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Dorsal periaqueductal gray ensembles represent approach and avoidance states

  1. Fernando MCV Reis  Is a corresponding author
  2. Johannes Y Lee
  3. Sandra Maesta-Pereira
  4. Peter J Schuette
  5. Meghmik Chakerian
  6. Jinhan Liu
  7. Mimi Q La-Vu
  8. Brooke C Tobias
  9. Juliane M Ikebara
  10. Alexandre Hiroaki Kihara
  11. Newton S Canteras
  12. Jonathan C Kao  Is a corresponding author
  13. Avishek Adhikari  Is a corresponding author
  1. Department of Psychology, University of California, Los Angeles, United States
  2. Department of Electrical and Computer Engineering, University of California, Los Angeles, United States
  3. Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, Brazil
  4. Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Brazil
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Cite this article as: eLife 2021;10:e64934 doi: 10.7554/eLife.64934

Abstract

Animals must balance needs to approach threats for risk assessment and to avoid danger. The dorsal periaqueductal gray (dPAG) controls defensive behaviors, but it is unknown how it represents states associated with threat approach and avoidance. We identified a dPAG threatavoidance ensemble in mice that showed higher activity farther from threats such as the open arms of the elevated plus maze and a predator. These cells were also more active during threat avoidance behaviors such as escape and freezing, even though these behaviors have antagonistic motor output. Conversely, the threat approach ensemble was more active during risk assessment behaviors and near threats. Furthermore, unsupervised methods showed that avoidance/approach states were encoded with shared activity patterns across threats. Lastly, the relative number of cells in each ensemble predicted threat avoidance across mice. Thus, dPAG ensembles dynamically encode threat approach and avoidance states, providing a flexible mechanism to balance risk assessment and danger avoidance.

Introduction

Behavioral variables and emotional states are thought to be represented in neural activity (for review, see Salzman and Fusi, 2010; Anderson and Adolphs, 2014). Such representations must be specific enough to differentiate across behaviors, yet general enough to maintain functional cohesion across diverse threatening situations (Gründemann et al., 2019). A large body of evidence has shown that defensive behaviors related to threat exposure are represented in dorsal periaqueductal gray (dPAG) activity (Deng et al., 2016; Esteban Masferrer et al., 2020; Evans et al., 2018; Watson et al., 2016), as dPAG activity correlates with escape and freeze. Additionally, dPAG optogenetic and electrical stimulation induce these behaviors, as well as aversion (Brandão et al., 1982; Carvalho et al., 2015; Carvalho et al., 2018; Deng et al., 2016; Tovote et al., 2016). Furthermore, pharmacological manipulations of dPAG activity impact open arm exploration in the elevated plus maze (EPM), a traditional measure of rodent anxiety (Fogaça et al., 2012). Lastly, PAG activity in humans correlates positively with threat imminence (Mobbs et al., 2007; Mobbs et al., 2010). These reports show that the dPAG is a central node orchestrating defensive responses.

However, it is unknown how the dPAG represents moment-to-moment changes in brain states during threat exposure. The two main behavioral states observed during exposure to threats are approach and avoidance (Stankowich, 2019). In the approach state, animals voluntarily go near the threat and perform risk assessment behaviors. In this state, the exploratory risk evaluation drive is stronger than the motivation to avoid danger. By contrast, in the avoidance state, animals perceive high risk, and thus attempt to minimize exposure to danger by escaping, freezing, and maintaining distance to the threat. No reports to date have investigated whether the dPAG consistently encodes approach and avoidance states across distinct threats.

Key questions regarding the neural representation of approach and avoidance states remain unanswered. Do dPAG cells respond uniformly to transitions between higher and lower threat imminence? What is the overlap between the dPAG encoding of two completely distinct threats? Does dPAG neuronal activity encode moment-to-moment changes regarding defensive approach and avoidance states? Addressing these questions would require population-level analysis of dPAG cells recorded longitudinally across threat modalities. Here, we report experimental data and analyses that directly address these questions.

Results and discussion

We performed microendoscopic calcium imaging of dPAG neurons expressing GCaMP6s (Figure 1A and Figure 1—figure supplement 1; Cai et al., 2016) during EPM and rat exposure assays. During the EPM test, we recorded 107 ± 19 cells per mouse (n = 8 mice; 857 cells were imaged; see Materials and methods). As expected, mice spent more time in the closed arms of the EPM (Figure 1B). They also displayed exploratory head dips (average frequency of events = 27.0 ± 9.7; n = 8 mice) over the edges of the open arms (Figure 1B–C). During EPM exploration cells often showed preferential activity either in closed or open arms (Figure 1D–E). To identify EPM arm-type modulated neurons, we defined an ‘arm score metric’ ranging from −1 to +1, in which the +1 indicates that cell activity in the open arm is greater than activity in the closed arm, and vice versa. The arm score distribution in the observed data is wider than expected by chance, indicating that dPAG cells show robust preference for EPM arm types (Figure 1F, left panel). We defined neurons as belonging to one of the ensembles if activity in each arm-type was significantly greater than the pooled activity in the opposite arm type (Figure 1F, right panel, see Materials and methods). The results showed that cells fired similarly in arms of the same type, as cell activity in one open arm was highly correlated to activity in the other open arm (Figure 1G, top panel). Conversely, cell activity in closed and open arms was negatively correlated (Figure 1G, bottom panel).

Figure 1 with 1 supplement see all
Dorsal periaqueductal gray (dPAG) neuronal ensembles encode arm type in the elevated plus maze (EPM).

(A) Gradient refractive index (GRIN) lens implantation, virus expression strategy, and example Ca2+ signals of neurons in the dorsal periaqueductal gray (dPAG). (B) Example mouse exploration path recorded in the EPM. Mice spent significantly more time in the closed arms compared to the open arms (data are represented as mean ± SEM; W = 0, p=0.012, Wilcoxon signed rank test, n = 8 mice). (C) Mean percentage of time in which mice engaged in head dips during the whole EPM session (n = 8 mice). (D) dPAG dF/F traces from the same mouse that display preferential activity in the closed (upper trace) and open (lower trace) arms of the EPM (open and closed arm-preferring cells). Epochs corresponding to exploration of the closed and open arms are shown respectively as green and blue shaded areas. (E) Activity heatmaps for corresponding example neurons shown in (D). (F) The open arm preference score was calculated for each neuron (orange bars; see Materials and methods), as was the distribution of open arm preference score for shuffled data (red line). Bars show the distribution of open arm preference score for open, closed, and neither cells (n = 857 cells). (G) Scatterplots showing correlations between neural activity across the two open arms (top) and between open and closed arms of the EPM (bottom). Each point represents one cell (n = 857 cells, r = Pearson’s correlation coefficient). (H) Pie chart shows the percent of all recorded neurons that were classified as open, closed, or neither cells (n = 857 cells). (I) For each subplot, each row depicts the mean normalized activity of an open, closed, or neither arm-preferring cell during behavior-aligned arm transitions (n = 857 cells).

Based on the distribution of cells per arm score, roughly half of the dPAG neurons were classified as arm-modulated cells (49%, with 26% closed- and 23% open-modulated cells) (Figure 1H), which suggests these ensembles are functionally relevant dPAG populations. During transitions between arms, we identified opposite changes in activity levels of these two major, non-overlapping populations of dPAG neurons (Figure 1I and Figure 2A–B). To ensure that positive results were not due to cell categorization itself, in Figure 2A–C,G, cell categorization was done on training data and results were plotted for separate testing data (see Materials and methods). For example, the closed arm-activated ensemble showed a decrease in activity when mice traversed from a closed arm to an open arm. Moreover, open and closed cells showed increased and decreased activity, respectively, during exploratory head dip behavior (Figure 2C). Aggregate activity of dPAG cells in the EPM did not show significantly higher activity in the open arms, showing that in the entire population, the closed and open cell patterns counterbalance each other (Figure 2—figure supplement 1C). Importantly, 95% of dPAG cells showed relatively low correlations of speed and neural activity, between −0.17 and +0.2. These results suggest that arm-related activity preferences are not driven by variations in velocity (Figure 2D). Instead, if EPM arm type is prominently represented in dPAG ensemble activity, then it may be possible to use dPAG activation patterns to differentiate mouse location in the EPM. Indeed, upon training support vector machine (SVM) decoders on dPAG activity, we obtained significantly higher than chance performance in identifying whether the mouse was in an open or closed arm (Figure 2E, see Materials and methods).

Figure 2 with 1 supplement see all
Dorsal periaqueductal gray (dPAG) population activity predicts elevated plus maze (EPM) exploration.

(A) Traces show the mean z-scored activity (±1 SEM) of all open, closed, and neither cells, behavior-aligned to arm transitions (respectively the blue, green, and gray traces). Cell categorization and calculated results were performed on non-overlapping training and testing data (n = 180 open cells, n = 513 neither cells, n = 164 closed cells). (B) Bars depict the change in z-scored dF/F for entries to closed left) and open (right) arms, separately for open, closed, and neither cells (data are represented as mean ± SEM; both closed and open arms; n of cells same as (A); closed arm U = 10.6, ***p<0.001, open arm U = 10.8, p<0.001, Wilcoxon rank sum test, n = 8 mice). (C) Average activity traces for open, closed, and neither cells relative to onset of head dips in the EPM and quantification of changes in activity for all cell types (0–2.5 s after minus 2.5–5.0 s before head dip onset) (data are represented as mean ± SEM; n of cells same as (A); U = 3.9, ***p<0.001, Wilcoxon rank sum test). (D) Histograms depict the distribution of the Pearson’s correlation of dF/F with speed for each cell type in the EPM (n of cells same as Figure 1I. (E) Prediction of arm-type mouse position in the EPM from neural data using a linear support vector machine (SVM). The blue and green areas represent the actual arm-type occupancy label (open and closed arm, respectively), and the black trace represents the prediction of arm location by the SVM hyperplane projection. If the trace was above 0 a.u., then that period was classified as open arm exploration, otherwise, it was classified as closed arm occupancy. The pink and purple represent the data split (training and testing data, respectively). The Matthews correlation coefficient for real and permuted shuffled training data are shown to the right (mean ±1 SEM; n = 8 mice, U = 4.9, p<0.001, Wilcoxon rank sum test). (F) (Left) Example of EPM location index where 1 and −1 correspond, respectively, to the extreme end of the open and closed arms and (right) prediction of labeled EPM location index from dPAG neural data for an example mouse (scatterplot displays testing data that was not used for training, r = Pearson’s correlation coefficient). (G) (Left) Correlation of example closed and open cell activity and (right) mean correlation of dF/F with EPM location index (data are represented as mean ± SEM; n of cells same as (A); U = 13.7, ***p<0.001, Wilcoxon rank sum test, r = Pearson’s correlation coefficient).

Next, we investigated whether dPAG activity could also predict specific mouse positions within the arms. An acceptable interpretation of EPM behavior is that mice avoid the open arms because they are more vulnerable to potential threats in open spaces (Montgomery, 1955; Walf and Frye, 2007). This view suggests that the beginning of the open arms is only slightly threatening, as if a dangerous stimulus is detected the mouse can quickly retreat to the safety of the closed arms. Conversely, it takes a longer time for the mouse to retreat to the closed arms if they are at the extreme end of the open arms. Thus, we argue that distance from the center of the maze is related to a gradient of threat, with the highest threat levels at the end of the open arms. The animal’s behavior supports this view, as mice avoid the extreme end of the open arm more robustly than the beginning of the open arm (Figure 2—figure supplement 1B). We therefore defined the EPM location index to be a linearly varying metric between −1 and +1 (at closed and open arms extremities, respectively), assigning 0 to the center of the maze (Figure 2F, see Materials and methods). We then fitted a linear regression model to predict the EPM location index from dPAG cell activity. Interestingly, the model showed significantly higher than chance performance in predicting EPM location index, suggesting that the identified dPAG ensembles may not only encode arm type, but rather a risk perception and threat exposure gradient (Figure 2F–G and Figure 2—figure supplement 1A). Though there are closed and open arm-preferring cells, aggregate bulk dPAG activity did not show different activation levels across EPM arms (Figure 2—figure supplement 1C).

The above results show that the identified ensembles encode approach and avoidance toward the threatening locations in the EPM. To investigate whether dPAG population coding of approach-avoidance states generalizes to exploratory behavior across different threatening contexts, we recorded the same dPAG neurons during exposure to a live predator (Figure 3A–B and Figure 3—figure supplement 1). Mice were allowed to freely explore a context, a long chamber, in the presence of a rat, which was tethered with a harness to one end of the chamber (see Materials and methods). All behavioral data from synchronized videos underwent automated behavior scoring (Mathis et al., 2018). Mice spent most of the trial away from the rat (Figure 3A), indicating aversion from perceived threat. Consistent with previous threat imminence theories and the array of defensive behaviors evoked in the presence of a predator (Blanchard et al., 2011; McNaughton and Corr, 2004; Perusini and Fanselow, 2015; Stankowich, 2019), mice presented defensive strategy repertoires composed of approach and avoidance-related behaviors (i.e., escape and freeze) (Figure 3A). Freeze bouts lasted 2.1 ± 1.3 s on average. Notably, average dPAG activity increased with rat proximity, rat movement onset, and escape, and decreased during approach, while no significant change was observed during freezing (Figure 3C–E). Importantly, mice displayed no signs of aversion nor differences in dPAG activity with proximity to a toy rat (Figure 3—figure supplement 2).

Figure 3 with 5 supplements see all
Arm-specific ensembles maintain functions across threatening situations.

(A) Illustration of the rat exposure assay (top) and example track (bottom), with labels depicting the area to which the rat is confined (rat zone) as well as areas near to and far from the rat (safe side). In all figures depicting this assay the rat area will be shown to the right. (B) Example imaging field of view with dorsal periaqueductal gray (dPAG) cells co-registered between elevated plus maze (EPM) and rat exposure sessions. (C) Heatmap depicts the mean z-scored dF/F at each position of the rat exposure assay (left, n = 713 cells). Mean dF/F is significantly greater in the threatening zone than in the safe zone (n = 713 cells, n = 7 mice, U = 2.24, p=0.03, Wilcoxon rank sum test). (D) Change in dF/F (0–2.5 s after minus 0–2.5 s before) activity for all dPAG cells for behaviors in the rat exposure assay (data are represented as mean ± SEM; n of cells for approach, rat movement, escape = 714, n of cells for freeze = 640; approach t = −2.65, **p=0.008, rat movement t = 7.87, ***p<0.001, escape t = 3.28, **p=0.001, freeze t = 0.39, p=0.69, one-sample t-test). (E) Traces show the mean z-scored activity of all cells (±1 SEM), aligned to onset of various behaviors (onset is indicated by the red vertical line) in the rat exposure assay (n of cells same as D). (F) Bars depict the mean z-scored dF/F of cells on the safe side and threatening side of the enclosure (data are represented as mean ± SEM; n = 64 open cells, n = 166 neither cells, n = 87 closed cells; n = 7 mice, safe U = −3.82, ***p=0.0001, threatening U = 3.05, **p=0.002, Wilcoxon rank sum test). (G) Traces show the mean z-scored activity of open, closed, and neither cells (±1 SEM), aligned to exit of the safe side of the enclosure (far from the rat). (H) Bars show the mean change in z-scored dF/F (0–2.5 s after minus 0–2.5 s before) aligned to safe side exit for open, closed, and neither cells (data are represented as mean ± SEM; n = 64 open cells, n = 166 neither cells, n = 87 closed cells; U = 3.29, ***p=0.001, Wilcoxon rank sum test). (I) Traces show the mean z-scored activity of open, closed, and neither cells (±1 SEM), aligned to behaviors in the rat exposure assay (approach, rat movement, escape: n = 64 open cells, n = 87 closed cells, n = 166 neither cells; freeze: n = 50 open cells, n = 67 closed cells, n = 132 neither cells). Onset of behaviors is indicated by a red vertical line. (J) Bars depict the change in z-scored dF/F (0–2.5 s after minus 0–2.5 s before) for behaviors in the rat exposure assay, separately for open, closed, and neither cells (data are represented as mean ± SEM; n of cells same as (I); approach U = 2.45, *p=0.014, rat movement U = −3.70, ***p=0.0002, escape U = −2.12, *p=0.034, freeze U = −3.62, ***p=0.0003, Wilcoxon rank sum test). (K) A generalized linear model (GLM) to predict single-cell activity was constructed using approach, escape, and freeze behaviors as variables. Bar plots show average GLM weights for approach and freeze for open, closed, and neither cells (data are represented as mean ± SEM; n = 62 open cells, n = 155 neither cells, n = 83 closed cells; approach U = 4.17, ***p<0.001, p=0.11, freeze U = 3.02, ***p<0.0002, Wilcoxon rank sum test).

We then explored whether the activity of dPAG closed and open cell ensembles identified in the EPM also represent risk evaluation in the rat assay. A positive result would show that the ensembles are likely responding not only to the original sensory biases (i.e., closed and open arms features), but potentially representing behavioral states that generalize across threats. Indeed, open cells showed higher activation than closed cells near the rat even though all cell types were positively activated (Figure 3F–H and Figure 3—figure supplement 3). These data agree with reports showing dPAG activation with predator proximity (Deng et al., 2016; Esteban Masferrer et al., 2020). The present results also showed that proximity to a live predator activated the neither cell ensemble. As the relatively low threat open arms did not significantly activate it, this ensemble of neurons may require a higher degree of threat to be activated relative to open cells.

Closed cells showed increased activity following onset of both escape and freeze, despite these behaviors having opposite motor outputs (Figure 3I–K). Additionally, even though freezing and approach onset occur similarly far from the rat (Figure 3A), open and closed ensembles showed opposite activity patterns and different generalized linear model (GLM) weights (Figure 3I–K). The freezing data in Figure 3I excludes freeze bouts that happened within 10 s of an escape. Thus, the activity patterns plotted during freezing cannot be attributed to slow decay induced by escapes. Rat movement onset likely constitutes a threat signal and switches states from approach to avoidance, as predator movement is indicative of increased threat imminence. Indeed, rat movement is a significant predictor variable for less frequent approach and more occurrences of threat avoidance-related behaviors, such as escape and freezing (Figure 3—figure supplement 4A). Interestingly, rat movement bouts are not consistently directed toward the mouse, as it most often increases distance toward the mouse (Figure 3—figure supplement 4B–C). Nevertheless, rat movements predict escape and freezing, indicating it is perceived as a threat by the mouse. Accordingly, after rat movement onset, the threat avoidance-related closed arm ensembles displayed higher activity (Figure 3I–J). Notably, neither of the ensembles consistently resembled the overall average dPAG activity during rat exposure (Figure 3E), as each ensemble had its own functional profile (Figure 3I). Furthermore, dPAG cells also used shared patterns of neural activity across rat and EPM assays to represent threat imminence (measured as distance to threat) (Figure 3—figure supplement 5, see Materials and methods). These results suggest that dPAG neuronal activity can represent internal brain states using shared patterns of activity across different threats.

An approach state is associated with open arm entries, head dips in the EPM, and proximity to threat. Conversely, an avoidance state would be expected far away from threats and during actions that decrease threat exposure, such as closed arm exploration, escape, and freezing. Our results showed that closed cells were more active during higher distance from threat and threat avoidance-related behaviors, such as freezing and escaping, while open cells were more active during proximity to threats and exploratory head dips in the EPM (Figure 2 and Figure 3). The consistency of these results across behaviors and two different threat modalities indicates that dPAG closed and open cells were encoding threat avoidance and threat approach states, respectively.

To further investigate how dPAG cells use a shared representation to encode approach and avoidance states, we developed an avoidance/approach score ranging between −1 and 1 (see Materials and methods). The score gradually increases during approach to threat and during EPM head dips, reaching +1 when the mouse is adjacent to the rat or in the extreme end of the open arms. The score decreases when the mouse is retreating from threat and is assigned a value of −1 when the mouse is furthest from the rat, freezing, or in the extreme end of the closed arms (Figure 4A). To investigate how the avoidance/approach score is encoded in dPAG activity, we used k-means clustering, an unsupervised approach, to group the data points into clusters (Figure 4B, panel 1). We used the Akaike information criterion (AIC) to determine the optimal number of clusters for each assay, which were two clusters for EPM and three for the rat assay (see the Materials and methods section entitled ‘k-Means clustering of neural data’; see also AIC values for a range of cluster numbers in Figure 4—figure supplement 1). We then calculated the avoidance/approach score for each cluster (Figure 4B, panel 2). The clusters with the lowest and highest scores were classified as the ‘avoidance’ and the ‘approach’ cluster, respectively. Experimentally observed approach and avoidance clusters respectively had higher and lower scores than sampling distributions obtained from permuted behavioral labels, showing that our k-means approach identified activity patterns that strongly encode the avoidance/approach score (Figure 4C). The approach and avoidance cluster centroids from one assay were then applied to the other assay (i.e., centroids were defined by training on EPM and applied on previously unseen data from the rat assay, or vice versa). Cluster centroids defined from the training data in one assay were applied to the data from the other assay to assign cluster identity based on shortest Euclidean distance to the centroid. For example, the points in the testing dataset that were closest to the avoidance centroid, previously defined by the training dataset, were assigned to the avoidance cluster (Figure 4B, panels 3–4). Scores for approach and avoidance clusters for shuffled data were used to create a sampling distribution. Lastly, we show that approach and avoidance clusters trained on one assay and applied on the other assay result in significantly different scores, despite the two assays having different geometries and distinct threat modalities (Figure 4D). Importantly, these results were not found when computing approach and avoidance cluster centroids defined on the control toy rat assay but applied to the rat assay (Figure 4—figure supplement 2). These results indicate, using an unsupervised method, that approach and avoidance states are encoded using shared patterns of neural activity across assays. Similar results were also obtained by employing a hidden Markov model (HMM), showing that the results in Figure 4 can be found using a diversity of computational approaches (Figure 4—figure supplement 3). Importantly, dPAG activity reflects moment-to-moment changes in the behavioral states of the animal. Rather than merely encoding the defensive behavior movements, these activity patterns may indicate threat perception. Encoding of exploratory and defensive behavior has also been reported in the amygdala (Gründemann et al., 2019), indicating that multiple defensive nodes represent such brain states.

Figure 4 with 4 supplements see all
Dorsal periaqueductal gray (dPAG) displays a shared neural representation of avoidance and approach states across the elevated plus maze (EPM) and rat exposure assays.

(A) Example tracks in the EPM (left) and rat exposure assay (right), color-coded by avoidance/approach score (see Materials and methods). The approach score increased during movements toward the threat, reaching +1 when the animal reaches the end of the open arms or the rat. The score decreases during movement away from threat and reaches its minimum value of −1 when the mouse reaches the end of closed arms or the furthest point from the rat. This score was developed as a measure of avoidance/approach states. (B) Explanatory diagram depicting steps of the clustering analysis (see Materials and methods). (1) k-Means was used to find clusters in the neural data in an unsupervised manner. (2) The mean avoidance/approach score was calculated for each cluster defined in step 1. (3) The ‘avoidance’ and ‘approach’ clusters were identified as those with, respectively, the minimum or maximum mean avoidance/approach score calculated in step 2. (4) The approach and avoidance centroids defined in one assay were used to classify neural data from the other assay, based on the minimum Euclidean distance for each sample (as depicted by solid arrow). (C) Arrow depicts the experimentally observed mean avoidance/approach score for avoidance and approach clusters (EPM k = 2; rat k = 3, see Figure 4—figure supplement 1) across concatenated sessions (n = 7 mice). This mean was compared to a distribution of avoidance (top) or approach (bottom) cluster means, calculated by permuting the neural data 100 times (EPM cells n = 734, rat assay cells n = 713; for all, p<0.01). (D) (Left) Bars depict the mean rat and EPM avoidance/approach scores (±1 SEM) for approach and avoidance clusters across mice. (Right) As described in Materials and methods and Figure 4B, these cluster centroid locations, trained on one assay, were then used to define approach and avoidance timepoints in the other assay. Bars depict the corresponding mean avoidance/approach score (±1 SEM) for this testing data (train on EPM: avoidance cluster n = 31,938, approach cluster n = 22,630; test on rat: avoidance cluster n = 30,245, approach cluster n = 24,412; train on rat: avoidance cluster n = 14,658, approach cluster n = 10,514; test on EPM: avoidance cluster n = 15,319, approach cluster n = 2510 (n represents the number of timepoints, not cells); co-registered cells n = 317; Wilcoxon rank sum test, ***p<0.001).

Finally, we investigated if ensemble composition was related to threat avoidance traits across assays. Rat approach and open arm exploration were correlated across mice, indicating that these measures also reflected trait avoidance levels (Figure 4—figure supplement 4A). We then found that mice with a higher proportion of open cells in relation to closed cells displayed increased avoidance of open arms and rat (Figure 4—figure supplement 4B–C). A possible interpretation for these results is that, rather than causing open arm exploration, the open arm ensemble is highly responsive to risk assessment and proximity to threat. Considering this hypothesis, our results suggest that mice with more open arm cells display an increased sensitivity to risk evaluation reflected on increased avoidance of threat. These data indicate that in addition to encoding moment-to-moment changes in behavioral state, dPAG ensembles composition may integrate risk evaluation processes and influence individual mouse differences in threat avoidance traits.

The dPAG’s role in defense has been described as restricted to the initiation of escape (Evans et al., 2018). A proposed model is that excitatory neurons initiate escape (but not other defensive behaviors) in response to imminent threat (Evans et al., 2018). Accordingly, optogenetically stimulating vglut2-positive cells in dPAG caused escape and presenting looming stimuli while optogenetically inhibiting those cells caused freezing (Evans et al., 2018). Thus, threat information would be transmitted to the dPAG, resulting in initiation of escape and inhibition of freezing (Lefler et al., 2020; Tovote et al., 2016). However, recent data indicate that the dPAG’s influence on defense is not restricted to escape initiation. For example, optogenetic activation of dPAG CaMKIIα-positive neurons can cause both escape and freezing (Deng et al., 2016). Increasing the optogenetic stimulation frequency of CaMKIIα-positive dPAG neurons switches responses from freezing to escape (Deng et al., 2016). Supporting the view that the dPAG may promote freezing, activation of excitatory ventromedial hypothalamus projections to the dPAG induces freezing (Wang et al., 2015). Conversely, the hypothalamic dorsal premammillary nucleus projections to the dPAG control escape elicited by numerous innate threats (Wang et al., 2021). Recent work shows that the medial prefrontal cortex promotes aversion and might regulate defensive strategies through projections to the dPAG (Vander Weele et al., 2018). These findings indicate that through different inputs, the dPAG may integrate distinct aspects of emotional states, detect diverse threats, and coordinate a range of defensive responses, including freezing and escape.

We propose that when the subject can voluntarily approach the threat, the dPAG has a broader function in threat processing. It may then function as a risk estimator that represents approach- and avoidance-related states, and consequently influences a wide range of behaviors related to both approach and avoidance. In contrast, there are situations in which threats appear independently of voluntary approach, such as during the presentation of looming stimuli. In such cases, approach cannot be assessed as only avoidance behaviors such as freezing and escape are possible (Evans et al., 2018; Salay et al., 2018). Hence, these situations only allow assessing the dPAG’s involvement in avoidance, but not approach-related states.

Our study reveals novel features of dPAG neurons. First, dPAG cells differentiated between open and closed spaces in the EPM. Second, we show that closed cell activity patterns were positively correlated with threat perception represented by the predator’s movement. Furthermore, despite having opposite motor outputs, escape and freezing correlated with increased closed cell activity. Conversely, open cell activity was inversely correlated with distance to threat. Closed and open cells show positive z-scored activity following escape onset. However, open cell activity starts decreasing following escape onset, while closed cell activity increases after flight initiation (Figure 3J). Thus, our data highlight the importance of considering activity dynamics rather than only analyzing if cells display high or low activity during a behavior. Our observations suggest that dPAG is not simply a premotor area but rather reflects the subjects’ underlying emotional state, in agreement with previous work (Carvalho et al., 2015; Carvalho et al., 2018; Johansen et al., 2010; Kim et al., 2013; Kincheski et al., 2012; Nashold et al., 1969). This idea is consistent with the fact that dPAG activity represented location within the EPM (Figure 2F), and that the dPAG used shared patterns of neural activity across rat and EPM assays to represent threat imminence (Figure 3—figure supplement 5) and behavioral states (Figure 4). The finding that the dPAG uses a conserved representation of threat approach and avoidance across assays expands prior results using stimulation, calcium imaging, electrophysiology, and c-fos expression (Bittencourt et al., 2005; Brandão et al., 1982; Canteras and Goto, 1999; Deng et al., 2016; Esteban Masferrer et al., 2020; Evans et al., 2018). A role beyond motor output execution has also been proposed for the ventral PAG, as its neural activity reflects threat probability associated with specific cues (Wright and McDannald, 2019; Wright et al., 2019).

A possible mechanistic explanation for the differences between open and closed ensembles is that they might correspond to specific neural populations integrating different neurotransmitter systems with particular modulatory effects over defensive behaviors (e.g., glutamate, GABA, substance P, nitric oxide, etc.). Alternatively, these ensembles may have distinct input connectivity, leading to distinct activity patterns. Based on its connectivity, the dPAG is well positioned to have a privileged role in computing threat imminence and threat probability, as it receives input from sensory, limbic, and cognitive areas (Silva and McNaughton, 2019), and projects not only to centers that control motor actions (Ferreira-Pinto et al., 2018; Marchand and Hagino, 1983) but also to prosencephalic targets likely to mediate fear behavior (Gross and Canteras, 2012; Krout and Loewy, 2000; Motta et al., 2017). Future studies are needed to further dissect dPAG cell types based on genetic markers, connectivity, and functional differences. In summary, our study reveals two large functional neuronal ensembles of the dPAG representing internal states. By performing population-level analysis of dPAG neurons in two different threatening situations, we were able to demonstrate that dPAG cells are not only encoding specific behaviors or threat imminence (Deng et al., 2016; Esteban Masferrer et al., 2020; Evans et al., 2018; Watson et al., 2016), but also that dPAG ensemble activity reflects moment-to-moment changes in the approach-avoidance state of the animal. These findings expand on the oversimplified view of dPAG as a premotor output region and highlight it as a key node reflecting the internal brain states that prepare the organism to engage in approach or avoidance of threat.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(Mus musculus)
C57BL/6JJackson LabsRRID:IMSR_JAX:000664
Strain, strain background (Rattus norvegicus)Long-EvansCharles River LabsRRID:RGD-631593
Recombinant DNA reagentAAV9.Syn.GCaMP6s.WPRE.SV40Addgene100843-AAV9
Software, algorithmMATLABMathworksRRID:SCR_001622

Mice

Mice (Mus musculus) of the C57BL/6J strain (Jackson Laboratory stock No. 000664) were used for all experiments. Male mice between 2 and 5 months of age were used in all experiments. Mice were maintained on a 12 hr reverse light-dark cycle with food and water ad libitum. Sample sizes were chosen based on previous behavioral studies with miniaturized microscope recordings on defensive behaviors, which typically use 6–10 mice per group. All mice were handled for a minimum of 5 days prior to any behavioral task. In this work, analyses of the EPM environment used eight mice, while any analyses involving rat exposure used seven mice. Sample size was chosen based on prior dPAG calcium transient recordings (Evans et al., 2018). All procedures have been approved by the University of California, Los Angeles Institutional Animal Care and Use Committee, protocols 2017–011 and 2017–075.

Rats

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Male Long-Evans rats (250–400 g) were obtained from Charles River and were individually housed on a standard 12 hr light-dark cycle and given food and water ad libitum. Rats were only used as a predatory stimulus. Rats were handled for several weeks prior to being used and were screened for low aggression to avoid attacks on mice. No attacks on mice were observed in this experiment.

Surgeries

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Eight-week-old mice were anaesthetized with 1.5–3.0% isoflurane and placed in a stereotaxic apparatus (Kopf Instruments). AAV9.Syn.GCaMP6s.WPRE.SV40 were packaged and supplied by UPenn Vector Core at titers 7.5 × 1013 viral particles per ml and viral aliquots were diluted prior to use with artificial cortex buffer to a final titer of 5 × 1012 viral particles per ml. After performing a craniotomy, 100 nl of virus was injected into the dPAG (coordinates in mm, from skull surface): −4.20 anteromedial, −0.85 lateral, −2.3 depth, 15-degree angle. Five days after virus injection, the animals underwent a second surgery in which two skull screws were inserted and a microendoscope was implanted above the injection site. A 0.5 mm diameter, ~4-mm-long gradient refractive index (GRIN) lens (Inscopix, Palo Alto, CA) was implanted above the dPAG (−2.0 mm ventral to the skull surface) (Resendez et al., 2016). The lens was fixed to the skull with cyanoacrylate glue and adhesive cement (Metabond; Parkell, Edgewood, NY, USA). The exposed end of the GRIN lens was protected with transparent Kwik-seal glue and animals were returned to a clean cage. Two weeks later, a small aluminum base plate was cemented onto the animal’s head on top of the previously formed dental cement. Animals were provided with analgesic and anti-inflammatory (carprofen).

Behavioral timeline 

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Behavioral tests were combined in the following manner across days: EPM test, rat exposure environment habituation (no rat), toy rat exposure, and rat exposure. Each mouse was only exposed to each assay once, as fear assays cannot be repeated. Thus, there are no technical replicates. No outliers were found or excluded. Neural recordings were obtained from all mice in identical conditions, and thus they were all allocated to the same experimental group. There were no experimentally controlled differences across mice and there were no ‘treatment groups’.

EPM test

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Mice were placed in the center of the EPM facing one of the closed arms and could freely explore the environment for 20 min. The length of each arm was 30 cm, the width was 7 cm, and the height of the closed arm walls was 20 cm. The maze was 65 cm elevated from the floor by a camera stand. A total of eight mice were analyzed. 

Rat exposure assay

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Twenty-four hours after the EPM test, mice were habituated to a white rectangular box (70 cm length, 26 cm width, 44 cm height) for a 20 min session. On the following day, mice were exposed to the same environment for 20 min but in the presence of a toy rat. Twenty-four hours later, mice were exposed to an adult rat in this environment. The rat was secured by a harness tied to one of the walls and could freely ambulate only within a short perimeter. The mouse was placed near the wall opposite to the rat and freely explored the environment for 20 min. No separating barrier was placed between the mouse and the rat, allowing for close naturalistic encounters that can induce a variety of robust defensive behaviors. One mouse was removed from the analysis due to poor recording quality. This is why a total of seven mice were analyzed, instead of eight like in the EPM.

Behavior and miniscope video capture

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All videos were recorded at 30 frames/s using a Logitech HD C310 webcam and custom-built head-mounted UCLA miniscope (Aharoni and Hoogland, 2019). Open-source UCLA miniscope software and hardware (http://miniscope.org/) were used to capture and synchronize neural and behavioral video (Cai et al., 2016; Schuette et al., 2020). Neural recordings were sampled at 7.5 Hz.

Perfusion and histological verification

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Mice were anesthetized with Fatal-Plus and transcardially perfused with phosphate-buffered saline followed by a solution of 4% paraformaldehyde. Extracted brains were stored for 12 hr at 4°C in 4% paraformaldehyde. Brains were then placed in sucrose solution for a minimum of 24 hr. Brains were sectioned in the coronal plane in a cryostat, washed in phosphate-buffered saline, and mounted on glass slides using PVA-DABCO. Images were acquired using a Keyence BZ-X fluorescence microscope with a 10 or 20× air objective.

Data Analysis was performed using custom-written code in MATLAB and Python.

Miniscope postprocessing and co-registration

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 Miniscope videos were motion-corrected using the open-source UCLA miniscope analysis package (https://github.com/daharoni/Miniscope_Analysis) (Aharoni and Hoogland, 2019). They were spatially downsampled by a factor of 2 and temporally downsampled by a factor of 4, and the cell footprints and activity were extracted using the open-source package Constrained Nonnegative Matrix Factorization for microEndoscopic data (CNMF-E; https://github.com/zhoupc/CNMF_E) (Zhou et al., 2018). Neurons were co-registered across sessions using the open-source probabilistic modeling package CellReg (https://github.com/zivlab/CellReg) (Sheintuch et al., 2017). On average, 45.3 ± 19.4 cells (43.6 ± 15.9%) were co-registered across EPM and rat exposure assays for the seven mice analyzed for both assays.

Artifact suppression 

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For suppression of long time scale artifacts, for example, long time scale fluctuations in calcium fluorescence shared across many neurons due to bleaching or other factors, we used PCA (principal component analysis) to identify large variance principal components (PCs) (≥5% total variance) where the projected data exhibited artifacts. Artifacts are typically large in magnitude and occur across many neurons, resulting in dimensions of high variance that resemble artifacts. Cell activity was then reconstructed by excluding these PCs from reconstruction (O'Shea and Shenoy, 2018). This method was applied only to data for mouse 1 in the rat exposure assay.

Variance thresholding

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A minority of recorded candidate cells had very small variance over the course of an experimental session. To exclude these cells from analysis, we identified a reference cell for each trial. To choose the reference cell, cell variances were first sorted in decreasing order and then plotted. By visual inspection, the cell at the elbow of the plot was chosen as the reference cell. If there was no obvious elbow, the cell with the highest variance was chosen. Candidate cells with less than 10% of the reference cell’s variance were discarded. The remaining cells were z-scored and used for further analysis. Among the 375 total coregistered cells, 10 were excluded because of low variance in both the EPM and Rat, 11 were excluded because of low variance in the EPM but were not excluded in the Rat, and 38 were excluded in the Rat but not the EPM.

z-Scoring of activity

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Cell activity was z-scored once for the whole trace prior to use of the neural activity data in any analysis. We denote the cell activity as x, and the mean and standard deviation of the cell’s activity over the entire experimental session as μ and σ, respectively. The z-scored cell activity was then computed as (x-μ)/σ.

Behavior detection

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To extract the pose of freely behaving mice in the described assays, we implemented DeepLabCut (Mathis et al., 2018), an open-source convolutional neural network-based toolbox, to identify mouse nose, ear, and tailbase xy-coordinates in each recorded video frame. These coordinates were then used to calculate velocity and position at each timepoint, as well as classify defensive behaviors in an automated manner using custom MATLAB scripts. Freezing was defined as epochs when head and tailbase velocities fell below 0.25 cm/s for a period of 0.33 s. Approach and escape were defined as epochs when the mouse moved, respectively, toward or away from the rat at a velocity exceeding a minimum threshold of 3 cm/s.

We also measured the onset of prominent rat movements, as predator motion elicited defensive behavior in the mouse (Figure 3—figure supplement 4A). We identified all rat movements in which rat motion was above the 99.5th percentile of rat speed during the session. This approach ensured that only large predator movements were used, rather than minor changes in position, such as small rat tail movements. If multiple rat movements occurred within 5.33 s of each other, only the first was used for analysis. Rat movement direction was measured with respect to the position of the mouse at movement initiation. A 0-degree movement was toward the mouse’s initial position and a 180-degree movement was away from the mouse’s initial position; 90- and 270-degree movements were sideways from the mouse’s initial position.

Categorization of open, neither, and closed arm-preferring cells

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A cell was categorized as an open arm-preferring cell if activity in each individual open arm was significantly greater than the pooled activity in the closed arms (Wilcoxon rank sum test, p<0.05). Likewise, a closed arm-preferring cell was identified as a cell whose activity in each individual closed arm was significantly greater than the pooled activity in the open arms. The remaining cells were labeled as neither arm-preferring cells. The activity and correlations of open, closed, and neither arm cells in Figure 2A–C,G were computed on withheld data. The first 40% of each session was used for cell categorization while the last 60%, after a 10 s separation, was used to calculate activity traces and correlations. All other results used data from the whole session for cell categorization.

Behavior-aligned trace and ΔdF/F activity

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We calculated each cell’s behavior-aligned activity by computing the mean activity of the cell over all behavior occurrences, aligned to behavior onset. The mean peri-behavior trace for an ensemble (e.g., closed cells, open cells, or neither cells) was the average of peri-behavior activity across all cells in the ensemble. Change in mean activity after and before behavior was calculated by first subtracting the mean activity of each cell during the time frame [−2.5, 0] seconds relative to behavior onset from the mean activity of each cell in the time frame [0, 2.5] seconds. The overall difference in an ensemble, denoted ΔdF/F, was the average of the change in mean activity across all cells in the ensemble. For head dips, ΔdF/F was calculated using windows of [−5,–2.5] (before) and [0, 2.5] (after). In Figure 3, escapes and freezes which occurred within 10 s after the other behavior were excluded to reduce confounding due to slow GCaMP decay.

Interleaved training and testing data

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For analyses involving regression (EPM location index prediction, constrained correlation analysis [CoCA]), as well as SVM classification, testing data were interleaved with training data, with 60 s for each segment and 10 s of separation between data types, that is, [60 s training, 10 s excluded, 60 s testing, 10 s excluded, 60 s training, etc.]. We included gaps to minimize overlapping activity in the training and testing sets, which may arise due to dynamics in calcium activity.

Open arm preference score

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Cells with strong preference for higher activity in the closed arms or open arms respectively have open arm preference scores near −1 and +1. The arm score quantifies the separability of cell activity between arm types and is invariant to shifting and scaling of the cell activity. Excluding times when the mouse was in the center of the EPM, data points were labeled according to whether the mouse was in an open arm (positive label) or a closed arm (negative label). The open arm preference score was then defined as:

Openarmpreferencescore=2AUC1

where AUC is the area under the receiver operating characteristic (ROC) curve resulting from predicting which arm the mouse was in from single-cell activity. Predictions were made by thresholding cell activity. Cell activity greater than or equal to the threshold was predicted as being in the open arms while cell activity less than the threshold was predicted as the closed arms. We calculated the true positive (open) and false positive (open) rates by varying the threshold across every value of cell activity. These were then sorted to build the ROC curve and calculate the AUC. An open arm preference score of exactly +1 indicates that cell activity in the open arms is strictly greater than activity in the closed arms.

EPM score

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The EPM score (Adhikari et al., 2011) quantifies how differently a cell fires between closed vs. open arms. It is close to one when the cell has large differences in activity between arms of different types but is negative if the cell’s activity is more similar between different arm types than between same arm types. To calculate EPM score, we first compute the mean difference in z-scored activity between arms of different types (A) and arms of the same type (B). These are defined as:

A=0.25(|FC1FO1|+|FC1FO2|+|FC2FO1|+|FC2FO2|)
B=0.5(|FC1FC2|+|FO1FO2|),

where FO1 and FO2 are the mean z-scored activity of the cell in open arms 1 and 2, respectively, and FC1 and FC2 are the mean z-scored activity in closed arms 1 and 2, respectively. The EPM score is defined as:

EPMscore=(AB)/(A+B).

Cells with high EPM score if they have large differences in activity in different arm types (large A) and similar activity in same type arms (small B). The maximum score of 1.0 indicates no difference in cell activity across arms of the same type (B = 0). Cells with negative EPM scores have more similar activity across arms of different types than across arms of the same type.

EPM location index

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The EPM location index quantifies the position of the mouse in the EPM. It is close to 1 when the mouse is at the end of an open arm, and close to −1 when the mouse is at the end of the closed arms. To calculate the EPM location index, we first normalized the x and y position of the mice in the EPM to be in the range [−1, 1], where the x position is ±1 at the ends of the open arms and the y position is ±1 at the ends of the closed arms. We defined the EPM location index as:

EPMlocationindex=|x||y|.

The EPM location index is thus continuously varying between −1 and 1. Prediction of EPM location index (Figure 2F–G) was performed using linear regression with interleaved training and testing data. Outputs were clipped to the range [−1, 1] before final prediction was made.

Prediction of mouse position in the EPM from neural data using a linear SVM.

After z-scoring data, times when the mouse was in the center of the EPM were removed from training. The remaining data were separated into alternating blocks of 60 s training data and 60 s testing data with 10 s of separation between blocks. A linear SVM was fit on training using scikit-learn’s SVC function with balanced class weights (Pedregosa et al., 2011). Significance testing was performed using permutation tests with shuffled training labels for 100 random trials per mouse. The Matthews correlation coefficient was used to quantify the relation between predicted and observed arm-type occupancy because this metric was developed to assess correlations between binary values (such as arm type, which can only be closed or open arms).

Zones in the rat assay

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The safe zone was defined as the left 20% of the rat environment, based on x position. A mouse was labeled as being in the threatening zone whenever the distance to the rat was 14 cm or less.

Generalized linear model

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A GLM was fit for each cell. Each GLM mapped behavior variables to the cell’s z-scored calcium activity. In total, there were eight behavior variables for rat: distance to rat, mouse velocity, rat velocity, angle from mouse’s head to the rat, and occurrence of approaching, escaping, and freezing. Discrete behaviors were binary, labeled as one at all times in which they occurred and 0 otherwise. To enable behaviors to alter neural activity prior to and following the behavior, each binarized behavior was convolved with a log-time scaled raised cosine basis, from 5 s before behavior onset to 5 s after behavior offset. This kernel had the form:

fj(x)=12[cos(πxφj)+1],1πφj1<x1πφj+1
x=log(t+b+ε)
φj=log(b+ε)+12(j1)π

These kernels are non-zero when x is between [1πφj-1,1πφj+1], and are zero otherwise. Further, to enable historical kinematics to affect present neural data, the kinematics were convolved with a kernel, which used the same set of bases as the behavior, but only had responses after the onset within 5 s. These convolved behavior variables, denoted here as y1, y2, etc., were then modeled to produce the cell’s calcium fluorescence as:

x=β1y1+β2y2+...+βmym+c,

where βi is the coefficient for the ith behavior variable. The GLM was optimized by minimizing the mean-square error of the reconstruction between the GLM activity estimate, x, and the recorded calcium activity.

Cross-assay constrained correlation analysis

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To investigate if the dPAG uses shared patterns of neural activity to represent threat imminence across assays, we developed Constrained Correlation Analysis (CoCA). This model identifies the features used in a shared neural representation between different threatening situations. The CoCA technique defined a shared neural projection as a linear combination of the activity of individual neurons. Additionally, we constructed a behavioral projection for each assay, through a linear combination of each assay’s behavioral variables. Optimization via CoCA produced neural projection weights that were compared across open and closed cell ensembles.

We denote calcium fluorescence neural data as XRk × T and externally observed behavioral data as YRp × T, where k is the number of recorded cells for the corresponding mouse, shared across assays, p is the chosen number of behavioral variables for the corresponding assay, shared across mice, and T is the length of a recording session, unique for each session, but shared between neural and behavioral data for the same session. Behavioral variables contained both continuous kinematic variables (such as speed and distance from rat) and binary defensive behavior variables (such as the occurrence of freezing and escape). All variables were normalized to 0 mean and unit variance, except normalized |x| and |y| position in the EPM, which were instead in the range [0, 1] (variance < 0.25).

In order to find a common linear projection of threat across mice and assays, we performed the following optimization with mouse IDs i = 1, 2,…, 7 and assay IDs j = EPM, RAT. Calcium fluorescence traces of dPAG cells for mouse i were linearly combined after multiplying each cell with weights n1i to nki, where k is the number of cells that were co-registered in both assays. Taking the dot product of the calcium activity for mouse i in assay j, given by Xi,j, and the weights ni = [ni1...k] defined a neural projection for mouse i and assay j, given by Ni,j = (ni)TXi,j, neural data projection in red). For each mouse, the weights, ni, were the same across assays, so that each cell had the same weight in both assays. The behavioral variables for the EPM (such as x and y position, speed, etc.) were linearly combined with a set of weights b1 to b6 (as six behavioral variables were used for the EPM). These weights, bEPM = [b1EPM, b2EPM, …, b6EPM] were conserved across all mice. Linearly combining the EPM behavioral variables resulted in a behavioral projection for mouse i and assay EPM, given by Bi,EPM = (bEPM)TYi,EPM. Similarly, nine behavioral variables from the rat assay were linearly combined to produce a behavioral projection Bi,RAT = (bRAT)TYi,RAT using weights bRAT = [b1RAT, b2RAT, …, b9RAT]. We chose the neural weights, ni, and the behavioral weights, bEPM and bRAT, to optimize the correlations across all mice and assays:

maxijcorr(Ni,j,Bi,j)

where corr() is the Pearson’s correlation coefficient, and Ni,j and Bi,j are the linear projections of neural and behavioral data, respectively, given by Ni,j = (ni)TXi,j and Bi,j = (bj)TYi,j. The optimization variables ni, i = 1, 2,…, 7 and bj, j = EPM, RAT were simultaneously optimized using gradient descent via the Adam optimizer (Kingma and Ba, 2015) until convergence. Results presented use interleaved training and testing data. This method was implemented using PyTorch.

CoCA significance testing

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In order to test if correlations of testing data were better than expected by chance, correlations were computed between projected behavioral data (using weights fit by training data) and random projections of neural data (1000 trials). We emphasize that these correlations were applied to the testing data, and therefore it was possible for a random projection to have higher correlation than the CoCA projection. A one-tailed test was used (p<0.05).

Avoidance/approach score

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To calculate the continuous avoidance/approach score for each assay, the distance from safety was calculated (rat: distance from the safe wall; EPM: distance from the end of the closed arm) and normalized such that it ranged from 0 to 1 in the rat assay and 0 to 0.9 in the EPM assay. A binarized direction value was also assigned to each timepoint, indicating if the mouse was moving toward (+1) or away from (−1) the threat. To incorporate categorized behaviors, the avoidance/approach score for freeze samples equaled the minimum score of −1. For the EPM only, the avoidance/approach score was multiplied by 1.11 for head dip samples, such that a head dip at the end of the open arm would yield the maximum score of 1.

To calculate the score at each timepoint:

  • While approaching threat, avoidance/approach score = distance to safety × direction

  • While avoiding threat: avoidance/approach score = [1−distance to safety] × direction

k-Means clustering of neural data

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To determine if the avoidance/approach score is represented in the neural data, the k-means algorithm was used to cluster this data in an unsupervised manner. One of the simplest unsupervised classification algorithms, this approach identifies groupings of activity that are strongly represented in the neural data, without providing any additional information about animal behavior. If behavior then shows a significant relationship to these clusters, it is clearly represented as a prominent network motif.

For each assay, the Akaike information criterion (AIC) was calculated, which balances both the fit of the data (log-likelihood) and the model complexity, resulting in an optimal number of clusters that reasonably fit the data but are not too numerous. To measure AIC for k-means, we assumed the clusters were Gaussian-distributed with identity covariance matrices. The formula was as follows:

AIC=RSSmin(K)+2MK

where RSS is the residual sum of squares, M is the dimensionality of the dataset, and K is the number of clusters.

For each implementation of k-means, clusters were identified using 10 different randomized initializations; the set with the minimum sum of Euclidean distances was used. The approach and avoidance clusters then identified, for each session, as those with, respectively, the highest and lowest mean avoidance/approach scores. The overall mean avoidance/approach scores for approach and avoidance clusters were then calculated across mice. To determine if these approach and avoidance cluster scores were statistically significant, the actual mean was compared to a sampling distribution of means, calculated in an identical manner with permuted behavioral labels over 100 iterations. If the approach and avoidance score means were respectively greater than or less than 95% of this sampling distribution, they were considered significant. For the training/testing analysis, k-means was implemented on one assay as described above (the training assay), using only cells that co-registered between both assays. The cluster centroids identified in the training assay were then used to categorize approach and avoidance samples in the withheld testing assay. The mean avoidance/approach score was calculated for all approach and avoidance timepoints, across all training or testing sessions.

In a similar way, avoidance/approach states were identified by Hidden Markov Models (HMMs (4 states), using the top principal components of the neural data as input (accounting for ≥ 60% of the total variance). Similar to k-means, this approach finds ‘hidden states’ or states represented by the neural data in an unsupervised manner; unlike k-means, HMMs additionally consider the sequentiality of the neural data. The parameters of the HMM were found via expectation maximization. These states were analyzed in an identical manner to the k-means clusters described above. For the code, see ‘Expectation-Maximization for Hidden Markov Models using real-values Gaussian observations’ at Zoubin Ghahramani’s code base: http://mlg.eng.cam.ac.uk/zoubin/software.html.

Statistical analysis

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Significance values are included in the figure legends. Unless otherwise noted, all statistical comparisons were performed by either nonparametric Wilcoxon rank sum or signed-rank tests. With the exception of CoCA significance testing, all significance tests were two-tailed. Standard error of the mean was plotted in each figure as an estimate of variation of the mean. Correlations were calculated using Pearson’s method. Multiple comparisons were corrected with the false discovery rate method. All statistical analyses were performed using SciPy (Virtanen et al., 2020) and custom MATLAB scripts.

Data availability

All data was uploaded to dryad and all code was uploaded to github. https://datadryad.org/stash/share/4GezSjw4dvDJClAWa_zRoNWioH9qzGtDCJjLQ89HVoA, https://doi.org/10.5068/D1TM2G, https://github.com/schuettepeter/eLife_dPAG-ensembles-represent-approach-and-avoidance-states (copy archived at https://archive.softwareheritage.org/swh:1:rev:7c5aa29ff557bd8955eafb0a5699960a79aee4f7).

The following data sets were generated

References

  1. Conference
    1. Kingma DP
    2. Ba JL
    (2015)
    ADAM: a method for stochastic optimization. in ICLR
    International Conference on Learning Representations.
    1. Pedregosa F
    2. Varoquaux G
    3. Gramfort A
    4. Michel V
    5. Thirion B
    6. Grisel O
    7. Blondel M
    8. Prettenhofer P
    9. Weiss R
    10. Dubourg V
    (2011)
    Scikit-learn: machine learning in Python
    Journal of Machine Learning Research 12:2825–2830.
  2. Book
    1. Stankowich T
    (2019)
    Defensive Risk-Taking in Animals
    In: Choe J. C, editors. Encyclopedia of Animal Behavior. Academic Press. pp. 340–348.

Decision letter

  1. Mihaela D Iordanova
    Reviewing Editor; Concordia University, Canada
  2. Laura L Colgin
    Senior Editor; University of Texas at Austin, United States
  3. Mihaela D Iordanova
    Reviewer; Concordia University, Canada
  4. Philip Jean-Richard-dit-Bressel
    Reviewer; University of New South Wales, Australia
  5. Jonathan Fadok
    Reviewer; Tulane University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The manuscript is a thorough examination of dPAG activity in threat exposure. Some strengths of the paper include relating the open-closed sensory states to threatening states in a live rat study, and the elegant neural analyses that show support for the generalization of neural representation of threat approach and avoidance across procedures.

Decision letter after peer review:

Thank you for submitting your article "Dorsal Periaqueductal gray ensembles represent approach and avoidance states" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Mihaela D Iordanova as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Laura Colgin as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Philip Jean-Richard-dit-Bressel (Reviewer #2); Jonathan Fadok (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

The manuscript looks at how dPAG neurons represent threat approach and avoidance using the elevated plus maze and a live rat exposure situation. The data show that the dPAG populations split in terms of avoidance and approach when the mice are exposed to the EPM. These populations correspond to threat approach and avoidance in the live rat procedure. Machine learning algorithms further show support for the generalization of neural representation of threat approach and avoidance across procedures. The paper uses established behavioral methods as well as state-of-the-art neural methods and analyses. It presents a thorough examination of dPAG activity in threat exposure tasks. Although the study of the dPAG in these tasks has been reported in the past (e.g., Deng et al., 2016, DOI: 10.1523/JNEUROSCI.4425-15.2016; Masberrer et al., 2020, DOI: https://doi.org/10.1523/JNEUROSCI.0761-18.2020), which limits the novelty of the present paper, relating the open-closed sensory states to threatening states in the live rat study is a nice development.

Essential revisions:

The existing data mostly supports the main conclusions of the paper and I do not think added experimentation is needed. However, there are some concerns:

1. Concerns about the analyses, interpretation and representation of the data:

a. An argument is made in terms of threat and operationalized as a threat score and dPAG activity. However, this threat score seems to be determined where in the EPM the mouse is and has nothing to do with perception of threat derived from a behavioural response. So, if differential dPAG neurons fire to open vs closed arms in the EPM, then it seems hardly surprising that there would be a correlations between the 'threat score' and neural activity. What is more the threat score is more related to distance from centre as opposed to any actual threat. The latter is inferred. Correlation with head dip may be a better examination.

b. Cells were defined by their bias in firing patterns in open vs closed arms, which was then used to predict animal location. It would be more compelling if cell classification was determined using separate portions of the session than that used for prediction (akin to way training and testing is separated for SVM) to avoid circularity.

c. Emphasis is placed on how activity patterns are shared between incompatible behaviours such as freezing and escape, both of which are reactions to threat. While this might suggest threat-coding, it may also be the case that shared activity are an artifact of the slow kinetics of GCaMP6s coupled with these behaviours occurring in close temporal proximity to each other. Is there any evidence that the same patterns of activity are observed when the potential contribution of other behaviours are factored out? Perhaps GLM could be used to isolate event-related activity kernels.

d. For Figure 2D, define what is meant by strong correlation. What is the threshold?

e. Please include the EPM aggregate activity heatmap for all cells, as is reported for the REA in Figure 3C.

f. Is the overall activity bias near the rat in Figure 3C driven by novel threat preference in previously 'neither' cells, reduced activation of closed cells near the safe wall, or increased activation of open cells near the rat (relative to activity during EPM)?

g. The data in Figure 3-S3A do not appear to match the data Figures 3C and 3F and therefore are not representative.

h. The increased z-score to rat movement by closed-arm cells in Figure 3I-J is inconsistent with the interpretation that these neurons reflect threat proximity, since the threat is moving closer to the mouse and these cells are inhibited in proximity to threat in other scenarios. The data are better explained by the activity representing the mouse's state on the exploratory-defensive continuum.

i. There should be a zoomed-out image of PAG histology showing the spread of the GCaMP expression and the lens placement.

2. Methods. Methods should be written rigorously enough to promote reproducibility. Some specific questions are included below, but the authors should ensure that they provide a thorough account of the methods used.

a. Lack of clarity regarding shuffling and bootstrapping. It is unclear whether the authors are in fact applying bootstrapping (resampling from dataset with replacement) or another analysis method such as permutation tests (randomly relabelling cases). Please clarify and provide more details within Methods.

b. k = 10 seems arbitrary. It would be useful to know the relative strength of k = 2-10+ clusters (e.g. table of mean and minimum silhouette values) to confirm k = 10 is sensible.

c. The behavioural timeline, methods and procedures were confusing. More detail and clarity need to be provided in the Methods. This should account for the differential number of animals in the EPM and REA. Further, it was unclear when habituation took place.

d. Is the threat score binary (+1 or -1) or is it bounded by -1 to +1, with values in between? If that latter, how are values in between calculated? A liner a relationship is assumed between threat and distance travelled, which seems appropriate for the open arm but perhaps more uniformly low throughout the closed arms.

3. Discussion.

a. A discussion of the results and how they fit into the larger body of PAG literature is necessary.

b. The significance of the neither cells being activated in the REA.

c. Any insight the authors may have into the circuit or physiological differences of the two populations.

d. A discussion of the counterintuitive finding that a greater relative ratio of open:closed neurons correlates with less time in the open arm and threat approach.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Dorsal Periaqueductal gray ensembles represent approach and avoidance states" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Mihaela D Iordanova as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Laura Colgin as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Philip Jean-Richard-dit-Bressel (Reviewer #2); Jonathan Fadok (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

The reviewers agreed that the paper was greatly improved with the inclusion of necessary additions to methods, improvements to language, and addressing concerns around circular analyses and overlapping behaviour-related activity. The change in the semantics from EPM threat score to EPM location index was welcomed, although whether there is a linear gradient of threat in EPM remains up for debate. Some questions remain over the concerns raised previously. These are specified below.

Essential revisions:

1. Justification regarding the choice of 10 clusters has not been adequately addressed. A good solution would be to show that mean and minimum silhouette values for k = 10 is positive and higher/comparable to the range of other k-means solutions. This can be presented in a supplementary table. This is important because it goes a long way towards showing that the choice in clusters was valid. Currently the justification in the paper is not adequate and does not address why k=10 was chosen, rather is focused on why k=2 was not a good choice.

2. The added point that rat movements did not generally decrease distance to the mouse needs clarification. Did the increased distance between rat and mouse include movement made by the mouse? Given rat movement precipitates mouse escape, the increased distance is potentially due to mouse escape, not the rat moving away? Separately, was there a reason a 2 sec window was chosen? Rat movements towards the mouse followed by retreat (e.g. due to constrained movement) would easily elapse within 2secs, and be registered as movements away despite the mouse likely perceiving/responding to the advance. All relevant details about measuring rat movement should be included in Methods.

3. For variance thresholding, if this was performed per experimental session, were there cells that had high variance in one assay but low variance (and were thus excluded) in another assay? This would inflate the apparent degree of cross-assay coding. Details, i.e., a statement or Venn diagram, explaining how many cells were excluded on basis of EPM alone, REA alone, or both would be pertinent. If the number excluded on basis of only one assay is substantial, the text should acknowledge the impact this likely has on apparent cross-assay coherence.

https://doi.org/10.7554/eLife.64934.sa1

Author response

Essential revisions:

The existing data mostly supports the main conclusions of the paper and I do not think added experimentation is needed. However, there are some concerns:

1. Concerns about the analyses, interpretation and representation of the data:

a. An argument is made in terms of threat and operationalized as a threat score and dPAG activity. However, this threat score seems to be determined where in the EPM the mouse is and has nothing to do with perception of threat derived from a behavioural response. So, if differential dPAG neurons fire to open vs closed arms in the EPM, then it seems hardly surprising that there would be a correlations between the 'threat score' and neural activity. What is more the threat score is more related to distance from centre as opposed to any actual threat. The latter is inferred. Correlation with head dip may be a better examination.

The data in Figure 2E show that dPAG neural activity can be used to predict if the mouse is in the closed or open arms. Here, location is defined as a binary value, as the maze was divided into two locations (open and closed arms).

Our next analysis, in Figure 2F-G, shows dPAG activity can be used to predict not just which arm type the mouse is in, but where within that arm the mouse is. Is the mouse located in the beginning of the open arm (near the center) or is it in the end of the open arm? For this analysis the location of the mouse was not defined by a binary value (closed vs open), but with the Threat score, which is a continuous variable, corresponding to -1 in the end of the closed arms, 0 in the center and +1 in the end of the open arms. We created the EPM threat score to correlate neural activity with a continuous, non-binary, definition of mouse position. Figure 2F-G shows that dPAG neural activity does not simply encode arm type (as shown in 2E), but also finer grained location within an arm.

We believe this suggestion arose due to the name “EPM threat score”. This name suggests that the metric is directly reflecting threat, when in reality we simply used it as a number to describe the position of the mouse within the maze. We apologize for this misunderstanding, and thus have changed the name of the metric to “EPM location index”. We have altered the manuscript to remove any text that could indicate to the reader that this score is directly measuring threat.

The reason we investigated if the animal represents continuously varying locations within an arm type is because we believe that different locations within the open arm are more or less aversive, with the end of the open arms being the most aversive location in the maze. Our justification for this view is explained below.

As the reviewers correctly point out, the EPM location index is fully determined by the animal’s position in the maze, and as such this metric does not directly represent threat perception or behavioral response. However, we disagree that the location index has ‘nothing to do with perception of threat’. Regions of the maze with higher location index are correlated with higher avoidance, which is a behavioral response dictated by threat perception. Even within the open arms, mice avoid the regions with larger positive scores (corresponding to the end of the open arms) more than the regions with small positive scores, which are located in the beginning of the open arms, near the center of the maze. This behavioral gradient of avoidance within the open arms strongly indicates that the animal likely perceives higher threat in the extreme end of the open arms than in the beginning of the open arms. The most common interpretation of plus maze behavior is that mice avoid the open arms because they are more vulnerable to potential threats in open locations. This aversion facilitates thigmotaxis, a tendency to be close to vertical surfaces (i.e. closed arms). This view suggests that the beginning of the open arms are only slightly threatening, as if a dangerous stimulus is detected the mouse can quickly retreat to the safety of the closed arms. Conversely, it takes a longer time for the mouse to retreat to the closed arms if they are at the extreme end of the open arm. Thus, we argue that distance from the center of the maze is related to a gradient of threat, with the highest threat levels at the end of the open arms. The animal’s behavior supports this view, as mice avoid the extreme end of the open arm more robustly than the beginning of the open arm. We now include data showing that behavioral avoidance varies even within an arm type (Figure 2—figure supplement 1A, B), providing a stronger justification to investigate if dPAG cells represent finer-grained locations within an arm and discuss it in the text.

Although we support the interpretation that distance from center is a metric related to EPM aversiveness, we do not claim in the text that the EPM location index is directly reflecting threat perception or behavior, and we only used the EPM location index as a continuous, non-binary, metric to describe mouse location in the maze. Lastly, as mentioned above we changed the name of this score to “EPM location index” to avoid confusion.

It is unclear to us how to implement the Reviewer’s suggestion of using ‘head dips’ instead of the threat score. Head dips cannot be used as a gradual continuous metric of location within the maze.

b. Cells were defined by their bias in firing patterns in open vs closed arms, which was then used to predict animal location. It would be more compelling if cell classification was determined using separate portions of the session than that used for prediction (akin to way training and testing is separated for SVM) to avoid circularity.

We agree with this suggestion, and have therefore recalculated the results in Figure 2A-C, G to use the first 40% of each session for cell categorization and the last 60% for quantitative analyses. We altered the text and Methods to identify this. Interesting, we see similar results to before, indicating that cell responses to arm transitions and location in the EPM are robust during a session.

c. Emphasis is placed on how activity patterns are shared between incompatible behaviours such as freezing and escape, both of which are reactions to threat. While this might suggest threat-coding, it may also be the case that shared activity are an artifact of the slow kinetics of GCaMP6s coupled with these behaviours occurring in close temporal proximity to each other. Is there any evidence that the same patterns of activity are observed when the potential contribution of other behaviours are factored out? Perhaps GLM could be used to isolate event-related activity kernels.

This is an important point that was not addressed in the original submission. We thank the Reviewer for this question. In the rat assay the mouse is initially placed far from the rat. The mouse spends most of the time far from the rat, but sometimes it slowly approaches the rat, and then escapes in high velocity back to the safety of the opposite side of the chamber. After reaching the safer side, the mouse often displays many freezing bouts. Thus, freezing tends to occur after escaping. The analysis showed that the transition probability from freezing to escape is much lower than escape to freezing (0.01 and 0.61 respectively, n=44 mice). Thus, freezing tends to happen after escape (but escape rarely happens immediately after freezing).

As a consequence, it is possible that the freeze-related activity we identify could reflect slowly decaying GCaMP activity from a prior recent escape, rather than new “de novo” activity induced by freezing. To account for this confound, we calculated freeze bout-triggered activity traces excluding all freezes that happen within 10 seconds from an escape. These data show that even when excluding potentially confounding escape-related activity prior to freezing, closed cells and open cells show respective increases and decreases in activity during freezing (new Figure 3, panels D and E; I and J).

d. For Figure 2D, define what is meant by strong correlation. What is the threshold?

We apologize for not defining ‘strong’. We did not wish to create a binary distinction between cells with strong and weak correlation. Rather, we intended to emphasize that speed cannot explain the arm-type preference displayed by dPAG cells. We altered the text to read “Importantly, 95% of dPAG cells showed relatively low correlations of speed and neural activity, between -0.17 and +0.2. These results suggest that arm-related activity preferences are not driven by variations in velocity”.

e. Please include the EPM aggregate activity heatmap for all cells, as is reported for the REA in Figure 3C.

We have performed this new analysis as requested and added it to the manuscript (Figure 2—figure supplement 1). Aggregate dPAG does not show differential activation in closed and Open arms, as shown in Figure 2—figure supplement 1C.

f. Is the overall activity bias near the rat in Figure 3C driven by novel threat preference in previously 'neither' cells, reduced activation of closed cells near the safe wall, or increased activation of open cells near the rat (relative to activity during EPM)?

We thank the Reviewer for the opportunity to address this important question. We show in Figure 3F that all three cell types are more active near the rat than far from it. However, consistent with their activity patterns in the EPM, open cells show significantly higher activation near the rat than closed cells.

g. The data in Figure 3-S3A do not appear to match the data Figures 3C and 3F and therefore are not representative.

We apologize for this oversight. We now updated Figure 3-S3A to show representative examples of open and closed cells respectively displaying increases and decreases in activity as the mouse approaches the rat. This same result is shown averaged across cell ensembles in Figure 3G.

Figure 3C shows that dPAG cells in general (without separating into closed and open cells) are more active near the rat. This result is independent of the example individual heat maps for closed and open cells in Figure 3-S3A, and thus is not in disagreement (or agreement) with 3-S3A. Different results can be found when separating the cells into closed and open ensembles compared to pooling all cells together.

If the explanation above is not satisfactory, we ask the Reviewer to please explain in more detail what the discrepancy is between 3-S3A and 3C.

h. The increased z-score to rat movement by closed-arm cells in Figure 3I-J is inconsistent with the interpretation that these neurons reflect threat proximity, since the threat is moving closer to the mouse and these cells are inhibited in proximity to threat in other scenarios. The data are better explained by the activity representing the mouse's state on the exploratory-defensive continuum.

We apologize for not explaining more clearly the rat movement metric. As stated in the paper, the rat is constrained in its movement by a harness, which only allows it to move in a restricted area. Thus, for the most part, the rat cannot significantly change the distance to the mouse by moving. We now added a new panel in Figure 3—figure supplement 4 (panel B) showing that proximity to the rat does not generally increase during rat movements. The rat movement bouts are mostly changes from one resting position to another, or exploratory sniffing towards the walls, and may be in any direction. The rats were screened for low aggressiveness prior to their use, and consequently rat movement is very rarely motion towards the mouse, and the rat never displayed any attacking bouts.

Even though rat motions are not directed towards the mouse, the mouse tends to react to rat movements as sign of increased threat, and thus rat movement predicts decreased approach to rat, and increased escape and freezing (Figure 3-S4). These data show that rat movement is predictive of increased defensive behaviors such as escape and freezing. We argue that closed cells are more activated during threat-avoidance related defensive behaviors (i.e., escape and freezing). Consequently, it is expected that rat movement (which is a stimulus that provokes more escape and freezing) should be positively correlated with increases in closed cell activity.

Nevertheless, we strongly agree with the Reviewer’s view that the data is well explained by changes in the “exploratory-defensive continuum”. This continuum is reflected in both behavioral and non-behavioral features. Higher defensive states are displayed during increased threat perception, and one of the metrics influencing threat perception is threat distance, while another is movement of the predator.

i. There should be a zoomed-out image of PAG histology showing the spread of the GCaMP expression and the lens placement.

We agree with this comment and have added a zoomed out histology photo showing GCaMP expression spread and lens placement (Figure 1—figure supplement 1).

2. Methods. Methods should be written rigorously enough to promote reproducibility. Some specific questions are included below, but the authors should ensure that they provide a thorough account of the methods used.

We have significantly increased the level of detail in the methods to ensure reproducibility. In addition, all scripts have been posted on github.

a. Lack of clarity regarding shuffling and bootstrapping. It is unclear whether the authors are in fact applying bootstrapping (resampling from dataset with replacement) or another analysis method such as permutation tests (randomly relabelling cases). Please clarify and provide more details within Methods.

We apologize for the lack of clarity. The analyses were incorrectly described as using bootstrap resampling when they were permutation tests using randomly permuted labels. We have corrected the text and Methods to reflect this.

b. k = 10 seems arbitrary. It would be useful to know the relative strength of k = 2-10+ clusters (e.g. table of mean and minimum silhouette values) to confirm k = 10 is sensible.

We repeated the analysis for a range of k values and now summarize these results in the revised Figure 4—figure supplement 1. The mean avoidance and approach score values were respectively and significantly less than and greater than the distribution of avoidance/approach values built from permuted neural data for EPM and rat assay across a range of k values (EPM: k=2-12; rat: k=3-12). While k-means with k=2 did not significantly differentiate the avoidance score from the permuted distribution in the rat assay, this is not particularly surprising--the analysis is unsupervised and the most salient dPAG feature may not be avoidance/approach for this assay. However, given an additional cluster (k=3), the avoidance score fell significantly below the permuted distribution for the rat assay. The approach and avoidance clusters defined on one assay were, moreover, significantly different when applied to the other assay across this range of k values. This analysis worked for all k values, from 3 to 12. To avoid plotting so dozens of plots demonstrating the validity across k values, we show these results only for the minimum and maximum significant outputs of the tested range (Author response image 1).

Author response image 1
Approach and avoidance identified by k-means with a range of k values and Hidden Markov Model.

(A) (top) Arrow depicts the mean avoidance/approach score for avoidance and approach clusters (3 clusters), identified by k-means, across concatenated sessions (n = 7 mice). This mean was compared to a permuted distribution of avoidance/approach means, calculated by shuffling the neural data 100 times (n cells same as Figure 4C; p < 0.01). (bottom left) Bars depict the mean Rat and EPM avoidance/approach scores (+/- 1 s.e.m.) for approach and avoidance clusters across mice. (bottom right) As described in Methods and Figure 4B, these cluster centroid locations, trained on one assay, were then used to define approach and avoidance timepoints in the other assay. Bars depict the corresponding mean avoidance/approach score (+/- 1 s.e.m.) for this testing data. (Train on EPM: avoidance cluster n = 17753, approach cluster n= 13096; Test on Rat: avoidance cluster n = 15727, approach cluster n = 7724; Train on Rat: avoidance cluster n = 21092, approach cluster n = 10672; Test on EPM: avoidance cluster n = 9867, approach cluster n = 3028; n cells same as Figure 4D; Wilcoxon ranked-sum test) (B) Same as (A), but with 12 clusters. (Train on EPM: avoidance cluster n = 2614, approach cluster n= 3671; Test on Rat: avoidance cluster n = 1749, approach cluster n = 1488; Train on Rat: avoidance cluster n = 3693, approach cluster n = 2337; Test on EPM: avoidance cluster n = 2163, approach cluster n = 1323; n cells same as Figure 4D; Wilcoxon ranked-sum test) *** p < 0.001.

c. The behavioural timeline, methods and procedures were confusing. More detail and clarity need to be provided in the Methods. This should account for the differential number of animals in the EPM and REA. Further, it was unclear when habituation took place.

We agreed with the Reviewer and rewrote the behavioral timeline and methods in the Methods session in this new version of the manuscript.

d. Is the threat score binary (+1 or -1) or is it bounded by -1 to +1, with values in between? If that latter, how are values in between calculated? A liner a relationship is assumed between threat and distance travelled, which seems appropriate for the open arm but perhaps more uniformly low throughout the closed arms.

The threat score (now called EPM location index) is bounded between -1 to +1. A value of zero is assigned to the center of the maze, and values were linearly interpolated from zero to -1 or +1 respectively in the end of the closed arms and open arms. We are not assuming a linear distance between threat and distance, even though we agree that higher score values are associated with higher threat. This score was developed only to have a continuous metric of plus maze location to test if dPAG neural activity can predict location within an arm type, as explained in question “1A” above. We altered the text to make it clear that this score is only being used as a continuous metric of location within the maze, and we are in no way implying this score is a direct measure of threat perception. Please see our detailed explanation in question 1a above,

3. Discussion.

a. A discussion of the results and how they fit into the larger body of PAG literature is necessary.

As pointed out by the Reviewers earlier, prior papers have recorded dPAG activity during threat exposure. We now discuss this prior work in more detail and explain how our data advance understanding of the dPAG. We believe the novelty of this paper is in:

1. Tracking the same cells across two assays and finding that dPAG represent open spaces and proximity to a predator with shared activity patterns

2. Performing at population-level analysis of dPAG cells, rather than only quantifying the % of cells that are activated or inhibited by specific behaviors

3. Demonstrating that dPAG cells are not only encoding specific behaviors, but rather that dPAG ensemble activity reflects moment-to-moment changes in the approach-avoidance state of the animal. This is our conceptually most important result, and it indicates that the view that the dPAG is merely a motor output structure is likely oversimplified.

We have now altered the manuscript, discussing these findings in the context of prior papers.

b. The significance of the neither cells being activated in the REA.

Our interpretation of these data is that the neither ensemble may require a higher amount of threat to be activated. The relatively low threat open arms are not enough to activate them, while proximity to a live predator is more dangerous, and thus is enough to activate these cells. We have added this discussion to the text.

c. Any insight the authors may have into the circuit or physiological differences of the two populations.

We speculate that perhaps these two cell classes may correspond to populations expressing different dPAG markers, such as substance P, nitric oxide or cholecystokinin. Another possibility is that these two cell classes have distinct connectivity patterns. We added discussion related to this issue in the text, while making it clear that this work does not answer this question, and future studies are needed to further dissect dPAG cell types based on genetic and connectivity differences.

d. A discussion of the counterintuitive finding that a greater relative ratio of open:closed neurons correlates with less time in the open arm and threat approach.

Our results are very similar to the one previously reported by Jimenez et al., 2018 in Neuron (“Anxiety Cells in a Hippocampal-Hypothalamic Circuit”). In that paper the authors identify ventral hippocampal cells that are preferentially active in the open arms of the elevated plus maze. Increased open arm activity was correlated with decreased open arm exploration and inhibition of the ventral hippocampus (which would result in inhibition of these open arm cells) increased open arm exploration. Thus, both this published result in the hippocampus as well as our dPAG result suggest that increased activity of open arm cells is correlated with decreased open arm exploration.

In our view, a possible interpretation for these results is that rather than causing open arm exploration, open arm ensemble is highly responsive to risk-assessment and proximity to threat. Considering this hypothesis, the present results suggest that mice with more open arm cells display an increased sensitivity to risk-evaluation reflected on increased avoidance of threat. We now added text discussing our interpretation of this result.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1. Justification regarding the choice of 10 clusters has not been adequately addressed. A good solution would be to show that mean and minimum silhouette values for k = 10 is positive and higher/comparable to the range of other k-means solutions. This can be presented in a supplementary table. This is important because it goes a long way towards showing that the choice in clusters was valid. Currently the justification in the paper is not adequate and does not address why k=10 was chosen, rather is focused on why k=2 was not a good choice.

To address the Reviewers' concern regarding our choice of cluster number, we have added Figure 4—figure supplement 1, which depicts the mean (+/- s.e.m.) Akaike information criterion (AIC) for 2 to 10 clusters. The AIC score is a standard hyperparameter and model selection metric in machine learning. We chose the AIC compared to silhouette values because the AIC score prioritizes model fit and complexity, rather than cluster consistency. The AIC measures the log likelihood minus a model complexity term that reflects the number of model parameters. The AIC therefore balances both the fit of the data (log-likelihood) and the model complexity, resulting in an optimal number of clusters that fit the data reasonably but are not too numerous. To measure AIC for k-means, we assumed the clusters were Gaussian distributed with identity covariance matrices.

This supplemental figure shows that the data is best fit by a k-means model with 2 clusters for the EPM assay and either 2 or 3 clusters for the rat exposure assay, due to their significantly lower AIC than other tested values of k.

We thus no longer use 10 clusters, and instead updated Figure 4C-D and Figure 4-2 with the optimized k values mentioned above.

2. The added point that rat movements did not generally decrease distance to the mouse needs clarification. Did the increased distance between rat and mouse include movement made by the mouse? Given rat movement precipitates mouse escape, the increased distance is potentially due to mouse escape, not the rat moving away? Separately, was there a reason a 2 sec window was chosen? Rat movements towards the mouse followed by retreat (e.g. due to constrained movement) would easily elapse within 2secs, and be registered as movements away despite the mouse likely perceiving/responding to the advance. All relevant details about measuring rat movement should be included in Methods.

The distance presented did not include movement by the mouse and was relative to the mouse's position at the start of movement. Consequently events categorized as increasing rat to mouse distance are due to the rat moving away from the mouse, rather than due to the mouse escaping.

We added a polar plot displaying the angle of all rat movements relative to the mouse (Author response image 2 and also Figure 3, figure supplement 4C). An angle of zero corresponds to Rat movements towards the mouse, while an angle of 180 indicates movements away from the mouse. Note that movements away from the mouse are more common than movements towards the mouse. Taken together, these data show that the rat is not consistently moving towards the mouse, as indicated both by the polar angle plot and also by the distribution of mouse-rat distance changes during rat movements (Figure 3, figure supplement 4B).

A window of 2 seconds was used to allow sufficient time to include the entire rat movement bout. We also added detailed information in the Methods explaining the measurements of rat movements.

Author response image 2
Rat movements are not consistently directed towards mice.

(A) Polar plot showing the distribution of direction of rat movement relative to mouse position at time = 0 seconds. (B) Distribution of direction of average rat movement across 2 seconds after rat movement initiation relative to mouse position at the start of rat movement initiation. Zero degrees represents movement toward initial mouse position, while 180 degrees corresponds to movements away from the mouse.

3. For variance thresholding, if this was performed per experimental session, were there cells that had high variance in one assay but low variance (and were thus excluded) in another assay? This would inflate the apparent degree of cross-assay coding. Details, i.e., a statement or Venn diagram, explaining how many cells were excluded on basis of EPM alone, REA alone, or both would be pertinent. If the number excluded on basis of only one assay is substantial, the text should acknowledge the impact this likely has on apparent cross-assay coherence.

Among the 375 total co-registered cells, 10 were excluded because of low variance in both the EPM and Rat, 11 were excluded because of low variance in the EPM but were not excluded in the Rat, and 38 were excluded in the Rat but not the EPM. We opted to show a more informative color scatterplot instead of the suggested Venn diagram because the full range of the data can be seen in the scatterplot. This scatterplot (Author response image 3) reveals that even among cells that were excluded due to low variance in only one assay, very few cells had very large variance in the other assay. For example, very few of the green cells (excluded due to low variance in the EPM) had very high variance in the Rat assay.

Importantly, our metric of cross-assay representation is based on the same cells similarly representing concrete threat-related metrics across assays, such as avoidance or approach behaviors. Our metric of cross-assay representation is not a direct consequence of simply having overall higher or lower variance across assays. Thus, it is unlikely that the selection of cells based on variance could be artifactually increasing cross-assay representation.

Instead of mentioning in the text that the selection of cells could be influencing cross-assay coherence, as suggested by the Reviewers, we opted instead to directly demonstrate that all of our main results can be replicated without excluding any cells. These data show that cell selection by variance thresholding does not artifactually create our results.

Author response image 3
Raw variances of co-registered cells for both EPM and Rat assays across each entire session.

(n=375 cells).

https://doi.org/10.7554/eLife.64934.sa2

Article and author information

Author details

  1. Fernando MCV Reis

    Department of Psychology, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    Contributed equally with
    Johannes Y Lee
    For correspondence
    fmcvreis.84@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0121-2887
  2. Johannes Y Lee

    Department of Electrical and Computer Engineering, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Conceptualization, Formal analysis, Writing - original draft, Writing - review and editing
    Contributed equally with
    Fernando MCV Reis
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2420-4916
  3. Sandra Maesta-Pereira

    Department of Psychology, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6522-8311
  4. Peter J Schuette

    Department of Psychology, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Meghmik Chakerian

    Department of Psychology, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Jinhan Liu

    Department of Electrical and Computer Engineering, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Formal analysis, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  7. Mimi Q La-Vu

    Department of Psychology, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Brooke C Tobias

    Department of Psychology, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2043-9523
  9. Juliane M Ikebara

    Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Bernardo do Campo, São Paulo, Brazil
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  10. Alexandre Hiroaki Kihara

    Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Bernardo do Campo, São Paulo, Brazil
    Contribution
    Funding acquisition, Methodology
    Competing interests
    No competing interests declared
  11. Newton S Canteras

    Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
    Contribution
    Conceptualization, Funding acquisition
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7205-5372
  12. Jonathan C Kao

    Department of Electrical and Computer Engineering, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Avishek Adhikari
    For correspondence
    kao@seas.ucla.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9298-0143
  13. Avishek Adhikari

    Department of Psychology, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    Contributed equally with
    Jonathan C Kao
    For correspondence
    avi@psych.ucla.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9187-9211

Funding

National Institutes of Health (R00 MH106649)

  • Avishek Adhikari

National Institutes of Health (R01 MH119089)

  • Avishek Adhikari

Brain and Behavior Research Foundation (22663)

  • Avishek Adhikari
  • Fernando MCV Reis
  • Jonathan C Kao

Brain and Behavior Research Foundation (27654)

  • Fernando MCV Reis
  • Avishek Adhikari
  • Jonathan C Kao

National Science Foundation (DGE-1650604)

  • Peter J Schuette

Fundação de Amparo à Pesquisa do Estado de São Paulo (#2019/17677–0)

  • Juliane M Ikebara

Fundação de Amparo à Pesquisa do Estado de São Paulo (2017/08668-1)

  • Fernando MCV Reis

Fundação de Amparo à Pesquisa do Estado de São Paulo (2014/05432-9)

  • Newton S Canteras

Hellman Foundation

  • Avishek Adhikari

National Institutes of Health (F31 MH121050-01A1)

  • Mimi Q La-Vu

Achievement Rewards for College Scientists Foundation

  • Mimi Q La-Vu

Brain and Behavior Research Foundation (29204)

  • Avishek Adhikari
  • Fernando MCV Reis
  • Jonathan C Kao

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the National Institute for Mental Health (R00 MH106649 and R01 MH119089) (AA), the Brain and Behavior Research Foundation (Grants #22663, 27654, and 29204 respectively to AA, FMCVR, and JCK), the National Science Foundation (NSF-GRFP DGE-1650604, PJS), the UCLA Affiliates fellowship (PJS), and the Hellman Foundation (AA). Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Research Grant #2019/17677–0 (JMI), #2014/05432–9 (NSC). FMCVR was supported with FAPESP Grants #2015/23092–3 and #2017/08668–1. MQLV was supported by the Achievement Rewards for College Scientists Foundation and by the National Institute for Mental Health award F31 MH121050-01A1.

Ethics

Animal experimentation: All procedures have been approved by the University of California, Los Angeles Institutional Animal Care and Use Committee, protocols 2017-011 and 2017-075.

Senior Editor

  1. Laura L Colgin, University of Texas at Austin, United States

Reviewing Editor

  1. Mihaela D Iordanova, Concordia University, Canada

Reviewers

  1. Mihaela D Iordanova, Concordia University, Canada
  2. Philip Jean-Richard-dit-Bressel, University of New South Wales, Australia
  3. Jonathan Fadok, Tulane University, United States

Publication history

  1. Received: November 16, 2020
  2. Accepted: May 5, 2021
  3. Accepted Manuscript published: May 6, 2021 (version 1)
  4. Version of Record published: May 19, 2021 (version 2)

Copyright

© 2021, Reis et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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