A New Optogenetics Approach: Creation of a Rhodopsin/Mu-Opioid Receptor Chimera
Opioids are used clinically to treat many different pain conditions, but these drugs are problematic due to their abuse potential and side effects, including constipation, respiratory depression, and sedation. A better understanding of the mu-opioid receptor (MOR), the most frequent target of the majority of clinically used opioids, is needed to develop better analgesics without these adverse consequences. A new chimeric receptor, termed opto-MOR, that combines parts of the light-sensitive channel rhodopsin with parts of MOR will enable researchers to more precisely investigate opioid signaling and behavior, reports a new study led by Michael Bruchas, Washington University, St. Louis, US. The work was published May 20 in Neuron.
“Despite sharing relatively little sequence identity with the native receptor, opto-MOR appears to recapitulate the properties of MOR quite well. Activation by light engages many of the known sequelae, including inhibition of cyclic AMP, coupling to GIRK [G protein-coupled inwardly rectifying potassium] channels, and activation of phosphorylated ERK [extracellular signaling-regulated kinase],” wrote Liam Browne, Harvard Medical School, Boston, US, in an email to PRF. Browne, who was not involved with the new work, studies pain signaling using optogenetic tools.
“The fact that Cre-inducible opto-MOR can be targeted to discrete populations of neurons, and that MOR-like signaling can be activated specifically in these neurons to modulate behavior in the expected manner, is an important breakthrough,” wrote Diane Damez-Werno and Paul Kenny, Icahn School of Medicine, Mount Sinai Hospital, New York City, US, in a commentary published in the same issue of Neuron.
In trying to understand how MORs, which are expressed broadly throughout the nervous system, mediate the effects of opioids on neural activity and function, the Washington University team saw that a new research tool was required. “We needed a way to be able to fine-tune the signaling in various cell types and have better spatiotemporal control,” Bruchas said.
In the past, optogenetic approaches have been used to evaluate pain signaling (e.g., see PRF related news stories here and here), but opto-MOR is of particular note because of the more precise control of the native MOR it allows. Most current optogenetic tools affect ion channel activity, allowing for simple depolarization or hyperpolarization of neurons, said Bruchas. “We’re using a G protein-coupled receptor-based system that allows for [more nuanced] neuromodulatory control,” he explained.
At the crux of traditional optogenetic strategies is the light-gated ion channel rhodopsin, which is a member of the same family of G protein-coupled receptors to which MOR belongs and shares similar structural features with MOR. Because these two receptors are closely related, the researchers could take genetic pieces of each and fuse them together to make a chimera, Bruchas said. “We took components of the rhodopsin receptor to allow for light sensitivity and components of the MOR that face inside the cell to create a receptor that will respond to light but talk with MOR pathways,” he explained.
Left to right: Bryan Copits, Michael Bruchas, and Edward Siuda]
If it walks like a MOR, and it talks like a MOR….
To assess the chimeric receptor’s similarity to the native MOR, the study’s four co-first authors, Edward Siuda, Bryan Copits, Martin Schmidt, and Madison Baird, compared the downstream signaling pathways stimulated by activation of opto-MOR (with blue light) with those of wild-type rat MOR stimulated with the MOR agonist DAMGO. Activation of opto-MOR and wild-type MOR in human embryonic kidney 293 (HEK293) cells produced similar time-dependent reductions in forskolin-induced intracellular cyclic AMP levels. These results are consistent with the known cyclic AMP inhibition that results from the interaction of agonist-stimulated opioid receptors with inhibitory G proteins (Childers and Snyder, 1978; Al-Hasani and Bruchas, 2011).
Opto-MOR also coupled to another canonical opioid signaling pathway, namely, the mitogen-activated protein kinase (MAPK)/ERK pathway (Belcheva et al., 2005; Al-Hasani and Bruchas, 2011). Activation of opto-MOR or wild-type MOR in HEK293 cells produced similar rapid and transient increases in levels of phosphorylated ERK. Opto-MOR also appeared to be internalized via the typical clathrin-dependent machinery that MOR uses and exhibited similar internalization kinetics following activation as the native receptor.
The researchers then examined the function of opto-MOR in cultured dorsal root ganglion (DRG) neurons, a more physiologically relevant system. In order to express the chimeric receptor specifically in DRG neurons, a Cre-dependent virus containing opto-MOR was injected into cultured DRG neurons from mice expressing Cre under the control of the Advillin promoter. Receptor expression was observed beginning two days post-transduction with the virus. By five days, the researchers observed robust opto-MOR expression that partially co-localized with the synaptic marker synapsin, indicating that opto-MOR was successfully trafficked to presynaptic terminals. Similar to the findings in HEK293 cells, increases in phosphorylated ERK levels and receptor internalization were also observed.
(E) Unstimulated opto-MOR expressed in DRGs after 5 DIV [days in vitro] labeled with pERK (red inset). (F) Opto-MOR expressing DRGs (5 DIV) 5 minutes following photostimulation and labeled for pERK (red inset). Open arrows denote internalized punctate receptors. Image and caption reprinted from Siuda et al., with permission from Elsevier.
Viral-mediated expression of opto-MOR in the periaqueductal grey (PAG), a midbrain region rich in MOR-expressing neurons, revealed that the chimeric receptor could also inhibit neuronal activity. Whole-cell recordings from acute brain slices showed that outward currents were rapidly evoked in γ-aminobutyric acid GABAergic inhibitory neurons following light stimulation. This current induction was accompanied by a decrease in the cells’ input resistance. Both phenomena were reversed by the GIRK channel blocker barium, indicating that, similar to MOR, opto-MOR stimulation leads to GIRK channel activation (Vaughan et al., 2003). A parallel set of experiments following activation of MOR with DAMGO produced similar findings, again highlighting the functional similarity of the chimeric and native receptors.
In a final set of experiments, using a real-time place conditioning procedure, the researchers evaluated behavioral responses to opto-MOR stimulation of GABAergic neurons. They used two groups of mice expressing Cre under control of the vesicular GABA transporter promoter, with a fiber optic implant above the ventral tegmental area (VTA) in one group and in the ventral pallidum (VP) in the other; these are two midbrain structures involved in the reward pathway. In the first group, unlike control mice (injected with Cre-negative opto-MOR), those injected with Cre-positive opto-MOR in a subregion of the VTA, the rostral tegmental nucleus, preferred a chamber illuminated with light over one without light. This behavior was very similar to that seen with halorhodopsin-induced VTA inhibition achieved using more traditional optogenetic methods, providing further evidence that opto-MOR is a viable alternative optogenetic strategy. Conversely, mice from the second group in which the chimeric receptor was injected into the VP displayed an aversion to the illuminated chamber.
Strengths and limitations
Having demonstrated that opto-MOR behaves similarly to the native MOR, the researchers hope others will put the new tool to work by isolating the contribution of MOR-like signaling to particular behaviors or circuit dynamics.
The degree of real-time place preference of the mice was dependent on the power of the light used, which means that the new tool will afford researchers finer control over MOR-like signaling than has previously been available with traditional channel-based optogenetic tools. Those tools can turn neurons on and off, but they are not capable of the more subtle, sub-threshold effects of neuromodulators such as opto-MOR. “The way that the brain is connected isn’t just pluses and minuses. There is actually a lot of modulation (or gain) in these circuits that is dynamically controlled by G protein-coupled receptors,” said Bruchas.
The new tool is not without caveats, however. Browne wrote that, since many of the downstream effectors of MOR are quite ubiquitous throughout the nervous system, “it will be important in the future to ensure that opto-MOR is expressed with a normal spatiotemporal pattern to avoid non-specific effects.” In addition, the authors noted that achieving activation of one particular anatomic subregion in slice studies may be difficult because the high photosensitivity of opto-MOR will necessitate working in a fairly dark environment. They also acknowledged that because opto-MOR doesn’t bear the same sequence as the native MOR, the chimeric receptor may exhibit a different protein interactome than its wild-type counterpart.
In an email to PRF, Ajay Yekkirala, Browne’s colleague at Harvard Medical School, praised the study authors for addressing both the advantages and disadvantages of their new tool. But he also noted the paper’s lack of in vivo data relating to pain—something future studies will need to address.
Bruchas referred to a new kappa-opioid receptor-based Designer Receptor Activated Exclusively by Designer Drugs (DREADD) tool, also published in the same issue of Neuron (Vardy et al., 2015; that paper, too, is discussed in the commentary by Damez-Werno and Kenny). In combination, the two new tools could be useful for looking at differences between kappa- and mu-opioid-based pain circuitry, he said.
Allison Marin (Curley), PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.
Image credit: Siuda et al., with permission from Elsevier
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