DOI: 10.5068/d1tm2g
title: Dorsal Periaqueductal gray ensembles represent approach and avoidance states

description: 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 threat-avoidance
 ensemble in mice (Mus musculus) that showed higher activity far from
 threats such as the open arms of the elevated plus maze and a live
 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 approach/avoidance 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.
description: 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-hour 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 8 mice, while any analyses involving rat exposure used 7
 mice. All procedures conformed to guidelines established by the National
 Institutes of Health and have been approved by the University of
 California, Los Angeles Institutional Animal Care and Use
 Committee. Rats. Male Long-Evans rats (250-400 grams)
 were obtained from Charles River and were individually housed on a
 standard 12-hour 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. 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 x 103 viral particles per ml
 and viral aliquots were diluted prior to use with artificial cortex buffer
 to a final titer of 5 x 1012 viral particles per ml. After performing a
 craniotomy, 100nl 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). 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.
 Behavioral tests were combined in the following manner across days: EPM
 test, habituation 1, habituation 2, rat exposure. Three days after, the
 fear conditioning test was conducted in the following manner: habituation
 1, habituation 2, fear conditioning and retrieval.
 Elevated Plus Maze (EPM) test. Mice were placed in the center of
 the EPM facing one of the closed arms and were allowed to freely explore
 the environment for 20 minutes. 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 8 mice
 were analyzed. Rat Exposure Assay. Mice were habituated
 to a white rectangular box (70 cm length, 26 cm width, 44 cm height) for
 two consecutive days during 20-minute sessions. Mice were then exposed to
 an adult rat in this environment on the following day. 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 context for 20 minutes. 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. A total of 7 mice were analyzed. Behavior
 and miniscope video capture. All videos were recorded at 30 frames/sec
 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). Perfusion and histological verification.
 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 hours at 4°C in 4% paraformaldehyde.
 Brains were then placed in sucrose solution for a minimum of 24 hours.
 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 20X air objective. Data Analysis was performed
 using custom-written code in MATLAB and Python.
 Miniscope postprocessing and co-registration. 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 two and
 temporally downsampled by a factor of four, 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). Artifact suppression. For suppression of long
 timescale artifacts, e.g. long-time scale fluctuations in calcium
 fluorescence shared across many neurons due to bleaching or other factors,
 we used PCA to identify large variance PCs (≥ 5% total variance)
 reflecting these artifacts. Cell activity was then reconstructed using
 these PCs excluded from reconstruction (O'Shea and Shenoy, 2018).
 This method was applied only to data for mouse 1 in the rat exposure
 assay. Variance thresholding. A minority of recorded
 cells had very small variance over the course of an experimental session.
 To exclude these cells from analysis, we identified a representative cell
 for each trial. Cells with less than 10% of the representative cell’s
 variance were discarded. The remaining cells were used for further
 analysis. Behavior detection. 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 tail base xy-coordinates in each
 recorded video frame. These coordinates were then used to calculate
 velocity and position at each time point, as well as classify defensive
 behaviors in an automated manner using custom Matlab scripts. Freezing was
 defined as epochs of cessation of all movement except for breathing.
 Approach and escape were defined as epochs when the mouse moved,
 respectively towards or away from the rat at a velocity exceeding a
 minimum threshold.
description: Each numbered folder corresponds to a mouse.  Within each
 folder: 'neural_data.mat' is a struct
 containing 'C_raw', the raw CNMF-E output neural data.
 ______________________________________________________________________________________________ 'BehaviorMS.mat' contains a series of vectors or matrices.  Pertinent to this study are: Rat sessions-<br> -approachFrameMS and approachIndicesMS<br> -stretchFrameMS and stretchIndicesMS<br> -escapeFrameMS and escapeIndicesMS<br> -freezeFrameMS and freezeIndicesMS EPM sessions-<br> -openArmFrameMS and openArmIndicesMS<br> -closedArmFrameMS and closedArmIndicesMS<br> Additionally, the separate file 'headDip.mat' gives the headDipFrameMS and headDipIndicesMS for all <br> head dips over the edge of the open arms of the EPM. All '*FrameMS' files give in/out points, aligned to the neural data, for each behavior. 'In' is<br> column 1 and 'Out' is column 2. All '*IndicesMS' files give logical '0' or '1' values for whether a <br> behavior is happening during a frame of neural data. _______________________________________________________________________________________________ 'Tracking.mat' is a struct containing the pertinent fields:<br> FOR EPM, RAT, and TOY RAT, for each frame of neural data:<br> 'mouse_positionMS', which provides the xy coordinates of the point between the mouse ears.<br> 'mouseAngleMS' provides the angle in radians of mouse head direction.<br> 'mouseVelMS' provides the frame-by-frame velocity of the mouse, in pixels per frame. FOR RAT and TOY RAT only, for each frame of neural data:<br> 'rat_positionMS' provides the xy coordinates of the point between the rat ears.<br> 'angleDiffMouseHeadDirRatMS' provides the difference in radians between the head direction of the mouse<br> and the rat position.<br> 'ratVelMS' provides the frame-by-frame velocity of the rat, in pixels per frame.<br> 'distanceMouseRatMS' or 'distanceMouseToyRatMS' provides the distance in pixels between the mouse and rat.