A Forebrain Hub for Cautious Actions via the Midbrain
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Curated by eLife
eLife Assessment
This useful study uses fiber photometry, implantable lenses, and optogenetics to show that a subset of subthalamic nucleus neurons is active during movement, and that active but not passive avoidance depends in part on STN projections to substantia nigra. The strength of the evidence for these claims is solid, whereas evidence supporting the claims that STN is involved in cautious responding or the speed of avoidance is incomplete. This paper will be of interest to basic and applied behavioural neuroscientists working on avoidance if suitably streamlined to support the strongest claims.
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Abstract
Abstract
Adaptive goal-directed behavior requires dynamic coordination of movement, motivation, and environmental cues. Among these, cautious actions, where animals adjust their behavior in anticipation of predictable threats, are essential for survival. Yet, their underlying neural mechanisms remain less well understood than those of appetitive behaviors. Using calcium imaging in freely moving mice, we show that glutamatergic neurons in the subthalamic nucleus (STN) are robustly engaged during cue-evoked avoidance and exploratory behavior, encoding both contraversive movement and cautious responding. Targeted lesions and optogenetic manipulations reveal that STN projections to the midbrain, but not to the globus pallidus, are necessary for executing cued avoidance. Moreover, the frequency of STN activation governs response timing, accelerating the initiation of goal-directed actions to the point that it becomes incompatible with passive response, without being aversive. These findings identify a critical role for the STN in orchestrating adaptive goal-directed behavior by directing timely actions via its midbrain projections.
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eLife Assessment
This useful study uses fiber photometry, implantable lenses, and optogenetics to show that a subset of subthalamic nucleus neurons is active during movement, and that active but not passive avoidance depends in part on STN projections to substantia nigra. The strength of the evidence for these claims is solid, whereas evidence supporting the claims that STN is involved in cautious responding or the speed of avoidance is incomplete. This paper will be of interest to basic and applied behavioural neuroscientists working on avoidance if suitably streamlined to support the strongest claims.
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Reviewer #1 (Public review):
Summary:
The manuscript presents a robust set of experiments that provide new fundamental insights into the role of STN neurons during active and passive avoidance tasks. These forms of avoidance have received comparatively less attention in the literature than the more extensively studied escape or freezing responses, despite being extremely relevant to human behaviour and more strongly influenced by cognitive control.
Strengths:
Understanding the neural infrastructure supporting avoidance behaviour would be a fundamental milestone in neuroscience. The authors employ sophisticated methods, including calcium imaging and optogenetics, to delineate the functions of STN neurons during avoidance behaviours. The work is extremely thorough, and the evidence presented is compelling. Experiments are carefully …
Reviewer #1 (Public review):
Summary:
The manuscript presents a robust set of experiments that provide new fundamental insights into the role of STN neurons during active and passive avoidance tasks. These forms of avoidance have received comparatively less attention in the literature than the more extensively studied escape or freezing responses, despite being extremely relevant to human behaviour and more strongly influenced by cognitive control.
Strengths:
Understanding the neural infrastructure supporting avoidance behaviour would be a fundamental milestone in neuroscience. The authors employ sophisticated methods, including calcium imaging and optogenetics, to delineate the functions of STN neurons during avoidance behaviours. The work is extremely thorough, and the evidence presented is compelling. Experiments are carefully constructed, well-controlled, and the statistical analyses are appropriate.
Points for Authors' Consideration:
(1) Motoric role of STN:
The authors interpret their findings within the context of active avoidance, a cognitively demanding process. An alternative interpretation is that STN activation enhances global motoric tone, facilitating general movement rather than specifically encoding cautious avoidance. Experimentally, this could be evaluated by examining STN-induced motoric tone in non-avoidance contexts, such as open field tests with bilateral stimulations. Alternatively, or additionally, the authors could explicitly discuss evidence for and against the possibility that increased motoric tone may account for aspects of the observed behaviours.(2) Temporal Dynamics in Calcium Imaging (AA2 vs. AA1):
Based on previous work by this group, a delay (~1-2 sec) in neuronal response onset was anticipated in AA2 compared to AA1. Although a delay in peak response is observed, there is no clear evidence of a significant delay in response onset or changes in slope of neural activity. The authors could quantify calcium onset latencies and slopes and statistically compare these parameters across conditions.(3) Speed Differences (AA2 vs. AA1):
Given the increased latency in AA2, and based on previous work from the group, one would expect faster movements following initiation. However, such differences are not evident in the presented data. The authors might want to discuss the absence of an expected speed increase and clarify whether this absence is consistent with previous findings.(4) Behavioural Differences Across Neuronal Classes (Figure 7):
The manuscript currently does not compare responses of neuronal classes I, II, and III between AA1 and AA2 conditions separately or provide information regarding their activity during AA3.(5) Streamlining Narrative and Figures:
Given the extensive amount of material presented, the manuscript and figures would benefit from streamlining. Many data points and graphs could be moved to supplementary materials without affecting the core interpretation and simplifying the reading of the work by a non-expert audience. Similarly, the main text could be refined to more clearly emphasise the key findings, which would improve both readability and impact. At the same time, certain aspects would benefit from additional clarification. For example, it would be helpful to explain the key features of the AA1-AA3 tasks at the point of introduction, rather than referring readers to previous literature. Overall, enhancing clarity and accessibility would serve the authors well and broaden the impact of the work. -
Reviewer #2 (Public review):
Summary:
Zhou, Sajid et al. present a study investigating the STN involvement in signaled movement. They use fiber photometry, implantable lenses, and optogenetics during active avoidance experiments to evaluate this. The data are useful for the scientific community, and the overall evidence for their claims is solid, but many aspects of the findings are confusing and seemingly contradictory. For example, STN activity increases with contraversive turning in the fiber photometry experiments, but optogenetic stimulation of the STN evokes ipsiversive turning. While the authors present a huge collection of data, it is somewhat difficult to extract the key information and the meaningful implications resulting from this data.
Strengths:
The study is comprehensive in using many techniques, stimulation powers, …
Reviewer #2 (Public review):
Summary:
Zhou, Sajid et al. present a study investigating the STN involvement in signaled movement. They use fiber photometry, implantable lenses, and optogenetics during active avoidance experiments to evaluate this. The data are useful for the scientific community, and the overall evidence for their claims is solid, but many aspects of the findings are confusing and seemingly contradictory. For example, STN activity increases with contraversive turning in the fiber photometry experiments, but optogenetic stimulation of the STN evokes ipsiversive turning. While the authors present a huge collection of data, it is somewhat difficult to extract the key information and the meaningful implications resulting from this data.
Strengths:
The study is comprehensive in using many techniques, stimulation powers, frequencies, and configurations.
Weaknesses:
Here are the specific weaknesses of the paper.
(1) Vglut2 isn't a very selective promoter for the STN. Did the authors verify every injection across brain slices to ensure the para-subthalamic nucleus, thalamus, lateral hypothalamus, and other Vglut2-positive structures were never infected?
(2) The authors say in the methods that the high vs low power laser activation for optogenetic experiments was defined by the behavioral output. This is misleading, and the high vs low power should be objectively stated and the behavioral results divided according to the power used, not according to the behavioral outcome.
(3) In the fiber photometry experiments exposing mice to the range of tones, it is impossible to separate the STN response to the tone from the STN response to the movement evoked by the tone. The authors should expose the mouse to the tones in a condition that prevents movement, such as anesthetized or restrained, to separate out the two components.
(4) The claim 'STN activation is ideally suited to drive active avoids' needs more explanation. This claim comes after the fiber photometry experiments during active avoidance tasks, so there has been no causality established yet.
(5) The statistical comparisons in Figure 7E need some justification and/or clarification. The 9 neuron types are originally categorized based on their response during avoids, then statistics are run showing that they respond differently during avoids. It is no surprise that they would have significantly different responses, since that is how they were classified in the first place. The authors must explain this further and show that this is not a case of circular reasoning.
(6) The authors show that neurons that have strong responses to orientation show reduced activity during avoidance. What are the implications of this? The author should explain why this is interesting and important.
(7) It is not clear which conditions each mouse experienced in which order. This is critical to the interpretation of Figure 9 and the reduction of passive avoids during STN stimulation. Did these mice have the CS1+STN stimulation pairing or the STN+US pairing prior to this experiment? If they did, the stimulation of the STN could be strongly associated with either punishment or with the CS1 that predicts punishment. If that is the case, stimulating the STN during CS2 could be like presenting CS1+CS2 at the same time and could be confusing.
(8) The experiments in Figure 10 are used to say that STN stimulation is not aversive, but they only show that STN stimulation cannot be used as punishment in place of a shock. This doesn't mean that it is not aversive; it just means it is not as aversive as a shock. The authors should do a simpler aversion test, such as conditioned or real-time place preference, to claim that STN stimulation is not aversive. This is particularly surprising as previous work (Serra et al., 2023) does show that STN stimulation is aversive.
(9) In the discussion, the idea that the STN encodes 'moving away' from contralateral space is pretty vague and unsupported. It is puzzling that the STN activates more strongly to contraversive turns, but when stimulated, it evokes ipsiversive turns; however, it seems a stretch to speculate that this is related to avoidance. In the last experiments of the paper, the axons from the STN to the GPe and to the midbrain are selectively stimulated. Do these evoke ipsiversive turns similarly?
(10) In the discussion, the authors claim that the STN is essential for modulating action timing in response to demands, but their data really only show this in one direction. The STN stimulation reliably increases the speed of response in all conditions (except maximum speed conditions such as escapes). It seems to be over-interpreting the data to say this is an inability to modulate the speed of the task, especially as clear learning and speed modulation do occur under STN lesion conditions, as shown in Figure 12B. The mice learn to avoid and increase their latency in AA2 vs AA1, though the overall avoids and latency are different from controls. The more parsimonious conclusion would be that STN stimulation biases movement speed (increasing it) and that this is true in many different conditions.
(11) In the discussion, the authors claim that the STN projections to the midbrain tegmentum directly affect the active avoidance behavior, while the STN projections to the SNr do not affect it. This seems counter to their results, which show STN projections to either area can alter active avoidance behavior. What is the laser power used in these terminal experiments? If it is high (3mW), the authors may be causing antidromic action potentials in the STN somas, resulting in glutamate release in many brain areas, even when terminals are only stimulated in one area. The authors could use low (0.25mW) laser power in the terminals to reduce the chance of antidromic activation and spatially restrict the optical stimulation.
(12) Was normality tested for data prior to statistical testing?
(13) Why are there no error bars on Figure 5B, black circles and orange triangles?
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Reviewer #3 (Public review):
Summary:
The authors use calcium recordings from STN to measure STN activity during spontaneous movement and in a multi-stage avoidance paradigm. They also use optogenetic excitation, optogenetic inhibition, and lesion approaches to increase or decrease the activity of STN during the avoidance paradigm. The paper reports a large amount of data and makes many claims, some seem well supported to this Reviewer, others not so much.
Strengths:
Well-supported claims include data showing that during spontaneous movements, especially contraversive ones, STN calcium activity is increased using bulk photometry measurements. Single-cell measures back this claim but also show that it is only a modest minority of STN cells that respond strongly, with most showing no response during movement, and a similar number showing …
Reviewer #3 (Public review):
Summary:
The authors use calcium recordings from STN to measure STN activity during spontaneous movement and in a multi-stage avoidance paradigm. They also use optogenetic excitation, optogenetic inhibition, and lesion approaches to increase or decrease the activity of STN during the avoidance paradigm. The paper reports a large amount of data and makes many claims, some seem well supported to this Reviewer, others not so much.
Strengths:
Well-supported claims include data showing that during spontaneous movements, especially contraversive ones, STN calcium activity is increased using bulk photometry measurements. Single-cell measures back this claim but also show that it is only a modest minority of STN cells that respond strongly, with most showing no response during movement, and a similar number showing smaller inhibitions during movement.
Similar data during cued active avoidance procedures show that STN calcium activity sharply increases in response to auditory cues, and during cued movements to avoid a footshock. Optogenetic and lesion experiments are consistent with an important role for STN in generating cue-evoked avoidance. And a strength of these results is that multiple bi-directional approaches were used.
Weaknesses:
I found the experimental design and presentation convoluted and the results over-interpreted.
(1) I really don't understand or accept this idea that delayed movement is necessarily indicative of cautious movements. Is the distribution of responses multi-modal in a way that might support this idea, or do the authors simply take a normal distribution and assert that the slower responses represent 'caution'? Even if responses are multi-modal and clearly distinguished by 'type', why should readers think this that delayed responses imply cautious responding instead of say: habituation or sensitization to cue/shock, variability in attention, motivation, or stress; or merely uncertainty which seems plausible given what I understand of the task design where the same mice are repeatedly tested in changing conditions. This relates to a major claim (i.e., in the work's title).
(2) Related to the last, I'm struggling to understand the rationale for dividing cells into 'types' based the their physiological responses in some experiments (e.g., Figure 7).
(3) The description and discussion of orienting head movements were not well supported, but were much discussed in the avoidance datasets. The initial speed peaks to cue seem to be the supporting data upon which these claims rest, but nothing here suggests head movement or orientation responses.
(4) Similar to the last, the authors note in several places, including abstract, the importance of STN in response timing, i.e., particularly when there must be careful or precise timing, but I don't think their data or task design provides a strong basis for this claim.
(5) I think that other reports show that STN calcium activity is recruited by inescapable foot shock as well. What do these authors see? Is shock, independent of movement, contributing to sharp signals during escapes?
(6) In particular, and related to the last point, the following work is very relevant and should be cited: https://elifesciences.org/reviewed-preprints/104643#tab-content. Note that the focus of this other paper is on a subset of VGLUT2+ Tac1 neurons in paraSTN, but using VGLUT2-Cre to target STN will target both STN and paraSTN.
(7) In multiple other instances, claims that were more tangential to the main claims were made without clearly supporting data or statistics. E.g., claim that STN activation is related to translational more than rotational movement; claim that GCaMP and movement responses to auditory cues were small; claims that 'some animals' responded differently without showing individual data.
(8) In several figures, the number of subjects used was not described. This is necessary. Also necessary is some assessment of the variability across subjects. The only measure of error shown in many figures relates to trial-to-trial or event variability, which is minimal because, in many cases, it appears that hundreds of trials may have been averaged per animal, but this doesn't provide a strong view of biological variability. When bar/line plots are used to display data, I recommend showing individual animals where feasible.
(9) Can the authors consider the extent to which calcium imaging may be better suited to identify increases compared to decreases and how this may affect the results, particularly related to the GRIN data when similar numbers of cells show responses in both directions (e.g., Figure 3)?
(10) Raw example traces are not provided.
(11) The timeline of the spontaneous movement and avoidance sessions was not clear, nor was the number of events or sessions per animal nor how this was set. It is not clear if there was pre-training or habituation, if many or variable sessions were combined per animal, or what the time gaps between sessions were, or if or how any of these parameters might influence interpretation of the results.
(12) It is not clear if or how the spread of expression outside of the target STN was evaluated, and if or how many mice were excluded due to spread or fiber placements.
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