We observed the effect of morphine (10 µM) on the spontaneous firing frequency of VTA-DA neurons in rats. We select 10 µM of morphine because this concentration is commonly used in in vitro experiments as it elicits a significant effect useful for further analyses (Akaishi et al., 2000), and the effect of morphine at this concentration can be essentially abolished by the opioid receptor antagonist naloxone (Valentino and Dingledine, 1982). VTA-DA neurons were identified based on I_h currents (Figure 1A) and tyrosine hydroxylase (TH) staining (Figure 1B). Details of the identification method are given in the ‘Materials and methods’ section. Using original recordings of spontaneous firing (left panel of Figure 2A) and the time course of spontaneous firing (middle panel of Figure 2A) in VTA-DA neurons for comparison, we could see that morphine (10 μM) increased the frequency of spontaneous firing in VTA-DA neurons. The average frequency of spontaneous firing increased from 1.0 ± 0.3 Hz before to 1.4 ± 0.4 Hz for 10–15 min after morphine application (n = 6 cells from five rats, paired t test, p < 0.05, compared to control before morphine, right panel of Figure 2A). In order to determine the role of glutamatergic input in the morphine-induced increase in the spontaneous firing frequency of VTA-DA neurons, we observed the influence of the N-methyl-D-aspartic acid (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV) (50 μM) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6, 7-Dinitroquinoxalie-2, 3-dione (DNQX) (10 μM) on the effect of morphine. In the presence of APV and DNQX, morphine no longer increased spontaneous firing frequency (Figure 2B). The average frequency of spontaneous firing was 0.7 ± 0.1 Hz before and 0.8 ± 0.1 Hz for 10–15 min after morphine application in the presence of APV and DNQX (n = 6 cells from five rats, paired t test, p > 0.05, compared to control with APV and DNQX before morphine, right panel of Figure 2B). These results suggest that the morphine-induced increase in the spontaneous firing frequency of VTA-DA neurons requires AMPA and NMDA receptor-mediated glutamatergic input, consistent with a recent report using the NMDA antagonist APV and the AMPA receptor antagonist 6-Cyano-7-nitroquinoxaline-2, 3-dione (CNQX) in in vivo experiments (Jalabert et al., 2011).

Figure 1

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Figure 2

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In order to study the effect of morphine on glutamatergic input to VTA-DA neurons, we examined the effect of morphine on the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in VTA-DA neurons in rats. First, we observed the effect of morphine on the frequency of sEPSCs when the GABA_A receptor antagonist picrotoxin (PTX) was added to a bath solution to remove spontaneous inhibitory postsynaptic currents (sIPSCs). Consistent with earlier reports (Manzoni and Williams, 1999; Margolis et al., 2005), in the presence of extracellularly applied PTX (100 μM), morphine (10 μM) decreased the frequency of sEPSCs (Figure 3A). The average frequency of sEPSCs decreased from 4.2 ± 0.7 Hz before to 3.5 ± 0.7 Hz for 10–15 min after morphine application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before morphine, right panel of Figure 3A). However, bath application of GABA_A receptor antagonists can lead to a wide blocking effect on GABA_A receptors present on different types of neurons, including DA-neurons and GABA neurons, in VTA slices. To circumvent this wide influence and target the inhibition of GABA_A receptors to VTA-DA neurons, we added the GABA_A receptor antagonist PTX (100 µM) to the internal solution of microelectrodes as described by Akaike et al. (1985) when recording sEPSCs. To demonstrate the effectiveness of the blockade of GABA_A receptors by intracellularly applied PTX, we first examined the effect of intracellularly applied PTX (100 μM) on inhibitory postsynaptic currents (IPSC) in VTA-DA neurons. The results showed that under this experimental paradigm, IPSCs disappeared (top of panel 1 in Figure 3B, n = 5 cells from two rats), demonstrating that GABA_A receptors in the recorded VTA-DA neurons were blocked by intracellularly applied PTX. In addition, to further confirm that the spontaneous events we measured in the presence of intracellularly applied PTX were in fact sEPSCs, we observed the effect of the AMPA receptor antagonist DNQX on spontaneous events in the presence of intracellularly applied PTX. The results showed that the spontaneous events were completely blocked by DNQX (10 μM) (bottom of panel 1 in Figure 3B). We repeated the experiment in three cells from different slices and obtained similar results. On this basis, we observed the effect of morphine on the sEPSCs in the presence of intracellularly applied PTX. Raw current traces (panel 2 of Figure 3B) and the time course of sEPSCs (panel 3 of Figure 3B) before and after morphine application in the presence of intracellularly applied PTX showed that morphine (10 μM) increased the frequency of sEPSCs. The average frequency of sEPSCs increased from 4.8 ± 0.6 Hz before to 5.5 ± 0.6 Hz for 10–15 min after morphine application (n = 8 cells from five rats, paired t test, p < 0.05, compared to control before morphine, panel 4 of Figure 3B). However, morphine (10 μM) had no significant effect on the amplitude of sEPSCs. The average amplitude of sEPSCs was 16.6 ± 1.3 pA before and 15.3 ± 1.3 pA for 10–15 min after morphine application (n = 8 cells from five rats, paired t test, p > 0.05, compared to control before morphine, panel 5 of Figure 3B). To confirm that the increased spontaneous events we measured in the presence of intracellularly applied PTX after morphine were also in fact sEPSCs, we observed the effect of the AMPA receptor antagonist DNQX on spontaneous events after morphine application in the presence of intracellularly applied PTX. The results showed that the spontaneous events after morphine application were completely blocked by adding DNQX (10 μM) (panel 6 of Figure 3B). We repeated the experiment in three cells from different slices and obtained similar results. We also used the first excitatory postsynaptic current (EPSC) of paired pulse facilitation (PPF) as an index of EPSC (Maejima et al., 2001), and the PPF of paired EPSC as an indicator of presynaptic glutamate release (Zucker and Regehr, 2002) to confirm the effect of morphine on presynaptic glutamate release in VTA-DA neurons in the presence of intracellularly applied PTX. As shown in the left panel of Figure 3C, morphine (10 μM) increased the amplitude of the first EPSC, which was accompanied by a clear change in the presynaptic parameter PPF. The average amplitude of the first EPSCs was 124.1 ± 9.0 pA before and 161.9 ± 10.8 pA for 10–15 min after morphine application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before morphine, middle panel of Figure 3C). The average PPF was decreased from 1.5 ± 0.2 before to 1.2 ± 0.1 for 10–15 min after morphine application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before morphine, right panel of Figure 3C). These results supported the suggestion that morphine increased presynaptic glutamate release in VTA-DA neurons in the presence of intracellularly applied PTX. More importantly, when we clamped the membrane potential of VTA-DA neurons at the reversal potential of Cl⁻ channels to remove sIPSCs, as an alternative to the application of a GABA_A antagonist, either by the intracellular or bath approach, morphine still exerted a promoting effect on presynaptic glutamate release in VTA-DA neurons (Figure 3D). The average frequency of sEPSCs in this condition increased from 4.4 ± 0.3 Hz before to 5.5 ± 0.4 Hz for 10–15 min after morphine application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before morphine, right panel of Figure 3D).

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The mechanism underlying the promoting effect of morphine on presynaptic glutamate release in VTA-DA neurons may involve different processes. One suggestion is that morphine may directly act at glutamatergic terminals to promote glutamate release. To test this hypothesis, we studied the effect of morphine on glutamate release from the VTA synaptosomes of rats, which are sealed particles containing vesicles, viable mitochondria, and all components necessary to store, release, and retain neurotransmitters (Breukel et al., 1997), using on-line fluorometry. However, we did not find that morphine (10 μM) had a direct effect at glutamatergic terminals to promote glutamate release. The average concentration of glutamate was 8.1 ± 0.1 nmol/mg before and 8.5 ± 0.7 nmol/mg after morphine application (n = 6 samples from eight rats, paired t test, p > 0.05, Figure 4A). We also studied the effect of morphine on the frequency of sEPSCs from mechanically dissociated single VTA-DA neurons in rats, which retained functional synaptic terminals (Akaike and Moorhouse, 2003; Ye et al., 2004; Deng et al., 2009). The left top image of panel 1 of Figure 4B shows mechanically dissociated neurons from the VTA. These VTA-DA neurons were identified by only using I_h currents (the graph on the right of panel 1 of Figure 4B) without TH staining because in dissociated DA neurons, it was difficult to fix the cell after recording for TH staining. The results showed that morphine (10 µM) had no significant effect on the frequency of sEPSCs (panels 2 and 3 of Figure 4B). The average frequency of sEPSCs was 2.1 ± 0.3 Hz before and 2.0 ± 0.2 Hz for 10–15 min after morphine application (10 µM) (n = 6 cells from six rats, paired t test, p > 0.05, compared to control before morphine, panel 4 of Figure 4B). These results suggest that morphine may promote presynaptic glutamate release in VTA-DA neurons in an indirect way.

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To explore how morphine promoted presynaptic glutamate release in VTA-DA neurons, we hypothesized that glutamatergic input to VTA-DA neurons was inhibited by GABAergic interneurons and morphine disinhibited glutamatergic input by removing this inhibition, thus promoting glutamate release. To test this hypothesis, we first studied whether GABA could inhibit presynaptic glutamate release in VTA-DA neurons by examining the effect of exogenous applied of GABA on the frequency of sEPSCs of VTA-DA neurons in rats. From raw current traces (left panel of Figure 5A) and the time course of sEPSCs (middle panel of Figure 5A), we could see that GABA (10 μM) apparently decreased the frequency of sEPSCs. The average frequency of sEPSCs decreased from 6.7 ± 0.7 Hz before to 4.7 ± 0.6 Hz for 10–15 min after GABA application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before GABA, right panel of Figure 5A). Then, we observed whether the activation of intrinsic GABAergic neurons could inhibit presynaptic glutamate release of VTA-DA neurons in mice. To do this, AAV virus expressing a double floxed-stopped channelrhodopsin-2 (ChR2)-mCherry was stereotaxically injected into the VTA of mice expressing Cre recombinase in GABA neurons. 2 weeks after infection, expression of ChR2–mCherry was observed in the VTA (panel 1 of Figure 5B). We then performed whole-cell patch-clamp recording in GABA neurons of the VTA and observed the light-induced action potentials in GABA neurons in order to confirm that ChR2 was indeed expressed in the GABA neurons of the VTA in mice. The results showed that 470 nm light stimulation (20 Hz) elicited action potentials in GABA neurons (panel 2 of Figure 5B). On this basis, whole-cell patch-clamp recording was performed in VTA-DA neurons to observe the effect of 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX (100 µM). Following 5–10 min of baseline recording of sEPSCs, twenty 470 nm light pulses of 5 ms at 20 Hz were delivered every 4 s for 15 min. Raw current traces (panel 3 of Figure 5B) and the time course of sEPSCs (panel 4 of Figure 5B) before and after light stimulation showed that the light stimulation apparently decreased the frequency of sEPSCs. The average frequency of sEPSCs decreased from 4.1 ± 0.7 Hz before to 2.7 ± 0.3 Hz for 10–15 min after light stimulation (n = 6 cells from five mice, paired t test, p < 0.05, compared to control before light stimulation, panel 5 of Figure 5B).

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We also studied which kinds of GABA receptors (GABA_A or GABA_B receptors) mediated the decreasing effect of GABA on presynaptic glutamate release in VTA-DA neurons by examining the influence of the GABA_A or GABA_B receptor antagonist on the effect of GABA on the frequency of sEPSCs in rats. The results showed that the GABA_A receptor antagonist PTX (100 µM) had no significant influence on the effect of GABA (Figure 6A). The average frequency of sEPSCs still decreased from 4.0 ± 0.3 Hz before to 3.0 ± 0.2 Hz for 10–15 min after GABA application in the presence of PTX (n = 6 cells from four rats, paired t test, p < 0.05, compared to PTX before GABA, right panel of Figure 6A). The percentage of GABA-produced response in the presence of PTX (−25.8 ± 3.8%) was not statistically significant (n = 6, independent t test, p > 0.05) compared to that without PTX (−29.2 ± 6.0%). However, in the presence of the GABA_B receptor antagonist CGP54626, the effect of GABA on the frequency of sEPSCs disappeared (Figure 6B). The average frequency of sEPSCs was 3.8 ± 0.4 Hz before and 3.8 ± 0.4 Hz for 10–15 min after GABA application in the presence of CGP54626 (2 µM) (n = 6 cells from four rats, paired t test, p > 0.05, compared to CGP54626 before GABA, right panel of Figure 6B). These results suggest that it is GABA_B receptors, rather than GABA_A receptors, that mediate the decreasing effect of GABA on presynaptic glutamate release in VTA-DA neurons.

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To study the role of GABA_B receptors in intrinsic GABA-induced inhibition of presynaptic glutamate release in VTA-DA neurons, we observed the effect of the GABA_B receptor antagonist CGP54626 on the 470 nm light-induced inhibition of the frequency of sEPSCs of VTA-DA neurons in mice. From raw current traces (left panel of Figure 6C) and the time course of sEPSCs (middle panel of Figure 6C) before and after the 470 nm light stimulation in the presence of CGP54626 (2 µM), we could see that the inhibitory effect of the 470 nm light stimulation on the frequency of sEPSCs disappeared in the presence of CGP54626. The average frequency of sEPSCs was 4.2 ± 0.3 Hz before and 4.1 ± 0.2 Hz for 10–15 min after 470 nm light stimulation in the presence of CGP54626 (n = 6 cells from five mice, paired t test, p > 0.05, compared to control before 470 nm light stimulation in the presence of CGP54626, right panel of Figure 6C).

We examined the expression of GABA_B receptors in the presynaptic terminals of the VTA in rats. Western blotting indicated that GABA_B receptors were present in synaptosomes from the VTA (Figure 7A). Immunohistochemistry results for GABA_B receptors, VGLUT2 (a glutamatergic terminal marker) and TH (a DA neuron marker) showed that GABA_B receptors (green color, panel 1 of Figure 7B), VGLUT2 (red color, panel 2 of Figure 7B) and TH-positive neurons (blue color, panel 3 of Figure 7B) were present in VTA slices and the coexpression of GABA_B receptors and VGLUT2 (yellow color, panel 4 of Figure 7B) indicated that GABA_B receptors were present in glutamatergic terminals of the VTA in rats. Moreover, this coexpression was close to TH-positive neurons (panel 4 of Figure 7B).

Figure 7

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In addition, to determine whether there is basal GABAergic inhibitory control through GABA_B receptors on presynaptic glutamate release in VTA-DA neurons, we observed the effect of the GABA_B receptor antagonist CGP54626 on the frequency of sEPSCs in rats. The results showed that after the application of CGP54626, the frequency of sEPSCs significantly increased (Figure 8A). The average frequency of sEPSCs was 3.6 ± 0.3 Hz before and 4.5 ± 0.3 Hz for 10–15 min after CGP54626 application (2 µM) (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before CGP54626, right panel of Figure 8A). However, the GABA_A receptor antagonist PTX had no significant effect on the frequency of sEPSCs (Figure 8B). The average frequency of sEPSCs was 5.8 ± 1.0 Hz before and 5.7 ± 0.9 Hz for 10–15 min after PTX (100 µM) application (n = 6 cells from four rats, paired t test, p > 0.05, compared to control before PTX, right panel of Figure 8B). This is consistent with the above result showing that GABA-mediated inhibition of presynaptic glutamate release in VTA-DA neurons is via GABA_B receptors rather than GABA_A receptors.

Figure 8

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To study whether the morphine-induced increase in presynaptic glutamate release in VTA-DA neurons was via presynaptic disinhibition, we observed the effect of ‘closing’ local GABAergic interneurons on the morphine-induced increase in glutamate release in VTA-DA neurons using optogenetic methods in mice. AAV virus expressing a double floxed-stopped eNpHR3.0-EYFP was stereotaxically injected into the VTA of mice expressing Cre recombinase in GABA neurons. 2 weeks after infection, we performed whole-cell patch-clamp recording in GABA neurons of the VTA and observed light-induced inhibition of firing of action potentials in GABA neurons. The results showed that yellow light stimulation (590 nm) could reliably inhibit the current injection-induced firing of action potentials in GABA neurons (left panel of Figure 9A). On this basis, whole-cell patch-clamp recording was performed in VTA-DA neurons to observe the influence of the light-induced disinhibition of glutamatergic input on the effect of morphine. First, we observed whether this inhibition by light stimulation of GABA neurons affected the frequency of sEPSCs in VTA-DA neurons. As expected, light stimulation increased the frequency of sEPSCs in VTA-DA neurons (middle panel of Figure 9A). The average frequency of sEPSCs was 3.9 ± 0.4 Hz before and 4.5 ± 0.4 Hz for 10–15 min after light stimulation (n = 6 cells from five mice, paired t test, p < 0.05, compared to control before light stimulation, right panel of Figure 9A). Then, we observed the influence of the light-induced disinhibition of glutamatergic input on the effect of morphine. From raw current traces (left panel of Figure 9B) and the time course of sEPSCs (middle panel of Figure 9B), we could see that the effect of morphine on the frequency of sEPSCs disappeared in the presence of the light stimulation. The average frequency of sEPSCs was 4.5 ± 0.4 Hz before and 4.4 ± 0.5 Hz for 10–15 min after morphine application in the presence of the light stimulation (n = 6 cells from five mice, paired t test, p > 0.05, compared to control before morphine with light stimulation, right panel of Figure 9B). These results suggest that prior removal of the inhibition of GABAergic input on presynaptic glutamate release leads to disappearance of the effect of morphine on presynaptic glutamate release of VTA-DA neurons, indicating that morphine promotes presynaptic glutamate release of VTA-DA neurons via presynaptic disinhibition.

Figure 9

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In addition, we used the GABA_B receptor antagonist CGP54626 to remove inhibition of presynaptic glutamate release by GABAergic input and then observed its influence on the effect of morphine on glutamate release in rats. The results showed that after application of the GABA_B receptor antagonist CGP54626 (2 µM), the frequency of sEPSCs significantly increased (Figure 9C). The average frequency of sEPSCs was 2.5 ± 0.4 Hz before and 3.0 ± 0.4 Hz for 5–10 min after CGP54626 application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before CGP54626, right panel of Figure 9C), suggesting that CGP54626 induced a disinhibitory effect on presynaptic glutamate release. On this basis, morphine (10 µM) was applied in the same cell, but did not further increase the frequency of sEPSCs (Figure 9C). The average frequency of sEPSCs was 3.0 ± 0.4 Hz before and 2.8 ± 0.4 Hz for 5–10 min after morphine application in the presence of CGP54626 (n = 6 cells from four rats, paired t test, p > 0.05, compared to CGP54626 before morphine, right panel of Figure 9C).

We studied the influence of the selective presynaptic GABA_B receptor antagonist CGP36216 on the effect of morphine on the frequency of spontaneous firing of VTA-DA neurons in rats. First, we observed the effect of CGP36216 on the frequency of spontaneous firing in VTA-DA neurons. The results showed that CGP36216 (100 µM) could increase the frequency of spontaneous firing in VTA-DA neurons (Figure 10A). The average frequency of spontaneous firing increased from 1.8 ± 0.3 Hz before to 2.1 ± 0.3 Hz after CGP36216 application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before CGP36216, right panel of Figure 10A). Then, we observed the influence of CGP36216 on the effect of morphine on the frequency of spontaneous firing in VTA-DA neurons. As shown by raw spontaneous firing traces (left panel of Figure 10B) and the time course of spontaneous firing (middle panel of Figure 10B) in VTA-DA neurons, the effect of morphine (10 µM) disappeared in the presence of CGP36216 (100 µM). The average frequency of spontaneous firing in VTA-DA neurons was 1.3 ± 0.2 Hz before and 1.2 ± 0.2 Hz for 10–15 min after morphine application in the presence of CGP36216 (n = 6 cells from four rats, paired t test, p > 0.05, compared to CGP36216 before morphine, right panel of Figure 10B).

Figure 10

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Since enhanced DA function in the VTA has most often been assessed as increased locomotor activity at the behavioral level (Vezina, 2004), we also studied the contribution of the morphine-induced disinhibition of glutamatergic input in the VTA to behavioral changes produced by morphine. In behavioral experiments, to avoid morphine acting on other brain areas sensitive to morphine, we injected morphine locally into the VTA in rats. Injection sites were verified under light microscope (left panel of Figure 11A); the injection sites used for data analysis are shown in the middle and right panels of Figure 11A. We first observed the effect of CGP36216 on locomotor activity. The results showed that CGP36216 (20 μg/rat) increased locomotor activity. The distance traveled by rats was 6.3 ± 1.4 m in the control group compared with 12.2 ± 2.0 m in the CGP36216 group (n = 6 rats, independent t test, p < 0.05). Then we observed the influence of CGP36216 on the effect of morphine on locomotor activity in rats. As shown in Figure 11B, locomotor activity was significantly increased following intra-VTA injection of morphine (1 µg/rat). The distance traveled by rats was 6.3 ± 1.4 m in the control group compared with 37.6 ± 9.6 m in the morphine alone group (n = 6 rats, independent t test, p < 0.05, right panel of Figure 11B). However, in animals with intra-VTA injection of CGP36216 (20 µg/rat), the morphine-induced increase in locomotor activity disappeared (Figure 11B). The distance traveled by rats was 37.6 ± 9.6 m in the morphine alone group compared with 7.5 ± 1.4 m in the morphine plus intra-VTA CGP36216 group (n = 6 rats, independent t test, p < 0.05, right panel of Figure 11B). In addition, to evaluate the role of VTA GABA_B receptors in the overall effect of intraperitoneal (i.p.) morphine, we observed the effect of intra-VTA injected CGP36216 on i.p. morphine-induced increase in locomotor activity. As shown in Figure 11C, locomotor activity was significantly increased by i.p. injection of morphine (10 mg/kg) plus intra-VTA injection of saline. The distance traveled by rats was 5.4 ± 0.8 m in the control group compared with 28.3 ± 5.7 m in the i.p. morphine (10 mg/kg) plus intra-VTA saline group (n = 6 rats, independent t test, p < 0.05, right panel of Figure 11C). However, in animals with intra-VTA injection of CGP36216 (20 µg/rat), the i.p. morphine-induced increase in locomotor activity disappeared (Figure 11C). The distance traveled by rats was 28.3 ± 5.7 m in the i.p. morphine (10 mg/kg) plus intra-VTA saline group compared with 6.6 ± 2.3 m in the i.p. morphine plus intra-VTA CGP36216 group (n = 6 rats, independent t test, p < 0.05, right panel of Figure 11C).

Figure 11

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We also observed the effect of the presynaptic GABA_B receptor antagonist CGP36216 on morphine-induced conditioned place preference (CPP) in rats. Morphine (1 µg/rat) and CGP36216 (20 µg/rat) were locally injected into the VTA. Injection sites for data analysis are shown in Figure 12A. A schematic of the experimental design for CPP and drug application is shown in Figure 12B. As shown in Figure 12C, two-way ANOVAs conducted on the CPP score using treatment with different drugs as the between-subjects factors and test condition (preconditioning and postconditioning) as the within-subjects factor, revealed that there was a significant interaction of treatment (F_(3,40) = 4.45; p = 0.008) and test condition (F_(1,40) = 19.79; p < 0.001). Post-hoc analysis showed that after CPP training, the morphine group (n = 6 rats) exhibited greater CPP compared with the control group (n = 6 rats, p < 0.05) but in groups receiving CGP36216 (n = 6 rats), morphine-induced CPP was absent (p < 0.05). These results suggest that intra-VTA injection of a presynaptic GABA_B receptor antagonist abolishes CPP induced by intra-VTA injection of morphine.

Figure 12

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Male Sprague–Dawley rats (14–16 days old) or mice (4–5 weeks old) were anesthetized with chloral hydrate (400 mg/kg, i.p.). All experimental procedures conformed to Fudan University as well as international guidelines on the ethical use of animals. All efforts were made to minimize animal suffering and reduce the number of animals used. VTA slices were prepared according to procedures described previously (Hopf et al., 2007). The brain was removed rapidly from the skull and placed in modified ACSF containing 75 mM sucrose, 88 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 7 mM MgCl₂, 0.5 mM CaCl₂, 25 mM NaHCO₃, and saturated with 95% O₂ and 5% CO₂ at ∼0°C. Horizontal 250 μm midbrain slices containing VTA were cut on a vibratome (VT-1200, Leica, Wetzlar, Germany) and transferred to normal ACSF containing 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM MgSO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃, and 10 mM glucose at 32°C. Slices were incubated for at least 60 min before patch-clamp recording.

The medial terminal nucleus of the accessory optic tract (MT) was used as the anatomical structure to define the VTA (Hopf et al., 2007). VTA neurons were visualized on an upright microscope (BX50WI, Olympus, Tokyo, Japan) using infrared differential interference contrast or fluorescent optics. Whole-cell current- and voltage-clamp recordings were made using an EPC10 amplifier and PatchMaster 2.54 software (HEKA, Lambrecht, Germany). Electrodes had a resistance of 3–4 MΩ when filled with the patch pipette solution. The internal pipette solution contained 130 mM K-gluconate, 8 mM NaCl, 0.1 mM CaCl₂, 0.6 mM EGTA, 2 mM Mg-ATP, 0.1 mM Na₃-GTP, and 10 mM HEPES (pH 7.4). Lucifer yellow (2 mM) was added to the internal pipette solution for labeling the recorded neuron. Cells were held at 0 pA under a current-clamp mode to record spontaneous firing. Only neurons with regular spontaneous firing and an action potential amplitude greater than 60 mV were used for electrophysiological analyses. Cells were held at −70 mV under a voltage-clamp mode to record sEPSCs or evoked EPSC. A concentric stimulating electrode (FHC, Bowdoin, USA) was placed near the recorded cell. To observe PPF, two synaptic responses were evoked by a pair of stimulating pulses given at short intervals (50 ms) at 0.1 Hz. The series resistance (Rs) was monitored by measuring the instantaneous current in response to a 5 mV voltage step command. Rs compensation was not used, but cells where Rs changed by >15% were discarded.

After forming whole-cell recording mode, we first identified DA neurons based on electrophysiological characteristics, which included both a spontaneous pacemaker-like firing and expression of a hyperpolarization-induced current (I_h) in the voltage-clamp configuration, by 1 s hyperpolarizing voltage steps (−70 mV to −150 mV) (Grace and Onn, 1989; Margolis et al., 2006; Zhang et al., 2010; Chieng et al., 2011) (Figure 1A). Next, DA neurons were retrospectively confirmed by labeling the recorded neuron with Lucifer yellow (2 mM) in the internal pipette solution and subsequent TH staining of the recorded cell with Lucifer yellow. Slices were fixed immediately after electrophysiological recording in 4% formaldehyde for 2 hr and then washed with 0.01 M PBS solution. The slices were incubated for 2 hr at 4°C in a blocking solution containing 10% normal goat serum and 0.2% Triton X-100 in PBS and then incubated overnight at 4°C with primary rabbit anti-TH antibody (1:1000; Abcam, Cambridge, UK). The slices were washed thoroughly in PBS before being incubated for 2 hr at 4°C with goat anti-rabbit-Cy3 secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, USA). The recorded cells were identified as DA neurons based on the co-labeling by Lucifer yellow and TH (Figure 1B).

Male Sprague–Dawley rats (100–150 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.). Synaptosomes were prepared as described previously (Dong et al., 2005). The VTA was dissected and homogenized in 0.32 M sucrose solution at 4°C using the Art-Miccra D-8 tissue grinder with a motor-driven pestle rotating at 900 rpm. The homogenate was centrifuged at 3000×g for 3 min at 4°C. The supernatant (S1) was centrifuged at 14,500×g for 12 min at 4°C. The pellet (P2) was resuspended and loaded onto Percoll gradients consisting three steps of 23%, 10%, and 3% Percoll in 0.32 M sucrose additionally containing 1 mM EDTA and 250 μM DTT. The gradients were centrifuged at 32,500×g for 6.5 min at 4°C. Synaptosomes were harvested from the interface between the 23% and 10% Percoll layers and washed in Hanks' balanced salt solution (HBSS) containing 140 mM NaCl, 5 mM KCl, 5 mM NaHCO₃, 1 mM MgCl₂, 1.2 mM Na₂HPO₄, 10 mM glucose, and 20 mM HEPES, pH 7.4. Washed synaptosomes were centrifuged at 27,000×g for 15 min at 4°C.

Glutamate release from synaptosomes was assayed by on-line fluorimetry as described previously (Nicholls et al., 1987). Synaptosomal pellets were suspended again by adding 1.5 ml of incubation medium: 122 mM NaCl, 3.1 mM KCI, 0.4 mM KH₂PO₄, 5 mM NaHCO₃, 20 mM Na-TES, 1.2 mM MgSO₄, 16 pM bovine serum albumin (fatty acid free, type V), and 10 mM D-glucose, pH 7.4. Additionally, NADP⁺ (2 mM), glutamate dehydrogenase (50 units/ml), and CaCl₂ (1 mM) were added after 3 min. After a 5-min incubation period, morphine (10 μM) was added to observe its effect on glutamate release. Oxidative decarboxylation of released glutamate, leading to reduction in NADP⁺, was monitored by measuring NADPH fluorescence at excitation and emission wavelengths of 340 nm and 460 nm, respectively. Data points were obtained at 60 s intervals. An exogenous glutamate standard (5 nmol) was added at the end of each experiment. The fluorescence change produced by the addition of exogenous glutamate (5 nmol) was used to calculate the released glutamate in nmol/mg.

VTA-DA neurons with functional presynaptic terminals attached were obtained by a mechanical dissociation method as previously described (Akaike and Moorhouse, 2003; Ye et al., 2004; Deng et al., 2009). First, VTA slices were prepared as described above. Then the slice was transferred to a 35-mm culture dish (Sigma, USA) and held down with a flat U-shaped wire. The dish was filled with standard external solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose (pH 7.4). The MT was used as the anatomical structure to define the VTA (Hopf et al., 2007) under a stereomicroscope (SZ61, Olympus). A fire-polished glass pipette, lightly touching the surface of the VTA, was vibrated horizontally at 50–60 Hz for 1–2 min by a home-made device. The slice was then removed. The isolated neurons adhered to the bottom of the dish within 20 min and were then ready for electrophysiological experiments.

Immunoblot analysis of GABA_B R1 receptors was performed on the synaptosomes obtained from the VTA. The synaptosomal pellets were homogenized in a buffer containing 100 mM Tris-HCl (pH 6.7), 1% SDS, 143 mM 2-mercaptoethanol, and 1% protease inhibitor. The lysate was centrifuged at 12,000 rpm for 10 min at 4°C. The samples were treated with the SDS sample buffer at 95°C for 5 min, loaded on a 10% SDS polyacrylamide gel, and blotted to a PVDF membrane. Each blot was incubated with a rabbit anti-GABA_B R1 (1:100; Alomone Labs, Jerusalem, Israel). Following extensive washing, membranes were incubated with IRDye 800CW goat anti-rabbit secondary antibodies (1:20,000; LI-COR, Lincoln, USA) for 1 hr, and images were acquired on a LI-COR Odyssey system.

Sprague–Dawley rats (15–20 days old) were perfused transcardially with 0.01 M PBS followed by 4% paraformaldehyde. After perfusion, brains were removed and postfixed in 4% paraformaldehyde overnight. Horizontal brain slices (40-μm thick) containing VTA were prepared using a vibratome (VT-1000S, Leica). Slices were pre-blocked for 2 hr at 4°C in a blocking solution containing 10% horse serum and 0.2% Triton X-100 in PBS and again washed three times in PBS for 5 min. Slices were incubated overnight at 4°C with primary antibody mouse anti-TH (1:1000; Millipore, Billerica, USA), rabbit anti-GABA_B R1 (1:100; Alomone Labs), and guinea pig anti-VGLUT2 (1:3000; Millipore) dissolved in the blocking solution. Afterwards, slices were washed three times in PBS for 5 min and then incubated with the following secondary antibodies: horse anti-rabbit-Alexa 488 (1:200; Jackson ImmunoResearch Laboratories), horse anti-mouse-Cy5 (1:200; Jackson ImmunoResearch Laboratories), and horse anti-guinea pig-Cy3 (1:200; Jackson ImmunoResearch Laboratories) for 2 hr at room temperature in 2% horse serum and 0.2% Triton X-100 in PBS. Subsequently, slices were washed three times in PBS for 5 min, and sections were mounted on glass slides using aqua-mount mounting medium (Thermo Fisher Scientific, Waltham, USA). Confocal images of sections were obtained using confocal microscopy (FV1000, Olympus) with a 60× oil-immersion lens.

The optogenetic approach was based on procedures described previously (van Zessen et al., 2012). Bilateral injections of purified and concentrated pAAV-EF1a-double floxed-hChR2 (H134R)-mCherry or pAAV-EF1a-double floxed-eNpHR 3.0-EYFP virus (2.05 × 10¹² vector genomes/ml; Neuron Biotech Company, Shanghai, China) were stereotaxically performed in 3-week-old male Gad2-IRES-Cre mice (B6N.Cg-Gad2^tm2(cre)Zjh/J; Jackson Laboratories, USA). Each side of the VTA (final coordinates: AP, −3.1 mm; ML, ±0.4 mm; DV, −4.2 mm from the skull surface) was injected with 0.5 µl AAV for 10 min followed by an additional 10 min to allow diffusion of viral particles away from the injection site. About 10–14 days after AAV virus injection, VTA slices were prepared according to the procedures described above. GABA neurons were stimulated by a 5 ms, 470 nm light or a constant 590 nm light delivered via an optical fiber (core diameter 200 µm, NA 0.39; ThorLabs, USA) coupled to an LED light source (Mightex, California, USA). The end of the fiber optic cannula (stainless ferrule, core diameter 200 µm, length 20 mm; ThorLabs, New Jersey, USA) connected to the optical fiber through a mating sleeve (ThorLabs) was placed 500 µm above the recording cell.

Male Sprague–Dawley rats (270–300 g) were used. Animals were housed singly under a 12 hr light/dark cycle (lights on 7:00 AM to 7:00 PM) in a temperature- and humidity-controlled environment with food and water freely available. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in stereotaxic instruments (Stoelting, Wood Dale, USA). Two 24-gauge stainless steel guide cannulae were implanted bilaterally 2 mm above the VTA. The coordinates were: AP, −5.8 mm; ML, ±2.5 mm; DV, −8.0 mm from the skull surface with a 14° lateral angle. The cannulae were secured to the skull with two anchoring screws and dental cement. To prevent occlusion, 30-gauge wire plugs were inserted into the cannulae. After the surgery, the animals were housed individually and were allowed to recover for over a week.

The locomotor activity test was conducted as described previously with some modifications (Borgland et al., 2006). The locomotor activity of animals was monitored with a near infrared video camera within the operant chambers (Med Associates, St. Albans, USA). Distance traveled was measured using Open Field Activity Software (Med Associates) and analyzed locomotion estimates based on movement over a given distance and resting delays (movement in a given period of time). All animals were habituated to the test room for 2 hr prior to the start of the experiment. Rats were further habituated to the operant chambers for 30 min prior to the 120 min testing session. For bilateral intra-VTA microinjection, 30-gauge injection needles were inserted into the cannulae. The injection needles were connected to a 1-μl microsyringe (Hamilton, Reno, USA) by a polyethylene tube and controlled by a syringe pump (Harvard Apparatus, Holliston, USA). After injection, the needles were left in place for another 1 min. Rats were bilaterally injected with 0.2 μl morphine (0.5 μg/side) or CGP36216 (10 μg/side) for 1 min. After drug administration, the rats were placed in the chambers for the 120 min testing session. On days 1, 2, and 3, all rats were only given saline (bilateral intra-VTA microinjections or intraperitoneal injections) to habituate them to the test protocol. On day 4, rats were given drugs according to experimental group. After the behavioral tests, all rats were anesthetized with an overdose of chloral hydrate and perfused with 0.9% saline. The brain was removed and fixed in 4% paraformaldehyde for 24 hr. Coronal sections (80 μm) were cut by a vibratome and stained with cresyl violet. Injection sites were verified under light microscope. Animals where the injection site was outside the VTA were discarded.

The CPP test was conducted using a three-compartment place conditioning apparatus (Med Associates) with distinct tactile environments to maximize contextual differences. The procedure for CPP testing was similar to that described previously (Phillips and LePiane, 1980; Bardo and Neisewander, 1986; Wang et al., 2008), with some modifications. On day 1, rats underwent a preconditioning test: they were placed in the middle neutral area and were allowed to freely access both sides of the apparatus for 15 min. Rats with a strong preference (60%) for any compartment were discarded. Conditioning was performed using an unbiased, balanced protocol. On day 2, rats were microinjected with saline, morphine, CGP36216, or CGP36216 plus morphine 30 min before they were confined to the conditioning chambers for 45 min (drug-paired). On day 3, rats received a microinjection of saline and were then confined to the other chamber for 45 min. The day after conditioning, rats were tested for drug-induced (postconditioning test) CPP in the same conditions as for the preconditioning test. The place preference score (CPP score) was defined as the time spent in the drug-paired chamber minus that spent in the drug-unpaired (saline-paired) chamber. Injection sites were verified under light microscope as described above.

Methyl sulfoxide (DMSO), PTX, K-gluconate, Lucifer yellow, K₂-ATP, Na₃-GTP, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (Na-TES), nicotinamide adenine dinucleotide phosphate (NADP⁺), glutamate dehydrogenase, sodium dodecyl sulfate (SDS), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), Triton X-100, and 0.01 M PBS were purchased from Sigma. [S-(R*,R*)]-[3-[[1-(3,4-Dichlorophenyl)ethyl]amino]-2-hydroxypropyl] (cyclohexylmet-hyl) phosphinic acid (CGP54626) and (3-aminopropyl) ethylphosphinic acid hydrochloride (CGP36216) were purchased from Tocris. Morphine was from Shenyang No. 1 Pharmaceutical Factory, China. Percoll was purchased from Amersham Biosciences Corporation. Other AR grade reagents were from Shanghai Chemical Plant. PTX or CGP54626 was dissolved in DMSO and others were dissolved in ddH₂O. When DMSO was used as the vehicle, drugs were initially dissolved in 100% DMSO and then diluted into ASFC at a final DMSO concentration of less than 0.5%, which had no detectable effects on the parameters we observed.

Numerical data were expressed as the mean ±s.e.m. Off-line data analysis was performed using the Mini Analysis Program (Synaptosoft, Fort Lee, USA), Clampfit (Axon Instruments, Sunnyvale, USA), SigmaPlot (Systat Software, San Jose, USA), and Origin (Microcal Software, Northampton, USA). Spontaneous firing was analyzed using the Event Detection function of Clampfit. sEPSCs were analyzed using the Mini Analysis Program. Detection criteria were set at >8 pA, <1 ms rise-time, and <3 ms decay-time for sEPSCs (Borgland et al., 2006). Statistical significance was determined using Student's t-test for comparisons between two groups or ANOVAs followed by the Student–Newman–Keuls test for comparisons among three or more groups. In the patch-clamp studies, n refers to the number of cells. Every cell was from a different slice, and a group of cells in each experiment was from at least four animals. Two-way ANOVAs were performed on the data from CPP with the between-subjects factors treatment (different drugs). All post-hoc comparisons were made using Tukey's test. Results with p < 0.05 were accepted as being statistically significant.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We would like to thank Dr Jun Chen and Dr Ruth Anne Stetler at the University of Pittsburgh for critically reading this manuscript and offering many suggestions.

Animal experimentation: All experimental procedures conformed to Fudan University as well as international guidelines on the ethical use of animals and all efforts were made to minimize the number of animals used and their suffering.

© 2015, Chen et al.

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