Abstract
The ventral pallidum is centrally positioned within mesocorticolimbic reward circuits, and its dense projection to the ventral tegmental area (VTA) regulates neuronal activity there. However, the ventral pallidum is a heterogeneous structure, and how this complexity affects its role within wider reward circuits is unclear. We found that projections to VTA from the rostral ventral pallidum (RVP), but not the caudal ventral pallidum (CVP), were robustly Fos activated during cue-induced reinstatement of cocaine seeking—a rat model of relapse in addiction. Moreover, designer receptor–mediated transient inactivation of RVP neurons, their terminals in VTA or functional connectivity between RVP and VTA dopamine neurons blocked the ability of drug-associated cues (but not a cocaine prime) to reinstate cocaine seeking. In contrast, CVP neuronal inhibition blocked cocaine-primed, but not cue-induced, reinstatement. This double dissociation in ventral pallidum subregional roles in drug seeking is likely to be important for understanding the mesocorticolimbic circuits underlying reward seeking and addiction.
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Acknowledgements
We thank P. Do, M.J. Gilstrap and E.C. Lin for assistance with behavioral testing and immunohistochemistry and B.L. Roth (Department of Pharmacology, University of North Carolina) for DREADD constructs and consultation on DREADDs. CNO was supplied by US National Institutes of Health (NIH) National Cancer Institute (NCI) under the auspices of NS064882-01 and by the National Institute of Mental Health (NIMH) Chemical Synthesis and Drug Supply Program. Research was supported by NIH grants F32 DA026692, K99 DA035251 (S.V.M.), F31 DA030891 (J.T.B.), R21 DA025837 (G.A.-J. and S.P.W.), R01 DA013951 (J.J.W.), R37 DA006214 and P50 DA015369 (G.A.-J.). This project was supported by the National Center for Research Resources and the Office of the Director of the National Institutes of Health through grant number C06 RR015455.
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S.V.M. attained funding, designed and conducted experiments, performed surgeries, analyzed data and wrote the manuscript. E.M.V. designed and conducted anesthetized electrophysiology experiments, analyzed these data and wrote the manuscript. J.T.B. designed and conducted slice electrophysiology experiments, analyzed these data and wrote the manuscript. C.R.K. conducted behavioral experiments and wrote the manuscript. E.M.M. performed surgeries for and conducted behavioral and immunohistochemical experiments. J.K. conducted immunohistochemical and confocal microscopy experiments. S.P.W. attained funding and contributed the Syn-GFP viral construct. K.D. contributed the TH∷Cre transgenic rat line. J.J.W. attained funding, designed and conducted slice electrophysiology experiments and wrote the manuscript. G.A-J. attained funding, designed experiments and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Double dissociation for rostral vs. caudal VP in cued vs. primed reinstatement.
a) RVP, CVP, and surrounding structures are represented in the horizontal plane. Boxed area is enlarged in panels b&c. ac: anterior commissure; IPAC: interstitial nucleus of the posterior limb of the anterior commissure; Lat. Shell: lateral nucleus accumbens shell; LH: lateral hypothalamus; POA: preoptic hypothalamic area; SLEA: sublenticular extended amygdala. b) Top panel shows m±SEM active lever pressing during cue-induced reinstatement in animals with containment of hM4Di expression >95% within RVP or CVP, after vehicle or CNO (20 mg/kg, i.p.). *p=0.015. Bottom panel represents effects in individual animals of CNO microinjections into various parts of VP on cued reinstatement. Location of each oval represents site of virus injections in behaviorally tested animals (bilateral virus placement represented on a unilateral map; two ovals/animal). Size and shape of ovals represents the average zone of hM4Di expression around virus injection sites [m±SEM spread: dorso-ventral: m=1.18(0.02) mm; medio-lateral: m=0.72(0.02); rostro-caudal: m=0.95(0.02)]. Color of ovals represents change in cue-induced reinstatement after CNO injection (expressed as percentage of vehicle day lever pressing in the same animal). Blues indicate decreases from vehicle day due to hM4Di inhibition, yellows and reds represent increases in reinstatement responding over vehicle day. c) Using the same symbol logic as panel b, effects of CNO (20 mg/kg, i.p.) on cocaine-primed reinstatement are shown for RVP and CVP hM4Di animals. Bars=m±SEM. *p<0.05
Supplementary Figure 2 Cue-induced reinstatement elicits Fos in VTA-projecting neurons in RVP, not CVP.
a) Coronal 40 μm thick section from RVP (∼0.6 mm rostral of bregma) showing typical staining for Fos (blue-black nuclear stain) and CTb (brown somatic stain, indicating VTA-projecting neurons). Scale bar=1 mm. Expression typical of behaviorally tested CS+ group, n=8. b) Coronal 40 μm thick section from CVP (∼0.4 mm caudal of bregma) showing typical staining for Fos and CTb. Scale bar=1 mm. Expression typical of behaviorally tested CS+ group, n=8. c) Higher magnification view of Fos and CTb staining in RVP. Arrows indicate co-labeled cells. Scale bar=50 μm. d) Fos activation of VTA-projecting cells (%CTb cells that are Fos+; y-axis) in brain slices taken from various rostro-caudal levels of VP (mm rostral or caudal of bregma; x-axis). Symbols indicate the behavioral test prior to sacrifice (red diamonds: CS+ reinstatement, blue squares: control CS- cue, pink triangles: extinction test, black Xs: novel environment locomotor test). Note that the samples in which the greatest proportion of VTA-projecting neurons expressed Fos were in CS+ animals, and were located rostral of bregma (in RVP).
Supplementary Figure 3 Projection patterns of RVP and CVP to VTA.
a) RVP and CVP projections to rostral and caudal VTA are illustrated in the horizontal plane. b) Mean±SEM CTb+ cells/sample located in RVP (left) or CVP (right), projecting to either rostral or caudal VTA. CTb injections were localized to VTA either entirely rostral of the interpeduncular nucleus (blue bars), or entirely caudal of it (red bars). These animals also supplied data for a previous publication23, where additional details on CTb injection sites can be found. c) Mean±SEM total CTb+ cells/slice at various levels throughout the rostro-caudal axis of VP after CTb injection in VTA. Lines=m±SEM. d) Example of axonal labeling for the HA-tagged hM4Di receptor in midbrain after injection of Syn-hM4Di-HA-GFP into RVP. Strong axonal hM4Di expression (black) was seen in lateral VTA, and ventromedial SN (pars compacta). Scale bar=500 μm. Inset shows 60X magnification of axon terminals expressing HA (black) in the vicinity of VTA cells (red Nissl stained), scale bar=50 μm. The sample sizes of experimental groups with equivalent axonal DREADD expression in ventral midbrain are listed in Results. (e) Example of axonal labeling for the HA-tagged hM4Di receptor in midbrain after injection of Syn-hM4Di-HA-GFP into CVP. The strongest axonal hM4Di expression was seen in SN (especially pars reticulata), though fibers were also observed in VTA (inset). Scale bar=500 μm. Inset shows 60X magnification of axon terminals expressing HA (black) in the vicinity of VTA cells (red Nissl stained), scale bar=50 μm. The sample sizes of experimental groups with equivalent axonal DREADD expression in ventral midbrain are listed in Results.
Supplementary Figure 4 No changes in VTA dopamine neuron inputs caused by CNO in GFP control virus rats.
a) Syn-GFP control lentivirus used to express GFP under a neuronal-specific human synapsin promoter (Syn) in RVP. b) Representative traces from VTA dopamine neurons are shown in baseline conditions (left, black), and after CNO (right, red; scale=20 pA, 200 ms). c) CNO had no effect on mean sIPSC amplitude. d) CNO had no effect on mean sIPSC frequency. Lines=individual raw values.
Supplementary Figure 5 Midbrain CNO microinjection sites.
Midbrain cannulae placements for microinjections of CNO into VTA or SN in animals tested for reinstatement of cocaine seeking. Animals with placements within VTA borders are shown with filled black circles, animals with placements outside VTA (including in SN) are shown with unfilled (white) circles.
Supplementary Figure 6 Example hM4Di expression in VTA TH+ neurons and processes.
Confocal photomicrographs of VTA in TH::Cre rats after injection of DIO-Syn-hM4Di-mCherry. Equivalent expression seen in behavioral tested animals, n=9. Dopamine cells are labeled for TH (green, top left), and nearly all co-express the mCherry-tagged hM4Di receptor (red, bottom left). Yellow cells indicate cells and their processes co-expressing dopamine and DREADDs. Scale bar=50 μm.
Supplementary Figure 7 Disconnection of RVP from VTA or VTA Glutamate.
a) Animals received unilateral RVP Syn-hM4Di-HA-GFP, and a chronic cannula into contralateral VTA, and were tested for cue-induced reinstatement on separate days after i.p. 10 mg/kg CNO and intra-VTA vehicle (Veh-CNO; unilateral RVP inactivation control group), i.p. CNO plus unilateral intra-VTA cocktail of the AMPA/NMDA antagonists CNQX/AP5 (A/C-CNO: RVP/VTA glutamate disconnect group), or i.p. CNO plus unilateral intra-VTA cocktail of GABAA/B agonists baclofen and muscimol (B/M-CNO: RVP/VTA disconnect group). Some animals also received unilateral intra-VTA B/M + i.p. veh. (B/M-Veh: Unilateral VTA inactivation control group). b) Disconnection of RVP from VTA reduced reinstatement relative to unilateral RVP inhibition control, but neither unilateral VTA inactivation, nor disconnecting RVP from glutamate inputs did so. *p<0.05 Bars=m±SEM
Supplementary Figure 8 Inhibiting RVP inputs does not affect spontaneous excitatory inputs to VTA dopamine neurons.
a) Representative traces from VTA dopamine neurons are shown in baseline conditions (black), and b) after CNO (red; scale=20 pA, 200 ms). c) CNO had no effect on mean sIPSC amplitude. d) CNO had no effect on mean sIPSC frequency. Lines=individual raw values.
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Mahler, S., Vazey, E., Beckley, J. et al. Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking. Nat Neurosci 17, 577–585 (2014). https://doi.org/10.1038/nn.3664
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DOI: https://doi.org/10.1038/nn.3664
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