Behavioral experiments using inverse agonists and antagonists, characterized in our electrophysiological recordings from DRG neurons, reveal that constitutively active μ receptors in β-arr2-/- mice delay withdrawal responses to noxious heat. By contrast, there was no detectable differences in either the threshold of mechanical pain or the aversive effects of naloxone, in β-arr2+/+ and β-arr2-/- mice. These data suggest that an absence of β-arr2 has pathway specific behavioral effects mediated by constitutively active μ receptors in β-arr2-/- mice.
Our demonstration that constitutively active μ receptors play a role in thermal nociception in β-arr2-/- mice provides a mechanism for the phenomenon of prolonged basal tail withdrawal latencies observed in previous studies using these animals [26
]. It is well established that μ opioid receptors are located on primary afferent neurons that transmit pain evoked by noxious heat [32
]; therefore it is perhaps not surprising that enhanced constitutive μ receptor activity affects thermal nociception. It is interesting that, unlike the antinociceptive effect of morphine, which succumbs to tolerance [18
], analgesia mediated by constitutively active μ receptors appears to persist providing a long lasting basal analgesic tone in β-arr2-/- mice.
It is likely that the lack of effect of constitutive μ receptor activity on paw withdrawal using the von Frey assay in β-arr2-/- mice can be explained by an absence of μ receptors in mechanical nociceptive neurons which appear to predominantly express delta receptors [33
]. However, pathway specific effects of constitutive μ receptor activity may also be explained by a disparate influence of β-arr2 in different neuronal populations. The ability of morphine to induce μ receptor internalization in striatal neurons, but not spinal neurons, for example, has been attributed to differing properties of β-arr2 in these cell types [34
A previous study also observed differences in basal nociception in β-arr2-/- mice using different behavioral assays [26
]. In keeping with our findings, tail withdrawal from immersion in hot water was prolonged in β-arr2-/- compared to β-arr2+/+ mice. However, there was no difference in paw responses to heat generated using a hotplate. The latter assay quantifies the response to heat by scoring paw lifting and licking and the basal levels of these behaviors appear somewhat variable between studies [18
]. The different anatomical locations of the pain does not account for differences between tail and paw responses since we observed prolonged basal paw withdrawal in β-arr2-/- compared to β-arr2+/+ mice using the Hargreaves assay. However, it is likely that the more complex pain behavior generated by the hot-plate assay involves a greater input from the CNS perhaps recruiting neuronal pathways that are unaffected by constitutive μ receptor activity. This could account for a lack of effect of genotype on basal responses to the hotplate.
The possibility that constitutive μ receptors in β-arr2-/- mice play a greater role in peripherally-or spinally-mediated responses compared to centrally-mediated responses is supported by our findings using the conditioned place aversion assay of basal hedonic tone. Stimulation of hedonic tone by prolonged exposure to morphine causes an enhanced aversive response to the inverse agonist naloxone but not to the neutral competitive antagonists [11
]. By contrast, there was no difference between the aversive responses to naloxone of β-arr2-/- and β-arr2+/+ mice. These data suggest that the nature of the regulation μ receptors by β-arr2 within the reward pathway differs from that of the peripheral pain pathway. In keeping with this idea β-arr2-/- mice exhibit delayed analgesic tolerance to chronic morphine without altered centrally-mediated morphine dependence [35
Our study reveals that there are key differences between ligand-independent constitutive signaling and agonist-induced μ receptor activity. The former causes a low level basal inhibition of VDCC activity in DRG neurons and this is associated with a modest but sustained antinociception in β-arr2-/- mice. By contrast, the latter produces a more profound VDCC inhibition [17
] resulting in strong analgesia, the duration of which is limited by the adaptive process of tolerance and the opponent process of hyperalgesia [14
]. Furthermore, unlike μ receptor activation by morphine, which can lead to reward and dependence, constitutive μ receptor activity, induced by the absence of β-arr2, appears not to disrupt hedonic tone.
We previously demonstrated that inhibition of Src in β-arr2+/+ neurons mimics the enhancement of μ receptor constitutive activity seen in β-arr2-/- neurons [17
]. β-arr2, Src and Akt form a signaling complex in vivo that is disrupted in β-arr2-/- mice [37
]. μ Receptor activation stimulates Akt [38
] a process implicated in cross talk with the NMDA receptor and tolerance [39
]. Further studies are required to examine whether targeting the signaling pathway mediated by the β-arr2 complex can produce sustained analgesia through constitutive μ receptor activity.