Figures and data
SCIN burst-pause response to activation of thalamic terminals.
A. Experimental design. B. Micrograph showing the thalamic region transfected with ChR2-YFP (left) and a striatal region (z-stack maximal projection) showing tdTomato-reported SCIN and thalamic terminals expressing ChR2-YFP (right). C. Schematic representation of optogenetic stimulation of thalamic inputs to a SCIN recorded in cell-attached configuration. Picrotoxin (PIC, 100 uM; GABA-A antagonist) was present in the bath in all experiments. D. Representative recording of a SCIN that responded to optogenetic activation of thalamic terminals with 1 spike. Inset: Detail of the measurements of x1 and x2 values. x1: time from the last spike before the optogenetic stimulation and the start of the stimulation; x2: time from the initiation of stimulation to the following spike. E. x2/Baseline ISI vs x1/Baseline ISI for single spike responses to optogenetic activation of thalamic terminals (Nonlinear fit, R2=0.06237; Pearson correlation *p=0.0034). F. Representative recordings of the pause response of a SCIN to optogenetic stimulation of thalamic terminals in cell-attached configuration before and after bath application of CNQX (20 uM; AMPA receptor antagonist). G. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals (One-way RM ANOVA, treatment 2 vs 3 spikes: *p=0.0032; 2 vs 4 spikes: *p=0.0017). H. Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals (One-way RM ANOVA, ns). I. Representative recordings of SCIN stimulated with one train of optogenetic activation of thalamic terminals (top) or with two trains (bottom). J. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, with one or two trains of stimulation pulses. The second train occurs 350 ms after the first train. (Two-way RM ANOVA, interaction ns; treatment *p<0.0001; Elicited spikes *p<0.0001; 2 vs 4 spikes *p=0.0042). Mean +/- SEM; n=11-18 cells per group, from >5 mice.
Contribution of the Kv1 current to the pause response.
A. Schematic representation of the tested hypotheses. DP: depolarization; HP: hyperpolarization; Glu: glutamate; DA: dopamine. B. Representative recordings of SCIN in response to optogenetic activation of thalamic terminals, with or without Margatoxin (MgTx; Kv1.3 channel blocker) or dendrotoxin (DTx; Kv1.1 and Kv1.6 channels blocker) in the bath. C. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, with or without MgTx 3nM, 30 nM, or DTx 100 nM, in the bath (Two-way RM ANOVA interaction ns, treatment *p=0.0051; Control-MgTx 30nM: *p=0.0005; Control-MgTx 3nM: ns; Control-DTX: *p=0.0462; MgTx 30nM-MgTx 3nM: *p<0.0001; MgTx 30nM-DTX: ns; MgTx 3nM-DTX: *p=0.0027). D. Baseline ISI of SCIN recorded with or without MgTx 3nM, 30 nM, or DTx (One-way ANOVA, ns). E. Burst duration of SCIN that responded with 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, with or without MgTx 3nM, 30 nM or DTx 100 nM, in the bath (Two-way RM ANOVA, ns). F. Representative recordings of SCIN in response to optogenetic activation of thalamic terminals, with or without Ba2+ (10 uM; Kir2.2 channel blocker at this concentration) or XE 991 (10 uM; Kv7 channel blocker) in the bath. G. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, with or without 10 uM XE 991 or 10 uM Ba2+ (Two-way RM ANOVA, ns). H. Baseline ISI of SCIN recorded with or without Ba2+ or XE 991 (One-way ANOVA, ns). I. Burst duration of SCIN that responded with 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, for the above conditions (Two-way RM ANOVA, ns). Mean +/- SEM; n=7-16 cells per group, from >5 mice.
Modulation of the pause response by dopamine receptors.
A. Schematic representation of the tested hypotheses. DP: depolarization. B. Representative responses of SCIN to optogenetic activation of thalamic terminals, with SKF81297 (2 uM; D1/D5 selective agonist), SCH23390 (10 uM; D1/D5 selective antagonist) or SKF81297 + SCH23390 in the bath. C. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (Two-way RM ANOVA, interaction p=0.0381, Dunnett’s multiple comparisons test: Control vs SKF81297: 3 spikes *p=0.0001; 4 spikes *p=0.0010.). D. Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (One-way ANOVA, ns). E. Burst duration of SCIN that responded with 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (Two-way RM ANOVA, ns). F. Representative responses of SCIN to optogenetic activation of thalamic terminals, with Sulpiride (10 uM; D2-type receptor selective antagonist) or Mecamylamine (10 uM; nicotinic receptors antagonist) in the bath. G. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (Two-way RM ANOVA, interaction p=0.0369, Dunnett’s multiple comparisons test: Control vs Sulpiride: 4 spikes *p=0.0056; Control vs Mecamylamine: 4 spikes *p=0.0167.). H. Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (One-way ANOVA, ns). I. Burst duration of SCIN that responded with 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (Two-way RM ANOVA, ns). Mean +/- SEM; n=7-15 cells per group, from >5 mice.
D5R modulation independent of Ih current.
A. Schematic representation of the tested hypotheses. B. Time matched representative responses of SCIN to hyperpolarizing current steps with or without ZD7288 (30 uM; HCN channel blocker) in the recording pipette, and with or without SKF81297 (2 uM; D1/D5 selective agonist) in the bath. C. Sag evaluated in the hyperpolarizing step, under the above conditions (One-way ANOVA), p<0.0001; Tukey’s multiple comparisons test: Control vs ZD7288 *p<0.0001; Control vs ZD7288+SKF81297 *p<0.0001). D. Representative responses in whole cell configuration to optogenetic activation of thalamic terminals, under the above conditions. E. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (Two-way RM ANOVA, interaction ns, treatment p=0.0373; Control vs ZD7288+SKF81297: *p=0.0005; ZD7288 vs ZD7288+SKF81297: *p=0.0067). F. Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (One-way ANOVA, ns). G. Burst duration of SCIN that responded with 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (Two-way RM ANOVA, ns). Mean +/- SEM; n=13/14 cells per group, from >5 mice.
SCIN from L-DOPA-treated parkinsonian mice show a reduced pause response.
A. Experimental design. B. Micrographs showing TH immunostaining at the level of the substantia nigra. C-E. Rotation index (C), forelimb asymmetry (D) and latency to fall from the rotarod (E) for sham and 6-OHDA mice (C-D: unpaired t test, **p<0.0001; E: Two-way RM ANOVA, interaction: *p=0.0284, n=5 sham and 22 6-OHDA mice). F. Abnormal involuntary movement (AIM) score of chronically L-DOPA-treated 6-OHDA mice. (Two-way RM ANOVA, interaction: p=0.0081; post-hoc: Day 1 vs Day 4 *p<0.03). G. Representative cell-attached recordings of the pause response of SCIN to optogenetic stimulation of thalamic terminals, in a sham and a dyskinetic mouse in the OFF L-DOPA condition, with or without MgTx 30 nM in the bath. H. Pause duration/Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals (Two-way RM ANOVA, interaction ns, treatment *p=0.0227; Sham vs OFF L-DOPA: *p=0.0301; OFF L-DOPA vs OFF L-DOPA+MgTx: ns; Sham vs OFF L-DOPA+MgTx: *p=0.0032). I. Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals under the above conditions (One-way ANOVA, *p=0.0254; Sham vs OFF L-DOPA: *p=0.0201; OFF L-DOPA vs OFF L-DOPA+MgTx: ns; Sham vs OFF L-DOPA+MgTx: ns). J. Burst duration of SCIN that responded with 2, 3 or 4 spikes to optogenetic activation of thalamic terminals under the above conditions (Two-way RM ANOVA, ns). Mean +/- SEM; n=14-27 cells per group, from >5 mice.
D1-like receptors inverse agonist restores the pause response in SCIN from OFF L-DOPA parkinsonian mice.
A. Schematic representation of the tested hypothesis. B. Representative responses of SCIN from OFF L-DOPA dyskinetic mice to optogenetic activation of thalamic terminals, with or without SKF81297, Clozapine (10 uM; D1/D5 receptor inverse agonist), SCH23390 (10 uM; D1/D5 selective antagonist), Clozapine + SCH23390 or Clozapine + MgTx, in the bath. C. Pause duration/Baseline ISI of SCIN from OFF L-DOPA dyskinetic mice that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals (Two-way RM ANOVA, interaction *p=0.0489, Dunnett’s multiple comparisons test: 4 elicited spikes: OFF L-DOPA vs Clozapine: *p=0.0181). D. Baseline ISI of SCIN that responded with 1, 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (One-way ANOVA, ns). E. Burst duration of SCIN from dyskinetic mice OFF L-DOPA that responded with 2, 3 or 4 spikes to optogenetic activation of thalamic terminals, under the above conditions (Two-way RM ANOVA, interaction ns, treatment ns). Mean +/- SEM; n=6-17 cells per group, from >5 mice. F. Schematic representation of conclusion. Left, in physiologic conditions the balance of D2 versus D5 activation emerges as a determinant of the Kv1-dependent pause expression; right, in parkinsonian mice an imbalance towards D5 signaling abolishes the pause (top), which can be reset with D5 inverse agonism (bottom).
