Abstract
µ-Opioid peptide receptor (MOPR) stimulation alters respiration, analgesia and reward behaviour, and can induce substance abuse and overdose1,2,3. Despite its evident importance, the endogenous mechanisms for MOPR regulation of consummatory behaviour have remained unknown4. Here we report that endogenous MOPR regulation of reward consumption in mice acts through a specific dorsal raphe to nucleus accumbens projection. MOPR-mediated inhibition of raphe terminals is necessary and sufficient to determine consummatory response, while select enkephalin-containing nucleus accumbens ensembles are engaged prior to reward consumption, suggesting that local enkephalin release is the source of the endogenous MOPR ligand. Selective modulation of nucleus accumbens enkephalin neurons and CRISPR–Cas9-mediated disruption of enkephalin substantiate this finding. These results isolate a fundamental endogenous opioid circuit for state-dependent consumptive behaviour and suggest alternative mechanisms for opiate modulation of reward.
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Data availability
The behavioural dataset supporting the current study is available from the author upon request. Source data are provided with this paper.
Code availability
Custom MATLAB analysis and code was created to appropriately organize, process, and combine photometry and single-photon recording data with associated behavioural data. Analysis code for photometry and single-photon imaging from Figs. 2, 4 will be made available on Github (https://github.com/BruchasLab).
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Acknowledgements
We thank L. Lawson, D. Blumenthal, M. Chung, T. Hobbs and C. Pizzano for animal colony maintenance; the Bruchas laboratory, Stuber laboratory and multiple trainees and faculty within the NAPE Center for helpful discussions; B. Kieffer for the Oprm1fl/fl mice; and G. Scherrer for the Penk-Cre mice. D.C.C. was funded by NIH grants NS007205, DA043999, DA049862 and DA051489. C.E.P. was funded by NIH grant DA051124. M.A.R. was funded by NIH grant DK121883 and a NARSAD Young Investigator Award. J.A.M. was funded by NIH grants DA041781, DA042499 and DA045463. G.D.S. was funded by DA032750, DA038168 and DA048736. M.R.B. was funded by NIH grants R37DA033396 and R61DA051489 and the Mallinckrodt Endowed Professorship. M.R.B., G.D.S. and L.S.Z. were funded by NIH grant P30DA048736.
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Contributions
D.C.C. designed and performed experiments, collected and analysed data, and wrote the manuscript. C.S.O., E.T.Z. and A.G. performed experiments and collected data. M.A.R. collected and analysed electrophysiological data. A.C.H. and L.S.Z. designed and analysed CRISPR virus. C.E.P designed and analysed fibre photometry data. S.C.P. designed and analysed one-photon data. M.R.B. and J.A.M. facilitated resources for generation of Oprm1fl/fl × Penk and Oprm1fl/fl × Pdyn mouse lines. J.A.M., L.S.Z. and G.D.S. helped to design experiments, discuss results and write the manuscript. M.R.B. helped lead the design, analysis, oversight of experiments, discuss results, provide resources and write the manuscript.
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Peer review information Nature thanks Brigitte Kieffer, Christian Lüscher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Endogenous MOPR activation in mNAcSh is necessary for potentiating state-dependent consummatory behavior.
a. Placement map of each microinjector tip (blue = intake suppression compared to vehicle-deprived, green = intake enhancement compared to vehicle-deprived). b. Schematic of vehicle (ACSF, gray, top) or drug (CTAP, blue, bottom) microinjections into areas surrounding nucleus accumbens (NAc) medial shell. c. CTAP (blue) had no effect on ad libitum or hunger enhanced intake compared to vehicle (gray) control days when injections were outside NAc medial shell (n = 8). d. In situ hybridization of Pdyn, Penk and Oprm1 in NAc medial shell (scale bar = 200µm). e, f. Quantification of MOPR expression in mNAcSh. g. Schematic of Oprm1fl/fl x Pdyn-Cre mouse line cross. h, i. In situ hybridization (h) and quantification (i) of Pdyn, Penk and Oprm1 in NAc medial shell (scale bar = 200 µm) in Oprm1fl/fl x Pdyn-Cre mouse line. j. Loss of MOPRs on Pdyn-Cre+ neurons did not disrupt normal ad libitum or food deprived enhanced intake compared to Pdyn-Cre- littermate control mice (n = 9 Cre-, 10 Cre+). k and l. In situ hybridization (k) and quantification (l) of Pdyn, Penk and Oprm1 in NAc medial shell (scale bar = 200 µm) in Oprm1fl/fl x Penk-Cre mouse line. m. Schematic and image of rAAV5-CMV-Cre-GFP injections into NAc medial shell of Oprm1fl/fl mice. n. Schematic of combined viral spread map of local MOPR deletion. o. Schematic (top) and image (bottom) of AAV2retro-CMV-myc-NLS-Cre or AAV2retro-GFP-Cre injections into NAc (left); retrogradely labeled cells in dorsal raphe nucleus (right). All error bars represent ± SEM and n = biologically independent mice or cells (f, i, l). Medians marked with orange bar. Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (c, j).
Extended Data Fig. 2 Endogenous MOPR activation in mNAcSh is necessary for potentiating state-dependent avoidance behavior.
a. Schematic of elevated zero maze (EZM) test. Mice were tested after habituation to the test room (Unrestrained) or after 30 min of restraint stress (Restraint). b. Example heat plots of time spend in the open arms of the EZM in wildtype (WT, left) or Oprm1 KO (KO, right) after no restraint (top) or 30 min of restraint (bottom). c. Unrestrained WT mice spent ~30% of the EZM test in the open arms. Mice exposed to restraint stress significantly reduced their exploration to 10%. Pretreatment with naloxone prevented Restraint induced avoidance. Oprm1 KO mice did not display open arm avoidance after Restraint. Penk-Cre x cKO mice displayed normal avoidance of the open arms after Restraint (n = 9 WT Unrestrained, 9 WT Restrained, 8 Oprm1 KO Unrestrained, 7 Oprm1 KO Restrained, 8 Oprm1fl/fl x Penk-Cre- Restrained, 9 Oprm1fl/fl x Penk-Cre+ Restrained). d. Schematic of food intake assay after food deprivation or Restraint. e. Food deprived mice showed normal increase in intake relative to their ad libitum test day. Mice exposed to Restraint did not increase food intake relative to their Unrestrained test day (n = 11 deprived, 10 Restrained). All error bars represent ± SEM and n = biologically independent mice. Medians marked with orange bar. Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (c, e).
Extended Data Fig. 3 LDRNPenk-NAc projections express MOPRs.
a. Retrograde fluorescently tagged cells in amygdala of Penk-Cre+ mouse after injections into nucleus accumbens medial shell. b. (left) In situ hybridization of Oprm1, Slc32a1 and Slc17a6 in DRN (scale bar = 200 µm). Zoomed in and channel separated images (right) of the red square in the left panel. c. Quantification of in situ from panel b. d, e. Schematic and image of a local injection of AAV2retro-GFP-Cre into nucleus accumbens shell (scale bar = 200 µm). Zoomed in images of e are designated as red (i) and yellow (ii) boxes. f. (left) In situ hybridization of Oprm1, Penk and Cre in dorsal raphe nucleus (scale bar = 200 µm). Zoomed in and channel separated images (right) of the red square in the left panel. g. Quantification of in situ from panel f. h. Schematic of CTb experiment. i. Fluorescently tagged CTb was injected into mNAcSh (scale bar = 200 µm). j. CTb tagged cells were observed in dorsal raphe nucleus, including the lateral sites in which enkephalin neurons were labeled in Fig. 2a (scale bar = 200 µm). Red square shows zoomed in image (right) with labeled cells (depicted by white arrows). All error bars represent ± SEM and n = biologically independent cells.
Extended Data Fig. 4 Non-DRN sites do not mediate food deprived potentiation of sucrose consumption.
a. Local MOPR deletion in paraventricular thalamus of Oprm1fl/fl mice did not reduce food deprived enhanced intake (n = 8, paired t-test t(7) = 9.634, p < 0.001). b. Schematic of local caspase ablations in either ventral pallidum or basomedial amygdala. c. Local and cell-type specific ablation of enkephalin neurons in VP or BMA did not reduce food deprived enhanced intake relative to Cre- control mice (n = 8 Cre-, 9 VP, 7 BMA). d. Caspase injection site confirmation in ventral pallidum (scale bar = 200 µm). AAV2-FLEX-taCas3-TEPp and AAV5-hsyn-EYFP were coinfused for cell-type specific deletion, and non-specific labeling.e, f. Schematic and image of a local injection of AAV5-Ef1a-DIO-EYFP into DRNPenk (scale bar = 200 µm). g. Image of dorsal raphe projection fibers from e (scale bar = 200 µm). h. oEPSC amplitude was reduced by the application of DNQX (n = 8). Blue shaded region indicates duration of optical stimulation. i. oIPSC amplitude was reduced by the application of gabazine (n = 5). All error bars represent ± SEM and n = biologically independent mice or cells (h, i). Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (c) or two tailed paired t-test (h, i).
Extended Data Fig. 5 LDRNMOPR-mNAcSh projection activity is negatively modulated by sucrose consumption in an opioid receptor dependent manner.
a. Schematic of photometry and voluntary sucrose consumption paradigm. b. Expression of GCaMP6s in DRNPenk (left, scale bar = 200 µm) and fiber placement in mNAcSh (scale bar = 200 µm). c. FD increases food intake (green) relative to ad libitum intake (white), and is reduced by systemic naloxone (blue, n = 8). d. Eating microstructure across ad libitum/saline (white), food deprived/saline (green) and food deprived/naloxone (blue) test days. After food deprivation, the majority of sucrose pellets consumed during multi-pellet bouts (left), and more pellets are eaten per multi-pellet bout (right). This shift in eating behavior is blunted by naloxone. e. Example of raw 405nm and 470nm channels from photometry experiments. f. Example of raw df/f trace highlighting specific single pellet (black) or multi-pellet (green) intake events during food deprived test day. Colored boxes on top expand color matched portions of the trace below. Orange lines indicate onset of pellet consumption. g. Average Z-scored trace aligned to onset of multi-pellet consumption of LDRNPenk-mNAcSh terminals in FD/saline condition (green) and FD/naloxone condition (blue). h. Average Z-scored trace (dark line) and error in on the ad libitum test day. When GCaMP activity is aligned to the onset of each pellet eaten in the ad libitum condition, no significant deviation in activity is observed (black, top). When aligned to only multi-pellet bout onset, there is no deviation from baseline activity (brown, bottom). i. Schematic of regulated food intake paradigm. Two pellets were non-contingently delivered every 90-150 s for 30 min. Mice were tested in either FD/saline or FD/naloxone conditions. j. Total number of sucrose pellets eaten in the regulated intake paradigm. Mice ate significantly more pellets on the food deprivation/saline test day (green), which was reduced to baseline levels after systemic naloxone (blue) (n = 10) k. Average Z-scored trace aligned to onset of multi-pellet consumption of LDRNPenk-mNAcSh. Food deprived/saline trace (green) shows rapid and sustained inhibition. Food deprived/naloxone trace (blue) shows blunted response. l. Heatmap of individual trials across all tested mice in food deprived/saline (green) or food deprived/naloxone test days. Orange lines indicate onset of pellet consumption. m. Quantification of the average Z-score twenty seconds prior to the onset of multi-pellet pellet bouts versus twenty seconds after the onset. FD/saline traces (green) show significant reductions in GCaMP6s activity whereas FD/naloxone traces (blue) do not (n = 10). All error bars represent ± SEM and n = biologically independent mice. Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (c, j, m).
Extended Data Fig. 6 MOPR activation on LDRNMOPR-mNAcSh is sufficient to enhance consummatory behavior.
a. Schematic of the Tail Immersion Test. Mice were tested at time 0, were injected with morphine (5mg/kg, s.c.), then tested every 10 min for up to 60 min, then again at 90 min. b. WT, Oprm1 KO, and Oprm1 KO x Penk-Cre rescue mice all show similar baseline responses at time 0. After morphine administration, WT mice significantly increase their latency to flick their tail, whereas Oprm1 KO and Oprm1 KO x Penk-Cre show no analgesic response to morphine mice (n = 5 WT, 2 Oprm1 KO, 6 Oprm1 KO x Penk-Cre rescue; WT/T0 vs Oprm1 KO/T0 p = 0.116, WT/T0 vs Oprm1 KO x Penk-Cre rescue/T0 p = 0.063, WT/T10 vs Oprm1 KO/T10 p < 0.001, WT/T10 vs Oprm1 KO x Penk-Cre rescue/T10 p < 0.001, WT/T90 vs Oprm1 KO/T90 p < 0.001, WT/T90 vs Oprm1 KO x Penk-Cre rescue/T90 p < 0.001). Statistical differences between WT and Oprm1 KO designated as (***) and differences between WT and Oprm1 KO x Penk-Cre rescue designated as (+++). c. Raster plots of individual licking events for one mouse, separated by trial. Only trials in which mice licked were included. d. Schematic of ChR2 experiments. e. Expression of EYFP-tagged ChR2 in LDRNPenk cell bodies (left, scale bar = 200 µm) and fiber placement in mNAcSh (right, scale bar = 200 µm). f. Penk-Cre- and Penk-Cre+ mice licked similar amounts for a sucrose solution in the ad libitum/No Laser condition. ChR2 photo-stimulation did not reduce licking (n = 7 Cre-, 6 Cre+). g. Penk-Cre- and Penk-Cre+ mice licked similar amounts for a sucrose solution in the WD/No Laser condition. By contrast, ChR2 photo-stimulation significantly reduced Cre+ licking, but not Cre- licking. h. Raster plots of individual licking events for one Penk-Cre+ mouse in the WD condition, separated by trial. Only trials in which mice licked were included. ChR2 photo-stimulation disrupted lick bout behavior compared to No Laser test days. All error bars represent ± SEM and n = biologically independent mice or cells (a). Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (c, f, g).
Extended Data Fig. 7 mNAcSh enkephalinergic ensembles are modulated by physiological state and potentiate consummatory behavior.
a. Sucrose consumed during ad libitum and food deprived test days (n = 5). Miniscope headmount did not disrupt normal intake behaviors. b. Examples of individual cell traces aligned to initiation of a multi-pellet bout. c. Average trace of all tracked cells aligned to bout consumption on food deprived test day. d. TSNE plot of clusters for multi-pellet bouts during the ad libitum state. e. Average trace of Onset activated neurons (cluster 1). f. Average trace of Pre-onset activated neurons (cluster 2). g. Total proportion and overlap of enkephalin neuron subpopulations modulated by multi-pellet bouts (pink), food sniffs (orange), rearing (blue), and grooming (brown). h. TSNE (left) and mean Z-scored traces (right) of food sniffing behavior sorted by kmeans clustering. i. TSNE (left) and mean Z-scored traces (right) of rearing behavior sorted by kmeans clustering. j. TSNE (left) and mean Z-scored traces (right) of grooming behavior sorted by kmeans clustering. All error bars represent ± SEM (a) or SEM is represented by the shaded region surrounding the trace (c, e, f, h, i, j).
Extended Data Fig. 8 Modulation of mNAcSh or POMC-containing neurons during sucrose consumption.
a. Schematic of hM3D(Gi) (left) and hM3D(Gq) (right) DREADD experiments. b. Fluorescent micrograph of mCherry-tagged enkephalin cells in mNAcSh for hM3D(Gi) (left) and hM3D(Gq) (right) experiments (scale bar = 200µm). c. CNO injections suppressed hunger enhanced intake in Cre+ mice, but had no effect in Cre- mice (n = 15 Cre-, 12 Cre+). d. CNO injections increased intake above the already elevated food deprived intake in Penk-Cre+ mice, but had no effect in Penk-Cre- mice (n = 7 Cre-, 10 Cre+). e. Systemic CNO administration (3mg/kg, i.p.) suppressed the already low ad libitum intake in Penk-Cre+ mice, but did not reduce intake in Penk-Cre- mice. f. Systemic CNO administration (3mg/kg, i.p.) had no effect on ad libitum intake in Penk-Cre- or Penk-Cre+ mice). g. Gq or Gi DREADD injections into arcuate nucleus of POMC-Cre mice. h. Micropictograph of mCherry-tagged, DREADD-expressing cells in arcuate nucleus (scale bar = 200 μm). i–k. Neither Gi nor Gq stimulation had an effect on ad libitum or food deprived intake in Cre- or Cre+ mice (n = 7 Cre-, 7 Gq, 9 Gi). All error bars represent ± SEM and n = biologically independent mice. Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (c, d, e, f, i, j, k).
Extended Data Fig. 9 Endogenous mu-opioid peptide within mNAcSh is necessary for potentiating consummatory behavior.
a. FISH (scale bar = 200 µm) of mNAcSh caspase injections. b. FISH quantification of (a). c. FISH (scale bar = 200 µm) of mNAcSh CRISPR injections. d. FISH quantification of (c). e. Schematic of CRISPR virus development and validation. f. Sequencing of GFP+ nuclei: (Top) sgPenk sequence with PAM underlined and SaCas9 cut site indicated by black arrow. (Middle) Sanger sequencing results displaying multiple peaks beginning at the SaCas9 predicted cut site. (Bottom) Top ten mutations at cut site with the percent of occurrence on the left. Insertions: underlined. Deletions: marked with “-“. Affected sites after SaCas9 insertion: shaded brown. g. Percent of wild type (black), deletions (brown), insertions (pink), and base changes (white) as percent of total reads for GFP+ and GFP- nuclei. h. Frequency distribution of insertions (pink) and deletions (brown) for Penk from GFP+ nuclei. i. Sanger sequencing results displaying no unusual peaks beginning at the SaCas9 predicted cut site. j. Unilateral hits or bilateral misses of NAc medial shell with the CRISPR mediated deletion of enkephalin did not reduce food deprived enhanced intake (n = 4). k. Fluorescent micrograph of CRISPR virus expression in dorsal raphe nucleus. l. Deletion of enkephalin from dorsal raphe nucleus did not reduce food deprived enhanced intake (n = 8) m. Deletion of dynorphin in NAc medial shell did not reduce food deprived enhanced intake (n = 7). n. Heatmap of individual trials across all tested mice in CRISPR/saline (orange) or Control/saline (gray) test days. Orange lines indicate onset of pellet consumption. o. Average Z-scored trace aligned to onset of multi-pellet consumption of LDRNPenk-mNAcSh after systemic naloxone injections. Control-treated mice (blue) show blunted inhibition. CRISPR-expressing trace (brown) shows negligible and phasic inhibition. p. Heatmap of individual trials across all tested mice in CRISPR/naloxone (orange) or Control/naloxone (blue) test days. q. Quantification of the average Z-score twenty seconds prior to the onset of multi-pellet pellet bouts versus twenty seconds after the onset. Neither Control traces (blue) nor CRISPR traces (brown) show significant deviations in GCaMP activity. Some mice did not lick on naloxone treated days and were therefore not included in this analysis (CRIPSR n = 3, Control n = 3). All error bars represent ± SEM and n = biologically independent mice or cells (b, d). Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (q) or two-tailed paired t-test (j, l, m).
Extended Data Fig. 10 A LDRNMOPR-mNAcShPenk circuit mediates potentiation of consummatory behaviors.
Schematic of voluntary sucrose consumption task (upper left) and LDRN-mNAcSh projection (upper right). Effects of LDRNMOPR-mNAcShPenk manipulations on behavior (lower left) and schematic of hypothesized physiology (lower right).
Supplementary information
Supplementary Discussion
This text considers how the data described in this manuscript are contextualized by known and unknown information. It examines potential circuits, psychological functions, and intracellular signalling mechanisms of interest.
Supplementary Video 1
This video demonstrates endoscopic recording of GCaMP6s labelled Penk-cre neurons in mNAcSh in an awake and behaving mouse. Behavioural and imaging videos are temporally synchronized.
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Castro, D.C., Oswell, C.S., Zhang, E.T. et al. An endogenous opioid circuit determines state-dependent reward consumption. Nature 598, 646–651 (2021). https://doi.org/10.1038/s41586-021-04013-0
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DOI: https://doi.org/10.1038/s41586-021-04013-0
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