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. 2017 Sep 5;6:e26231. doi: 10.7554/eLife.26231

Figure 1. Striatal output reconfiguration following pharmacological inhibition of FSIs directly opposes substrates for habitual behavior.

(A) Schematic of calcium imaging approach. Top: SPN activity was evoked by electrical stimulation of cortical afferent fibers in an acute parasaggital brain slice. Bottom: Evoked SPN firing was imaged in the direct and indirect pathways simultaneously using a transgenic direct pathway reporter mouse line (left), calcium indicator dye fura-2 (middle) and two-photon laser scanning microscopy (right, see scanning vector in overlay). (B) Experimental approach. Striatal microcircuitry was manipulated in tissue from untrained animals in order to reproduce the known circuit substrate for habitual behavior (described in O'Hare et al., 2016) and thereby identify a candidate microcircuit mechanism. (C) Representative heat maps of dSPN (x) and iSPN (●) calcium transient amplitudes before (left) and after (right) pharmacological inhibition of FSIs using IEM-1460 show a selective reduction in cells with the strongest (bright red) initial responses. (D) Left: Representative SPN calcium transient waveforms before and after wash-in of IEM-1460. SPNs were grouped into ‘high-firing’ (red) or ‘low-firing’ (blue) clusters based solely on their baseline response amplitudes using a Gaussian mixture model. SPNs with strong baseline responses (red, ‘high firing’) show weaker responses after wash-in whereas those with initially weak responses (blue, ‘low firing’) are unaffected. Right: Evoked calcium transient amplitudes for all imaged SPNs before (-) and after (+) wash-in of IEM-1460. For both cell types, high-firing SPNs showed decreased responses after IEM-1460 wash-in (dSPNs: t(22) = 6.43, p=0.0000018, n = 23 cells; iSPNs: t(17) = 3.43, p=0.0032, n = 18 cells) whereas low-firing SPNs did not (dSPNs: p=0.24, n = 64 cells; iSPNs: p=0.21, n = 34 cells). (E) Linear regression and correlational analyses show that the inhibitory effect of IEM-1460 on SPN responses (post – baseline difference) is a linear function of baseline response amplitudes for both dSPNs (red; r(86) = −0.87, p=2.20×10−28, n = 87 cells) and iSPNs (green; r(51) = −0.80, p=1.59×10−12, n = 52 cells). (F) Relative pathway timing, as measured by latency to peak detection, before and after inhibition of FSIs using IEM-1460. Indirect pathway activation precedes direct pathway activation by a greater margin after wash-in of IEM-1460 (t(102) = 2.42, p=0.017, n = 52 independent dSPN/iSPN pairs). *p<0.05. Dotted error bands indicate 95% confidence interval. Error bars indicate SEM. Effects of IEM-1460 on FSI and SPN spike probability are shown in Figure 1—figure supplement 1. Electrophysiological assessment of IEM-1460’s effect on evoked multi-AP SPN responses is included in Figure 1—figure supplement 2. GMM parameters and calcium transient amplitude source data can be found in Figure 1—source data 1.

Figure 1—source data 1. GMM parameters and source data for SPN calcium transient amplitudes (MATLAB).
GMMs contains parameters for the Gaussian mixture model fits on pre-IEM-1460 calcium transient amplitude data by cell type. Amplitude values are included for high- and low-firing dSPNs and iSPNs in dSPNs_high, dSPNs_low, iSPNs_high, and iSPNs_low. Matrices are N x 2 with column 1 containing pre-drug amplitudes and column 2 containing paired measurements after drug wash-in. Data can be combined within cell type and analyzed using source code file PrePostGMM.m to reproduce the clustering shown in Figure 1D (see comments in code).
elife-26231-fig1-data1.zip (12.2KB, zip)
DOI: 10.7554/eLife.26231.006

Figure 1.

Figure 1—figure supplement 1. IEM-1460 inhibits evoked FSI firing but does not affect SPN spike probability.

Figure 1—figure supplement 1.

(A) Probability of evoked FSI action potential firing, as measured in cell-attached recordings, before and after wash-in of IEM-1460. Drug wash-in significantly inhibited FSI firing (t(5) = 4.08, p=0.0096, n = 6 cells). (B) Spike probability for dSPNs (red) and iSPNs (green) before (filled) and after (open) wash-in of IEM-1460 in 2PLSM calcium imaging experiments. Drug wash-in did not affect spike probability for dSPNs (p=0.055, n = 87) or iSPNs (p=0.11, n = 52). *p<0.05. Error bars represent SEM.

Figure 1—figure supplement 2. IEM-1460 selectively inhibits evoked multi-action potential SPN responses ex vivo.

Figure 1—figure supplement 2.

Cell-attached electrophysiological recordings showing selective effect of IEM-1460 for multi-action potential SPN responses to afferent stimulation. Left: example trace showing multi-action potential SPN response to single-pulse stimulation of cortical afferents (top) and response to same stimulus after drug wash-in (bottom). Right: Effect of IEM-1460 (left) and vehicle (right) as a function of mean # APs fired prior to drug wash-in. IEM-1460 consistently reduced SPN responses to singlets (r(7) = 0.94, p=0.00060, n = 8 cells) whereas vehicle had no such effect (mean effect = 0.28 ± 0.66; p=0.89 for correlational analysis, n = 8 cells). *p<0.05. Dotted error bands indicate 95% confidence interval.