Article Figures & Data
Figures
- Figure 1.
Optogenetic activation of striatal cholinergic interneurons elicits complex PSPs in FSIs. A1–A4, Striatal slices in ChAT-Cre::Lhx6-GFP mouse injected with AAV-DIO-ChR2-mCherry. Immunohistochemistry for Lhx6-GFP (A1), mCherry (A2), ChAT (A3), and all three (A4). Scale bar, 50 μm. B, Left, Recording configuration. Whole-cell current-clamp recordings from mCherry-positive cholinergic interneurons. Membrane potential responses to injection of ± 100 pA (left) or a train of 5 ms pulses of blue light (470 nm) at 5 Hz (right, blue bar). C, Left, Schematic of recording configuration. Representative light-evoked synaptic potentials in whole-cell current-clamp recordings from striatal FSIs before (center) and after (right) addition of picrotoxin (picro; 50 μm). D, Upper left, Presumed glutamatergic responses recorded in the presence of picrotoxin, scopolamine (scop; 10 μm), mecamylamine (mec; 5 μm), and methyllycaconitine (MLA; 10 nm). Lower left, Presumed cholinergic responses recorded in the presence of picrotoxin, NBQX, and APV. Right, Average PSP amplitude attributable to glutamate (Glu), acetylcholine (ACh), or GABA, and the total PSP. Arrow indicates resting membrane potential.
- Figure 2.
Optogenetic stimulation of cholinergic interneurons triggers VGLUT3-dependent glutamatergic currents. Recordings performed in picrotoxin, mecamylamine, scopolamine, and methyllycaconitine to isolate glutamatergic currents. A, Whole-cell voltage-clamp recordings of FSIs from ChAT-Cre::Lhx6-GFP mice injected with AAV-DIO-ChR2-mCherry. Left, Recording configuration. Representative light-evoked EPSC recorded at −70 mV, before (black) and after (gray) application of the AMPA antagonist NBQX (5 μm). Center, Representative light-evoked EPSCs at holding potentials of −80 mV (black) and +60 mV (gray). Right, Current–voltage relationship of EPSCs before (open symbols; n = 6 neurons) and after (filled symbols) application of APV (50 μm). B, Whole-cell voltage-clamp recordings from striatal FSIs in Lhx6-GFP mice following cortical injection of AAV-hSyn-ChR2-YFP. Left, Recording configuration. Center, Representative light-evoked EPSCs at holding potentials of −80 mV (black) and +60 mV (gray). Right, Current–voltage relationship of EPSCs (n = 6 neurons). C, Light-evoked currents in ChAT-Cre::Lhx6-GFP::VGLUT3 KO mice following striatal injection of AAV-DIO-ChR2-mCherry. Left, Recording configuration. Center, Representative light-evoked EPSC at −70 mV. Right, Glutamatergic EPSC amplitudes in WT (n = 19 neurons, small black circles) versus VGLUT3 KO mice (n = 8 neurons, red circle). Large filled symbols show average ± SEM in each group. Ctl, control. *p ≤ 0.05.
- Figure 3.
Optogenetic stimulation of cholinergic interneurons triggers VGLUT3-dependent monosynaptic nicotinic currents. Whole-cell voltage-clamp recordings from striatal FSIs recorded in the presence of picrotoxin, NBQX, and APV to isolate cholinergic currents. A, ChAT-cre::Lhx6-GFP mice injected with AAV-DIO-ChR2-mCherry in dorsal striatum. Left, Recording configuration. Center, Representative light-activated currents recorded from an FSI at −70 mV before (black) and after (orange) application of mecamylamine. Right, EPSC amplitudes before and after addition of mecamylamine (mec; n = 6 neurons); filled symbols represent average ± SEM. B, ChAT-cre::Lhx6-GFP::VGLUT3 KO mice injected with AAV-DIO-ChR2-mCherry. Left, Recording configuration. Center, Representative light-activated current at a holding potential of −70 mV. Right, Cholinergic EPSC amplitudes in FSIs from WT (black symbols; n = 9 neurons) and VGLUT3 KO mice (red symbols, n = 6 neurons). Filled symbols represent average ± SEM. *p ≤ 0.05.
- Figure 4.
Light-evoked disynaptic inhibitory currents in MSNs depend on VGLUT3. ChAT-Cre animals injected with AAV-DIO-ChR2-mCherry in dorsal striatum. Whole-cell recordings of striatal MSNs measured in the presence of NBQX and APV to isolate GABAergic currents. Voltage-clamp recordings with cesium chloride-based internal solution, resulting in inward inhibitory currents at −70 mV. A, Left, Recording configuration. Center, Representative light-evoked IPSC from a WT mouse. Right, Light-evoked voltage response during spiking (induced with direct current injection). B, Left, Representative light-evoked IPSC in a VGLUT3 KO. Middle, Light-evoked voltage response during spiking (induced with direct current injection). Right, Light-evoked IPSC amplitudes in MSNs from WT (black symbols, n = 7 neurons) and VGLUT3 KO (red symbols, n = 16 neurons) animals. Filled symbols represent average ± SEM. In current-clamp traces, arrow indicates resting membrane potential. *p ≤ 0.05.
