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Mu and Delta Opioid Receptors: Where Are They, and Do They Interact?

New study finds MOR and DOR co-expression limited to small populations of dorsal and ventral horn neurons, and that these opioid receptor subtypes function independently to regulate pain

by Dara Bree


3 May 2018


PRF News

Figure8_vF

New study finds MOR and DOR co-expression limited to small populations of dorsal and ventral horn neurons, and that these opioid receptor subtypes function independently to regulate pain

Opioid receptors mediate opioid analgesia and are located throughout the central nervous system (CNS). How they are organized—the cell populations and neural circuits in which they are present—and whether receptor subtypes work together or independently remain poorly understood. Now, a new study provides a comprehensive analysis of the organization and function of delta and mu opioid receptors (DOR and MOR) in CNS circuits that are crucial for the transmission and processing of nociceptive signals.

 

Research led by Dong Wang and Gregory Scherrer, Stanford University, Palo Alto, US, shows that MOR and DOR are expressed in neurons throughout the dorsal horn of the spinal cord and in brain regions involved in processing nociceptive information. However, the team shows that co-expression of these two receptor subtypes on neurons is rare, and when there is co-expression both receptors act independently, indicating a functional divergence of MOR and DOR. Furthermore, the results show that DOR selectively regulates mechanical pain in mice by controlling the excitability of dorsal horn interneurons.

 

The study “brings together a lot of different experimental expertise to shed light at the functional level on both spinal neurons and brain neurons that express either delta opioid or mu opioid receptors, or indeed both receptors together, in the pain pathway,” said Louis Gendron, Université de Sherbrooke, Canada, who was not involved in the study. “Although these receptors can be expressed together on neurons, it appears that they may act on different kinds of pain, namely mechanical and heat pain.”

 

The findings were published online March 22 in Neuron.

 

Is DOR present in spinal cord neurons?

The expression of MOR in particular has been examined previously throughout the CNS. In the spinal cord, MOR is primarily confined to laminae I-II, which receive sensory information from primary afferent nerve fibers innervating the skin and deeper tissues of the body. This incoming information reaches projection neurons in the dorsal horn that transmit it to many brain regions involved in nociceptive processing.

 

Regarding DOR, previous rodent work using an antibody directed against this receptor revealed expression only on primary afferent nerve terminals in the dorsal horn, with no labeling throughout the rest of the spinal cord. Moreover, when this afferent input to the spinal cord was interrupted, no DOR expression was observed (Dado et al., 1993). This led to the assumption that DOR is not expressed by intrinsic spinal cord neurons, but only at the central terminals of peripheral sensory neurons. In contrast, subsequent studies did show DOR binding sites and messenger RNA (mRNA) in the rodent spinal cord (Mennicken et al., 2003). But a comprehensive analysis of receptor expression that might resolve such a discrepancy hadn’t been undertaken.

 

To gain a clearer idea of whether or not DOR is expressed in the spinal cord, the current study employed a genetically engineered reporter mouse in which the receptor is tagged with green fluorescent protein (DORGFP), a commonly used expression marker. Using immunohistochemistry, the researchers saw diffuse expression of DORGFP in the spinal cord, particularly in lamina II. This provided the most definitive evidence to date for the presence of DOR-expressing neurons in the spinal cord. Complementary techniques in wild-type mice, including in situ hybridization and electrophysiology, confirmed the finding.

 

“Nobody had really searched for these types of cells before, as most people thought that they didn’t exist based on the previous experiments that used antibody staining,” explained senior author Gregory Scherrer. “With the availability of new experimental tools, we were able to re-examine some of these previous conclusions.”

 

The researchers further characterized DOR-expressing spinal neurons using tissue staining of spinal cord slices from DORGFP mice along with an additional fluorescent marker for the vesicular glutamate transporter 2 (VGLUT2), showing that the majority of these cells co-expressed the transporter. As glutamate is the primary excitatory neurotransmitter in the CNS, this suggests that the DOR-expressing spinal neurons are excitatory in nature. Electrophysiological experiments provided further evidence of this, as the neurons displayed a more negative resting membrane potential compared to inhibitory neurons, and a pattern of single action potential firing, both of which are characteristic of excitatory neurons.

 

Mechanical pain regulation

The majority of DOR-expressing spinal neurons also expressed the peptide hormone somatostatin (SOM), which functions as a neurotransmitter in the nervous system. This piqued the team’s interest, as a previous study reported a critical role of SOM-expressing excitatory interneurons in mechanical pain (Duan et al., 2014here). Given the co-expression of DOR and SOM on lamina II neurons, they hypothesized that DOR-expressing neurons would also regulate mechanical pain responses.

 

Indeed, upon noxious mechanical stimulation of the hind paw of mice, about 40 percent of DORGFP lamina II neurons exhibited an increase in c-Fos, a marker of neuronal activation. More than half of these neurons also expressed SOM, strongly suggesting that DOR is present on spinal neurons that process mechanical pain information. The majority of these neurons received synaptic input from A-delta and A-beta fibers, which are important regulators of mechanosensation.

 

Next, the team generated transgenic mice in which DOR was selectively deleted from SOM-expressing spinal interneurons. They discovered that the ability of the DOR agonist deltorphin II to decrease sensitivity to mechanical stimulation in models of inflammatory and neuropathic pain was profoundly reduced in these animals. These results suggested that DOR present on SOM-expressing interneurons is essential for the analgesia provided by DOR agonists. Interestingly, DOR knockout had no effect on the ability of deltorphin II to relieve heat pain.

 

Scherrer and colleagues also asked if DOR was expressed in the ventral horn of the spinal cord, which contains large numbers of neurons that control movement. DOR was indeed expressed there. For example, microinjection of a retrograde tracer, which traces neurons from the synapse to the cell body, into the cerebellum (a region intimately involved in motor control) labeled DOR-expressing cells in laminae VII/X of the spinal cord. This region contains Clarke’s column neurons, which are involved in proprioception.

 

“It’s actually quite interesting, as many of the pain tests we use in the lab, such as the tail flick assay, often have a motor response, and the use of an opioid agonist will increase the latency for tail withdrawal. The extent to which this represents antinociception exclusively or if there is a motor component is something we are going to follow up on,” said Scherrer.

 

Together at last—but not that often

Previous work suggested that interactions between DOR and MOR regulate opioid analgesia by a process known as receptor heteromerization, which involves the formation of a new protein complex by the two receptors. Prior findings also indicated that activation of DOR by DOR agonists causes internalization and degradation of MOR, and thus contributes to morphine tolerance (He et al., 2011). However, the extent of co-expression of these two receptors in spinal neurons remained unknown, so the team set about addressing this question.

 

 

Co-expression was frequently observed in lamina I projection neurons. The majority of these projection neurons express MOR, and of these, 58 percent also expressed DOR. But projection neurons only account for approximately 5 percent of lamina I neurons and are a very small percentage of all spinal neurons, making co-expression of both receptors on spinal neurons relatively rare. Small populations of excitatory lamina II dorsal horn neurons also exhibited co-expression of MOR and DOR. Co-expression was also rare in brain circuits involved with pain affect, including the lateral parabrachial nucleus, amygdala, and anterior cingulate cortex.

 

Based on the findings of this paper, “it’s true that under normal conditions the mu and delta opioid receptors are not highly co-expressed on neurons, and there may well be a functional significance to this,” said Gendron. “This paper certainly increases our knowledge of these receptors in spinal and supraspinal areas that are involved in pain processing. Why certain cells are expressing both receptors and whether we can find a way to specifically target them are two important outstanding questions.”

 

The identification of neurons that co-express both receptors enabled the researchers to ask if internalization of MOR in response to DOR internalization occurred in nociceptive spinal neurons in vivo. Using the DOR agonist SNC80 to induce DOR internalization and staining spinal cord slices for MOR, the investigators showed that DOR internalization had no impact on the distribution of MOR, which remained on the cell surface. Similarly, the MOR agonist DAMGO caused internalization of MOR without affecting DOR expression.

 

Finally, the distinct trafficking of these two opioid receptor subtypes raised the question of whether MOR and DOR have different functions in pain relief from opioids. To address this, the researchers gave deltorphin II (the DOR agonist) to both wild-type and DOR knockout mice, and assessed their responses to heat and mechanical pain. Deltorphin II alleviated both heat and mechanical pain in wild-type mice. The benefit on mechanical pain was lost in the DOR knockout, but interestingly, heat pain relief remained intact, suggesting that in vivo, deltorphin II may cross-activate other opioid receptors in addition to activating DOR. In support of this hypothesis, giving a MOR antagonist at the same time as deltorphin II to the knockout mice blocked noxious heat antinociception from deltorphin II. This showed that the beneficial effects of deltorphin II on heat pain depended on MOR.

 

As for future work, Scherrer envisions a two-pronged approach. “We would like to understand the logic of why some cells express both receptors and others only have one, and what each receptor is doing to modulate cell excitability or neurotransmitter release. The expression in motor circuits is also a surprise, as we found these receptors in places we didn’t expect to see them based on what is known about opioids. So another direction we can go is to try and interrogate their function [in those motor circuits] with pharmacology and behavioral studies.”

 

Dara Bree is a postdoctoral fellow at Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, US.

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