Simultaneous optical and electrical in vivo analysis of the enteric nervous system

Abstract

The enteric nervous system (ENS) is a major division of the nervous system and vital to the gastrointestinal (GI) tract and its communication with the rest of the body. Unlike the brain and spinal cord, relatively little is known about the ENS in part because of the inability to directly monitor its activity in live animals. Here, we integrate a transparent graphene sensor with a customized abdominal window for simultaneous optical and electrical recording of the ENS in vivo. The implanted device captures ENS responses to neurotransmitters, drugs and optogenetic manipulation in real time.

Figure 1. In vivo chronic multiphoton microscopy of ENS with abdominal window.

(a) Schematic of in vivo multiphoton microscopy. (b–e) Days 2, 5, 8 and 11 after abdominal surgery, respectively, of in vivo multiphoton imaging in Wnt1-cre:tdTomato mice. The ENS is labelled with tdTomato (red), and the vasculature is labelled with FITC-dextran (green). Scale bar, 100 μm.

Figure 2. Simultaneous optical/electrical recording in vivo using integrated graphene sensor and abdominal window.

(a) Schematic of in vivo optical/electrical recording. The graphene sensor is in close proximity to the small intestine, within the titanium ring of the surgical implant. (b) Mouse with integrated graphene sensor and abdominal window, several days after abdominal window implantation surgery. (c) The implantable abdominal window integrated with the graphene sensor. (d) Graphene electrode implanted in Pirt-GCaMP3 mouse. One graphene electrode (500 μm by 500 μm, white box) in contact with the surrounding nerves (green, identified with white arrows) and surrounding collagen tissue (blue). Scale bar, 100 μm. (e) Simultaneous multiphoton microscopy and electrical potential (EP) recording in vivo, in Pirt-GCaMP3 mouse. GCaMP3 fluorescence (green) and electrical potential (blue) are plotted, with absolute value of each signal normalized to 100%. (f) A high-temporal resolution waveform of an in vivo recorded AP.

Figure 3. Unpaired analysis of fluorescent and electrical potential response to chemical stimuli.

(a) Frames from time-lapse recording of GCaMP3-expressing nerves during serotonin stimulation. The activated nerves fluoresce (green) due to stimulation, before fading back to low fluorescence levels. Scale bar, 50 μm. (b–d) Average GCaMP3 fluorescence (green) and average percentage of electrical recording (blue), after (b) acetylcholine, (c) serotonin or (d) PBS stimulations plotted relative to the time of stimulation (n=3 and 5 for GCaMP3 and electrical potential respectively; error bars show standard deviation (s.d.)). Stimulus was applied at time 0 (red dashed line).

Figure 4. Electrical response to chemical stimuli in vivo.

(a–d) Neural response to in vivo stimulation by (a) acetylcholine, (b) bethanechol, (c) serotonin and (d) tegaserod. Stimulus occurs at 120 s (red dashed line), and the electrical potential (black) and spikes (red circles) are plotted over time. (e–h) Waveforms of representative APs from above recordings (blue box) are shown at higher temporal resolution. (i) The effect of chemical stimulation is compared between acetylcholine, bethanechol, serotonin, tegaserod and a PBS control group. The average percentage of spikes that occur before stimulation (green) and the average percentage that occur after (blue) are plotted for each group (n=4; error bars show s.d.). All repeats consisted of recordings lasting 240 s, with stimulation at 120 s. Measurements of individual recordings used to calculate the average percentages are shown in Supplementary Fig. 7. *P<0.05; ***P<0.005; ****P<0.001, one sided t-test.

Figure 5. Optogenetic application of integrated graphene sensor and abdominal window.

(a) A schematic for in vivo stimulation of Nos1-creERT2:Chr2 mice with 470 nm light. (b) Mechanistic schematic for 470 nm inhibition in Nos1-creERT2:Chr2 mice. (c) Representative in vivo recording with 470 nm stimulation in Chr2-expressing mice. 470 nm light stimulation occurs at 300 s (red dashed line). Electrical potential (black) is plotted over time. (d) The effect of in vivo 470 nm light stimulation is compared between Nos1-creERT2:Chr2 mice (Chr2) and wild-type mice (WT). The average percentage of spikes that occur before stimulation (green), and the average percentage that occur after (blue) are plotted for each group (n=4; error bars show s.d.). Only data 120 s before and after stimulation are analysed during repeats. ***P<0.005, one sided t-test.