Abstract Body:

Background：
Deciphering neural circuits is a primary objective of contemporary research in neuroscience, often necessitating invasive methodologies or ex vivo detection techniques. Reporter gene imaging with positron emission tomography (PET) is a versatile tool for biological and medical research, yet there remains a lack of reporter genes available for imaging neuroscience. Tyrosinase (TYR) is a rate-limiting enzyme regulating the production of melanin, which can be imaged using PET. Given the low expression level of TYR in the brain, and the favorable blood-brain barrier permeability of the melanin-targeting probe ¹⁸F-picolinamide-benzamide (¹⁸F-FPABZA) reported recently, TYR shows promise for utilization in brain imaging and evaluation of neural circuits when employed in conjunction with traditional neurotropic virus tools.
Objectives:
The purpose of this study was to evaluate the potential of TYR reporter genes in brain imaging, and to develop visualization techniques for evaluating neural circuit in vivo.
Methods: 
Hela cell lines were transfected with lentivirus carrying TYR in vitro, and rat brain tissues were transfected with adeno-associated virus (AAV) encoding TYR in vivo. ¹⁸F-FPABZA PET was performed to validate the imaging effect of TYR reporter gene in vitro and in vivo, as well as the availability in brain imaging. To explore the potential of TYR PET in tracing neural circuits, a retrograde subtype AAV carrying TYR was constructed and injected in right somatosensory cortex of rats, with ¹⁸F-FPABZA PET was used to image connected brain regions. To explore the potential of TYR PET in imaging neural activity, an AAV carrying TYR drove by c-fos was constructed and injected in right dorsomedial prefrontal cortex (dmPFC) of rats, with Freuxe’s complete adjuvant (FCA) injected in left hinder paw to induce the c-fos expression in dmPFC, and ¹⁸F-FPABZA PET performed to image the expression of TYR. 
Results:
Hela-TYR cells showed significantly higher expression of TYR mRNA, TYR protein, and melanin than the control Hela-Con cells. Hela-TYR cells presented higher ¹⁸F-FPABZA PET signals, which correlated well with the multiplicity of infection, and number of imaged cells. In brain tissues with persistently expressed TYR, uptake of ¹⁸F-FPABZA was obvious, with background signals decreased over time. In rats transfected with AAVretro-TYR-GFP virus in the right somatosensory cortex, ROI-based group analysis revealed enhanced signals across multiple brain regions such as right somatosensory cortex, bilateral motor cortex, and bilateral cingulate gyrus, etc., while voxel-based analysis demonstrated various radiotracer distribution pattern in each rat. Ex vivo staining results further corroborate ¹⁸F-FPABZA PET signals. Injection of AAV virus carrying c-fos-driven TYR resulted in significantly increased ¹⁸F-FPABZA uptake in dmPFC after FCA treatment. Ex vivo staining experiments verified the differences in the expression of c-fos, TYR and melanin in pre- and post- stimulation rats.
Conclusions:
This study suggested that TYR is a valuable reporter gene available for brain imaging, and the combination use of ¹⁸F-FPABZA PET with classical neurotropic virus tools would enable the evaluation of neural circuit in vivo.

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