Fig. 5: High spatiotemporal resolution electrophysiologic acquisition permits identification of putative single neurons in the enteric nervous system.

a Custom implantable wire was designed to facilitate chronic device implantation. The wire has two symmetric plugs / connectors, used to insert into a zero-insertion force (ZIF)-type plug (right), which, via a custom-printed circuit board (PCB), can be interfaced with an Intan headstage for recording. The other end of the wire has an identical geometry but is bonded onto the microfabricated implant (left), which has the same replicated connector on the device itself. b Inset showing plug with intermediate wiring visible on the left of the image. The intermediate wiring is double-sided to reduce footprint and bifurcated to allow for the placement of sutures around the wire. Small tabs connect the bifurcated wires across the length of the wire, enabling the wire to be anchored at the point where it crosses the peritoneal wall for access to the colon. c Schematic detailing the placement of wiring and device within the rat. For the surgery, the wiring was routed subcutaneously between the percutaneous port down to the ventrolateral flank of the rat, where it crossed the peritoneal wall, allowing for implantation of the microfabricated portion of the device into the colon. d Illustration of the tetrode arrangement of four electrodes in one device in a chronically implanted rat. e Sample high-pass filtered traces obtained during a recording session conducted 12 days post-surgery from three implanted electrodes (color refers to matching electrode in (e) during (right; scale bar, 100 ms and 30 µV). f Averaged extracellular spike waveforms for three sample putative single neurons recorded from the implanted electrodes presenting acceptable (<500 kΩ) electrical impedance, which also presented good signal quality confirmed through offline analysis in the same chronic recording session demonstrating distinct and anatomically consistent localization across electrodes (n = 4718 spikes/neuron 1, 4748 spikes/neuron 2, 2497 spikes/neuron 3; trace, 3.5 ms, right; scale bar 3.5 ms and 10 µV). g Autocorrelations of spike occurrence for putative single neurons shown in b demonstrate physiologic refractory periods. h Cross-correlations of spike occurrence between putative single neurons reveal different co-activation patterns. i Spatiotemporal features of putative single neuron activity form distinct clusters in principal component space. j Spike occurrence attributable to putative single neurons is stable across a 30-min recording session as visualized using example channel-projected principal components.