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. 2017 Sep 1:123:420-432.
doi: 10.1016/j.neuropharm.2017.06.019. Epub 2017 Jun 21.

Two delta opioid receptor subtypes are functional in single ventral tegmental area neurons, and can interact with the mu opioid receptor

Elyssa B Margolis  1 , Wakako Fujita  2 , Lakshmi A Devi  2 , Howard L Fields  3
Affiliations

Two delta opioid receptor subtypes are functional in single ventral tegmental area neurons, and can interact with the mu opioid receptor

Elyssa B Margolis et al. Neuropharmacology. .

Abstract

The mu and delta opioid receptors (MOR and DOR) are highly homologous members of the opioid family of GPCRs. There is evidence that MOR and DOR interact, however the extent to which these interactions occur in vivo and affect synaptic function is unknown. There are two stable DOR subtypes: DPDPE sensitive (DOR1) and deltorphin II sensitive (DOR2); both agonists are blocked by DOR selective antagonists. Robust motivational effects are produced by local actions of both MOR and DOR ligands in the ventral tegmental area (VTA). Here we demonstrate that a majority of both dopaminergic and non-dopaminergic VTA neurons express combinations of functional DOR1, DOR2, and/or MOR, and that within a single VTA neuron, DOR1, DOR2, and MOR agonists can differentially couple to downstream signaling pathways. As reported for the MOR agonist DAMGO, DPDPE and deltorphin II produced either a predominant K+ dependent hyperpolarization or a Cav2.1 mediated depolarization in different neurons. In some neurons DPDPE and deltorphin II produced opposite responses. Excitation, inhibition, or no effect by DAMGO did not predict the response to DPDPE or deltorphin II, arguing against a MOR-DOR interaction generating DOR subtypes. However, in a subset of VTA neurons the DOR antagonist TIPP-Ψ augmented DAMGO responses; we also observed DPDPE or deltorphin II responses augmented by the MOR selective antagonist CTAP. These findings directly support the existence of two independent, stable forms of the DOR, and show that MOR and DOR can interact in some neurons to alter downstream signaling.

Keywords: Ca(v)2.1; D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (PubChem CID: 10418702); Delta opioid receptor; H-Tyr-Tic-psi(CH(2)NH)Phe-Phe-OH (TIPP-psi) (PubChem CID: 5311481); Ventral tegmental area; [D-Ala(2), Glu(4)]deltorphin (deltorphin II) (PubChem CID: 123795); [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-Enkephalin acetate salt (DAMGO) (PubChem CID: 5462471); [D-Pen(2), D-Pen(5)]enkephalin (DPDPE) (PubChem CID:104787); mu opioid receptor.

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Figures

Fig. 1
Fig. 1. The DOR1 agonist DPDPE inhibits and excites different subsets of VTA neurons
Example recordings of identified VTA dopamine neurons filled with biocytin (red) and cytochemically labeled for TH (green) that were (A) inhibited by, (B) depolarized by, or (C) insensitive to bath application of DPDPE (1 μM). Insets, all 3 example neurons were Ih(+). Scale bars 100 ms and 200 pA. Summary data showing the time course of the DPDPE induced hyperpolarizations (D) and depolarizations (E) in quiescent VTA neurons. Across populations, more reversal was evident during DPDPE washout following hyperpolarizations compared to depolarizations. Example recording (F) showing that the DOR selective antagonist TIPP-Ψ (100 nM) completely blocked the response to DPDPE. In control neurons, a second application of DPDPE induces a response of the same magnitude as the first application (G, left, n = 8), however when the second application is completed in the presence of TIPP-Ψ (100 nM) DPDPE responses are blocked (G, right, n = 9) Circles show individual neurons, grey bars indicate means. ****p ≤ 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2. The DOR2 agonist deltorphin II inhibits some and excites other VTA neurons
Summary data showing the time course of deltorphin II induced hyperpolarizations (A) and depolarizations (B) in quiescent VTA neurons. Across all neurons, deltorphin II induced depolarizations tended to be followed by more rapid and complete reversal during washout than hyperpolarizations. Example recording (C) showing that 100 nM of the DOR selective antagonist TIPP-Ψ completely blocked the response to deltorphin II in VTA neurons. (D) Summary data showing that in control neurons reapplication of deltorphin II causes a second response of equal magnitude to the first response (right), however when the second deltorphin II application is performed in the presence of TIPP-Ψ (100 nM), the deltorphin II response is eliminated (right). Circles show individual neurons, grey bars indicate means. ****p ≤ 0.0001.
Fig. 3
Fig. 3. Distributions of DOR1 and DOR2 effects in different subpopulations of VTA neurons
Neither DPDPE (blue) nor deltorphin II (orange) effects in VTA neurons appear to sort with TH content or Ih expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4. Both DOR subtypes hyperpolarize VTA neurons via K+ channel activation and excite VTA neurons through activation of Cav2.1
(A) In control experiments, VTA neurons respond similarly to the first and second application of the same DOR agonist, regardless of the magnitude or direction of the response. (1 μM DPDPE, n = 7; 1 μM deltorphin II, n = 5) (B) In neurons responding to DPDPE or deltorphin II with a hyperpolarization, the K+ channel blocker BaCl2 (100 μM) prevented a hyperpolarization in response to a second agonist application (DPDPE n = 6; deltorphin II n = 9). (C) In neurons where the initial response to DPDPE (n = 5) or deltorphin II (n = 5) was a depolarization, this response was blocked by the Cav2.1 blocker ω-agatoxin-IVA (100 nM). In an additional neuron that first responded to deltorphin II with a hyperpolarization, the response was larger in the presence of ω-agatoxin-IVA. Paired t-tests, *p < 0.05; **p < 0.01 (D) Example VTA recording of a spontaneously firing neuron where bath application of DPDPE (1 mM) increased the firing rate of the cell, and this increase was prevented by 100 nM of ω-agatoxin-IVA. Inset: this cell was filled with biocytin (red) during recording and cytochemically identified as TH(+) (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5. Individual VTA neural responses to DOR1, DOR2, or MOR do not predict responses to the other agonists
(A) Example VTA neuron that was excited by DPDPE (1 μM) and inhibited by deltorphin II (1 μM). (B) Example recorded VTA neuron tested with all three ligands. This neuron was filled with biocytin during recording (red), and cytochemically identified as TH(+) (green). (C) Among quiescent neurons tested for responses to DPDPE and deltorphin II, subsets of neurons responded to just one or the other agonist. Further, among neurons tested for responses to DPDPE and DAMGO (D) or deltorphin II and DAMGO (E), no clear relationship between responses to the two subtype agonists were observed. (F) Among quiescent neurons tested for responses to all three agonists, no relationship between responses was observed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6. Different DPDPE and deltorphin II responses were distributed throughout the VTA
Recordings were made in horizontal midbrain slices and distributed throughout the VTA. Changes from baseline membrane potential are color coded in quiescent neurons here. There is no observed relationship between DOR response and recording location. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7
Fig. 7. MOR and DOR interact in a subset of VTA neurons
(A) Example VTA neuron recording in which a small DAMGO-induced hyperpolarization (saturating dose of 500 nM) is substantially larger when DAMGO was re-applied in the presence of the DOR selective antagonist, TIPP-Ψ (100 nM). (B) Time course average across 8 neurons in which this experiment was performed. (C) The population of DAMGO responses is shifted to larger than baseline DAMGO responses when the second application is in the presence of TIPP-Ψ. (D) Example recording in which the MOR selective antagonist CTAP (500 nM) augmented the hyperpolarization response to DPDPE (1 μM). (E) Summary across cells showing that DPDPE applied in the presence of CTAP tends to have an augmented response compared to the first response to DPDPE alone. (F) Example recording in which CTAP augmented a hyperpolarization response to deltorphin II (1 μM). (G) Summary across cells shows that CTAP markedly changes a cell’s response to deltorphin II, either increasing the magnitude of the response or even switching the effect from hyperpolarizing to depolarizing (indicated by negative values here). Light grey circles are cells where the first response was a hyperpolarization; dark grey circles are cells where the first response was a depolarization.
Fig. 8
Fig. 8. Hypothesis to explain how DOR subtypes and MOR independently produce either direct excitations or inhibitions in VTA neurons
We suggest that the DOR1, DOR2, and MOR signaling observed here results from the segregation of these different receptors and their respective signaling channels to separated neural compartments. The long dendrites and bipolar geometry of many VTA neurons may facilitate separation into such relatively isolated domains. For instance, in A, B, and C, homodimers of DOR1, DOR2, or MOR are localized to separate cellular domains and signal differentially through the GIRK or Cav2.1 specifically associated with that particular opioid receptor. (D) A proposed organization of MOR homodimers and MOR-DOR heterodimers that elicit a GIRK conductance at the MOR homodimers in the absence of TIPP-Ψ. When TIPP-Ψ is bound to the MOR-DOR heterodimer, the MOR agonist acting at the heterodimer more effectively opens the GIRK, resulting in a more pronounced hyperpolarization.
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