Doi:10.1016/j.psyneuen.2006.07.00

Psychoneuroendocrinology (2006) 31, 1087–1097 Serotonin regulation of the human stress response Sean D. Hooda,Ã, Dana A. Hincea,b, Hayley Robinsona,Melita Cirilloa, David Christmasa, Joey M. Kayec aSchool of Psychiatry and Clinical Neurosciences (M521), University of Western Australia,QEII Medical Centre, Perth, Nedlands, Western Australia 6009, AustraliabPsychopharmacology Unit, University of Bristol, Bristol, UKcDepartment of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Perth, Australia Received 5 May 2006; received in revised form 6 July 2006; accepted 16 July 2006 Acute tryptophan depletion (ATD) is a technique that has been used to evaluate the effects on humans of acutely reducing serotonin neurotransmission. We have developed a model using a single breath of 35% CO axis and produces autonomic and behavioural arousal, thus modelling a stress response. This study combines ATD and single breath 35% CO2 inhalation to study A randomised, double-blinded, placebo-controlled, cross-over trial involving 14 healthy adult volunteers aged between 18 and 65 years was undertaken. Subjectsunderwent double-blind tryptophan depletion over 2 days and were then crossedover 1 week later. During each study day, at the time of peak depletion, participantswere single blinded to receive a single breath of 35% CO2 or air. This was followed40 min later by the other gas. Psychological outcomes were assessed with theSpielberger State Anxiety Inventory (SSAI), Visual Analogue Scales (VAS), PanicInventory (PI), Panic and Agoraphobia Scale (PSI) and Beck Depression Inventory(BDI). Physiological outcome was measured by serial plasma cortisol, prolactin andtryptophan levels, pulse and blood pressure.
Tryptophan depletion did not exacerbate 35% CO2 inhalation effects on anxiety symptoms. Single breath CO2 robustly increased plasma cortisol levels in comparisonto an air inhalation; this was less certain for prolactin levels. ATD influenced the HPAaxis (associated with higher cortisol levels), apparently independent of CO2 or airinhalation stressors. ATD and 35% CO2 inhalation both induced a pressor response andbradycardia in these normal volunteers.
Thirty-five percent CO2 inhalation and ATD independently activate the human stress response, but do not appear to produce synergistic effects when combined, atleast for the conditions produced in this study.
& 2006 Elsevier Ltd. All rights reserved.
ÃCorresponding author. Tel.: +61 8 9346 2393; fax: +61 8 9346 3828.
E-mail address: sean.hood@uwa.edu.au (S.D. Hood).
0306-4530/$ - see front matter & 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.psyneuen.2006.07.001 dietary manipulation that causes a rapid reductionin plasma tryptophan levels (typically 70–80% Stress leads to subjective anxiety, endocrine activation (especially of the hypothalamo-pituitary There have been several studies of the effect of adrenal (HPA) axis) and cardiovascular changes 5-HT manipulation on CO2 challenge in both volun- (Sinha et al., 1999). Existing tests of the human teer and patient groups. One study (Miller et al., stress response are limited because they (a) are not 2000) that measured salivary cortisol reported no reproducible in laboratory settings (b) are variable changes in response to 5% CO2 inhalation after in effect and involve painful stimuli or (c) are tryptophan depletion, which our studies suggest is confounded by other emotional effects. It is too low a dose to increase cortisol (Kaye et al., therefore essential to develop a technique that 2004a). Two studies found that tryptophan depletion may be employed to reliably and safely induce resulted in more 35% CO2-induced increases in stress responses in subjects in a controlled labora- anxiety symptoms measured with the Panic Symp- tom List Rating scale, in both volunteers (Klaassen et The 35% carbon dioxide (CO2) single inhalation al., 1998), and patients with panic disorder paradigm fulfils these requirements (Kaye et al., (Schruers et al., 2000). Reducing brain 5-HT function 2004a). Firstly, it robustly activates the HPA axis by another means, using the 5-HT antagonist and produces a rapid and profound sympathetic metergoline, also significantly enhanced the anxio- activation as well as significantly increasing anxiety genic effect of 35% CO2 (Ben Zion et al., 1999).
(Argyropoulos et al., 2002; van Duinen et al., 2005; Taken together, the studies discussed above Wetherell et al., 2006). For example, in the suggest that agents that decrease serotonergic Argyropoulos et al. study, 13 of 14 subjects showed throughput may enhance CO2 stress responses in an elevation of plasma cortisol in response to the both patients and volunteers. However, the neu- 35% CO2 challenge, and salivary cortisol has roendocrine and autonomic variables have not been recently been shown to be elevated by 35% CO2 assessed under altered serotonergic conditions, and inhalation (van Duinen et al., 2005). Also in the an important question is whether manipulating Argyropoulos et al. study, cardiovascular measure- serotonergic function alters one or both of these ments indicated autonomic arousal with immediate parameters in this paradigm. The aim of the activation of the sympathetic system demonstrated present study, therefore, was to assess neuroendo- by an immediate increase in blood pressure.
crine, autonomic and psychological responses to Secondly, we have found that these effects show single breath 35% CO2 inhalation in healthy volun- good test–retest reliability over a period of several teers after altering serotonergic function using the weeks (Kaye et al., 2004a). And thirdly, the effects tryptophan depletion technique. It was hypothe- of CO2 on cortisol are dose related in that sised that tryptophan depletion would increase the inhalation of CO2 below a concentration of 25% psychological (increase in subjective anxiety mea- evokes little response, whereas at concentrations sures), neuroendocrine (e.g. cortisol and prolactin above 35%, glottal spasm is induced which in turn increases) and autonomic (e.g. bradycardia and impairs performance in the test. The 35% CO2 pressor response) responses to single breath 35% challenge therefore appears to be the optimal CO2 in normal volunteers. Prolactin was also challenge in clinical research settings (Kaye et al., measured as it is known to be stress responsive There are several lines of evidence suggesting a role for the serotonin system in the stress response.
Serotonergic neurones within the rostral medullamay have close associations with cerebral arteries and function as primary chemosensory cells (Brad-ley et al., 2002). In addition, studies in animals suggest that serotonergic projections to the peri-aqueductal grey region play an important role in This was a randomized, double blind, placebo- suppressing the behavioural and autonomic corre- controlled, balanced cross-over study. Fourteen lates of panic (Deakin and Graeff, 1991). Further, healthy volunteers were tested on two similar the hypothalamus and pituitary are densely inner- days, 1 week apart. On the 1st day they were vated by 5-HT neurons. It is possible to decrease randomized in a double blind fashion into either 5-HT function by using the technique of tryptophan tryptophan depletion (ATD) or non-tryptophan depletion (acute tryptophan depletion (ATD)) (Bell depletion (nTD) arms. At the time of peak deple- et al., 2005; Hood et al., 2005). This involves a tion subjects were challenged, in a single blind fashion, with one forced breath of either 35% CO2, diet the day before the test, and fasted from or placebo (air). The other gas was administered in midnight. On each test day the volunteers arrived the same fashion 40 min later. Psychological out- at the testing unit at 0900 h where they were rested comes were measured at set times using standar- in a semi-supine position on a bed. The testing room was designed to be a non-stimulating environment depression. Neuroendocrine outcomes were mea- minimising excitement and stress. Subjects remained sured by blood samples taken from an intravenous in the test room for the duration of the experiment, cannula that had been inserted at 0900 h to control resting quietly, reading or performing similar activ- for the effect of repeated venipuncture as a ities, and were allowed to drink water for the confounding stressor. Cardiovascular physiological duration of the study. By 0930 h baseline ratings of outcomes (blood pressure and heart rate) were symptoms and cardiovascular measures were re- measured using a non-invasive automated machine.
corded, and blood samples taken. Subjects then The subjects were then crossed over into the other consumed a tryptophan-free amino acid drink (the arm of the study 1 week later for which their ATD test) or a control drink (the nTD test) containing tryptophan status remained double blinded and the 2.3 g of tryptophan. The other amino acids included experiment continued to the same schedule. This in both drinks (in the same proportion as human milk) study was prospectively registered with the Inter- are L-alanine, 5.5 g; L-arginine, 4.9 g; L-cysteine, national Standard Randomised Controlled Trial 2.7 g; glycine, 3.2 g; L-histidine, 3.2 g; L-isoleucine, Number Register (ISRCTN77876347). The protocol 8 g; L-leucine, 13.5 g; L-lysine, 11 g; L-methionine, 3 g; was approved by the University of Western Aus- tralia Human Research Ethics Committee.
6.9 g; L-threonine, 6.9 g; L-tyrosine, 6.9 g; and L-valine 8.9 g. The order of the ATD and nTD testswas randomly allocated by unweighted blocking.
Women consumed only 80% of these drinks by virtueof their smaller body mass (Smith et al., 1997).
Volunteers were recruited via the UWA staff e-mail Challenge testing was performed 5 h after consump- database and by using personal contacts of the tion of the TD or nTD drink, as this is the point of peak research team. Informed consent was gained. Parti- depletion (Hood et al., 2005). At the end of each test cipants were screened by a medical practitioner for day patients were given a meal and after having been psychiatric and physical morbidity. This was achieved assessed as recovered were allowed home. Subjects using clinical interview, physical examination, a MINI were given access to telephone contact over the v5 semi-structured interview (Sheehan et al., 1998), following 24 h and were discharged thereafter.
Panic and Agoraphobia Scale (PAS) (Bandelow, 1995),Panic Inventory (PI) (Coupland et al., 1997), BeckDepression Inventory (BDI) (Beck and Steer, 1987), 2.3.2. Thirty-five percent CO2 inhalation Spielberger State (SSAI) and Spielberger Trait Anxiety Inventories (STAI) (Spielberger et al., 1983). The The CO2 provocation followed established proce- Swedish Personality Questionnaire (SSP) (Gustavsson dure (Argyropoulos et al., 2002). Gas delivery was et al., 2000) and 24 h heart rate monitor (Stampfer, single-blind, placebo controlled (air and 35% CO2).
1998) were additional screening measures. Exclusion Order of delivery was randomised by unweighted criteria were: age o18 or 465, a history of epilepsy, blocking was the same for ATD and nTD occasions.
head injury, serious medical disorder, substance The inhalation tests were performed at +5 and +5 h abuse, current pregnancy or lactation, a psychiatric 40 min on both ATD and nTD days to minimise the disorder within the last 6 months, arrhythmias, effects of diurnal variability of neuroendocrine tachycardia, hypertension and SSP results outside measures and maximise the tryptophan depletion one standard deviation from normal range. Preme- effects. The gas mixtures were delivered via an oral nopausal women were asked to present for testing in gas regulator using the one vital capacity inhalation the first 2 weeks of their menstrual cycles.
technique (van den Hout et al., 1987).
The tryptophan depletion procedure was performed The three subjective ratings of anxiety were: according to our standard protocol (Hood et al.,2005) based upon the established technique (Young 1. Visual Analogue rating Scales (VAS), measured on et al., 1985). Subjects observed a low tryptophan 100 mm line, anchored from 0: ‘‘not at all’’ to 100: ‘‘the most y ever’’. Subjects were trained Thayer, 1987), the Huyhn-Feldt epsilon (when in the use of these scales (during screening and Greenhouse-Geisser epsilon40.75) or the Green- on the morning of each session) as integer house-Geisser epsilon (otherwise) was used to measures; these scales have been extensively correct degrees of freedom when this assumption used in similar settings (Bell et al., 2002; was violated (Girden, 1992). A logarithmic trans- formation was applied to cortisol and prolactin 2. The Panic Symptom Inventory (PSI): lists 34 data. The Bonferroni correction was applied to symptoms related to a panic attack with the post-hoc pair-wise tests. p-values less than 0.05 option of rating 0 ¼ not at all, 1 ¼ slight, were considered statistically significant.
2 ¼ moderate, 3 ¼ severe, 4 ¼ very severe andhas been used in studies of anxiety provocation(e.g. Nutt et al., 1990).
3. The Spielberger State Anxiety Inventory (SSAI).
Each questionnaire was administered at baseline, 1.5 and 4 h post amino acid drink, 10 min prior to The sample consisted of 14 subjects, of which nine and 6 min post each gas inhalation, and at the end were female. All the participants in this study were of the study (30 min after eating a meal). For the between the ages of 21 and 60 years with a mean 6 min post inhalation time point participants were age of 34.5 years. The baseline variables shown in instructed to complete the questionnaires accord- Table 1 were the mean (SD) for the first time point ing to how they felt at the peak effect of the The SSP profile of this cohort showed all 13 parameters within 71 standard deviation of the Serum prolactin was measured at baseline (0900 h),1400 h, 1 min before and 20 min after each gas challenge. A further final measurement was takenat 1630 h, which was 30 min after eating a meal.
As shown in Fig. 2, the tryptophan depletion Serum cortisol was measured 20 and 1 min prior procedure reduced free tryptophan (fTRP) serum as well as 20 min after each gas challenge. Base- levels in the ATD condition, and increased fTRP line, middle and endpoint values were also mea- levels in the nTD condition. These observations were supported by a significant depletion condition Serum tryptophan was measured at 0900, 1510 by time interaction (F (2, 26) ¼ 79.39, po0.001).
There was no difference between the groups attime 0 (pre-drink) (F (1, 13) ¼ 0.11, p40.75).
At time 2 (5 h post amino acid drink and coinci- Heart rate and blood pressure were measured at À19, À5, À4, À3, À2, À1 before and +1, +2, +3, had decreased by 68% from baseline under the +4, +5, +19 min after each gas challenge. These ATD condition, and had increased by 76% on parameters were also measured at 0930, 1130, the nTD occasion (F (1, 13) ¼ 192.2, po0.001). At 1402 and 1632 h to check variation throughout eachtest day.
Statistical analysis was conducted using SPSS v12.
Missing values in this dataset determined to be missing-at-random were estimated using the multi- ple imputation method using NORM software (Schafer and Graham, 2002). Repeated measures analysis of variance (ANOVA) was the primary statistical technique used with time, depletion status and gas inhalation type as within-subject factors. As violation of the assumption of sphericityleads to increased type I error rates (Vasey and Figure 1 Mean SSP personality profile for the sample (n ¼ 14). All are within 1 SD of the mean (À10 to +10).
(mcg/mL)
Figure 2 Free tryptophan levels for acute tryptophan depleted and non-depleted conditions at baseline (pre- drink), 5 h post ingestion and after a meal. ***po0.001 nTD vs. ATD. Error bars represent the standard error of PSI total score
the end of the study the fTRP levels between the two conditions were still significantly different (F (1,13) ¼ 187.7, po0.001). Similar results were Figure 3 Total SSAI (A) and total PSI (B) scores pre and found for total tryptophan levels (data not shown).
post gas inhalation under ATD and nTD conditions.
***po0.001 AIR vs. CO2, nTD condition. **po0.01, AIRvs. CO2, ATD condition. +++po0.001, AIR vs. CO2, ATDand nTD condition. Error bars represent the standard To assess the impact of CO2 inhalation and tryptophan depletion on psychological measures Figure 3A displays the mean SSAI scores for the ATD of anxiety, SSAI scores, PSI total scores, and VAS and nTD days following AIR and CO2 inhalation. CO2 scores for the items ‘‘Anxiety’’, ‘‘Palpitations’’ and inhalation significantly increased mean total SSAI ‘‘Something bad is just about to happen’’ were scores compared with air inhalation (F (1, 13) ¼ analysed using time (pre- vs. post-inhalation), 25.12, po0.001) and there was a trend towards depletion status (nTD vs. ATD) and gas (AIR vs.
higher SSAI scores in the ATD compared to the CO2) as factors. Treatment orders and gender nTD condition (F (1, 13) ¼ 3.89, p ¼ 0:07). How- effects are only reported if statistically significant.
ever, this analysis failed to find evidence of an interaction between gas inhalation and depletion main effect of time (F (1, 13) ¼ 41.8, po0.001), of status (F (1, 13) ¼ 0.30, p ¼ 0:595). That is, CO2 gas (F (1,13) ¼ 40.0, po0.001) and a significant gas significantly increased mean SSAI scores to a similar by time interaction (F (1,13) ¼ 39.0, po0.001). PSI degree in both nTD (F (1, 13) ¼ 31.0, po0.001) and scores increased post CO2 inhalation compared with ATD (F (1, 13) ¼ 12.6, po0.01) conditions, com- pre inhalation means in both the nTD (F (1, 13) ¼ A depletion order effect was found on total SSAI po0.001) conditions but were not affected by AIR scores, with participants depleted on the 1st day inhalation in either of the depletion conditions. The showing significantly lower SSAI scores compared to main effect of depletion, and the interactions those depleted on the 2nd day (ATD day 1: involving depletion did not reach significance (all M ¼ 28:7, 95% CI ¼ 25.3 to 32.1; ATD day 2: p40.3). A similar pattern of results was seen in PSI M ¼ 34:2, 95% CI ¼ 30.9 to 37.6; (F (1, 12) ¼ 6.4, somatic and PSI psychological subscales (data not po0.05). Inspection of the data found that this was likely a consequence of four of the seven subjectsrandomised to the ATD condition on day 2 having 3.3.3. VAS-‘‘anxiety’’, ‘‘palpitations’’ and STAI (trait) scores in the low clinical range (30 or ‘‘something bad is going to happen’’ above). To assess whether this failure of randomi- Table 2 displays the mean VAS-anxiety scores pre- sation had an impact on the pattern of effects seen and post-inhalation for nTD and ATD conditions.
on the SSAI, the group was split according to normal Analysis of the mean VAS scores revealed a (20–30; n ¼ 9) or low clinical (31+; n ¼ 5) trait significant gas by time interaction for VAS-anxiety anxiety (STAI) scores and this factor was added (F (1, 13) ¼ 25.6, po0.001), VAS-palpitations to the ANOVA model. As expected, a main effect (F (1, 13) ¼ 14.6, po 0.01) and VAS-‘‘something of trait anxiety was seen on the SSAI means bad is going to happen’’ (F (1, 13) ¼ 8.8, po0.05).
(F (1, 12) ¼ 5.1, po0.05), with people with high All three measures increased significantly following trait anxiety reporting more state anxiety. More CO2 inhalation, but not after air inhalation (see importantly, normal vs. low clinical state anxiety Table 2). Tryptophan depletion had no effect on any did not interact with any of the other factor (all of these measures, as the main effect of and all p’s40.1). Thus, it appears unlikely this order interactions involving depletion status were not effect can account for effects seen in the overall 3.3.2. PSIFigure 3B shows the mean PSI scores immediately The mean cortisol levels, prolactin levels and pre- and post-inhalation for both the ATD and nTD cardiovascular measures across the test day are conditions. Similar to that seen in the SSAI means, displayed in Figs. 4 and 5. Baseline (t ¼ 0) analysis of the mean total PSI scores measured differences can be assumed not present unless immediately pre and post gas inhalation revealed a VAS mean (SD) for pre- and post-inhalation for each depletion status/inhalation condition.
t rate (bpm)
tisol (micr
BASELINE
PRL (micr
t rate (1 min post-1 min pre)
BASELINE
Figure 4 Cortisol (A) and prolactin (B) means as a function of depletion status and inhalation challenge.
Figure 5 Top: Mean heart rate for ATD and nTD *po0.05 pre 1 vs. post 20 in the CO2/ATD condition. Error conditions for AIR and CO2 inhalation challenges. Bottom: bars represent the standard error of the mean.
Distribution and mean (horizontal bar) of change in heartrate post inhalation challenge.
3.4.1. CortisolFigure 4A plays the mean cortisol levels for the nTDand ATD conditions at baseline, 20 and 1 min pre-, po0.05). Post hoc ANOVA conducted at À20, À1 and 20 min post-, gas inhalation. Inspection of the and +20 min separately found this interaction to be graph demonstrates that cortisol decreased across the consequence of a significant depletion status by the day, in accordance with its usual circadian gas interaction at post 20 min (F (1, 12) ¼ 9.5, rhythm. The analysis of mean log cortisol levels 20 po0.01), but not at the other two time points.
and 1 min pre- and 20 min post-gas inhalation found However, pair-wise comparisons between air and cortisol levels to be significantly higher under the CO2 conditions for nTD and ATD days were not ATD condition compared with the nTD condition (main effect of depletion status (F (1, 13) ¼ 4.7,po0.05)). The gas by time interaction (F (1.3,16.9) ¼ 5.5, po0.05) reflected the increase in cortisol following CO2 inhalation, but not followingair inhalation, irrespective of depletion status. No other main effects or interactions were significant.
Figure 5A displays the mean heart rate recordedbetween 19 min pre- to 19 min post-inhalation for ATD and nTD conditions. Analysis of these means Analysis of plasma prolactin levels pre (À20 and found a near significant main effect of depletion À1 min) and post (+20 min) gas inhalation for status (F (1, 13) ¼ 4.4, po0.06), with higher heart both depletion conditions. As anticipated, women rates observed under the ATD condition. To assess had higher levels of prolactin compared to men the effect of the inhalation challenge on heart (women: M ¼ 2:4, 95% CI ¼ 2.6–2.5; men: M ¼ 2:2, rate, the difference between heart rate 1 min pre 95% CI ¼ 2.1–2.3; F (1, 12) ¼ 16.5, po0.01). Sex and 1 min post breath was calculated. These did not interact significantly with any other factor.
difference scores are seen in Fig. 5B. Analysis of A trend towards a main effect of tryptophan the difference scores revealed a trend towards a main effect of gas (F (1, 13) ¼ 3.5, p ¼ 0:08), with p ¼ 0:06), with prolactin levels tending to be higher CO2 producing a greater reduction in heart rate on the ATD day compared to the nTD day (see compared to air inhalation. No other effects were Fig. 4B). A significant depletion status by gas by significant. One subject showed an increase in HR time interaction was also seen (F (1.4, 16.3) ¼ 5.2, by 14 beats/min in the nTD/CO2 condition. This was (r ¼ 0:28, p40.3, n ¼ 14) or the nTD (r ¼ 0:12, score ¼ 2.57). Removal of this participant from the analysis rendered the main effect of challengesignificant (F (1, 12) ¼ 5.2, po0.05).
The aim of the present study was to assess Figure 6A displays the mean systolic blood pressure psychological, neuroendocrine and cardiovascular recorded between 19 min pre- to 19 min post- inhalation for ATD and nTD conditions. Analysis of healthy volunteers after altering serotonergic these means found no main effects or interactions function. The tryptophan depletion procedure to be significant. To assess the effect of the produced a 68% reduction in free tryptophan levels.
inhalation challenge on systolic blood pressure, This degree of reduction is congruent with reduc- the difference between systolic blood pressure tions reported in the literature that are associated 1 min pre- and 1 min post-breath was calculated with increased anxiety in clinical populations (Bell for each subjects under each condition. These et al., 2005; Hood et al., 2005). Psychological, difference scores are seen in Fig. 6B. Analysis of the neuroendocrine and cardiovascular responses in difference scores found no main effects or inter- healthy volunteers were differentially affected by actions to be significant. Furthermore, one partici- ATD (no effect on self-reported anxiety in contrast pant showed an extreme increase in SBP under the with ATD-related increased in cortisol, prolactin ATD/CO2 condition (standard score ¼ 2.75). Re- and heart rate), but all measures were similarly moval of this participant from the analysis did not response was indicative of a stress response.
Contrary to prediction, however, the present data 3.6. Cortisol reactivity and pressor response provides very little evidence for a synergisticincrease in stress response in all three domains To assess the relationship between changes in cortisol and the pressor response post CO2, sepa-rate correlations were computed for these vari- ables in the ATD and nTD condition. The change incortisol did not predict a significant amount of the The 35% CO2 inhalation procedure (compared with variance in the pressor response in either the ATD air inhalation) was seen to be a reliable anxiogenictrigger as measured by SSAI, PSI and VAS scales, as anticipated. Reduced serotonergic throughput via tryptophan depletion (1) did not produce increasedanxiety ratings prior to CO2 challenge and (2) did not augment self-rated anxiety produced by single breath 35% CO2 inhalation in healthy volunteers.
The lack of synergistic effect between CO2 and tryptophan depletion is unlikely to be a conse- quence of a ceiling effect. The highest scores recorded were at the most 2/3 of the maximum score obtainable on all the psychological scales These findings are consistent with previous research. Firstly, studies that explicitly studied the effect of ATD on anxiety in normal volunteers report that ATD alone had little effect on anxiety ratings (Goddard et al., 1995; Klaassen et al., 1998; Miller et al., 2000), albeit small increases in SBP (1 min post - 1 min pre)
‘‘nervousness’’ were reported on VAS (Goddard et al., 1995) and STAI (Klaassen et al., 1998)measures. Two positive studies reported minor Figure 6 Top: Mean systolic blood pressure for ATD andnTD conditions for AIR and CO increases in anxiety ratings in male subjects who Bottom: Distribution and mean (horizontal bar) of change also reported an increase in depressive symptoma- in systolic blood pressure post inhalation challenge.
tology (Smith et al., 1987; Benkelfat et al., 1994); other studies that included anxiety ratings in the activity of the HPA axis. Serotonin is known to influence the HPA axis through direct actions at the et al., 1985; Abbott et al., 1992; Park et al., hypothalamic, pituitary and adrenal levels (Chaoul- 1994; Cleare and Bond, 1995; Oldman et al., 1995; off, 2000) with serotonergic systems having both Ellenbogen et al., 1996; Smith et al., 1997; Barr et facilitatory and inhibitory actions on the axis al., 1997; Moore et al., 1998). Secondly, Klaassen (Lowry et al., 2005). Further, these systems may and colleagues found that volunteers exposed to also act through the hippocampal negative feed- single-blind 35% CO2 exhibited higher scores on the back regulation of the HPA axis causing disinhibition Panic Symptom Inventory on ATD vs. control of the axis and a rise in cortisol levels (Lowry, occasions; no ATD by 35% CO2 effect was seen on 2002). Despite the higher baseline cortisol levels on a VAS-anxiety item (Klaassen et al., 1998). The the ATD day, and the possibility of HPA axis same group did, however, demonstrate a signifi- disinhibition, the cortisol rise following CO2 was cantly greater anxiety response to the CO2 chal- not enhanced as might be anticipated under these lenge in tryptophan depleted panic disorder circumstances. This is also consistent with our patients compared with patients who were trypto- findings in volunteers with both physiological phan replete (Schruers et al., 2000). Of interest, (during lactation) and pharmacological (naltrexone premedication of healthy volunteers with the premedication) disinhibition of the HPA axis. In known anxiogenic agents CCK-4 (Pols et al., 1999) both circumstances, baseline cortisol levels were or yohimbine (Pols et al., 1994) had no synergistic significantly greater than control or placebo condi- effect on anxiety response following the 35% CO2 tions, but the increase in cortisol following 35% CO2 challenge. No notable interaction between ATD and was no greater (Kaye et al., 2004b; Kaye, 2005).
5% CO2 inhalation was seen in another group of It is not clear why ATD produced a neuroendo- volunteers (Miller et al., 2000), although this may crine stress response in volunteers in the absence reflect an inadequate CO2 concentration (Kaye of a psychological stress response. This finding demonstrates that, although often co-occurring,the different dimensions of the stress response donot necessarily need to appear together. This highlights the importance of assessing the stressresponse in all 3 domains.
Single breath 35% CO2 robustly increased plasmacortisol levels in comparison with air inhalation.
These results are congruent with previous findings in healthy volunteers and indicates HPA axisactivation (Argyropoulos et al., 2002; van Duinen The results presented here go someway to replicate et al., 2005; Wetherell et al., 2006). Tryptophan previous work concerning 35% CO2 effects upon depletion did not facilitate this effect, but pro- heart rate and blood pressure (Argyropoulos et al., duced an increase in cortisol levels compared to 2002; Kaye et al., 2004a, 2006; Wetherell et al., the nTD condition. Although not statistically sig- 2006). CO2 inhalation produced significant brady- nificant pre inhalation, the consistently higher cardia compared with air inhalation, consistent cortisol levels across time in this group became with previous findings. This was not influenced by significant post inhalation. This is in keeping with tryptophan depletion or related to plasma cortisol the known mechanism of tryptophan depletion, levels and is likely a parasympathetic response that is, central 5-HT gradually decreases with time, discrete from either sympathetic, HPA or arousal and is maximally effective near the time of gas centres (Kaye et al., 2006; Wetherell et al., 2006).
Although visual inspection of the data suggested It is of some interest that ATD itself acted as a that systolic blood pressure was increased by the stressor, as measured by plasma cortisol (and to a 35% CO2 inhalation (and perhaps air inhalation lesser extent prolactin) increases. It may be that under ATD conditions: see Fig. 6), we failed to find ATD produces an aversive state that is perceived as these effects to be statistically significant. This stressful and indirectly results in increased cortisol may be a consequence of limited temporal grada- levels. We found little evidence for this however, as tion of the equipment used to measure blood participants did not report the ATD day as being pressure. The results of Kaye et al. (2006) suggest significantly more stressful or aversive than the nTD that the maximum pressor response is approxi- condition on the basis of self-report measures.
mately 45 s post CO2 inhalation—we were only Another explanation might be that reduced central able to measure blood pressure every minute.
5-HT via tryptophan depletion may be modulating Furthermore, Kaye et al. considered the maximum change in blood pressure post breath, which takes Argyropoulos, S.V., Bailey, J.E., Hood, S.D., Kendrick, A.H., Rich, into account individual differences in this response, A.S., Laszlo, G., Nash, J.R., Lightman, S.L., Nutt, D.J., 2002.
thereby increasing the power to detect an effect.
Inhalation of 35% CO2 results in activation of the HPA axis inhealthy volunteers. Psychoneuroendocrinology 27, 715–729.
This was only possible at 1 min intervals in the Bandelow, B., 1995. Assessing the efficacy of treatments for present study, and therefore it may still be the case panic disorder and agoraphobia. II. The Panic and Agorapho- that the maximum response for an individual was bia Scale. Int. Clin. Psychopharmacol. 10, 73–81.
not detected. Therefore, future work should Barr, L.C., Heninger, G.R., Goodman, W., Charney, D.S., Price, ideally use a beat-to-beat measurement of blood L.H., 1997. Effects of fluoxetine administration on mood response to tryptophan depletion in healthy subjects. Biol.
Psychiatry 41, 949–954.
The trend towards higher blood pressures seen on Beck, A.T., Steer, R.A., 1987. Beck Depression Inventory Manual.
the ATD day suggests a modulatory role for The Psychological Corporation, Harcourt Brace Jovanovic, serotonin on the stress-induced pressor response.
There is some recent evidence that a reduction in Bell, C., Forshall, S., Adrover, M., Nash, J., Hood, S., central serotonin by tryptophan depletion is asso- Argyropoulos, S., Rich, A., Nutt, D., 2002. Does 5-HT restrainpanic? A tryptophan depletion study in panic disorder ciated with significant excess pressor response in patients recovered on paroxetine. J. Pharmacol. 16, 5–14.
anxious patients given a stress challenge (Davies Bell, C., Hood, S.D., Nutt, D.J., 2005. Acute tryptophan depletion. Part II: clinical effects and implications. Aust. N.
One limitation of the present study was that the capacity of the inhalation breath was not mea- Ben Zion, I.Z., Meiri, G., Greenberg, B.D., Murphy, D.L., sured. It is possible also that the reduced effect of Benjamin, J., 1999. Enhancement of CO2-induced anxiety inhealthy volunteers with the serotonin antagonist metergo- CO2 on blood pressure is a consequence of line. Am. J. Psychiatry 156, 1635–1637.
incomplete inhalations, leading to a reduced CO2 Benkelfat, C., Ellenbogen, M.A., Dean, P., Palmour, R.M., Young, ‘‘dose’’. Although possible, this explanation is S.N., 1994. Mood-lowering effect of tryptophan depletion.
unlikely as cortisol levels increased following CO Enhanced susceptibility in young men at genetic risk for major affective disorders. Arch. Gen. Psychiatry 51, 687–697.
Bradley, S.R., Pieribone, V.A., Wang, W., Severson, C.A., Jacobs, An explanation of the differential effects of ATD on R.A., Richerson, G.B., 2002. Chemosensitive serotonergic patient groups but not on healthy volunteers might neurons are closely associated with large medullary arteries.
be that patients have a vulnerability to serotonergic manipulation that healthy populations do not. The established finding of SSRI effectiveness for these patient groups could therefore be viewed as a means Cleare, A.J., Bond, A.J., 1995. The effect of tryptophan depletion and enhancement on subjective and behavioural of promoting serotonergic resiliency. Further delinea- aggression in normal male subjects. Psychopharmacology tion of the ways by which serotonergic systems may modulate the human stress response is an active area Coupland, N.J., Lillywhite, A., Bell, C.E., Potokar, J.P., Nutt, D.J., 1997. A pilot controlled study of the effects offlumazenil in posttraumatic stress disorder. Biol. Psychiatry41, 988–990.
Davies, S.J., Hood, S.D., Argyropoulos, S.V., Morris, K., Bell, C., Witchel, H.J., Jackson, P.R., Nutt, D.J., Potokar, J., 2006.
Depleting serotonin enhances both cardiovascular and psy- Unlike studies of tryptophan depletion in patient chological stress reactivity in recovered patients with anxietydisorders. J. Clin. Psychopharmacol. 26 (4), 414–418.
groups (e.g. panic disorder, depression, etc.), ATD Deakin, J.F., Graeff, F.G., 1991. 5HT and mechanisms of defence.
did not augment the psychological response to a 35% CO2 stressor in healthy controls. Furthermore, there Ellenbogen, M.A., Young, S.N., Dean, P., Palmour, R.M., appears to be no additive effect of ATD on endocrine Benkelfat, C., 1996. Mood response to acute tryptophan or cardiovascular responses to this challenge. A novel depletion in healthy volunteers: sex differences and tempor-al stability. Neuropsychopharmacology 15, 465–474.
finding from the present study was that ATD was Freeman, M.E., Kanyicska, B., Lerant, A., Nagy, G., 2000.
itself a neuroendocrine stressor, the mechanism of Prolactin: structure, function, and regulation of secretion.
which requires further investigation.
Girden, E.R., 1992. ANOVA: Repeated Measures. Sage, Newbury Goddard, A.W., Charney, D.S., Germine, M., Woods, S.W., Heninger, G.R., Krystal, J.H., Goodman, W.K., Price, L.H.,1995. Effects of tryptophan depletion on responses to Abbott, F.V., Etienne, P., Franklin, K.B., Morgan, M.J., Sewitch, yohimbine in healthy human subjects. Biol. Psychiatry 38, M.J., Young, S.N., 1992. Acute tryptophan depletion blocks morphine analgesia in the cold-pressor test in humans.
Gustavsson, J., Bergman, H., Edman, G., Ekselius, L., von Psychopharmacology (Berl) 108, 60–66.
Knorring, L., Linder, J., 2000. Swedish universities Scales of Personality (SSP): construction, internal consistency and Pols, H., Griez, E., Verburg, K., van der, W.D., 1994.
normative data. Acta Psychiatr. Scand. 102, 217–225.
Yohimbine premedication and 35% CO2 vulnerability in Hood, S.D., Bell, C., Nutt, D.J., 2005. Acute tryptophan healthy volunteers. Eur. Arch. Psychiatry Clin. Neurosci.
depletion. Part I: rationale and methodology. Aust. N. Z.
Schafer, J.L., Graham, J.W., 2002. Missing data: our view of the Kaye, J., 2005. Mechanisms and clinical implications of the state of the art. Psychol. Methods 7, 147–177.
neuroendocrine response to a novel carbon dioxide stressor in Schruers, K., Klaassen, T., Pols, H., Overbeek, T., Deutz, N.E., man. Ph.D. Thesis, The University of Western Australia.
Griez, E., 2000. Effects of tryptophan depletion on carbon Kaye, J., Buchanan, F., Kendrick, A., Johnson, P., Lowry, C., dioxide provoked panic in panic disorder patients. Psychiatry Bailey, J., Nutt, D., Lightman, S., 2004a. Acute carbon dioxide exposure in healthy adults: evaluation of a novel Sheehan, D.V., Lecrubier, Y., Sheehan, K.H., Amorim, P., Janavs, means of investigating the stress response. J. Neuroendocri- J., Weiller, E., Hergueta, T., Baker, R., Dunbar, G.C., 1998.
The Mini-International Neuropsychiatric Interview (M.I.N.I.): Kaye, J., Soothill, P., Hunt, M., Lightman, S., 2004b. Responses the development and validation of a structured diagnostic to the 35% CO challenge in postpartum women. Clin.
psychiatric interview for DSM-IV and ICD-10. J. Clin.
Kaye, J.M., Young, T.M., Mathias, C.J., Watson, L., Lightman, Sinha, S.S., Coplan, J.D., Pine, D.S., Martinez, J.A., Klein, D.F., S.L., 2006. Neuroendocrine and behavioural responses to CO2 Gorman, J.M., 1999. Panic induced by carbon dioxide inhalation in central versus peripheral autonomic failure.
inhalation and lack of hypothalamic–pituitary–adrenal axis activation. Psychiatry Res. 86, 93–98.
Klaassen, T., Klumperbeek, J., Deutz, N.E., van Praag, H.M., Smith, K.A., Fairburn, C.G., Cowen, P.J., 1997. Relapse of Griez, E., 1998. Effects of tryptophan depletion on anxiety depression after rapid depletion of tryptophan. Lancet 349, and on panic provoked by carbon dioxide challenge.
Smith, S.E., Pihl, R.O., Young, S.N., Ervin, F.R., 1987. A test of Lowry, C.A., 2002. Functional subsets of serotonergic neurones: possible cognitive and environmental influences on the mood implications for control of the hypothalamic–pituitary–adre- lowering effect of tryptophan depletion in normal males.
nal axis. J. Neuroendocrinol. 14, 911–923.
Psychopharmacology (Berl) 91, 451–457.
Lowry, C.A., Johnson, P.L., Hay-Schmidt, A., Mikkelsen, J., Spielberger, C.D., Gorsuch, R.L., Lushene, R., Vagg, P.R., Jacobs, Shekhar, A., 2005. Modulation of anxiety circuits by seroto- G.A., 1983. Manual for the State-Trait Anxiety Inventory.
nergic systems. Stress 8, 233–246.
Consulting Psychologists’ Press, Palo Alto, CA.
Miller, H.E., Deakin, J.F., Anderson, I.M., 2000. Effect of acute Stampfer, H.G., 1998. The relationship between psychiatric tryptophan depletion on CO2-induced anxiety in patients with illness and the circadian pattern of heart rate. Aust. N. Z.
panic disorder and normal volunteers. Br. J. Psychiatry 176, van den Hout, M.A., van der Molen, G.M., Griez, E., Lousberg, Moore, P., Gillin, C., Bhatti, T., DeModena, A., Seifritz, E., Clark, H., Nansen, A., 1987. Reduction of CO2-induced anxiety in C., Stahl, S., Rapaport, M., Kelsoe, J., 1998. Rapid patients with panic attacks after repeated CO2 exposure. Am.
tryptophan depletion, sleep electroencephalogram, and mood in men with remitted depression on serotonin reuptake van Duinen, M.A., Schruers, K.R., Maes, M., Griez, E.J., 2005.
inhibitors. Arch. Gen. Psychiatry 55, 534–539.
CO2 challenge results in hypothalamic–pituitary–adrenal Nutt, D.J., Glue, P., Lawson, C., Wilson, S., 1990. Flumazenil activation in healthy volunteers. J. Psychopharmacol. 19, provocation of panic attacks. Evidence for altered benzodia- zepine receptor sensitivity in panic disorder. Arch. Gen.
Vasey, M.W., Thayer, J.F., 1987. The continuing problem of false positives in repeated measures ANOVA in psychophy- Oldman, A., Walsh, A., Salkovskis, P., Fairburn, C.G., Cowen, P.J., 1995. Biochemical and behavioural effects of acute tryptophan depletion in abstinent bulimic subjects: a pilot Wetherell, M.A., Crown, A.L., Lightman, S.L., Miles, J.N., Kaye, study. Psychol. Med. 25, 995–1001.
J., Vedhara, K., 2006. The four-dimensional stress test: Park, S.B., Coull, J.T., McShane, R.H., Young, A.H., Sahakian, psychological, sympathetic–adrenal–medullary, parasympa- B.J., Robbins, T.W., Cowen, P.J., 1994. Tryptophan depletion thetic and hypothalamic-pituitary-adrenal responses follow- in normal volunteers produces selective impairments in ing inhalation of 35% CO2. Psychoneuroendocrinology 31, learning and memory. Neuropharmacology 33, 575–588.
Pols, H., Griez, E., Bourin, M., Schruers, K., 1999. Effect of CCK-4 Young, S.N., Smith, S.E., Pihl, R.O., Ervin, F.R., 1985. Trypto- on a 35% carbon dioxide challenge in healthy volunteers. Prog.
phan depletion causes a rapid lowering of mood in normal Neuropsychopharmacol. Biol. Psychiatry 23, 1345–1350.
males. Psychopharmacology (Berlin) 87, 173–177.

Source: http://gc.nesda.com.br/Conteudo/Arquivos/Biblioteca/Artigos%20T%C3%A9cnicos/Artigos%20B%C3%A1sicos%20de%20Neuroimunomodula%C3%A7%C3%A3o/serotonina%20no%20stress%20humano.pdf