Volume 27, Issue 13, Pages 1900-1914.e4 (July 2017) Differential Control of Dopaminergic Excitability and Locomotion by Cholinergic Inputs in Mouse Substantia Nigra  Jasem Estakhr, Danya Abazari, Kaitlyn Frisby, J. Michael McIntosh, Raad Nashmi  Current Biology  Volume 27, Issue 13, Pages 1900-1914.e4 (July 2017) DOI: 10.1016/j.cub.2017.05.084 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Lateral SNc Expressed Mainly Excitatory Glutamatergic- and Nicotinic-Mediated Cholinergic Neurotransmission (A) Direct nAChR EPSCs. (A1 and A2) Blue light activation (5 ms pulse duration, blue bars) of cholinergic terminals in the lateral SNc elicited nAChR currents that were insensitive to DNQX and AP5 but completely inhibited by the nAChR inhibitor cocktail of MEC, MLA, and DHβE. (A3) Current traces of nAChR responses at different holding potentials (−100 to +40 mV) and the corresponding I-V relationship (A4). (A5) Supportive evidence that nAChR EPSCs were monosynaptic because 4-AP (100 μM) was able to restore the blue-light-evoked response after abolishment with TTX (1 μM). (B) Indirect glutamatergic EPSCs. (B1 and B2) Disynaptic glutamatergic EPSCs mediated by presynaptic nAChRs as evidenced by inhibition with CNQX and AP5 and the cocktail of nAChR antagonists. (B3) Blue-light-evoked disynaptic glutamatergic responses at different holding potentials and I-V plot (B4) at the earlier (black circles) and latter (red triangles) time points displayed AMPA and NMDA currents, respectively. (B5) Supportive evidence that glutamatergic EPSCs were disynaptic because 4-AP was unable to restore the blue-light-evoked response after inhibition with TTX. (C) Injection of a floxed ChR2-YFP AAV into the PPT of a ChAT-cre mouse showing expression of ChR2-YFP in the PPT and fibers in the SNc. Blue-light-evoked nAChR EPSCs were inhibited by a cocktail of nAChR antagonists. (D) Injection of AAV floxed ChR2-YFP into the LDT of ChAT-cre mice, showing expression of ChR2-YFP in the LDT and in the fibers in the SNc. Also shown are blue-light-evoked disynaptic glutamatergic EPSCs that were reversibly inhibited by DNQX and AP5. Summary data are reported as mean ± SE. See also Figures S1–S3 and Tables S1 and S2. Current Biology 2017 27, 1900-1914.e4DOI: (10.1016/j.cub.2017.05.084) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Medial SNc Expressed Mainly Cholinergic-Mediated GABAergic Neurotransmission and Biphasic GABAergic and nAChR Currents, Including Coreleased GABA and ACh Responses (A and B) Blue light activation of cholinergic fibers in the medial SNc elicited a fast IPSC mediated by presynaptic nicotinic receptors (Vh = −20 mV). The disynaptic (indirect) response was blocked by both GABAA receptor and nAChR antagonists. (C and D) Monosynaptic (direct) GABA currents held at different potentials in the presence of nAChR antagonists (C) and its corresponding I-V plot (D). (E) Bicuculline-sensitive monosynaptic (direct) GABAA current (Vh = −20 mV) and nAChR antagonist cocktail (MEC, MLA, and DHβE)-sensitive nicotinic current (Vh = −70 mV) recorded in the same DA neuron, indicating corelease of ACh and GABA. The lack of inhibition of GABAA currents with nAChR inhibitors suggests release of GABA from cholinergic neurons. (F) Direct monosynaptic GABAA currents were significantly reduced in ChAT-ChR2-VGAT KO mice as compared to ChAT-ChR2-VGAT WT. No differences were found in blue-light-evoked disynaptic (indirect) GABAA currents between ChAT-ChR2-VGAT WT and ChAT-ChR2-VGAT KO mice. (G) Traces of biphasic direct and indirect GABAA-mediated IPSCs with their corresponding nAChR currents. (H) Onset latencies for direct and indirect IPSCs over different blue light stimulation intensities. (I) The mean latency of current onset for nAChR currents was significantly greater than that of direct GABAergic currents, whereas the mean latency of current onset for nAChR currents was significantly less than that of indirect GABAergic currents. (J) Further evidence that coreleased nAChR EPSCs and GABAergic IPSCs are monosynaptic because 4-AP was able to restore the EPSC and IPSC following abolishment with TTX. (K) Meanwhile, indirect GABA IPSCs were disynaptic because 4-AP was unable to rescue the elimination of the response with TTX. (L) Injection of AAV-floxed ChR2-YFP into the PPT of ChAT-cre mice resulted in blue-light-evoked indirect GABAergic IPSCs that were reversibly inhibited by nAChR inhibitors. The PPT-injected mouse also expressed coreleased direct GABAergic IPSCs and nAChR EPSCs that were blocked by GABAzine and nAChR inhibitors. Summary data are reported as mean ± SE. See also Figures S1–S3 and Tables S1 and S2. Current Biology 2017 27, 1900-1914.e4DOI: (10.1016/j.cub.2017.05.084) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Colocalization of ACh and GABA in Cholinergic Terminals in the Medial SNc and Colocalization of ACh and GABA in PPT and LDT Neurons (A–C) Immunostaining of cholinergic (VAChT) (A) and GABAergic (VGAT) (B) markers and merged image (C) in presynaptic terminals in the medial SNc. DA neurons were immunolabeled against tyrosine hydroxylase (TH) (C). (D) Quantification of colocalization of VAChT and VGAT by calculating the Manders coefficients (M1 and M2). M1 colocalization index was significantly greater than rotated M1 when one of the two color image channels was rotated 90 degrees relative to the other (p < 0.0001; Wilcoxon rank-sum test); ditto for M2 (p < 0.0001; Wilcoxon rank-sum test). (E–G) Immunohistochemistry of PPT brain slices from ChAT-tdTomato mice showing some neuronal soma with GABA (E) and ChAT (F) and colocalized (arrowheads) GABA and ChAT in the merged image (G). (H) Graph quantifying the number of neurons in the PPT with cholinergic, GABAergic, or both markers. (I–K) Immunohistochemistry of LDT brain slices from ChAT-tdTomato mice also showed neurons with GABA (I) and ChAT (J) and colocalized (arrowheads) GABA and ChAT in the merged image (K). (L) Graph quantifying the number of neurons in the LDT with cholinergic, GABAergic, or both markers. (M–P) Immunostaining of PPT from a brain section of a ChAT-ChR2-YFP-VGAT WT mouse showing GABA (M), VGAT (N), ChR2-YFP (O), and the merged image (P) showing VGAT colocalization in the cell bodies positive and negative for ChAT. (Q–T) However, in the PPT of a brain section from a ChAT-ChR2-YFP-VGAT KO mouse showing GABA (Q), VGAT (R), ChR2-YFP (S), and the merged image (T), there was a lack of colocalization of VGAT and ChAT, though there was colocalization of VGAT and GABA in ChAT-negative neurons. (P and T) Empty magenta arrowheads, colocalized GABA and VGAT; empty white arrowheads, ChAT only; white filled arrowheads, colocalized GABA, VGAT, and ChAT; yellow filled arrowheads: colocalized GABA and ChAT, but not VGAT. Summary data are reported as mean ± SE. Current Biology 2017 27, 1900-1914.e4DOI: (10.1016/j.cub.2017.05.084) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Frequency-Dependent Changes in GABAergic and nAChR Currents Mediated by Cholinergic Neurotransmission in the Medial SNc (A) Voltage-clamp recordings of medial SNc DA neurons receiving coreleased GABA and ACh showed that, with repeated blue light stimulation at 5 Hz with 5 ms pulse duration, there was a robust repeatable outward GABAergic current (held at −20 mV) and a weak nAChR current (held at −70 mV), which facilitated. (B) At 15 Hz stimulation, there was a depression of GABAergic currents, whereas nAChR currents facilitated. (C and D) For medial SNc neurons displaying indirect GABAergic and nAChR currents, both 5 Hz (C) and 15 Hz (D) stimulation resulted in facilitation of GABAA and nAChR currents. Summary data are reported as mean ± SE. See also Figure S4. Current Biology 2017 27, 1900-1914.e4DOI: (10.1016/j.cub.2017.05.084) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Frequency-Dependent Changes in DA Neuronal Excitability in the Medial SNc (A–D) Current-clamp recordings of medial SNc DA neurons with coreleased ACh and GABA. (A1) Recordings from a ChAT-ChR2-VGAT WT mouse brain showing a current-clamp trace at 5 Hz blue light stimulation train (5 ms pulse duration), which inhibited action potential firing. (A2) Raster plot showing the time course of nine DA neurons with 5 Hz blue light stimulation, which produced inhibition of neuronal excitability. (A3) Histogram of the time course of mean firing frequency across all nine DA neurons. (B1) Recordings from a ChAT-ChR2-VGAT WT mouse brain section showing a current-clamp trace in which DA neuronal excitability initially showed an inhibition and then an enhancement in action potential firing when the frequency of blue light stimulation was increased to 15 Hz. (B2) Raster plot of six DA neurons and (B3) histogram of the mean firing frequency. (C1) Histograms of the mean firing frequency of five or six neurons over a range of different blue light stimulation frequencies from 5 to 60 Hz. Graphs summarizing the mean peak and baseline firing (C2) and change in peak firing (C3) over different frequencies of blue light stimulation. (D1) Current-clamp trace from a ChAT-ChR2-VGAT KO mouse showing an increase in neuronal excitability during blue light stimulation, the raster plot (D2), and histogram showing the time course of the mean firing frequency of the five DA neurons (D3). (E and F) Although DA neurons having nAChR and indirect currents are rare, stimulation at 5 Hz (E) results in a very sharp and brief increase in neuronal excitability, which was also found at 15 Hz stimulation (F). Summary data are reported as mean ± SE. Current Biology 2017 27, 1900-1914.e4DOI: (10.1016/j.cub.2017.05.084) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Frequency-Dependent Changes in nAChR Current and Neuronal Excitability of Lateral SNc DA Neurons (A) A voltage-clamp trace of a lateral SNc DA neuron at 5 Hz blue light stimulation. (B) Plot of mean current amplitude showing that nAChR currents undergo a moderate depression at 5 Hz stimulation. (C) A voltage-clamp trace of a lateral SNc DA neuron at 15 Hz blue light stimulation. (D) Evoked nAChR EPSCs at 15 Hz optical stimulation showed a more severe depression of the nAChR EPSCs. (E–J) Current-clamp recordings of the lateral SNc DA neurons. (E) A current-clamp trace showing that 5 Hz blue light stimulation increased firing of the DA neuron. (F) Histogram showing the average firing frequency of six DA neurons. (G) Raster plot. (H) A current-clamp trace at 15 Hz stimulation showing increased firing of DA neuron. (I) Histogram time course of the mean firing frequency of eight DA neurons. (J) Raster plot of action potential events. Summary data are reported as mean ± SE. Current Biology 2017 27, 1900-1914.e4DOI: (10.1016/j.cub.2017.05.084) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Optogenetic Stimulation of the Lateral SNc Increased Locomotion, whereas Stimulation of the Medial SNc Depressed Locomotion (A) Experimental design performed on mice implanted with fiber optics showing the 5 min periods of baseline, blue light stimulation at 5 Hz (blue segments), recovery no stimulation, blue light stimulation at 15 Hz, and then followed by recovery no stimulation. (B) In ChAT-ChR2 mice with fiber optics implanted into the lateral SNc, the top trajectory plots show baseline activity (black trace) and activity during blue light stimulation at 5 Hz and 15 Hz (blue traces). Graph shows that photostimulation of lateral SNc in ChAT-ChR2 mice induced significant enhancement in locomotion at 5 Hz (p = 0.012; paired t test; Bonferroni’s correction; n = 5 mice) and 15 Hz stimulation (p = 0.005; paired t test; Bonferroni’s correction; n = 5 mice). (C) Open-field locomotion during 5 Hz blue light stimulation of the lateral SNc was compared to baseline in mice injected (+MEC) or not injected (−MEC) with mecamylamine (5 mg/kg). There was significant increase in locomotion with blue light stimulation with uninjected mice (p = 0.008; Tukey’s HSD; n = 9 mice). Injected mice showed no significant change in locomotor activity (p = 0.98; Tukey’s HSD; n = 4 mice). (D) Blue light photostimulation of α4YFP mice with fiber optic implants into the lateral SNc did not significantly alter locomotion. (E) With fiber optics implanted into the medial SNc of ChAT-ChR2 mice, photostimulation at 5 Hz decreased locomotor activity (p = 0.038; paired t test; Bonferroni’s correction; n = 5 mice), but not at 15 Hz stimulation, which showed significantly greater locomotion than with 5 Hz (p = 0.02; paired t test; Bonferroni’s correction; n = 5 mice). (F) Mecamylamine-injected ChAT-ChR2 mice did not show any significant change in open-field locomotion (p = 0.93; Tukey’s HSD; n = 2 mice), whereas the uninjected mice decreased their locomotor activity (p = 0.028; Tukey’s HSD; n = 8 mice) with blue light. (G) Blue light stimulation of the medial SNc of ChAT-ChR2-VGAT KO mice resulted in a significant increase in locomotion (p = 0.003; paired t test; n = 4 mice). (H) A schematic diagram of the cholinergic circuitry in the SNc summarizing cholinergic-mediated postsynaptic currents, their effect on DA neuronal excitability, and locomotion. Summary data are reported as mean ± SE. See also Figure S5. Current Biology 2017 27, 1900-1914.e4DOI: (10.1016/j.cub.2017.05.084) Copyright © 2017 Elsevier Ltd Terms and Conditions