Effects of Tryptophan Depletion on Brain Processing of Emotions in Patients With Mood Disorders

Major depressive disorder (MDD) has been associated with abnormally reduced function of
central serotonergic systems by various types of evidence. One instructive paradigm for
investigating the relationship between serotonergic function, cognition and depression has
involved the mood response to tryptophan depletion (TD), achieved by oral loading with all
essential amino acids excepting the 5-HT precursor, tryptophan. Subjects who are depressed
show altered behavioral and neural response to affectively valenced stimuli, similar to those
seen under TD.

More, specifically, increased response latencies to happy words have been noted in both
depressed patients and healthy controls following TD using the Affective Go/No go (AGNG)
test, in which subjects are required either to respond or inhibit a response to a series of
emotionally valenced words.

The first round of our study employed fMRI imaging of BOLD response and arterial spin
labeling to investigate response to affectively valenced words under TD in two groups,
remitted MDD (rMDD) patients and healthy controls with no family history of depression. We
have thus far examined the effects of TD depletion in our healthy control group. We found
that emotional information processing was indeed altered by 5-HT depletion.

A behavioral bias toward positive stimuli was attenuated following depletion, which was
accompanied by increased hemodynamic responses during the processing of emotional words in
several subcortical structures, including the ventral striatum, hippocampal cortex, anterior
insula, superior temporal gyrus and posterior cingulate. Further, inter-individual
differences in tryptophan depletion-elicited anxiety correlated positively with caudate bias
toward negative stimuli. On the basis of these data, we suggest that the bias towards
positive stimuli might represent a normal suppression of attention to negative stimuli; a
process which is abrogated in conditions, such as depression, which are characterized by a
serotonergic dysfunction. In the second round of this study, we are examining whether such
biases also occur beneath the level of conscious awareness.

One of the weaknesses of these data is that the patients were consciously aware of the verbal
stimuli that were presented to them and likely possess learned, individualized associations
to the verbal stimuli in question. We have developed a conditioning task which obviates this
problem through the presentation of emotionally-valenced visual stimuli below the threshold
of conscious awareness.

Since embarking on the first two phases of this study, a new paradigm has been developed
which may provide further insight into affective behavioral biases. The Split Reversals task
is a pavlovian reversal-learning task which allows the assessment of responses to expected
and unexpected punishment and reward cues. Previous research with this task has demonstrated
that reduction of serotonin in healthy individuals removes a disproportionate inability to
predict punishment at baseline. In other words, serotonin reduction removes a positive bias
on this task. . This is consistent with the observed removal of a positive bias on the AGNG
following TD. However, on the split reversals task this positive bias is revealed by an
inability to process negative stimuli at baseline, which is improved, rather than a bias
towards positive stimuli which is diminished, by TD. As such, the new task provides a
different direction from which to consider the emotional biases observed in affective
disorders. We have suggested that the bias on the new task reflects a serotonin-mediated
suppression of responses to negative stimuli in healthy individuals which, in turn, promote
resilience to affective disorders. Serotonin reduction via TD hence removes this bias. That
this task may therefore be sensitive to cognitive biases resulting from resilience
mechanisms.

Similarly, fear potentiated startle which has been shown to be sensitive to the symptoms of
anxiety has been shown to be reduced by the elevation of 5-HT via two week citalopram
treatment. This is consistent with reduced processing of negative information following 5-HT
increase (Grillon et al 2009). We predict that reducing 5-HT via TD will have the opposite
effect and increase the duration of fear potentiated startle processing. This would provide
an indirect link between reduced 5-HT and anxiety disorders. As such, we include this task in
a new phase of this protocol.

In addition these behavioural TD findings, this task has been neuroimaged (fMRI) in healthy
individuals. As predicted, the striatum was shown to be involved in reward processing, whilst
the amygdala was shown to be involved in punishment processing. A subproject of this study
will therefore attempt to extend the prior findings from the split reversals task (and
reconcile them with the AGNG findings) by examining healthy individuals completing the task
whilst undergoing both serotonin manipulation (TD) and fMRI. This subproject will be
initially restricted to healthy individuals and should provide us with further insight into
the neural substrates involved in serotonergic inhibition of aversive processing highlighted
by the first phase of this study. This will also provide a basis from which to consider
potential future study with the same task in MDD subjects undergoing TD.

There may well be a genetic basis to these phenomena. It is known that the functional variant
in the promoter region of the serotonin transporter gene (5-HTTLPR) is partly responsible for
determining how individuals respond to TD. In fact, recently reported that carriers of the
short 5-HTTLPR allele showed impaired recognition of fearful faces following TD.

- INCLUSION CRITERIA:

Healthy Volunteers

Right-handed subjects (ages 18-50) will be selected who have not met criteria for any major
psychiatric disorder, have no known first-degree relatives with mood disorders, and have a
current score on the Hamilton Depression Rating Scale (HDRS; 17 item) in the not depressed
range ( 7). Control subjects will be matched to depressed subjects and their relatives for
age, gender, and education.

MDD Samples

Right-handed subjects (ages 18-50) will be selected with a past history of MDD by DSM-IV
criteria.

Healthy Relatives

Healthy, right-handed, first-degree relatives of patients with MDD will be recruited.

EXCLUSION CRITERIA:

Subjects must not have taken antidepressant drugs for at least 3 months (4 months for
fluoxetine) prior to the fMRI studies or other medications likely to alter monoamine
neurochemistry or cerebrovascular and cardiovascular function for at least 3 weeks prior to
imaging. However, effective medications will not be discontinued for the purposes of this
study. Subjects will also be excluded if they have:

1. Psychosis

2. Medical or neurological illnesses likely to affect physiology or anatomy, i.e.
hypertension, cardiovascular disorders

3. A history of drug (including benzodiazepines (BZD)) abuse within 1 year or a lifetime
history of drug dependence (DSM IV criteria)

4. A history of alcohol abuse within 1 year or a lifetime history of alcohol dependence
(DSM IV criteria)

5. Current pregnancy (as documented by pregnancy testing at screening or at days of the
challenge studies)

6. Current breast feeding

7. Are smokers

8. Serious suicidal ideation or behavior. In this study, there is a small risk of
transient depressive symptoms occurring after ingesting the amino acid mixture.
Therefore, if volunteers manifest evidence of serious suicidal ideation or behavior,
they will be excluded from participating. Criteria for meeting suicidal ideation
include but are not limited to: 1) thoughts of suicide within the past 3 months which
are accompanied by intent to harm oneself, serious consideration of means or plan to
attempt suicide, evidence of arranging for a suicide attempt (e.g. giving away prized
possessions or updating a will), or clear desire to commit suicide, 2) severity of
past suicide events, if applicable or 3) a current plan for harming themselves. All
assessments will be conducted by an experienced, NIMH credentialed health
professional, such as a psychiatrist or psychiatric nurse who will use their expert
knowledge and experiences in determining the authenticity of a participant s
information and handle the situation accordingly.

9. General MRI exclusion criteria

Subjects must exhibit no or only moderate alcohol use. Subjects with current or previous
regular use (greater than 4 weeks) of BZDs and excessive use of alcohol (greater than 8
ounces/day for men and greater than 6 ounces/day for women) in the past or present are
ineligible for participation, as such drug use may confound the results. Smokers (regular
use within the last 3 months) are ineligible because of the evidence for interactions
between nicotine and depression, and the possibility of withdrawal symptoms that may affect
behavioral and neural responses to TD.

Subjects beyond age 50 are excluded to address the biological heterogeneity encompassed by
the MDD criteria, since depressives whose age-at-onset is later than 50 have a far greater
likelihood of having MRI correlates of cerebrovascular disease than age-matched healthy
controls or age-matched early-onset depressives. Subjects whose first major depressive
episodes arose temporally after other medical or psychiatric conditions will also be
excluded, since their functional imaging results generally differ from those reported in
primary MDD.