Mapping serotonergic dynamics using drug-modulated molecular connectivity

Understanding the complex workings of the brain is one of the most significant challenges in neuroscience, providing insights into the healthy brain, diseases, and the effects of potential therapeutics. A major challenge in this field is the limitations of traditional brain imaging techniques, which often deliver only a part of the complex puzzle of brain function. Our research employs a novel approach named "Molecular Connectivity" (MC), which merges the strengths of various imaging methods to offer a comprehensive view of how molecular imaging readouts interact across different areas of the brain.

This innovative technique bridges the gap between functional magnetic resonance imaging (fMRI), known for its ability to monitor brain activity by tracking blood flow, and positron emission tomography (PET), which can depict specific molecular changes. By integrating these methods, we can better understand the far-reaching impacts of drugs on the brain. Our study focuses on the application of dynamic [11C]DASB PET scans to map the distribution of serotonin transporters, a key player in regulating mood and emotions, and examines how these are altered following the use of methylenedioxymethamphetamine (MDMA), commonly known as ecstasy.

Through a detailed analysis comparing MC with traditional measures of brain connectivity, we uncover significant patterns that closely align with physiological changes. Our results reveal clear changes in molecular connectivity after a single dose of MDMA, establishing a direct link between the drug's effects on serotonin transporter occupancy and changes in the brain's functional network.

This work not only offers a novel methodology for the in-depth study of brain function at the molecular level but also opens new pathways for understanding how drugs modulate brain activity.

Methods
For the baseline dataset (cited from Ionescu T.M. et al. 2021 Striatal and prefrontal D2R and SERT distributions contrastingly correlate with default-mode connectivity, Neuroimage 243)

"Anesthesia was induced in knock-out boxes by delivering 3% isoflurane in regular air until reflex tests indicated sufficient sedation. For the following preparation steps the concentration of isoflurane was reduced to 2%. The weights of the animals were measured and a catheter was placed into a tail vein using a 30G needle for tracer administration. Subsequently, the rats were transferred onto a dedicated feedback temperature-controlled rat bed (Medres, Cologne, Germany). A rectal probe was positioned to monitor and maintain a stable body temperature at 36.5°C and a breathing pad was used to observe respiration rates. Finally, the animals were introduced into the PET/MRI scanner and the isoflurane concentration was reduced to 1.3 % during the scan.

The scans were acquired using a small-animal 7 T ClinScan scanner (Bruker BioSpin MRI, Bruker, Ettlingen, Germany) with a linearly polarized RF coil (Bruker) of 72 cm in diameter for transmission and a four channel rat brain coil (Bruker) for reception. Localizer scans were first acquired to accurately position the rat brains into the center of the PET/MRI field of view. Subsequently, local field homogeneity was optimized by measuring local magnetic field maps. Anatomical reference scans were then performed using T2-weighted MRI sequences (TR: 1800 ms, TE: 67.11 ms, FOV: 40 x 32 x 32 mm3, image size: 160 x 128 x 128 px, Rare factor: 28, averages: 1). Finally, T2*-weighted gradient echo EPI sequences (TE: 18 ms, TR: 2500 ms, 0.25 mm isotropic resolution, FoV 25 x 23 mm2, image size: 92 x 85 x 20 px, slice thickness: 0.8 mm, 20 slices) were acquired for functional MR imaging.

A small-animal PET insert developed in cooperation with Bruker (Bruker Biospin, Ettlingen Germany) was used for [11C]DASB and [11C]raclopride acquisitions. This insert is the second generation of a PET insert developed in-house described previously (Judenhofer et al., 2008). Both generations of PET inserts have similar technical specifications. The radioactive tracers were applied via a bolus plus constant infusion protocol with a Kbol of 38.7 min using an initial bolus of (...) 152 ± 44 MBq for [11C]DASB in a volume of 0.48 ml over 20 s, followed by a constant infusion of 15 µl/min until the end of the scan. PET/fMRI acquisition was started simultaneously with the tracer injection and was performed over a period of 80 min. The PET data were saved as list-mode files and reconstructed using an ordered-subsets expectation maximization 2D (OSEM-2D) algorithm written in-house and then converted to NiFTI format."
For the MDMA dataset (cited from Ionescu T.M. et al. Neurovascular Uncoupling: Multimodal Imaging Delineates the Acute Effects of 3,4-MethylenedioxymethamphetamineJ Nucl Med 64:466–471):

"The animals were scanned under 1.3% isoflurane and constant monitoring of breathing rate and temperature (Supplemental Fig. 1) using a 7-T small-animal MRI scanner (ClinScan; Bruker). T2-weighted anatomic reference scans and fMRI scans (repetition time, 2,000 ms; echo time, 18 ms) were obtained using a linearly polarized radiofrequency coil for transmission and a 4-channel surface rat brain coil for reception. The PET scans were acquired simultaneously using an in-house–developed insert and reconstructed into 100 frames of 1 min using an ordered-subsets expectation-maximization 2-dimensional algorithm. The MDMA challenge (3.2 mg/kg) was applied 40 min after the start of the acquisition."