Abstract There are significant limitations in investigating complex neural circuits in vivo , including drawbacks to midline-adjacent surgeries, limited accessibility to deep brain regions and number of feasible regional targets for simultaneous recordings, and analytical or experimental biases from recording one columnar plane. On the other hand, recording extracellular neural signals ex vivo or in vitro using planar microelectrode arrays (MEAs) only permits slice surface recordings, and since conventional slices under 400 μm-thick or dissociated cultures are used, no experiments contain a physiological multi-region circuit, drastically limiting conclusions about connectivity and pharmacology. Using thick, tract-preserving acute brain slices to record otherwise unassailable neural circuits ex vivo combines the strengths of both types of experiments, but is assumed to precipitate ischemic injury due to oxygen scarcity within the slice. Here, we report the first application of custom, multi-region silicon neural probe arrays to record spontaneous activity &amp; optogenetically-induced functional connectivity acrosshe mesocorticolimbic pathway within tract-preserving 800 μm sagittal mouse brain slices, compared with 400 μm slices, among three brain regions: the ventral tegmental area (VTA), ventral striatum (VS), &amp; medial prefrontal cortex (mPFC). We show that most single-unit signals are an order of magnitude below the noise floor seen using silicon probes in vivo , providing unit yields far higher than previously assumed, allowing for a deep functional understanding of acute slice condition compared to the assumed deterioration due to ischemia. Overall, our method allows for acute circuit manipulations beyond what is available in vivo, with far more information than conventional slice preparations.

Investigation of neural functional connectivity in thick acute mouse brain slices with novel multi-region 3D neural probe arrays