"Nitric oxide (NO) is a gaseous, freely diffusible free-radical which functions as an intercellular signaling molecule in the central nervous system (CNS). ... NO is produced mainly in postsynaptic structures as a response to the activation of the NMDA receptors an activates soluble guanylyl cyclase, causing an increase in cGMP concentration" (da Silva, et. al., 2000). Experimental evidence has also shown that NO is involved in other neuronal functions that include synaptic plasticity which includes long-term potentiation, long-term depression, and learning and memory.

As of 2000, most studies done have focused on the effects of NO by changing the enzyme NO synthase (NOS), through the use of NOS synthase inhibitors. The findings from the study done by D. Jefferys, and J. Funder (1996), A. Harkin et. al. (1999), and F. Yildiz et. al. (2000) (as cited by da Silva et. al., 2000) showed that "NOS inhibitors L-NNA, L-NAME, and 7-nitroindazole exhibit antidepression-like properties in the forced swimming test (FST), which are blocked by pretreatment with the L-Arg".

The animals used in this experiment were male Swiss mice between 30-35g. They had free access to food and water and were kept on a 12:12 hour light:dark cycle. The drugs tested were L-Arg, D-Arg, D-NNA, and L-NNA. They were dissolved in saline and given to the mice in a volume of 10 ml/ kg of body weight 30 minutes before the FST or the TST. The control mice received only the saline solution.

"In the FST, mice are forced to swim in a restricted space from which there is no escape, and will, after periods of agitation, cease attempts to escape and become immobile. ... It measures the ability of antidepressants to reduce the occurrence of immobility after exposure to swimming stress" (da Silva et. al., 2000). According to R. Porsolt, A. Bertin, and M. Jalfre (1977) (as cited by da Silva et. al., 2000), "The FST is one of the most frequent behavioral models used to evaluate compounds with potential antidepressant activity". In this experiment, mice were individually forced to swim in an open cylindrical container that has a diameter of 10 cm, a height of 25 cm, and water filled to 19 cm. Immobility was judged when the mouse stopped struggling and floated in the water, only making movements that would allow it to keep it's head above water. Immobility was measured for 6 minutes.

In the TST, mice were isolated from sound and sight and suspended 50 cm above the ground by tape placed about 1 cm from the tip of the tail. Immobility time was recorded during a 6 minute period.

The ambulatory behavior of the mice was determined in an open-field test which consisted of a wooden box measuring 40 x 60 x 50 cm. The floor of the box had 12 squares. Ambulatory behavior was assessed by the number of squares the mice crossed with all 4 paws during a 6 minute session.

This experiment showed that at a dose of 250 mg/kg and 500 mg/kg of L-Arg, but not at 100, 750, or 1000 mg/kg of L-Arg, there was a significant decrease in the immobility time during the FST and TST test. At doses between 100-1000 mg/kg, no changes occurred in ambulation in mice tested in a separate experiment. D-Arg, on the other hand, did not have any effect in FST or TST. L-Arg is dose dependent because at 250 mg/kg and 500 mg/kg it increased nitrate and nitrate levels in the brain by about 50%. L-NNA, like L-Arg, also decreased the time of immobility in FST at doses except of .3 and 30 mg/kg and decreased the time of immobility in TST at doses of .3 and 3 mg/kg. These were the doses that didn't interfere with locomotor activity. L-NNA didn't produce any significant change in ambulatory motion in a separate experiment. D-NNA, like D-Arg, didn't produce any effect in FST or TST. In addition to these results, post hoc analysis showed "the anti-immobility effect of L-Arg was completely reversed by pretreatment with L-NNA but not with D-NNA. ... Post hoc analysis revealed that the reduction in the duration of immobility caused by L-NNA was completely reversed by L-Arg, but not by D-Arg" (da Silva, et. al., 2000).

This study was showed "The ability of the NOS inhibitor, L-NNA, but not D-NNA in preventing the anti-immobility effect of L-Arg in the FST seems to be mediated by the production of NO through the activation of NOS. Systemic administration of L-Arg in rats has been shown to increase NO production in the brain" (da Silva et. al., 2000). This study has also shown the anti-immobility effects in both FST and TST are dependent upon the dose of L-Arg being used.

Overall, this experiment showed that "the precursor of NO, L-Arg, and the inhibitor of NO synthesis, L-NNA, but not the inactive isomers D-Arg and D-NNA produced antidepressant-like effects in FST and TST at doses that didn't interfere with locomotor activity...The antidepressant-like effect of L-Arg was completely blocked by L-NNA administered at a dose that produced no anti-immobility effect. The anti-immobility effect of L-NNA was completely blocked by L-Arg at a dose which had no effect when administered alone" (da Silva et. al., 2000).