Measuring serotonin synthesis: from conventional methods to PET tracers and their (pre)clinical implications

The serotonergic system of the brain is complex, with an extensive innervation pattern covering all brain regions and endowed with at least 15 different receptors (each with their particular distribution patterns), specific reuptake mechanisms and synthetic processes. Many aspects of the functioning of the serotonergic system are still unclear, partially because of the difficulty of measuring physiological processes in the living brain. In this review we give an overview of the conventional methods of measuring serotonin synthesis and methods using positron emission tomography (PET) tracers, more specifically with respect to serotonergic function in affective disorders. Conventional methods are invasive and do not directly measure synthesis rates. Although they may give insight into turnover rates, a more direct measurement may be preferred. PET is a noninvasive technique which can trace metabolic processes, like serotonin synthesis. Tracers developed for this purpose are α-[11C]methyltryptophan ([11C]AMT) and 5-hydroxy-L-[β-11C]tryptophan ([11C]5-HTP). Both tracers have advantages and disadvantages. [11C]AMT can enter the kynurenine pathway under inflammatory conditions (and thus provide a false signal), but this tracer has been used in many studies leading to novel insights regarding antidepressant action. [11C]5-HTP is difficult to produce, but trapping of this compound may better represent serotonin synthesis. AMT and 5-HTP kinetics are differently affected by tryptophan depletion and changes of mood. This may indicate that both tracers are associated with different enzymatic processes. In conclusion, PET with radiolabelled substrates for the serotonergic pathway is the only direct way to detect changes of serotonin synthesis in the living brain

Sex differences of brain serotonin synthesis in patients with irritable bowel syndrome using α-[ 11 C]methyl-L-tryptophan, positron emission tomography and statistical parametric mapping

BACKGROUND: Irritable bowel syndrome (IBS) is the most common functional bowel disorder and has a strong predominance in women. Recent data suggest that the brain may play an important role in the pathophysiology of IBS in the brain-gut axis. It is strongly suspected that serotonin (5-HT), a neurotransmitter found in the brain and gut, may be related to the pathophysiology of IBS. It is reported that a 5-HT 3 antagonist is effective only in female patients with diarrhea-predominant IBS. OBJECTIVE: In the present study, 5-HT synthesis was measured using positron emission tomography, with α-[ 11 C]methyl-L-tryptophan as the tracer, in patients with IBS. The aim of the present study was to compare 5-HT synthesis in the IBS patients with that in the controls, and to compare 5-HT synthesis between male and female IBS patients. METHODS: Six male and six female nonconstipated IBS patients were scanned. Age-matched healthy volunteers were scanned as controls. Eighty minute dynamic scans were performed. Functional 5-HT synthesis images were analyzed using statistical parametric mapping. RESULTS: 5-HT synthesis was greater only in the female IBS patients in the right medial temporal gyrus (multimodal sensory association cortex) compared with the female controls (P<0.001). CONCLUSIONS: The greater brain 5-HT synthesis in the female IBS patients than in the controls may be related to the pathological visceral pain processing of the IBS patients, a larger female predominance of the disorder, and the sex difference of the efficacy of the 5-HT 3 antagonist in treatment. Key Words: Irritable bowel syndrome; Positron emission tomography; Serotonin; Sex differences Différences de sexe quant à la synthèse de la sérotonine dans le cerveau chez des patients souffrant du syndrome du côlon irritable, relevées à la tomographie par émission de positrons avec l'alpha-[ 11 C]méthyl-L-tryptophane et à la cartographie paramétrique statistique CONTEXTE : Le syndrome du côlon irritable (SCI) est le trouble fonctionnel intestinal le plus fréquent et il se rencontre surtout chez les femmes. Selon des données récentes, le cerveau jouerait un rôle important dans la physiopathologie du syndrome le long de l'axe cerveau-tube digestif. On croit fermement que la sérotonine (5-HT), un neurotransmetteur présent à la fois dans le cerveau et le tube digestif, n'est pas étrangère à la physiopathologie de l'affection. Des rapports indiquent que l'antagoniste des récepteurs 5-HT 3 n'est efficace que chez les femmes souffrant du SCI à prédominance diarrhéique. OBJECTIF : Nous avons mesuré, dans la présente étude, la synthèse de la 5-HT au moyen de la tomographie par émission de positrons en utilisant l'alpha-[ 11 C]méthyl-L-tryptophane comme traceur chez des patients atteints du SCI. L'étude visait à comparer la synthèse de la 5-HT chez des patients souffrant du syndrome et chez des témoins ainsi qu'entre patients de sexe différent. MÉTHODE : Six hommes et six femmes atteints du SCI sans constipation ont été soumis à la tomographie, de même que des témoins volontaires en bonne santé, appariés selon l'âge. Nous avons d'abord procédé à un enregistrement dynamique de données durant 80 minutes, puis analysé les images fonctionnelles de la synthèse de la 5-HT au moyen de la cartographie paramétrique statistique. RÉSULTATS : Seules les femmes souffrant du SCI ont présenté une augmentation de la synthèse de la 5-HT dans la deuxième circonvolution temporale droite (aire des associations plurisensorielles) par rapport aux femmes témoins (P<0,001). CONCLUSION : La synthèse accrue de la 5-HT cérébrale chez les femmes souffrant du SCI comparativement aux témoins peut être liée au processus pathologique de douleur viscérale, trouble à forte prédominance féminine, et à la différence d'efficacité du traitement à l'antagoniste des récepteurs 5-HT 3 . I rritable bowel syndrome (IBS) is a common functional bowel disorder, and is characterized by abdominal pain and alterations of defecation (1,2) in the absence of biochemical or structural abnormalities that could normally explain the symptoms. The adult prevalence of the disorder is approximately 20% in western populations (3). IBS is described by multiple factors such as affective factors, including cognitive, and physiological and behavioural factors. In addition, all of these different factors are interconnected, making it even harder to describe the definitive importance of each of them in the generation and/or as a consequence of IBS symptoms. The central nervous system modulates peripheral intestinal motor or sensory activity and vice versa. These domains interact through the bidirectional parallel circuits, the so-called brain-gut axis ORIGINAL ARTICLE ©2003 Pulsus Group Inc. All rights reserved which links the visceral afferent sensation and intestinal motor function with higher cortical centres (4-7). Recent advances in the investigation of the brain-gut axis suggest that the brain plays an important role in the pathophysiology of IBS (4-7). For example, stress often induces or exacerbates major symptoms in IBS patients Serotonin (5-HT) is one of the neurotransmitters found in the brain and gut, that may regulate and/or modulate activities related and/or resulting from IBS (11). 5-HT terminals are widely distributed throughout the brain from the cell bodies found in the brainstem (12). Because of the widely spread distribution of 5-HT projections, it is not surprising that serotonergic neurotransmission is involved in many physiological as well as pathophysiological processes in the brain. In addition, there is a great interconnection between many brain structures, which suggests the possibility that 5-HT synthesis in some brain structures may be affected directly by brain-gut interactions, while in other structures, the effect could be an indirect one. Indeed, it has been reported that in diarrhea-predominant IBS patients, postprandially, the serum levels of 5-HT are higher than in the normal population (13). However, it is difficult to relate the plasma levels of 5-HT or 5-HT metabolites to any actions of 5-HT in the brain It has also been reported that alosetron, a 5-HT 3 antagonist, may have therapeutic potential in female diarrhea-predominant IBS patients (15,16). The 5-HT 3 receptors are present on the peripheral and central neurons It has been reported, in community samples, that IBS symptoms have a two to one predominance in women (3). Recent advances in neuroimaging techniques revealed that patients with IBS may exhibit abnormal integration of painful stimulation centrally has been developed as a tracer for the measurement of brain 5-HT synthesis rates in living mammals (14). This tracer has been used to measure 5-HT synthesis rates in the human brain using positron emission tomography (PET). The synthesis rates are calculated from the blood-to-brain clearance constant. The method is based on the unidirectional uptake of α-[ 11 C]MTrp, which is transported and, in part, trapped in the brain. It has been shown that in the rat brain, it is converted, in part, to α-methyl-5-HT, and that the brain trapping of α-MTrp correlates with the brain conversion of tryptophan to 5-HT but not with the tryptophan incorporation into proteins or tryptophan transport through the blood brain barrier (14). Tissue radioactivity images of α-[ 11 C]MTrp can be easily converted to functional images representing the regional rates of 5-HT synthesis in the brain using the Patlak plot approach (27). The functional images can then be used for statistical comparisons, such as statistical parametric mapping (SPM). SPM analyses have the advantage over the region of interest comparisons because they remove the intersubject variability, and standardize the images to the Talairach space (28) -the procedures that facilitate comparisons of regional differences. In addition, the observer cannot introduce any bias to the comparison. The authors previously reported on the use of SPM for comparisons between male and female normal subjects (29) as well as between patients with psychiatric disorders, such as borderline personality disorder (30). In the present study, brain 5-HT synthesis was measured with PET using α-[ 11 C]MTrp as the tracer in 12 patients with IBS, and compared with respective age-matched healthy controls using SPM. On the basis of the possible involvement of 5-HT in IBS, as described above, it was hypothesized that there may be regional differences in brain 5-HT synthesis between IBS patients and the controls; and that there may be sex specific differences in brain 5-HT synthesis among the IBS patients. It could also be hypothesized that these differences in 5-HT synthesis may be related to the brain regions involved in the processing of visceral sensation in IBS patients. METHODS Subjects Six male (mean age 44±9 years; mean ± SD) and six female (mean age 52±11 years) nonconstipation-predominant IBS patients were scanned. The diagnoses of IBS were made based on the Rome I criteria for IBS and each patient underwent a basic evaluation to exclude organic disease. Psychiatric evaluations were also conducted using the Diagnostic and Statistical Manual of Mental Disorders (DSM)-III-R (31). The patients did not take any prohibited medication, including IBS treatments, antidepressants, anxiolytics, narcotics, 5-HT3 antagonists and tryptophan supplements, for more than seven days before the PET scan. Agematched male (n=6, mean age of 40±13 years) and female (n=7, mean age 52±9 years), right-handed, healthy volunteers were scanned as controls. There is no statistical significance in age between the control and patient and sex groups. Exclusion criteria for the subjects included evidence of a past or present Axis-I or II DSM-III-R diagnosis in the subjects or in the subjects' first-degree relatives, or any significant medical illness, including gastrointestinal diseases. McGill University, and Centre Hospitalier de l'Université de Montréal. All the subjects provided written informed consents before the study. PET procedure T he radiopharmaceutical α-[ 11 C]MTrp was prepared by the method reported by Mzengeza et al (32). The day before the PET study, all subjects received a standard low-protein diet to reduce the variability in the plasma concentrations of amino acids, including tryptophan, that may be attributable to individual differences in diet. The tracer was injected over 2 min, and 80-min dynamic PET scans were performed commencing at the beginning of the tracer injection. The duration of the acquisition frames progressively increased from 1 to 10 min. Blood samples drawn from the antecubital vein were taken at progressively increased time intervals (15 s to 10 min). Five additional venous samples were drawn during the PET scans for high performance liquid chromatography analysis to measure the free and total plasma concentration of tryptophan. The PET scans were obtained with an ECAT EXACT HR+ wholebody tomography scanner (CTI/Siemens, USA) that has 63 image slices at an intrinsic resolution of 5.0×5.0×5.0 mm fullwidth half-maximum value. The scans were acquired and reconstructed three dimensionally. The images were blurred, for use with SPM, with a Gaussian filter to a final resolution of 14 mm. The transmission scans were performed using 68 Ga for the attenuation correction. Each subject underwent a high resolution magnetic resonance imaging (MRI) scan (160 slices, 1 mm thick) obtained with a Siemens Vision (1.5T) (CTI/Siemens, Mississauga, Ontario). The coregistration of the individual PET and MRI images was performed using an automatic procedure (33). The MRI images from each subject were transferred into Talairach space (28) and coregistered with the functional PET images (34). Comparisons between groups were done using the SPM program. To calculate 5-HT synthesis rates with the venous samples, normalized input functions were estimated, using a previously reported procedure (35). Briefly, the input function for the initial 20 min was derived from time-radioactivity curves (TACs) obtained from the sagittal sinus normalized to the venous plasma radioactivity at 20 min. After 20 min, the venous plasma TAC was used. TACs from the sagittal venous sinus were established by drawing small regions of interest on two to three slices at the level of the confluence of sinuses. Calculations of 5-HT synthesis rate images were then made with normalized venous sinus TACs derived from a combination of the two curves. As was previously reported, this approach provides results not significantly different from those obtained using the arterial input function (34,35). The rate of 5-HT synthesis (R, pmol/g/min) is an algebraic product of the net blood to brain clearance of the tracer (K*, mL/g/min) and the availability of the endogenous 5-HT precursor, L-tryptophan (14,36,37). To calculate K*, tissue data, in the form of distribution volumes (mL/g), were fitted as a function of exposure time (min). K* values were then determined using the Patlak plot method (27) applied to the data points obtained from 20 min to 60 min as previously reported Statistical analysis Plasma free and total tryptophan were compared statistically by an analysis of variance (ANOVA). Statistical significance was defined as P<0.05. The functional PET data was analyzed with SPM analysis, using the SPM99 software (39-42). Before the SPM comparison, all of the images were smoothed with the Gaussian filter (see above) to increase the signal-to-noise ratio and to reduce the effect of the individual variability in the cortical gyral anatomy of the brain (39-41). The functional images were normalized using proportional scaling to the global mean (global mean = 50 pmol/g/min; close to the mean 5-HT synthesis rate or in the case of K* -images to 10 mL/g/min). SPM comparisons were performed at a threshold of 80% of the peak value (39). The t-test was applied pixel by pixel to compare the regional differences in 5-HT synthesis images between the men and women. The SPM{t} data for each pixel was then transformed to the unit normal distribution (SPM{z}), which makes parametric maps independent of the effective degrees of freedom RESULTS In The SPM comparison between the male and female healthy controls did not have any significant differences in brain 5-HT synthesis when the above mentioned criteria were used (a clus- ter of 100 voxels and a height at P<0.001). There were also no clusters reaching these criteria for significance between the different sexes in the IBS group (data not shown). In the SPM analysis of the data sets after separating the subjects into sex specific groups, we only observed significant differences in brain 5-HT synthesis in the female comparisons. The brain areas with significant differences in 5-HT synthesis that are superimposed on the three dimensional images of the Talairach brain or overlayed on the representative MRI slices are shown in In contrast to the results of the comparison between the female patients, there is no significant difference between male IBS patients and the age-matched male control subjects. In a comparison between all the IBS patients (males and females pooled together) and the pooled control subjects, there was no significant brain region in which 5-HT synthesis differed between the pooled control subjects and the pooled IBS patients at this threshold. DISCUSSION In the present study, we demonstrated for the first time that there are significant differences in regional 5-HT synthesis in the brain between female IBS patients and female controls. This is the first study evaluating 5-HT synthesis in IBS patients and it possibly indicates that brain-gut interactions are related to 5-HT synthesis in such patients. There was no significant difference between the male patients and the respective controls. The plasma total tryptophan in the male controls was significantly higher than that in the female controls, and also higher than in the male and female IBS patients. The plasma free tryptophan in the female controls was significantly higher than that in the female IBS patients. Lower plasma total and free tryptophan levels in the women than in the men were observed in our previous study (44) and in studies reported previously by others In contrast to the comparisons between female patients and female controls, there are no significant differences in the male comparisons and in the comparisons between the male and female IBS patients in this study. This may be due to the small sample size and/or strict statistical threshold we used. With a less stringent threshold at P<0.005 and 100 voxels, however, the male IBS patients also showed higher 5-HT synthesis than that of the age-matched controls in the multimodal sensation association cortex, although the significance and size of clusters are less and smaller than the female comparisons with the same threshold (data not shown). Further, at this threshold, the female IBS patients also showed higher 5-HT synthesis in the prefrontal cortex than the female controls (data not shown). Everyday experiences, including sensory information, unfold in multiple modalities (52). Chronic symptoms of IBS, including visceral sensation through the neuronal network, which is mentioned above, could affect the multimodal convergence from sensation to cognition in this cortex, as the brain-gut axis. The results of our study may suggest the important role of this cortex in the pathophysiology of IBS and may provide an explanation of the central effects of alosetron in IBS through the 5-HT 3 antagonism in the multimodal sensation association cortex with sex differences. CONCLUSIONS Our preliminary data presented here suggest that chronic IBS symptoms result in an increase in 5-HT synthesis in the brain, and these increases appear to be sex specific. The higher 5-HT synthesis in the brains of female IBS patients than in the controls may be related to the pathological visceral pain processing of IBS patients, a larger predominance and the efficacy of the 5-HT 3 antagonist in women

Brain Serotonin Synthesis in Adult Males Characterized by Physical Aggression during Childhood: A 21-Year Longitudinal Study

Adults exhibiting severe impulsive and aggressive behaviors have multiple indices of low serotonin (5-HT) neurotransmission. It remains unclear though whether low 5-HT mediates the behavior or instead reflects a pre-existing vulnerability trait.C-AMT bilaterally in the orbitofrontal cortex and self-reported more impulsiveness. Despite this, in adulthood there were no group differences in plasma tryptophan levels, genotyping, aggression, emotional intelligence, working memory, computerized measures of impulsivity, psychosocial functioning/adjustment, and personal and family history of mood and substance abuse disorders.These results force a re-examination of the low 5-HT hypothesis as central in the biology of violence. They suggest that low 5-HT does not mediate current behavior and should be considered a vulnerability factor for impulsive-aggressive behavior that may or may not be expressed depending on other biological factors, experience, and environmental support during development

Serotonin synthesis, release and reuptake in terminals: a mathematical model

<p>Abstract</p> <p>Background</p> <p>Serotonin is a neurotransmitter that has been linked to a wide variety of behaviors including feeding and body-weight regulation, social hierarchies, aggression and suicidality, obsessive compulsive disorder, alcoholism, anxiety, and affective disorders. Full understanding of serotonergic systems in the central nervous system involves genomics, neurochemistry, electrophysiology, and behavior. Though associations have been found between functions at these different levels, in most cases the causal mechanisms are unknown. The scientific issues are daunting but important for human health because of the use of selective serotonin reuptake inhibitors and other pharmacological agents to treat disorders in the serotonergic signaling system.</p> <p>Methods</p> <p>We construct a mathematical model of serotonin synthesis, release, and reuptake in a single serotonergic neuron terminal. The model includes the effects of autoreceptors, the transport of tryptophan into the terminal, and the metabolism of serotonin, as well as the dependence of release on the firing rate. The model is based on real physiology determined experimentally and is compared to experimental data.</p> <p>Results</p> <p>We compare the variations in serotonin and dopamine synthesis due to meals and find that dopamine synthesis is insensitive to the availability of tyrosine but serotonin synthesis is sensitive to the availability of tryptophan. We conduct <it>in silico </it>experiments on the clearance of extracellular serotonin, normally and in the presence of fluoxetine, and compare to experimental data. We study the effects of various polymorphisms in the genes for the serotonin transporter and for tryptophan hydroxylase on synthesis, release, and reuptake. We find that, because of the homeostatic feedback mechanisms of the autoreceptors, the polymorphisms have smaller effects than one expects. We compute the expected steady concentrations of serotonin transporter knockout mice and compare to experimental data. Finally, we study how the properties of the the serotonin transporter and the autoreceptors give rise to the time courses of extracellular serotonin in various projection regions after a dose of fluoxetine.</p> <p>Conclusions</p> <p>Serotonergic systems must respond robustly to important biological signals, while at the same time maintaining homeostasis in the face of normal biological fluctuations in inputs, expression levels, and firing rates. This is accomplished through the cooperative effect of many different homeostatic mechanisms including special properties of the serotonin transporters and the serotonin autoreceptors. Many difficult questions remain in order to fully understand how serotonin biochemistry affects serotonin electrophysiology and vice versa, and how both are changed in the presence of selective serotonin reuptake inhibitors. Mathematical models are useful tools for investigating some of these questions.</p