Neuropeptide Y in the rostral ventromedial medulla reverses inflammatory and nerve injury hyperalgesia in rats via non-selective excitation of local neurons

Abstract

Chronic pain reflects not only sensitization of the ascending nociceptive pathways, but also changes in descending modulation. The rostral ventromedial medulla (RVM) is a key structure in a well-studied descending pathway, and contains two classes of modulatory neurons, the ON-cells and the OFF-cells. Disinhibition of OFF-cells depresses nociception; increased ON-cell activity facilitates nociception. Multiple lines of evidence show that sensitization of ON-cells contributes to chronic pain, and reversing or blocking this sensitization is of interest as a treatment of persistent pain. Neuropeptide Y (NPY) acting via the Y1 receptor has been shown to attenuate hypersensitivity in nerve-injured animals without affecting normal nociception when microinjected into the RVM, but the neural basis for this effect was unknown. We hypothesized that behavioral anti-hyperalgesia was due to selective inhibition of ON-cells by NPY at the Y1 receptor. To explore the possibility of Y1 selectivity on ON-cells, we stained for the NPY-Y1 receptor in the RVM, and found it broadly expressed on both serotonergic and non-serotonergic neurons. In subsequent behavioral experiments, NPY microinjected into the RVM in lightly anesthetized animals reversed signs of mechanical hyperalgesia following either nerve injury or chronic hindpaw inflammation. Unexpectedly, rather than decreasing ON-cell activity, NPY increased spontaneous activity of both ON- and OFF-cells without altering noxious-evoked changes in firing. Based on these results, we conclude that the anti-hyperalgesic effects of NPY in the RVM are not explained by selective inhibition of ON-cells, but rather by increased spontaneous activity of OFF-cells. Although ON-cells undoubtedly facilitate nociception and contribute to hypersensitivity, the present results highlight the importance of parallel OFF-cell-mediated descending inhibition in limiting the expression of chronic pain.

Keywords: NPY; OFF-cells; ON-cells; descending pain modulation; nociception; raphe magnus.

Figure 1

Location of RVM injections and cell recordings. Left are the locations at which NPY was microinjected into the RVM in behavioral studies. Right are the locations for which cells were recorded during simultaneous NPY microinjection. Numbers on the left represent distances from the interaural line (mm).

Figure 2

Identification of TPH+ and Y1-LI+ neurons in the RVM. A. Outline of area considered the RVM, and shown in B, C, and D, with higher magnification view shown in E, F, and G. Y1-LI: red, TPH-LI: green, overlay: yellow. H: number of RVM cells with Y1-LI, TPH-LI, or both at different distances from the interaural line.

Figure 3

NPY microinjection (1 to 10 μg) into the RVM reverses mechanical hyperalgesia in SNL and CFA models of chronic pain. In normal animals, microinjection of NPY did not significant change responses to mechanical stimuli (p = 0.098, n = 7, Wilcoxon test). NPY also failed to alter response thresholds for the uninjured paw in either model. However, hyperalgesia ipsilateral to nerve-injury and inflammation was reversed by RVM NPY (Compared to pre-injection baseline, SNL: n = 6, p = 0.009; CFA: n = 9, p = 0.014. Kruskal-Wallis analysis of variance by rank, followed by Dunn’s post-hoc test.)

Figure 4

NPY microinjection (0.5 μg) in the RVM slightly increases the thermal withdrawal threshold compared to baseline, while aCSF had no effect. Repeated-measures ANOVA with Dunnett’s post-hoc test for comparison to baseline. No significant changes occurred with aCSF microinjection. **p < 0.01 vs. middle baseline value. NPY: F_{11, 220} = 5.50, n = 21; aCSF: F_{11, 209} = 1.77, n = 20.

Figure 5

Dose response curve for thermal antinociception for NPY. Circles show the average change in nociceptive threshold over the 45 minutes following NPY injection into the RVM, and the line shows a non-linear dose-response curve fit to these points. (From left to right, n’s = 20,4,3,21,15, and 3 in the different dose groups).

Figure 6

RVM microinjection of NPY increased spontaneous neuronal activity without compromising the pause or the burst. In representative examples, (A) RVM microinjection of 1 μg NPY increased spontaneous activity of neurons from each cell class. (B) Across all cells tested, NPY significantly increased spontaneous activity (Wilcoxon pre- vs. post-drug; n = 10, p = 0.006 for NEUTRAL cells; n = 11, p = 0.001 for OFF-cells; n = 7, p = 0.016 ON-cells), whereas aCSF did not change the activity of any cell class (n = 7, p = 0.11 for NEUTRAL cells; n = 6, p = 0.18 for OFF-cells; n = 7, p = 0.68 for ON-cells). (C) When looking at the relative change in firing at the withdrawal, NPY did not significantly change the magnitude of the ON-cell increase (p = 0.94) or the OFF-cell decrease in activity (p = 0.054).