Concurrent activation of striatal direct and indirect pathways during action initiation

Abstract

The basal ganglia are subcortical nuclei that control voluntary actions, and they are affected by a number of debilitating neurological disorders. The prevailing model of basal ganglia function proposes that two orthogonal projection circuits originating from distinct populations of spiny projection neurons (SPNs) in the striatum--the so-called direct and indirect pathways--have opposing effects on movement: activity of direct-pathway SPNs is thought to facilitate movement, whereas activity of indirect-pathway SPNs is presumed to inhibit movement. This model has been difficult to test owing to the lack of methods to selectively measure the activity of direct- and indirect-pathway SPNs in freely moving animals. Here we develop a novel in vivo method to specifically measure direct- and indirect-pathway SPN activity, using Cre-dependent viral expression of the genetically encoded calcium indicator (GECI) GCaMP3 in the dorsal striatum of D1-Cre (direct-pathway-specific) and A2A-Cre (indirect-pathway-specific) mice. Using fibre optics and time-correlated single-photon counting (TCSPC) in mice performing an operant task, we observed transient increases in neural activity in both direct- and indirect-pathway SPNs when animals initiated actions, but not when they were inactive. Concurrent activation of SPNs from both pathways in one hemisphere preceded the initiation of contraversive movements and predicted the occurrence of specific movements within 500 ms. These observations challenge the classical view of basal ganglia function and may have implications for understanding the origin of motor symptoms in basal ganglia disorders.

Figure 1. Optical measurement of neural activity-dependent fluorescence changes in direct- and indirect-pathway SPNs in freely moving mice

a, Schematic of TCSPC-based fiber optic system. 1: single-mode fiber for excitation. 2: multimode fiber for photon collection. 3: optional electrophysiological cable. 4: right lever. 5: food magazine. 6: left lever. Insert illustrates hybrid fiber probe (8) lowered into dorsal striatum and fixed in place by dental acrylic (7). b, Time-resolved spectrum of GCAMP3 expressed in striatal SPNs. c, d, Fluorescence spectrum (c) and lifetime (d) of GCAMP3, GFP and brain autofluorescence measured from dorsal striatum of GCAMP3-expressing A2A-Cre, D2-GFP and wildtype mice, respectively. e, g, GCAMP3 fluorescence from acutely cut brain slices showing GCAMP3 selectively expressed in direct- and indirect-pathway SPNs in D1-Cre (e) and A2A-Cre (g) mice, respectively. White arrowheads indicate fiber probe tract. f, h, Examples of striatal GAMP3 fluorescence recorded in two freely moving D1-Cre (f) and A2A-Cre (h) mice. i, Examples of striatal GFP fluorescence recorded in two freely moving D2-GFP mice. j, k, Example traces showing that isoflurane anesthesia (j) and intra-striatal TTX injection (k) abolished GCAMP3 transients in A2A-Cre mice. l, Summary of isoflurane and TTX effects on GCAMP3 transient frequency. ***P<0.001, unpaired t-test, n=5 bins under each condition per animal, bin size=1 min. Error bars indicate ±S.E.M.

Figure 2. Sensory stimuli evoked brief activation in both direct- and indirect-pathway SPNs

a, b, Examples of GCAMP3 fluorescence recorded in the first minute of a lever-pressing session. Vertical ticks indicate timestamps of behavioral events. Arrows indicate session start with complex visual (light on, both levers extended) and auditory (sound of lever extension) stimuli. c-e, Averaged transients in D1-Cre (c), A2A-Cre (d) and D2GFP mice (e) aligned to session start. ***P<0.001, paired t-test between baseline (averaged between -1s and session start) and peak value (averaged between 250 ms and 350 ms after session start), n = 11 trials from 4 D1-Cre mice, 11 trials from 4 A2A-Cre mice, and 4 trials from 2 D2-GFP mice. Grey lines indicate ± S.E.M.

Figure 3. GCAMP3 fluorescence transients in both direct- and indirect-pathway SPNs during action initiation in a lever-pressing task

a, b, Examples of GCAMP3 fluorescence changes in direct- (a) and indirect-pathway (b) SPNs during a two-lever free choice operant task. Vertical ticks are timestamps for operant actions (lever presses + magazine entries). c, d, Comparison of CGAMP3 transient frequency during ‘inactive’ states and ‘active’ states in direct- (c) and indirect-pathway (d) SPNs. Main effect of action states, direct pathway F_1,12=212, P<0.001; indirect pathway F_1,12=108, P<0.001; Posthocs tests **P<0.01, ***P<0.001, n = 5 trials for each mouse. e, f, Peri-event time histograms (PETHs) of actions (bottom panel) and corresponding GCAMP3 fluorescence (top panel) of direct-pathway SPNs (e) and indirect-pathway SPNs (f) aligned to the first action in an action sequence. Black squares indicate individual actions. Horizontal black bars are 5 s pauses between 60 s acquisitions.

Figure 4. Both direct- and indirect-pathway SPNs showed strong activation immediately before and during contraversive movement

a, Illustration of four action types analyzed in (b) and (c): initiation of movement from left lever (LL) to food magazine (M), from M to LL, from right lever (RL) to M, from M to RL. b, c, GCAMP3 fluorescence in direct- and indirect-pathway SPNs measured in the left striatum, aligned to initiation of the corresponding actions illustrated in (a). Top row: multiple trials showing color-coded GCAMP3 fluorescence and average response from a single D1-Cre (b) and A2A-Cre (c) mouse. Bottom row: averaged responses from 3 D1-Cre (b) and 4 A2A-Cre mice (c). *P<0.05, paired-comparison between baseline and GCAMP3 peak. d, g, multiple trials of GCAMP3 fluorescence and average response aligned to the threshold of each detected fluorescence transient from a single mouse. The two boxes indicate ‘Pre’ and ‘Post’ time windows analyzed in panels (e), (f), (h), and (i). e, h, Probability analysis of different types of locomotor activities before and after onset of fluorescence transients in direct- (e) and indirect-pathway (h) SPNs in left striatum. Interaction between action states and Pre/Post, Direct-pathway F_4,10=233, P<0.001; indirect-pathway F_4,10=39.4, P<0.001; Posthocs, *P<0.05, **P<0.01, ***P<0.001. f, i, Probability analysis of two categories of locomotor activity based on whether a rightward movement was made within the defined time window. Right move- was pooled from ‘inactive’, ‘left turn’ and ‘straight’ in (e) and (h). Right move+ was pooled from ‘right turn’ and ‘complex’ in (e) and (h). N = 3 for D1-Cre, N = 4 for A2A-Cre mice. Error bars represent ±S.E.M.