Voltage imaging with fluorescent dyes affords the opportunity to map neuronal activity in both time and space. One limitation to imaging is the inability to image complete neuronal networks: some fraction of cells remains outside of the observation window. Here, we combine voltage imaging, post hoc immunocytochemistry, and patterned microisland hippocampal culture to provide imaging of complete neuronal networks. The patterned microislands completely fill the field of view of our high-speed (500 Hz) camera, enabling reconstruction of the spiking patterns of every single neuron in the network. Cultures raised on microislands develop similarly to neurons grown on coverslips and display similar composition of inhibitory and excitatory cell types. The principal excitatory cell types (CA1, CA3, and dentate granule cells, or DGC) are also present in similar proportions in both preparations. We calculate the likelihood that action potential firing in one neuron to trigger action potential firing in a downstream neuron in a spontaneously active network to construct a functional connection map of these neuronal ensembles. Importantly, this functional map indicates preferential connectivity between DGC and CA3 neurons and between CA3 and CA1 neurons, mimicking the neuronal circuitry of the intact hippocampus. We envision that patterned microislands, in combination with voltage imaging and methods to classify cell types, will be a powerful method for exploring neuronal function in both healthy and disease states. Additionally, because the entire neuronal network is sampled simultaneously, this strategy has the power to go further, revealing all functional connections between all cell types.Significance StatementIn vitro model systems provide unsurpassed control and access for exploring the molecular and cellular details of neurobiology. We developed a patterned microisland system for culturing rat hippocampal neurons that recapitulates the features of bulk hippocampal cultures, but with the added benefit of allowing access to high-speed imaging of entire neuronal ensembles using voltage imaging. By using far-red voltage-sensitive fluorophores, we map the functional connections across all cells in the neuronal ensemble, revealing that several important functional synapses present in the intact hippocampus are recapitulated in this microisland system. We envision the methods described here will be a powerful complement to ongoing research into basic neurobiological mechanisms and the search for therapies to treat diseases arising from their dysfunction.

The surge in interest in nanomaterials in the past decade is ascribable, in large part, to the specialized properties due to size, shape, and structure at the nanoscale. Catalysts are a good example of this almost atom-by-atom dependence, with the number and coordination of each atom within a particle impacting the performance. A recent study even has shown that the simple rearrangement of 25 gold atoms from a spherical to cylindrical shape can significantly impact the efficacy of that catalyst [1]. The length scale of such materials makes characterization of these materials more difficult. Most studies must rely on ensemble measurements such as powder x-ray diffraction (XRD) or small angle x-ray scattering (SAXS) and assume a homogenous population such that the ensemble measurement is representative of each particle. This means that information on structural heterogeneity in samples is completely lost. The only technique which can give local structural information on a nanoparticle by nanoparticle basis is highresolution transmission electron microscopy (HRTEM) or high resolution scanning transmission electron microscopy (HR-STEM). HRTEM is generally extremely low throughput due to constraints on data analysis. How to make HRTEM a more high-throughput process has become a goal for the TEM community and has large implications for the understanding of structure-property relationships in nanomaterials. 150

Voltage imaging with fluorescent dyes affords the opportunity to map neuronal activity in both time and space. One limitation to imaging is the inability to image complete neuronal networks: some fraction of cells remains outside of the observation window. Here, we combine voltage imaging, post hoc immunocytochemistry, and patterned microisland hippocampal culture to provide imaging of complete neuronal ensembles. The patterned microislands completely fill the field of view of our high-speed (500 Hz) camera, enabling reconstruction of the spiking patterns of every single neuron in the network. Cultures raised on microislands are similar to neurons grown on coverslips, with parallel developmental trajectories and composition of inhibitory and excitatory cell types (CA1, CA3, and dentate granule cells, or DGC). We calculate the likelihood that action potential firing in one neuron triggers action potential firing in a downstream neuron in a spontaneously active network to construct a functional connection map of these neuronal ensembles. Importantly, this functional map indicates preferential connectivity between DGC and CA3 neurons and between CA3 and CA1 neurons, mimicking the neuronal circuitry of the intact hippocampus. We envision that patterned microislands, in combination with voltage imaging and methods to classify cell types, will be a powerful method for exploring neuronal function in both healthy and disease states. Additionally, because the entire neuronal network is sampled simultaneously, this strategy has the power to go further, revealing all functional connections between all cell types.