Rostral and Caudal Ventral Tegmental Area GABAergic Inputs to Different Dorsal Raphe Neurons Participate in Opioid Dependence

•Rostral and caudal VTA GABAergic neurons target different DRN neurons•Rostral and caudal VTA→DRN pathways function oppositely in reward•The rostral VTA→DRN pathway is depressed by repeated usage of morphine•Chronically activating the rVTA→DRN inhibitory pathway disrupts morphine reward Both the ventral tegmental area (VTA) and dorsal raphe nucleus (DRN) are involved in affective control and reward-related behaviors. Moreover, the neuronal activities of the VTA and DRN are modulated by opioids. However, the precise circuits from the VTA to DRN and how opioids modulate these circuits remain unknown. Here, we found that neurons projecting from the VTA to DRN are primarily GABAergic. Rostral VTA (rVTA) GABAergic neurons preferentially innervate DRN GABAergic neurons, thus disinhibiting DRN serotonergic neurons. Optogenetic activation of this circuit induces aversion. In contrast, caudal VTA (cVTA) GABAergic neurons mainly target DRN serotonergic neurons, and activation of this circuit promotes reward. Importantly, μ-opioid receptors (MOPs) are selectively expressed at rVTA→DRN GABAergic synapses, and morphine depresses the synaptic transmission. Chronically elevating the activity of the rVTA→DRN pathway specifically interrupts morphine-induced conditioned place preference. This opioid-modulated inhibitory circuit may yield insights into morphine reward and dependence pathogenesis. Both the ventral tegmental area (VTA) and dorsal raphe nucleus (DRN) are involved in affective control and reward-related behaviors. Moreover, the neuronal activities of the VTA and DRN are modulated by opioids. However, the precise circuits from the VTA to DRN and how opioids modulate these circuits remain unknown. Here, we found that neurons projecting from the VTA to DRN are primarily GABAergic. Rostral VTA (rVTA) GABAergic neurons preferentially innervate DRN GABAergic neurons, thus disinhibiting DRN serotonergic neurons. Optogenetic activation of this circuit induces aversion. In contrast, caudal VTA (cVTA) GABAergic neurons mainly target DRN serotonergic neurons, and activation of this circuit promotes reward. Importantly, μ-opioid receptors (MOPs) are selectively expressed at rVTA→DRN GABAergic synapses, and morphine depresses the synaptic transmission. Chronically elevating the activity of the rVTA→DRN pathway specifically interrupts morphine-induced conditioned place preference. This opioid-modulated inhibitory circuit may yield insights into morphine reward and dependence pathogenesis. The dorsal raphe nucleus (DRN) is a well-organized hindbrain structure containing half of brain total serotonergic neurons (Jacobs and Azmitia, 1992Jacobs B.L. Azmitia E.C. Structure and function of the brain serotonin system.Physiol. Rev. 1992; 72: 165-229Crossref PubMed Scopus (2126) Google Scholar). DRN serotonergic neurons are involved in affective control (Dayan and Huys, 2009Dayan P. Huys Q.J. Serotonin in affective control.Annu. Rev. Neurosci. 2009; 32: 95-126Crossref PubMed Scopus (241) Google Scholar); however, the role of serotonin in reward-related behaviors is still controversial. Recent research has shown that activation of DRN serotonergic neurons is reinforcing (Li et al., 2016Li Y. Zhong W. Wang D. Feng Q. Liu Z. Zhou J. Jia C. Hu F. Zeng J. Guo Q. et al.Serotonin neurons in the dorsal raphe nucleus encode reward signals.Nat. Commun. 2016; 7: 10503Crossref PubMed Scopus (222) Google Scholar, Liu et al., 2014Liu Z. Zhou J. Li Y. Hu F. Lu Y. Ma M. Feng Q. Zhang J.E. Wang D. Zeng J. et al.Dorsal raphe neurons signal reward through 5-HT and glutamate.Neuron. 2014; 81: 1360-1374Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), whereas other studies indicate that DRN serotonergic neurons are activated by both reward and punishment (Cohen et al., 2015Cohen J.Y. Amoroso M.W. Uchida N. Serotonergic neurons signal reward and punishment on multiple timescales.eLife. 2015; 4: e06346Crossref PubMed Scopus (205) Google Scholar, Ren et al., 2018Ren J. Friedmann D. Xiong J. Liu C.D. Ferguson B.R. Weerakkody T. DeLoach K.E. Ran C. Pun A. Sun Y. et al.Anatomically defined and functionally distinct dorsal raphe serotonin sub-systems.Cell. 2018; 175: 472-487.e420Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Such differences may be explained by the existence of subpopulations of DRN serotonergic neurons and/or circuits related to the DRN. It is widely accepted that the ventral tegmental area (VTA) plays an important role in the control of motivated behaviors (Bromberg-Martin et al., 2010Bromberg-Martin E.S. Matsumoto M. Hikosaka O. Dopamine in motivational control: rewarding, aversive, and alerting.Neuron. 2010; 68: 815-834Abstract Full Text Full Text PDF PubMed Scopus (1391) Google Scholar). Interaction between the VTA and DRN is of long-standing interest and may be of great importance for discovering the role of the DRN in reward and aversion. According to previous study, DRN serotonergic neurons strongly innervate the VTA (Watabe-Uchida et al., 2012Watabe-Uchida M. Zhu L. Ogawa S.K. Vamanrao A. Uchida N. Whole-brain mapping of direct inputs to midbrain dopamine neurons.Neuron. 2012; 74: 858-873Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). However, the characteristics and functions of the circuits projecting from the VTA to DRN remain unclear (Ogawa et al., 2014Ogawa S.K. Cohen J.Y. Hwang D. Uchida N. Watabe-Uchida M. Organization of monosynaptic inputs to the serotonin and dopamine neuromodulatory systems.Cell Rep. 2014; 8: 1105-1118Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, Pollak Dorocic et al., 2014Pollak Dorocic I. Fürth D. Xuan Y. Johansson Y. Pozzi L. Silberberg G. Carlén M. Meletis K. A whole-brain atlas of inputs to serotonergic neurons of the dorsal and median raphe nuclei.Neuron. 2014; 83: 663-678Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, Weissbourd et al., 2014Weissbourd B. Ren J. DeLoach K.E. Guenthner C.J. Miyamichi K. Luo L. Presynaptic partners of dorsal raphe serotonergic and GABAergic neurons.Neuron. 2014; 83: 645-662Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). To further understand the controversial results regarding the role of serotonin in reward-related behaviors in the DRN, it is critical to dissect the architecture and function of the subcircuits between the VTA and DRN and study how they are differentially regulated. The VTA is the main target of multiple addictive drugs, including opioids, psychostimulants, and cannabinoids (Lüscher and Malenka, 2011Lüscher C. Malenka R.C. Drug-evoked synaptic plasticity in addiction: from molecular changes to circuit remodeling.Neuron. 2011; 69: 650-663Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar). Interestingly, opioids specifically target and inhibit VTA GABAergic neurons through μ-opioid receptors (MOPs), leading to the disinhibition of VTA dopaminergic neurons (Fields and Margolis, 2015Fields H.L. Margolis E.B. Understanding opioid reward.Trends Neurosci. 2015; 38: 217-225Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, Johnson and North, 1992Johnson S.W. North R.A. Opioids excite dopamine neurons by hyperpolarization of local interneurons.J. Neurosci. 1992; 12: 483-488Crossref PubMed Google Scholar). However, how opioids modulate VTA projection GABAergic neurons remains unclear. Intriguingly, GABAergic transmissions in serotonergic neurons in the DRN increase under chronic morphine administration, resulting in a decrease in serotonin efflux (Jolas et al., 2000Jolas T. Nestler E.J. Aghajanian G.K. Chronic morphine increases GABA tone on serotonergic neurons of the dorsal raphe nucleus: association with an up-regulation of the cyclic AMP pathway.Neuroscience. 2000; 95: 433-443Crossref PubMed Scopus (99) Google Scholar). Thus, we wondered whether VTA-DRN circuits could be modulated by opioids. In the current study, we found that VTA projections in the DRN were mainly GABAergic, not dopaminergic. Unexpectedly, the rostral VTA (rVTA) preferentially innervated DRN GABAergic neurons, whereas the caudal VTA (cVTA) tended to target DRN serotonergic neurons. Second, rVTA and cVTA GABAergic projections in the DRN exhibited opposite functions in reward-related behaviors, probably by contrasting regulation of DRN serotonergic neuronal activity. Third, MOPs were differentially distributed in the rostral and caudal VTA GABAergic neuronal terminals within the DRN, resulting in different modulation of these two circuits by chronic morphine exposure. Finally, chronically elevating the activity of the rVTA→DRN inhibitory pathway during morphine administration interrupted morphine reward. To investigate the monosynaptic inputs from the VTA to DRN serotonergic and GABAergic neurons, we injected a mixture of helper viruses (AAV9-CAG-DIO-RVG and AAV9-CAG-DIO-GFP-TVA, 1:1) into the DRN of ePet1-Cre and Gad2-IRES-Cre mice followed by EnvA-pseudotyped and rabies virus glycoprotein (RVG)-deleted rabies virus RV-EnvA-dsRed at the same coordinates 2 weeks later (Figure 1A; Method Details). Only neurons expressing both the RVG and EnvA cognate receptor TVA (starter cells) allowed the retrograde spreading of the rabies virus to presynaptic neurons (input cells) (Wickersham et al., 2007Wickersham I.R. Lyon D.C. Barnard R.J. Mori T. Finke S. Conzelmann K.K. Young J.A. Callaway E.M. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons.Neuron. 2007; 53: 639-647Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar). Starter cells were co-stained with the serotonin marker tryptophan hydroxylase 2 (Tph2) to confirm the specificity of the virus. We found 91.9% ± 3.0% of the starter cells in ePet1-Cre mice were Tph2 positive (Figures S1A, S1B, and S1E), with no overlap observed between Tph2-positive and starter cells in Gad2-IRES-Cre mice (Figures S1C–S1E). Starter cells in a series of coronal sections matched the expression patterns of DRN serotonergic and GABAergic neurons (Weissbourd et al., 2014Weissbourd B. Ren J. DeLoach K.E. Guenthner C.J. Miyamichi K. Luo L. Presynaptic partners of dorsal raphe serotonergic and GABAergic neurons.Neuron. 2014; 83: 645-662Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) (Figures S1F and S1G). Control experiments confirmed that the rabies-mediated transsynaptic tracing system was dependent on Cre and RVG expression (Figures S1I–S1L; Method Details). In the midbrain, input cells were concentrated in the VTA, and both DRN serotonergic and GABAergic neurons received dense projections from the VTA (Figures 1B and 1C, top panel). Immunohistochemical analysis was performed to identify types of input cells in the VTA. As the GABAA receptor (GABAAR) α1 subunit (GABAAR α1) is selectively clustered on VTA GABAergic neurons (Tan et al., 2010Tan K.R. Brown M. Labouèbe G. Yvon C. Creton C. Fritschy J.M. Rudolph U. Lüscher C. Neural bases for addictive properties of benzodiazepines.Nature. 2010; 463: 769-774Crossref PubMed Scopus (268) Google Scholar), we stained sections with GABAAR α1 and tyrosine hydroxylase (TH) antibodies to label VTA GABAergic and dopaminergic neurons (Figures 1B and 1C, bottom panel). Very few VTA afferents to the DRN were TH positive (Figures 1D and 1E). In contrast, the VTA inputs to DRN serotonergic and GABAergic neurons were mainly GABAAR α1 positive and TH negative (Figures 1D and 1E), indicating that these neurons were mainly GABAergic. Thus, these data suggest that the VTA sent strong GABAergic afferents to both serotonergic and GABAergic neurons in the DRN. The VTA is a large heterogeneous area divided into rostral and caudal parts (Sanchez-Catalan et al., 2014Sanchez-Catalan M.J. Kaufling J. Georges F. Veinante P. Barrot M. The antero-posterior heterogeneity of the ventral tegmental area.Neuroscience. 2014; 282: 198-216Crossref PubMed Scopus (70) Google Scholar). We sectioned the VTA from rostral to caudal regions after the rabies virus injection to determine whether the distribution patterns of input cells between the rostral and caudal VTA were different (Figure 1A). The number of input cells was divided by the number of starter cells, and the ratios in the rVTA and cVTA were separately compared between the two mouse lines. In the rVTA, the input/starter ratio was higher in Gad2-IRES-Cre mice (Figures 1F, 1G, S1H, and S2). In contrast, the input/starter ratio was higher in the cVTA in ePet1-Cre mice (Figures 1F, 1G, S1H, and S2). Thus, the rVTA may send more projections to DRN GABAergic neurons, whereas the cVTA may innervate more serotonergic neurons in the DRN. To confirm this connectivity pattern, we injected AAV5-ef1α-DIO-ChR2-mCherry into the rostral or caudal (R-C) VTA in Gad2-IRES-Cre mice to separately express channelrhodopsin-2 (ChR2) in rVTA (Figure 2A) or cVTA (Figure 2B) GABAergic neurons and performed electrophysiology to record light-evoked inhibitory postsynaptic currents (eIPSCs) on DRN slices. Immunohistochemistry were performed to confirm the specificity of ChR2 expression (Figures S3A–S3D). In vitro whole-cell recordings (Figure S3E) and in vivo local field potential (LFP) recordings (Figure S3G) were performed to confirm the effectiveness of ChR2. In addition, dense ChR2-mCherry-positive signals were observed in the DRN (Figure S3H), and photoactivation of cVTA GABAergic terminals in the DRN induced local neuronal responses (Figure S3I), further suggesting that the VTA sends GABAergic afferents to the DRN. Gad2-IRES-Cre mice were crossed with Ai9 mice to label GABAergic neurons with tdTomato. 4–6 weeks after injecting AAV5-ef1α-DIO-ChR2-mCherry into the R-C VTA, slices containing the DRN were subjected to in vitro electrophysiology. When rVTA GABAergic terminals in the DRN were photoactivated (Figure 2C), eIPSCs with larger amplitudes and a greater number of connections were recorded in DRN GABAergic neurons than in non-GABAergic neurons (Figure 2D). The eIPSCs were inhibited by the GABAAR antagonist picrotoxin (PTX), indicating that GABAAR mediated neurotransmission (Figure 2E). Biocytin staining showed that the recorded putative GABAergic neurons were all co-localized with tdTomato (Figures S3J and S3L). In contrast, when expressing ChR2 in cVTA GABAergic neurons (Figure 2F), eIPSCs recorded in DRN non-GABAergic neurons had larger amplitudes and a greater number of connections (Figure 2G). PTX inhibited the eIPSCs, indicating that GABAAR mediated neurotransmission (Figure 2H). From biocytin staining, 82% of the recorded non-GABAergic neurons were Tph2 positive (Figures S3K and S3L), indicating that they were serotonergic neurons. Light-evoked IPSCs recorded in the R-C VTA→DRN circuits were blocked by tetrodotoxin (TTX) but rescued by 4-aminopyridine (4-AP) (Figures S3M and S3N), indicating that R-C VTA GABAergic neurons monosynaptically inhibited DRN cells. It has been reported that VTA GABAergic neurons co-release glycine (Polter et al., 2018Polter A.M. Barcomb K. Tsuda A.C. Kauer J.A. Synaptic function and plasticity in identified inhibitory inputs onto VTA dopamine neurons.Eur. J. Neurosci. 2018; 47: 1208-1218Crossref PubMed Scopus (24) Google Scholar). Therefore, we bath applied the glycine receptor blocker strychnine to test whether it could attenuate the eIPSCs in the R-C VTA→DRN pathways. Bath application of bicuculline, but not strychnine, blocked the eIPSCs in both pathways (Figures S3O and S3P), indicating that the neural transmissions were mediated by GABA rather than by glycine. To further confirm that the cVTA-targeted non-GABAergic neurons were serotonergic, we crossed ePet1-Cre mice with Ai9 mice to label serotonergic neurons with tdTomato and injected AAV5-Syn-ChR2-mCherry into the R-C VTA. Excitatory neurotransmission was blocked by bath application of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). In agreement with previous results, eIPSCs with larger amplitudes and a greater number of connections were recorded in non-serotonergic neurons when the rVTA terminals in the DRN were photoactivated (Figures 2I and 2J); photoactivation of the cVTA terminals evoked IPSCs with larger amplitudes and a greater number of connections in the DRN serotonergic neurons (Figures 2L and 2M). PTX blocked the eIPSCs recorded in both serotonergic and non-serotonergic neurons in the DRN (Figures 2K and 2N). These data provide strong evidence that the rVTA targets and exerts inhibitory effects on DRN GABAergic neurons, whereas the cVTA mainly innervates and inhibits DRN serotonergic neurons. We next recorded the firing rates of DRN GABAergic and non-GABAergic neurons during photoactivation of R-C VTA ChR2-expressing GABAergic terminals in slices containing the DRN to evaluate the effects of R-C VTA GABAergic projections on DRN neuronal activity. Membrane potentials were held at −45 mV to induce action potentials, with the firing rates of DRN GABAergic and non-GABAergic neurons then recorded before, during, and after photostimulation. Light significantly inhibited the firing rates of GABAergic neurons when rVTA GABAergic terminals were photoactivated (Figures 3A and 3B ). This inhibition was diminished by PTX application (Figure 3B), further confirming that DRN GABAergic neurons received direct inhibition from rVTA GABAergic neurons. In contrast, the firing rates of biocytin-identified serotonergic neurons were increased when rVTA GABAergic terminals were photoactivated (Figures 3C and 3D). Based on this result, together with the finding that DRN serotonergic neurons were inhibited by local GABAergic neurons (Huang et al., 2017Huang L. Yuan T. Tan M. Xi Y. Hu Y. Tao Q. Zhao Z. Zheng J. Han Y. Xu F. et al.A retinoraphe projection regulates serotonergic activity and looming-evoked defensive behaviour.Nat. Commun. 2017; 8: 14908Crossref PubMed Scopus (50) Google Scholar) (Figures S4A–S4C), we hypothesized that activation of rVTA GABAergic neurons could disinhibit DRN serotonergic neurons by directly inhibiting local GABAergic interneurons. The disinhibition model was further supported by blockade of the increase in firing rates by PTX (Figure 3D). When cVTA GABAergic terminals in the DRN were photoactivated, the firing rates of biocytin-identified serotonergic neurons were reduced by light (Figures 3E and 3F) and the reduction could be blocked by PTX (Figure 3F), indicating GABAAR-mediated inhibition. No effect was observed on GABAergic neurons when the cVTA→DRN circuit was photoactivated (Figures 3G and 3H). Thus, the cVTA mainly inhibited DRN serotonergic neurons, in agreement with the results shown in Figures 2G and 2M. The spontaneous activity of serotonergic neurons in the DRN is driven by the norepinephrine system in vitro (Vandermaelen and Aghajanian, 1983Vandermaelen C.P. Aghajanian G.K. Electrophysiological and pharmacological characterization of serotonergic dorsal raphe neurons recorded extracellularly and intracellularly in rat brain slices.Brain Res. 1983; 289: 109-119Crossref PubMed Scopus (405) Google Scholar). We restored the spontaneous firing of DRN serotonergic neurons by adding DL-norepinephrine hydrochloride (NE) (30 μM) to the recording buffer and recorded the spontaneous firing rates of DRN ePet1-tdTomato-positive neurons in cell-attached mode while photoactivating R-C VTA GABAergic inputs (Figures 3I and 3K). Consistent with our results in Figures 3D and 3F, the firing rates of DRN serotonergic neurons were increased by activation of rVTA GABAergic inputs (Figure 3J) but were inhibited by activation of cVTA GABAergic innervations (Figure 3L). This indicates that the rVTA and cVTA oppositely regulated serotonergic neuronal activities in the DRN. To further test the hypothesis that rVTA GABAergic neurons disinhibited DRN serotonergic neurons, we recorded spontaneous IPSCs (sIPSCs) in DRN non-GABAergic neurons before and during photoactivation of inhibitory inputs from the rVTA (Figure 3M). The frequencies of sIPSCs recorded in DRN non-GABAergic neurons were significantly decreased after 30 s of 20-Hz blue light illumination (Figure 3N). Based on this result, the inhibitory inputs to DRN non-GABAergic neurons were diminished when most DRN GABAergic neurons were inhibited by rVTA GABAergic neurons. The frequency of miniature IPSCs (mIPSCs) was not altered by light, indicating that the decrease in sIPSC frequency was activity dependent (Figures 3O and 3P). To study the local circuits in the DRN, we expressed ChR2 in DRN GABAergic and serotonergic neurons. Light-evoked IPSCs were recorded in non-GABAergic neurons when photoactivating ChR2-expressing DRN GABAergic neurons (Figure S4A). All neurons recorded eIPSCs in the presence of TTX and 4-AP, and eIPSCs could be blocked by PTX (Figures S4B and S4C). When expressing ChR2 in DRN serotonergic neurons (Figure S4D), we could not record any eEPSCs, eIPSCs, or mixed responses following a 5-ms single pulse (Figure S4E). Serotonin-mediated postsynaptic potentials following 5 s of 20-Hz light illumination were not detected either (Figure S4F). These results indicate that DRN GABAergic neurons inhibited serotonergic neurons directly, but serotonergic neurons may not influence local GABAergic neurons directly. VTA GABAergic neurons participate in reward-related behaviors by inhibiting local dopaminergic neurons (Tan et al., 2012Tan K.R. Yvon C. Turiault M. Mirzabekov J.J. Doehner J. Labouèbe G. Deisseroth K. Tye K.M. Lüscher C. GABA neurons of the VTA drive conditioned place aversion.Neuron. 2012; 73: 1173-1183Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, van Zessen et al., 2012van Zessen R. Phillips J.L. Budygin E.A. Stuber G.D. Activation of VTA GABA neurons disrupts reward consumption.Neuron. 2012; 73: 1184-1194Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) or projecting to other brain areas that control motivated behaviors (Root et al., 2014Root D.H. Mejias-Aponte C.A. Zhang S. Wang H.L. Hoffman A.F. Lupica C.R. Morales M. Single rodent mesohabenular axons release glutamate and GABA.Nat. Neurosci. 2014; 17: 1543-1551Crossref PubMed Scopus (209) Google Scholar, Stamatakis et al., 2013Stamatakis A.M. Jennings J.H. Ung R.L. Blair G.A. Weinberg R.J. Neve R.L. Boyce F. Mattis J. Ramakrishnan C. Deisseroth K. Stuber G.D. A unique population of ventral tegmental area neurons inhibits the lateral habenula to promote reward.Neuron. 2013; 80: 1039-1053Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The DRN is also involved in affective control (Dayan and Huys, 2009Dayan P. Huys Q.J. Serotonin in affective control.Annu. Rev. Neurosci. 2009; 32: 95-126Crossref PubMed Scopus (241) Google Scholar, Liu et al., 2014Liu Z. Zhou J. Li Y. Hu F. Lu Y. Ma M. Feng Q. Zhang J.E. Wang D. Zeng J. et al.Dorsal raphe neurons signal reward through 5-HT and glutamate.Neuron. 2014; 81: 1360-1374Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Therefore, we hypothesized that modulation of the VTA→DRN circuit would result in a reward-related phenotype. To test this hypothesis, we expressed ChR2 or N. pharaonis halorhodopsin (NpHR) in R-C VTA GABAergic neurons (Figures 2A, 2B, S3F, and S5), with optical fibers implanted above the DRN to allow light-mediated activation or inhibition of R-C VTA inhibitory inputs in the DRN (Figures 4A and 4G ). We used real-time place preference (RTPP) or aversion (RTPA) paradigms (see Method Details). The mice, as compared with littermate controls, showed avoidance of the chamber receiving photoactivation of rVTA→DRN inhibitory inputs (Figures 4B and 4C). The inhibition of this circuit elicited a preference for the photostimulation-paired chamber (Figures 4B and 4D). In contrast to the rVTA, activation of cVTA→DRN inhibitory inputs resulted in a preference for the photostimulation-paired chamber (Figures 4H and 4I), and the inhibition of this circuit resulted in avoidance of the photostimulation-paired chamber (Figures 4H and 4J). Activation of neural terminals might induce backpropagating action potentials in the soma. Thus, the behavior changes may be due to activation of VTA GABAergic neurons and inhibition of dopaminergic neurons. To test this, we microinjected PTX into the DRN through a guide cannula. Microinjecting PTX into the DRN blocked the avoidance or preference for the photostimulation-paired chamber (Figures 4E, 4F, 4K, and 4L), indicating that this behavioral outcome was dependent on GABAAR within the DRN but was not due to activation of pass-by fibers or cell bodies retrogradely. These behaviors were not due to the change in motor function or state of anxiety, as photoactivation of the R-C VTA→DRN inhibitory circuits had no influence on total distance traveled in the open field test or time spent in the open arm in the elevated plus maze (Figures S6A–S6E). Opioid receptors are expressed at high levels in brain areas related to reward (Le Merrer et al., 2009Le Merrer J. Becker J.A. Befort K. Kieffer B.L. Reward processing by the opioid system in the brain.Physiol. Rev. 2009; 89: 1379-1412Crossref PubMed Scopus (656) Google Scholar). MOPs on VTA GABAergic neurons are thought to be critically involved in opioid reward (Fields and Margolis, 2015Fields H.L. Margolis E.B. Understanding opioid reward.Trends Neurosci. 2015; 38: 217-225Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). We sought to determine whether MOPs were also expressed in the reward-related inhibitory circuits between the R-C VTA and DRN. To directly verify the locations of MOPs, we injected AAV5-ef1α-DIO-mGFP into the R-C VTA of Gad2-IRES-Cre mice and evaluated the coexistence of R-C VTA GABAergic terminals and MOPs in the DRN 2 weeks later. MOPs were highly co-localized with rVTA GABAergic terminals in the DRN (Figure 5A, top panel, and Figure 5B), but few MOPs were co-localized with cVTA GABAergic terminals (Figure 5A, bottom panel, and Figure 5B). These results suggest that in the DRN, MOPs were mainly expressed on rVTA GABAergic terminals, but not on cVTA terminals, even on DRN local neurons. The resting membrane potentials of DRN GABAergic and non-GABAergic neurons were not affected by bath perfusion of [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO) (Figures S6F–S6H), indicating that DRN local neurons were not sensitive to MOP agonists, consistent with the above staining results. To further investigate the different effects of MOPs on R-C VTA terminals, we expressed ChR2 in R-C VTA GABAergic neurons, perfused slices containing the DRN with the MOP agonist DAMGO (1 μM), and recorded eIPSCs. DAMGO incubation elicited a depression of eIPSCs in the rVTA→DRN circuit (Figure 5C), with an increase in the paired-pulse ratio (PPR) (Figure 5D), but not in the cVTA→DRN circuit eIPSCs or PPR (Figures 5E and 5F). Based on these findings, MOP activation decreased GABA release at rVTA→DRN synapses, but not at cVTA→DRN synapses, further indicating that MOPs existed on rVTA→DRN GABAergic terminals, but not on cVTA→DRN GABAergic terminals. DAMGO bath perfusion at a concentration of 1 μM may also activate δ-opioid receptors (DOPs) (Banghart et al., 2015Banghart M.R. Neufeld S.Q. Wong N.C. Sabatini B.L. Enkephalin disinhibits Mu opioid receptor-rich striatal patches via delta opioid receptors.Neuron. 2015; 88: 1227-1239Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To investigate if DAMGO-mediated inhibition of the rVTA→DRN pathway was MOP specific, we perfused a cocktail of the DOP antagonist ICI 174,864 (3 μM) and DAMGO (1 μM) followed by a cocktail of the MOP-specific antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) (1 μM) and DAMGO (1 μM). ICI 174,864 did not affect the percentage of inhibition by DAMGO, but CTOP fully reversed the DAMGO-induced inhibition of the rVTA→DRN pathway (Figure S6I), indicating that MOP, but not DOP, existed at the rVTA→DRN GABAergic synapses. As activation of the rVTA→DRN inhibitory pathway produced aversion (Figure 4C), we speculated that microinjecting MOP agonist DAMGO into the DRN might attenuate the aversive behavioral outcomes by depressing rVTA→DRN GABA transmission. To address this assumption, we microinjected DAMGO (1 mM) into the DRN 10 min before the RTPA experiment, in which mice received photoactivation of the rVTA→DRN inhibitory pathway. Place aversion was blocked by DAMGO pretreatment (Figure S6J), consistent with the electrophysiology results. In repeated morphine-treated mice, the RTPA was no longer inhibited by DAMGO (Figure S6K), reflecting the development of tolerance. Based on these results, we proposed that within the DRN, MOPs were located on rVTA GABAergic terminals, but not cVTA GABAergic terminals. Activation of presynaptic MOPs inhibited rVTA GABAergic transmission in the DRN and blocked aversive outcomes induced by activating the rVTA→DRN pathway, whereas DRN local neurons were not sensitive to MOP agonists. The different distributions of MOPs in R-C VTA terminals might lead to different effects of opioids on the two circuits. Morphine is a commonly used clinical opioid with high affinity toward MOPs and is strongly associated with addiction and abuse. We evaluated the influence of chronic morphine administration on the synaptic plasticity of the R-C VTA→DRN circuits. We intraperitoneally (i.p.) injected mice with morphine (15 mg/kg) for 5 days. The mice were sacrificed 12 hr after the last injection, brain slices that contained the VTA and DRN were prepared (Figure 6A), and synaptic transmission between the R-C VTA and DRN was evaluated. First, we recorded sIPSCs in DRN GABAergic and non-GABAergic neurons after morphine exposure to evaluate inhibitory transmission in different DRN neurons. Compared with saline-treated mice, morphine-treated mice showed a decrease