Slice electrophysiological recordings
Coronal sections (300 μm thick) containing dorsolateral striatum were prepared from brains of 3–5 week-old male and female Lhx6-EGFP mice on an FVB genetic background. Sections were prepared on a Leica VT1000S vibratome in an ice-cold sucrose cutting solution (in mM): 79 NaCl, 23 NaHCO3, 68 sucrose, 12 glucose, 2.3 KCl, 1.1 NaH2PO4, 6 MgCl2, and 0.5 CaCl2. Sections were bisected to generate left and right hemisphere slices. and transferred to a chamber filled with warmed carbogenated ACSF containing (in mM): 125 NaCl, 26 NaHCO3, 2.5 KCl, 1 MgCl2, 2 CaCl2, 1.25 NaH2PO4, and 12.5 glucose.
All recordings were performed at 31 – 33°C in ACSF (see above), in the presence of 50 μM picrotoxin to block GABAA-mediated currents. For experiments where rectification of AMPAR-mediated EPSCs was measured, 50 μM APV was also included in the external medium. To isolate NMDAR-mediated EPSCs in MSNs (), neurons were voltage clamped at +40 mV and 5 μM NBQX was included in the bath to block AMPAR-mediated currents. In most experiments, EPSCs were evoked using a glass stimulating electrode positioned in the striatum within 100 – 200 μM of the recorded neuron. The tissue was stimulated with 0.2 ms electrical pulses, every 20 s (2 – 6 μA for FSIs and MSNs and 10 – 20 μA for PLTS). Because stronger stimuli were required to elicit EPSCs onto cholinergic interneurons, a concentric bipolar stimulating electrode was used for these experiments. The stimulating electrode was positioned within the striatum > 500 μm away from the recorded neuron. The tissue was stimulated with 0.2 ms electrical pulses, every 20 s (10 – 20 mA).
Figure 1 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 (more ...)
Internal solution contained (in mM): 120 CsMeSO3, 15 CsCl, 8 NaCl, 0.5 EGTA, 10 Hepes, 2 Mg-ATP, 0.3 Na-GTP, 5 QX-314, pH 7.3. For experiments measuring NMDA currents in MSNs we added 10 mM BAPTA, and for experiments measuring rectification we added both 10 mM BAPTA and 0.1 mM spermine.
Data were collected with a MultiClamp 700B amplifier (Molecular Devices) and ITC-18 A/D board (HEKA) using Igor Pro software (Wavemetrics) and custom acquisition routines (mafPC, courtesy of M.A. Xu-Friedman). Recordings were filtered at 2 kHz and digitized at 10 kHz. Electrodes were made from borosilicate glass (pipette resistance, 2 – 4 MΩ). During recordings, Rseries was monitored and recordings were terminated if Rseries changed by >30%.
FSIs and PLTS interneurons were identified using GFP fluorescence, and distinguished using Rin
in whole cell recordings as previously established (Gittis et al., 2010
). Cholinergic interneurons were identified by their large somata and lack of GFP fluorescence in the Lhx6-EGFP line.
In vivo electrophysiological recordings
Both male and female mice were used for in vivo
experiments. Two mice were implanted with 64-channel silicon probes (8×8 grid, 200 μm spacing; NeuroNexus Technologies, Ann Arbor, Michigan) with the long axis of the probe aligned at 25° from the anterior-posterior direction so that all contacts were in left dorsal/lateral striatum. Infusion cannulae (Plastics One, Roanoke, Virginia) had their tips near the center of the probe grids (tip AP +0.4 mm, ML +/−2.2 mm, DV 2.5 mm). Following recovery from surgery, wideband data (1–9000 Hz) were recorded continuously from all sites at 31,250 Hz as described previously (Wiltschko et al., 2010
). Signals were referenced to a skull screw over the midline cerebellum. During each session, a 15 min baseline recording was followed by infusion of ACSF (0.25 μl/min for 2 min, with the cannula left in place for 2 minutes post-infusion). After 30 minutes, 1 mM IEM-1460 dissolved in ACSF was infused using the same parameters. The recording continued for at least 90 min.
To extract single unit activity, the wideband signals were wavelet-filtered offline using custom Matlab scripts (Wiltschko et al., 2008
), and spikes were clustered in Offline Sorter (Plexon, Inc.). The resulting spike timestamps were used to extract mean wideband waveforms for each unit. To classify units as FSIs or MSNs (Berke et al., 2004
; Gage et al., 2010
), the full width at half-maximum (FWHM) and peak-to-valley (P-V) times were used (see ; FSI: FWHM 50 to 150 ms, P-V 50 to 455 ms; MSN: FWHM 150 to 450 ms, P-V 560 to 1500 ms). None of the cells classified as MSNs or FSIs had the regular tonic firing characteristic of cholinergic interneurons. Any units that did not meet these waveform criteria were labeled as “unclassified.” Inverted units (units with a more prominent positive than negative waveform) were automatically labeled as unclassified, as the FWHM and P-V times have no clear meaning for these units.
Figure 2 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 = (more ...)
Three groups of mice were implanted with infusion cannulae. For each group, half of the implants were in the right hemisphere. For the first group (“Group 1”, Black circles in , n=6) the target was dorsolateral striatum (AP +0.4 mm, ML +/− 2.4 mm, DV 2.8 mm relative to Bregma). After 2 days for recovery, the mice were infused daily with one of 3 concentrations (250 mM, 1 mM, or 2.5 mM) of IEM-1460 dissolved in ACSF or ACSF alone. Doses were delivered in a counterbalanced order over four successive days.
Figure 3 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 (more ...)
The second group (“Group 2”, Grey circles in , n=4) was implanted with cannulae targeted to the same coordinates, but received combined infusions of mecamylamine (a nicotinic receptor antagonist) and scopolamine (a non-specific muscarinic antagonist). Two doses were used: 5 mg mecamylamine with 10 mg scopolamine, and 10 mg mecamylamine with 20 mg scopolamine. These values were chosen to be on the high end of doses previously found to cause learning impairments in rats and mice (Klinkenberg and Blokland, 2010
; Schildein et al., 2002
The third group (“Group 3”, Open circles in , n=4) was implanted with cannulae in dorsomedial striatum (AP +0.4 mm, ML +/− 1.25 mm, DV 2.4 mm relative to Bregma), and received infusions of ACSF and 2.5 mM IEM-1460 in counterbalanced order.
All drug infusions were performed at a rate of 0.25 μl/min for 2 min. The infusion cannula was left in place for a further 2 minutes to allow drug diffusion, followed by 90 minutes of video recording. 60-second segments were scored every 5 minutes for the first 30 minutes after the infusion, and for every 10 minutes thereafter by a clinical neurologist (DKL) blind to the treatments. Two previously described semi-quantitative rating scales (Shirley et al., 2008
) were used: a “dyskinesia” score that characterized the range of movement types and body parts involved, and an “impairment” score that characterized the severity of the movements. Briefly, a “1” indicated minor motor impairment, “2” indicated moderate abnormalities with at most infrequent falls, “3” indicated significant impairment with frequent falls and severely impaired locomotion, and “4” indicated severe impairment with almost no ambulation (Shirley et al., 2008
). The sole difference to the previously reported rating scales is that we also noted laterality for each relevant movement type (for example, neck twisting could be scored as ipsilateral or contralateral to the infusion, but neck flexion could not).
Data Analysis and Statistics
The area under each dyskinesia (or impairment) score vs. time curve was calculated for each behavioral session. The areas under the curve (AUC) in Group 1 at each dose were compared using ANOVA with post-hoc Bonferroni corrections for pairwise comparisons. AUCs for Group 2 were analyzed in the same manner. AUCs at the 2.5 mM IEM dose for dorsomedial and dorsolateral striatum (Groups 1 & 3) were compared with a 2-sample t-test. All tests were considered significant at p<0.05.
Single-unit firing rates were calculated for the period beginning 10 minutes after each infusion to 5 minutes before the next infusion (the baseline period began with the beginning of the recording; the IEM-1460 period ended with the end of the recording). The overall firing rate of each cell was computed as the number of spikes recorded in an epoch divided by the epoch duration. Firing rates of distinct cell populations in the baseline, ACSF, and IEM-1460 epochs were compared using Friedman’s test with post-hoc pairwise comparisons (SPSS 18.0). To determine if an individual unit changed its firing rate with each intervention, firing rates were computed in 1 s non-overlapping windows during the ACSF and IEM-1460 epochs, creating a distribution of firing rates for each epoch. These rates were compared using paired t-tests, with p < 0.01 considered to indicate a significant change in firing rate.