Spiral laser scanning photoacoustic microscopy for functional brain imaging in rats.

Significance: There are many neuroscience questions that can be answered by a high-resolution functional brain imaging system. Such a system would require the capability to visualize vasculature and measure neural activity by imaging the entire brain continually and in rapid succession in order to capture hemodynamic changes. Utilizing optical excitation and acoustic detection, photoacoustic technology enables label-free quantification of changes in endogenous chromophores, such as oxyhemoglobin, deoxyhemoglobin, and total hemoglobin. Aim: Our aim was to develop a sufficiently high-resolution, fast frame-rate, and wide field-of-view (FOV) photoacoustic microscopy (PAM) system for the purpose of imaging vasculature and hemodynamics in a rat brain. Approach: Although the most PA microscopy systems use raster scanning (or less commonly Lissajous scanning), we have developed a simple-to-implement laser scanning optical resolution PAM system with spiral scanning (which we have named "spiral laser scanning photoacoustic microscopy" or sLS-PAM) to acquire an 18 mm diameter image at fast frame rate (more than 1 fps). Such a system is designed to permit continuous rat brain imaging without the introduction of photobleaching artifacts. Conclusion: We demonstrated the functional imaging capability of the sLS-PAM system by imaging cerebral hemodynamics in response to whisker and electrical stimulation and used it for vascular imaging of a modeled brain injury. We believe that we have demonstrated the development of a simple-to-implement PAM system, which could become an affordable functional neuroimaging tool for researchers.

Quantification of prefrontal cortical neuronal ensembles following conditioned fear learning in a Fos-LacZ transgenic rat with photoacoustic imaging in Vivo.

Understanding the neurobiology of complex behaviors requires measurement of activity in the discrete population of active neurons, neuronal ensembles, which control the behavior. Conventional neuroimaging techniques ineffectively measure neuronal ensemble activity in the brain in vivo because they assess the average regional neuronal activity instead of the specific activity of the neuronal ensemble that mediates the behavior. Our functional molecular photoacoustic tomography (FM-PAT) system allows direct imaging of Fos-dependent neuronal ensemble activation in Fos-LacZ transgenic rats in vivo. We tested four experimental conditions and found increased FM-PAT signal in prefrontal cortical areas in rats undergoing conditioned fear or novel context exposure. A parallel immunofluorescence ex vivo study of Fos expression found similar findings. These findings demonstrate the ability of FM-PAT to measure Fos-expressing neuronal ensembles directly in vivo and support a mechanistic role for the prefrontal cortex in higher-order processing of response to specific stimuli or environmental cues.

Direct measurement of neuronal ensemble activity using photoacoustic imaging in the stimulated Fos-LacZ transgenic rat brain: A proof-of-principle study.

Measuring neuroactivity underlying complex behaviors facilitates understanding the microcircuitry that supports these behaviors. We have developed a functional and molecular photoacoustic tomography (F/M-PAT) system which allows direct imaging of Fos-expressing neuronal ensembles in Fos-LacZ transgenic rats with a large field-of-view and high spatial resolution. F/M-PAT measures the beta-galactosidase catalyzed enzymatic product of exogenous chromophore X-gal within ensemble neurons. We used an ex vivo imaging method in the Wistar Fos-LacZ transgenic rat, to detect neuronal ensembles in medial prefrontal cortex (mPFC) following cocaine administration or a shock-tone paired stimulus. Robust and selective F/M-PAT signal was detected in mPFC neurons after both conditions (compare to naive controls) demonstrating successful and direct detection of Fos-expressing neuronal ensembles using this approach. The results of this study indicate that F/M-PAT can be used in conjunction with Fos-LacZ rats to monitor neuronal ensembles that underlie a range of behavioral processes, such as fear learning or addiction.

Traumatic Stress, Chronic Ethanol Exposure, or the Combination, Alter Cannabinoid System Components in Reward and Limbic Regions of the Mouse Brain.

The cannabinoid system is independently affected by stress and chronic ethanol exposure. However, the extent to which co-occurrence of traumatic stress and chronic ethanol exposure modulates the cannabinoid system remains unclear. We examined levels of cannabinoid system components, anandamide, 2-arachidonoylglycerol, fatty acid amide hydrolase, and monoacylglycerol lipase after mouse single-prolonged stress (mSPS) or non-mSPS (Control) exposure, with chronic intermittent ethanol (CIE) vapor or without CIE vapor (Air) across several brain regions using ultra-high-performance liquid chromatography tandem mass spectrometry or immunoblotting. Compared to mSPS-Air mice, anandamide and 2-arachidonoylglycerol levels in the anterior striatum were increased in mSPS-CIE mice. In the dorsal hippocampus, anandamide content was increased in Control-CIE mice compared to Control-Air, mSPS-Air, or mSPS-CIE mice. Finally, amygdalar anandamide content was increased in Control-CIE mice compared to Control-Air, or mSPS-CIE mice, but the anandamide content was decreased in mSPS-CIE compared to mSPS-Air mice. Based on these data we conclude that the effects of combined traumatic stress and chronic ethanol exposure on the cannabinoid system in reward pathway regions are driven by CIE exposure and that traumatic stress affects the cannabinoid components in limbic regions, warranting future investigation of neurotherapeutic treatment to attenuate these effects.