A plus maze apparatus consisting of two open arms (16 × 5 cm) and two enclosed arms (16 × 5 cm with 12 cm high walls) that extended from a central platform (5 × 5 cm) was placed 40 cm above the floor. Each mouse was placed in the center of the elevated plus maze (EPM) facing an open arm away from the experimenter and allowed to freely explore the maze for 5 min in dim lighting. Movement was tracked using SMART v3.0 (Panlab, Barcelona, Spain). The box was cleaned with 70% ethanol and water between tests. The number of entries and time spent in the closed and open arms were monitored for 10 min and analyzed using SMART v3.0 (Panlab, Barcelona, Spain). The percentage of open arm entries and percentage of open arm time were used as indices of anxiety-like behavior. The percentage of open arm entries was calculated as the number of open arm entries divided by total entries (open arm entries plus closed arm entries). The open arm time percentage was calculated as the amount of time spent in open arms divided by the total amount of time spent in both open and closed arms. An anxiety index ranging from 0 (low anxiety) to 1 (high anxiety) was also calculated based on the following formula [20 (link)]: anxiety index = 1 − [(time spent in open arm/test duration + number of entries to the open arms/total number of entries)/2].
Smart v3
Manufactured by Harvard Apparatus
Sourced in Spain, United States, Germany
The SMART v3.0 is a versatile lab equipment product designed for various experimental applications. It features a compact and durable design, with a digital display and intuitive controls. The SMART v3.0 is capable of performing a range of functions, but a detailed description of its core function is not available at this time.
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121 protocols using smart v3
1
Elevated Plus Maze Anxiety Assessment
2
Exploring Rodent Locomotor Activity
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Open Field tests were done in a wooden box (Open Field arena) of dimensions: 50 cm × 50 cm × 45 cm in which the internal surface was coated with white polish. The Open Field arena was custom made in JNCASR. Mice were trained in an Open Field arena for two consecutive days prior to the day of actual tests. During training or tests, a single mouse was left in the Open Field arena for 5-min to explore. The activity of mouse was recorded with the help of a digital camera (SONY® color video camera, Model no. SSC-G118) supported by the software SMART v3.0.04 (Panlab Harvard Apparatus, Holliston, MA, USA). At the end of 5-min, the mouse was removed and returned to its home cage. The Open Field arena was cleaned with 70% ethanol and dried before placing the next mouse in it. The distance traveled in the zone periphery was calculated by SMART v3.0.04 software (Panlab Harvard Apparatus, Holliston, MA, USA). The data were compared, analyzed and represented as bar diagrams by using the software GraphPad prism5.
3
Automated Open Field Test for Rat Depression
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The rats were placed individually in an unfamiliar open field box (80 cm × 80 cm × 60 cm) and allowed to freely explore for 5 min (Meng et al., 2023 (link)). Their spontaneous activity within the past 5 min was recorded using an automated video tracking system (Smart V3.0, Panlab). The locomotor activity of the rats was defined as a measure of depression. Lower activity indicated higher levels of depression. To eliminate the odor left by previous rats, their feces and urine were cleaned with 75% alcohol before each rat underwent the open field test (OFT).
4
Open Field Maze Behavior Analysis
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The mice were placed in the center of an open field maze (50 × 50 × 50 cm), and their movements were recorded using a video camera; behaviors, total distance, and time in the center and periphery zones were tracked for 5 min using the SMART video tracking system (SMART v3.0, Panlab SL, Barcelona, Spain).
5
Elevated Plus Maze for Anxiety Evaluation
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Anxiety-like behaviors were measured in the EPM. The maze was elevated 50 cm above the floor and consisted of a central part (5 cm × 5 cm), two opposing open arms (30 cm × 5 cm), and two opposing closed arms (30 cm × 5 cm) with a 15 cm height. Each animal was placed in the central part of the maze facing an open arm and allowed free exploration for 5 minutes under ambient room light. The maze was cleaned with 75% ethanol and dried between usage. All processes were videotaped, and the number of entries to open and closed arms and the time spent in each arm were measured by the SMART video tracking system (SMART v3.0, Panlab, Spain). The lower percent of time and entries in the open arm indicates more anxiety-like behavior in rats.
6
Evaluating Animal Locomotion and Behavior
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An open-field test was designed to evaluate our animals’ basal activity and its change across time in response to our pharmacological treatments in accordance with the procedure described in a previous study [22 (link)]. Video tracking with recording was conducted under dim lighting condition to examine the movement pattern within the peripheral and central zones in an open field for 30 min. Movement patterns were quantified based on the distance traveled and time spent in each zone using a video tracking system (SMART V3.0; Panlab, Barcelona, Spain).
7
Spatial Learning and Memory Assessment
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After 14 days of continuous feeding, the mice were brought to the testing room to adapt to the environment for 30 min before the behavioral test. The Morris water maze (MWM) test was performed with minor adjustments as previously described. The test consisted of 5 days of learning trials and a 1-day probe test. The mice were given 1 min to find the platform. If a mouse found the platform within 1 min and stayed for 3 s, we considered the mouse to have successfully found the platform and ended the experiment. Otherwise, something was used to guide the mouse to the platform and stay for 20 s. In the probe test, the platform was removed, and the mice were allowed to swim for 1 min. All mouse movements were recorded and analyzed by Smart v3.0 (Panlab, Spain), a computerized tracking system.
8
Open Field Test for Anxiety Assessment
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Exploratory behavior and emotional reaction were observed in the OFT. The OFT was conducted based on our laboratory procedure which was published in a previous study [25 (link)]. The apparatus (90 cm × 90 cm × 45 cm) was divided into 25 equal squares. Rats in each group were put in the center of the open-field and observed for 5 minutes. The duration of time spent in the center squares (squares except peripheral squares along the side), line crossings (frequency with which the rat crossed one of the grid lines with all 4 paws), frequency of grooming (the number of times when the rat spent licking or scratching itself while stationary), and frequency of rearing (frequency with which the rat stood on their hind legs on the ground) were recorded for 5 minutes by the SMART video tracking system (SMART v3.0, Panlab, Spain). The low frequencies of the above behaviors indicated low levels of locomotion and exploration but high level of anxiety.
9
Anticonvulsant Effects of Duvelisib in Rat PTZ Model
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The animals were randomly assigned into 4 groups via simple randomization (n = 5/group) as follow:
Over the course of the study, fresh solutions of vehicle, phenobarbital, and duvelisib were injected intraperitoneally (IP) in a constant volume of 4 mL/kg. The doses of PTZ, PHB, and DUV were chosen based on previous studies on experimental animal models.22 (link)
-24 (link, link) Also, the prolonged administration of DMSO via IP route at this concentration did not result in any detrimental physiological effects as evidenced in previous studies.25 (link),26 (link) An upper cut-off time of 30 minutes was used in measuring behavior after each rat had been exposed to PTZ. PTZ convulsive behavior was graded on a 5-point scale based on Racine’s stages as, Stage 1: ear and facial twitching; Stage 2: head nodding and myoclonic jerks; Stage 3: unilateral forelimb clonus with lorditic posture; Stage 4: bilateral forelimb clonus with rearing and falling; and Stage 5: generalized tonic-clonic seizure (GTCS) with loss of postural tone.27
Video of rat seizures was captured using camera in the center of the chamber and software (SMART V3.0, Panlab, Barcelona). The latency to each stage (S1-5),27
number of myoclonic jerks, and GTCS duration was also recorded in a blind fashion with help of 2 independent observer.
Over the course of the study, fresh solutions of vehicle, phenobarbital, and duvelisib were injected intraperitoneally (IP) in a constant volume of 4 mL/kg. The doses of PTZ, PHB, and DUV were chosen based on previous studies on experimental animal models.22 (link)
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Video of rat seizures was captured using camera in the center of the chamber and software (SMART V3.0, Panlab, Barcelona). The latency to each stage (S1-5),27
number of myoclonic jerks, and GTCS duration was also recorded in a blind fashion with help of 2 independent observer.
10
Spatial Memory and Learning in Rats using the Morris Water Maze
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The MWM was used to determine the spatial memory and the learning ability of rats (37 (link)). The apparatus was a circular black tank (120 cm diameter and 50 cm height) and was filled with warm water (22°C). The software assigned four quadrants to the surface of the water. The platform (13 cm diameter and 29 cm height) was placed at the center of the target quadrant (quadrant II) and was 1–2 cm below the surface of the water. The movement of each rat was tracked and recorded using the SMART video tracking system (SMART v3.0, Panlab, Spain). A training period was carried out for 5 consecutive days, four times a day. The rats were gently placed into a random quadrant facing the wall and were allowed to swim freely to find the platform within 60 s, followed by a 20-s reprieve on the platform. Rats that failed to find the platform were manually placed on the platform for a 20-s break. The sixth day was the testing period; the rats were placed into quadrant IV and allowed to swim freely for 60 s in the maze without the platform. The number of across the target quadrant and the platform were recorded.
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