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Articles, Systems/Circuits

Direct Bidirectional μ-Opioid Control of Midbrain Dopamine Neurons

Elyssa B. Margolis, Gregory O. Hjelmstad, Wakako Fujita and Howard L. Fields
Journal of Neuroscience 29 October 2014, 34 (44) 14707-14716; DOI: https://doi.org/10.1523/JNEUROSCI.2144-14.2014
Elyssa B. Margolis
1Department of Neurology, The Wheeler Center for the Neurobiology of Addiction, Ernest Gallo Clinic and Research Center, University of California, San Francisco, California 94143, and
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Gregory O. Hjelmstad
1Department of Neurology, The Wheeler Center for the Neurobiology of Addiction, Ernest Gallo Clinic and Research Center, University of California, San Francisco, California 94143, and
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Wakako Fujita
2Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York 10029
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Howard L. Fields
1Department of Neurology, The Wheeler Center for the Neurobiology of Addiction, Ernest Gallo Clinic and Research Center, University of California, San Francisco, California 94143, and
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Abstract

The ventral tegmental area (VTA) is required for the rewarding and motivational actions of opioids and activation of dopamine neurons has been implicated in these effects. The canonical model posits that opioid activation of VTA dopamine neurons is indirect, through inhibition of GABAergic inputs. However, VTA dopamine neurons also express postsynaptic μ-opioid peptide (MOP) receptors. We report here that in Sprague Dawley rat, the MOP receptor-selective agonist DAMGO (0.5–3 μm) depolarized or increased the firing rate of 87 of 451 VTA neurons (including 22 of 110 dopamine neurons). This DAMGO excitation occurs in the presence of GABAA receptor blockade and its EC50 value is two orders of magnitude lower than for presynaptic inhibition of GABA release on to VTA neurons. Consistent with a postsynaptic channel opening, excitations were accompanied by a decrease in input resistance. Excitations were blocked by CdCl2 (100 μm, n = 5) and ω-agatoxin-IVA (100 nm, n = 3), nonselective and Cav2.1 Ca2+ channel blockers, respectively. DAMGO also produced a postsynaptic inhibition in 233 of 451 VTA neurons, including 45 of 110 dopamine neurons. The mean reversal potential of the inhibitory current was −78 ± 7 mV and inhibitions were blocked by the K+ channel blocker BaCl2 (100 μm, n = 7). Blockade of either excitation or inhibition unmasked the opposite effect, suggesting that MOP receptors activate concurrent postsynaptic excitatory and inhibitory processes in most VTA neurons. These results provide a novel direct mechanism for MOP receptor control of VTA dopamine neurons.

  • calcium channel
  • midbrain
  • opioid
  • VTA

Introduction

While μ-opioid peptide (MOP) receptors are widely distributed in the brain, their expression in the ventral tegmental area (VTA) is required for MOP receptor-mediated positive reinforcement. Local microinfusion of MOP receptor agonists into the VTA produces conditioned place preference (Bozarth and Wise, 1984) and rodents self-administer MOP receptor agonists directly into the VTA (Bozarth and Wise, 1981; but see Jhou et al., 2012). Importantly, the rewarding effect of systemically administered MOP receptor agonists is blocked by VTA inactivation of neurons, injection of MOP receptor-selective antagonists, and downregulation of MOP receptor expression (Olmstead and Franklin, 1997; Moaddab et al., 2009; Zhang et al., 2009). Because selective activation of VTA dopamine neurons produces positive reinforcement (Tsai et al., 2009; Witten et al., 2011; Steinberg et al., 2013) and dopamine receptor antagonists in the ventral striatum can block reinforcement produced by VTA MOP receptor activation in opioid-dependent rodents (Shippenberg et al., 1993; Nader and van der Kooy, 1997; Laviolette et al., 2002), it is widely accepted that MOP receptor agonists produce positive motivational actions through excitation of VTA dopamine neurons.

Although the synaptic and local circuit mechanisms responsible for MOP receptor reinforcement are unknown, the dominant current view is that the excitatory action of MOP receptors on VTA dopamine neurons is indirect, due to somadendritic hyperpolarization of local GABA interneurons or inhibition of GABA release from terminals. Several lines of indirect evidence support this idea: local GABA neurons synapse on and inhibit VTA dopamine neurons (Johnson and North, 1992a; Omelchenko and Sesack, 2009; van Zessen et al., 2012), a subset of VTA GABA neurons is hyperpolarized by MOP receptor activation (Steffensen et al., 2006; Chieng et al., 2011; Margolis et al., 2012), and GABA terminals in the VTA are inhibited by MOP receptor-selective agonists (Johnson and North, 1992a; Bonci and Williams, 1997; Matsui and Williams, 2011; Hjelmstad et al., 2013). Therefore, the local circuitry required for disinhibition exists in the VTA. However, there is no direct evidence that inhibition of GABA inputs by MOP receptor agonists is required for excitation of dopamine neurons. Arguments supporting the disinhibition model depend in part upon the assumption that the VTA consists of only two types of neurons: GABA interneurons uniformly inhibited by MOP receptor agonists and dopamine projection neurons not directly affected by MOP receptor agonists. In fact, MOP receptor agonists have a variety of synaptic actions on different neuronal types in the VTA. MOP receptor agonists inhibit GABA inputs to both dopamine and nondopamine VTA neurons (Margolis et al., 2008; Xia et al., 2011; Hjelmstad et al., 2013). MOP receptor activation also inhibits glutamate release onto most VTA neurons (Bonci and Malenka, 1999; Margolis et al., 2005). Even more problematic, direct inhibition of VTA dopamine neurons by MOP receptor agonists has been observed in guinea pigs, mice, and rats (Cameron et al., 1997; Margolis et al., 2003; Ford et al., 2006). To directly examine the synaptic mechanism(s) by which MOP receptor agonists excite VTA dopamine neurons, we systematically investigated the effect of the MOP receptor-selective agonist DAMGO across a large sample of neurons throughout the VTA.

Materials and Methods

Animal care and all experimental procedures were in accordance with guidelines from the National Institutes of Health and approved in advance by the Ernest Gallo Clinic and Research Center and University of California, San Francisco institutional animal care and use committees.

Slice preparation and electrophysiology.

Recordings were made in control male Sprague Dawley rats (postnatal day 22–56). A small number of experiments were completed in adult male Sprague Dawley rats (250–350 g). Some data were obtained in neurons also used for previously reported experiments (Margolis et al., 2003, 2006, 2012). Rats were thoroughly anesthetized with isoflurane and then decapitated. Horizontal brain slices (150 μm thick) were prepared using a vibratome (Leica Instruments). Slices were prepared in ice-cold Ringer's solution (in mm: 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1.0 NaH2PO4, 2.5 CaCl2, 26.2 NaHCO3, and 11 glucose saturated with 95% O2–5% CO2) and allowed to recover at 33–35°C for ≥1 h. Slices were visualized under a Zeiss Axioskop or Axioskop FS 2 Plus with differential interference contrast optics and near infrared illumination, using a Zeiss Axiocam MRm and Axiovision 4 (Zeiss) or Microlucida (MBF Biosciences) software. Whole-cell recordings were made at 33°C using 2.5–5 MΩ pipettes containing the following (in mm): 123 K-gluconate, 10 HEPES, 0.2 EGTA, 8 NaCl, 2 MgATP, 0.3 Na3GTP, and 0.1% biocytin, pH 7.2, osmolarity adjusted to 275. Liquid junction potentials were not corrected during recordings. Hyperpolarization-activated cation current (Ih) was measured by voltage-clamping cells and stepping from −60 to −40, −50, −70, −80, −90, −100, −110, and −120 mV. Input (Ri) and series resistances were monitored with hyperpolarizing pulses (0.1 Hz) throughout each experiment.

Recordings were made using an Axopatch 1-D (Molecular Devices), filtered at 2 kHz, and collected at 5 kHz, or filtered at 5 kHz, and collected at 20 kHz using IGOR Pro (Wavemetrics). For all current-clamp experiments, I = 0; for voltage-clamp experiments, V = −60 or −70 mV. Most VTA neurons were selected in an unbiased manner from throughout the VTA by superimposing a grid on the slice, numbering each grid location, and using a random number generator to choose the grid location for recording. The closest healthy cell to the randomly generated grid location was patched.

In all cases, action potentials (APs) were collected by holding the cell in current clamp at I = 0, and only spontaneously occurring APs collected within the first 2 min of gaining whole-cell access were analyzed for waveform data. Neurons were required to be firing spontaneously and stably for ≥2 min with firing rates >0.25 Hz to be included in the firing rate data.

Agonists, antagonists, salts, ATP, and GTP were obtained from Sigma-Aldrich or Tocris Bioscience. ω-Agatoxin-IVA was purchased from Alomone Labs.

Cell-type identification and immunocytochemistry.

All cells were labeled with biocytin during whole-cell recording. Slices were fixed immediately after recording in 4% formaldehyde for 2 h and then stored at 4°C in PBS. Slices were preblocked for 2 h at room temperature in PBS plus 0.2% BSA and 5% normal goat serum, then incubated at 4°C with a rabbit anti-TH polyclonal antibody (1:100). Slices were then washed thoroughly in PBS with 0.2% BSA before being agitated overnight at 4°C with Cy5 anti-rabbit secondary antibody (1:100) and FITC streptavidin (6.5 μl/ml). Sections were rinsed and mounted on slides using Bio-Rad Fluoroguard Antifade Reagent mounting media and visualized under a Zeiss LSM 510 META microscope or with an Axioskop FS2 Plus with an Axiocam MRm running Neurolucida (MBF Biosciences). Primary antibodies were obtained from Millipore Bioscience Research Reagents or Millipore, secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, and all other reagents were obtained from Sigma Chemical.

In neurons where immunocytochemistry was inconclusive, Ih− neurons were classified as nondopaminergic (Margolis et al., 2006). While false negatives are possible using immunocytochemical techniques, we have previously demonstrated that the experimental methods for both recording and cytochemistry used here produce reliable TH results (Margolis et al., 2010). Further, our percentage of TH+ neurons out of the total population of cytochemically identified neurons when Ih− neurons are included in the total (52%, 110 of 211 neurons; Table 1) is remarkably similar to those obtained in the systematic anatomical analysis of sections cytochemically processed for TH and NeuN (Margolis et al., 2006).

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Table 1.

Postsynaptic MOP receptor agonist effects do not sort by neurotransmitter content

Single-cell qRT-PCR.

Individual neurons were recorded in whole-cell configuration for ≥3 min. At the termination of recording, the cytoplasm of the neuron was aspirated into the recording pipette, the pipette was retracted from the slice, and the pipette contents were ejected into an RNase-free centrifuge tube prechilled to −20°C. Samples were stored at −80°C until they were processed. cDNA was synthesized from single-cell VTA neurons using the MessageBOOSTER cDNA Synthesis from Cell Lysates Kit (MBCL90310, Epicentre) according to the manufacturer's protocol. Real-time PCR was performed using the Power SYBR Green qPCR Master Mix (Applied Biosystems). The PCR template source was 4 μl of 10× diluted first-strand cDNA. Amplification was performed with an ABI Prism 7900HT sequence detection system (Applied Biosystems). After an initial denaturation step at 95°C for 10 min, amplification was performed using 45 cycles of denaturation (95°C for 15 s), annealing (55°C for 30 s), and extension (72°C for 30 s). We amplified GAPDH, a housekeeping gene, as control. The data were analyzed using the sequence detection system software (version 2.2.1, Applied Biosystems). The software generates the baseline-subtracted amplification plot of normalized reporter values (ΔRn) versus cycle number. The amplification threshold was set at 6–7 of ΔRn linear dynamic range (50–60% of maximum ΔRn). The fractional cycle at which the intersection of amplification threshold and the plot occurs is defined as the threshold cycle (Ct-value) for the plot. Samples that gave a Ct-value within 45 cycles were considered to be positive for the mRNA expression. The samples for which Ct-values were not observed within 45 cycles (i.e., undetected) were considered to be negative for the mRNA expression. The OPRM1 primers were confirmed as specific by comparing brain tissue samples from wild-type (WT) mice and OPRM1-knock-out (KO) mice. These samples were quantified using ImageJ band intensity analysis following gel visualization. Using this approach, midbrain and cortex WT samples were measured at 35.3 and 29.1 mean band intensity, respectively, compared with 3.7 and −1.9 mean band intensity in samples from the same brain regions in the KO mice. Further, RNase/DNase-free water yielded 0.0 signal for all primers.

The forward (F) and reverse (R) primers are as follows: GAPDH-F, TCAAGAAGGTGGTGAAGCAG; GAPDH-R, AGGTGGAAGAATGGGAGTTG; OPRM1-F, TCGGTCTGCCTGTAATGTTC; OPRM1-R, CAGATTTTGAGCAGGTTCTCC; TH-F, AGGGGTACAAAACCCTCCTC; TH-R, CGCACAAAATACTCCAGGTG.

Data analysis.

At least 10 APs from each neuron were averaged together, and the resulting trace was measured between when the membrane potential (Vm) crossed threshold (when the slope of the Vm first rise exceeded 5 V/s for data collected at 5 kHz or 10 V/s for data collected at 20 kHz) to when the Vm recrossed the threshold following the AP peak. Ih magnitude was measured as the difference between the initial capacitative response to a voltage step from −60 to −120 mV and the final current during the same 200 ms step. Neurons were considered Ih− if the slope of the I–V curve for hyperpolarizing steps from −60 to −90, −100, −110, and −120 mV was 0. The reported firing rates are averages of the instantaneous firing rate over at least 2 and up to 10 min, usually at the beginning of the experiment.

Results are presented as mean ± SEM. DAMGO effects were statistically evaluated in each neuron by binning data into 30 s data points and comparing the last eight baseline data points to the last eight data points during drug application using Student's unpaired t test. In neurons that were firing spontaneously, firing rate was analyzed. In neurons that were quiescent, membrane potential was analyzed. In experiments where DAMGO was applied multiple times, this analysis was applied to each individual DAMGO application, where baseline data were the 4 min preceding each individual DAMGO application. Therefore, when DAMGO was applied in the presence of blockers, the baseline period consisted of 4 stable minutes of recording during aCSF plus blocker application. Effects of blockers were assessed with Student's paired t tests comparing the response to the first DAMGO application in control aCSF compared with the second DAMGO application in the presence of the blocker. To assess whether intracellular GDP-β-s interfered with DAMGO-induced effects, we performed permutation analysis of the SDs of changes in Vm with DAMGO of quiescent neurons. The one-tailed p value is given because the hypothesis being tested is directional: did the GDP-β-s make the variability of responses smaller? Statistical comparisons between groups of neurons were made using one-way ANOVAs. p < 0.05 was required for significance in all analyses.

Results

Using whole-cell current-clamp recording, we measured responses of VTA neurons to bath application of the MOP receptor agonist DAMGO. Significant numbers of VTA neurons were either excited or inhibited: 87 of 451 (19%) of VTA neurons showed a depolarization or an increase in firing rate in response to a saturating dose of DAMGO (500 nm–3 μm; Fig. 1; Fig. 3). The EC50 value was in the low nanomolar range (Fig. 5I). Surprisingly, a larger percentage (233 of 451, 52%) was inhibited (hyperpolarized or decreased firing rate) by DAMGO (500 nm–3 μm; Figs. 2, 3). The dose–response curve for inhibitions overlapped that of the excitations (EC50 value in the low nanomolar range; Fig. 5I). Furthermore, contrary to previous interpretations, the proportions of excited and inhibited VTA neurons and the magnitudes of these effects were similar in identified dopamine (TH+) and nondopamine (TH−) neurons (Table 1). We also observed inhibitions and excitations in neurons that were Ih− (Table 1), which are uniformly TH− (Margolis et al., 2006), and possibly GABAergic or glutamatergic. Both excitations and inhibitions were reversed by the MOP receptor-selective antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Pen-Thr-NH2 (CTAP, 100–500 nm; Figs. 1D, 2C), indicating that both effects require actions at the MOP receptor. There was no apparent topographic organization within the VTA of neurons exhibiting excitations or inhibitions (Fig. 4). While most observations were made in young rats (postnatal days 22–56), we confirmed these observations in VTA tissue taken from adult rats (250–350 g; Table 1).

Figure 1.
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Figure 1.

MOP receptors excite VTA dopamine neurons by activating an inward current. A, Example VTA neuron that depolarized and started firing in response to the MOP receptor-selective agonist DAMGO (500 nm; application time indicated by horizontal bar above trace). B, Pooled membrane potential data from all quiescent neurons showing a significant depolarization following bath application of the DAMGO (0.5–3 μm). C, Example of a confirmed dopamine neuron excited by DAMGO. Left column, Biocytin fill of recorded neuron (top, red) with post hoc TH immunostaining (middle, green) and merged image (bottom, overlap indicated in yellow). Right column, Current-clamp experiment of this neuron (I = 0) showing response to 500 nm DAMGO. Upper trace, This neuron responded to the MOP receptor agonist DAMGO (500 nm) with a transient increase in firing rate. Lower trace, The concurrent membrane potential in the same neuron illustrating a sustained depolarization following DAMGO application. D, A different example neuron where the MOP receptor-selective antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Pen-Thr-NH2 (CTAP; 100 nm) reversed a DAMGO-induced depolarization. E, Example voltage-clamp (Vm = −70 mV) experiment where DAMGO activated an inward current in the presence of picrotoxin (100 μm), and this current was accompanied by an increase in conductance (blue). F, Another example neuron in which DAMGO was applied twice, first in voltage clamp, resulting in an inward current, and subsequently in current clamp, yielding a depolarization.

Figure 2.
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Figure 2.

MOP receptors inhibit VTA dopamine neurons by activating an outward current. A, Pooled data from all quiescent neurons that showed a significant hyperpolarization following bath application of the MOP receptor-selective agonist DAMGO. B, Example current-clamp (I = 0) experiment in a neuron filled with biocytin (red, left) and post hoc identified as dopaminergic with immunocytochemistry against TH (green, middle; merged image in yellow on right). DAMGO (500 nm) hyperpolarized this neuron and decreased its input resistance (blue). This neuron was Ih+ and had a short-duration AP (top and bottom insets, respectively). Inset scale bars: top, 100 pA, 50 ms; bottom, 10 mV, 2 ms. C, Example neuron where the MOP receptor-selective antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Pen-Thr-NH2 (CTAP; 100 nm) reversed a DAMGO-induced hyperpolarization. D, Example voltage-clamp (Vm = −70 mV) recording where DAMGO activated an outward current in the presence of picrotoxin (100 μm), and this current was accompanied by an increase in conductance (blue).

Figure 3.
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Figure 3.

Distribution of significant (p < 0.05) DAMGO-elicited physiological responses in VTA neurons. A, DAMGO-induced changes in membrane potential in neurons not firing spontaneously. B, Among these quiescent neurons, decreases in input resistance (top) were observed in most neurons with significant DAMGO-induced change in membrane potential, including those that were depolarized by DAMGO. A smaller number of neurons showed either an increase (middle) or no change (bottom) in input resistance. C, Distribution of DAMGO-induced increases (top) or decreases (middle) in firing rate in neurons that were firing spontaneously before DAMGO application.

Figure 4.
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Figure 4.

Topographical distribution of sampled neurons within the VTA. Top row, Locations in horizontal slices of cytochemically identified dopamine neurons tested with DAMGO. Bottom row, Identified nondopamine neurons, includes (●) TH− and (▿) Ih− without cytochemistry. Inset, SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; MT, medial terminal nucleus of the accessory optic tract; IPN, interpeduncular nucleus; A, anterior; P, posterior; L, lateral; M, medial.

Excitations are postsynaptic, not disinhibition

One immediate question raised by these data is as follows: are the excitations due to removal of GABA inputs, i.e., disinhibition, as posited in the canonical model? MOP receptor agonists robustly inhibit GABA release onto all VTA neurons in ex vivo recordings (Bonci and Williams, 1997; Margolis et al., 2008), and bath application of the GABAA receptor Cl− channel blocker picrotoxin (100 μm) can depolarize neurons in the slice (5 of 5 neurons, where all neurons had baseline membrane potentials more depolarized than the Cl− equilibrium potential; data not shown) indicating that there is tonic GABA release in VTA slice preparations. However, arguing against the disinhibition model, we observed that most of the DAMGO-induced depolarizations were associated with a concurrent increase in conductance (Fig. 3B). Furthermore, the magnitudes of the excitations were directly and significantly correlated with the increase in conductance (r = 0.66, p = 0.0005). This is consistent with opening a channel, not disinhibition through reduction of an inhibitory input that would decrease membrane conductance through channel closings. To test the GABA disinhibition model directly, responses to DAMGO were measured in voltage clamp (Vm = −60 or −70 mV) in the presence of picrotoxin (100 μm). In 6 of 24 neurons (25%), we observed an inward current in response to DAMGO, a proportion similar to that of excitations observed in control aCSF in current-clamp experiments (Fig. 1E,F). Furthermore, of four neurons where DAMGO induced an inward current in voltage clamp, depolarizations were observed in three when they were tested again with DAMGO after switching into current clamp (Fig. 1F). To provide additional evidence that the MOP receptor excitations were not due to presynaptic inhibition of GABA terminals, we compared the DAMGO dose–response curve for the presynaptic inhibition of GABA release with that for the depolarization. If the two effects were due to activation of MOP receptors on GABA terminals, the dose–response curves for the two actions should overlap. Instead, we found that the EC50 value for DAMGO presynaptic inhibition of GABA release was two orders of magnitude higher than that for the somadendritic hyperpolarizations/depolarizations (Fig. 5I). Thus three independent lines of evidence argue strongly against disinhibition as a major factor in this excitation of VTA neurons: it is associated with a conductance increase, it requires a much lower MOP receptor agonist concentration than presynaptic inhibition of GABA release, and it is insensitive to pharmacological blockade of GABAA receptors.

Figure 5.
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Figure 5.

DAMGO excitations require Cav2.1. A, Example neuron where DAMGO (500 nm) application caused a depolarization and increase in AP firing frequency. In the presence of highly selective Cav2.1 (P/Q type) Ca2+ channel blocker ω-agatoxin IVA (100 nm), the same dose of DAMGO caused an inhibition. B, Time course data across seven neurons where either the nonselective Ca2+ channel blocker CdCl2 (100 μm) or ω-agatoxin IVA (100 nm) was used to block a DAMGO-induced depolarization. The neuron in A was excluded from this plot because of the nonlinearity of membrane potential changes when neurons start and stop firing. C, Control data show that repeated applications of DAMGO within the same cell elicited responses of similar magnitudes for both depolarizations and hyperpolarizations. D, In neurons with an initial depolarizing response to DAMGO, a mixture of ionotropic glutamate receptor antagonists did not block DAMGO excitations. E, The nonselective Ca2+ channel blocker CdCl2 (100 μm) completely blocked DAMGO-induced excitations and in most cases unmasked hyperpolarizations. In gray, a small hyperpolarizing effect of DAMGO was made bigger by CdCl2, suggesting an underlying but masked depolarization. F, The highly selective Cav2.1 (P/Q type) Ca2+ channel blocker ω-agatoxin IVA (100 nm) also completely blocked the excitatory effect of DAMGO, unmasking an inhibitory action. In gray, a small hyperpolarizing effect of DAMGO was made bigger by ω-agatoxin IVA. G, In neurons that responded to DAMGO with a hyperpolarization, the K+ channel blocker BaCl2 (500 μm) eliminated the hyperpolarizations, and in most neurons revealed a depolarization (inset). H, In voltage clamp, voltage ramps were used to estimate the reversal potential for the DAMGO effect in neurons that hyperpolarized ≥8 mV in response to DAMGO (n = 10). I, Dose responses were measured for the postsynaptic excitations and inhibitions, as well as for the inhibition of electrically stimulated GABA release (evIPSCs). Dose–response relationships were indistinguishable for postsynaptic DAMGO excitations (blue) and inhibitions (red) but the EC50 value was two orders of magnitude lower than for DAMGO inhibition of evIPSCs (green). n = 4–8 for each concentration. J, Including GDP-β-s (500 μm) in the recording pipette to block G-protein signaling in the recorded cell eliminated all DAMGO responses. GDP-β-s significantly decreased the variation in responses to DAMGO (permutation analysis, p < 0.0005), consistent with a blockade of both excitations and inhibitions.

Many VTA neurons fire spontaneously in the slice and this leads to local GABA, glutamate, and dopamine neuron crosstalk in the VTA (Ford et al., 2009; Omelchenko and Sesack, 2009; Dobi et al., 2010). Because more neurons than expected responded to DAMGO, it is possible that the observed effects in some neurons could be indirect. If the DAMGO effects on the recorded neuron were indirect, i.e., due to a direct receptor action on the firing of other neurons in the slice, the voltage-gated Na+ channel blocker tetrodotoxin (TTX; 500 nm–1 μm) should decrease or eliminate DAMGO responses. In fact, DAMGO-induced hyperpolarizations persisted in the presence of TTX (control response: −9.7 ± 1.9 mV; in TTX: −6.7 ± 2.0 mV; n = 4). Because TTX alone often caused VTA neurons to depolarize, we tested for DAMGO-induced excitations in the presence of TTX in voltage clamp, holding the cells at Vm = −60 mV, further from the reversal potential of a depolarizing conductance, and closer to the basal membrane potential of typical VTA neurons. Under these conditions, 8 of 21 neurons responded to DAMGO with a significant inward current (control: −91 ± 33 pA, n = 3; TTX: −248 ± 131 pA, n = 8). These observations render it unlikely that the inhibitions or excitations produced by DAMGO are indirect.

What MOP receptor-activated conductance(s) depolarize VTA neurons?

Another possibility is that MOP receptor activation increases glutamate release that is independent of AP activity, either by a direct effect on nerve terminals (Velásquez-Martinez et al., 2012) or via an effect on glia (Moussawi et al., 2011). A MOP receptor-induced increase in glutamate release from nerve terminals is unlikely since synaptic experiments have demonstrated that MOP receptor activation inhibits glutamate release onto VTA neurons (Bonci and Malenka, 1999; Margolis et al., 2005). MOP receptors have been observed on microglia and astrocytes (Ruzicka et al., 1995; Dever et al., 2012; Merighi et al., 2013), and activation of microglia can cause glutamate release (Noda et al., 1999). To test whether glutamate is involved in MOP receptor-mediated excitations, we first found a VTA neuron that exhibited an excitation in response to DAMGO, then retested the neuron with DAMGO in the presence of DNQX (10 μm) and d(−)-2-amino-5-phosphonopentanoic acid (AP-V, 50 μm) to block excitatory glutamate receptors. In control experiments where DAMGO was applied twice, the magnitude and direction of membrane potential change was consistent between applications (Fig. 5C). DAMGO effects in the presence of glutamate antagonists were not different from controls (Fig. 5D).

In isolated Purkinje neurons, MOP receptor activation increases a Cav2.1 conductance (Iegorova et al., 2010). Cav2.1 composes ∼40% of voltage-dependent Ca2+ currents in VTA dopamine neurons (Cardozo and Bean, 1995). To test whether a Ca2+ channel is involved in the MOP receptor-induced depolarizations in the VTA, we measured responses to DAMGO in the presence of the Ca2+ channel blocker CdCl2 (100 μm) in neurons that were excited under control conditions. CdCl2 not only prevented excitations, but in 5 of 5 neurons CdCl2 treatment revealed an inhibition in response to DAMGO (Fig. 5E). To test whether Cav2.1 channels were involved, in another set of neurons we retested DAMGO in the presence of the selective blocker ω-agatoxin IVA (100 nm); this also prevented the DAMGO-induced excitations and revealed inhibitions, similar to CdCl2 (Fig. 5A,F). Finally, we also tested whether the excitations depended upon G-protein signaling in the recorded neuron by replacing intracellular GTP with the non-hydrolyzable GDP-β-s (500 μm). None of the five cells tested showed any response to DAMGO after GDP-β-s was delivered into the cell via the recording electrode (Fig. 5J), despite the fact that four of these neurons were tested with and responded to DAMGO in cell-attached configuration before establishing whole-cell access and therefore GDP-β-s delivery, two increasing and two decreasing in firing rate. Consistent with intracellular GDP-β-s eliminating DAMGO signaling, the variability of the differences in Vm between baseline and in DAMGO application in these cells was significantly smaller than across the population of quiescent neurons (permutation analysis of SD, p < 0.0005). These data indicate that excitations require G-protein signaling in the recorded neuron and are consistent with the interpretation that DAMGO causes a direct excitation of a subset of VTA neurons, including dopamine neurons, through G-protein-dependent activation of Cav2.1.

Inhibitions

Our finding that approximately half of all VTA neurons, including 41% of all confirmed dopamine neurons, were inhibited by MOP receptor activation contradicts the assumptions that support the canonical disinhibition model. Postsynaptic MOP receptor-induced inhibitions are typically mediated by the opening of G-protein-activated inwardly rectifying K+ channels (GIRKs). In neurons that were hyperpolarized by DAMGO, we reapplied DAMGO in the presence of the K+ channel blocker BaCl2 (500 μm) to directly test that opening K+ channels mediates the DAMGO-induced inhibitions. Not only did BaCl2 treatment block the inhibitions, in six of seven neurons it uncovered small excitations (Fig. 5G). We further observed that both CdCl2 and ω-agatoxin IVA made DAMGO-induced hyperpolarizations larger (Fig. 5E,F). By applying a voltage ramp in voltage clamp, we determined that the reversal potential for the DAMGO-induced inhibitions is −78 ± 7 mV (n = 8) and correcting for the liquid junction potential estimated at −15 mV yields a reversal potential of −93 mV (Fig. 5H). This is somewhat depolarized from the estimated K+ reversal potential given the aCSF and internal solutions used here (−103 mV), possibly due to the contribution of the excitatory component of MOP receptor signaling in these neurons. The inhibitions are thus most likely an example of the well known opioid receptor activation of a GIRK. It is important to note that although blockade of the DAMGO inhibition by BaCl2 reveals an excitation, intracellular blockade of G-protein signaling with GDP-β-s does not unmask an excitation. This supports the conclusion that both excitations and inhibitions are G-protein mediated and are specific to the recorded neuron. Furthermore, together with the observation that the inhibitions and excitations have the same dose–response curve, this observation provides additional support for the idea that DAMGO concurrently produces both effects directly on the recorded neuron.

MOP receptors are expressed in most VTA dopamine neurons

Together, our observations that the majority (71%) of both dopamine and nondopamine VTA neurons respond to DAMGO (by either excitation or inhibition) indicate that most VTA neurons express the MOP receptor. This was unexpected given that previous studies have reported only modest MOP receptor protein or mRNA expression in the VTA (Mansour et al., 1994, 1995). Furthermore, while some VTA dopamine neurons have previously been reported to express MOP receptor (Garzón and Pickel, 2001) and to be inhibited by MOP receptor activation (Cameron et al., 1997; Margolis et al., 2003; Ford et al., 2006), our proportions of MOP receptor-responsive VTA neurons are higher than predicted. To provide an independent line of support for the conclusion that many VTA neurons express MOP receptor, we measured TH and MOP receptor mRNA from individual VTA neurons with RT-PCR. Of the 21 sampled neurons, 12 (57%) expressed TH mRNA; this proportion is consistent with anatomical findings of proportions of TH+ VTA neurons (Margolis et al., 2006; Nair-Roberts et al., 2008). We found that 10 of 12 neurons that expressed mRNA for TH also expressed MOP receptor mRNA. Among the nine neurons in which TH mRNA was not detectable, MOP receptor mRNA was detected in five. Together with the electrophysiological observations described above, these RT-PCR results provide strong support for the concept that the majority of VTA dopaminergic neurons express MOP receptors.

Discussion

Here we report that a subset of both dopamine and nondopamine VTA neurons are excited by DAMGO. This occurred in the absence of GABAA receptor signaling and required Cav2.1 activity. Consistent with a direct postsynaptic effect, the excitations were associated with an increase in conductance and were blocked by intracellular application of GDP-β-s. A larger proportion of both dopamine and nondopamine neurons were directly inhibited through activation of a GIRK. We confirmed that both excitatory and inhibitory effects of DAMGO occur in VTA neurons from adult rats as well. Blockade of either excitation or inhibition revealed or enhanced the opposite effect, indicating that MOP receptor agonists concurrently activate both GIRKs and Cav2.1 channels in the same neuron.

Direct excitation or disinhibition?

Although excitations of dopamine neurons were frequent, we found no evidence of MOP receptor-mediated disinhibition. This was unexpected because presynaptic inhibition of evoked GABA release by MOP receptor agonists is robust in this preparation and there is significant ongoing tonic GABAA receptor activity as revealed by picrotoxin-induced depolarizations. In other words, even though (1) a reduction of GABA input in this ex vivo preparation does excite VTA dopamine neurons and (2) activation of MOP receptor inhibits GABA neurons and their terminals, inhibition of GABA input is not required for the excitation of VTA neurons by locally acting MOP receptor agonists. However, disinhibition could be significant if the nervous system is intact. There are MOP receptor-sensitive GABAergic terminals in the VTA that arise from neurons in other structures, including the ventral pallidum (Hjelmstad et al., 2013) and the rostromedial tegmental nucleus (Matsui and Williams, 2011), and while these synapses may be very active in vivo, their activity would be minimized in our preparation where their terminals are severed from their cell bodies. Consistent with this idea, spontaneous GABA IPSCs occur at very low rates in VTA neurons in horizontal brain slices (∼1–4 Hz; Margolis et al., 2008; Theile et al., 2008; Xiao and Ye, 2008), and similar rates have been reported for AP-independent miniature IPSCs (Bonci and Williams, 1997; Melis et al., 2002). Furthermore, when measured directly, IPSC frequency is only reduced by ∼30% by TTX (our unpublished observations). Despite the low spontaneous IPSC event rates, there must be a source for the significant tonic GABAA receptor signaling revealed by picrotoxin. One possibility is that there is ambient GABA from a non-neuronal source, such as astrocytes (Jiménez-González et al., 2011), which could explain why it is not significantly regulated by MOP receptors. On the other hand, DAMGO excitations were associated with an increase in conductance. Consequently, although the present results do not rule out a contribution of disinhibition to MOP receptor activation of VTA neurons, they strongly support the conclusion that direct excitation contributes significantly in vivo when MOP agonists are systemically administered.

MOP receptor expression and function is ubiquitous in the VTA

Approximately 20% of VTA neurons, both TH+ and TH−, were excited by MOP receptor activation. Such a Cav2.1-dependent MOP receptor excitatory effect has been reported in cerebellar neurons (Iegorova et al., 2010). Although the Cav2.1-selective blocker ω-agatoxin IVA abolished the excitations, some of the neurons were relatively hyperpolarized at baseline, with membrane potentials at which Cav2.1 channels are reportedly inactive. Consequently, an indirect mechanism could be involved, such as presynaptic MOP receptor enhancement of Ca2+ channel-dependent vesicular release of an excitatory substance. However, MOP receptor activation inhibits release from glutamate terminals in the VTA (Bonci and Malenka, 1999; Margolis et al., 2005), and the excitations we observed were not blocked by glutamate receptor antagonists. Alternatively, MOP receptors might be expressed on glia or at neuronal sites where another depolarizing agent may be released that acts at a G-protein-coupled receptor, such as orexin, substance P, cholecystokinin, or neurotensin. However, the fact that the dose–response curves for the excitations and inhibitions are virtually identical is consistent with a single postsynaptic receptor binding site capable of mediating both excitations and inhibitions. Therefore, the most parsimonious explanation for our data is that these DAMGO-induced excitations are due to postsynaptic MOP receptors on the recorded neurons that open Cav2.1 channels.

We also observed that postsynaptic excitations and inhibitions occurred at much lower concentrations than the presynaptic inhibition of VTA GABA release at terminals. This relationship is consistent with the presynaptic and postsynaptic potency differences reported by Johnson and North for Met-enkephalin in the VTA (1992a). Such differences could be caused by a wide variety of conditions that would affect the affinity of the receptor for the ligand. For instance, the conformation of the receptor may be influenced by the proximity of a G-protein (Kuszak et al., 2009; Malik et al., 2013) or by the local ion concentrations (Wong et al., 1994). There is also evidence that heterodimerization of opioid receptors alters ligand binding (Jordan and Devi, 1999) and the regulator of the G-protein-signaling protein family can alter opioid receptor activation (Georgoussi et al., 2006; Wang et al., 2009). The fact that the postsynaptic receptors are more sensitive to not only DAMGO, as demonstrated here, but also Met-enkephalin raises the possibility that endogenous opioid release acts more potently at postsynaptic than presynaptic sites.

A particularly intriguing observation is that DAMGO-induced excitations were unmasked in most inhibited cells following blockade of K+ currents, and conversely, most excited neurons responded with an inhibition when Ca2+ channels were blocked. The purpose this serves in an individual neuron is not obvious, especially if each receptor is coupled to both downstream ion channels. On the other hand, this observation might make sense if the excitatory and inhibitory actions were initiated by spatially segregated MOP receptors, such that certain dendrites are controlled by MOP receptors coupled to K+ channels, while in other dendrites the MOP receptors are coupled primarily to Ca2+ channels. A similar possibility is that the different MOP receptor effects may segregate according to the source of the nearby synaptic inputs, or may depend upon which other receptors are nearby in the plasma membrane.

While opioid receptors are generally thought of as inhibitory, there is a growing literature supporting the idea that the signaling pathways they employ are actually more varied. In fact, evidence of seven transmembrane domain receptor/G-protein coupled receptor signaling concurrently through several different pathways has been accumulating. In addition to MOP receptors activating Cav2.1 channels in the cerebellum (Iegorova et al., 2010), a variety of stimulatory effects of opioid receptor activation has been reported (Harrison et al., 1998). For example, morphine increases adenylyl cyclase in rat corpus striatum (Puri et al., 1975), and endogenous opioid peptides increase adenylyl cyclase in the rat olfactory bulb in a Gi/o-dependent manner (Onali and Olianas, 1991). In cell culture, there is also evidence that in cells expressing both the MOP receptor and the δ-opioid receptor, DAMGO increases intracellular Ca2+ in a Gi/o-dependent manner (Charles et al., 2003). These and other observations in a variety of brain regions and in different reduced systems lend credence to the interpretation that in the VTA, MOP receptor activation can directly excite neurons by opening a Ca2+ channel.

The high proportion of confirmed dopamine neurons directly inhibited by MOP receptor activation was surprising. At first glance these results differ from the seminal electrophysiological work by Johnson and North (1992b). However, Johnson and North identified “dopamine” neurons as those inhibited by dopamine and not opioids; “GABA” neurons were identified as those insensitive to dopamine and inhibited by opioids. Subsequent work has shown that these criteria are unreliable. However, when we applied the Johnson and North criteria to our neuronal cohort, the electrophysiological properties of the two datasets are strikingly similar (Table 2). However, 28% of our neurons that would have been classified as GABAergic by Johnson and North were TH+. Furthermore, others have shown that some VTA dopamine neurons are inhibited by MOP receptor agonists (Cameron et al., 1997; Ford et al., 2006; but see Chieng et al., 2011). One possible reason that we found more MOP receptor-inhibited dopamine neurons is that our sample size is much larger and includes recordings in the medial VTA, which is not sampled in many ex vivo studies. While we did not observe any topographical sorting of MOP receptor-inhibited dopamine neurons, we excluded substantia nigra pars compacta neurons, which are reportedly MOP receptor insensitive (Lacey et al., 1987). Further, some of the inhibitions were relatively small (Fig. 3) and may have been ignored in previous studies. However, we found that these changes were significant compared with baseline, time locked to drug application, reproducible within cells, and blocked by BaCl2.

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Table 2.

Electrophysiological properties of rat VTA neurons sorted according to Johnson and North's 1992 criteriaa

VTA MOP receptors and behavior

The physiologic data are compelling that virtually every VTA neuron is either presynaptically or postsynaptically modulated by MOP receptor activation, and any of these actions may contribute to reward. In fact, even MOP receptor actions at nondopamine neurons can be rewarding, as in naïve rodents where VTA MOP reward is unaffected by dopamine antagonists (Nader and van der Kooy, 1997). The extent of MOP receptor-mediated inhibition of dopamine neurons is also consistent with a growing appreciation of the diversity of VTA dopamine neuron connectivity and function. Particularly noteworthy is evidence that some dopamine neurons are activated by aversive stimuli (Brischoux et al., 2009; Bromberg-Martin et al., 2010). Also, activation of PFC-projecting dopamine neurons can cause conditioned place aversion (Lammel et al., 2012). One possibility is that the MOP receptor-inhibited neurons we report here represent a subpopulation of dopamine neurons that respond to noxious stimulation.

Given the variety of synaptic actions of the MOP receptor in the VTA, it is difficult to predict the relative contribution of GABA terminal inhibition versus somadendritic dopamine neuron excitation to an increase in dopamine neuron firing in an awake behaving rat. This will depend upon a variety of factors, including the activity of specific GABAergic inputs, the membrane potential of the neuron, and the concentration of nonopioid neurotransmitters and modulators. Behaviorally effective systemic doses of morphine do suggest that the more potent postsynaptic effects reported here are significant to behavioral responses: following a typical rodent analgesic dose of morphine in rat (ED50 value, ∼10 mg/kg), brain concentrations of ∼140 ng/g or 0.5 μm are achieved (Patrick et al., 1975). Therefore, a systemic dose of 3 mg/kg morphine, which is sufficient to produce place preference (Shippenberg and Herz, 1987), yields a brain concentration of 150 nm. Assuming equivalent dose responses for morphine and DAMGO, this is a saturating dose for the somadendritic action but is closer to the EC50 value for inhibition of GABA release, raising the possibility that postsynaptic effects dominate in the VTA for systemic opioid administration. Consequently, it is likely that the direct MOP receptor-mediated excitatory effect on VTA dopamine neurons contributes significantly to the rewarding action of MOP receptor agonists. This conclusion is supported by the findings of Zhang et al. (2009) showing that siRNA knockdown of MOP receptors intrinsic to the VTA/substantia nigra region reduces the rewarding effect of systemic MOP receptor agonist administration.

In summary, we report a novel mechanism for neuronal activation by MOP receptor in the VTA, i.e., the opening of somadendritic Cav2.1 channels, an effect seen on both dopamine and nondopamine neurons. MOP receptor-mediated postsynaptic inhibition is more common than excitation even among VTA dopamine neurons. Both postsynaptic excitations and inhibitions are G-protein mediated and both occur concurrently in the same neuron at much lower concentrations of agonist than presynaptic inhibition of GABA release. These observations challenge the canonical disinhibition model and are consistent with the idea that more than one synaptic mechanism can contribute to MOP reward.

Footnotes

  • This work was supported by National Institute on Drug Abuse Awards R01 DA030529 to E.B.M. and DA008863 to Lakshmi A. Devi, and funds provided by the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco. We thank Mirabel Lim, Joseph Driscoll, Adam Ferris, Peter Fong, Junko Ishikawa, Hagar Lock, and Kelly Pollack for technical support; Antonello Bonci and Lakshmi A. Devi for scientific discussions; and Kevin Bender and Roger Nicoll for feedback on the manuscript.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Elyssa B. Margolis, 675 Nelson Rising Lane, Box 0444, San Francisco, CA 94143. elyssa.margolis{at}ucsf.edu

References

  1. ↵
    1. Bonci A,
    2. Malenka RC
    (1999) Properties and plasticity of excitatory synapses on dopaminergic and GABAergic cells in the ventral tegmental area. J Neurosci 19:3723–3730, pmid:10234004.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Bonci A,
    2. Williams JT
    (1997) Increased probability of GABA release during withdrawal from morphine. J Neurosci 17:796–803, pmid:8987801.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Bozarth MA,
    2. Wise RA
    (1981) Intracranial self-administration of morphine into the ventral tegmental area in rats. Life Sci 28:551–555, doi:10.1016/0024-3205(81)90148-X, pmid:7207031.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Bozarth MA,
    2. Wise RA
    (1984) Anatomically distinct opiate receptor fields mediate reward and physical dependence. Science 224:516–517, doi:10.1126/science.6324347, pmid:6324347.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Brischoux F,
    2. Chakraborty S,
    3. Brierley DI,
    4. Ungless MA
    (2009) Phasic excitation of dopamine neurons in ventral VTA by noxious stimuli. Proc Natl Acad Sci U S A 106:4894–4899, doi:10.1073/pnas.0811507106, pmid:19261850.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Bromberg-Martin ES,
    2. Matsumoto M,
    3. Hikosaka O
    (2010) Dopamine in motivational control: rewarding, aversive, and alerting. Neuron 68:815–834, doi:10.1016/j.neuron.2010.11.022, pmid:21144997.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Cameron DL,
    2. Wessendorf MW,
    3. Williams JT
    (1997) A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids. Neuroscience 77:155–166, doi:10.1016/S0306-4522(96)00444-7, pmid:9044383.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Cardozo DL,
    2. Bean BP
    (1995) Voltage-dependent calcium channels in rat midbrain dopamine neurons: modulation by dopamine and GABAB receptors. J Neurophysiol 74:1137–1148, pmid:7500139.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Charles AC,
    2. Mostovskaya N,
    3. Asas K,
    4. Evans CJ,
    5. Dankovich ML,
    6. Hales TG
    (2003) Coexpression of delta-opioid receptors with micro receptors in GH3 cells changes the functional response to micro agonists from inhibitory to excitatory. Mol Pharmacol 63:89–95, doi:10.1124/mol.63.1.89, pmid:12488540.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Chieng B,
    2. Azriel Y,
    3. Mohammadi S,
    4. Christie MJ
    (2011) Distinct cellular properties of identified dopaminergic and GABAergic neurons in the mouse ventral tegmental area. J Physiol 589:3775–3787, doi:10.1113/jphysiol.2011.210807, pmid:21646409.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Dever SM,
    2. Xu R,
    3. Fitting S,
    4. Knapp PE,
    5. Hauser KF
    (2012) Differential expression and HIV-1 regulation of μ-opioid receptor splice variants across human central nervous system cell types. J Neurovirol 18:181–190, doi:10.1007/s13365-012-0096-z, pmid:22528479.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Dobi A,
    2. Margolis EB,
    3. Wang HL,
    4. Harvey BK,
    5. Morales M
    (2010) Glutamatergic and nonglutamatergic neurons of the ventral tegmental area establish local synaptic contacts with dopaminergic and nondopaminergic neurons. J Neurosci 30:218–229, doi:10.1523/JNEUROSCI.3884-09.2010, pmid:20053904.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Ford CP,
    2. Mark GP,
    3. Williams JT
    (2006) Properties and opioid inhibition of mesolimbic dopamine neurons vary according to target location. J Neurosci 26:2788–2797, doi:10.1523/JNEUROSCI.4331-05.2006, pmid:16525058.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Ford CP,
    2. Phillips PE,
    3. Williams JT
    (2009) The time course of dopamine transmission in the ventral tegmental area. J Neurosci 29:13344–13352, doi:10.1523/JNEUROSCI.3546-09.2009, pmid:19846722.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Garzón M,
    2. Pickel VM
    (2001) Plasmalemmal mu-opioid receptor distribution mainly in nondopaminergic neurons in the rat ventral tegmental area. Synapse 41:311–328, doi:10.1002/syn.1088, pmid:11494402.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Georgoussi Z,
    2. Leontiadis L,
    3. Mazarakou G,
    4. Merkouris M,
    5. Hyde K,
    6. Hamm H
    (2006) Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the mu- and delta-opioid receptors regulate opioid receptor signaling. Cell Signal 18:771–782, doi:10.1016/j.cellsig.2005.07.003, pmid:16120478.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Harrison C,
    2. Smart D,
    3. Lambert DG
    (1998) Stimulatory effects of opioids. Br J Anaesth 81:20–28, doi:10.1093/bja/81.1.20, pmid:9771269.
    OpenUrlFREE Full Text
  18. ↵
    1. Hjelmstad GO,
    2. Xia Y,
    3. Margolis EB,
    4. Fields HL
    (2013) Opioid modulation of ventral pallidal afferents to ventral tegmental area neurons. J Neurosci 33:6454–6459, doi:10.1523/JNEUROSCI.0178-13.2013, pmid:23575843.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Iegorova O,
    2. Fisyunov A,
    3. Krishtal O
    (2010) G-protein-independent modulation of P-type calcium channels by mu-opioids in Purkinje neurons of rat. Neurosci Lett 480:106–111, doi:10.1016/j.neulet.2010.06.015, pmid:20541588.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Jhou TC,
    2. Xu SP,
    3. Lee MR,
    4. Gallen CL,
    5. Ikemoto S
    (2012) Mapping of reinforcing and analgesic effects of the mu opioid agonist endomorphin-1 in the ventral midbrain of the rat. Psychopharmacology 224:303–312, doi:10.1007/s00213-012-2753-6, pmid:22669129.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Jiménez-González C,
    2. Pirttimaki T,
    3. Cope DW,
    4. Parri HR
    (2011) Non-neuronal, slow GABA signalling in the ventrobasal thalamus targets delta-subunit-containing GABA(A) receptors. Eur J Neurosci 33:1471–1482, doi:10.1111/j.1460-9568.2011.07645.x, pmid:21395866.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Johnson SW,
    2. North RA
    (1992a) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483–488, pmid:1346804.
    OpenUrlAbstract
  23. ↵
    1. Johnson SW,
    2. North RA
    (1992b) Two types of neurone in the rat ventral tegmental area and their synaptic inputs. J Physiol 450:455–468, pmid:1331427.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Jordan BA,
    2. Devi LA
    (1999) G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399:697–700, doi:10.1038/21441, pmid:10385123.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Kuszak AJ,
    2. Pitchiaya S,
    3. Anand JP,
    4. Mosberg HI,
    5. Walter NG,
    6. Sunahara RK
    (2009) Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2. J Biol Chem 284:26732–26741, doi:10.1074/jbc.M109.026922, pmid:19542234.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Lacey MG,
    2. Mercuri NB,
    3. North RA
    (1987) Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J Physiol 392:397–416, pmid:2451725.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Lammel S,
    2. Lim BK,
    3. Ran C,
    4. Huang KW,
    5. Betley MJ,
    6. Tye KM,
    7. Deisseroth K,
    8. Malenka RC
    (2012) Input-specific control of reward and aversion in the ventral tegmental area. Nature 491:212–217, doi:10.1038/nature11527, pmid:23064228.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Laviolette SR,
    2. Nader K,
    3. van der Kooy D
    (2002) Motivational state determines the functional role of the mesolimbic dopamine system in the mediation of opiate reward processes. Behav Brain Res 129:17–29, doi:10.1016/S0166-4328(01)00327-8, pmid:11809491.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Malik RU,
    2. Ritt M,
    3. DeVree BT,
    4. Neubig RR,
    5. Sunahara RK,
    6. Sivaramakrishnan S
    (2013) Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells. J Biol Chem 288:17167–17178, doi:10.1074/jbc.M113.464065, pmid:23629648.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Mansour A,
    2. Fox CA,
    3. Thompson RC,
    4. Akil H,
    5. Watson SJ
    (1994) mu-Opioid receptor mRNA expression in the rat CNS: comparison to mu-receptor binding. Brain Res 643:245–265, doi:10.1016/0006-8993(94)90031-0, pmid:8032920.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Mansour A,
    2. Fox CA,
    3. Burke S,
    4. Akil H,
    5. Watson SJ
    (1995) Immunohistochemical localization of the cloned mu opioid receptor in the rat CNS. J Chem Neuroanat 8:283–305, doi:10.1016/0891-0618(95)00055-C, pmid:7669273.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Margolis EB,
    2. Hjelmstad GO,
    3. Bonci A,
    4. Fields HL
    (2003) κ-Opioid agonists directly inhibit midbrain dopaminergic neurons. J Neurosci 23:9981–9986, pmid:14602811.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Margolis EB,
    2. Hjelmstad GO,
    3. Bonci A,
    4. Fields HL
    (2005) Both kappa and mu opioid agonists inhibit glutamatergic input to ventral tegmental area neurons. J Neurophysiol 93:3086–3093, doi:10.1152/jn.00855.2004, pmid:15615834.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Margolis EB,
    2. Lock H,
    3. Hjelmstad GO,
    4. Fields HL
    (2006) The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons? J Physiol 577:907–924, doi:10.1113/jphysiol.2006.117069, pmid:16959856.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Margolis EB,
    2. Fields HL,
    3. Hjelmstad GO,
    4. Mitchell JM
    (2008) δ-Opioid receptor expression in the ventral tegmental area protects against elevated alcohol consumption. J Neurosci 28:12672–12681, doi:10.1523/JNEUROSCI.4569-08.2008, pmid:19036960.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Margolis EB,
    2. Coker AR,
    3. Driscoll JR,
    4. Lemaître AI,
    5. Fields HL
    (2010) Reliability in the identification of midbrain dopamine neurons. PLoS One 5:e15222, doi:10.1371/journal.pone.0015222, pmid:21151605.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Margolis EB,
    2. Toy B,
    3. Himmels P,
    4. Morales M,
    5. Fields HL
    (2012) Identification of rat ventral tegmental area GABAergic neurons. PLoS One 7:e42365, doi:10.1371/journal.pone.0042365, pmid:22860119.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Matsui A,
    2. Williams JT
    (2011) Opioid-sensitive GABA inputs from rostromedial tegmental nucleus synapse onto midbrain dopamine neurons. J Neurosci 31:17729–17735, doi:10.1523/JNEUROSCI.4570-11.2011, pmid:22131433.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Melis M,
    2. Camarini R,
    3. Ungless MA,
    4. Bonci A
    (2002) Long-lasting potentiation of GABAergic synapses in dopamine neurons after a single in vivo ethanol exposure. J Neurosci 22:2074–2082, pmid:11896147.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Merighi S,
    2. Gessi S,
    3. Varani K,
    4. Fazzi D,
    5. Stefanelli A,
    6. Borea PA
    (2013) Morphine mediates a proinflammatory phenotype via μ-opioid receptor-PKCε-Akt-ERK1/2 signaling pathway in activated microglial cells. Biochem Pharmacol 86:487–496, doi:10.1016/j.bcp.2013.05.027, pmid:23796752.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Moaddab M,
    2. Haghparast A,
    3. Hassanpour-Ezatti M
    (2009) Effects of reversible inactivation of the ventral tegmental area on the acquisition and expression of morphine-induced conditioned place preference in the rat. Behav Brain Res 198:466–471, doi:10.1016/j.bbr.2008.11.030, pmid:19073220.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Moussawi K,
    2. Riegel A,
    3. Nair S,
    4. Kalivas PW
    (2011) Extracellular glutamate: functional compartments operate in different concentration ranges. Front Syst Neurosci 5:94, doi:10.3389/fnsys.2011.00094, pmid:22275885.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Nader K,
    2. van der Kooy D
    (1997) Deprivation state switches the neurobiological substrates mediating opiate reward in the ventral tegmental area. J Neurosci 17:383–390, pmid:8987763.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Nair-Roberts RG,
    2. Chatelain-Badie SD,
    3. Benson E,
    4. White-Cooper H,
    5. Bolam JP,
    6. Ungless MA
    (2008) Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat. Neuroscience 152:1024–1031, doi:10.1016/j.neuroscience.2008.01.046, pmid:18355970.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Noda M,
    2. Nakanishi H,
    3. Akaike N
    (1999) Glutamate release from microglia via glutamate transporter is enhanced by amyloid-beta peptide. Neuroscience 92:1465–1474, doi:10.1016/S0306-4522(99)00036-6, pmid:10426500.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Olmstead MC,
    2. Franklin KB
    (1997) The development of a conditioned place preference to morphine: effects of microinjections into various CNS sites. Behav Neurosci 111:1324–1334, doi:10.1037/0735-7044.111.6.1324, pmid:9438801.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Omelchenko N,
    2. Sesack SR
    (2009) Ultrastructural analysis of local collaterals of rat ventral tegmental area neurons: GABA phenotype and synapses onto dopamine and GABA cells. Synapse 63:895–906, doi:10.1002/syn.20668, pmid:19582784.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Onali P,
    2. Olianas MC
    (1991) Naturally occurring opioid receptor agonists stimulate adenylate cyclase activity in rat olfactory bulb. Mol Pharmacol 39:436–441, pmid:1673223.
    OpenUrlAbstract
  49. ↵
    1. Patrick GA,
    2. Dewey WL,
    3. Spaulding TC,
    4. Harris LS
    (1975) Relationship of brain morphine levels to analgesic activity in acutely treated mice and rats and in pellet implanted mice. J Pharmacol Exp Ther 193:876–883, pmid:1151736.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Puri SK,
    2. Cochin J,
    3. Volicer L
    (1975) Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum. Life Sci 16:759–767, doi:10.1016/0024-3205(75)90352-5, pmid:164598.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Ruzicka BB,
    2. Fox CA,
    3. Thompson RC,
    4. Meng F,
    5. Watson SJ,
    6. Akil H
    (1995) Primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mRNA. Brain Res Mol Brain Res 34:209–220, doi:10.1016/0169-328X(95)00165-O, pmid:8750824.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Shippenberg TS,
    2. Herz A
    (1987) Place preference conditioning reveals the involvement of D1-dopamine receptors in the motivational properties of mu- and kappa-opioid agonists. Brain Res 436:169–172, doi:10.1016/0006-8993(87)91571-X, pmid:2961413.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Shippenberg TS,
    2. Bals-Kubik R,
    3. Herz A
    (1993) Examination of the neurochemical substrates mediating the motivational effects of opioids: role of the mesolimbic dopamine system and D-1 vs. D-2 dopamine receptors. J Pharmacol Exp Ther 265:53–59, pmid:8386244.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    1. Steffensen SC,
    2. Stobbs SH,
    3. Colago EE,
    4. Lee RS,
    5. Koob GF,
    6. Gallegos RA,
    7. Henriksen SJ
    (2006) Contingent and non-contingent effects of heroin on mu-opioid receptor-containing ventral tegmental area GABA neurons. Exp Neurol 202:139–151, doi:10.1016/j.expneurol.2006.05.023, pmid:16814775.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Steinberg EE,
    2. Keiflin R,
    3. Boivin JR,
    4. Witten IB,
    5. Deisseroth K,
    6. Janak PH
    (2013) A causal link between prediction errors, dopamine neurons and learning. Nat Neurosci 16:966–973, doi:10.1038/nn.3413, pmid:23708143.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Theile JW,
    2. Morikawa H,
    3. Gonzales RA,
    4. Morrisett RA
    (2008) Ethanol enhances GABAergic transmission onto dopamine neurons in the ventral tegmental area of the rat. Alcohol Clin Exp Res 32:1040–1048, doi:10.1111/j.1530-0277.2008.00665.x, pmid:18422836.
    OpenUrlCrossRefPubMed
  57. ↵
    1. Tsai HC,
    2. Zhang F,
    3. Adamantidis A,
    4. Stuber GD,
    5. Bonci A,
    6. de Lecea L,
    7. Deisseroth K
    (2009) Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324:1080–1084, doi:10.1126/science.1168878, pmid:19389999.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. van Zessen R,
    2. Phillips JL,
    3. Budygin EA,
    4. Stuber GD
    (2012) Activation of VTA GABA neurons disrupts reward consumption. Neuron 73:1184–1194, doi:10.1016/j.neuron.2012.02.016, pmid:22445345.
    OpenUrlCrossRefPubMed
  59. ↵
    1. Velásquez-Martinez MC,
    2. Vázquez-Torres R,
    3. Jiménez-Rivera CA
    (2012) Activation of alpha1-adrenoceptors enhances glutamate release onto ventral tegmental area dopamine cells. Neuroscience 216:18–30, doi:10.1016/j.neuroscience.2012.03.056, pmid:22542873.
    OpenUrlCrossRefPubMed
  60. ↵
    1. Wang Q,
    2. Liu-Chen LY,
    3. Traynor JR
    (2009) Differential modulation of mu- and delta-opioid receptor agonists by endogenous RGS4 protein in SH-SY5Y cells. J Biol Chem 284:18357–18367, doi:10.1074/jbc.M109.015453, pmid:19416973.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    1. Witten IB,
    2. Steinberg EE,
    3. Lee SY,
    4. Davidson TJ,
    5. Zalocusky KA,
    6. Brodsky M,
    7. Yizhar O,
    8. Cho SL,
    9. Gong S,
    10. Ramakrishnan C,
    11. Stuber GD,
    12. Tye KM,
    13. Janak PH,
    14. Deisseroth K
    (2011) Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement. Neuron 72:721–733, doi:10.1016/j.neuron.2011.10.028, pmid:22153370.
    OpenUrlCrossRefPubMed
  62. ↵
    1. Wong CS,
    2. Su YF,
    3. Watkins WD,
    4. Chang KJ
    (1994) Opioid agonist binding affinity is increased by magnesium in the presence of guanosine diphosphate but decreased by magnesium in the presence of guanyl-5′-yl imidodiphosphate. J Pharmacol Exp Ther 268:653–661, pmid:8113975.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Xia Y,
    2. Driscoll JR,
    3. Wilbrecht L,
    4. Margolis EB,
    5. Fields HL,
    6. Hjelmstad GO
    (2011) Nucleus accumbens medium spiny neurons target non-dopaminergic neurons in the ventral tegmental area. J Neurosci 31:7811–7816, doi:10.1523/JNEUROSCI.1504-11.2011, pmid:21613494.
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Xiao C,
    2. Ye JH
    (2008) Ethanol dually modulates GABAergic synaptic transmission onto dopaminergic neurons in ventral tegmental area: role of mu-opioid receptors. Neuroscience 153:240–248, doi:10.1016/j.neuroscience.2008.01.040, pmid:18343590.
    OpenUrlCrossRefPubMed
  65. ↵
    1. Zhang Y,
    2. Landthaler M,
    3. Schlussman SD,
    4. Yuferov V,
    5. Ho A,
    6. Tuschl T,
    7. Kreek MJ
    (2009) Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin. Neuroscience 158:474–483, doi:10.1016/j.neuroscience.2008.09.039, pmid:18938225.
    OpenUrlCrossRefPubMed
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Direct Bidirectional μ-Opioid Control of Midbrain Dopamine Neurons
Elyssa B. Margolis, Gregory O. Hjelmstad, Wakako Fujita, Howard L. Fields
Journal of Neuroscience 29 October 2014, 34 (44) 14707-14716; DOI: 10.1523/JNEUROSCI.2144-14.2014

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Direct Bidirectional μ-Opioid Control of Midbrain Dopamine Neurons
Elyssa B. Margolis, Gregory O. Hjelmstad, Wakako Fujita, Howard L. Fields
Journal of Neuroscience 29 October 2014, 34 (44) 14707-14716; DOI: 10.1523/JNEUROSCI.2144-14.2014
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  • A Direct Excitatory Postsynaptic Action of Mu Opioid Agonists
    Howard Fields, Elyssa Margolis and Gregory O. Hjelmstad
    Published on: 13 March 2015
  • Direct excitatory action of opioids?
    John T Williams
    Published on: 25 February 2015
  • Published on: (13 March 2015)
    Page navigation anchor for A Direct Excitatory Postsynaptic Action of Mu Opioid Agonists
    A Direct Excitatory Postsynaptic Action of Mu Opioid Agonists
    • Howard Fields, Professor, UCSF
    • Other Contributors:
      • Elyssa Margolis
      • Gregory O. Hjelmstad
    In response to our report that mu opioids can directly excite a subset of ventral tegmental area (VTA) neurons, John Williams and colleagues posted a comment citing numerous studies in a variety of brain regions that did not report a direct excitatory action of mu opioid agonists. They conclude; "The work of Margolis et al. will need to be independently validated and the mechanism critically explored before the notion of...
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    In response to our report that mu opioids can directly excite a subset of ventral tegmental area (VTA) neurons, John Williams and colleagues posted a comment citing numerous studies in a variety of brain regions that did not report a direct excitatory action of mu opioid agonists. They conclude; "The work of Margolis et al. will need to be independently validated and the mechanism critically explored before the notion of a direct opioid receptor dependent depolarization can be incorporated into our general understanding of opioid actions on single neurons." We agree that validation of novel and important observations is essential for scientific progress, however, we also believe the evidence presented in our paper to document the phenomenon and to explain its mechanism is sufficient to permit confidence that the observation is correct.

    Importantly, Iegorova et al. (2010) reported a similar direct excitatory action in cerebellar Purkinje neurons; i.e. activation of a P-type (Cav2.1) Ca2+ channel at low nanomolar concentrations of DAMGO. In contrast to our result, this action was not G-protein mediated, however, it was a direct excitation.

    The critical test of an observation that is inconsistent with extant literature is failure to replicate the data when the experiment is performed as described. Our reading of the methods of the quoted literature indicates to us that there were significant differences in the recording methods and the locations of the neurons studied. We remain confident that if looked for in this neuronal population using our sampling and recording methods, our finding will be replicated.

    The Williams et al. comment also raises some other issues as potential reasons to question the validity of our observation.

    First is the small size and variable duration of the depolarizations. This might be a reason why the excitatory effect was not previously observed, however, small depolarizations can have large effects on firing frequency if a neuron's membrane potential is close to the action potential threshold. Figure 1 shows an example of a significant increase in firing rate, furthermore, in 54 quiescent neurons, the mean depolarization was approximately 5 mV, and specifically 4.2 +/- 1.2 mV for TH(+) neurons (n=12, Table 1). We also displayed the range of effects in both firing and quiescent neurons in Figure 3. While the duration of the effect was variable, as stated in our paper, neurons showing a depolarization exhibited hyperpolarizations with a second application of DAMGO with bath applied CdCl2 or omega-agatoxin indicating that in most cells DAMGO elicited a variable balance of excitations and inhibitions. This could account for the variability in duration of the effect in an individual neuron.

    Second, the extreme sensitivity of the neurons to DAMGO (i.e. low nanomolar) is indicated as a reason to doubt the effect. Our view is the opposite: in addition to reversal by a selective antagonist, a lower required concentration for an effect increases confidence that it is receptor selective. The authors state "It is only in experiments carried out in cell lines that over express receptors that the EC50 of DAMGO approaches concentrations that range from 0.5-30 nM, which is surely attributable to overexpression of receptors (Table 2)." This statement is at odds not only with what we (and Iegorova et al., 2010) report, but we refer Williams to his own paper (Osborne and Williams, 1995). In that paper he reports that the EC-50 for a DAMGO induced postsynaptic outward current is about 20 nM in locus coeruleus neurons. Importantly, in that thorough paper the [Met5] enkephalin EC-50 is at least an order of magnitude greater than that for DAMGO, illustrating the well-known point that different ligands for the same receptor can have markedly different potency.

    Third, objections are raised to the possibility that an increase in calcium conductance could cause a significant depolarization especially since opioids are known to inhibit calcium conductance in other neurons. This objection is at odds with reports documenting large soma-dendritic depolarizations mediated by p-type Calcium channels (see for example Cavelier et al 2002). We propose that the mu opioid ligand has lowered the activation threshold for the Cav2.1 Ca2+ channel. The Cav2.1 Ca2+ channel blocker omega-agatoxin IVA is highly selective and it blocked the mu opioid excitation in our preparation and in cerebellar Purkinje cells (Iegorova et al., 2010).

    Of the papers cited as failing to report a direct postsynaptic excitatory effect several are of relevance to this controversy. Fourteen papers are cited as studying substantia nigra/VTA mu opioid actions. Of these, five were ex vivo studies in rat and so are directly relevant. It is important to point out that the 1992 Johnson and North paper (Johnson and North, 1992) DID show excitations of VTA dopamine neurons by bath applied DAMGO (1 uM, Figure 4). They concluded that the dopamine neuron excitation was due to disinhibition via inhibition of GABA release, but did not show that the excitation is blocked by GABAA receptor (GABAAR) blockade. The excitation we observed was preserved in the presence of picrotoxin. Johnson and North did not report whether the excitation they observed in dopamine neurons was associated with a conductance change. Therefore, though disinhibition is certainly a possibility the actual mechanism of the excitatory effect they observed is uncertain. Manzoni & Williams (1999) reported no change in holding current in dopamine neurons when DAMGO was applied. This is surprising in view of other reports from the Williams laboratory in guinea pig (Cameron et al., 1997) and mouse (Ford et al., 2006) that opioids activate a GIRK in a subset of midbrain dopamine neurons. That said, the Manzoni and Williams paper was studying presynaptic actions of mu opioids and they were not specifically looking for postsynaptic actions.

    Another critical issue is that with whole cell recording the internal solution of the recording electrode changes the intracellular milieu. For example, use of high concentrations of Calcium buffers (e.g. 10 mM BAPTA), will reduce the effect of Ca2+ currents on membrane potential. Another variable is the Cl- concentration in the internal solution of the recording electrode. For example, the two Matsui papers (Matsui and Williams, 2011; Matsui et al., 2014) were primarily investigations of presynaptic actions but there is no mention of holding current; in these papers, high Cl- internal solutions were used to improve the resolution of GABAAR mediated currents, and it has been reported that high internal Cl- can interfere with other postsynaptic currents, such as GIRKs (Lenz et al., 1997; Lopantsev and Schwartzkroin, 1999).

    Not mentioned or quoted is a study by Lacey, Mercuri, and North (Lacey et al., 1987) of substantia nigra pars compacta (SNc) neurons, in which they reported that dopamine neurons were insensitive to mu opioid agonists. However, Lacey et al. (1987) show that putative GABA neurons of the SNc are hyperpolarized by DAMGO, and the effect of 1 uM DAMGO washed out in 0.5 - 2 min, consistent with our observations in the VTA. In fact, both DAMGO induced excitatory and inhibitory effects washed out quickly according Johnson and North's seminal VTA paper as well (1992). Since the boundary between VTA and SNc can be difficult to discern, one of the reasons many people may have failed to see the excitations we observed is that they were unintentionally recording in the SNc. Unfortunately, none of the SNc/VTA studies of opioid action reported the locations of their recorded neurons. The locations of neurons in our studies were plotted (Figure 4) and were distributed throughout the rostrocaudal and medio-lateral extent of the VTA.

    In summary, we believe our evidence supporting a direct postsynaptic excitatory effect of DAMGO is substantial, that it likely depends on both G protein signaling and activation of a Cav2.1 Ca2+ channel. We suggest that success in replication of our findings will benefit from close attention to experimental variables such as the internal solution of the recording electrodes in whole cell studies and precise location of the neurons studied. The use of less invasive approaches such as cell attached recording and calcium imaging might also be informative.


    Cameron DL, Wessendorf MW, Williams JT (1997) A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids. Neuroscience 77:155-166.

    Ford CP, Mark GP, Williams JT (2006) Properties and opioid inhibition of mesolimbic dopamine neurons vary according to target location. J Neurosci 26:2788-2797.

    Cavelier P, Pouille F, Desplantez T, Beekenkamp H, Bossu JL. (2002) Control of the propagation of dendritic low-threshold Ca(2+) spikes in Purkinje cells from rat cerebellar slice cultures. J Physiol. ;540(Pt 1):57-72.

    Hjelmstad GO, Xia Y, Margolis EB, Fields HL (2013) Opioid modulation of ventral pallidal afferents to ventral tegmental area neurons. J Neurosci 33:6454-6459.

    Iegorova O, Fisyunov A, Krishtal O (2010) G-protein-independent modulation of P-type calcium channels by mu-opioids in Purkinje neurons of rat. Neurosci Lett 480:106-111.

    Johnson SW, North RA (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483-488.

    Lacey MG, Mercuri NB, North RA (1987) Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J Physiol 392:397-416.

    Lenz RA, Pitler TA, Alger BE (1997) High intracellular Cl- concentrations depress G-protein-modulated ionic conductances. J Neurosci 17:6133-6141.

    Lopantsev V, Schwartzkroin PA (1999) GABAA-Dependent chloride influx modulates GABAB-mediated IPSPs in hippocampal pyramidal cells. J Neurophysiol 82:1218-1223.

    Matsui A, Williams JT (2011) Opioid-sensitive GABA inputs from rostromedial tegmental nucleus synapse onto midbrain dopamine neurons. J Neurosci 31:17729-17735.

    Matsui A, Jarvie BC, Robinson BG, Hentges ST, Williams JT (2014) Separate GABA afferents to dopamine neurons mediate acute action of opioids, development of tolerance, and expression of withdrawal. Neuron 82:1346-1356.

    Osborne PB, Williams JT (1995) Characterization of acute homologous desensitization of mu-opioid receptor-induced currents in locus coeruleus neurones. Br J Pharmacol 115:925-932.
     

    Conflict of Interest:

    None declared

    Show Less
    Competing Interests: None declared.
  • Published on: (25 February 2015)
    Page navigation anchor for Direct excitatory action of opioids?
    Direct excitatory action of opioids?
    • John T Williams, Senior Scientist
    • Other Contributors:
      • Veronica Alvarez, Elaina Bagley, Christopher Bailey, Billy Chieng, MacDonald J Christie, Vu Dang, Christopher Ford, Graeme Henderson, Shane Hentges, Susan Ingram, Carl Lupica, Olivier Manzoni, Carlos Paladini, Christopher Vaughn

    The recent article from Margolis et al (Direct bidirectional mu-opioid control of midbrain dopamine neurons, J Neurosci 34:14707-14716) presented work suggesting that opioids can directly depolarize neurons in the ventral tegmental area in brain slices from rat. Beginning in the late 1970s, many studies have shown that opioids inhibit neurons through postsynaptic mechanisms but can increase excitability through disinhibito...

    Show More

    The recent article from Margolis et al (Direct bidirectional mu-opioid control of midbrain dopamine neurons, J Neurosci 34:14707-14716) presented work suggesting that opioids can directly depolarize neurons in the ventral tegmental area in brain slices from rat. Beginning in the late 1970s, many studies have shown that opioids inhibit neurons through postsynaptic mechanisms but can increase excitability through disinhibiton (inhibition of synaptic inhibition). This article posits a direct action that has not been reported previously. The question is how could such an action be missed for so many years? This question was asked of people in the field that have extensive experience with the study of opioid receptor dependent processes and the response is summarized here.

    Preparations where opioids have been studied on single neurons vary widely as do the species in which the experiments were carried out (Table 1 is not comprehensive but with a few exceptions lists papers in which the authors were directly contacted). Species include rat, mouse and guinea pig. Neurons in brain slices of the locus coeruleus (LC), ventral midbrain (substantia nigra and VTA), hippocampus, raphe magnus, rostro-medial tegmental area (RMTg), spinal trigeminal nucleus, spinal dorsal horn, periaqueductal gray, parabrachial nucleus, hypothalamic neurons that include the pro-opiomelanocortin (POMC) neurons, hippocampus, rostral ventral medulla and central nucleus of the amygdala have all been studied over many years.

    These studies were carried out with a variety of methods that include field potential recordings, intracellular recordings with sharp electrodes and whole cell recordings using a number of different internal solutions. A number of opioid agonists were examined over a wide range of concentrations. In none of these studies involving thousands of recordings was an excitatory action of opioids reported or even observed in passing.

    There are several aspects of the study by Margolis, et al. that require further confirmation. First, both the size of the depolarization was small and in some cases declined rapidly in the presence of DAMGO. In other cases the action washed out of the slice quickly. Such a high affinity drug is not expected to washout of a brain slice as quickly as illustrated in many figures.

    Second, the extreme sensitivity of the neurons to the mu opioid agonist, DAMGO (EC50, 1-3 nM) presented in this paper stands alone. Reports of the EC50 of DAMGO in multiple neuronal preparations range from 50-300 nM. It is only in experiments carried out in cell lines that over express receptors that the EC50 of DAMGO approaches concentrations that range from 0.5-30 nM, which is surely attributable to overexpression of receptors (Table 2). There are also technical issues that surround the measurement of responses to very low concentrations of agonists. The time course of equilibration of low drug concentrations is considerable and requires very long-lasting, stable recordings, particularly when measuring small responses. To reach a steady state to a concentration of DAMGO that is less than 10 nM requires many minutes and a reliable measure can only be obtained with little or no drift in baseline current or firing rate. The baseline shown in several of the illustrations within the Margolis paper are distinctly unsteady. In addition, there were also no illustrations of experiments using a concentration of DAMGO below 500 nM.

    Third, the inhibition of the DAMGO-dependent inward current/depolarization in the presence of calcium channel toxins also stands alone. It is difficult to account for depolarization (with a significant conductance increase) solely through an increase in calcium conductance. This mechanism is particularly difficult to understand given the well-established opioid receptor mediated inhibition of voltage dependent calcium conductance.

    A wise man once said ???when you are reporting something that goes against dogma, the data has to be better than anything else on the subject.??? The work of Margolis et al. will need to be independently validated and the mechanism critically explored before the notion of a direct opioid receptor dependent depolarization can be incorporated into our general understanding of opioid actions on single neurons.

    References

    Alvarez VA, Arttamangkul S, Dan V, Salem A, Whistler J, von Zastrow M, Grandy D, Williams JT (2002) mu-opioid receptors: ligand dependent activation of potassium conductance, desensitization and internalization. J Neurosci 22:5769-5776.

    Arttamangkul S, TorrecillaM, KobayashiK, Okano H, Williams JT (2006) Separation of mu opioid receptor desensitization and internalization: Endogenous receptors in primary neuronal cultures. J Neurosci 26:4118-4125.

    Arttamangkul S, Quillinan N, Low M, von Zastrow M, Pintar J, Williams JT (2008) Differential Activation and Trafficking of Mu-opioid Receptors in Brain Slices. Mol. Pharm. 74:972-979.

    Arttamangkul S, Lau EK, Lu H-W, Williams JT (2011) Desensitization and trafficking of mu-opioid receptors in locus coeruleus neurons: Modulation by kinases. Mol Pharm 81:348-355.

    Bagley EE, Chieng BC, Christie MJ, Connor M (2005a) >Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine.Br J Pharmacol. 146:68-76.

    Bagley, EE, Gerke, MB, Vaughan, CW, Hack, SP, Christie, MJ (2005b). GABA transporter currents activated by protein kinase A excite midbrain neurons during opioid withdrawal. Neuron 45:433-45.

    BaileyCP, Couch D, Johnson E, Griffiths K, Kelly E, Henderson G (2003) Mu-opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine.J Neurosci 23:10515-10520.

    BaileyCP, Kelly E, Henderson G.(2004) >Protein kinase C activation enhances morphine-induced rapid desensitization of mu-opioid receptors in mature rat locus ceruleus neurons.Mol Pharmacol 66:1592-1598.

    BaileyCP, Llorente J, Gabra BH, Smith FL, Dewey WL, Kelly E, Henderson G.(2009a) >Role of protein kinase C and mu-opioid receptor (MOPr) desensitization in tolerance to morphine in rat locus coeruleus neurons.Eur J Neurosci. 29:307-318.

    BaileyCP, Oldfield S, Llorente J, Caunt CJ, Teschemacher AG, Roberts L, McArdle CA, Smith FL, Dewey WL, Kelly E, Henderson G. (2009b)>Involvement of PKC alpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons.Br J Pharmacol. 158:157-164

    Banghart MR, Sabatini BL. (2012)>Photoactivatable neuropeptides for spatiotemporally precise delivery of opioids in neural tissue.Neuron 73:249-259.

    Banghart MR, Williams JT, Shah RC, Lavis LD, Sabatini BL (2013) Caged naloxone reveals opioid signaling deactivation dynamics. Mol Pharm 84:687-695.

    Blanchet C, Sollini M, L??scher C. (2003) >Two distinct forms of desensitization of G-protein coupled inwardly rectifying potassium currents evoked by alkaloid and peptide mu-opioid receptor agonists.Mol Cell Neurosci 24:517-523

    Bonci A, Williams JT (1997) Increased probability of GABA release during withdrawal from morphine. J. Neurosci. 17:796-803.

    Borgland SL, Connor M, Osborne PB, Furness JB, Christie MJ (2003) Opioid agonists have different efficacy profiles for G protein activation, rapid desensitization, and endocytosis of mu-opioid receptors. J Biol Chem 278:18776-18784.

    Brundege JM, Williams JT (2002) Differential modulation of nucleus accumbens synapses. J. Neurophysiol. 88:142-151.

    >Charpak S(1998) mu-Opioid peptides inhibit thalamic neurons.J Neurosci. 18:1671-1678.

    Cameron DL, Wessendorf MW, Williams JT (1997) A subset of VTA neurons are inhibited by dopamine, 5-HT and opioids. Neuroscience. 77:155-166.

    >ChiengB, Christie MJ (1994) Hyperpolarization by opioids acting on mu-receptors of a sub-population of rat periaqueductal gray neurones in vitro.Br J Pharmacol. 113:121-128.

    Chieng BC, Christie MJ, Osborne PB (2006) Characterization of neurons in the rat central nucleus of the amygdala: cellular physiology, morphology and opioid sensitivity. J Comp Neurol 497:910-927.

    Chieng B, Azriel Y, Mohammadi S, Christie MJ. (2011) >Distinct cellular properties of identified dopaminergic and GABAergic neurons in the mouse ventral tegmental area.J Physiol 589:3775-3787.

    Chiou LC, Huang LY (1999) Mechanism underlying increased neuronal activity in the rat ventrolateral periqueductal grey by a mu-opioid. J Physiol 518:551-559.

    Christie MJ, Williams JT, North RA (1987) Cellular mechanisms of opioid tolerance: studies in single brain neurons. Mol Pharmacol 32:633-638.

    Christie, M.J. and R.A. North (1988) Agonists at mu-opioid, M2-muscarinic and GABA-B-receptors increase the same potassium conductance in rat lateral parabrachial neurons. Brit. J. Pharmacol 95:896-902.

    ClarkMJ, Traynor JR (2006) >Mediation of adenylyl cyclase sensitization by PTX-insensitive GalphaoA, Galphai1, Galphai2 or Galphai3.J Neurochem 99:1494-504.

    >Henderson G(1996) delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells.Br J Pharmacol 117:333-340.

    Christie MJ, Williams JT, North RA (1987) Cellular mechanisms of opioid tolerance: studies in single brain neurons. Mol Pharmacol 32:633-638.

    Dacher M, Nugent FS (2011) Morphine-induced modulation of LTD at GABAergic synapses in the ventral tegmental area. Neuropharmacology 61:1166-1171.

    Dang VC. Williams JT (2005) Morphine-induced mu-opioid receptor desensitization. Mol Pharm 68:1127-1132.

    Fiorillo CD, Williams JT (1996) Opioid desensitization: interactions with G protein-coupled receptors in the locus coeruleus. J Neurosci 15:1479-1485.

    Ford CP, Mark GP, Williams JT (2006) Properties and opioid inhibition of mesolimbic dopamine neurons vary according to target location. J Neurosci 26:2788-2797.

    Ford CP, Beckstead MJ, Williams JT (2007) Kappa opioid inhibition of somatodendritic dopamine inhibitory postsynaptic currents. J Neurophysiol 97:883-891.

    >Ingram SL(2010)Tolerance to the antinociceptive effect of morphine in the absence of short-term presynaptic desensitization in rat periaqueductal gray neurons.J Pharmacol Exp Ther 335:674-680.

    Glickfeld LL, Atallah BV, Scanziani M (2008) Complementary modulation of somatic inhibition by opioids and cannabinoids. J Neurosci 28:1824-1832.

    Grudt TJ, Williams JT (1994) mu-opioid agonists inhibit spinal trigeminal substantia gelatinosa neurons in guinea pig and rat. J Neurosci 14:1646-1654.

    Grudt TJ, Henderson G (1998) Glycine and GABA-A receptor-mediated synaptic transmission in rat substantia gelatinosa: inhibition by?? mu-opioid and GABA-B agonists. J Physiol 507:473-483.

    >J Neurosci11:2574-2581.

    Heinricher MM, Morgan MM, Tortorici V, Fields HL (1994) Disinhibition of off-cells and antinociception produced by an opioid action within the rostral ventromedial medulla. Neuroscience 63:279-288.

    Hjelmstad GO, Xia Y, Margolis EB, Fields HL. (2013) >Opioidmodulation of ventral pallidal afferents to ventral tegmental area neurons.J Neurosci 33:6454-6459.

    Ingram S, Wilding TJ, McCleskey EW, Williams JT (1997) Efficacy and kinetics of opioid action on acutely dissociated neurons. Mol Pharm 52:136-143.

    Ingram SL, Vaughan CW, Bagley EE, Connor M, Christie MJ (1998) Enhanced opioid efficacy in opioid dependence is caused by an altered signal transduction pathway. J Neurosci 18:10269-10276.

    Jalabert M, Bourdy R, Courtin J, Veinante P, Manzoni OJ, Barrot M, Georges F. (2011)>Neuronal circuits underlying acute morphine action on dopamine neurons.Proc Natl Acad Sci U S A 108:16446-16450.

    Johnson SW, North RA (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483-488.

    Johnson EE, Chieng B, Napier I, Connor M (2006) >Decreased mu-opioid receptor signaling and a reduction in calcium current density in sensory neurons from chronically morphine-treated mice.Br J Pharmacol 148:947-955.

    Kelly MJ, Loose MD, Ronnekleiv OK (1990) Opioids hyperpolarize beta-endorphin neurons via mu-receptor activation of a potassium concuctance. Neuroendocrinology 62:268-275.

    Knapman A, Abogadie F, McIntrye P, Connor M (2014) >A real-time, fluorescence-based assay for measuring mu-opioid receptor modulation of adenylyl cyclase activity in Chinese hamster ovary cells.J Biomol Screen 19:223-231.

    Koyama S, Akaike N. (2008) >Activation of mu-opioid receptor selectively potentiates NMDA-induced outward currents in rat locus coeruleus neurons.Neurosci Res 60:22-28.

    Lecca S, Melis M, Luchicchi A, Muntoni AL, Pistis M. (2012) Inhibitory inputs from rostromedial tegmental neurons regulate spontaneous activity of midbrain dopamine cells and their responses to drugs of abuse.Neuropsychopharmacology. 37:1164-7116.

    Llorente J, Lowe JD, Sanderson HS, Tsisanova E, Kelly E, Henderson G, Bailey CP. (2012) >mu-Opioid receptor desensitization: homologous or heterologous?Eur J Neurosci 36:3636-3642

    Levitt ES, Purington LC, Traynor JR (2011)>Gi/o-coupled receptors compete for signaling to adenylyl cyclase in SH-SY5Y cells and reduce opioid-mediated cAMP overshoot.Mol Pharmacol 79:461-471.

    Levitt ES, Williams JT Morphine desensitization and cellular tolerance are distinguished in rat locus coeruleus neurons. (2012) Mol Pharmacol 82:983-992.

    Lowe JD, Bailey CP (2015) Functional selectivity and time-dependence of mu-opioid receptor desensitization at nerve terminals in the mouse ventral tegmental area. Br J Pharmacol 172:469-481.

    Madhavan A, Bonci A, Whistler JL. (2010) >Opioid-Induced GABA potentiation after chronic morphine attenuates the rewarding effects of opioids in the ventral tegmental area.J Neurosci 30:14029-14035.

    Manzoni O, Williams JT (1999) Presynaptic regulation of glutamate release in the ventral tegmental area during morphine withdrawal J. Neurosci. 19:6629-6636.

    Matsui A, Williams JT (2010) Activation of mu-opioid receptors and block of K-ir3 potassium channels and NMDA receptor conductance by l- and d-methadone in rat locus coeruleus. Brit J Pharm 161:1403-1413.

    Matsui A, Williams JT (2011) Opioid-sensitive GABA inputs from Rostromedial Tegmental Nucleus synapse onto midbrain dopamine neurons. J Neurosci 31:17729-17735.

    Matsui A, Jarvie BC, Robinson BG, Hentges ST, Williams JT (2014) Separate GABA afferents to dopamine neurons mediate acute actions of opioid, development of tolerance and expression of withdrawal. Neuron 82:1346-1356.

    Nicoll RA, Alger BE, Jahr CE (1980) Enkephalin blocks inhibitory pathways in the vertebrate CNS. Nature 287:22-25.

    North RA, Williams JT (1985) On the potassium conductance increased by opioids in rat locus coeruleus neurones. J Physiol 364:265-280.

    Osborne PB, Williams JT (1995) Characterization of acute homologous desensitization of mu-opioid receptor-induced current in locus coeruleus neurones. Brit J Pharmacol 115:925-932.

    Osborne PB, Williams JT (1996) Forskolin enhancement of opioid currents in rat locus coeruleus neurons. J Neurophysiol 76:1559-1565.

    Osborne PB, Vaughan CW, Wilson HI, Christie MJ. (1996) Opioid inhibition of rat periaqueductal grey neurones with identified projections to rostral ventromedial medulla in vitro. J Physiol 490:383-389.

    Pan ZZ, Williams JT, Osborne P (1990) Opioid actions on single nucleus raphe magnus neurons from rat and guinea pig in vitro. J Physiol 427:519-532.

    Pan Y-Z, Li D-P, Chen S-R, Pan H-L (2004) Activation of ??-opioid receptor excites a population of locus coeruleus-spinal neurons through presynaptic disinhibition Brain Res 997:67-78.

    Pedersen NP, Vaughan CW, Christie MJ. (2011) >Opioid receptor modulation of GABAergic and serotonergic spinally projecting neurons of the rostral ventromedial medulla in mice.J Neurophysiol 106:731-740.

    Pennock RL, Hentges ST (2011) Differential expression and sensitivity of presynaptic and postsynaptic opioid receptors regulating hypothalamic proopiomelanocortin neurons. J Neurosci 31:281-288.

    Pepper CM, Henderson G (1980) Opiates and opioid peptides hyperpolarize locus coeruleus neurons in vitro. Science 209:394-395.

    Rogers H, Henderson G (1990) Activation of mu- and delta-opioid receptor present on the same nerve terminals depresses transmitter release in the mouse hypogastric ganglion Brit J Pharmacol 101:505-512.

    Seward E, Hammond C, Henderson G (1991) Mu-opioid-receptor-mediated inhibition of the N-type calcium-channel current. Proc Biol Sci 244:129-135.

    Shoji Y, Delfs J, Williams JT (1999) Presynaptic inhibition of GABA-B-mediated synaptic potentials in the VTA during morphine withdrawal. J Neurosci 19:2347-2355.

    Svoboda KR, Lupica CR (1998) Opioid inhibition of hippocampal interneurons via modulation of potassium and hyperpolarization-activated cation (Ih) currents. J Neurosci 18:7084-7098.

    Torrecilla M, Marker CL, Cintora SC, Stoffel M, Williams JT, Wickman K (2002) G protein-gated potassium channels containing GIRK2 and GIRK3 subunits mediate the acute opioid inhibition of locus coeruleus neurons. J Neurosci 22:4328-4334.

    Torrecilla, M, Quillian N, Williams JT, Wickman K (2008) Pre- and postsynaptic regulation of locus coeruleus neurons after chronic morphine treatment: a study of GIRK knockout mice. E J Neurosci 28:618-624.

    Travagli RA, Dunwiddie TV, Williams JT (1995) Opioid inhibition in locus coeruleus.?? J Neurophysiol 74:519-528.

    Travagli RA, Wessendorf M, Williams JT (1996) The dendritic arbor of locus coeruleus neurons contributes to opioid inhibition. J Neurophysiol 75:2029-2035.

    Vaughan CW, Ingram SL, Connor MA, Christie MJ (1997) How opioids inhibit GABA-mediated neurotransmission. Nature?? 390:611-614.

    Vaughan, CW, Connor, M, Bagley, EE, Christie, MJ (2000). Actions of cannabinoids on membrane properties and synaptic transmission in rat periaqueductal gray neurons in vitro.Mol Pharmacol 57:288-95

    Virk MS, Williams JT (2008) Agonist-Specific Regulation of mu-Opioid Receptor Desensitization and Recovery from Desensitization Mol Pharm 73: 1301-1308.

    Virk MS, Arttamangkul S, Birdsong WT, Williams JT (2009) Buprenorphine is a weak partial agonist that inhibits opioid receptor desensitization. J Neurosci 29:7341-7348.

    Walwyn W, John S, Maga M, Evans CJ, Hales TG (2009) Delta receptors are required for full inhibitory coupling of mu-receptors to voltage-dependent Ca(2+) channels in dorsal root ganglion neurons. Mol Pharmacol 76:134-143.

    Williams JT, Egan TM, North RA (1982) Enkephalin opens potassium channels in mammalian central neurones. Nature 299:74-77.

    Williams JT, North RA (1984) Opiate-receptor interactions on single locus coeruleus neurones. Mol Pharm 26:489-497.

    Williams JT, Christie MJ, North RA (1987) Potentiation of enkephalin action by peptidase inhibitors in rat locus coeruleus in?? vitro. J Pharmacol Expt Ther 243:397-401.

    Williams JT, North RA, Tokimasa T (1988) Inward rectification of resting and opioid-activated potassium currents in rat locus coeruleus neurons. J Neurosci 8:4299-4306.

    Williams JT (2014) Desensitization of functional mu-opioid receptors increases agonist off-rate. Mol Pharmacol 86:52-61.

    Wuarin JP, Dudek FE (1990) Direct effects of an opioid peptide selective for mu-receptors: Intracellular recordings in the paraventricular and supraoptic nuclei of the guinea-pig. Neuroscience 36:291-298.

    Yoshimura M, North RA (1983) Substantia gelatinosa neurones hyperpolarized in vitro by enkephalin. Nature 305:529-530.

    Zieglgansberger W, French ED, Siggins GR, Bloom FE (1979) Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons. Science 205:415-417.

    ??

    Table 1
    Locus Coeruleus
    Guinea Pig Intracellular Pepper, Henderson 1979
    Rat Intracellular Williams, Egan, North 1982
    Williams, North 1984
    North, Williams 1985
    Williams, Christie, North 1987
    Christie, Williams, North 1987
    Williams, North, Tokimasa 1988
    Harris, Williams 1991
    Oleskevich, Clements, Williams 1993
    Osborne, Williams 1995
    Travagli, Dunwiddie, Williams 1995
    Fiorillo, Williams, 1996
    Whole cell Travagli, Wessendorf, Williams 1996
    Osborne, Williams 1996
    Ingram, Wilding, McCleskey, Williams 1997
    Bailey et al., 2003
    Blanchet, Lollini, Luscher 2003
    Bailey et al., 2004
    Dang, Williams 2005
    Virk, Williams 2008
    Koyama, Akaike 2008
    Bailey et al., 2009a,b
    Virk et al., 2009
    Matsui, Williams 2011
    Llorente et al., 2012
    Levitt, Williams 2012
    Banghart, Sabatini 2012
    Banghart et al., 2013
    Williams 2014
    Disinhibition Pan et al., 2004
    Mouse Whole cell Torrecilla et al., 2002
    Alvarez et al., 2002
    Arttamangkul et al., 2006
    Torrecilla et al., 2008
    Bailey et al., 2009a
    Arttamangkul et al., 2011
    Substantia Nigra/VTA
    Guinea pig Intracellular Cameron, Wessendorf, Williams 1997
    Shoji, Delfs, Williams 1999
    Whole cell Bonci, Williams 1997
    Rat Intracellular Johnson, North 1992
    Extracellular Jalabert et al., 2011
    Whole cell Manzoni, Williams 1999
    Whole cell Lecca et al., 2012
    Matsui, Williams 2012
    Matsui et al., 2014
    Mouse Whole cell Ford, Mark, Williams 2006
    Ford, Beckstead, Williams 2007
    Madhavan, Bonci, Whistler 2010
    Dacher, Nugent 2011
    Chieng et al., 2011
    Hjelmstad et al., 2013
    Lowe, Bailey 2015
    Hippocampus
    Rat Extracellular Zieglgansberger et al., 1979
    Intracellular Nicoll, Alger, Jahr, 1980
    Svoboda, Lupica 1998
    Whole cell Glickfeld, Atallah, Scanziani 2008
    Spinal Cord/Trigeminal nucleus
    Rat/frog Intracellular Nicoll, Alger, Jahr, 1980
    Rat Intracellular Yoshimura, North 1983
    Rat/Guinea pig Intracellular Grudt, Williams 1994
    Rat Whole Cell Grudt, Henderson 1998
    Raphe Magnus
    Rat Intracellular Pan, Williams, Osborne 1990
    Rat Extracellular Heinricher et al., 1994
    Rat Whole cell Pedersen, Vaughan, Christie 2011
    Periaqueductal Gray
    Rat Whole cell Osborne et al., 1996
    Vaughan et al., 1997
    Chiou, Huang 1999
    Perf Patch Vaughan, et al., 2000
    Mouse Perf Patch Bagley et al., 2005
    Hypothalamic neurons
    Guinea Pig Intracellular Wuarin, Dudek 1990
    Guinea Pig Intracellular Kelly, Loose, Ronnekleiv 1990
    Mouse Whole cell Pennock, Hentges 2011
    Central Nucleus of the Amygdala
    Rat Whole Cell Chieng et al., 2006
    Table 2: DAMGO EC50
    Cell Measure EC50 (nM) Reference
    Postsynaptic
    LC GIRK (intracellular) 91 Christie et al., 1987
    Parabrachial GIRK (intracellular) 55 Christie, North 1988
    PAG GIRK (intracellular) 80 Chieng, Christie 1994
    Spinal / Trigeminal GIRK (intracellular) 70 Grudt, Williams 1994
    LC GIRK (intracellular) 20 Osborne, Williams 1995
    Thalamus GIRK (intracellular) 300 Brunton, Charpak 1998 POMC
    POMC GIRK (whole cell) 350 Pennock, Hentges 2011
    Sh-Sy5Y Calcium release 145-270 Connor, Henderson 1996
    Sh-Sy5Y Calcium release 11 Seward et al., 1991
    AtT20 G-Calcium 31 Borgland et al., 2003
    PAG G-Calcium 150 Bagley et al., 2005
    Trigeminal G-Calcium 190 Johnson et al., 2006
    DRG G-Calcium 161 Walwyn et al., 2009
    LC I-NMDA facilitation 620 Koyama, Akaike 2008
    Presynaptic
    Hypogastric EPSCs 38 Rogers, Henderson 1990
    VTA EPSCs 100 Manzoni, Williams 1999
    Spinal / Trigeminal EPSCs 52 Grudt, Williams 1994
    Accumbens EPSCs 240 Brundege, Williams 2002
    RVM IPSCs 70 Pan, Williams 1990
    VTA IPSCs 365 Shoji et al., 1999
    PAG IPSCs 163 Fyfe et al., 2010
    POMC IPSCs 80 Pennock, Hentges 2011
    VTA IPSCs 64 Matsui, Williams, 2011
    Cell lines
    C6-mu GTP-g-S 51 Clark, Traynor 2006
    Sh-Sy5Y AC inhibition 15 Levitt, Traynor 2011
    Sh-Sy5Y GTP-g-S 121 Levitt, Traynor 2011
    OVER-expression
    HEK AC inhibition 2.5 Levitt, Traynor 2009
    HEK GTP-g-S 17 Levitt, Traynor 2009
    AtT20 Voltage Dye 5 Knapman et al., 2013

    Conflict of Interest:

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    Competing Interests: None declared.

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