The overall objective of the proposed project is to create a compact, robust, and easy to use device that is capable of sensitive and quantitative detection of fluorescence lifetime indicators of physiological and biochemical dynamics deep in the brain of a freely moving mouse. In contrast to fluorescence intensity based methods, fluorescence lifetime can delineate spectrally overlapping fluorescent signals. Our focus is on measurements related to alcohol, addiction, and behavior in the striatum and other structures deep inside the brain. Most in vivo mouse brain studies are based on the fluorescence intensity measurements. However, for measurements of more than one fluorophore we face challenges of mixed or overlapping emission spectra, which complicates the quantitative interpretation of the results. In 2013, Cui et al. (Nature 2013) was the first to introduce fluorescence lifetime asa measurement parameter in examining the relationship between neural activities in a freely moving mouse and behavior deep in the brain area of the striatum. It has provided the first definitive evidence that direct- and indirect-pathway striatal neurons are co-activated during movement initiation, and are inactive when the animal is not moving. Inspired by the success of their device, we will collaborate with the original authors to develop this instrument to a device for general applicability in neuroscience labs to investigate alcohol addiction.
Aim 1 is to develo a prototype instrument and in-house testing. This includes constructing main body of the device by integrating the time-correlated single photon counting (TCSPC) electronics, the pulse laser source, and single photon detector;incorporating innovative in vivo probe designs and fiber coupling technology;and to perform rigorous testing and calibration to validate the new design.
Aim 2 is to have the device tested at neuroscience labs for the iterative process of testing and modification (Months 16 - 24). Three aspects make our device truly innovative and the first of its kind. Firstly, fluorescence lifetime detection makes it possible to distinguish the source of fluorescent signals despite their mixed or overlapping emission spectra. Secondly, our fiber optics-based in vivo probes will be designed to shape the excitation and detection volume for efficient signal throughput, and be combined with the latest technology in highest efficiency coupling to offer a flexible on/off connection to the implantable in vivo probe. Thirdly, our desig will result in a compact, robust, and easy-to-use device, with versatile interchangeable in vivo probes. We anticipate that this device will make fluorescence lifetime detection technology the method of choice for investigations where it can add a definitive contrasting mechanism and specificity. We envision that the combination of fluorescence lifetime detection with fluorescent proteins allows for a wide range of applications and will be used for studies involving photoactivation, voltage-sensing, redox-sensing, and calcium sensing and release, and to examine protein-protein interactions (using fluorescence-lifetime and Firster resonance energy transfer (FLIM-FRET).
We propose to develop a compact, robust, easy-to-use device that is capable of measuring dynamic biochemical and electrical signals in the brain of a freely moving rodent. Understanding the relationship between neural activities in the brain of an experimental model system and its behavior will be a step forward in understanding how that relationship might work in human. The device that we have proposed in this application will implement fluorescence lifetime detection using time-correlated single- photon counting as its core technology. This method has a distinct advantage in resolving complex fluorescence signals from more than one source, and will lead to more accurate interpretation of biological signals in the brain. The immediate application of this device is in understanding the signaling pathways that control behavior related to drug seeking and their roles in alcohol addiction. However, the device will find a broad range of applications in the studies related to Parkinson's disease, Alzheimer's disease, and other neurological based diseases.
