CEST Imaging of the Serotonin Pathway
Rafal Janik1, Lynsie A.M. Thomason2, and Greg J. Stanisz1,2,3

1Medical Biophysics, University of Toronto, Toronto, ON, Canada, 2Physical Sciences, Sunnybrook Research Institute, Toronto, ON, Canada, 3Department of Nerurosurgery and Pediatric Neurosurgery, Medical University of Lublin, Lublin, Poland


A novel method for the detection of brain 5-HT, tryptophan, and 5-HIAA is presented. The method relies on the chemical exchange of an amide proton which is shifted outside the normal range for amide protons. This is demonstrate in-vivo in a rat model of 5-HT increase.


In this work we present what we believe to be the first detection of serotonin along with it’s derivatives and substrate, in vivo using a noninvasive technique. Serotonin (5-HT) is uniquely amenable to being measured using CEST. 5HT, along with it’s derivative 5HIAA and its substrate tryptophan, have a amide proton which is shifted far outside (10.5ppm referenced to tetramethylsilane) the usual range of exchangeable protons (fig. 1), and 5-HT remains in the slow exchange regime over the range of physiological pH values 1. 5-HT, 5-HIAA, and tryptophan concentrations in the brain range from 10-100µM 2, and as a result cannot be directly and noninvasively measured. 5-HT is currently implicated in the patho-physiology of major depressive and anxiety disorders3, and it is the target of many pharmacological interventions such as mono amine oxidase inhibitors (MAOIs), and serotonin selective reuptake inhibitors (SSRIs)3.


All experiments where conducted on a Bruker (Ettlingen, Germany) 70/30 Biospec MRI system. Phantom experiments: Chemical exchange rates of 5-HT phantoms of varying concentration (25mM, 15mM, 5mM, and 1mM) were measured using a continuous wave (CW) saturation prepared PRESS (repetition time/echo time=10,000/25ms, saturation time=4,800ms) sequence with variable saturation pulse powers (0.1-3.0µT) over a range of saturation offsets (-2,500Hz to 3,000Hz, with 20Hz spacing). T1 and T2 relaxation time constants of the phantoms were measured using an inversion prepared fast spin echo sequence (repetition time/echo time=10,000/42ms, FSE factor=8) and a CPMG sequence (repetition time/echo time spacing=10,000/5ms, number of echoes=128) respectively. The water line areas for each saturation time were fit using full Bloch equation, including chemical exchange, simulations. Measured T1 and T2 of the water pool were provided as known parameters. In total four parameters were fit: the parameters CEST exchange rate constant, Rex, proton fraction of the CEST pool, Mob, its longitudinal, T1b and transverse, T2b relaxation times. Phantom parameters were then used for in vivo data analysis. Animal experiments: CEST spectra were measured in 4 naïve rats and 5 rats treated with MAOIs and tryptophan (IP injection 20 mg/kg tranylcypromine followed by 100mg/kg L-tryptophan, animals were imaged once they started exhibiting behavioral symptoms of 5-HT overload ~1.5 hours)2, in order to increase brain 5-HT, before and after treatment. In vivo CEST experiments were carried out using a saturation prepared echo planar imaging sequence (repetition time/echo time=5,000/21ms, saturation time=4800ms, saturation offsets= -2,000Hz to 2,500Hz, with 20Hz spacing, saturation power=1.0µT). B1 mapping before and after the CEST acquisition was performed using WASSR. Reference frames at a saturation offset frequency of 20 kHz were acquired every other. CEST spectra for each voxel were interpolated and shifted according to the B1 maps and averaged for anatomical ROI (cortical GM, WM, hippocampus, and other subcortical GM). The average CEST spectra were fit using five Lorentzian peaks, to account for the direct effect, semi-solid MT, NOE, amide CEST, and amine CEST plus a sixth peak (5-HT) with fixed position and width determined by the phantom experiments. 5-HT CEST peak areas were compared with 5-HT concentrations were assessed ex-vivo using an enzyme-linked immunosorbent assay (ELISA) (Rocky Mountain Diagnostics, Colorado, USA).


The exchange rate of the amide proton, Rex on 5-HT (pH=7.4, T=37°) was measured. In phantoms the area of the CEST peak correlated with 5-HT concentration (R=0.94, p<0.01). Naïve animals showed no statistically significant change in the area of the 5-HT CEST peak between imaging sessions. Animals treated with MAOIs and tryptophan after the first imaging session showed a statistically significant increase (14±3% p<0.05) in the 5-HT CEST peak in the cortical gray matter ROI, consistent with the expected increase in brain 5-HT levels. The increase in cortical 5-HT CEST can be seen in individual animals as shown in figure 2. Data for both naïve and treated animals is summarized in figure 3. The area of the 5-HT CEST peak shows good linearity with ex vivo concentration of 5-HT. This work represents the first in vivo MR measurement of 5-HT along with its substrate, tryptophan, and derivative, 5-HIAA. We believe that this measurement will serve as an important biomarker in future studies investigating the roll of serotonin in major brain disorders.


No acknowledgement found.


1. Ward, K. M. & Balaban, R. S. Determination of pH using water protons and chemical exchange dependent saturation transfer (CEST). Magn Reson Med 44, 799–802 (2000).

2. Fernstrom, J. D. & Wurtman, R. J. Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 173, 149–152 (1971).

3. Nemeroff, C. B. & Owens, M. J. The role of serotonin in the pathophysiology of depression: as important as ever. Clinical chemistry (2009).


Serotonin (5-HT) has an exchangeable proton on the aromatic rings as shown in Panel A label A with a chemical shift far from the usual range of amide and amine protons as shown in the proton spectrum (Panel B peak A). The proton exhibits a strong CEST effect (C).

T2 weight anatomical image showing the imaging slice (A). EPI image a reference saturation offset of 20kHz with cortical GM mask overlaid in red (B). Average cortical GM spectra are shown in (C) for an individual animal before and after treatment with MAOI + Tryptophan. An increase in the area of the CEST peak is evident even in an individual animal.

Box plot showing the changes in the area of the 5-HT CEST peak before and after treatment with MAOIs and Tryptophan. No change is seen in naïve animals but there is a statistically significant increase of 14±3% p<0.05 in treated animals.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)