Muhammad Shan Sohail is a graduate researcher in the Milosevic Lab at the Department of Biomedical Engineering, University of Toronto, specializing in dual-color optical interrogation of GABAergic signaling in mammalian motor control. His research combines fiber-photometry, optogenetic stimulation, and fluorescent biosensing to investigate how inhibitory neural circuits regulate locomotion in freely moving animals. Muhammad’s work bridges neuroscience and optical engineering, with a focus on simultaneous multi-wavelength recording and stimulation through a single fiber using Mightex’s OASIS platform.

This study aimed to validate the dual-wavelength functionality of the Optical Activation, Stimulation, and Imaging System (OASIS) for performing simultaneous fiber-photometry and optogenetic stimulation through a single patch cable and optical fiber in freely moving animals. The OASIS platform integrates 405 nm and 488 nm LEDs for photometric excitation and a 638 nm diode laser for optogenetic manipulation. These wavelengths were strategically selected to minimize optical crosstalk between channels and to isolate the functional contributions of both the biosensor and the opsin by maintaining a large spectral separation. Furthermore, the two excitation paths are optically isolated using dichromatic filters, allowing the biosensor’s fluorescence emission to be captured by the CMOS camera while effectively filtering out the 638 nm stimulation light. This configuration enables validation of both the biosensor’s functionality during optogenetic activation and the opsin’s efficacy under biosensor monitoring.

To demonstrate this capability, wild-type mice were intracranially infused with AAV vectors encoding ChrimsonR and iGABASnFR, followed by implantation of a single optical fiber targeting the motor faculty. Behavioral experiments were conducted in an open-field arena under a repeated 60 s ON / 60 s OFF 638 nm laser paradigm (Figure 1), during which both photometry signals and locomotor activity were continuously recorded.

With the dual-wavelength capacity of the OASIS, we have demonstrated the implication of time-locked GABA release during optogenetically mediated locomotion. The prospective work will interrogate the origin and target of GABA release within the faulty by leveraging OASIS’s capacity to identify different inhibitory sources.

Figure 1. Dual-wavelength Opto-Photometry overlay trace: Open field imaging of iGABASnFR in rodent with optical excitation for 60 second for every other 60 seconds. The blue highlights are the ChrimsonR opto-stimulation events. a. iGABASnFR trace, overall increase the signal in every stimulation events. b & c. Kinematics of the animals plotting its velocity (cm/s) and timeimmobile (<0.1cm/s). d. Square waveform of the 638nm optogenetics paradigm. 60s on – 60 off – 60 on cycles.

During the 638 nm ON phase (see video – 638nm ON), animals exhibited a robust increase in fluorescence intensity, accompanied by enhanced locomotor velocity and a corresponding reduction in immobility time. These observations confirm the efficacy of OASIS in achieving artifact-free, dual-wavelength recording during optogenetic activation in behaving animals.