Tryptophan depletion resulted in significant reductions in both plasma tryptophan levels and the TRP:ΣLNAA ratio. A repeated-measures ANOVA revealed a significant two-way interaction between treatment (tryptophan depletion, placebo) and time point (baseline, +5.5h), resulting from significant reductions in total tryptophan levels (F(1,21) = 73.166, p < 0.0001) and the TRP:ΣLNAA ratio (F(1,21)= 45.968, p < 0.0001), 5.5h following tryptophan depletion relative to placebo. Simple effects analyses showed a significant decrease in plasma tryptophan levels (t(21) = 15.648, p < 0.0001) on the tryptophan depletion session, averaging 71%. There was also a significant decrease in TRP:ΣLNAA ratios (t(21) = 12.710, p < 0.001) on the tryptophan depletion session, averaging 85%. On the placebo session, plasma tryptophan levels increased by an average of 64% (t(21) = −6.385, p < 0.0001), and there was a trend toward increased TRP:ΣLNAA ratios (t(21) = −1.924, p = 0.07), averaging 25%.
Lowering serotonin does not affect motor response inhibition
Across all task conditions, manipulating serotonin had no effect on motor response inhibition, as measured by commission error rates (main effect of Treatment: F(1,21) = 0.783, p = 0.386; see ).
Figure 2 Effect of tryptophan depletion on motor response inhibition, measured by percentage of commission errors (incorrect Go responses), in the four experimental conditions. Error bars depict the standard error of the difference of the means (SED), the index (more ...)
Motor response inhibition did vary as a function of response-outcome contingencies. When the payoff schedule biased subjects' responding toward “Go” (in Reward-Go and Punish-NoGo), participants made a higher proportion of commission errors than when the payoff schedule biased subjects' responding toward “No-go” (in Reward-NoGo and Punish-Go) (main effect of Bias: F(1,21) = 14.676, p = 0.001). However, this effect was not modulated by tryptophan depletion (Treatment × Bias interaction, F(1,21) = 2.779, p = 0.11).
It is possible that our primary measure of motor response inhibition, commission errors, was not sufficiently sensitive to detect changes in response inhibition resulting from tryptophan depletion. We therefore analyzed RTs as a more sensitive measure of motor response inhibition. RTs were also sensitive to response-outcome contingencies, in a similar manner to commission errors. When the payoff schedule biased responding toward “Go”, RTs were faster than when payoffs biased subjects' responding toward “No-go” (main effect of Bias: F(1,21) = 24.397, p < 0.001).
If lowering serotonin impairs motor response inhibition, then we might expect tryptophan depletion to produce a general speeding of RTs, and/or to eliminate slowing in conditions biased away from “Go”. Neither of these effects was observed. Tryptophan depletion did not induce general speeding in the experimental conditions (main effect of Treatment on RT: F(1,21) = 1.435, p = 0.244) or in the initial neutral block (t(21) = 0.580, p = 0.568). Furthermore, tryptophan depletion did not modulate the effect of Bias on RTs (Treatment × Bias interaction, F(1,21) = 0.940, p = 0.343).
Lowering serotonin abolishes punishment-induced inhibition
Our analysis of the RT data revealed a significant interaction between Treatment and Feedback on response speed (F(1,21) = 5.210, p = 0.033). On placebo, participants were slower to respond in punished conditions (mean ± SE, 0.059 ± 0.102) than in rewarded conditions (mean ± SE, −0.139 ± 0.127; t(21) = −2.177, p = 0.041; see ). However, this punishment-induced inhibition of responding was absent following tryptophan depletion; responses were not slower in punished conditions (mean ± SE, −0.203 ± 0.182) compared to rewarded conditions (mean ± SE, −0.181 ± 0.149) (t(21) = 0.254, p = 0.802; see ).
Figure 3 Effect of tryptophan depletion on punishment-induced inhibition, assessed by comparing reaction times (RTs) for correct Go responses in punished conditions, relative to rewarded conditions. All RTs were normalized against a neutral baseline. Error bars (more ...)
The data indicate that tryptophan depletion likely abolished slowing in punished conditions, rather than enhancing speeding in rewarded conditions; RTs in punished conditions showed a trend toward being significantly slower following placebo, compared to tryptophan depletion (t(21) = −1.932, p = 0.067), but there was no significant difference between RTs in rewarded conditions when comparing placebo to tryptophan depletion within subjects (t(21) = −0.305, p = 0.764).
We next tested whether the reduction of slowing in punished conditions following tryptophan depletion was specific to trials that immediately followed the receipt of punishment. To do this, we broke down the RT data in the punished conditions into trials following correct responses (non-punished) and trials following incorrect responses (punished), and conducted a repeated-measures ANOVA with Treatment and Prior Punishment as within-subjects factors. This analysis revealed a significant main effect of Treatment (F(1,21) = 4.806, p = 0.040), but no significant interaction between Treatment and Prior Punishment (F(1,21) = 2.529, p = 0.127). Across all trials in punished conditions, participants were faster to respond following tryptophan depletion, compared to placebo. The general reduction in slowing across all trials in punished conditions following tryptophan depletion suggests that lowering serotonin dampened the behavioral effects of expectations of punishment, leading to faster responding.
Finally, we examined whether the reduction in punishment-induced inhibition depended on the degree to which tryptophan depletion reduced serotonin levels. We performed a linear regression with the RT difference between punished and rewarded conditions as the dependent variable, and change in plasma TRP:ΣLNAA as the predictor variable. The effect of plasma TRP:ΣLNAA on slowing in punished conditions, relative to rewarded conditions, was highly significant (r = 0.558, t(21) = −2.992, p = 0.007). Higher reductions in the ratio of plasma TRP:ΣLNAA (i.e., greater degree of depletion) corresponded to reduced slowing in punished conditions, relative to rewarded conditions.
The plasma TRP:ΣLNAA data also indicate that tryptophan depletion abolished slowing in punished conditions, rather than enhancing speeding in rewarded conditions. Greater biochemical depletion following tryptophan depletion significantly predicted larger reductions in slowing following tryptophan depletion (compared to placebo) in punished conditions (t(21) = 2.853, p = 0.011) but did not predict RT differences between the tryptophan depletion and placebo treatments in rewarded conditions (t(21) = 1.087, p = 0.291).
Lowering serotonin does not affect sensitivity to aversive outcomes
Given serotonin's potential role in reporting aversive outcomes, we tested whether manipulating serotonin influenced sensitivity to punishments. First, we examined whether tryptophan depletion affected participants' ability to discriminate between aversive response-outcome contingencies. In the Punish-Go condition, the “Go” response is punished more severely than the “No-go” response, so if punishment discrimination is intact, subjects should become biased toward the “No-go” response; in the Punish-NoGo condition, the “No-go” response is punished more severely than the “Go” response, so if punishment discrimination is intact, subjects should become biased toward the “Go” response.
If serotonin reports aversive outcomes, we might expect tryptophan depletion to reduce differences in response bias between the Punish-Go and Punish-NoGo conditions. This was not observed. We conducted a repeated-measures ANOVA on the response bias data from the punished conditions only. Following both treatments, subjects were biased toward “Go” in the Punish-NoGo condition, and biased toward “No-go” in the Punish-Go condition (main effect of Bias: F(1,21) = 11.853, p = 0.002). There was no significant interaction between Treatment and Bias (F(1,21) = 0.377, p = 0.546). These results indicate that tryptophan depletion did not affect subjects' sensitivity to punishment contingencies; following tryptophan depletion they were equally likely to favor the less-punished response, compared with placebo (see ).
Figure 4 Effect of tryptophan depletion on punishment sensitivity, assessed by comparing response bias in the Punish-Go condition to the Punish-NoGo condition. Response bias was assessed by the natural log of β from signal detection theory; more negative (more ...)
We also examined whether tryptophan depletion altered the emotional impact of punishment on responding. Our analysis of RTs on trials following punishment vs. trials following non-punishment showed a main effect of Prior Punishment (F(1,21) = 23.024, p < 0.001); subjects were slower to respond on trials that followed punishment (mean ± SE, 0.303 ± 0.177) than on trials that followed non-punishment (mean ± SE, −0.234 ± 0.131). But the interaction between Treatment and Prior Punishment was not significant (F(1,21) = 2.529, p = 0.127); tryptophan depletion did not abolish subjects' tendency to be slower on trials immediately following punishment, compared to trials following non-punishment. Subjects' responses were significantly slower on trials following punishment, compared to non-punishment, on both tryptophan depletion (t(21) = −3.019, p = 0.007) and placebo (t(21) = −4.085, p = 0.001).