convincing as an approach to understand contextual representation via chronic optical stimulation. The utility of optogenetics should ideally derive from its temporal specificity, and as such, non-specific pulses applied throughout the session would take away from that core strength. Indeed, it seems to me that were the authors particularly invested in this chronic stimulatory or inhibitory approach intersecting with a vector-based targeting that DREADDs would likely present a superior option for these populations. Building on my last comment, this approach would also gain value from being able to target selected populations (e.g., hippocampal or DRN projections) via intersectional strategies.
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Reviewer #1:
The manuscript by Sachella examines the role of the lateral habenula (LHb) in learning to associate a context and a cue with an aversive event. The methods use pharmacological and optogenetic modulation of LHb function. The data show that inactivation of the LHb impairs contextual fear conditioning (CFC) as well as cued fear conditioning (when testing occurs in a novel context). The disruption in context but not cued FC is also obtained when testing occurs in the context of conditioning (A) 7 days after training but the deficit in both is evident when testing occurs 21 days after training. Overall, similar results are obtained with cue-specific optogenetic inhibition using ArchT and more sustained optogenetic excitation across the entire training session with oChiEF. Finally, exposure to the context and tone 24hrs prior to …
Reviewer #1:
The manuscript by Sachella examines the role of the lateral habenula (LHb) in learning to associate a context and a cue with an aversive event. The methods use pharmacological and optogenetic modulation of LHb function. The data show that inactivation of the LHb impairs contextual fear conditioning (CFC) as well as cued fear conditioning (when testing occurs in a novel context). The disruption in context but not cued FC is also obtained when testing occurs in the context of conditioning (A) 7 days after training but the deficit in both is evident when testing occurs 21 days after training. Overall, similar results are obtained with cue-specific optogenetic inhibition using ArchT and more sustained optogenetic excitation across the entire training session with oChiEF. Finally, exposure to the context and tone 24hrs prior to the test rescued cued but not contextual fear.
The present paper provides an interesting set of studies looking at the role of the LHb in fear conditioning. There are many strengths to the paper. The variation in testing and training conditions is great. It allows to examine memory to the conditioning context when it is the only stimulus the animals learn about, as well as to examine the memory for the cue when tested in a novel context in the absence of influence from the conditioning context (i.e., cue test in context B), as well as in the context of conditioning (i.e., context A). This allows the authors to rule out overshadowing as an interpretation. For example, the LHb-inactivated animals do not present an augmented case of overshadowing in the cued and contextual fear training conditions. If that was the case in the CFC alone experiment, LHb inactivation would not have disrupted learning, but it did. Further, if the LHb had a specific role in summation of context and cued fear (this could account for the data in Fig 3 as ceiling levels could mask performance differences in 3B), then it would not modulate contextual and cued FC when examined independently (Fig 1 and 2). The authors allude to this briefly in line 226. Other strengths of the manuscript include excellent anatomical controls.
Despite the strengths, there are a number of weaknesses that need to be addressed. The major one, I believe, lies in the necessity for additional data to support the conclusions. Although there are a lot of data presented in the manuscript, together they are not a convincing set that speaks to one interpretation. Specifically, the idea that LHb inactivation/stimulation leads to weakening of the memory strength is interesting, but it also requires additional investigation to show that under conditions when the CFC is strengthened, LHb inactivation has a less devastating effect. Further, the authors concede on line 252-253 that more experiments are needed to determine whether LHb inactivation disrupts the associative or representation components of CFC. I agree but feel this should have been done in the present paper instead of the reconsolidation studies which are also incomplete. The authors argue 'under inactivation of the LHb, a cued FC memory is formed whose retrieval depends on the context in which the cue is presented'. However, the disruption of contextual fear makes this interpretation difficult to accept. If the correct context is needed for cued fear to be expressed then this suggests either a possible generalization decrement effect that is ameliorated by being placed in the same context or a context-gating effect. Both require some knowledge of the context where the cued fear learning occurred. Yet, this is difficult to reconcile with the consistent disruption in context fear.
The reconsolidation experiments, although interesting, lack clarity and the vehicle controls. A systematic investigation of exposure to the conditioned context or the conditioned cue (in context B) on fear to the conditioned context, the cue and both would help dissociate how retrieval-based reconsolidation acts in the current preparation. This may warrant an independent investigation/publication.
Some other arguments that I didn't find convincing: The equivalent reduction in exploration in the OF for the vehicle and muscimol animals is argued to suggest that similar contextual representation are formed between the groups and therefore the CFC differences are unlikely to be due to deficits in context encoding. The OF data are insufficient to argue this. Many aspects can modulate activity in the OF from the traditional anxiety argument (here similar reduction in anxiety) to a sense of familiarity. There is no evidence for similar contextual encoding.
Some additional comments:
The way the 24hr and 7d data are presented is a little odd. While the authors justify this, it seems strange from the reader's perspective to see the 7d test data before the 24hr test data. In addition, the 24hr tests data are referred to as long term memory, which can be perceived as odd relative to the longer 7d test. This section just needs to be revised for clarity in the presentation.
Does the difference in cued fear at the 24hr interval persist if conditioning differences are used as a covariate in the analysis and if a difference score is calculated from the baseline difference?
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Summary: The manuscript examined the role of the lateral habenula (LHb) on contextual and cued fear conditioning with tests occurring at different time points since acquisition. The investigation provided important controls and systematic examination of testing and training conditions in places. The findings are interesting and likely of broad significance. However, the reviewers felt that the investigation lacked focus; that is, a more hypothesis-driven examination of the potential role of the LHb in the differential disruption of contextual and cued fear was viewed as necessary to make a major impact on a broad range of readers. Currently, there is not a clear and strong interpretation of the data, and more studies are necessary to further explore some of the options put forward by the authors.
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