Rapid multi-directed cholinergic transmission in the central nervous system

In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale ‘non-synaptic’ mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a ‘tripartite’ structure facilitates a ‘multi-directed’ form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.

: Evoked cholinergic currents are slow to rise relative to spontaneous EPSCs regardless of the level of stimulation, making it difficult to ascertain whether they are mediated by paracrine or synaptic mechanisms. a. Two-photon image stack showing the morphology of a connected starburst and a DSGC, recovered after a paired whole-cell patch-clamp recording during which they were dialyzed with fluorescent dyes. Scale bar = 50 μm. b. Brief voltage pulses delivered to the starburst (0 mV, for 17 ms) voltage-clamped at -60 mV, evoked robust cholinergic EPSCs in the DSGC (blue). Weaker pulses (-10 mV, for 17ms) evoked smaller amplitude responses, less reliably (inset; red). c. A comparison of the mean peak (left), rise time (middle) and decay constant (right) of the sEPSCs with evoked EPSCs (red: weak stimulation; blue: strong stimulation; n = 6 starburst-DSGC pairs from 4 retinas). The kinetics of the evoked EPSCs was slow compared to the sEPSCs. (Supplementary Table 1; peak: p = 0.0009; rise: p = 0.004; decay: p = 0.031; paired ttests). Data represented as mean ± SEM. Source data are provided as a Source Data file for Supplementary fig. 4b, c.
These experiments illustrate why results from previous studies examining cholinergic connections between starbursts and DSGCs could not be used to infer synaptic or non-synaptic nature of ACh transmission in this circuit. Supplementary Fig. 5: Kinetic model illustrating the potential pre-and postsynaptic factors that promote multi-directed transmission. a. A plot showing the concentration transients of ACh after a single vesicle release at peripheral 'preferred' sites (r = 1.1µm from the release site; blue) and proximal 'null' sites (r = 0 µm from release site; red) for the cases where ACh spreads in 2D (between pre-and postsynaptic membranes) or in 3D (porous medium), using equations described by Barbour  Predicted activity at preferred sites (blue) and null sites (red) for 2D diffusion model, when α7-nAChRs are employed. The ratio of peak receptor activity at preferred and null sites was 75%. d. Similar to c, but when α3-nAChRs were employed. The ratio of activity at preferred and null sites was 20%. e. Similar to c, but the desensitization of α7-nAChRs was reduced 4-fold (α7-nAChRs*). In this case, the ratio of preferred-null activity was more than 80%. Subtle differences in amplitude and rise time would be undetectable in our experiments, where somatic whole cell recordings were used to estimate the cholinergic response kinetics. f. A comparison of the α7-nAChRs* activity at preferred and null sites when the 3D diffusion model was used.

Supplementary Fig. 6: Heterogeneous ACh release properties across starburst varicosities.
a. An image stack showing the morphology of a starburst (red channel) that was loaded with a red dye through the patch electrode used to stimulate it. Markers indicate the heterogeneity in ACh release across starburst varicosities, with * indicating sites which elicited responses upon starburst stimulation, and  indicating sites which did not elicit responses (One such window was recorded in each of the 12 starbursts used in this experiment).
b. An image of the peak ΔF after brief depolarizations of the starburst (single stim.) shown in a (average of 7 trials). The white space indicates regions without ACh3.0 expression. A zoomed section of the bottom right is shown in Fig. 6c. c. Peak change in fluorescence (average of 3 trials) across the same field of view evoked by a moving spot of light. This stimulus activates the whole starburst network resulting in widespread changes in ACh3.0 fluorescence (Whole network stim.). d. The spatial profile of sites which respond to electrical stimulation of the starburst. The average fluorescence in the red channel (left) is shown weighted to the peak ACh3.0 signals evoked by stimulating single starburst (middle) (n = 81 sites from 12 cells; same as Fig. 6d). The right panel shows the average ACh3.0 signal in a subset of these sites when stimulated with a moving spot of light (n = 53 sites from 7 cells). The ACh sensor signals during light stimulation were widespread in the same regions where signals from electrical stimulation were highly localized. Hence the localized activity observed during electrical stimulation accurately reports on ACh release, and is not an artefact of the sensor expression patterns. e. Similar to d, but for sites which do not respond to electrical stimulation of the starburst (n = 63 sites from 6 cells). Note that these sites responded to stimulating the whole starburst network with light, indicating that the ACh sensor was functional. These observations indicate that these varicosities do not release detectable ACh. Orange arrow in f, an example of a "looping" dendritic arbor (green, filled dendrite; magenta, ChAT). A magnified image of the dendritic stratification (white box in g) is shown in h (left). Right, the fluorescence intensity profile (green, filled dendrite; magenta, ChAT). This confirmed the observations in a-c that the EYFP cells co-stratified with starburst cells, suggesting that these cells were DSGCs. However, this line of analysis was not repeated multiple times, as more direct evidence in the form of their light responses confirmed these EYFP cells to be DSGCs (see m, n). i. Density recovery profile of Oxtr-T2A-Cre labeled cells. Note that the cells overlapped with ChAT were excluded from the calculation. The effective radius was 48.4 um. Data represented as mean ± SD. j. Histogram of the nearest-neighbor distance in the Oxtr-T2A-Cre labeled cells. k. Top, example firing responses to a static flash stimulus (gray shaded, 300 µm spot, 2 s duration, 50% contrast) recorded from a labeled cell. Bottom, firing raster in three trials, and a peri-stimulus time histogram. l. Summary of peak firing rates before stimulus, light ON, and light OFF phases (gray, 6 cells; black, average). m, n. Directional tunings of the labeled cells (gray, 6 cells; arrow, vector sums in the individual cells) in responses to motion stimulus (n; 500 µm in diameter, 1000 µm/s, 50% contrast). The labeled cells showed high direction selective index (DSI; m) with an overall preference for nasal direction. Source data are provided as a Source Data file for Supplementary fig. 7e, h, i, j, l, m, n.

SUPPLEMENTARY METHODS
In the kinetic model used to estimate cholinergic currents ( Supplementary Fig. 5), a series of states were connected by a set of rates that allowed receptors to transition between states. At each time step, the population of receptors that will move from a given state to an adjacent state was calculated by simply multiplying the proportion of receptors in that state with the rate constant and the duration of the time step (1 μs). Each of the rates leaving a given state is applied independently, giving the proportion of receptors that will be transferred to the corresponding adjacent states. Note that moving from unbound to bound states (C0C1 or C1C2) is dependent on the concentration of ACh, and hence, in these cases, the transitioning receptor population was calculated by further multiplying rate constants with the ACh concentration. After calculating the transitioning population for each state, the receptor proportions were updated, and this process was repeated for the next time step. At any given time, the proportion of receptors in the open state relative to the total receptor population is used as the model output.
The transition rate constants were replicated from Coggan et al. 3 (2005) and are tabulated below. α7-nAChRs* refers to receptors similar to α7-nAChRs but with a slower desensitization rate.
The time course of ACh concentration was modeled using either 2D or 3D diffusion models, replicated from Barbour and Hausser 2 (1997), and described using the following equations.
[ACh](t) 2D = 4ℎ where [ACh](t)2D and [ACh](t)3D represents the ACh concentration at the time t for the 2D and 3D diffusion models, M is the quantity of molecules released (10000), h denotes the height of the disc (20nm), D is the diffusion coefficient (4 x 10 -6 cm 2 /s) 4 , r is the distance from the release site (either 0 or 1.1 was used), α corresponds to the fraction of the extracellular space relative to the total volume (0.21), and λ denotes the tortuosity of the extracellular space (1.55).
To identify the morphology of the labeled EYFP cells in Oxtr-T2A-Cre X Thy1-STOP-EYFP retinas, the cells were filled with neurobiotin. After the recordings, the retinas were fixed for 30 minutes in 4% paraformaldehyde in PBS, and washed with PBS overnight at 4 0 C on a shaker. The retinas were incubated in 30% sucrose in PBS for at least 3 hours at room temperature (RT).
To enhance the penetration of antibodies, retinas were transferred in the sucrose buffer and