Our studies reveal an important brain region-specific role for RGS4 in opiate responses. Since several RGS proteins are expressed in neurons that influence drug reward, dependence, and analgesia, it is important to understand the function of each of these proteins in particular networks and cell types. A large body of clinical and preclinical work links RGS4 to neuropsychiatric disorders such as schizophrenia, a syndrome linked by some to striatal dysfunction. Although RGS4 is moderately expressed in striatum, compared to other regions such as prefrontal cortex, it appears to exert important modulatory effects in this brain region on several GPCRs including muscarinic M2 autoreceptors and D1 and D2 dopamine receptors (4
). While evidence from in vitro studies suggests that RGS4 may associate with MOR (52
), and electrophysiological studies implicate RGS4 in opiate actions in LC slices (11
), there is to date no information about the way RGS4 modulates opiate actions in vivo. In the present study, we use inducible and constitutive gene knockout models to elucidate the role of RGS4 in opiate actions. Given the lack of pharmacological tools for the study of RGS4 in vivo, and the wide distribution of this protein in the CNS, inducible knockout models permit local alterations in RGS activity in particular brain regions of the adult mouse. Indeed, we show that AAV-CreGFP injection into the NAc of floxed RGS4 mice mediates a near complete, but selective, loss of RGS4 from this brain region. Conversely, we selectively overexpressed RGS4 in NAc by use of viral vectors. These data are further supported by biochemical assays in striatal tissue, showing that RGS4 participates in MOR-dependent signal transduction complexes.
Our findings demonstrate that RGS4 action in the NAc controls behavioral and biochemical responses to morphine. In particular, we show that local knockout of RGS4 from the NAc of adult mice increases sensitivity to the rewarding and locomotor activating effects of morphine. Therefore, like RGS9-2 (12
), RGS4 is a negative regulator of morphine action in the NAc. However, RGS9-2 has a more potent effect on reward sensitivity, as deletion of the RGS9 gene results in a tenfold increase in sensitivity to morphine in the CPP paradigm, whereas knockout of RGS4 causes a less dramatic shift. Interestingly, RGS4 overexpression in the NAc of RGS9 knockout mice cannot compensate for the loss of RGS9, which supports the involvement of independent pathways (12
). It remains to be elucidated whether RGS9-2 and RGS4 act in the same cell types, and if they participate in the same biochemical signaling complexes. The use of constitutive RGS4 knockout mice in the CPP test revealed the opposite phenotype. The decreased sensitivity to morphine CPP in RGS4 knockout mice presumably reflects RGS4 actions in brain regions outside the NAc (e.g., prefrontal cortex, amygdala, hippocampus) that also regulate morphine reward or influence associations with cues required for CPP. Although the global loss of RGS4 had no effect on fear conditioning, different aspects of cognitive function could be affected. On the other hand, both constitutive and NAc-specific RGS4 knockout mice are less sensitive to the analgesic actions of fentanyl in the hot plate test, but the global knockout shows a more dramatic impairment likely due to RGS4 actions in other CNS regions.
Another important finding from our study concerns the actions of RGS4 in the LC. A large literature implicates changes in LC firing activity in contributing to aspects of opiate physical dependence and withdrawal (46
). We have shown that alterations in gene expression in LC following chronic morphine are distinct from those observed in reward related networks like the NAc (53
). Morphine-induced changes in LC firing activity occur in part from adaptive responses in signal transduction pathways downstream of the MOR. One of the most robust adaptations is upregulation of adenylyl cyclase (AC) activity, particularly the AC1 and AC8 isoforms (55
), molecules highly regulated by G protein βγ subunits (57
). It is, therefore, expected that proteins like RGS4, which regulate α and βγ subunit availability to effectors, play a prominent role in modulation of LC activity. Here, we report that, although inhibition of LC firing upon acute exposure to MOR agonists is unaffected by the loss of RGS4, firing provoked by activation of the cAMP pathway is greatly enhanced by the absence of this protein. These findings support earlier studies on the role of RGS4 in the cellular adaptations of LC neurons to chronic morphine (11
), and provide a better understanding of the neuron-specific signaling events that contribute to opiate dependence. In accord with the electrophysiology findings, RGS4 knockout mice exhibit more severe withdrawal compared to their wildtype littermates. These experiments were only performed using constitutive RGS4 knockouts, as it was not feasible to reliably target the LC with our AAV vectors. Analysis of NAc-specific RGS4 knockout mice suggested that the morphine withdrawal phenotype is not related to RGS4 actions in the NAc.
Although RGS4 negatively modulates morphine reward and physical dependence, it does not affect morphine analgesia or the development of morphine analgesic tolerance. These findings are in agreement with earlier studies of a different line of RGS4 mutant mice (58
). However, the study by Grillet and colleagues found no effect of RGS4 deletion on morphine withdrawal. This discrepancy might be a result of genetic background or related to the use of a morphine treatment protocol in the earlier study that leads to maximal withdrawal intensity in wildtype animals. In striking contrast to RGS4, RGS9-2 negatively modulates morphine analgesia and analgesic tolerance. This difference between RGS9-2 and RGS4 could be explained by the distinct localization of these proteins in the CNS, but may also reflect their distinct functions. Specifically, the difference between RGS9-2 and RGS4 with respect to morphine analgesia may lie in distinct selectivity for Gα subunits (59
). In vitro data indicate that RGS4 functions as part of a G protein receptor kinase 2 (GRK2)/Gαq complex without preventing GRK2 action (61
). Our data indicate that RGS4 is a necessary component of signaling complexes mediating the analgesic actions of those opiates that, in addition to activating Gαi subunits, also activate Gαq subunits (62
), since RGS4 knockout mice show decreased sensitivity to the analgesic actions of fentanyl and methadone in the hot plate assay. Earlier in vitro studies have indicated an interaction between RGS4 and opioid receptors (52
) but there is no information about RGS4 containing complexes in the brain, as this type of assay is not easy to perform due to the modest density of MOR and RGS4 in striatum. Here, we used IP’s to better understand the mechanisms underlying the agonist selective interactions between MOR and RGS4 in striatum. These interactions appear to have a great impact on hot plate analgesia, as behavioral responses to fentanyl are diminished in RGS4 knockout mice. We hypothesize that loss of RGS4 permits greater association between MOR and Gαq. These data further support the notion that although many opiate analgesics activate MOR, and then recruit several Gα subunits including Gαi and Gαq, morphine does not recruit Gαq and, therefore, its actions are not affected by the absence of RGS4. Future studies are needed, however, to conclusively determine the role of Gαq signaling in morphine analgesia. It should also be mentioned that as morphine acts at both MOR and DOR, while fentanyl is MOR selective, and RGS4 does not affect DOR actions (64
), the lack of a morphine analgesia phenotype may be related to the fact that DOR responses were unaltered in RGS4 knockout mice.
This study thereby provides a better understanding of the signal transduction mechanisms underlying the different actions of opiate agonists. Earlier studies revealed that deletion of the β-arrestin-2 gene affects opiate analgesia and tolerance (65
) but not physical dependence, while deletion of the spinophilin gene increases reward sensitivity (40
), promotes the development of morphine tolerance, and decreases responsiveness to all MOR agonists in the hot plate assay. On the other hand, RGS9-2 has a very potent role in morphine reward and dependence, and may also delay the development of tolerance in the hot plate assay (12
). All of these findings point to a very precise role of signal transduction molecules downstream of MOR in different opiate actions. The fact that RGS9-2 is a negative modulator of morphine’s rewarding and analgesic actions makes it a difficult pharmacological target, as improving analgesia by decreasing RGS9-2 activity might also increase abuse potential. In contrast, RGS4 represents a better target for analgesia, since increasing RGS4 function would promote opiate analgesic responses while reducing reward and dependence liability.
In conclusion, RGS4 is a negative regulator of opiate reward and physical dependence via actions in the NAc and LC, respectively, and likely other brain regions as well. Moreover, as a Gαq-associated protein, RGS4 promotes responses to opiate analgesics such as fentanyl and methadone that recruit Gαq. These findings provide a better understanding of the molecular mechanisms of the diverse actions of opiates on the nervous system and reveal specific actions of RGS4 in opiate reward, dependence, and analgesia.