bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446169; this version posted May 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Simultaneous fMRI and fast-scan cyclic voltammetry bridges oxygenation and neurotransmitter dynamics across 2 spatiotemporal scales 3 Lindsay R Waltona,b,c* 4 Matthew Verbera,b,d 5 Sung-Ho Leea,b,c 6 Tzu-Hao Chaoa,b,c 7 R. Mark Wightmand 8 Yen-Yu Ian Shiha,b,c* 9 aCenter for Animal MRI, University of North Carolina at Chapel Hill, Chapel Hill, NC USA 10 bBiomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, NC USA 11 cDepartment of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC USA 12 dDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC USA 13 *Corresponding authors contact information: [email protected] (LR Walton); [email protected] (YY 14 Shih) 15 Abstract: 16 The vascular contributions of neurotransmitters to the hemodynamic response are gaining more attention in 17 neuroimaging studies, as many neurotransmitters are vasomodulatory. To date, well-established electrochemical 18 techniques that detect neurotransmission in high magnetic field environments are limited. Here, we propose an 19 experimental setting enabling simultaneous fast-scan cyclic voltammetry (FSCV) and blood oxygenation-dependent 20 functional magnetic imaging (BOLD fMRI) to measure both local tissue oxygen and dopamine responses, and global 21 BOLD changes, respectively. By using MR-compatible materials and the proposed data acquisition schemes, FSCV 22 detected physiological analyte concentrations with high spatiotemporal resolution inside of a 9.4 T MRI bore. We 23 found that tissue oxygen and BOLD correlate strongly, and brain regions that encode dopamine amplitude 24 differences can be identified via modeling simultaneously acquired dopamine FSCV and BOLD fMRI time-courses. 25 This technique provides complementary neurochemical and hemodynamic information and expands the scope of 26 studying the influence of local neurotransmitter release over the entire brain. 27 28 Introduction 29 Coupling between neuronal activity and cerebral hemodynamic changes ensures that healthy energetic 30 homeostasis is maintained within the active brain1,2. Classic blood oxygenation-level dependent functional magnetic 31 resonance imaging (BOLD fMRI) data interpretation assumes that hemodynamic responses to stimuli are 32 proportional to neuronal activity, such that hemodynamic response functions (HRF)1 can be derived to translate 33 neuronal activity into BOLD signal predictions3. However, these assumptions are not always valid. Neurotransmitters 34 have been receiving more attention as contributors to the hemodynamic response, as many are vasomodulatory 35 (e.g., dopamine, nitric oxide, norepinephrine)1,4 and dysregulated in pathologies where neurovascular coupling is 36 also dysregulated, such as schizophrenia and Parkinson’s1,5. It is paramount to understand how specific 37 neurotransmitters influence vascular responses and accurately interpret neuroimaging data throughout the brain. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446169; this version posted May 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 38 Dopamine is a vasomodulatory neurotransmitter concentrated in the striatum4,6, and plays a strong role in 39 motivation, movement, addiction, learning, and reward prediction7. Dopamine receptors are located on 40 microvasculature within the striatum, thalamus, and cortex, as well as on astrocytes6,8. Though dopamine has been 41 studied intensely for decades, few studies have delved into its vascular modulation properties. Amphetamine and 42 phencyclidine challenges show a linear relationship between evoked striatal hemodynamic changes and dopamine9– 43 11, and D1- and D2-like dopamine receptor agonists show diametrically opposed vascular responses6. Though fMRI 44 responses to cocaine and amphetamine challenge have been identified10, existing HRF models do not consider 45 dopaminergic influence from shorter release events where higher affinity, presynaptic receptor binding would have 46 more of an impact9. Dissecting how dopamine influences striatal vasculature is an important first step into 47 understanding how whole-brain hemodynamics are affected by dopaminergic neurotransmission. 48 Popular methods of monitoring dopamine or other neurotransmitter kinetics in vivo are electrochemical, 49 such as fast-scan cyclic voltammetry (FSCV) and amperometry12, though advances are being made with fluorescent 50 sensor and magnetic resonance-based detection13–16, among others17. FSCV is especially desirable because it is 51 chemically selective and quantifiable, and multiple, different neurotransmitters can be detected using well- 52 established modifications12,17. It has high spatiotemporal resolution and minimal tissue damage, especially versus 53 competing techniques like positron emission tomography and microdialysis17. FSCV translates directly to human 54 use18–20, a challenge for fluorescent sensor-based techniques, and is therefore a promising method for studying 55 neurotransmission during human behavior19, improving therapeutic deep-brain stimulation21, and interpreting 56 human pharmacological fMRI studies22. Though FSCV can detect both tonic23 and phasic dopamine changes, and 57 oxygen changes24–27, its high spatial resolution limits the scope of interpretation. To reveal the influence of local 58 dopamine release on whole brain circuits requires pairing FSCV with fMRI to acquire neurotransmitter and brain- 59 wide hemodynamic data simultaneously. 60 In this work, we develop and characterize a simultaneous FSCV-fMRI technique. We address the challenges 61 of developing an MR-compatible FSCV recording system, circumventing high-frequency electronic noise from MR 62 imaging gradients, and synchronizing multimodal data acquisition. We perform in vivo FSCV-fMRI during an oxygen 63 inhalation challenge using an oxygen-sensitive FSCV waveform and electrical deep brain stimulation using both 64 oxygen- and dopamine-sensitive FSCV waveforms at an electrode implanted into the nucleus accumbens (NAc). To 65 compare hemodynamic measurements at different spatial scales, we collected tissue oxygen and BOLD fMRI data 66 concurrently. We derive an HRF from high-resolution dopamine data acquired during simultaneous fMRI, and for 67 the first time, we demonstrate that this simultaneous FSCV-fMRI recording platform can identify brain regions that 68 encode dopamine amplitude changes. This method should contribute significantly to the understanding of local 69 neurotransmission on dynamic changes of brain activity. 70 71 Results 72 fMRI and FSCV compatibility 73 A major step to achieve simultaneous FSCV-fMRI is ensuring that FSCV material components do not produce 74 imaging artifacts. Traditional glass capillary microelectrodes, like those used for FSCV, produce minor artifacts28, but 75 polyimide-fused silica further enhances MR-compatibility29 and is also used with FSCV microelectrodes (Fig.1A)30. 76 Both in agarose phantoms and in vivo, polyimide-fused silica electrodes showed minimal MR artifacts (Fig.1A-C); 77 however, modifications were necessary to maximize MR compatibility. Standard fabrication connects the carbon 78 fiber directly to a connection pin via silver epoxy, which provides structural support within the headcap and makes 79 the electrode suitable for chronic implantation30. Unfortunately, commercially available pins contain trace amounts 80 of nickel, a magnetic material with large susceptibility artifacts31. Here, we silver epoxied the carbon fiber directly 81 onto silver wires. Following implantation, 1 cm of silver wire from both working and Ag/AgCl reference electrodes 82 was left exposed above the headcap during recovery (Fig.1D). To protect the free-standing wires, plastic shields were 83 cemented to the headcaps (Fig.1D). We performed flow-through analysis to assess whether these modifications 84 affected sensitivity. In our hands, the calibration factors for oxygen and dopamine on the oxygen-sensitive waveform 85 were -0.19 nA/µM/100 µm and 4.8 nA/µM/100 µm, respectively (Supplemental Fig.1). Both calibration factors fall 86 between values reported for glass microelectrodes (normalized to 100 µm)24. On the dopamine-sensitive waveform, 87 dopamine sensitivity was 34 nA/µM/100 µm, in agreement with the literature30 (Supplemental Fig.1). These data 88 indicate that our modified polyimide-fused silica electrodes are MR compatible and can detect analytes of interest 89 with high sensitivity. A B Hz C D 50 0 -50 40 Stimulating Electrode Working Electrode SE (RARE) SE (RARE) Hz Ag/AgCl GE (FLASH) Hz 0 -40 50 50 GE (EPI) 90 - 91 Fig.1. Modifying FSCV materials improves MRI compatibility. (A) Left: Polyimide-fused silica capillary 92 microelectrode (scale bar=100 µm). Right: Electrode axial cross-section scanned inside an agarose phantom using 93 spin echo (SE) rapid acquisition with relaxation enhancement (RARE), and gradient echo (GE) fast low angle shot 94 (FLASH) sequences (scale bar=1 mm). (B)