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. Author manuscript; available in PMC: 2020 Sep 27.
Published in final edited form as: Nano Lett. 2020 May 12;20(6):4073–4083. doi: 10.1021/acs.nanolett.9b04877

Figure 3. ACh sensors spatiotemporally profile cholinergic transmission at non-neuronal cells.

Figure 3.

(A) Schematic drawing of the design of stimulation-imaging experiments with iAChSnFR in acute mouse MEC slices. HP: Hippocampus; MEC: Medial entorhinal cortex.

(B-C) Snapshots of fluorescence ΔF/F responses (B left panel), heatmap displays of time-dependent spatial ΔF/F responses (B right panel) and three-dimensional spatiotemporal ΔF/F profiling (C) of distal processes of an iAChSnFR expressing entorhinal astrocyte in response to local electrical stimuli. Note one isolated release site indicated by pink arrow in C. Note that the astrocytic cell body, localized below the image, was trimmed to highlight the responses at distal processes.

(D) Plot of relative maximal ΔF/F of each pixel against its distance to the pixel with largest maximum ΔF/F at the isolated release site indicated by pink arrow in C. Fitting the data points in this plot with a single exponential decay function (pink line) yields an estimated ACh spread length constant of 1.01 μm.

(E) Summary plot of volume spread length constants obtained from putative single release sites and the average volume spread length constant of 1.00±0.08 μm for cholinergic transmission at entorhinal astrocytes (n = 14 from 8 neurons from 6 animals). Note the average single exponential decay function fitting curve in black.

(F) Schematic drawing of the design of stimulation-imaging experiments with GACh2.0 using an in vivo viral expression and in vitro mouse pancreatic and adrenal slice preparations. Inserts show transmitted light (left), fluorescence microscopic (middle) and overlay (right) images of GACh2.0 expressing pancreatic and adrenal cells.

(G-H) Heatmap snapshots of fluorescence ΔF/F responses (G upper panel), time-dependent spatial ΔF/F responses (G lower panel) and three-dimensional spatiotemporal ΔF/F profiling (H) of a GACh2.0 expressing pancreatic cell in response to local electrical stimuli. Note one isolated release site indicated by pink arrow in H.

(I) Plot of relative maximal ΔF/F of each pixel against its distance to the pixel with largest maximum ΔF/F at the isolated release site indicated by pink arrow in H. Fitting the data points in this plot with a single exponential decay function (pink line) yields an estimated ACh spread length constant of 1.31 μm.

(J) Summary plot of spread length constants obtained from putative single release sites and the average volume spread length constant of 1.13±0.07 μm for cholinergic transmission at the pancreatic and adrenal cells (n = 16 from 8 neurons from 8 animals). Note the average single exponential decay function fitting curve in black.

(K) Values for transmitter ACh spread length constants obtained with iAChSnFR at entorhinal stellate neurons (U = 115.0, p = 0.859; see data in Fig 2F), iAChSnFR under 60x objective at entorhinal stellate neurons (U = 74.0, p = 0.505; see data in Fig 2K), iAChSnFR at amygdalar neurons (0.96±0.02 pm; n = 12 from 4 neurons from 4 animals; U = 121.0, p = 0.225), iAChSnFR at entorhinal astrocytes (U = 124.0, p = 0.633; see data in Fig 3E), GACh2.0 at pancreatic and adrenal cells (U = 148.0, p = 0.462; see data in Fig 3J) compared to that obtained with GACh2.0 entorhinal stellate neurons (Mann-Whitney Rank Sum tests; see data in Fig 1F).