Article Figures & Data
- Figure 1.
Imaging of the striatal cholinergic network in freely moving mice reveals both somatic and neuropil signals. A, Immunohistochemical analysis of dorsal striatum of ChAT-cre mice stereotactically injected with AAVs harboring floxxed GCaMP6s demonstrates its selective expression in CINs. B, A 1-mm-diameter GRIN lens is implanted into dorsolateral striatum following aspiration of cortical tissue. C, Implanted mouse with a microendoscope mounted on its head moves freely in a behavior chamber. D, Image via lens in freely moving mouse reveals a GCaMP6s signal from 44 identifiable somata and from the surrounding neuropil. E, 3-D rendition of a patch of the ΔF/F signal surrounding a soma reveals that an elevation in the neuropil signal precedes elevation of the somatic signal (region of soma indicated by a red circle). F, Illustration of the sampling of a somatic ROI (central circle) and a surrounding (white) annular region.
- Figure 2.
Cholinergic neuropil signal in dorsolateral striatum in freely moving mouse. A, Color-coded matrix of activity of neuropil ΔF/F signal sampled from the annuli surrounding 44 somata scattered in the FoV. Time is represented along the horizontal axis. Each row corresponds to an individual annulus. B, Population average of signals from all annuli (red, using annuli associated with the somata; gray, using randomly located circular ROIs that are far from any soma). Arrows above represent peaks of strong network activation (see Materials and Methods). C, Color-coded activity of the 44 somata ΔF/F signals, after subtraction of the surrounding annular signal.
- Figure 3.
Annular (neuropil) signal precedes somatic signal in freely moving mice. A, Calcium (ΔF/F) signal from a soma–annulus pair. B, Average calcium signal from the soma and its corresponding annulus averaged over soma–annulus pairs triggered on the somatic calcium events. Shaded areas mark the 95% confidence intervals. C, Boxplot of decay time constants of somatic versus annular calcium signals. Bold line is the median and the whiskers are the 25th and 75th percentiles.
- Figure 4.
2PLSM imaging of CIN somata in acute striatal slices. A, 2PLSM image of CINs expressing GCaMP6s in an acute striatal slice. Nine individual CINs are identified and color-coded with arrows. B, Color-coded traces of calcium (ΔF/F) signals from the nine cells depicted in A. C, 2PLSM image from another mouse, with three identified and color-coded CINs. D, Three repetitions of calcium imaging of the three CINs depicted in C, a few minutes apart.
- Figure 5.
Estimation of autonomous discharge of CINs using 2PLSM GCaMP6 imaging is limited to bursting or to slow regular firing neurons. A, 2PLSM imaging of somatic and dendritic calcium (ΔF/F) signals in conjunction with electrophysiological recording from GCaMP6s-expressing CIN. B, Somatic (blue) and dendritic (pink) ΔF/F signals and membrane potential of CIN depicted in A. C, 2PLSM somatic imaging and electrophysiological recording from another CIN. D, Somatic imaging and corresponding electrophysiological recording from CIN firing autonomously at a lower rate (top) or driven to fire faster at a higher rate (bottom). E, STA of somatic ΔF/F signal (from panel D) for slow (top) and fast (bottom) discharge. F, Amplitude of STA versus the firing rate of regularly firing CINs. Solid blue line, Fit of phenomenological model (see text).
- Figure 6.
Wide-field one-photon imaging of CIN somata in acute striatal slices. A, Wide-field image of CINs expressing GCaMP6s in an acute striatal slice. Five individual CINs are identified and color-coded with arrows. B, Calcium (ΔF/F) signal from the five cells depicted in A. C, Wide-field image from another cell recorded in cell attached mode. D, Calcium (ΔF/F) in conjunction with spike raster of the CIN depicted in panel C.
- Figure 7.
Dendritic GCaMP6 signals detectable using 2PLSM imaging are limited to bAPs. A, 2PLSM imaging of somatic and dendritic calcium (ΔF/F) signals in conjunction with electrophysiological recording from GCaMP6s-expressing CIN surrounded by PfN fibers expressing ChR2 (not shown). B, Calcium (ΔF/F) signals in response to optogenetic activation of PfN synapses elicits either a subthreshold (green) or suprathreshold (red) synaptic response (bottom) in CIN depicted in A. Only the spiking response elicits a detectable ΔF/F signal. Subthreshold depolarization (in another cell) does not elicit a detectable response (cf. Goldberg et al., 2009). C, Distribution of integrated ΔF/F response in response to spontaneous bAPs, subthreshold EPSPs, and subthreshold depolarization.
- Figure 8.
Spike-triggered averages of calcium transients elicited by bAPs at various distances from the soma. 2PLSM line scans performed at various linear distances from the soma of a GCaMP6s-expressing CIN.
- Figure 9.
Dendritic calcium transients are larger than somatic ones. A, 2PLSM imaging of somatic and dendritic calcium (ΔF/F) signals in conjunction with electrophysiological recording from GCaMP6s-expressing CIN. B, Calcium (ΔF/F) responses in soma (black) and proximal dendrite (pink) during spontaneous discharge (bottom). C, STA of proximal dendritic (pink) calcium signals is larger than the somatic (black) signal. Right, Paired comparison of the amplitude of somatic and dendritic STAs.
Data structure Type of test Power a Ranks Two-tailed Wilcoxon signed rank test for paired replicates 3.7 × 10−4 b Ranks Two-tailed Wilcoxon signed rank test for paired replicates 7.8 × 10−3 c Ranks Two-tailed Wilcoxon signed rank test for paired replicates 0.0195 d Ranks Two-tailed Wilcoxon signed rank test for paired replicates 7.35 × 10−4
- Movie 1.
Synchronous spatiotemporal patterns in striatal cholinergic neuropil of a freely moving mouse. Microendoscopic imaging of the DLS of a freely moving mouse expressing the calcium indicator GCaMP6s selectively in CINs. The size of the visualized area is ∼600 ×900 µm, and the movie is presented in real time. Imaging reveals fluctuations in the fluorescence of cholinergic neuropil characterized by rapid bursts of activation that permeate the entire FoV and decay slowly. The movie includes three examples of activation events.
- Movie 2.
The cholinergic neuropil calcium signal precedes the somatic signal. Surface plot visualization of the microendoscopic calcium imaging signal in the DLS of a freely moving mouse (as in Movie 1). Visualized area is a 40 × 60 µm patch consisting of a single CIN soma surrounded by cholinergic neuropil. Example of an activation event demonstrating that somatic fluctuations are superimposed upon the temporal fluctuations of surrounding pixels and are slightly preceded by them.
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