Supplementary Information
Supplementary Materials and Methods
Discrete paired-trial variable-delay T-maze task. Naïve COMT+/+ dys+/+, COMT+/- dys+/+,
COMT-/- dys+/+, COMT+/+ dys+/-, COMT+/+ dys-/-, COMT+/- dys+/-, and COMT-/- dys-/littermates were tested in this T-maze task as described previously1-3. Animals were presented
with a sequence of randomly chosen forced runs that were each followed by a choice run. This
required the integration of information held online (the forced run) with the learned rule (nonmatch to sample). The T-maze apparatus was constructed from transparent Plexiglass (0.5 cm
thick; dimensions of the alleys: 40x10.2x17.5 cm. Light levels were: 20±2 lux in the main alley;
and 10±2 lux in the side alleys). A recessed cup at the end of each side alley concealed the food
reinforcement from view. In addition, care was taken to remove all visual cues that could be used
by the animal to guide response: behavioral studies were conducted in a room without any visual
landmarks or windows and the two goal arms presented the same cues symmetrically disposed.
After a week of single housing, ad libitum body weight and 24-hour food intake was recorded for
3 consecutive days. Mice were then food restricted throughout the experiment to maintain 8590% of their ad libitum body weight. During the first week of food-restriction, each mouse was
also habituated to the food reinforcer (14 mg, 5TUL, TestDiet, Richmond, IN) for three
consecutive days. After, mice were habituated to the T-maze apparatus and shaped to retrieve the
food reinforcement for 10 minutes/day for two consecutive days. After this, the mice were
exposed to 1 day of 10 forced-alternation runs. The mouse was placed in the T-maze with one
goal arm closed off and had up to 2 minutes to locate and eat the food reinforcer (14 mg, 5TUL)
1
in the open arm. After consuming the food pellet, the mouse was given an inter-trial period of at
least 20 minutes in the home cage, and then placed back in the maze for another forced run.
Beginning on the following day, the discrete-trial delayed alternation training began. Following a
randomly chosen forced run, and a 4-second delay interval in the home cage, the mouse was
placed back in the maze with access to both arms. The food reinforcer was located in the
opposite arm entered in the previous forced trial. After an inter-trial period of at least 20 minutes,
the animal was placed back in the maze for another forced run-choice run paired trial, for a total
of ten paired trials per day. A different pseudo-randomly chosen pattern of forced runs (e.g., RR-L-R-L-L-R-L-R-L) was used every day, but on a given day the same pattern was used for all
animals. The apparatus was cleaned with water and ethanol 70% after each trial with special
attention to the choice point of the T-maze. Mice were trained at a 4-second intra-trial delay and
20-minute inter-trial delay for 20 days, or until the mouse successfully performed 8 correct out of
10 daily trials (80% choice accuracy) for 3 consecutive days.
2
Supplementary Discussion
Theoretical inverted-U model describing the predicted epistatic effects of COMT and
dysbindin genotypes on PFC-dependent working memory function in relationship to
cortical dopamine levels and D2/D1 activation.
Healthy human adults with relative increased COMT enzyme activity (e.g. humans homozygote
for the functional COMT Val single nucleotide polymorphism) have been shown to manifest
relatively less efficient PFC function and working memory deficits4-7. This is presumably based
on the role of COMT in prefrontal cortical dopamine flux and is consistent with animal and
computational models of reduced PFC dopamine signaling8-10. Thus, these subjects are
considered displaced relatively leftwards and on the “downside” of the inverted U curve
representing the relationship between PFC dopamine signaling and PFC-dependent function11-13
(see also Figure 1, white circle). Similarly, adult mice with relative increased COMT enzyme
activity and less cortical synaptic dopamine (e.g. transgenic mice overexpressing the COMT Val
polymorphism) have been shown to take longer to achieve working memory performance criteria
and to manifest performance deficits in a mPFC-dependent working memory T-maze paradigm
as well as in an executive function attentional set shifting task9. Thus, analogous to the human
COMT genotype associated with enhanced COMT activity, the COMT Val-tg mice are
considered displaced relatively leftwards and downwards on the inverted-U model (Figure 1:
white circle). Interestingly, both humans and mice with relative increased COMT enzyme
activity (i.e. humans COMT Val homozygote and mice COMT Val-tg) show improvement in
their cognitive abilities following amphetamine administration9, 14, a finding consistent with the
3
expectation that increasing cortical dopamine levels in these genotype groups will rescue their
relative diminished basal dopamine signaling and functional state. These data further illustrate
the inverted-U-shaped relationship between cognitive performance and cortical dopamine levels
and its modulation by COMT, and the potential to move individuals along the x axis of the
graph, changing their position on the curve.
Conversely, healthy adult humans with relatively decreased COMT enzyme activity (e.g.
humans COMT Met homozygotes) have been shown to manifest more efficient PFC function
and/or working memory improvements compared to individuals with relatively reduced COMT
enzyme activity4-7, 15. Thus, these subjects, at least at baseline conditions, are considered placed
at more optimal levels near the peak of the inverted-U model (Figure 1: blue square). Similarly,
adult mice with relative decreased COMT enzyme activity or complete absence of COMT (i.e.
COMT+/- and -/- knockout mice, respectively) have been shown to have improved working
memory acquisition and performance in an mPFC-dependent working memory T-maze paradigm
as well as in an executive function attentional set shifting task9, 16. Importantly, these COMT
knockout mutant mice have selective increased dopamine levels and decreased dopamine
elimination from the extracellular space in the PFC17-18. Thus, analogous with their human
counterparts, the COMT knockout mice are considered to be placed at the more optimal position
on the inverted-U model (Figure 1: blue square). Also, consistent with the notion that subjects
can be moved rightwards along the x axis onto the downslope of the U curve by increasing
dopamine signaling, individuals with COMT Met homozygote genotypes show deterioration in
cognitive function when given amphetamine14 or COMT inhibitors19.
Optimal cortical dopamine signaling presumably is mediated by optimal activation of D1
and D2 receptors. The former has been shown to be important for maintaining the stability of
4
cortical microcircuits during working memory while the latter is especially important in circuit
flexibility and set shifting11-13,
20-21
. Because cortical D1 receptors are expressed primarily
perisynaptically on the necks of spines and have lower affinity for dopamine than do D2
receptors22-23,24-25, D1 signaling is more sensitive to being compromised in states of diminished
dopamine innervation, such as in normal ageing or in Parkinson’s disease. In contrast, because
D2 receptors are oriented around synapses at the crowns of spines and have a greater affinity for
dopamine22,
24-25
, they are more susceptible to being excessively activated during
hyperdopaminergic states (e.g. stress, amphetamine psychosis). Thus, presumably both D1
hypofunction and D2 hyperfunction will result in displacement from the optimum peak of the
dopamine signaling-PFC function curve and result in deteriorated function, albeit in different
directions and via different mechanisms.
Dysbindin is involved in the intracellular trafficking of D2 receptors via the lysosomes
and related organelles systems, thus modulating dopamine activated levels of D2 receptors at the
cell surface26-28. Prior studies in cell culture26 and in dysbindin knockout mice1, 27 have shown
that diminished dysbindin expression leads to increased D2 expression on the cell surface and
increased D2 signaling. Thus, healthy adults (both humans and mice) with relatively decreased
dysbindin levels (e.g. humans carrying the Bray-risk haplotype29 and mice dysbindin +/- and -/knockouts1,
30
) should show, at least at baseline, relatively increased D2 signaling, and a
relatively rightward shift in the inverted U model of dopamine activity and PFC function (see
Figure 1: green square). In particular, it might be expected that under some conditions they
would have a relative advantage in cortically mediated new learning. This is consistent with
previous findings showing that dysbindin knockout mice present relatively enhanced D2
signaling and improved working memory acquisition of a mPFC-dependent working memory T5
maze paradigm1,
27, 30
. However, these earlier data also suggest that subjects with relative
decreased dysbindin are situated to the right side of the optimal peak level of the inverted-U
model as working memory performance deficits emerged after introducing proactive
interference, longer delays and greater handling stress1, 31-32. These findings suggest that subjects
with reduced dysbindin are relatively closer to the downside of the curve, as even small
increments in dopamine signaling associated with stress and greater cognitive load lead to
depreciation in cognitive function1.
Finally, consistent with these basic mechanisms and these earlier observations, we
predicted that healthy adults (both humans and mice) with both relative increases in synaptic
dopamine (based on decreases of COMT enzyme activity, i.e. human COMT Met homozygotes
and mice COMT+/- and -/- knockouts) and relative decreases in dysbindin levels (i.e. humans
carrying the Bray-risk haplotype and mice dysbindin +/- and -/- knockouts) would show an
exaggerated rightward shift on the x axis and displacement to the downslope of the inverted U
curve, resulting in less efficient PFC function and/or working memory deficits, consistent with
excessive dopamine/D2 signaling overdrive (red triangle in the Figure 1). Indeed, this
phenomenon represents a lawful expression of the biological effects of a combination of
increased synaptic dopamine levels in the PFC arising from reduced COMT expression7, 9, 17-18
and increased D2 signaling produced by the dysbindin genetic disruption1, 26-27, 30.
6
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