Background & Aims
The rostral ventromedial medulla (RVM) is a key hub for descending pain modulation. It receives input from the periaqueductal grey (PAG) and projects to the dorsal horn of the spinal cord[1]. Opioids act in part through µ opioid receptors (MORs) in the RVM to modulate pain sensation. Injection of opioid agonists into the RVM produces potent analgesia[2,3,4,5], and RVM inactivation reduces opioid efficacy[6]. MORs are widely expressed in the RVM, present on a majority of GABAergic neurons[7] and subsets of glutamatergic and serotonergic neurons. MORs may also identify a heterogenous set of RVM neurons called on-cells, which are activated by painful stimuli and inhibited by opioids[1,8,9]. Despite the rich history of RVM research, the precise role of MORs on distinct RVM neuronal subtypes for descending pain modulation remains unclear. In this study, we used Cre/Lox and CRISPR/Cas9 gene editing strategies to knock out RVM MORs and clarify their roles in descending pain modulation.
Methods
Histology: We injected Complete Freund’s Adjuvant (CFA) or saline bilaterally into the hind paws of 6 week old C57 mice. 24 hours later, we administered a hind paw pinch 30 minutes before perfusion. The brains were sectioned and in situ hybridization was performed to identify pain-activated neurons using a cFos probe, MOR+ neurons, and either GABAergic, glutamatergic, or serotonergic neurons.
Pain Behavior: For conditional and retrograde MOR knockout, we injected virus expressing eGFP or eGFP-Cre into the RVM of 6 week old Oprm1fl/fl mice. 6 weeks later, we performed hot plate before and after morphine administration, Hargreaves before and after hind paw CFA administration, and von Frey assays. For cell-type specific MOR knockout, we injected virus expressing a guide RNA against the MOR or a control guide and a Cre-dependent fluorophore into mice expressing Cre and Cas9 specifically in GABAergic, glutamatergic, or serotonergic neurons[10]. We then performed the same assays as above.
Results
Preliminary histology results show that 86.9% of GABAergic RVM neurons, 58.8% of glutamatergic RVM neurons, and 54.7% of serotonergic RVM neurons were MOR+. Furthermore, the overall number of cFos+ RVM neurons was greater in CFA-treated animals vs. saline controls (CFA: 47.6 neurons/mm2; Sal: 18.0 neurons/mm2; p<0.0001). We found that non-specific MOR knockout (KO) in RVM cell bodies increases Hargreaves latency compared to controls (KO: 15.0s; Control: 10.7s; p=0.002), but that there is no difference in latency following CFA injection. Conversely, retrograde MOR KO targeting presynaptic terminals in the RVM decreased hot plate response latency in edited animals compared to controls (KO: 4.8s; Control: 7.0s; p=0.02). Neither local nor retrograde MOR KO affected morphine analgesia in hot plate tests. Preliminary data on MOR knockout in GABAergic RVM neurons revealed decreased hot plate response latency in KO animals compared to control (KO: 15.51s; Control: 20.36s; p=0.04).
Conclusions
Our results demonstrate widespread MOR expression among GABAergic, glutamatergic, and serotonergic RVM neurons, supporting a need to study MOR function in each population. We also found that pain induces dramatic cFos activation in the RVM. Our upcoming results will quantify the fraction of pain-activated cells among each RVM subpopulation and directly examine the overlap between pain-activated and MOR+ neurons.
The findings that MOR KO on RVM cell bodies increases Hargreaves latency, but presynaptic and GABAergic MOR KO decrease thermal pain thresholds, further support our hypothesis that different RVM cell types have divergent effects on descending pain modulation. Thus, it is possible that non-specific MOR KO masks this variability. It is also possible that presynaptic MORs from interneurons or the PAG play a distinct role in descending pain modulation. Our upcoming results will begin to dissect these questions to better understand how the RVM affects descending pain modulation.
References
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Presenting Author
Jamie Moffa
Poster Authors
Jamie Moffa
Washington University in St. Louis
Lead Author
Vani Kalyanaraman
Washington University in St. Louis
Lead Author
Monique Heitmeier
Washington University in St. Louis
Lead Author
Heather Grossman
Washington University in St. Louis
Lead Author
Bryan A Copits
PhD
Washington University School of Medicine, St. Louis, MO
Lead Author
Topics
- Mechanisms: Biological-Molecular and Cell Biology