An automated evaluation system for analyzing antinociceptive effects on intracolonic capsaicin-induced visceral pain-related licking behavior in mice.

INTRODUCTION: The development of automated detection systems for animal behaviors is increasing in value in terms of saving time, objective analysis and reducing the need for well-trained experimenters. SCLABA(®) (Noveltec Inc., Kobe, Japan) is a commercially available analysis system originally developed for analyzing scratching behaviors in rodents, based on distances between points in videotaped images. Here, we used this software to automate analysis of abdominal licking behavior associated with visceral pain in mice. METHODS: Yellow and green spots were applied to the snout and the lower abdominal region of mice respectively to provide reference points for automated analysis of video recordings. Abdominal licking behavior after intracolonic administration of 0.3% capsaicin solution as a measure of visceral pain was determined based on changes in the inter-spot distance. RESULTS: A distance threshold between the colored spots was chosen based on manual measurements showing that 99% of minimal distances were below this threshold. Using this threshold, the number of licks determined by the automated analysis significantly and positively correlated with that determined by manual observation (R(2)=0.95 and p<0.001). The neurokinin-1 receptor antagonist GR205171A dose-dependently inhibited capsaicin-induced licking detected by automated analysis. DISCUSSION: We demonstrated that visceral pain-related licking behaviors after intracolonic capsaicin treatment can be automatically detected by applying commercially available image analysis software. This automated experimental system is very efficient and useful to evaluate antinociceptive effect of a test compound on visceral pain.

Automated experimental system capturing three behavioral components during murine forced swim test.

AIMS: An automated experimental system applying a commercially available video image analyzer was developed for the simultaneous detection and measurement of three behavioral components; immobility, swimming (horizontal movements) and climbing (vertical movements) that occur in the murine forced swim test (FST). The system was validated using four typical antidepressants. MAIN METHODS: System validity was confirmed by demonstrating no significant difference in 6 min time course of control group and imipramine-dosed group (30 mg/kg) between manual examinations and automated digital analysis for all the three behaviors (i.e., correlation coefficients were 0.96, 0.83 and 0.94 for immobility, swimming and climbing, respectively). The effects of acute single treatment with four antidepressants in clinical use, i.e., imipramine, desipramine, bupropion and fluvoxamine were evaluated at doses of 15, 30 and 60 mg/kg using the system. KEY FINDINGS: In 2-4 min time span analysis, all four antidepressants reduced immobility and increased climbing significantly, desipramine and bupropion increased swimming significantly, while imipramine and fluvoxamine did not. SIGNIFICANCE: The automated experimental system enabled efficient and accurate analysis of the three murine behaviors during FST at once. Climbing could be more sensitive parameter to detect anti-depressant-like effect than immobility in this system.

Pharmacologically distinctive behaviors other than burying marbles during the marble burying test in mice.

In the marble burying test, we focused on the 5 distinctive behavioral parameters of mice other than burying marbles, i.e. digging, latency to the first digging, exploration around marbles, rearing and locomotor activity. Typical anxiolytics or antidepressants with different mechanisms, fluvoxamine (30 mg/kg, selective serotonin reuptake inhibitor), bupropion (60 mg/kg, noradrenaline and dopamine reuptake inhibitor), imipramine (60 mg/kg, tricyclic antidepressant) and diazepam (10 mg/kg, benzodiazepine) were used to examine whether these behavioral parameters are sensitive to pharmacological treatments. Each of the drugs demonstrated an individual action pattern on the 4 behavioral parameters (latency to the first digging, exploration around marbles, rearing and locomotor activity). On the other hand, all 4 drugs reduced burying marbles and digging, which were correlated with each other. These results suggest that the former 4 behavioral parameters are sensitive to pharmacological treatment and that pharmacological regulation mechanisms of them may be different from burying marbles and digging. They could be useful to identify the type of action of a test drug like selective serotonin reuptake inhibitor, noradrenaline and dopamine reuptake inhibitor, tricyclic antidepressant or benzodiazepine.

Optimized analysis of the forced swim test using an automated experimental system: detailed time course study in mice.

INTRODUCTION: In most publications on the forced swim test studies, duration of immobility is measured during the last 4 min of the 6-min testing period as a marker for depression. However, it is not clear if 4-min span best captures antidepressant-like drug effects. In the present study, six typical antidepressants in clinical use were evaluated over time. Imipramine (60 mg/kg, PO), desipramine (60 mg/kg, PO), reboxetine (20 mg/kg, IP), bupropion (60 mg/kg, PO), fluoxetine (20 mg/kg, PO) and fluvoxamine (60 mg/kg, PO) were characterized over 1-min intervals for the 6-min testing period in order to identify more sensitive periods than the last 4-min span to detect drug effects, using the automated experimental system. METHODS: One day before the testing, wire rings were attached to the hind paws of male CD-1 mice. On the test day, after attaching a small magnet to the wire rings, each animal was placed for 6 min in a glass cylinder filled with water, which is surrounded by a coil. The duration of immobility was measured by the previously validated automated detection system, MicroAct, i.e. electrical signals generated by limb movements served as the marker for swimming behavior. RESULTS: In the 1-min interval examination, it was only in 2-3-min and 3-4-min spans that all of the six antidepressants reduced the duration of immobility with statistical significance. In the 2-4-min analysis, all of the six antidepressants demonstrated statistically significant reduction in the duration of immobility, while in the 2-6-min analysis, the reduction by fluvoxamine was not statistically significant. DISCUSSION: The detailed time course analysis of the FST in mice using the automated system revealed that the duration of immobility between 2 and 4 min is the optimum period to capture the antidepressant-like effect of test compounds.

Inhibition of phosphodiesterase type 4 decreases stress-induced defecation in rats and mice.

BACKGROUND/AIMS: Phosphodiesterase type 4 (PDE4) has been previously shown to regulate colonic contractile activity in vitro. In this study, the effects of PDE4 inhibition were assessed in a model of stress-induced defecation previously demonstrated to be due to increased colonic transit/evacuation. METHODS: Rats were individually placed in a mild restraint cage and placed into a 12 degrees C environment (cold-restraint stress) for 60 min. Mice received restraint (only) stress at room temperature for 30 min. Loperamide (positive control compound) or two different PDE4 inhibitors (rolipram and roflumilast) were administered orally at several doses to the rodents 1 h before stress began. Vehicle alone was administered for comparison. The number of fecal pellets expelled during stress (fecal pellet output), total fecal pellet wet weight and total fecal water content were measured. RESULTS: Loperamide produced a dose-related decrease (ID(50)s in mg/kg) in fecal pellet output (rat = 7.4, mouse = 0.7) and significantly decreased fecal wet weight (72.9%) and decreased fecal percent water content (9.4%). The two PDE4 inhibitors produced a similar dose-related inhibition of fecal pellet output. Rolipram exhibited ID(50)s in rat and mouse of 14.1 and 27.1, respectively. Rolipram significantly decreased fecal wet weight (58.8%) but increased fecal percent water content (15.0%). For roflumilast, ID(50)s were 24.2 mg/kg and 12.4 in the rat and mouse, respectively. Although roflumilast also significantly (p < 0.05) decreased fecal wet weight (47.2%), it did not significantly increase fecal percent water content. CONCLUSIONS: These data indicate that PDE4 inhibition is effective in reducing rodent stress-induced defecation, provides the first functional data on a potential role for PDE4 activity in the colonic evacuation response to stress, and indicates the potential utility of PDE4 inhibitors in functional bowel disease such as irritable bowel syndrome requires further evaluation.

A new automated and high-throughput system for analysis of the forced swim test in mice based on magnetic field changes.

INTRODUCTION: MicroAct (Neuroscience, Tokyo, Japan) was originally developed as a new system for an automated analysis of scratching behaviors in mice. This system is based on the detection of electric current in the coils according to the movement of the magnets implanted in the hind paws. We applied and improved this system to establish an automated analysis system of forced swimming behaviors in mice, which is used as an animal model of depression. METHODS: One day before the test, male CD-1 mice were attached with a wire ring to their hind paws under inhalation anesthesia. After attaching a small magnet to both of the wire rings, each animal was placed for 6 min in a glass cylinder filled with water, which is surrounded by a coil. The swimming behaviors of the mouse were analyzed for the measurement of duration of immobility, a major marker of depression in rodents, by the detection system (MicroAct). The duration of immobility was also determined by manual measurement using the swimming behavior-recorded videotapes produced at the same time as the automated analysis. RESULTS: The difference of the duration of immobility between naïve mice and mice with the rings was not significant. The dose-response effect of imipramine (tricyclic antidepressant, 0, 7.5, 15, 30 and 60 mg/kg, p.o.) on the duration of immobility in the last 4-min of the 6-min testing period determined by MicroAct was similar to that assessed by the manual measurement. These data from the two different methods were significantly correlated (r=0.8805). Moreover, throughput of the automated analysis was 15 times more efficient than that of the manual analysis. DISCUSSION: These results suggest that the automated analysis system of forced swimming of mice using MicroAct can be used as a high-throughput method to examine antidepressive activity of a compound with objectivity and reliability.

Kv7.2-7.5 voltage-gated potassium channel (KCNQ2-5) opener, retigabine, reduces capsaicin-induced visceral pain in mice.

K(v)7.2-7.5 voltage-gated potassium channels (KCNQ2-5) are associated with M-current and known to distribute in the nociceptive sensory pathway (e.g., dorsal root ganglia and spinal cord). Opening of these channels leads to cell membrane hyperpolarization that results in decreased neuronal action potentials. Since, KCNQ/M-current is located in the visceral sensory system, we examined the anti-nociceptive effect of the KCNQ opener, retigabine, on visceral pain induced by an intracolonic injection of capsaicin in mice. Intraperitoneal administration of retigabine (1, 3 and 10 mg/kg) dose-dependently suppressed visceral pain behavior (i.e., the number of licking) induced by the capsaicin treatment and prolonged the latency to first licking. These data provide the first evidence that increased KCNQ channel conductance plays an inhibitory role in the visceral pain pathway.