Mental health disorders, such as post-traumatic stress disorder (PTSD), are complex, leaving challenges for the development of effective interventions. Increasing our detailed understanding of these disorders, by studying the micro-neurocircuitry behind them, can help provide individualized solutions to these complicated conditions. This micro-neurocircuitry can be investigated by studying small groups of highly-active neurons, collectively known as neuronal ensembles, that are responsible for unique behavioral responses, e.g. fear learning, in PTSD. Behavioral studies examining neuronal ensembles have thus far been largely ex vivo, using immediate early genes associated with neuronal activity (e.g. c-Fos). In vivo imaging methods are currently limited (very invasive or low resolution), and generally measure indirect neuronal activity, such as the blood oxygen dependent response. To address this knowledge gap, we propose a new high-resolution imaging approach to detect and quantify fear-related neuronal activity in vivo, with the goal of discovering more detailed roles of the neuronal ensembles that mediate fear learning associated with PTSD. Specifically, we propose using photoacoustic (PA) imaging to map activated neurons in a Fos/LacZ transgenic rat model. Fusion of Fos, which is induced by neuronal activity, with the lacZ gene gives active (Fos+) cells the ability to cleave pro-chromogenic substrates, such as X-Gal, enzymatically, into PA active dyes. My phantom and ex vivo pilot studies in rat brains demonstrate high-contrast PA images using these dyes and image reconstructions with a very high signal-to-noise ratio. We hypothesize that during fear acquisition, unique neuronal ensembles will be activated and detectable in the medial prefrontal cortex (a region tightly associated with fear behavior) using a novel Fos/LacZ PA imaging system, providing a means to track longitudinal changes in these ensembles in vivo. To test this hypothesis, we will use male/female Fos/LacZ transgenic rats that will be administered cortical X-Gal upon exposure to footshock (fear). The X-Gal product will be PA imaged (Aim 1) and these images will be validated with traditional methods for immunohistochemical detection of Fos and ?-galactosidase in brain slices (Aim 2). Significance: This project will provide deeper insight into the role of fear-related neuronal ensembles and provide a new, low cost, high-resolution methodological approach to measure Fos expression longitudinally (representative of neuronal ensembles) in vivo. This will be broadly useful for behavioral research, not only in PTSD, but also in a variety of psychiatric science by allowing us to better define mechanisms behind complex mental health disorders. My training will focus on three main areas: molecular neuroscience and microscopy (led by sponsor, Dr. Conti), behavioral/animal modeling (led by co-sponsor, Dr. Perrine), and use of PA imaging (led by key training team member, Dr. Avanaki), facilitating my path towards my neuroradiology physician-scientist career goal.
A hallmark of post-traumatic stress disorder (PTSD) is impaired fear learning, however our understanding of fear behavior is limited, due to our inability to analyze the selective microcircuitry that underlies it, in a translationally-relevant manner. This proposal seeks to implement a novel neuroimaging technique, photoacoustic imaging, which will allow, for the first time, highly-resolved direct imaging of microcircuit (neuronal ensemble) activation associated with fear learning throughout the medial prefrontal cortex, in vivo. The identification and longitudinal analysis of these neuronal ensembles will provide novel information on the development of maladaptive fear learning processes in those that suffer from PTSD, with the ultimate goal of targeted therapy development.
