Introduction 

The Zantiks AD unit is an automated system that is ideal for measuring the behavior of small fish and small rodents. It includes two versions, one for aquatic and one for terrestrial animals. Model organisms used with this system include zebrafish, mice, and bees. It is a fully integrated behavioral testing environment that can perform live video tracking and recording of animal behavior. The Zantiks AD unit is suitable for various experimental protocols, including tracking fish and mice in operant tasks with food rewards, tracking fish in experimental environments such as an Open-Field Arena or Y-Maze, and tracking multiple singly housed fish for paradigms such as Conditioned Place Preference or Pavalonian Avoidance Learning. 

It consists of a computer, camera, overhead lights, a screen for visual stimuli, and a motor for food delivery. Both solid and liquid food can be delivered as well as chemical liquid stimuli. It can easily be modified according to experimental needs; for example, the testing environment can be split up into multiple arenas by adding tank inserts for tracking individual subjects easily. Each Zantiks AD unit comes with a router and Zantiks software controlled via a web browser. Therefore, users can operate it using their computer, laptop, tablet, or phone. Experimenters can use the behavioral data recorded to easily understand individual subjects’ behavior and compare behaviors within treatment groups. 

Since the Zantiks AD unit is automated, little experimental intervention is needed. Although the included equipment for each version allows extensive research to be performed, add-on equipment such as a Y-Maze insert for fish or a side screen can also be provided. 

Equipment 

Each Zantiks AD unit has an overall external unit size of approximately 220 mm in width, 300 mm in length, and 500 mm in height. A tank for fish or cage for rodents is placed inside the external unit that measures 140 mm in width, 200 mm in length, and 150 mm in height. Each unit is integrated and includes a computer, camera, overhead lights, screen for visual stimuli, and a motor for food delivery. The motor can operate a peristaltic pump to deliver liquid food or solid food. Apart from the food delivery, which serves as a stimulus, the overhead illumination and visual screen also serve as stimuli. The overhead illumination’s brightness, color, and duration can be specified with ms accuracy. It can be used for circadian rhythm entrainment, ambient lighting, and light flashes. The visual screen is placed underneath the tank and supplies visual stimuli specified with colors, shapes, sizes, locations, and bitmaps. Moreover, the visual stimuli’ update times and response times can be controlled up to the 60Hz refresh rate of the display. 

The Zantiks AD unit also includes a 12V power cable, Ethernet cable, and router. The first unit is supplied with a router, and subsequent units can be networked to the same router. The unit also comes with the Zantiks software called Zanscript. 

Included Equipment for Fish 

A tank with a mesh lid, opaque white sides, and a clear bottom. A mild shocking stimulus with controllable duration and voltage is present in the tank comprising two stainless steel shocking plates with cable and testing LED. The tank can be supplied with both solid and liquid food. Solid food is delivered via a brass feeder mechanism that attaches to the motor. On the other hand, liquid food is delivered via a peristaltic pump that also attaches to the motor. Silicone tubing (2 mm internal diameter and 4 mm external diameter) is recommended for the pump. The external light with an extra connector is also included with the tank. 

The following tank inserts can be added to create multiple arenas for fish:

1. Arena widthwise dividing inserts × 3
2. Arena lengthwise dividing inserts × 3

The arena dividers can create up to four compartments for tracking four separate fish using one unit.

1. CPP widthwise inserts × 2
2. CPP lengthwise inserts × 2

The CPP inserts create four separate arenas for tracking four separate fish in each arena using one unit. 

1. 5-aperture insert × 1 – It creates five distant entry points at the opposite side of food delivery in the tank.
2. Light-dark inserts × 1 – It creates a dark box within the tank and divides it into a light and dark compartment. A gap is present at the bottom of the insert to allow fish to move between light and dark zones. 
3. Novel tank diving inserts × 1 – It includes a tank, a funnel, a stand, and a foam door. 
4. Food magazine insert × 1 – It creates an area for the fish to enter and eat the food reward. It also creates an entry point to initiate trials for discrimination tasks. 

A two Y-maze insert can also be provided to create a testing environment for two separate fish using one unit. Additionally, aside screen for the lateral presentation of images on the front and back wall of the unit can also be added. The side screen operates in the same way as the built-in screen under the tank. It is supplied with a black tank with a clear base and a back wall. The back screen can be split into two different zones by a two-choice insert. 

Included Equipment For Mice 

A cage measuring 150 mm in height, 200 mm in depth, and 140 mm in width with a lid, clear bottom, and opaque white slides. A pellet feeder is present that attaches to the motor to deliver solid food. Additionally, liquid food can also be delivered via a peristaltic pump unit that also attaches to the motor. Similar to the fish version, silicone tubing (2 mm internal diameter and 4 mm external diameter) is also recommended. External light with an extra connector is also included with the cage. 

 The cage can also be divided into multiple arenas using the following inserts

1. 5-aperture insert x 1
2. Food magazine insert x1
3. Two-choice insert x 1

Experiment and Set-up

The Zantiks AD unit can be used for various studies, including Aversion learning, Reversal learning, 5-choice serial reaction time task (5-CSRTT), Y-Maze, Novel tank diving, and  Conditioned place preference. 

Each Zantiks AD unit is supplied with a router and controlled via a web browser. Therefore, users can use their computer, laptop, tablet, or phone to control it. The Zantiks AD unit needs to be connected to the same network as the user’s laptop or phone to operate. The unit’s IP address (displayed on the AD’s screen after turning on the unit or available on the delivery note) must be entered in the browser to open the Zantiks Control Console. All the networked units can then be operated from the home page. The web browser can be used to view live video, start and stop chosen services, edit scripts, and download results in .csv format. 

The Zantiks unit is set up by following the procedure below:

1. Place the AD unit on a stable surface away from external disturbances and near a power socket. 
2. At the back of the Zantiks unit, plug the Ethernet cable and then plug it into a slot in the LAN section of the router. 
3. In the back socket of the unit, below the Ethernet cable, insert the 12V power supper cable. Plug the cable into the mains socket. 
4. Turn on the mains socket of the Zantiks unit. Turn on the router. Wait for the text screen to load and scroll across the screen after the unit powers on. 
5. After the screen is turned on, the unit’s IP address will display on it, which is used to identify the unit of the Zantiks Control Console. 
6. On a browser-enabled device 
   1. Go to the network settings and connect to the “Zantiks” wifi network
   2. Type in the network password. The initial password is set up as: behavior
   3. Type in the IP address of the Zantiks unit on the address bar of the web browser
   4. The web browser should display the Zantiks Control Console page

Data Analysis 

The Zantiks AD unit can perform tracking and real-time video recording of small fish and rodents. The unit uses the Zanscript software, which can be used to make scripts or use script templates. The scripts deliver real-time processed results that can be downloaded as .csv files from the unit. The following parameters can be collected using the Zantiks AD unit:

1. Time spent in arenas/zomes
2. Number of times the subject entered a zone
3. Distance moved by the subject 

Strengths and Limitations 

The Zantiks AD unit can effectively measure the behavior of small fish and rodents. It generates a live video view and provides real-time video recording, allowing animal behavior to be easily analyzed. It enables high throughput and easy-to-run experiments for protocols such as Conditioned Placed Preference or Pavalonian Avoidance Learning. The unit includes different equipment specific to fish or rodents. It has an integrated camera, experimental chamber, computer, and built-in stimuli. Moreover, inserts to separate the unit into multiple arenas can also be provided, or add-on equipment such as Y-Maze inserts can also be purchased. Each unit is supplied with a router that networks the unit and allows it to be controlled via the web browser of any device. The unit is compact and takes up little bench space. Although the Zancscript software includes several template scripts that can be used with experiments, users will need to learn the Zanscript language to make their own scripts. 

Summary and Key Points 

1. The Zantiks AD unit is an automated system that is ideal for measuring the behavior of small fish and small rodents.
2. It is an integrated unit and includes a camera, experimental chamber, built-in stimuli, and a computer. 
3. It allows live tracking and real-time video recording of animal behavior.
4. It uses Zanscript software that uses scripts to deliver real-time processed results. 
5. It can be used for various studies, including Reversal learning, Aversion Learning, and Conditioned place preference.