In the current study, we used a touch screen–based visual reversal task to study the role of 5-HT and the 5-HTT in mediating reversal learning, a form of cognitive flexibility, in mice. The major findings were that 1) chronic treatment with the 5-HTT blocker fluoxetine improved reversal learning, specifically during the early phase of the task, 2) constitutive loss of the 5-HTT also led to improved reversal learning, and 3) neither acute pharmacological depletion of brain 5-HT (or NE) nor constitutive loss of brain 5-HT in Pet-1 null mutant mice demonstrably impaired reversal.
5-HTT KO and HET mice exhibit gene dosage-dependent reduced 5-HT clearance and a corresponding elevation of extracellular 5-HT levels in various cortical, hippocampal, and striatal areas studied (Mathews et al. 2004
; Daws et al. 2006
). Chronic fluoxetine treatment is also expected to significantly augment extracellular 5-HT levels in these regions in the mouse (Cryan et al. 2004
). In this context, improved reversal learning following 5-HTT null mutation or fluoxetine treatment is generally consistent with the observation of an inverse relationship between central 5-HT levels and performance on other forms of executive control, such as impulsivity (Linnoila et al. 1983
; Brigman et al. 2008
). Although this represents a novel and important finding, a number of caveats should be considered.
First, in contrast to the aforementioned link between 5-HT and impulsivity (Linnoila et al. 1983
; Chamberlain et al. 2006
), there is little direct evidence linking 5-HT levels with variability in measures of cognitive flexibility in human subjects. That is, there is, to our knowledge, little direct empirical precedent for our current finding that increased 5-HT availability would promote cognitive flexibility in human subjects. Second, the preclinical literature on the effects of genetic and pharmacological 5-HTT inactivation on cognitive flexibility and impulsivity has actually been rather mixed. For example, rats, in which the 5-HTT has been constitutively inactivated by a different gene knockout method (N
-nitrosourea chemical mutagenesis), show attenuated impulsivity but are normal on a visuospatial reversal task (Homberg et al. 2007
). Moreover, rhesus macaques carrying the putatively lesser functioning orthologue of the human 5-HTT–linked polymorphic region (5HTTLPR
) showed impaired rather than facilitated object reversal (Izquierdo et al. 2007
). On the other hand, in agreement with our current data, another laboratory has recently shown that the same variant was associated with improved reversal (Jedema et al. 2009
). Similarly, monkeys with a different putatively lesser functioning form of the 5-HTT gene (T1970, G1991, and T2327 haplotype in 3′ untranslated region) also showed improved reversal (Vallender et al. 2008
). Taken together with the current data, these findings suggest that, as with other phenotypes such as stress-related behaviors, the penetrance of 5-HTT gene variation on reversal likely depends upon interactions with other factors, including training history, task specifics, genetic background, and environmental factors (Holmes and Hariri 2003
; Caspi and Moffitt 2006
; Uher and McGuffin 2008
5-HTT null mutants made fewer errors to reach the final performance criterion for the reversal task (but were statistically equivalent to WT in attaining and retaining the initial discrimination). This phenotype was evident throughout the task, and not restricted to either the relatively early or late phases of reversal, when behavior is relatively dominated by perseveration and learning processes, respectively (Jones and Mishkin 1972
; Chudasama and Robbins 2003
; Brigman et al. 2008
). By contrast, improved reversal performance in fluoxetine-treated C57BL/6J mice was specifically restricted to the earlier, relatively perseverative phase (again, in the absence of any effects on discrimination retention). The lack of fluoxetine's effects on later reversal was unlikely an artifact of tolerance to the drug's behavioral effects, as the drug retained antidepressant-like effects after completion of the reversal task. Thus, these data might suggest that although both 5-HTT manipulations improved cognitive flexibility, the predominant action of chronic pharmacological inhibition specifically appeared to reduce perseveration, whereas constitutive gene deletion had a more generalized effect on the perseverative and learning components of the task.
Reversal performance is the net manifestation of multiple processes, including detection of a change in stimulus–reward, inhibition of a previously learned prepotent response, sensitivity to negative reinforcement following perseverative responding, and learning of a new stimulus–reward contingency (Roberts 2006
). Alterations in any one or more of these processes could contribute to the improved reversal we observed. However, given evidence that loss of 5-HTT gene functions is associated with increased anxiety, stress reactivity, and neural response to negative stimuli (Caspi and Moffitt 2006
; Hariri and Holmes 2006
; Uher and McGuffin 2008
), it is tempting to speculate that improved reversal in the 5-HTT null mutants may be driven by heightened sensitivity to negative reinforcement that served to better guide subsequent choices. This could be one aspect of the enhanced performance monitoring posited to underlie the improved performance on a probability discounting task in monkeys (Jedema et al. 2009
) and a risky gambling task in humans (Roiser et al. 2006
). Additional studies will be needed to more directly probe this hypothesis, for example, by testing whether the mutants fail to show improved reversal on a task in which incorrect responding is not negatively reinforced.
An interesting observation was that although chronic fluoxetine treatment in nonmutant C57BL/6J mice also improved reversal, the profile of effects differed from those seen in the 5-HTT null mutants in that drug effects were specific to the early (relatively preservative) phase of the task. This profile is reminiscent of the type of effect produced by neuronal or 5-HT lesions of the orbitofrontal cortex in nonhuman primate effect (albeit, in this case, impairing rather than facilitating), which also tend to be specific to the perseverative phase (Clarke et al. 2004
; Murray et al. 2007
). Other researchers have suggested that this type of profile primarily reflects alterations in inhibitory response control rather than shifts in perception of stimulus–reward contingency change (Boulougouris et al. 2008
). This raises the interesting possibility that increased inhibitory control could underlie the improved reversal performance of fluoxetine-treated mice. This will be another important avenue for future work. Whatever the precise nature of the effects of 5-HTT gene loss and fluoxetine treatment on reversal, the finding that the nature of the 2 effects differed is not unexpected. Genetically driven 5-HTT loss not only impacts 5-HTT function but also produces neurodevelopmental alterations in key (e.g., cortical and amygdala) neuroanatomical nodes within the reversal-mediating circuitry (Esaki et al. 2005
; Hariri and Holmes 2006
; Wellman et al. 2007
), and improved reversal in monkeys was associated with reduced PFC gray matter volume rather than alterations in 5-HTT binding (Jedema et al. 2009
Previous work has shown that various means of reducing brain 5-HT, including removing dietary tryptophan, 5-7-DHT–induced lesions, and PCPA treatment, impair reversal on various tasks in humans, nonhuman primates, and rats (Rogers et al. 1999
; Clarke et al. 2004
; Lapiz-Bluhm et al. 2009
) (for review, see Clark et al. 2004
). Although we are unaware of earlier analogous studies in mice, a somewhat unexpected finding of the current study was that neither constitutive genetically driven loss nor acute neurochemical depletion of brain 5-HT produced demonstrable effects on reversal. The reasons for these negative effects remain to be determined. We confirmed that PCPA-treated mice had levels of 5-HT tissue content in the mPFC and hippocampus that were still only approximately 30% of those in controls 6 days after treatment. It does remain possible, however, that the magnitude of PCPA-induced depletion was insufficient to produce significant impairment on our task or that homeostatic alterations in the 5-HT system mitigated the effects of acute depletion. Compensatory 5-HT alterations could also account for the intact reversal phenotype in the Pet-1 null mutants. This would in-of-itself be a remarkable demonstration of the capacity to mitigate the effects of 5-HT loss, given the life-long loss of near 90% of 5-HT raphe neurons and forebrain 5-HT in these mice (Hendricks et al. 2003
). Nonetheless, it should be noted that there are a number of reports that global depletion of 5-HT via dietary tryptophan depletion did not impact reversal in rats (van der Plasse and Feenstra 2008
) and has not always produced significant reversal deficits in humans (Park et al. 1994
; Evers et al. 2005
; Talbot et al. 2006
; Finger et al. 2007
). A parsimonious explanation for our negative data is that, in contrast to the more marked effects of selective ablation of 5-HT in orbitofrontal cortex (Clarke et al. 2004
), the net effects of brain-wide loss of 5-HT may be less disruptive in our paradigm.
In summary, the major findings of the current study were that either chronic pharmacological inhibition or constitutive genetic loss of the 5-HTT improved performance on a touch screen–based assay for cognitive flexibility in mice. By contrast, we were unable to detect effects of pharmacological or genetically driven depletion (via Pet-1 KO) of brain 5-HT. Our findings in 5-HTT null mutants add to growing evidence that although loss-of-function 5-HTT gene variation can increase sensitivity to stress, it may be advantageous for certain cognitive processes that benefit from greater performance monitoring and sensitivity to negative feedback. Similar processes may contribute to the improved reversal learning we saw following chronic fluoxetine treatment. Collectively, these findings further support an important role for the 5-HT system in modulating cognitive flexibility, with implications for understanding the pathophysiology and treatment of neuropsychiatric disorders characterized by executive dysfunction, such as OCD and depression.