To examine the effect of astrocyte Gi-DREADD stimulation on fear learning (Experiment 3), the HW-EFL paradigm was conducted. This procedure has been previously described at length (Parekh et al. 2020 ). Briefly, animals were randomly assigned to drug (heroin or saline) treatment. Animals undergo chronic escalating heroin administration and withdrawal in their home cage. Seven days after the start of withdrawal, animals were placed into a novel context for 15 min of habituation. On Day 8, animals were placed into the same context for a single scrambled foot shock (1 mA, 1s) at 3 min, 12 s. On days 9, 10, 15, 22 (Test Days 1, 2, 7, and 14) animals were placed into the same context for 8 min, 32 s and behavior was recorded to measure freezing behavior, a measure of learned fear. Ethovision XT video tracking software (Noldus Information Technology Inc.) was used to analyze freezing behavior. The activity analysis feature (Activity Threshold = 10) was used and described previously (Jones et al. 2018a (link), b (link)). This feature detects the entire arena and asks what percentage of pixels in the entire arena are changing or the same. To calculate the percent of time each animal was inactive during each contextual fear test and at baseline we used the % of pixels that were inactive through the activity analysis feature in Ethovision XT.
Ethovision xt video tracking software
Manufactured by Noldus
Sourced in Netherlands
Ethovision XT is a video tracking software developed by Noldus. It is designed to automatically track and analyze the behavior of animals in a variety of experimental settings. The software can be used to record and analyze the movement, position, and other behavioral parameters of animals in real-time.
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23 protocols using ethovision xt video tracking software
1
Assessing Astrocyte G-DREADD Impact on Fear
2
Elevated Plus Maze Anxiety Assay
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EPM was used to assay anxiety-related behavior. An applied apparatus is composed of two open arms and two closed arms (29 × 8 cm, with walls of 16 cm height), which was set up 30 cm above the floor. Each mouse was placed in the central square of the maze and allowed to explore the elevated plus maze for 5 min. The animals' movement track was recorded with EthoVision XT video-tracking software (Noldus, Netherlands). The percentage of time spent in open arms and numbers of arm entries were analyzed. Each arm was cleaned with alcohol before the next test.
3
Novel Object Recognition Memory Task
4
Open Field Exploration in Mice
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To assess whether prenatal inflammation affected the offsprings' explorative activity in a novel environment, the mice were exposed to a so called open field test. Mice were placed in an open field arena with a diameter of 85 cm and height of 30 cm. The arena was subdivided in a center zone and border zone. Mice were introduced in the arena at the outer wall, were allowed to explored the arena for 5 min and were then returned to their home cage. Between sessions the arena was cleaned with 30% ethanol for removal of olfactory cues. The movement of the animals was tracked using ethovision XT videotracking software (Noldus, The Netherlands).
5
Elevated Plus Maze for Anxiety Assessment
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The elevated plus maze was used as an additional measure of anxiety‐like behavior.30 , 31 A plus‐shaped maze was suspended 40 cm above the ground. Two arms were enclosed with acrylic walls and two arms were unenclosed. Each arm was 30 cm × 5 cm and stemmed from a 5 cm × 5 cm central platform. The maze was isolated in a temperature, light and noise‐controlled room. Each subject was placed in the center of the maze and allowed to freely explore the maze for 10 min without an experimenter present. Ethovision XT video tracking software (Noldus, Netherlands) was used to record the time each subject spent in the open and closed arms, as well as track the number of entries into the arms.
6
Conditioned Place Preference Assay with Optogenetics
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The CPP was performed in a custom-made two-chamber box (length × width × height: 40 × 20 × 30 cm3): the right chamber had vertical black-and-white stripes on the walls and a smooth floor, whereas the left chamber had horizontal black-and-white stripes on the walls and a mesh floor. The CPP test was performed according to a previous study with some modifications [20 (link)].
Day 1 was the preconditioning test (precondition) day. Mice were given free access to the 2 chambers and the time that the mice spent in each chamber was recorded. On days 2 to 4, the mice were restricted to 1 chamber for 20 min and received optogenetic modulation. At least 4 h later, the mice were restricted to the other chamber for 20 min without optogenetic modulation. On day 5 (around 18 h after the last conditioning), the mice were allowed to freely explore the 2 chambers for 20 min and the time spent in each chamber was recorded. On the precondition and test days, the animal’s movement was video-tracked and analyzed online or offline with an EthoVision XT video tracking software (Noldus Information Technology, Wageningen, the Netherlands) [56 (link),57 (link)]. We calculated the time spent in the light-paired side on the precondition and test days. Mice were not used if they spent more than 75% of the total time in 1 chamber on the pre-test day.
Day 1 was the preconditioning test (precondition) day. Mice were given free access to the 2 chambers and the time that the mice spent in each chamber was recorded. On days 2 to 4, the mice were restricted to 1 chamber for 20 min and received optogenetic modulation. At least 4 h later, the mice were restricted to the other chamber for 20 min without optogenetic modulation. On day 5 (around 18 h after the last conditioning), the mice were allowed to freely explore the 2 chambers for 20 min and the time spent in each chamber was recorded. On the precondition and test days, the animal’s movement was video-tracked and analyzed online or offline with an EthoVision XT video tracking software (Noldus Information Technology, Wageningen, the Netherlands) [56 (link),57 (link)]. We calculated the time spent in the light-paired side on the precondition and test days. Mice were not used if they spent more than 75% of the total time in 1 chamber on the pre-test day.
7
Colonic Motility Assessment with Propofol
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The whole colon was harvested and placed in ice cold Krebs buffer solution. The mesentery was trimmed using fine scissors and the whole colon was then loosely pinned in a Sylgard-lined flow bath, allowing a lateral movement of approximately 0.5 cm about the mid-line and perfused with oxygenated Krebs buffer solution at 37 ± 1 °C at a flow rate of 8 ml min -1 . A small (2 mm) incision was made in both ends of the colon and the openings pinned flat to facilitate pellet insertion and its expulsion at the distal end. If spontaneous evacuation was not achieved, the faecal pellets were removed from the isolated colon after 30 minutes, by gently flushing the lumen of the colon with warmed Krebs buffer solution. The colon was then left to stabilise for 15 minutes, prior to recordings of pellet motility.
Measurements of motility were carried out using an epoxy-coated natural faecal pellet to prevent disintegration. The coated faecal pellet was inserted 3-4 mm into the proximal end of the bowel using a fire-polished glass capillary and the movement of the pellet was monitored using a video camera. Pellet motility was tracked using EthoVision XT video tracking software (Noldus). Following a successful trial, the experiment was repeated two further times and the average response was utilised in the presence and absence of 5 µM propofol.
Measurements of motility were carried out using an epoxy-coated natural faecal pellet to prevent disintegration. The coated faecal pellet was inserted 3-4 mm into the proximal end of the bowel using a fire-polished glass capillary and the movement of the pellet was monitored using a video camera. Pellet motility was tracked using EthoVision XT video tracking software (Noldus). Following a successful trial, the experiment was repeated two further times and the average response was utilised in the presence and absence of 5 µM propofol.
8
Conditioned Place Preference for Spontaneous Pain
9
Elevated Plus-Maze for Mice Exploration
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The plus-shaped maze consisted of two enclosed arms and two open arms elevated 60 to 70 cm above the ground. Furthermore, tape was attached to the ends of the open arms (5 cm from the end of the arm) to limit falls. After 1 hour of habituation, mice were placed at the center of the maze facing an open arm. Mice could freely explore the four arms for 5 min. Time and distance traveled in each arm and center area were video recorded and tracked using EthoVision XT video tracking software (Noldus Information Technology Inc., Leesburg, VA). The apparatus was cleaned with 70% alcohol between mice.
10
Social Interaction and Locomotor Behavior
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Social behavior was measured using a 3-chambered social arena as described previously (Moy et al., 2008 (link)). Briefly, mice received a 10 min habituation session to the arena in the presence of two wire cages, one in each side of the chamber. A 10 min social preference test followed in which the mice were allowed to explore the arena containing a wired cage with a conspecific mouse (stranger mouse) or a wire cage with an inanimate object in the two lateral chambers. The placement of social and non-social targets was counterbalanced between animals. The measured parameters of social interaction and locomotor activity were the interaction time (sec) with each target (during the social preference test session) and the total distance (cm) moved (during habituation and social preference test sessions) respectively, calculated by Ethovision XT video tracking software (Noldus).
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