A Neural Circuit Mechanism for Encoding Aversive Stimuli in the Mesolimbic Dopamine System - PubMed (original) (raw)

A Neural Circuit Mechanism for Encoding Aversive Stimuli in the Mesolimbic Dopamine System

Johannes W de Jong et al. Neuron. 2019.

Abstract

Ventral tegmental area (VTA) dopamine (DA) neurons play a central role in mediating motivated behaviors, but the circuitry through which they signal positive and negative motivational stimuli is incompletely understood. Using in vivo fiber photometry, we simultaneously recorded activity in DA terminals in different nucleus accumbens (NAc) subnuclei during an aversive and reward conditioning task. We find that DA terminals in the ventral NAc medial shell (vNAcMed) are excited by unexpected aversive outcomes and to cues that predict them, whereas DA terminals in other NAc subregions are persistently depressed. Excitation to reward-predictive cues dominated in the NAc lateral shell and was largely absent in the vNAcMed. Moreover, we demonstrate that glutamatergic (VGLUT2-expressing) neurons in the lateral hypothalamus represent a key afferent input for providing information about aversive outcomes to vNAcMed-projecting DA neurons. Collectively, we reveal the distinct functional contributions of separate mesolimbic DA subsystems and their afferent pathways underlying motivated behaviors. VIDEO ABSTRACT.

Keywords: aversion; dopamine; dorsal raphe; lateral hypothalamus; nucleus accumbens; reward; ventral tegmental area.

Copyright © 2018 Elsevier Inc. All rights reserved.

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Figures

Figure 1.

Figure 1.. Response of vNAcMed and NAcLat DA terminals to aversive stimuli.

(A) Schematic of experimental design. (B) GCaMP6m (green), tyrosine hydroxylase (TH; red) and DAPI (blue) immunofluorescence in the VTA (IPN: interpeduncular nucleus; Scale bar 500 μm). Inset pie chart: 99.8% of the VTA neurons that expressed GCaMP6m were also immunopositive for TH. Inset fluorescence image shows higher magnification (Scale bar 20 μm). (C) Schematic of aversive conditioning procedure and fiber photometry setup (RI: random interval). (D) Top: Representative heat maps for NAcLat DA terminals showing individual Z scores for trials in which a 2s tone was followed by a 2s electrical foot shock (first to last shock trial). Bottom: Example of responses to the tone and foot shock before (first shock trial, red) and after conditioning (last shock trial, blue). (E) Before (first shock trial, red) and after conditioning (last shock trial, blue), both tone and foot shock decrease DA terminal activity in the NAcLat (quantified as area under the curve, AUC, during each 2 s epoch; data represent means ± SEM). (F) Comparison of Z score averages for NAcLat GCaMP6m fluorescence for trials in which a tone was followed by a foot shock (red) or was omitted (green; experiments were performed 24h after conditioning and consisted of 30 trials; 10 out of 30 tones (randomly assigned) were not followed by an electric foot shock; 67%-foot shock probability). Inset shows significant increase in activity (quantified as AUC) in no-shock trials compared with shock trials (*** p < 0.001; data represent means ± SEM). (G-I) Same as in (D-F) but for DA terminal activity in the vNAcMed. Note in (I) that although quantification of AUC does not yield significant differences between shock and noshock conditions, a peak is observed following shock onset, which is absent in no-shock trials (Figure S1D; * p < 0.05; data represent means ± SEM). (J, K) Comparison of Z-score averages for DA terminals in the dorsal NAcMed (dNAcMed; J) and NAcCore (K) in response to shock (red) and omission (green) trials. Inset shows AUC during shock versus no-shock trials (** p < 0.01; data represents means ± SEM). (L) Schematic of the anatomical locations of individual optical fiber implants from all animals. Different colors indicate the response to foot shock (red: excitation, green: inhibition, yellow: no response, blue hexagons highlight the examples shown in Figure S1E).

Figure 2.

Figure 2.. Phasic responses to reward-predictive cues dominate in NAcLat DA terminals.

(A) Schematic of experimental design. (B) Schematic of fiber implant locations in the vNAcMed and NAcLat. (C) Top: Representative raster plot of licks around cue presentation and reward delivery in the first 50 trials before (first session, left) and after training (fifth session, right). Bottom: Average lick rate of all mice during the first 50 trials of the first (left) and fifth session (right; data represent means ± SEM). (D) Average lick rate during cue presentation (* p < 0.05; data represent means ± SEM). (E) Top: Representative heat maps for NAcLat DA terminals showing individual Z-scores during the first 20 successful trials before (first session, above) and after training (fifth session, below). Bottom: Z score averages of the above heat maps (Data represent means ±SEM). (F) Mean response to the CS before (red) and after (blue) training (* p < 0.05; quantified as AUC during cue onset; data represent means ± SEM). (G) Mean AUC during reward anticipation (delay period) before (red) and after (blue) training (** p < 0.01; data represent means ± SEM). (H) Comparison of Z score averages for NAcLat GCaMP6m fluorescence during reward (green) and omission trials (red; 80% reward probability; data represent mean ± SEM); recorded during the last conditioning day (day 5). (I) Mean AUC during reward delivery (quantified as AUC) for reward (green) and omission (red) trials (** p < 0.01; data represent means ± SEM). (J-N) Same as in (E-I) but for DA terminals in the vNAcMed (* p < 0.05; data represent means ± SEM).

Figure 3.

Figure 3.. DRVGLUT3 inputs to VTA activate NAcLat-projecting DA neurons and promote reward.

(A) Schematic of experimental design. (B) Anatomical distribution of vNAcMed- (left) and NAcLat-projecting (right) starter cells. Note the clear anatomical separation of the two subtypes and their locations in the medial VTA (mVTA) and lateral VTA (lVTA), respectively (green: RV-ΔG-GFP, red: TVA-mCherry, blue: TH; Scale bar 25 μm). (C) Horizontal and sagittal views of processed whole brains displaying brain-wide inputs to vNAcMed- (left) and NAcLat- (right) projecting DA neurons. (D) Quantification of inputs to vNAcMed- (light blue) and NAcLat- (dark blue) projecting DA neurons. Data are presented as a percentage of total input (px) counted in each individual brain. Color code indicates different brain structures shown in C. Abbreviations shown in legend of Figure S3 (Data represent means ± SEM). (E) Schematic of experimental design. (F) ChR2-eYFP expressing DRVGLUT3 terminals (green) are more frequently detected in the lVTA adjacent to retrogradely labeled (beads, red) TH-immunopositive (blue) cells projecting to NAcLat (left) than in the mVTA (right; scale bars 10 μm). (G) Mean fluorescence intensity of ChR2-eYFP expression in lVTA and mVTA (* p < 0.05; data represent means ± SEM). (H, I) EPSCs generated by stimulation of DRVGLUT3 inputs in retrogradely labeled (beads, red) VTA neurons projecting to (H) vNAcMed or (I) NAcLat. Cells were filled with neurobiotin (NB, green) and are TH-immunopositive (blue; scale bars: 50 pA/10 ms, 10 μm; data represent means ± SEM). (J) Mean EPSC amplitudes and response probabilities generated by light stimulation of DRVGLUT3 inputs (*** p < 0.001; data represent means ± SEM). (K) Spontaneous firing in vNAcMed- (top) and NAcLat-projecting (bottom) DA neurons and 4 Hz stimulation of DRVGLUT3 inputs (scale bar: 20 mV/1 s). (L) Relative increase in firing rate during 4 Hz and 20 Hz DRVGLUT3 terminal stimulation for vNAcMed- and NAcLat-projecting DA neurons (* p < 0.05; data represent means ± SEM). (M) Schematic of experimental design. (N) Schematic of real-time place preference assay. (O) Trajectory of an animal that received 4 Hz light stimulation in one compartment (Phase 1, blue, top panel) for the initial 10 min period followed by stimulation in the other compartment (Phase 2, blue, lower panel) for an additional 10 min. (P) Mean time mice spent in the compartment paired with 4 Hz light stimulation and the compartment that was not paired with light stimulation for mice expressing ChR2 or eYFP in LHVGLUT2 neurons. (** p < 0.01; data represent means ± SEM).

Figure 4.

Figure 4.. Bidirectional modulation of aversion behavior by LHVGLUT2 inputs to VTA.

(A) Schematic of experimental design. (B) ChR2-eYFP (green) expression in LH neurons (left; EP: entopeduncular nucleus, f: fornix, scale bar 250 μm) and in LH terminals in the VTA (right; red: TH; IPN: interpeduncular nucleus; scale bar 500 μm). (C) Schematic of real-time place preference assay. (D) Trajectory of an animal that received 4 Hz stimulation in one compartment (Phase 1 (P1), blue, top panel) for the initial 10 min period and then in the other compartment (Phase 2 (P2), blue, lower panel) for an additional 10 min. (E) Time spent in individual compartments (non-stimulated side: white; stimulated side: blue) plotted as a function of time over the course of the experiment (1 min intervals). Dashed line indicates switching of compartment stimulation after 10 min (data represent means ± SEM). (F) Mice expressing ChR2, but not eYFP, in LHVGLUT2 neurons spent significantly less time on the side of the chamber paired with 4 Hz optical stimulation (** p < 0.01; data represent means ± SEM). (G, H) Same as in (A, B), but for targeting eNpHR3.0 to LHVGLUT2 neurons. (I) Schematic of approach/avoidance assay (F: formaldehyde, form). (J) Heat maps (top: NpHR, bottom: control animal) show normalized time spent in different areas of the chamber (warmer colors indicate more time spent). (K) Mean time control (white) and NpHR (orange) mice spent in different zones (safe [i.e., greatest distance to aversive stimulus], center, form) of the chamber (* p < 0.05, *** p < 0.001; data represent means ± SEM). (L) Mean difference scores ([time spent in form zone] – [time spent in safe compartment]) for NpHR and control (Ctrl) mice (* p < 0.05; data represent means ± SEM). (M) Mean total distance traveled for NpHR and Ctrl mice (p > 0.05; data represent means ± SEM).

Figure 5.

Figure 5.. Activation of LHVGLUT2 inputs to the VTA by unconditioned and conditioned aversive stimuli.

(A) Schematic of experimental design. (B) GCaMP6m (green) expression in LHVGLUT2 cell bodies (scale bar 200 μm). (C) Optical fiber tract location in the VTA and LHVGLUT2 terminals expressing GCaMP6m (green) in the mVTA (red: TH; scale bar 500 μm). (D) Schematic of approach/avoidance assay and fiber photometry setup (F: formaldehyde). (E) Example responses to interaction with formaldehyde (top, blue) or to a novel object (bottom, orange). Red arrows: stimulus interaction (scale bars 5% ΔF/F/1 min). (F) Example trajectories for interaction with formaldehyde (F; top) or novel object (N; bottom). Red dots: stimulus interaction. (G) Heat maps for mean response intensity distribution in the open field area for interaction with formaldehyde (top) or novel object (bottom; warmer colors indicate increased activity). (H) Mean response intensity during the first five stimulus interactions (time = 0, dashed line) with formaldehyde (blue) or a novel object (orange). Area of light shading represents SEM. (I) %ΔF/F for individual stimulus interactions shows significantly greater responses for formaldehyde compared with novel object interaction. Note that the response intensity decreases significantly between first and fifth formaldehyde interaction (** p < 0.01; data represent means ± SEM). (J) Schematic of experimental design. (K) Schematic of aversive conditioning paradigm. (L) Representative sample of LHVGLUT2 terminal activity in response to tone and foot shock. Note that both the ‘tone-shock’ trials and ‘tone-only’ (omission) trials increase activity in LHVGLUT2 terminals in the VTA (recorded during the omission session, i.e., 24h after conditioning). (M) Top: Representative heat maps showing the individual Z scores for trials in which a 2 sec tone was followed by a 2 sec electrical foot shock (ordered from first to last shock trial). Bottom: Example responses to the tone and foot shock in an unconditioned animal (first shock trial, red) and after conditioning (last shock trial, blue). (N) Mean AUC during tone and shock before (red) and after (blue) aversive conditioning (* p < 0.05; data represent means ± SEM). (O) Comparison of Z score averages for trials in which a tone was followed by a foot shock (red) or was omitted (green; 67%-foot shock probability). Inset shows significantly increased activity (quantified as AUC) in omission (no-shock) compared with shock trials (*** p < 0.001; data represent means ± SEM).

Figure 6.

Figure 6.. LHVGLUT2 neurons preferentially target and activate vNAcMed-projecting DA neurons.

(A) Schematic of experimental design to analyze VGLUT2 mRNA expression in LH neurons synapsing on vNAcMed-projecting DA neurons in DAT-Cre mice. VGLUT2-Cre and GAD2-Cre mice were used to determine connectivity of glutamatergic and GABAergic VTA neurons with LHVGLUT2 neurons. (B) Sample images showing VGLUT2-positive (red arrow) and VGLUT2-negative (white arrow) LH neurons (GFP-positive, green) that make monosynaptic connections onto vNAcMed- (top) or NAcLat-projecting DA neurons (bottom; scale bars 20 μm). (C) Mean percentage of presynaptic VGLUT2-expressing LH neurons for different VTA cell populations (data represent means ± SEM). (D) Schematic of experimental design. (E) ChR2-eYFP expressing glutamatergic LH terminals (green) are more frequently in the mVTA adjacent to retrogradely labeled (beads, red) TH-immunopositive (blue) cells projecting to vNAcMed (top) than in the lVTA (bottom; scale bars 10 μm). (F) Mean fluorescence intensity (analyzed as mean pixel intensity) of ChR2-eYFP expression in LH terminals in the mVTA compared with lVTA (** p < 0.01; data represent means ± SEM). (G-J) EPSCs generated by stimulation of LH inputs in retrogradely labeled (beads, red) VTA neurons projecting to (G) vNAcMed (red trace: after CNQX application) or (H) NAcLat and in VTA neurons expressing (I) GAD2 (tdTomato-positive, red) or (J) VGLUT2 (tdTomato-positive, red). Cells were filled with neurobiotin (NB, green) and are TH-immunopositive (blue) for (G), (H), and TH-immunonegative for (I), (J; scale bars: 20 pA/10 ms; 10 μm). (K) Mean EPSCs amplitudes and response probabilities generated by stimulation of LH inputs in 4 VTA cell populations (same color code as in G-J; * p < 0.05; data represent means ± SEM). (L) Mean EPSC amplitudes recorded in vNAcMed-projecting DA neurons before (grey) and after (red) bath application of 10 μM CNQX (* p < 0.05; data represent means ± SEM). (M) Spontaneous firing in vNAcMed- (top) and NAcLat-projecting (bottom) DA neurons and 4 Hz stimulation of LHVGLUT2 terminals (recorded in the same slice; scale bar 20 mV/1 s). (N) Relative increase in firing rate during 4 Hz and 20 Hz LHVGLUT2 terminal stimulation for vNAcMed- and NAcLat-projecting DA neurons (* p < 0.05; data represent means ± SEM).

Figure 7.

Figure 7.. LHVGLUT2 neurons activate vNAcMed-projecting DA neurons to regulate aversive behaviors.

(A) Top: Schematic of experimental design. Bottom: Representative heatmaps of individual Z scores during stimulation trials. (B) Z score averages of stimulation (green) and no-stimulation (red) trials (data represent means ± SEM). (C) Mean AUC in the vNAcMed during stimulation (green) and no stimulation (red; *** p < 0.001; data represent means ± SEM). (D-F) Same as in (A-C) but for NAcLat (data represent means ± SEM). (G) Schematic of experimental design, which involves infusion of D1 (SCH23390 [SCH]) and D2 (raclopride [RAC]) receptor antagonists in to the vNAcMed and optogenetic stimulation of LHVGLUT2 terminals in the VTA. (H) Trajectories of animals that received SCH and RAC infusion into the vNAcMed and LHVGLUT2 terminal stimulation in VTA. (I) Mean time spent in non-stimulated minus stimulated side for different experimental conditions (one data point outside axis limits; * p < 0.05, ** p < 0.01; data represent means ± SEM). (J) Mean total distance animals traveled during the experiment (data represent means ± SEM).

Figure 8.

Figure 8.. Encoding of future aversive outcomes involves LHVGLUT2 neurons and increased dopamine transients in the vNAcMed.

(A) Schematic of experimental design. (B) Schematic of aversive conditioning paradigm in head-fixed mice. (C) Comparison of Z score averages for trials in which a tone was followed by a foot shock for mice expressing CASP (blue) or mCherry (red) in LHVGLUT2 neurons. (D, E) Mean AUC during tone (D) and shock (E) after aversive conditioning for mice expressing CASP and mCherry in LHVGLUT2 neurons (* p < 0.05; data represent means ± SEM). (F-H) Same as in (C-E), but for trials in which a predicted electrical foot shock was omitted (67%-foot shock probability) (* p < 0.05; data represent means ± SEM). (I) Schematic of experimental design. (J) Schematic of aversive conditioning paradigm in head-fixed mice and fiber photometry of DA transients in the vNAcMed and NAcLat. (K) Comparison of Z score averages for DA transients in the NAcLat for trials in which a tone was followed by a foot shock (red) or was omitted (green; 67%-foot shock probability). (L) AUC during shock delivery versus shock omission (no-shock trials) for DA transients in the NAcLat (** p < 0.01; data represents means ± SEM). (M, N) Same as in (K, L) but for DA transients in the vNAcMed (** p < 0.01; data represents means ± SEM).

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