New Technology for In Vivo Monitoring the Brain Extracellular Proteome at High Spatial Resolution in Substance Abuse Models

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Project Summary. The overall objective of this project is to develop novel approaches to sample, identify, and quantify proteins secreted into the brain extracellular space in relation to substance use disorders (SUD). The progression to compulsive drug consumption is associated with changes in neuronal structure and neurotransmitter release in response to drugs. Evidence suggests that proteins in the extracellular space play a role in these changes. For example, brain derived neurotrophic factor (BDNF) released to the extracellular space may stimulate neuronal changes that are associated with adaptation to drugs of abuse; however, practically no direct measures of proteins in the brain extracellular space have been made. The ability to track release of certain neurotransmitters in vivo has dramatically advanced over the past 2 decades and led to profound increases in our understanding of how their release is altered by and contributes to SUD. Opening a window onto how proteins dynamically change will provide even greater insights. A powerful approach to monitoring is in vivo sampling coupled to analytical techniques. This approach, exemplified by microdialysis sampling, has been instrumental in uncovering dynamics of dopamine and other small molecules in brain function and substance abuse. This method has been less successful for tracking proteins due to low recoveries of these molecules. Low recovery is due to low diffusion of proteins to the sampling device and adsorption to dialysis membranes. Such probes are also too large to sample many brain nuclei. Here we describe novel sampling systems that will have higher recovery for proteins at spatial resolution that is 1000- fold better than microdialysis. We will develop a new sampling method that induces electrically driven flow through the brain to drive proteins into a sampling capillary. This electroosmotic push-pull perfusion (EOPPP) probe will be tested for its ability to recover proteins. The effectiveness of this method will be compared to another new probe based on low-flow push-pull perfusion. In this method, a microfabricated probe, tens of micrometers in width and thickness, has a single channel to withdraw sample and a second to deliver replacement fluid at ~100 nL/min. After testing and development of these sampling probes, we will then develop proteomic methods to analyze the extracellular proteome. These methods will be based on microscale sample preparation to digest proteins to component peptides, advanced high-resolution separation of peptides, and state-of-the art proteomic analysis by mass spectrometry and informatics. We will identify extracellular proteins in the nucleus accumbens and prefrontal cortex for the first time. We will then compare the extracellular proteome in controls to that found in animals trained to self-administer cocaine, a validated model of SUD. This work will uncover new neural substrates of SUD and set the stage for further exploration of the brain proteome in SUD models. .