Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons

  1. Prithviraj Rajebhosale
  2. Mala R Ananth
  3. Ronald Kim
  4. Richard Crouse
  5. Li Jiang
  6. Gretchen López-Hernández
  7. Chongbo Zhong
  8. Christian Arty
  9. Shaohua Wang
  10. Alice Jone
  11. Niraj S Desai
  12. Yulong Li
  13. Marina R Picciotto
  14. Lorna W Role
  15. David A Talmage

  • Curated by eLife

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    This important study examines the existence of a fear memory engram in acetylcholine neurons of the basal forebrain and seeks to link this to modulation of amygdala for fear expression. Using a combination of techniques including genetic access to cFos expressing neurons, in-vivo chemogenetics, and optical detection of acetylcholine (ACh), the authors present solid evidence that posteriorly-located amygdala projecting basal forebrain cholinergic neurons participate in cue-specific threat learning and memory. This paper will be of interest to those studying circuit-level mechanisms of learning and emotion regulation.

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Abstract

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SI p ) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can ‘learn’ the association between a naive tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24 hr later (recall). In the NBM/SI p cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SI p cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SI a cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

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  1. Version published to 10.7554/elife.86581 on eLife
  2. Version published to 10.21203/rs.3.rs-3938016/v1 on Research Square
  3. eLife

    Author Response

    Reviewer #1 (Public Review):

    “The authors use hM4Di to "silence" Fos-tagged neurons in the basal forebrain, but they have not validated the efficiency or the possible various effects of this reagent.

    It is possible that hM4Di actually has a relatively small effect on suppressing the AP activity of neurons. Nevertheless, hM4Di might still be an effective manipulation, because it was shown to additionally reduce transmitter release at the nerve terminal (see e.g. Stachniak et al. (Sternson) 2014, Neuron). Thus, the authors should evaluate in control experiments whether hM4Di expression plus CNO actually electrically silences the AP-firing of ChAT neurons in the BF as they seem to suggest, and/or if it reduces ACh release at the terminals. For example, one experiment to test the latter would be to perfuse CNO locally in the BLA; after expressing hM4Di in the cholinergic neurons of the BF. At the very least, the assumed action of hM4Di, and the possible caveats in the interpretation of these results should be discussed in the paper.”

    We find that activation of hM4Di with clozapine in basal forebrain cholinergic neurons results in clear alterations to neuronal activation in projection targets and in behavior (Figures 3, Figure 3-Supplement 1, Figure 5, Figure 5-Supplement 1, Figure 5-Supplement 2, Figure 6-Supplement 1 and Figure 8). Previous studies demonstrated that activation of hM3Dq or hM4di in cholinergic neurons results in changes to electrical activity and behavioral response (Zhang et al. 2017 & Jin et al. 2019). Though we are unable to distinguish whether the effects on behavior in our experiments are a result of decreases in ACh release at terminals, inhibition of action potential firing, or both, our behavioral findings are consistent with demonstrations that inhibition of basal forebrain cholinergic neurons can alter behavior. See Page 17 Lines 488-493 for a discussion.

    “The names of brain areas like "NBM/SIp" and "VP-SIa" need to be better introduced, and somehow contextualized (in the Introduction, and also at first reading in the Results).”

    We agree that our prior presentation of these regions was confusing and in general the boundaries of these regions are not well-defined in the field. We have included a description of anatomical landmarks and bregma coordinates to clarify our definitions of the regions NBM/SIp (Page 4 Line 103-104) and VP/SIa (Page 4 Line 107-108).

    “Figure 3C: Application of CNO on the memory recall day leads to a strong reduction in CS-driven freezing. However, in this experiment, and also in Fig. S7, the pre-tone value of freezing is also strongly reduced. This would indicate that the activity of NBM/SIp cells (or else, ACh-release from these cells - see also Major point 1), also influences contextual learning. The authors should, first, statistically, test these effects (I am not sure this was done). If these differences are significant, a possible role of ACh in contextual fear learning should be discussed. Has it been shown before whether ACh is involved in contextual fear learning? Does this indicate the involvement of another target area of ACh neurons (e.g., the hippocampus?).”

    We statistically compared the pre-tone freezing response between Sham and hM4Di groups across our experiments and found no significant differences in pre-tone freezing between the groups (Figure 3D- Sham vs. ADCD-hM4Di, Pre-tone p=0.3544; Figure 5B- Sham vs. hM4di, Pre-tone p=0.0679; Figure 5C- Sham vs. hM4Di, Pre-tone p=0.0966; Figure 5-Supplement 2A- Sham vs. hM4Di, Pre-tone p>0.99). These comparisons can also be reviewed in the statistical reporting table uploaded along with the manuscript.

    “The discussion could be improved by better comparing what they found, to the wider literature. For example, previous papers studying other neuromodulatory systems found evidence for a modulation of neuromodulator release after learning, e.g. see Martins and Froemke 2015 Nat. Neuroscience for the noradrenergic system, Tang et al. (Schneggenburger lab) 2020 J. Neuroscience for the dopaminergic system and fear learning; and Uematsu et al., 2017, Nat. Neuroscience for the noradrenergic system and fear learning. Maybe the authors could include these and similar references when revising their discussion to take into account a broader view of previous findings related to other neuromodulatory systems.”

    Our study joins the growing body of literature demonstrating stimulus-encoding and rapid stimulus-contingent responses in various neuromodulatory systems in learning and memory recall. We have now added a substantial discussion, detailing both the similarities and differences between our findings and those found in the dopaminergic, serotonergic, noradrenergic, and oxytocinergic systems in fear learning. See Pages 20-21 Lines 575-605.

    Reviewer 2 (Public Review):

    “Throughout the paper, the authors use comparisons of cell activity between groups to address questions about projection-specific and cue-specific cell activation and reactivation. However, statistical comparisons are sometimes done between biological replicates (e.g. Fig. 5A), whereas a lot of them are done between technical replicates (e.g. Fig. 2B, 5B, 7B). Adding statistics that compare biological replicates would help increase confidence in the results.”

    We have replotted our data as a comparison of biological replicate (by individual animal) in new versions of Figures 1-8, and Figure 1-Supplements 1-3, Figure 5-Supplements 1 & 2, Figure 6-Supplements 1 & 2, Figure 7-Supplement 1, and Figure 8-Supplement 1. Correspondingly, all statistical analyses have been conducted comparing biological replicates. To note, these changes have not changed the overall conclusions of each figure. The sample size, statistical test and p-values for our comparisons are included in the figure legends and in the newly included statistical reporting table.

    "To demonstrate engram-like specificity, in figure 4C the authors show fold change in cholinergic reactivation in low and high responders (animals that show low and high defensive freezing upon cue presentation) as normalized by cell activity while sitting in the home cage. However, the authors also collected a better control for this comparison, which is shown in figure S4, where the animals were exposed to an unconditioned tone cue. Comparing fold change to this tone-alone condition would provide stronger evidence for the authors' point, as this would directly compare the specificity of cholinergic reactivation to a conditioned vs an unconditioned cue. A discussion of the same comparison is relevant for figure 2 (and is shown in figure S4) but is not mentioned in the text.”

    We have evaluated the cholinergic response to the tone using GRABACh3.0 as a readout of ACh release in the BLA, and using IEG expression as a readout of cholinergic neuron activation. We find no significant increase in ACh release in the BLA in response to tone presentation (Figure 1C-left, 1D-left) and no significant increase in tone associated reactivation of cholinergic neurons (using IEG as a readout, 2C/D, Figure 1-Supplement 2, Figure 1-Supplement 3, Figure 6-Supplement 1A) unless the tone has been previously paired with a foot shock(see Figure 1C-right, 2C, 3D). In addition, we find no statistically significant differences between home cage and tone alone conditions (Figure 2C – home cage-home cage condition vs. tone-tone condition, p=0.5012; Based on these analyses, we use the home cage group as our control group for comparison.

    “The significant correlation between cue-evoked percent change in defensive freezing from pretone and fold change in cholinergic cell activity relative to the home cage that is shown in figure 4D is somewhat confusing. Is the correlation considering all the points shown (high and low responders as depicted by black and grey points)? It's first reported as one correlation but then is discussed as two populations that have different results. Further, is the average amount of reactivation for the home-cage controls used here the same denominator for each reported animal? Similarly to the point above, a correlation looking at fold change from tonealone would also be helpful to determine the degree to which cholinergic reactivation is specific to threat-association learning versus the more general attentional component that this system is known for.”

    We have substantially modified this figure, now new Figure 6, to clarify our point. Along with this revision, we have removed the correlation plots and corresponding analyses from the revised version of the manuscript and figures.

    Figure 6 now begins with behavior data from a distinct cohort of mice outlining our criteria for high vs. low responders (Figure 6A/B). In Figure 6C, conducted in a separate cohort of mice that only underwent behavioral testing to clarify the definition of high vs. low responders, we note via schematic that ADCD labeling was carried out during the recall session (unlike Figure 2). In panel D, we show fold change of activated cholinergic neurons stratified by High vs. Low responder status. This fold change is normalized to the average activation from the home cage control animals in each experimental cohort. Taken together we find animals with a ~2 fold increase in activation of cholinergic neurons display significant, distinguishable freezing in response to the tone as compared to pretone freezing. We find that this cluster of activated neurons is segregated to the anterior NBM/SIp (Figure 6E).

    Regarding the involvement of cholinergic reactivation tone response (attention) rather than learning - in Figure 1-Supplement 3, we evaluate ACh release and behavioral response in mice that were exposed to three shocks alone (no tone) on day 1 and then exposed to a single (novel) tone on day 2. In these mice we find no significant change in ACh release in the BLA in response to tone, and no significant increase in freezing behavior in response to the tone. In Figure 2D, we evaluate reactivation of cholinergic neurons in a similar context and find that this group does not significantly differ from the home cage → home cage group. Further, we present that this home cage group does not significantly differ from Low Responders. As such, we find significant reactivation of cholinergic neurons in animals with increased responsiveness to the CS tone during the recall session (High Responders).

    “The compelling argument of this paper is that the authors are separating out the general attention role typically attributed to the cholinergic system from a more specific, engram-based role. Given the importance of untangling this, it would useful to see the recorded traces and behavioral scoring for the data shown in figure S2B. For example, was the higher slope in the recorded cholinergic response during unconditioned tone 1 also accompanied by an increase in freezing, which later went away with additional non-reinforced tones? Given that the animals were not habituated to tones (according to the Methods), this activity could be related to a habituation/general attention response, which may then be weaker than the learned response.”

    We include individual traces of GRABACh3.0 release in the BLA in response to the unconditioned tone from a protocol with 3x tone presentation on Day 1 and tone presentation on Day 2 (Figure 1-Supplement 2C). We have also included average + SEM traces for the entire duration of the tone presentation for the three unconditioned tones in this paradigm along with an inset showing 1s before and after tone onset (Figure 1Supplement 2D). Finally, we include individual traces of GRABACh3.0 release in the BLA in response to the first (naïve) tone from mice that underwent the training (tone + shock) followed by recall (tone) paradigm in Figure 1-Supplement 4C, left. None of the unconditioned tone responses were statistically significantly different from the preceding baseline. Instead, we find the learned response is significantly higher than the response baseline (Figure 1D).

  4. eLife

    eLife assessment

    This important study examines the existence of a fear memory engram in acetylcholine neurons of the basal forebrain and seeks to link this to modulation of amygdala for fear expression. Using a combination of techniques including genetic access to cFos expressing neurons, in-vivo chemogenetics, and optical detection of acetylcholine (ACh), the authors present solid evidence that posteriorly-located amygdala projecting basal forebrain cholinergic neurons participate in cue-specific threat learning and memory. This paper will be of interest to those studying circuit-level mechanisms of learning and emotion regulation.

  5. eLife

    Reviewer #1 (Public Review):

    Previous work by this group has established that cholinergic projections from the forebrain to the basolateral amygdala (BLA) contribute to the acquisition of auditory-cued fear memories (Jiang et al., 2016). Here, the authors continue these studies, using a combination of techniques including genetic access to cFos expressing neurons, in-vivo optogenetics, and optical detection of acetylcholine (ACh) in the BLA. The main findings are that ACh is not only released during footshock presentation (the unconditioned stimulus, US, used in the fear learning) but that in addition, ACh is released upon CS presentation after fear learning. This implies that cholinergic neurons in the basal forebrain (BF) "learn" the response to tones and that they are recruited into a memory engram in the brain. The authors then follow up these ideas by showing with genetic, activity-dependent cFos labeling that BF ChAT+ neurons which are activated during the training session, are also re-activated by tone recall (Figure 2). Moreover, hM4Di- mediated block of the activity of those ChAT neurons activated during the training session strongly suppresses tone(CS) - driven freezing behavior during recall (Figure 3), again suggesting that re-activation of ChAT neurons in the BF is an important element for the retrieval of fear memory (or else, for the expression of a fear memory). Overall, I think the paper convincingly shows that learning of a tone response occurs in a neuromodulatory system and that neuromodulatory neurons are recruited to a fear memory engram. This adds a new dimension to the circuit- and neuromodulatory mechanisms that underlie learning and memory.

    The paper, as it stands, has weaknesses in data presentation, data analysis, and statistical reporting. For most experiments, significantly more raw data should be shown (e.g. raw example traces for GRAB-ACh3.0), and also brain section images for almost all experiments (specific examples below). Raw data should also be shown in the Main Figures.

    Major point

    1. The authors use hM4Di to "silence" Fos-tagged neurons in the basal forebrain, but they have not validated the efficiency or the possible various effects of this reagent.
      It is possible that hM4Di actually has a relatively small effect on suppressing the AP activity of neurons. Nevertheless, hM4Di might still be an effective manipulation, because it was shown to additionally reduce transmitter release at the nerve terminal (see e.g. Stachniak et al. (Sternson) 2014, Neuron). Thus, the authors should evaluate in control experiments whether hM4Di expression plus CNO actually electrically silences the AP-firing of ChAT neurons in the BF as they seem to suggest, and/or if it reduces ACh release at the terminals. For example, one experiment to test the latter would be to perfuse CNO locally in the BLA; after expressing hM4Di in the cholinergic neurons of the BF. At the very least, the assumed action of hM4Di, and the possible caveats in the interpretation of these results should be discussed in the paper.

    Further specific points.

    1. The names of brain areas like "NBM/SIp" and "VP-SIa" need to be better introduced, and somehow contextualized (in the Introduction, and also at first reading in the Results).

    2. Figure 3C: Application of CNO on the memory recall day leads to a strong reduction in CS-driven freezing. However, in this experiment, and also in Fig. S7, the pre-tone value of freezing is also strongly reduced. This would indicate that the activity of NBM/SIp cells (or else, ACh-release from these cells - see also Major point 1), also influences contextual learning. The authors should, first, statistically, test these effects (I am not sure this was done). If these differences are significant, a possible role of ACh in contextual fear learning should be discussed. Has it been shown before whether ACh is involved in contextual fear learning? Does this indicate the involvement of another target area of ACh neurons (e.g., the hippocampus?).

    3. The discussion could be improved by better comparing what they found, to the wider literature. For example, previous papers studying other neuromodulatory systems found evidence for a modulation of neuromodulator release after learning; e.g. see Martins and Froemke 2015 Nat. Neuroscience for the noradrenergic system, Tang et al. (Schneggenburger lab) 2020 J. Neuroscience for the dopaminergic system and fear learning; and Uematsu et al., 2017, Nat. Neuroscience for the noradrenergic system and fear learning. Maybe the authors could include these and similar references when revising their discussion to take into account a broader view of previous findings related to other neuromodulatory systems.

  6. eLife

    Reviewer #2 (Public Review):

    In this manuscript, the authors use a number of approaches to show that a posterior subset of cholinergic neurons located in the nucleus basalis of myenert (NBV) and substantia innominata (SIp) region of the basal forebrain, and projecting to the basolateral nucleus of the amygdala (BLA), are part of the conditioned threat-memory engram that is associated with the defensive freezing response. The paper clearly demonstrates that NBM/SIp inputs to the BLA are selectively activated during cued-associative learning which is then reactivated upon cued memory retrieval, leading to cholinergic release in the BLA. Likewise, the authors also use in-vitro recordings of cue-activated vs inactivated cholinergic cells to demonstrate that activated neurons are more excitable (firing more action potentials) and with a lower rheobase. Collectively, these data support the notion that NBM/SIp is part of the memory engram for the learned association. To better characterize the importance of the cholinergic input to the amygdala for behavior, the authors delineate the segregation of function in cholinergic input to the BLA along the rostrocaudal axis. They show that inputs to the BLA originating from the more anterior NBM/SIa region mediate innate anxiety behavior whereas the more posterior cholinergic inputs are involved in associative fear conditioning.

    Overall, these findings make a significant contribution to our understanding of how the cholinergic system partakes in mediating cue-specific and non-specific emotional behavior. There are several group comparisons and statistical analyses that could strengthen the claims made in the paper.

    1. Throughout the paper, the authors use comparisons of cell activity between groups to address questions about projection-specific and cue-specific cell activation and reactivation. However, statistical comparisons are sometimes done between biological replicates (e.g. Fig. 5A), whereas a lot of them are done between technical replicates (e.g. Fig. 2B, 5B, 7B). Adding statistics that compare biological replicates would help increase confidence in the results.

    2. To demonstrate engram-like specificity, in figure 4C the authors show fold change in cholinergic reactivation in low and high responders (animals that show low and high defensive freezing upon cue presentation) as normalized by cell activity while sitting in the home cage. However, the authors also collected a better control for this comparison, which is shown in figure S4, where the animals were exposed to an unconditioned tone cue. Comparing fold change to this tone-alone condition would provide stronger evidence for the authors' point, as this would directly compare the specificity of cholinergic reactivation to a conditioned vs an unconditioned cue. A discussion of the same comparison is relevant for figure 2 (and is shown in figure S4) but is not mentioned in the text.

    3. The significant correlation between cue-evoked percent change in defensive freezing from pretone and fold change in cholinergic cell activity relative to the home cage that is shown in figure 4D is somewhat confusing. Is the correlation considering all the points shown (high and low responders as depicted by black and grey points)? It's first reported as one correlation but then is discussed as two populations that have different results. Further, is the average amount of reactivation for the home-cage controls used here the same denominator for each reported animal? Similarly to the point above, a correlation looking at fold change from tone-alone would also be helpful to determine the degree to which cholinergic reactivation is specific to threat-association learning versus the more general attentional component that this system is known for.

    4. The compelling argument of this paper is that the authors are separating out the general attention role typically attributed to the cholinergic system from a more specific, engram-based role. Given the importance of untangling this, it would useful to see the recorded traces and behavioral scoring for the data shown in figure S2B. For example, was the higher slope in the recorded cholinergic response during unconditioned tone 1 also accompanied by an increase in freezing, which later went away with additional non-reinforced tones? Given that the animals were not habituated to tones (according to the Methods), this activity could be related to a habituation/general attention response, which may then be weaker than the learned response.

  7. eLife

    Reviewer #3 (Public Review):

    This paper examines the existence of a fear memory engram in acetylcholine neurons of the basal forebrain and seeks to link this to the modulation of the amygdala for fear expression. Using genetically encoded ACh sensors, they show that ACh is released in the basolateral amygdala (BLA) in response to cues that had been paired with aversive shock (CS+) and by shock itself. They then use a cfos activity capture specifically of ACh neurons approach to show that an overlapping population of basal forebrain ACh neurons are activated during learning and recall, that chemogenetically silencing them reduced aversive memory recall, and that these cells have enhanced excitability. Moving on to examining the role of basal forebrain ACh neurons in regulating BLA, the authors show that chemogenetically inhibiting BLA projecting ACh neurons reduces memory recall-induced Fos activity in BLA neurons. Finally, they demonstrate the importance of these cells in producing freezing responses to both learned and innate aversive stimuli, though from different ACh populations.

    The identification of specific activity-defined acetylcholine neurons for aversive memory expression as well as the role of basal forebrain ACh neurons in regulating BLA to produce expression of defensive behaviors is important and interesting. However, the paper is missing important control groups and experiments that are necessary to adequately support the authors' claims.

  8. Version published to 10.1101/2021.05.02.442364v3 on bioRxiv
  9. Version published to 10.1101/2021.05.02.442364v1 on bioRxiv
  10. Version published to 10.1101/2021.05.02.442364v2 on bioRxiv