Tachykinin1-expressing neurons in the parasubthalamic nucleus control active avoidance learning

Reviewer #1 (Public review):

This study is focused on a population of neurons in the mouse parasubthalamic nucleus (pSTN) that express Tackhykinin1 (Tac1). This gene has been used before to target pSTN for functional circuit studies because it is fairly selective for pSTN in this region, though it targets only a subset of pSTN neurons. Prior work has shown that activity in these neurons can impact motivated behaviors, including feeding and drinking behaviors, and that their activity is associated with aversion or avoidance behaviors. While not breaking much new ground, this study adds to that work by making use of a 2-way active avoidance assay, where a CS predicts a US (footshock), that the mice can escape. Using fiber photometry the authors show convincing evidence that Tac1 neurons in pSTN increase their activity in response to a US footshock, and that after some pairings the neurons will start responding to the CS too, though to a lesser extent than the US. Their most important data shows that either ablation or optogenetic inhibition of these cells can hugely block the active avoidance (escape) behavior, suggesting these neurons are key for the performance of this task, which they interpret as key for learning the task (but see more below). They show that optogenetic stimulation is aversive in a real-time place assay, and when paired with footshock can enhance active avoidance behavior. Finally, they show that Tac1 pSTN axons in PVT recapitulate these effects while showing that axons in CEA or PBN may only recapitulate some of these effects (more below). Overall I think the data is solid and shows that the activity of Tac1 pSTN neurons in the 2 way active avoidance task is causally related to avoidance behavior in the direction that would be predicted by recent literature. However, I think the authors overstate the conclusions in the title, abstract, and text. I do not think the data make a strong case for a role for these cells in learning, at least in any classical sense, as used in the title and abstract and elsewhere. Also the statement in the abstract that the pSTN mediates its effects 'differentially' through its downstream targets is not convincingly supported by data.

Major concerns:

(1) The authors infer that the activity in the Tac1 pSTN neurons is necessary for aversive or avoidance 'learning'. But this is not well defined, what exactly does that mean and what types of evidence would support or falsify such a hypothesis? Moreover, the authors show convincingly, and in line with prior reports, that these cells are activated by aversive stimuli (here footshock), and that activation of these cells is sufficient to induce avoidance behavior. Because manipulation of these cells can serve as a primary negative reinforcer, it becomes even more challenging and important to explain how experiments that manipulate these cells while measuring behavior/performance can discriminate between changes in: (1) primary aversion, (2) motivation to avoid, (3) associative learning, or (4) memory/retrieval. The authors seem to favor #3, but they don't make a clear case for this point of view or else what they mean by 'avoidance learning'. In my opinion, the data do not well discriminate between possibilities 1 through 3. The authors should clarify their logic and temper their conclusions throughout.

(2) Abstract line 37 is not well supported. The authors focus mostly on pSTN projections to PVT and show that the measurements or manipulation of these axons recapitulates the effects seen with pSTN cell bodies. The authors do fewer studies of axons in CeA and PBN, but do find that they can recapitulate the effects with opsin inhibition, but detect no effects with opsin stimulation. However, the lack of effect with opsin stimulation in Figure S7a-e proves very little on its own. It could be technical, due to inadequate expression or functional efficacy. It is not supported by histological and functional evidence that the manipulation was effective. Overall I can only conclude that the projections to these regions might be very similar (based on the inhibition data), or might be a little different. The data are thus inadequate to support the authors' claim that the pSTN mediates learning differentially through its downstream targets.

Other concerns:

(3) Line 93 is not adequately supported by data in Figure 1b. Additional data is needed that shows expression across cases, including any spread that may be visible when zooming out from pSTN. Additional methods are needed to indicate what exclusion criteria were applied and how many mice were excluded. These data could help support the statement on line 93 that expression was largely restricted within pSTN.

(4) From the results and methods it is not clear where the GFP signal would come from in the mice expressing Casp3 for the ablation studies. It is therefore not clear if the absence of GFP should be taken as evidence of cell loss. For example, it is not clear if multiple vectors were used, if volumes and titers were carefully matched between control groups, or if competition/occlusion between AAVs could be ruled out. It is also not clear how this was quantified, that is how many sections/subjects and how counting was done. It is not clear how long was waited between the AAV infusion, behavior, and euthanasia, perhaps especially important for the ablation done after avoidance learning occurred.

(5) The authors should consider showing individual measurements and not just mean/sem wherever feasible, for example, to support the statement on line 141 that 'all ablated mice showed...'.

(6) S3 is an important control for interpreting data in Figure 2d-i. Something similar is needed to support the inferences made in 2j-u. The very strong effect showing a lack of active avoidance in response to CS or the US when pSTN Tac1 neurons are inhibited during CS or during US suggests that something gross may be going on, such as a gross motor or sensory response that supersedes the effect of footshock. The authors do not comment on whether there are any gross behavioral responses to the inhibition, but an experiment as in S3 is needed, for example, to show that behavior is intact during pSTN inhibition if delivered after the mice already learned to associate CS with US.

(7) The authors use 100 shocks of 0.8 mA for 7 days. I think this is quite strong and in the pSTN inhibition experiments it seems to be functionally 'inescapable' and could thus produce behaviors similar to 'learned helplessness'. Can the authors consider whether this might contribute to the striking findings they observed in their opsin inhibition assays?

(8) The description of the experiment in S5 is inadequate. What are the adjacent areas? Where do the authors see spread? The use of the word 'case' in figure S5 implies an individual case, but the legend says 5 mice were used for 'case 1' and 3 mice were used for 'case 2'. The use of the word 'off-target in the figure implies that the expression was of the intended target. But the text of results and methods implies it was intentional targeting of unnamed and unshown adjacent regions. This should be clarified.

(9) The authors suggest the CPA study is divergent from Serra et al 2023. Though I think this could be due to how the conditioning was done, it would be helpful for the authors to include less processed data. This would aid in possible interpretations for any divergences across studies. Can the authors include raw data (in seconds of time spent) in each compartment for each group across baseline and test days?

Reviewer #2 (Public review):

Summary:

The manuscript by Hu et. al presents a clearly-designed examination of the role of tachykinin1-expressing neurons in the parasubthalamic nucleus of the lateral posterior hypothalamus (PTSN) in active avoidance learning. These glutamatergic neurons have previously been implicated in responding to negative stimuli. This manuscript expands the current understanding of PTSNTac1 neurons in learned responses to threats by showing their role in encoding and mediating the active avoidance response. The authors first use bulk fiber photometry imaging to show the encoding of the active avoidance procedure, followed by cell-type specific manipulations of PTSNTac1 neurons during active avoidance. Finally, they show that encoding and mediation of active avoidance in a downstream target of PTSNTac1 neurons, the PVT/intermediodorsal nuclei of the dorsal thalamus (IMD), has the same effect as what was discovered in the cell body. This contrasts other output regions of the PTSN, such as the PBN and CeA, which were not found to promote active avoidance learning. The experiments presented were well-designed to support the conclusions of the authors, however, the manuscript is missing several key control experiments and supplemental information to support their main findings.

Strengths:

The manuscript provides information on a brain region and downstream target that mediates active avoidance learning. The manuscript provides valuable information via necessity and sufficiency experiments to show the role of the population of interest (PTSNTac1 neurons) in active avoidance learning. The authors also performed most behavior experiments in male and female mice, with adequate power to address potential sex differences in the control of active avoidance by PTSNTac1 neurons. Finally, the manuscript provides valuable information about the specificity of the PTSNTac1 downstream target in regulating active avoidance learning, identifying the PVT/intermediodorsal nuclei of the dorsal thalamus as the key target and ruling out the PBN and CeA.

Weaknesses:

However, several main conclusions of the paper must be interpreted carefully due to missing or inadequate control experiments and histological verification.

(1) Inadequate presentation of viral localization. The authors state that expression was "largely restricted within PSTN" however there is no quantification of the amount of viral expression beyond the target region. Given that Tac1 is expressed in neighboring regions, it is critical to show the viral expression and fiber implant location data for all animals included in the figures. Furthermore, criteria for inclusion and exclusion based on mistargeting should be delineated. This should also be clearly outlined for the experiments in Figure S5, where "behavioral effects of activation of sparsely Tac1-expressing neurons in two adjacent areas of PSTN" was tested but the location of viral expression in those cases is unclear.

(2) Lack of motion artifact correction with isosbestic signal for GCamp recordings. It is appreciated that the authors included a separate EGFP-expressing group to compare to the GCamp-expressing group, however, additional explanation is required for the methods used to analyze the raw fluorescent signal. Namely, were fluorescent signals isosbestic-corrected prior to calculating ΔF/F? If no isosbestic signal was used to correct motion artifacts within a recording session, additional explanation is needed to explain how this was addressed. The lack of motion artifacts in the EGFP signal in a separate cohort is inadequate to answer this caveat as motion artifacts are within-animal.

(3) Missing control experiment demonstrating intact locomotor performance in caspase ablation experiments. The authors use caspase ablation of PTSNTac1 neurons prior to active avoidance learning to appraise the necessity of this cell population. However, a control experiment showing intact locomotor ability in ablated mice was not performed.

(4) Missing control experiment demonstrating [lack of] valence with PTSN silencing manipulations. The authors performed a real-time and conditioned place preference experiments for ChR2-expressing mice (Fig 3M) and found stimulation to be negatively-valenced and generate an aversive memory, respectively. Absent this control experiment with silencing, an alternative conclusion remains possible that optogenetic silencing via GtACR2 created nonspecific location preferences in the active avoidance apparatus, confounding the interpretation of those results.

(5) Incomplete analysis of sex differences. Data in female mice is conspicuously missing from inhibition experiments. The rationale for exclusion from this dataset would be useful for the interpretation of the other noted sex differences.

Reviewer #3 (Public review):

Summary:

This study by Hu et al. examined the role of tachykinin1 (Tac1)-expressing neurons in the para subthalamic nucleus (PSTH) in active avoidance of electric shocks. Bulk recording of PSTH Tac1 neurons or axons of these neurons in PVT showed activation of a shock-predicting tone and shock itself. Ablation of these neurons or optogenetic manipulation of these neurons or their projection to PVT suggests the causality of this pathway with the learning of active avoidance.

Strengths:

This work found an understudied pathway potentially important for active avoidance of electric shocks. Experiments were thoroughly done and the presentation is clear. The amount of discussion and references are appropriate.

Weaknesses:

Critical control experiments are missing for most experiments, and statistical tests are not clear or not appropriate in most parts. Details are shown below.

(1) There are some control experiments missing. Notably, optogenetic manipulation is not verified in any experiments. It is important to verify whether neural activation with optogenetic activation is at the physiological level or supra-physiological level, and whether optogenetic inhibition does not cause unwanted activity patterns such as rebound activation at the critical time window.

(2) Neural ablation with caspase was confirmed by GFP expression. However, from the present description, a different virus to express EITHER caspase or GFP was injected, and then the numbers of GFP-expressing neurons were compared. It is not clear how this can detect ablation.

(3) In many places, statistical approaches are not clear from the present figures, figure legends, and Methods. It seems that most statistics were performed by pooling trials, but it is not described, or multiple "n" are described. For example, it is explicitly mentioned in Figure 4H, "n = 3 mice, n = 213 avoidance trials and n = 87 failure trials". The authors should not pool trials, but should perform across-animal tests in this and other figures, and "n" for statistical tests should be clearly described in each plot.

(4) It is also unclear how the test types were selected. For example, in Figure 1K and O with similar datasets, one is examined by a paired test and the other is by an unpaired test. Since each animal has both early vs late trials, and avoidance vs failure trials, paired tests across animals should be performed for both.

(5) It is also strange to show violin plots for only 6 animals. They should instead show each dot for each animal, connected with a line to show consistent increases of activity in late vs early trials and avoidance vs failure trials.

(6) To tell specificity in avoidance learning, it is better to show escape in the current trials with optogenetic manipulation.

(7) For place aversion, % time decrease across days was tested. It is better to show the original number before normalization, as well.

(8) For anatomical results in Figure S6, it is important to show images with lower magnification, too.

(9) Inactivation of either pathway from PSTH to PBN or to CeA also inhibits active avoidance, but the authors conclude that these effects are "partial" compared to the inactivation of PSTH to PVT. It is not clear how the effects were compared since the effects of PSTH-CeA inactivation are quite strong, comparable to PSTH-PVT inactivation by eye. They should quantify the effects to conclude the difference.

(10) Supplementary table 1: as mentioned above, n for statistical tests should be clearer.

Author response:

Reviewer #1 (Public review):

This study is focused on a population of neurons in the mouse parasubthalamic nucleus (pSTN) that express Tackhykinin1 (Tac1). This gene has been used before to target pSTN for functional circuit studies because it is fairly selective for pSTN in this region, though it targets only a subset of pSTN neurons. Prior work has shown that activity in these neurons can impact motivated behaviors, including feeding and drinking behaviors, and that their activity is associated with aversion or avoidance behaviors. While not breaking much new ground, this study adds to that work by making use of a 2-way active avoidance assay, where a CS predicts a US (footshock), that the mice can escape. Using fiber photometry, the authors show convincing evidence that Tac1 neurons in pSTN increase their activity in response to a US footshock, and that after some pairings the neurons will start responding to the CS too, though to a lesser extent than the US. Their most important data shows that either ablation or optogenetic inhibition of these cells can hugely block the active avoidance (escape) behavior, suggesting these neurons are key for the performance of this task, which they interpret as key for learning the task (but see more below). They show that optogenetic stimulation is aversive in a real-time place assay, and when paired with footshock can enhance active avoidance behavior. Finally, they show that Tac1 pSTN axons in PVT recapitulate these effects while showing that axons in CEA or PBN may only recapitulate some of these effects (more below). Overall I think the data is solid and shows that the activity of Tac1 pSTN neurons in the 2 way active avoidance task is causally related to avoidance behavior in the direction that would be predicted by recent literature. However, I think the authors overstate the conclusions in the title, abstract, and text. I do not think the data make a strong case for a role for these cells in learning, at least in any classical sense, as used in the title and abstract and elsewhere. Also, the statement in the abstract that the pSTN mediates its effects 'differentially' through its downstream targets is not convincingly supported by data.

We are very pleased that Reviewer 1 thought our data is solid.

Major concerns:

(1) The authors infer that the activity in the Tac1 pSTN neurons is necessary for aversive or avoidance 'learning'. But this is not well defined, what exactly does that mean and what types of evidence would support or falsify such a hypothesis? Moreover, the authors show convincingly, and in line with prior reports, that these cells are activated by aversive stimuli (here footshock), and that activation of these cells is sufficient to induce avoidance behavior. Because manipulation of these cells can serve as a primary negative reinforcer, it becomes even more challenging and important to explain how experiments that manipulate these cells while measuring behavior/performance can discriminate between changes in: (1) primary aversion, (2) motivation to avoid, (3) associative learning, or (4) memory/retrieval. The authors seem to favor #3, but they don't make a clear case for this point of view or else what they mean by 'avoidance learning'. In my opinion, the data do not well discriminate between possibilities 1 through 3. The authors should clarify their logic and temper their conclusions throughout.

Thank you Reviewer 1 for providing us insightful suggestions. Based on our fiber photometry data that the activities of PSTN Tac1+ neurons show a significant increase in CS-evoked calcium fluorescent signals in late trials relative to those in early trials (Figure 1H-K) and our optogenetic inhibition experiments during CS (Figure 2N-Q), these results illustrate that the activities of PSTN Tac1+ neurons are modulated by learning and are required for active avoidance learning. Moreover, PSTN Tac1+ neurons are activated by footshock and activation of these cells is sufficient to induce avoidance behavior. These findings demonstrate that PSTN Tac1+ neurons encode aversive information. Together, our current data support that PSTN Tac1+ neurons encode both aversive event and its predicting cue. We will clarify our conclusions in the revised manuscript.

(2) Abstract line 37 is not well supported. The authors focus mostly on pSTN projections to PVT and show that the measurements or manipulation of these axons recapitulates the effects seen with pSTN cell bodies. The authors do fewer studies of axons in CeA and PBN, but do find that they can recapitulate the effects with opsin inhibition, but detect no effects with opsin stimulation. However, the lack of effect with opsin stimulation in Figure S7a-e proves very little on its own. It could be technical, due to inadequate expression or functional efficacy. It is not supported by histological and functional evidence that the manipulation was effective. Overall, I can only conclude that the projections to these regions might be very similar (based on the inhibition data), or might be a little different. The data are thus inadequate to support the authors' claim that the pSTN mediates learning differentially through its downstream targets.

In the revised version of manuscript, we will provide more histological and functional evidence for the PSTN-to-CeA and PSTN-to-PBN circuits to support our conclusion on the functional roles of these downstream targets. Similar with our anterograde experiment that the PSTN densely projects to CeA and PBN (Figure S6), optogenetic activation and inhibition experiments showed dense axonal terminals in the CeA and PBN from the PSTN and this line of data will be included in the revised manuscript. In addition, we will further examine these circuits by investigating the functional roles of CeA-projecting or PBN-Projecting PSTN neurons during 2-way active avoidance task.

Other concerns:

(3) Line 93 is not adequately supported by data in Figure 1b. Additional data is needed that shows expression across cases, including any spread that may be visible when zooming out from pSTN. Additional methods are needed to indicate what exclusion criteria were applied and how many mice were excluded. These data could help support the statement on line 93 that expression was largely restricted within pSTN.

In the revised version of manuscript, we will provide larger example images containing pSTN and its adjacent areas to demonstrate that the viral expression is well restricted into this brain area. Moreover, we will provide detailed information on the exclusion criteria and the number of mice excluded in the Method section.

(4) From the results and methods it is not clear where the GFP signal would come from in the mice expressing Casp3 for the ablation studies. It is therefore not clear if the absence of GFP should be taken as evidence of cell loss. For example, it is not clear if multiple vectors were used, if volumes and titers were carefully matched between control groups, or if competition/occlusion between AAVs could be ruled out. It is also not clear how this was quantified, that is how many sections/subjects and how counting was done. It is not clear how long was waited between the AAV infusion, behavior, and euthanasia, perhaps especially important for the ablation done after avoidance learning occurred.

I totally agree with Reviewer 1’s concerns. We will perform immunohistochemistry or in situ hybridization for Tachykinin-1 itself and then measure colocalization of GFP with Tachykinin-1 inside and outside of the PTSN, and the degree of absence of Tachykinin-1 in Casp mice. In addition, we will provide more detailed experimental information in the revised manuscript.

(5) The authors should consider showing individual measurements and not just mean/sem wherever feasible, for example, to support the statement on line 141 that 'all ablated mice showed...'.

Thank you Reviewer 1 for this suggestion. We will re-plot the data as individual measurements in the revised manuscript.

(6) S3 is an important control for interpreting data in Figure 2d-i. Something similar is needed to support the inferences made in 2j-u. The very strong effect showing a lack of active avoidance in response to CS or the US when pSTN Tac1 neurons are inhibited during CS or during US suggests that something gross may be going on, such as a gross motor or sensory response that supersedes the effect of footshock. The authors do not comment on whether there are any gross behavioral responses to the inhibition, but an experiment as in S3 is needed, for example, to show that behavior is intact during pSTN inhibition if delivered after the mice already learned to associate CS with US.

Thank you Reviewer 1 for this insightful suggestion. During the review process, we have performed this line of experiment as in Figure S3. We measured the behavioral responses during pSTN optogenetic inhibition after the mice already learned to associate CS with US and found most GtACR-expressing mice showed unaffected avoidance learning. This data will be included in the revised manuscript.

(7) The authors use 100 shocks of 0.8 mA for 7 days. I think this is quite strong and in the pSTN inhibition experiments it seems to be functionally 'inescapable' and could thus produce behaviors similar to 'learned helplessness'. Can the authors consider whether this might contribute to the striking findings they observed in their opsin inhibition assays?

I agree with the Reviewer 1’s comment on the string findings in the optogenetic inhibition results. Indeed, based on the results on days 1 and 2, optogenetic inhibition of PSTN tac1+ neurons has significantly blocked GtACR-expressing animals’ behavioral performance during 2-way active avoidance task. To examine whether the effect by optogenetic inhibition of these neurons could possibly decline with prolonged training, we conducted additional 5-day training. We will discuss and add this comment in the revised manuscript.

(8) The description of the experiment in S5 is inadequate. What are the adjacent areas? Where do the authors see spread? The use of the word 'case' in figure S5 implies an individual case, but the legend says 5 mice were used for 'case 1' and 3 mice were used for 'case 2'. The use of the word 'off-target in the figure implies that the expression was of the intended target. But the text of results and methods implies it was intentional targeting of unnamed and unshown adjacent regions. This should be clarified.

We will add histological images and clarify these comments in the revised manuscript. The purpose of this experiment is to illustrate that even slightly spreading ChR2 viruses into Tac1+ neurons of the adjacent areas of the PSTN did not result in behavioral changes and this will indirectly support the main behavioral function caused by the PSTN tac1+ neurons rather than its neighboring areas. Because Tac1+ neurons outside the PSTN are sparsely expressed, it is quite difficult to completely restrict the viral expression in the PSTN from the anterior to the posterior. Thus, we will provide detailed information on the exclusion criteria and the number of mice excluded in the Method section.

(9) The authors suggest the CPA study is divergent from Serra et al 2023. Though I think this could be due to how the conditioning was done, it would be helpful for the authors to include less processed data. This would aid in possible interpretations for any divergences across studies. Can the authors include raw data (in seconds of time spent) in each compartment for each group across baseline and test days?

We will follow Reviewer 1’s suggestion to include raw data (in seconds of time spent) in each compartment for each group across baseline and test days in the revised manuscript.

Reviewer #2 (Public review):

Summary:

The manuscript by Hu et. al presents a clearly-designed examination of the role of tachykinin1-expressing neurons in the parasubthalamic nucleus of the lateral posterior hypothalamus (PTSN) in active avoidance learning. These glutamatergic neurons have previously been implicated in responding to negative stimuli. This manuscript expands the current understanding of PTSNTac1 neurons in learned responses to threats by showing their role in encoding and mediating the active avoidance response. The authors first use bulk fiber photometry imaging to show the encoding of the active avoidance procedure, followed by cell-type specific manipulations of PTSNTac1 neurons during active avoidance. Finally, they show that encoding and mediation of active avoidance in a downstream target of PTSNTac1 neurons, the PVT/intermediodorsal nuclei of the dorsal thalamus (IMD), has the same effect as what was discovered in the cell body. This contrasts other output regions of the PTSN, such as the PBN and CeA, which were not found to promote active avoidance learning. The experiments presented were well-designed to support the conclusions of the authors, however, the manuscript is missing several key control experiments and supplemental information to support their main findings.

Strengths:

The manuscript provides information on a brain region and downstream target that mediates active avoidance learning. The manuscript provides valuable information via necessity and sufficiency experiments to show the role of the population of interest (PTSNTac1 neurons) in active avoidance learning. The authors also performed most behavior experiments in male and female mice, with adequate power to address potential sex differences in the control of active avoidance by PTSNTac1 neurons. Finally, the manuscript provides valuable information about the specificity of the PTSNTac1 downstream target in regulating active avoidance learning, identifying the PVT/intermediodorsal nuclei of the dorsal thalamus as the key target and ruling out the PBN and CeA.

We highly appreciate that Reviewer 2 thought that our experiments presented were well-designed to support the conclusions and provided valuable information in several aspects.

Weaknesses:

However, several main conclusions of the paper must be interpreted carefully due to missing or inadequate control experiments and histological verification.

(1) Inadequate presentation of viral localization. The authors state that expression was "largely restricted within PSTN" however there is no quantification of the amount of viral expression beyond the target region. Given that Tac1 is expressed in neighboring regions, it is critical to show the viral expression and fiber implant location data for all animals included in the figures. Furthermore, criteria for inclusion and exclusion based on mistargeting should be delineated. This should also be clearly outlined for the experiments in Figure S5, where "behavioral effects of activation of sparsely Tac1-expressing neurons in two adjacent areas of PSTN" was tested but the location of viral expression in those cases is unclear.

Similar with questions 3 and 8 of Reviewer 1. We will provide the viral expression and fiber implant location data for all animals included in the figures and histological images in Figure S5 in the revised manuscript. Moreover, we will provide detailed information on the exclusion criteria and the number of mice excluded in the Method section.

1. Lack of motion artifact correction with isosbestic signal for GCamp recordings. It is appreciated that the authors included a separate EGFP-expressing group to compare to the GCamp-expressing group, however, additional explanation is required for the methods used to analyze the raw fluorescent signal. Namely, were fluorescent signals isosbestic-corrected prior to calculating ΔF/F? If no isosbestic signal was used to correct motion artifacts within a recording session, additional explanation is needed to explain how this was addressed. The lack of motion artifacts in the EGFP signal in a separate cohort is inadequate to answer this caveat as motion artifacts are within-animal.

We will follow Reviewer 2’s suggestion and perform isosbestic-correction for fluorescent signals prior to calculating ΔF/F. We will re-plot related figures and add this information in the revised manuscript.

(3) Missing control experiment demonstrating intact locomotor performance in caspase ablation experiments. The authors use caspase ablation of PTSNTac1 neurons prior to active avoidance learning to appraise the necessity of this cell population. However, a control experiment showing intact locomotor ability in ablated mice was not performed.

We will follow Reviewer 2’s suggestion to perform a control experiment showing intact locomotor ability in caspase 3-ablated mice and will include this data in the revised manuscript.

(4) Missing control experiment demonstrating [lack of] valence with PTSN silencing manipulations. The authors performed a real-time and conditioned place preference experiments for ChR2-expressing mice (Fig 3M) and found stimulation to be negatively-valenced and generate an aversive memory, respectively. Absent this control experiment with silencing, an alternative conclusion remains possible that optogenetic silencing via GtACR2 created nonspecific location preferences in the active avoidance apparatus, confounding the interpretation of those results.

Thank you Reviewer 2 for this useful suggestion. We will examine the valence with PTSN silencing manipulations by using a RTPP test and add this data in the revised manuscript.

(5) Incomplete analysis of sex differences. Data in female mice is conspicuously missing from inhibition experiments. The rationale for exclusion from this dataset would be useful for the interpretation of the other noted sex differences.

Thank you Reviewer 2 for this useful suggestion. During the review process, we have performed ablation and inhibition experiments in females, demonstrating similar behavioral effects as those in males. We will add these data in the revised manuscript.

Reviewer #3 (Public review):

Summary:

This study by Hu et al. examined the role of tachykinin1 (Tac1)-expressing neurons in the para subthalamic nucleus (PSTH) in active avoidance of electric shocks. Bulk recording of PSTH Tac1 neurons or axons of these neurons in PVT showed activation of a shock-predicting tone and shock itself. Ablation of these neurons or optogenetic manipulation of these neurons or their projection to PVT suggests the causality of this pathway with the learning of active avoidance.

Strengths:

This work found an understudied pathway potentially important for active avoidance of electric shocks. Experiments were thoroughly done and the presentation is clear. The amount of discussion and references are appropriate.

We are very pleased to have Reviewer 3’s positive comments on the manuscript.

Weaknesses:

Critical control experiments are missing for most experiments, and statistical tests are not clear or not appropriate in most parts. Details are shown below.

(1) There are some control experiments missing. Notably, optogenetic manipulation is not verified in any experiments. It is important to verify whether neural activation with optogenetic activation is at the physiological level or supra-physiological level, and whether optogenetic inhibition does not cause unwanted activity patterns such as rebound activation at the critical time window.

Thank you Reviewer 3 for this useful suggestion. We will perform in vitro slice recording experiments to verify optogenetic manipulations and add this line of evidence in the revised manuscript.

(2) Neural ablation with caspase was confirmed by GFP expression. However, from the present description, a different virus to express EITHER caspase or GFP was injected, and then the numbers of GFP-expressing neurons were compared. It is not clear how this can detect ablation.

Similar with question 4 of Reviewer 1. We will perform immunohistochemistry or in situ hybridization for Tachykinin-1 itself and then measure colocalization of GFP with Tachykinin-1 inside and outside of the PTSN, and the degree of absence of Tachykinin-1 in Casp-ablated mice. In addition, we will provide more detailed experimental information in the revised manuscript.

(3) In many places, statistical approaches are not clear from the present figures, figure legends, and Methods. It seems that most statistics were performed by pooling trials, but it is not described, or multiple "n" are described. For example, it is explicitly mentioned in Figure 4H, "n = 3 mice, n = 213 avoidance trials and n = 87 failure trials". The authors should not pool trials, but should perform across-animal tests in this and other figures, and "n" for should be clearly described in each plot.

We have provided all statistical information in the Supplementary Table 1. In the revised manuscript, we will perform across-animal tests, re-plot new figures and provide clear statistical information.

(4) It is also unclear how the test types were selected. For example, in Figure 1K and O with similar datasets, one is examined by a paired test and the other is by an unpaired test. Since each animal has both early vs late trials, and avoidance vs failure trials, paired tests across animals should be performed for both.

Following Reviewer 3’s suggestion, we will perform across-animal tests. In the first version of our manuscript, for fiber photometry experiments, we pooled trial data of each animal and performed statistics tests across trials. Because avoidance and failure trials were different, we thus selected an unpaired test for this kind of dataset.

(5) It is also strange to show violin plots for only 6 animals. They should instead show each dot for each animal, connected with a line to show consistent increases of activity in late vs early trials and avoidance vs failure trials.

Similar with question 4 of Reviewer 3, we pooled trial data of each animal and performed statistics tests across trials. We will perform across-animal tests and re-plot figures by connecting with a line to show consistent increases of activity in late vs early trials and avoidance vs failure trials for each animal.

(6) To tell specificity in avoidance learning, it is better to show escape in the current trials with optogenetic manipulation.

Thank you Reviewer 3 for this useful suggestion. We will follow this suggestion and add this analysis in the revised manuscript.

(7) For place aversion, % time decrease across days was tested. It is better to show the original number before normalization, as well.

Similar with question 9 of Reviewer 1, we will show the original number before normalization in the revised manuscript.

(8) For anatomical results in Figure S6, it is important to show images with lower magnification, too.

We will follow this suggestion and provide histological images with lower magnification in the revised manuscript.

(9) Inactivation of either pathway from PSTH to PBN or to CeA also inhibits active avoidance, but the authors conclude that these effects are "partial" compared to the inactivation of PSTH to PVT. It is not clear how the effects were compared since the effects of PSTH-CeA inactivation are quite strong, comparable to PSTH-PVT inactivation by eye. They should quantify the effects to conclude the difference.

We will quantify the effects of different downstream targets of the PSTN to make a precise conclusion.

(10) Supplementary table 1: as mentioned above, n for statistical tests should be clearer.

As mentioned above, we will perform across-animal tests and provide clear statistical information in the figure legends and supplementary table 1.