The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed. Video LinkThe video component of this article can be found at http://www.jove.com/video/3638/ Protocol 1. Materials and Method The water tanksThe cylindrical tanks (30 cm height x 20 cm diameters) required for the mouse forced swim test (FST) in our laboratory are constructed of transparent Plexiglas, as this material is able to withstand the frequent movement of the tanks and accidents better than glass. The water level is 15 cm from the bottom and should be marked on the tank to ensure that the volume of water is consistent across mice. The number of tanks should ideally be at least twice as many as the number of mice being tested at a time, so that the second water tank set can be filled while the first set is in use. The dimensions of the tanks should be selected in a way that the mice will not be able to touch the bottom of the tank, either with their feet or their tails, during the swimming test. The height of the tank should be high enough to prevent the mice from escaping from the tank. Please note that the diameter of tank and the depth of water are important parameters that can be adjusted to change the behavior of mice (for a detailed analysis of these issues see 1-3 ). ThermometerA water resistant infrared thermometer is preferable, since rapid measurement of temperature reduces the amount of time required to conduct the test. However, a glass mercury thermometer will also be sufficient for this task.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

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Following administration at subanesthetic doses, (R,S)-ketamine (ketamine) induces rapid and robust relief from symptoms of depression in treatment-refractory depressed patients. Previous studies suggest that ketamine's antidepressant properties involve enhancement of dopamine (DA) neurotransmission. Ketamine is rapidly metabolized to (2S,6S)-and (2R,6R)-hydroxynorketamine (HNK), which have antidepressant actions independent of Nmethyl-D-aspartate glutamate receptor inhibition. These antidepressant actions of (2S,6S;2R,6R)-HNK, or other metabolites, as well as ketamine's side effects, including abuse potential, may be related to direct effects on components of the dopaminergic (DAergic) system. Here, brain and blood distribution/clearance and pharmacodynamic analyses at DA receptors (D 1 -D 5 ) and the DA, norepinephrine, and serotonin transporters were assessed for ketamine and its major metabolites (norketamine, dehydronorketamine, and HNKs). Additionally, we measured electrically evoked mesolimbic DA release and decay using fast-scan cyclic voltammetry following acute administration of subanesthetic doses of ketamine (2, 10, and 50 mg/kg, i.p.). Following ketamine injection, ketamine, norketamine, and multiple hydroxynorketamines were detected in the plasma and brain of mice. Dehydronorketamine was detectable in plasma, but concentrations were below detectable limits in the brain. Ketamine did not alter the magnitude or kinetics of evoked DA release in the nucleus accumbens in anesthetized mice. Neither ketamine's enantiomers nor its metabolites had affinity for DA receptors or the DA, noradrenaline, and serotonin transporters (up to 10 mM). These results suggest that neither the side effects nor antidepressant actions of ketamine or ketamine metabolites are associated with direct effects on mesolimbic DAergic neurotransmission. Previously observed in vivo changes in DAergic neurotransmission following ketamine administration are likely indirect.

Currently approved antidepressant drug treatment typically takes several weeks to be effective. The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine has shown efficacy as a rapid-acting treatment of depression, but its use is associated with significant side effects. We assessed effects following blockade of the glycine B co-agonist site of the NMDA receptor, located on the GluN1 subunit, by the selective full antagonist 7-chloro-kynurenic acid (7-Cl-KYNA), delivered by systemic administration of its brain-penetrant prodrug 4-chlorokynurenine (4-Cl-KYN) in mice. Following administration of 4-Cl-KYN, 7-Cl-KYNA was promptly recovered extracellularly in hippocampal microdialysate of freely moving animals. The behavioral responses of the animals were assessed using measures of ketamine-sensitive antidepressant efficacy (including the 24-hour forced swim test, learned helplessness test, and novelty-suppressed feeding test). In these tests, distinct from fluoxetine, and similar to ketamine, 4-Cl-KYN administration resulted in rapid, dose-dependent and persistent antidepressant-like effects following a single treatment. The antidepressant effects of 4-Cl-KYN were prevented by pretreatment with glycine or the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). 4-Cl-KYN administration was not associated with the rewarding and psychotomimetic effects of ketamine, and did not induce locomotor sensitization or stereotypic behaviors. Our results provide further support for antagonism of the glycine B site for the rapid treatment of treatment-resistant depression without the negative side effects seen with ketamine or other channelblocking NMDA receptor antagonists.