SNr and GPe axons display distinct distribution patterns across the rostral and caudal PPN.

(A,C) Stereotaxic injection of AAV1 delivering hSyn-ChR2-eYFP to the SNr or GPe of ChAT-Cre/Ai9-tdTomato mice, respectively. (B, D) Confocal images of EYFP-filled SNr or GPe axons across the PPN, respectively. CNII: cranial nerve II, scp: superior cerebellar peduncle; PB: parabrachial nucleus

SNr inhibition of rostral and caudal ChAT+ PPN neurons.

(A) Experimental set up to identify red ChAT+ PPN neurons for whole-cell patch clamp while stimulating ChR2-filled SNr axons [N=6]. (B) White arrowheads pointing to neurobiotin-filled patched neurons within the PPN across three 200µm slices. (C) Example trace of the first five oIPSCs [blue] in the 2-second 20 Hz train inhibited by GABA-a receptor blocker, GABAzine [green], while holding the cell at −50mV. (D) Percent connected among patched neurons in the rostral and caudal regions. (E) Average oIPSC amplitude at each of 40 optogenetic light pulses in n=15 rostral neurons and n=20 caudal neurons. (F) Left, Individual cell data for the first oIPSC amplitude and, right, example current traces. (G) Cell mapping of patched neuron locations with the first oIPSC amplitude represented by the color scale. (H) Normalized current amplitudes in E. (I) Left, Individual cell data for the PPR between the first two oIPSC amplitudes in the train and, right, example current traces. (J) Example voltage traces of action potential firing during a 2-second 20 Hz train stimulation in rostral (left) and caudal (right) neurons. (K) Percent of pre-optical stimulation firing frequency during stimulation and rebound in n=14 rostral vs n=23 caudal neurons. (L) Individual cell data for the absolute change in frequency during optical stimulation [ΔFrq During Opto]. (M) Individual cell data for the absolute change in rebound frequency post-stimulation [ΔRebFrq]; rostral vs. caudal p=0.0142. (N) Correlation analysis, color scale representing Spearman r [-1,1] and size representing p-value [1,0]. (O) Negative correlation between the absolute change in frequency during stimulation and post-stimulation rebound; r=-0.372, p=0.039. * p<0.05; bar graph data represent mean ± SEM; box plots show median line with boxes showing IQR and whiskers showing 9th and 91st percentiles.

SNr inhibition of rostral and caudal Vgat+ PPN neurons.

(A) Experimental set up to identify red Vgat+ PPN neurons for whole-cell patch clamp while stimulating ChR2-filled SNr axons [N=6]. (B) Percent connected among patched neurons in the rostral and caudal regions. (C) Average oIPSC amplitude at each of 40 optogenetic light pulses in n=9 rostral neurons and n=13 caudal neurons. (D) Left, Individual cell data for the first oIPSC amplitude and, right, example current traces. (E) Cell mapping of patched neuron locations with the first oIPSC amplitude represented by the color scale. (F) Normalized current amplitudes in C. (G) Left, Individual cell data for the PPR between the first two oIPSC amplitudes in the train and, right, example current traces. (H) Percent of pre-optical stimulation firing frequency [% Pre-Opto Frq] during stimulation and and rebound in n=7 rostral and n=19 caudal neurons. (I) Individual cell data for the absolute change in frequency during optical stimulation [ΔFrq During Opto]. (J) Correlation analysis, color scale representing Spearman r [-1,1] and size representing p-value [1,0]. (K) Negative correlation between the absolute change in frequency during stimulation and first oIPSC amplitude; r=-0.755, p=0.001. (L) Negative correlation between the pre-optical stimulation firing frequency and the PPR; r =-0.706, p=0.002. Box plots show median line with boxes showing IQR and whiskers showing 9th and 91st percentiles.

SNr inhibition of rostral and caudal Vglut2+ PPN neurons.

(A) Experimental set up to identify red Vglut2+ PPN neurons for whole-cell patch clamp while stimulating ChR2-filled SNr axons [N=6]. (B) Percent connected among patched neurons in the rostral and caudal regions. (C) Average oIPSC amplitude at each of 40 optogenetic light pulses in n=13 rostral neurons and n=13 caudal neurons. (D) Left, Individual cell data for the first oIPSC amplitude and, right, example current traces; p=0.0035. (E) Cell mapping of patched locations with the first oIPSC amplitude represented by the color scale. (F) Normalized current amplitudes in C. (G) Left, Individual cell data for the PPR between the first two oIPSC amplitudes in the train and, right, example current traces. (H) Example voltage traces of action potential firing during a 2-second 20 Hz train stimulation in rostral and caudal neurons, top to bottom. (I) Percent of pre-optical stimulation firing frequency [% Pre-Opto Frq] during stimulation and rebound in n=13 rostral and n=17 caudal neurons. (J) Individual cell data for the absolute change in frequency during optical stimulation [ΔFrq During Opto]; p=0.0197. (K) Spontaneous frequency in n=11 rostral and n=16 caudal neurons; p=0.0343. (L) Correlation analysis, color scale representing Spearman r [-1,1] and size representing p-value [1,0]. (M) Positive correlation between the absolute change in frequency during stimulation and PPR, r=0.486, p=0.030. (N) Negative correlation between the absolute change in frequency during stimulation and first oIPSC amplitude; r=-0.841, p<0.00001. (O) Negative correlation between the absolute change in frequency during stimulation and pre-optical stimulation frequency; r=-0.791, p<0.0001. (P) Positive correlation between the first oIPSC amplitude and pre-optical stimulation frequency; r=0.818, p<0.0001. * p<0.05, ** p<0.01; box plots show median line with boxes showing IQR and whiskers showing 9th and 91st percentiles.

The SNr most strongly inhibits caudal glutamatergic PPN neurons.

(A,C) Individual cell data for the first oIPSC amplitude recorded in each cell type for rostral and caudal PPN neurons, respectively. (B,D) Individual cell data for the absolute change in frequency during stimulation in each cell type for rostral and caudal PPN neurons, respectively. (E) Graphical depiction of SNr stimulation results. * p<0.05, ** p<0.01, **** p<0.0001; box plots show median line with boxes showing IQR and whiskers showing 9th and 91st percentiles.

GPe inhibition of the three PPN cell types.

(A) Experimental set up to identify red ChAT+, Vgat+, and Vglut2+ PPN neurons for whole-cell patch clamp while stimulating ChR2-filled GPe axons [N=6]. (B) Example trace of the first five oIPSCs in the 2-second 20 Hz train [blue] inhibited by GABA-a receptor blocker, GABAzine [green], while holding the cell at −50mV. (C) Left, Percent connected among patched neurons in the rostral and caudal regions and, right, cell mapping of patched locations with the first oIPSC amplitude represented by the color scale. Top to bottom, i. ChAT+, ii. Vgat+, and iii. Vglut2+ datasets. (D) Average oIPSC amplitude at each of 40 optogenetic light pulses in n=6 ChAT+, n=19 Vgat+, and n=15 Vglut2+ caudal PPN neurons. (E) Left, Individual cell data for the first oIPSC amplitude and, right, example current traces. (F) Normalized current amplitudes in C. (G) Left, Individual cell data for the PPR between the first two oIPSC amplitudes in the train; p=0.0206. Right, top, example current trace of short-term synaptic facilitation in VgAT+ neurons. Right, bottom, example current traces of short-term synaptic depression in Vgat+ and Vglut2+ neurons. (H) Percent of pre-optical stimulation firing frequency [%Pre-Opto Frq] during and post-stimulation in n=25 ChAT+, n=18 Vgat+, and n=29 Vglut2+ caudal PPN neurons. (I) Individual cell data for the absolute change in frequency during stimulation [ΔFrq During Opto].

(J) Correlation analysis for Vgat+ neurons, color scale representing Spearman r [-1,1] and size representing p-value [1,0]. (K) Negative correlation between the absolute change in frequency during stimulation and first oIPSC amplitude; r=-0.627, p=0.044. (L) Correlation analysis for Vglut2+ neurons.

(M) Negative correlation between the absolute change in frequency during stimulation and the pre-stimulation firing frequency; r=-0.648, p=0.014. *p<0.05, **p<0.01; box plots show median line with boxes showing IQR and whiskers showing 9th and 91st percentiles.

In vivo activation of GPe and SNr axons in the PPN show differential effects on ocomotion and opposite effects on valence.

(A) Experimental set up to stimulate ChR2-filled SNr or GPe axons over the PPN in vivo. (B) Representative image of optical fiber tract overlaid with the pproximate optical fiber placement for SNr-[green] and GPe-[orange] injected mice. (C) Distance raveled over time in an open field with 1 minute 20 Hz 4.25 mW optical stimulations over the PPN in N=9 ontrol (Ctrl) mice (black circles), N=8 mice injected with ChR2 in the SNr (green diamonds), and N=9 mice injected with ChR2 in the GPe (orange hexagons); vertical blue lines represent periods of optical timulation. (D) Average distance traveled for each mouse across the six 1-minute optical stimulations in he high (4.25 mW) and low laser power (0.25 mW) conditions (E) Representative mouse track tracings uring real time place preference task in a three-chamber box and continuously stimulating EGFP- or ChR2-filled axons over the PPN at 20 Hz in SNr- and GPe-injected mice when the mice are in the timulation zone. (F) Percent time spent in the stimulation zone in N=16 control mice, N=9 mice injected with ChR2 in the SNr, and N=10 mice injected with ChR2 in the GPe with 4.25 or 0.25 mW laser power. G) Percent time spent in the stimulation zone during the first minute reintroduced to the RTPP box on ay 2 of RTPP with the laser off (no chamber is stimulated) Black line = median. * p<0.05, ** p<0.01, *** <0.001, **** p<0.0001; box plots show median line with boxes showing IQR and whiskers showing 9th nd 91st percentiles. See related supplemental Figures S2-S4 and Videos S1-S4.

(A) Representative image of SNr injected with ChR2-EYFP. Cell bodies and axons filled with ChR2 are represented in green. Post hoc staining of tyrosine hydroxylase (TH) to label dopamine neurons in the substantia nigra pars compacta. TH+ neurons are represented in magenta. (B) Representative image of GPe injected with ChR2-EYFP in a Vgat-Cre/Ai9-TdTomato mouse. Cell bodies and axons filled with ChR2 are represented in green. Vgat+ neurons and their axonal projections are represented in red. (C-E) Graphical depiction of virus spread in N=6 SNr and N=6 GPe injected mice in each mouse line: (C) ChAT-Cre/Ai9-TdTomato, (D) Vgat-Cre/Ai9-TdTomato, and (E) Vglut2-Cre/Ai9-TdTomato mice.

Speed measurements across age and sex.

(A) Speed of SNr-ChR2 mice during optical stimulation in the open field. Individual mouse traces are shown as green broken lines, with the group average represented by a solid green line with diamond markers. SNr-ChR2 mice exhibited increased speed during stimulation. (B) Speed of GPe-ChR2 mice during six 1-minute optical stimulations in the open field. Individual mouse traces are shown as orange broken lines, with the group average represented by a solid orange line with hexagon markers. GPe-ChR2 mice exhibited a reduction in speed during stimulation. Data are presented as mean ± SEM.(C) Distance traveled in the open field as a function of age in SNr-ChR2 and control mice. Older SNr-ChR2 mice tend to travel further, whereas older control mice travel less. Individual mouse traces are shown. (D) Comparison of locomotion between male and female SNr-ChR2 mice in the open field, showing a trend toward increased movement in female mice with SNr stimulation.

Opposite place preference despite similar motor responses to nigral axon stimulation in the PPN of DAT-Cre and wildtype mice.

(B) Distance traveled over time in an open field during 1-minute optical stimulations (20 Hz, 4.25 mW) over the pedunculopontine nucleus (PPN). Data are shown for N = 6 DAT-Cre mice and wildtype mice injected with ChR2 in the substantia nigra. SNr data from Figure 7. Data are presented as mean ± SEM.

(C) Average distance traveled for each mouse during stimulation across six 1-minute optical stimulations under high laser power (4.25 mW). (C) Average distance traveled for each mouse during stimulation across six 1-minute optical stimulations under low laser power (0.25 mW). (D) Representative movement traces from individual mice during a real-time place preference task in a three-chamber box. Mice received continuous optical stimulation (20 Hz) of EGFP-or ChR2-expressing axons over the PPN when in the stimulation zone. (E) Percentage of time spent in the stimulation zone under high laser power (4.25 mW) for N = 9 wildtype and N = 6 DAT-Cre mice injected with ChR2 in the substantia nigra. (F) Percentage of time spent in the stimulation zone under low laser power (0.25 mW) for the same groups.

Rostral vs caudal implant site alters SNr axon stimulation effects.

(A) Implant location (same as Figure 7B) with most rostral (purple) and most caudal (red) bilateral implant pairs circled for SNr axon stimulaton. (B) Distance traveled in open field in mouse with rostral implants (purple) and mouse with caudal implants (red).

Statistical details of exact p-values and critical values for SNr input to ChAT+ PPN neurons

Statistical details of exact p-values and critical values for SNr input to Vgat+ PPN neurons

Statistical details of exact p-values and critical values for SNr input to Vglut2+ PPN neurons

Statistical details of exact p-values and critical values for GPe input to ChAT+, Vgat+, and Vglut2+ PPN neurons