Dissecting the interaction of pain and respiration at the level of the rostral ventromedial medulla via cell-type specific manipulations

Abstract

Opioid drugs are highly effective for pain management, but they also cause respiratory depression. This life-threatening side-effect of opioid use occurs due to the interacting and/or overlapping neuronal circuits that regulate pain and breathing. Understanding the brain regions and cell-types that link pain and breathing will be essential to develop safer pain management strategies. Opioid analgesia involves altered firing patterns of neurons within the rostral ventromedial medulla (RVM). Although the RVM is known for its role in descending pain modulation, local administration of opioids within the RVM induces potent respiratory depression in addition to analgesia. The RVM has two main classes of neurons identified based on their firing response to pain stimuli. As their name suggests, “ON-cells” are activated during pain and “OFF-cells” are inhibited. Pharmacological and electrophysiological studies have shown that ON-cells express the μ-opioid receptor (MOR) and respond directly to opioids. Moreover, it is thought that the activity of ON-cells facilitates pain and respiration, and that opioid suppression of ON-cells contributes to respiratory depression. In contrast, OFF-cells do not appear to express MOR; however, activation of these neurons is required for opioid-induced analgesia. Therefore, changes in OFF-cells activity may more selectively affect pain thresholds. Due to a lack of selective pharmacological agents, directly testing these predictions has not been possible. Moreover, ON-cells can be excitatory or inhibitory, whereas OFF-cells are mostly inhibitory, adding another layer of complexity to their proposed functions. Here, we dissect these RVM subpopulations using modern intersectional genetic approaches to selectively manipulate RVM neurons based on expression of Oprm1 (the gene encoding MOR), as well as genetic markers for excitatory (Vglut2) or inhibitory (Vgat) neurotransmitter phenotypes. To examine the functional effects of Oprm1+ RVM neurons, an AAV designed to express Cre-dependent ChR2 was injected into the RVM of Oprm1Cre mice, followed by the implantation of an optical fiber. Respiratory activity of awake mice was then recorded using plethysmography during optogenetic stimulation of Oprm1+ RVM neurons. Consistent with the expected effects of ON-cells, breathing rate was increased to 5-6 Hz for the duration of the stimulation, and mice exhibited flinching and freezing behavior at light onset. In a second set of experiments, double transgenic Oprm1Cre; Vglut2FlpO mice were injected with an AAV that expresses ChR2 only when FlpO is present but Cre is absent (i.e. FlpO NOT Cre). Using this strategy, we were able to selectively target excitatory RVM neurons that do not express MOR. Optogenetic stimulations of these neurons during plethysmography revealed no changes in breathing or notable behavioral responses. However, paw withdrawal thresholds tested using Hargreaves’ method suggest that optogenetic activation of these neurons increases pain sensitivity. Our ongoing work will expand on these preliminary findings by using this approach to specifically manipulate Oprm1+;Vglut2+, Oprm1+;Vgat+, and Oprm1-;Vgat+ neurons to assess the roles of these RVM subpopulations in the regulation of pain and breathing. (NAB) R00HL145004; R01HL166317 (RSP) K01DA058543. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.