Imaging voltage in complete neuronal networks within patterned microislands reveals preferential wiring of excitatory hippocampal neurons

Abstract

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.

Optical Spike Detection and Connectivity Analysis With a Far-Red Voltage-Sensitive Fluorophore Reveals Changes to Network Connectivity in Development and Disease

“…For this study, Δ t = 10 ms for a window of 20 ms surrounding a single spike. This value was chosen by examining cross correlations of spike timings ( Walker et al, 2021 ). These were calculated by selecting spike data and running the function “sttc_gui.” The resulting STTC values for each ROI pair were then exported as an excel file and plotted in GraphPad for comparison.…”

Imaging Spontaneous Neuronal Activity with Voltage‐Sensitive Dyes

“…This allows us to record for 10 s continuously without any appreciable photobleaching. Good agreement between the optical and electrophysiology traces can be obtained by using a signal‐to‐noise threshold of 16 for the optical trace recorded by BeRST 1 under these imaging settings (Walker, Raliski, & Nguyen, et al., 2020). However, if a desired imaging experiment requires different imaging parameters, the agreement between the optical and electrophysiology traces will need to be reassessed.…”

“…To date, BeRST 1 displays the highest signal‐to‐noise ratios for action potential detection in dissociated culture (Huang et al., 2015). To handle the large amount of data that is generated in a spontaneous voltage imaging experiment, our lab has also developed a MATLAB program for semiautomated spike detection, termed SpikeConnect (Walker, Raliski, & Nguyen, et al., 2020). Thus, this protocol serves as guide to the spontaneous voltage imaging experiments that are becoming routine in our lab and in academic labs across the globe.…”

“…Thus, BeRST 1 displays high signal‐to‐noise ratios when measuring spontaneous voltage changes in dissociated culture. BeRST 1's properties enabled the imaging of spontaneous activity from up to 25 neurons simultaneously and the investigation of excitability and connectivity in bulk dissociated culture (Walker, Raliski, & Karbasi, et al., 2020) and microisland cultures (Walker, Raliski, & Nguyen, et al., 2020). However, one challenge that arose during these studies was the difficulty of processing large amounts of spontaneous activity imaging data.…”

“…However, one challenge that arose during these studies was the difficulty of processing large amounts of spontaneous activity imaging data. Therefore, along with developing BeRST 1 imaging experiments, our lab also developed a MATLAB program, SpikeConnect, to facilitate analysis of spontaneous activity imaging data (Walker, Raliski, & Nguyen, et al., 2020).…”