Cerebellar stimulation effectively elicits excitatory and inhibitory responses in nucleus accumbens.

A, Schematic diagram of recording setup. Stimulation protocol consisted of 10 trials (inter-trial interval: 15 s) of five 100-µA, 0.5-ms pulses at 200 Hz. NAc: nucleus accumbens; CB: cerebellum. B, Average spiking activity (spikes/s) across all recorded units (N = 42 mice). Inset shows zoom-in of first two seconds after stimulation. C,D,E, Examples of excitatory (C), inhibitory (D), and mixed (E) modulation in the NAc. Top, Peri-stimulus time histogram (PSTH) (10-ms bins) of the average firing rate across 10 trials. Bottom, Baseline-normalized firing rate (z-score) across time. Inset, Example average waveform of putative single unit. Scale bars: 1 ms, 10 µV. Shaded area marks response window. F, Distribution of response types elicited in NAc by DCN stimulation. Blue: Excitatory (E), Red: inhibitory (I), Purple: mixed, Gray: non-responders (NR). Number of recorded units per response-type (white) and corresponding percentages are shown.

Nucleus accumbens responses to cerebellar stimulation.

A, Recording sites in nucleus accumbens core (NAcCore) and medial shell (NAcMed). Numbers indicate distance (in mm) from bregma. B, Distribution of response types elicited by 30-, 100-, or 300-µA DCN stimulation. N30 = 14 mice, N100 = 42 mice, N300 = 40 mice. Outer ring (M): NAcMed; inner ring (C): NAcCore. Blue: excitatory (E), Red: inhibitory (I), Purple: mixed, Gray: non-responders (NR). C, Comparison between average baseline (Bsln) and peak firing rate (Resp) elicited by 100-µA DCN stimulation for excitatory responses in NAcCore (C1) and MAcMed (C2). Group averages (± SEM) are overlaid onto lines representing single units. D1,2, Same as C but for inhibitory responses.

The distribution of response-types in nucleus accumbens subregions is not due to topographical specialization within deep cerebellar nuclei.

A, The lateral (lat) or interposed (int) DCN (A1) was electrically stimulated. M: medial n. A2, Demarcation of anatomical planes: ML: medio-lateral; DV: dorsoventral; AP: antero-posterior. B,C, Stereotactic coordinates of DCN stimulation sites in ML-DV (B1,C1), ML-AP (B2,C2), and AP-DV (B3,C3) planes. Colored dots denote sites that evoked excitatory (excit.: blue), inhibitory (inh.: red), mixed (purple) or no responses (NR: gray) in NAcCore (B1-3) and NAcMed (C1-3).

Co-localization of nucleus accumbens-projecting neurons with cerebellar projections in VTA and intralaminar thalamus.

A, Schematic diagrams of stereotactic injections in DCN and NAc. B1, Expression of GFP at DCN injection sites. B2, Ctb-Alexa 568 injection site in NAc. C, Overlap of ctb-labeled NAc projectors and DCN axons in VTA. C1, VTA identification through TH immunostaining. C2, TH+ neuron. C3, Retrograde ctb-Alexa 568 labelling in NAc-projecting cell. C4, GFP-expressing DCN axons. C5, C2-C4 merged. D, Overlap of ctb retrograde label (red) in NAc projectors (blue; NeuN) and GFP-expressing DCN axons (green) in intralaminar thalamic nuclei. D1-D2, Overlap in parafascicular (PF) n. D3-D4, Overlap in centromedial (CM) n. Yellow boxes in C1,D1,D3 denote zoom-in areas depicted in C2-C5, D2 and D4, respectively. E-F, Airyscan confocal images from experiments similar to A but with only lateral DCN injected with AAV-GFP. E1, Ctb- labeled (red) NAc-projecting cell in the VTA. E2, TH+ cell (blue) with overlapping GFP-labeled axonal projection (green) from lateral DCN. E3, E1-2 merged. F1, GFP-labeled axon from lateral DCN (green) overlaps with ctb-labeled (red) NAc-projecting cell (blue: NeuN) in the PF n. F2, Same as in F1 but for neuron in the CM n. Scale bars: B1-2,C1,D1: 200 µm; C2-C5,D2,D4,E1-3,F1,F2: 5 µm; D3: 100 µm. N = 6 mice. Numbers denote distance from bregma.

Disynaptic cerebellum - nucleus accumbens connectivity is confirmed via anterograde transsynaptic viral tracing.

A, Schematic diagram of AAV1-mediated transsynaptic labeling approach. B, Schematic diagrams of stereotactic injections of floxed fluorophore in VTA and thalamic nodes. C1, The VTA is visualized via TH immunostaining (green). Yellow box denotes zoom- in area depicted in C2. Rn: red nucleus; ml: medial lemniscus. C2, Neurons that receive DCN input are labeled with tdTomato (red; white arrowheads). Green: TH+ neurons (white arrows). Yellow arrowhead: TH+, tdTomato+ neuron. C3, Projections of CB-VTA neurons in NAcCore and NAcMed. D1, Neurons that receive DCN input in thalamus are labeled with tdTomato (red). Blue: NeuN. CM: centromedial n.; PC: paracentral n.; CL: centrolateral n.; VL: ventrolateral n. D2, Same as D1, but for parafascicular n. (PF). fr: fasciculus retroflexus. D3, Projections of CB-thalamic neurons in NAcCore and NAcMed. E-F, Fluorescence images from experiments similar to A-B but with only lateral DCN injected with AAV1-Cre. E, NAc projections of VTA neurons receiving input from lateral DCN. F, Same as in E but for thalamic neurons. Scale bars below images: C1: 200 µm; C2: 100 µm; C3,D1- 3,E,F: 500 µm. N = 7 mice. Numbers denote distance from bregma.

Photostimulation of cerebellar projections in VTA and thalamus elicits responses in nucleus accumbens.

A, Schematic diagram of optophysiological approach. NAc: nucleus accumbens, CB: cerebellum. B, Example ChR2-EYFP expression in DCN. lat: lateral n.; ip: interposed n; m: medial n. C,D,E, Histology and NAc responses to optogenetic stimulation of VTA (C1,2), CM (D1,2), and PF (E1,2). C1,D1,E1: Representative images of optic fiber placement. ml: medial lemniscus; fr: fasciculus retroflexus. C2,D2,E2: Example single units. Left: Average spike waveform. Scale bars: 1 ms, 10 µV. Top: PSTH (10-ms bins) of firing rate (spikes/s). Bottom: Baseline-normalized firing rate (z-score) across time. Blue lines denote photostimulation; Shaded area marks response window. Numbers below or next to images indicate distance from bregma (in mm).

Node-dependent differences in nucleus accumbens responses to photostimulation of cerebellar projections.

A1,B1,C1, Average normalized activity (z-score) in NAcCore (top) and NAcMed (bottom) as a function of time, for cerebellar fiber photostimulation (blue lines) in VTA (A1), CM (B1) and PF (C1). A2,B2,C2, Distribution of response-types in NAcCore (C: inner ring) and NAcMed (M: outer ring) upon photostimulation of cerebellar projections in VTA (A2), CM (B2), and PF (C2). Blue: excitatory (E), Red: inhibitory (I), Purple: mixed, Gray: non-responders (NR). D, Average normalized activity (z-score) in NAcCore (top) and NAcMed (bottom) as a function of time, for no-opsin controls. E, Cumulative histogram of NAc response onset latencies upon cerebellar fiber photostimulation in VTA, CM and PF. Inset: 0-25 ms zoom-in. F,G,H, Violin plots of onset latencies of excitatory responses in NAcCore (Core) and NAcMed (Med), upon cerebellar fiber photostimulation in VTA (F), CM (G) and PF (H). Black lines: Median; White lines: interquartile range (Q1-Q3). VTA: N = 4 mice, nNAcCORE = 22 units, nNAcMED = 42 units; CM: N = 4 mice, nNAcCORE = 56 units, nNAcMED = 26 units; PF: N = 3 mice, nNAcCORE = 40 units, nNAcMED = 20 units. D, no-opsin controls: N = 4 mice, nNAcCORE = 5 units, nNAcMED = 36 units.

Percent responses in NAcCore and NAcMed elicited by photostimulation of cerebellar axons in VTA, CM and PF at different intensities.

Proportion of excitatory responses in NAcCore and NAcMed elicited by the first light pulse, at different photostimulation intensities.