Behavioral Studies

This test was performed as described by Porsolt et al³⁹ with the modifications proposed by Detke et al.⁴⁰ On the first day of the experiment, each animal was gently placed in Plexiglas cylinders (40-cm high, 18-cm in diameter) containing 30 cm of water, at a temperature of 23–25°C, for 15 min. On removal from water, the rat was placed for 30 min in a Plexiglas box under a 60-W bulb to dry. On the following day (24 h later), the rat was placed again in the cylinder and the total duration of immobility, swimming, and climbing was recorded during the 5-min test period. The swimming behavior entailed active swimming motions, eg, horizontal movement around the cylinder. The climbing activity included upward-directed movements of the forepaws of the rat along the side of the swim chamber. The immobility was assigned when the rat showed no additional activity other than that necessary to keep its head above the water. Fresh water was used for each animal.

This test was performed in a dark room using Motor Monitor System (Campden Instruments, Ltd., UK) consisting of two Smart Frame Open Field stations (dimensions 40×40×38 cm) with 16×16 beams, located in sound-attenuating chambers and connected to PC software by control chassis. Each vehicle- or drug-injected animal was gently placed in the center of the station. The total distance covered by the animal in 5 min was recorded by the automated Motor Monitor System.

This test was performed as described by Pellow and File.⁴¹ The plus-maze apparatus (Campden Instruments Ltd., UK), an automated device used for this experiment, was made of durable, high-density, non-porous black plastic, elevated to a height of 50 cm. It consisted of two open arms (50×10 cm) and two closed arms (50×10 cm, and 30-cm high walls), arranged in such a way that the two arms of each type were opposite to each other. The floor of the plus-maze was made of an infrared transparent material, and thus were no visible sensors. The apparatus was connected to PC software by control chassis. The experiment was conducted in a dark room, with only the center of the maze illuminated with a low-intensity light (30 lx measured at the maze level). During the experiment, each rat was gently placed in the center of the plus-maze, facing one of the closed arms, immediately after 5-min adaptation in a plastic black box (dimensions 60×60×35 cm), to increase its overall activity in the EPM. During a 5-min test period, the number of entries of the rat into the closed and open arms and the time it spent in either type of arms were measured by an automated Motor Monitor System. The device counted an effective arm-entry when the four paws of the rat were into any arm. The maze was thoroughly cleaned after each trial. The number of open-arm entries of the rat, the total time spent by the rat in the open arms, and the percentages of these parameters were used as indications of anxiolytic-like activity.

To assess the influence of the tested compound on the general exploratory activity of rats and control possible changes within, the total number of entries (into open and closed arms) and the total distance covered by the rats during a 5-min test period (ie, the time equal to the observation period in the EPM test) were measured. The experiment was performed using the EPM apparatus described above.

This test was performed as described by Vogel et al⁴² using Anxiety Monitoring System “Vogel test” (TSE Systems). The apparatus consisted of a polycarbonate cage (dimensions 26.5×15×42 cm), equipped with a grid floor made of stainless steel bars and a drinking bottle containing tap water. The two experimental chambers were connected to PC software by control chassis and a device that generates electric shocks. In the “conditional” model, an electric shock was applied as a noxious stimulus. The testing procedure consisted of a 2-day habituation/adaptation and the exact test. On the first day of the experiment, the rats were adapted to the test chamber for 10-min (adaptation period), during which they had free access to the drinking bottle, followed by a 24-h water deprivation period. Afterward, the rats were allowed free access to water for 30-min (free-drinking session) in their home cages. This protocol of a 24-h deprivation and adaptation period was repeated on the second day. On the third day, the animals were placed again in the test chamber 60 min after the administration of the vehicle or the tested compounds and were allowed free access to the drinking bottle for 5 min. Recording of data started immediately after the first lick, and after every 20 licks, the rats were punished with an electric shock (0.5 mA, lasting 1 s). The impulses were released via the spout of the drinking bottle. The number of shocks received by an animal during the 5-min experimental session was recorded automatically and used as an indication of the anti-conflict activity.

To exclude the possibility of drug-induced changes in shock sensitivity or an increasing influence on thirst drive which can lead to false-positive results in the Vogel conflict drinking test, stimulus threshold and water consumption during the free-drinking session were determined for separate groups of rats. In both studies, the rats were manipulated similarly to the Vogel conflict drinking test, including two 24-h water deprivation periods separated by 10-min adaptation session in experimental cages and 30-min of water availability in their home cages. In the free-drinking test, each animal was allowed to drink freely from the drinking bottle and the amount of water (g) consumed during 5 min was recorded. The pain threshold was evaluated in rats using the hot plate test.⁴³ The plate (Commat Ltd, Turkey) was enclosed in a transparent Plexiglass cylinder (35-cm high) to keep the animal on the heated surface of the plate. The latency to pain reaction (licking of a hind paw or jumping) was measured when the rat was placed on the hot plate (52.5±0.5°C, 19-cm diameter). The rat was removed from the plate immediately upon a visible pain reaction or if no response was observed within 30 s.