A functional topography within the cholinergic basal forebrain for encoding sensory cues and behavioral reinforcement outcomes

  1. Blaise Robert
  2. Eyal Y Kimchi
  3. Yurika Watanabe
  4. Tatenda Chakoma
  5. Miao Jing
  6. Yulong Li
  7. Daniel B Polley

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    Evaluation Summary:

    This paper will be of interest to neuroscientists studying the effects of cholinergic modulation throughout the brain. It provides strong support for the view that activity in the basal forebrain cholinergic system is not monolithic, but varies across the rostrocaudal axis, consistent with previous reports of differential connectivity of these areas. Strong evidence for regional differences in cholinergic responses collected simultaneously under multiple behavioral conditions provides valuable context for interpreting variability in existing and future studies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Basal forebrain cholinergic neurons (BFCNs) project throughout the cortex to regulate arousal, stimulus salience, plasticity, and learning. Although often treated as a monolithic structure, the basal forebrain features distinct connectivity along its rostrocaudal axis that could impart regional differences in BFCN processing. Here, we performed simultaneous bulk calcium imaging from rostral and caudal BFCNs over a 1-month period of variable reinforcement learning in mice. BFCNs in both regions showed equivalently weak responses to unconditioned visual stimuli and anticipated rewards. Rostral BFCNs in the horizontal limb of the diagonal band were more responsive to reward omission, more accurately classified behavioral outcomes, and more closely tracked fluctuations in pupil-indexed global brain state. Caudal tail BFCNs in globus pallidus and substantia innominata were more responsive to unconditioned auditory stimuli, orofacial movements, aversive reinforcement, and showed robust associative plasticity for punishment-predicting cues. These results identify a functional topography that diversifies cholinergic modulatory signals broadcast to downstream brain regions.

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  1. Version published to 10.7554/elife.69514 on eLife
  2. Version published to 10.1101/2021.04.16.439895v2 on bioRxiv
  3. eLife

    Evaluation Summary:

    This paper will be of interest to neuroscientists studying the effects of cholinergic modulation throughout the brain. It provides strong support for the view that activity in the basal forebrain cholinergic system is not monolithic, but varies across the rostrocaudal axis, consistent with previous reports of differential connectivity of these areas. Strong evidence for regional differences in cholinergic responses collected simultaneously under multiple behavioral conditions provides valuable context for interpreting variability in existing and future studies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Although previous studies of the circuitry and activity of the basal forebrain (BF) in mice suggests that it does not function as a single monolithic unit, these studies have often focused on the dynamics of cholinergic modulation in a single behavioral paradigm, and variability in recording sites across studies has made it difficult to systematically evaluate these results. In this study, the authors perform simultaneous fiber photometric measurements of cholinergic cells in two areas of the basal forebrain: the horizontal limb of the diagonal band (HDB), and from the posterior tail of the basal forebrain in globus pallidus and substantia innominata (GP/SI). Importantly they examine the activity of these two regions of the BF both during spontaneous conditions, with unconditioned visual stimuli, and across a longer period of training on a sensory reversal task. They find a number of salient differences in the bulk cholinergic activity in these two areas, most notably a prominent enhancement of responses in the GP/SI to conditioned stimuli associated with punishment that was absent in the HDB.

    This study provides some of the strongest evidence to date for the differential involvement of regions of the BF in behavior, in opposition to the view that ACh is a single knob that can be turned to control global arousal throughout the brain.

    One weakness that the authors acknowledge is that fiber photometry provides only an aggregate view of activity in the two regions of the BF, and provides no information about more complicated dynamics that may occur at the level of individual cells. Another limitation which they don't discuss in depth is the limited temporal resolution of un-deconvolved GCaMP signals, which may give a false appearance of sustained activity because of the slow decay of GCaMP, which in some cases is longer than epochs in their behavioral trials. However, due to the challenges involved in getting cholinergic-specific measurements at two locations simultaneously in the BF, this data is still extremely useful and provides strong support for the main conclusions of the paper.

    Given the number of papers that record from or stimulate "the" basal forebrain, this kind of systematic approach to assessing regional differences across a variety of behavioral conditions is extremely valuable, and is likely to help convince researchers studying the cholinergic system that in future studies we need to be more nuanced about exactly where we are recording and intervening.

  5. eLife

    Reviewer #2 (Public Review):

    In this interesting manuscript, Robert and colleagues use simultaneous fiber photometry recordings from cholinergic neurons in two distinct basal forebrain nuclei, characterizing their differential activity during different sensory and behavioral events. They find largely qualitative distinctions between the functional properties of cholinergic neurons in the HDB and GP/SI, which primarily project to rostral and caudal sensory areas of the cortex, respectively. HDB cholinergic neurons display more tightly locked activity to pupil diameter fluctuations (proxy for arousal), and higher activity upon reward omission. GP/SI cholinergic neurons, in turn, respond more to lower-level behavioral variables: auditory stimuli outside of a task context, licking, punishment, and punishment-predicting sensory stimuli. The work significantly adds to a an important and timely discussion in the field about the heterogeneity of cholinergic signals conveyed to the cortex. The writing is very clear and the experimental design is excellent. I also appreciate the thoroughly documented methods. With a few exceptions that I detail below, the data and their analysis support the main claims.

    1. An important confound that needs to be more explicitly ruled out is the inadvertent imaging of striatal cholinergic interneurons. Specifically, if the basal ganglia structures near the GP/SI imaging site contribute to the signal, this could artificially inflate the functional differences in this nucleus compared to the HDB. This concern is particularly relevant for the photometry data, which lack cellular resolution. The results from auditory cortical GRAB sensor imaging help (Figs. 1H-J), but the lack of a comparison with cortical recordings from more frontal areas makes those experiments inconclusive. In the discussion the authors do mention lack of GCaMP expression in cholinergic striatal interneurons. Can they show quantification of this?

    2. Related to my point above, I would suggest showing quantification of fiber lesion locations for all animals. This is particularly important because anatomical heterogeneity is key to their findings.

    3. Regarding the findings in Figs. 2E-G showing very fast (1-trial) adaptation of sensory responses, I believe another confound should be ruled out. Given that the response is higher only in the very first stimulus presentation, an alternative explanation is that, rather than reflecting subsequent adaptation, this instead reflects a startle response to that stimulus. This would be compatible with the accompanying pupil diameter data. Can the authors exclude that possibility? Is there any evidence of a startle response, for instance in body posture?

  6. eLife

    Reviewer #3 (Public Review):

    In this study, Blaise et al. investigated sensory responsiveness and differences between cholinergic neurons in two anatomically distinct regions of the basal-forebrain (BFCN). They chose regions at the rostral and caudal extremes of the BFCN: the rostral/horizontal limb of the diagonal band (HDB bregma+0.3) vs the caudal globus pallidus/substantia innominata (GP/SI bregma -1.5mm). They assessed the responses to both auditory and visual stimuli and with respect to their validity as predictive cues in association with emotionally salient stimuli (both reward and punishment). A major strength ( as well as complexity) of this study is that all measurements were performed in these 2 regions in the same animal across time and with all of the behavioral paradigms tested.

    The authors provide a comprehensive introduction, reviewing previous studies of sensory responses within the cholinergic basal-forebrain. They point out that technical differences between the studies and heterogeneity within the basal-forebrain cholinergic system make it difficult to draw generalizable conclusions about functional organization from the existent literature. The authors of this study attempted to overcome some of these limitations by performing repeated calcium imaging studies using a genetically encoded calcium sensor (GCaMP) targeted for expression in cholinergic neurons using transgenic mouse lines. This approach overcomes the potential variability in targeting of different subsets of neurons using viral approaches and yield, given that cholinergic neurons generally make up {less than or equal to}10% of the total neurons in these regions.

    Repeated fiber photometric recordings of pooled activity in specific regions of the cholinergic basal forebrain are collected over 1 month in repeated sessions with the same 11 mice during which they test sensory responses and examine associative learning.

    In sum:

    1. The authors performed simultaneous long-term recording of calcium dynamics within cholinergic neurons of the HDB and caudal GP/SI across multiple conditions and sessions.

    2. The dynamics of this (presumably*) stable pool of neurons to multiple (distinguishable) auditory and visual stimuli was compared to pupillary dilation as a measure of general (+/-) arousal.

    3. Ca dynamics were also assessed based on pairing distinct auditory stimuli with either reward, reward omission, or punishment.

    Findings included:

    4.) Cholinergic neurons in both the HDB and caudal GP/SI, show responses to auditory stimuli. These responses were larger in GP/SI than in the HDB but showed similar decay upon repeated presentation, indicating habituation and potential encoding of stimulus novelty within both regions.

    1. Upon pairing three distinct auditory stimuli with reward, the authors characterized the responses of HDB vs GP/SI cholinergic neurons on trials where animals correctly licked in response to the cue to obtain reward (= hit) vs. miss trials where the mice did not perform and hence did not receive the reward. They found that cholinergic neurons in both HDB and GP/SI respond to sound onset on both types of trials but their activity during the pre-sound period distinguishes hit and miss trials. Curiously, the difference is that miss trials are characterized by higher calcium activity immediately prior to the onset of the sound cue.

    2. Cholinergic activity did not generally relate to motor movements such as licking with the exception of a lick-offset activity within the HDB only on trials where the mice made >7 licks (which was the criterion for reward delivery) indicating potential encoding of perception of successful motor execution and/or some component of motivation.

    3. The authors next assigned each of the three auditory stimuli to signal reward, omission of reward, or punishment (electrical shock) respectively (the latter 2 = rule reversal).

    - HDB - but not GP/ SI - neurons responded by increased activity locked to the offset of the licking bout elicited in response to the cue-predicted omission of a reward.

    - both HDB and GP/SI cholinergic neurons showed strongest responses to shocks and weakest responses to rewards.

    -GP/SI (but not HDB cholinergic neurons) showed learning-related enhanced responses to auditory cues upon successive pairings with shock.

    - The authors interpret their findings on learning related enhancement in response to reward as negative for both HDB and GP/SI.

    The major conclusion drawn is that HDB and GP/ SI cholinergic neurons are distinct in some of their functional roles and similar in others. It appears that distinctions between HDB and GP/ SI are not as pronounced as one might have expected. This could be due to a number of factors that may warrant deeper consideration/ additional experiments.

    a. the measured signal has relatively low cellular resolution and slow kinetics (effects of faster and/or more discrete events within the pooled signal)

    b. the possible role of (poly?) synaptic interconnections between HDB and GP/ SI

    c. the need for more data on how HDB vs GP/ SI Ca signaling activity relates to ACh release, behavioral output and/or engagement of their target domains (ie prefrontal cortex vs auditory cortex). Perhaps selective inhibition of Ca signaling in the cholinergic BFCN would enhance resolution of functional heterogeneity? In sum, how do the subtle differences in calcium dynamics between HDB and GP/SI cholinergic neurons functionally relate to the behavior of the animal?

    While the conclusions as stated by the authors are mostly supported by the data, the conclusion that there are no reward-related learning responses in either BFCN region requires further substantiation. The authors do note that their findings are not necessarily at odds with a previous demonstration of reward-learning related enhancement within the cholinergic basal forebrain because prior studies focused on anatomically distinct populations of BFCNs (i.e. more rostral NBM and GP/SI neurons that project to the amygdala compared to caudal GP/SI neurons that project to the auditory cortex).

    There may be other important differences: in Crouse et al (2020) learning related enhancement emerged from comparisons of calcium activity in cholinergic axons in the BLA between pre-training, training and post-training/acquisition periods. The authors in this study do not show data from pre-training (i.e. days 5-7 Fig 2A- operant shaping). The negative result of the current study would be further substantiated if the authors were to show that there are no enhancements as the mice initially learn to associate tones with rewards during this period. In the absence of such evidence, softening the conclusion that there is no reward-learning related enhancement might be advisable.

    An additional note: upon examining heatmaps shown in Fig 3D and E for hit trials, it appears that in later trials -while there isn't an enhancement to tone responses-, there is a decrease in activity following the reward predictive tone-related activity, which isn't apparent during early trials or on miss trials (~3 seconds following tone onset). Authors should comment on whether this decrease was statistically significant.

  7. Version published to 10.1101/2021.04.16.439895v1 on bioRxiv