IEM-1460 selectively reduces FSI activity in vivo. A. Schematic of in vivo recording configuration and drug infusion. An 8 × 8 chronic recording array was placed in dorsolateral striatum with an infusion cannula angled towards the array. Ctx = cortex; Str = striatum. B. Scatter plot of mean spike waveform parameters used to classify FSIs (red) and MSNs (blue). Cells were classified based on the width at half-height (x) and peak-to-valley time (y). Cells that could not clearly be sorted as FSI or MSNs were called ‘unclassified’ (black; cells with inverted waveforms not shown). Inset in bottom right of the graph shows examples of extracellular waveforms (negative voltage upwards) recorded from a presumed FSI (red) and presumed MSN (blue). Vertical scale bar = 0.1 mV; horizontal scale bar = 1 ms. C. Example of firing rates recorded simultaneously from an FSI and MSN during infusion of ACSF and then IEM-1460 (1 mM). IEM-1460 strongly suppressed spiking of the FSI and slightly increased spiking of this MSN. D. Summary of firing rate changes observed for all FSIs, MSNs, and unclassified neurons after IEM-1460 infusion. Number of neurons is listed above each bar. 54% (13/24) of MSNs increased their firing rates; 38% (9/24) decreased their firing rates; 8% (2/24) showed no change. 100% of FSIs (12/12) decreased their firing rates. 33% (5/15) of unclassified neurons increased their firing rates; 60% (9/15) decreased their firing rates; and 7% (1/15) showed no change. E. Average firing rates for the population of FSIs (n = 12) and MSNs (n = 24), after ACSF or IEM-1460 infusion. Note distinct y-axis scales for FSIs and MSNs. ACSF infusion did not significantly affect the firing rates of either population, whereas IEM-1460 significantly decreased the firing rates of FSIs, but not MSNs. * p < 0.01 compared both to baseline and to ACSF. Error bars show s.e.m.

IEM-1460 reduces EPSCs in FSIs but not MSNs. A. I–V curves of AMPAR-mediated currents in FSIs (n = 5) or MSNs (n = 5). Rectification index (current at +60 mV/current at −60 mV) was 0.11 ± 0.11 for FSIs and 0.54 ± 0.08 for MSNs. Insets show representative AMPAR-mediated currents from an FSI (red) and an MSN (blue) at V_hold = −80 mV (inward current) and V_hold = +60 mV (outward current). Stimulus artifacts were removed for clarity. For both insets, vertical scale bar = 400 pA; horizontal scale bar = 10 ms. B. IEM-1460 (50 μM) reduces EPSC amplitudes in FSIs (n = 6) but not MSNs (n = 6). Insets show EPSCs recorded from a representative MSN and FSI, respectively; solid line is the average trace 0–5 min before IEM-1460; dotted line is the average trace 25–30 min after IEM-1460. EPSCs were measured every 20 s and binned in 1 min bins. EPSCs were normalized by the average amplitude recorded during a 10 min baseline before drug application (−10 to 0 min). C. IEM-1460 (50 μM) does not affect NMDAR-mediated EPSCs in MSNs (V_hold = +40 mV). (left) Example of NMDA-mediated currents recorded from an MSN 0–5 min before (solid line) 25–30 min after (dotted line) bath application of IEM-1460. (right) Average change in NMDA-mediated currents in MSNs after 30 minutes of IEM-1460 (n = 4). D. Summary of the change in AMPAR-mediated EPSCs by IEM-1460 (EPSC amplitudes 25–30 min after IEM-1460 application/EPSC amplitudes 0–5 min before IEM-1460 application) in each cell type. E. (left) Spontaneous, extracellularly recorded action potentials from a representative cholinergic interneuron in slice 0–5 min before and 25–30 min after IEM-1460 application. Horizontal scale bar = 1 s; vertical scale bar = 1 mV. (right) Change in spontaneous firing rate of cholinergic interneurons recorded after IEM-1460 application. Bars represent the average and circles are individual cells.

IEM-1460 causes hyperkinetic motor impairments in awake, unrestrained mice. A. Picture of a mouse during a twisting episode after infusion of IEM-1460. Note the abnormal posture of the right (contralateral) arm and foot. B. Average normalized dyskinesia score by body region, split by ipsilateral/contralateral side following infusion of 1 mM IEM-1460. C. Average dyskinesia score in 10 min bins observed during 1.5 hrs after infusion of ACSF for different concentrations of IEM-1460. D. Average impairment score after infusion of ACSF for different concentrations of IEM-1460. E. Schematic of striatum showing infusion sites for behavioral experiments. Black circles are cannula placements for “Group 1”, dorsolateral IEM-1460 infusions; grey circles are cannula placements for “Group 2”, cholinergic antagonists, then IEM-1460 infusions; open circles are cannula placements for “Group 3”, dorsomedial IEM-1460 infusions. F. Scatter plot showing strong correlation (r² = 0.81, p = 1.1 × 10⁻⁵) between the medial-lateral position of the infusion cannula and the area under the dyskinesia score vs time curve (AUC) for all 2.5 mM IEM infusions. G. Average dyskinesia score AUC following infusion of ACSF, IEM-1460, or mecamylamine/scopolamine (M/S) (see Methods for doses) into dorsomedial or dorsolateral striatum. Circles indicate individual data points for each mouse.