Introduction
Fish have nociceptors with properties that are very similar to those found in mammals. fish respond to noxious stimulus in a variety of ways depending on the stimulus and fish species. This response is referred to as a nocifensive response. Immersion anesthesia in fish is analogous to gaseous inhalant anesthesia in terrestrial animals. The fish ventilates the anesthetic dissolved in the water, which enters the bloodstream mainly through the gills. A variety of anesthetic drugs have been investigated for their properties to reduce the nocifensive responses. There are few studies investigating or demonstrating antinociceptive activity of drugs in fish. Nefopam is believed to work in the brain and spinal cord to relieve pain. Firstly it increases the activity of the serotonin, norepinephrine and dopamine, neurotransmitters involved in pain signaling. Secondly, it modulates sodium and calcium channels, thereby inhibiting the release of glutamate, a key neurotransmitter involved in pain processing. Our hypothesis was that a noxious stimulus decreases zebrafish swimming activity and the subsedative doses of Nefopam will prevent this behavioral change after the fish are no longer exposed to Nefopam.

Methods
Large adult zebrafish (Danio rerio) were used. Each fish was placed individually in a 1.6 L video arena (17 x 8 x12 cm). The behavioral parameters in untreated fish (pre-exposure) was recorded for 35 seconds. Online records were stored on the computer and later analyzed for activity. Swimming distance was calculated as the change in position in two dimensions, and velocity as this distance per time. Median velocity, freeze time, high-speed swimming, and vertical distance. Each fish was immediately anesthetized with lidocaine and treated with an injection of 5% acetic acid (noxious stimulus). The injection was 5 μL into the face, between the nostrils, using a Hamilton syringe. Each fish was transferred to a 100 mL beaker containing Nefopam in tank water (0, 1, 2, 5, or 10 mg/L) where it was exposed to the drug for 45 minutes. Five minutes after exposure of Nefopam, each fish was transferred to the video arena, and swimming behavior was recorded (post-exposure) for 35 seconds. Exactly 30 seconds of the approximate 35 second video clip was analyzed (n=9).

Results
The noxious stimulus resulted in a decrease in median velocity, vertical distance and high-speed swimming, and an increase in freeze time. The decrease swimming behavior was recovered with postexposure of Nefopam 5 and 10 mg/L.

Conclusions
The change in the behavior of zebrafish associated with a noxious stimulus can be monitored and is a good model for studying anti-nociceptive effect in fish. The response after a noxious stimulus was recovered with postexposure of Nefopam 5 and 10 mg/L. Thus we could recommend the use of Nefopam as an anti-nociceptive drug.