Significant progress has been made to extend neuronal cultures from 2D into 3D. However, it still remains a great challenge to detect neuronal activity with high spatial and temporal resolution in 3D. The thickness of the structures within conventional 3D neuron cultures are too large for ideal signal detection and may impede neuron growth. Our research efforts in this area are focused on completely mapping the neural activity in the 3D neuron culture space with high temporal and spatial resolution. We will also introduce microfluidic “vasculature” and extend the 3D neuronal culture’s dimensions.