Elevated serotonin in mouse spinal dorsal horn is pronociceptive

Abstract

Serotonergic neurons in the rostral ventral medulla (RVM) contribute to bidirectional control of pain through modulation of spinal and trigeminal nociceptive networks. Deficits in this pathway are believed to contribute to pathological pain states, but whether changes in serotonergic mechanisms are pro or anti-nociceptive are debated. We used a combination of optogenetics and fiber photometry to examine these mechanisms more closely. We find that optogenetic activation of RVM serotonergic afferents in the spinal cord of naïve mice produces mechanical hypersensitivity and conditioned place aversion. Neuropathic pain, produced by chronic constriction injury of the infraorbital nerve (CCI-ION), evoked a tonic increase in serotonin concentrations within the spinal trigeminal nucleus caudalis (SpVc), measured with liquid chromatography-tandem mass spectroscopy (LC-MS/MS). By contract, CCI-ION had no effect on the phasic serotonin transients in SpVc, evoked by noxious pinch, and measured with fiber photometry of a serotonin sensor. These findings suggest that serotonin release in the spinal cord is pronociceptive and that an increase is sustained serotonin signaling, rather than phasic or event driven increases, potentiate nociception in models of chronic pain.

Figure 1:

Activation of serotonergic afferents in spinal cord is aversive. (A) Example of serotonergic neurons in RVM transfected to express channelrhodopsin and the fluorescent reporter eYFP. (B) In vitro verification that trains of optical stimuli (20 Hz) can entrain ChR expressing neurons in RVM. (C) Optical stimulation (4 repetitions of 10 pulses, 4 ms durations, at 20 Hz with 10 seconds between trains) of spinal serotonergic afferents that express channelrhodpsin produces mechanical allodynia in naïve mice (blue) but not in animals expressing eGFP (black). (D) Optical stimulation of spinal 5HT axons evokes conditioned place avoidance only in mice that express ChR and not eGFP controls. ** = p < 0.01, repeated measures ANOVA in (C) and paired t-test in (D). N = 5 per group.

Figure 2:

Phasic release of serotonin in SpVc is unaffected by CFA-induced inflammatory pain. (A) Changes in fluorescence of a serotonin sensor expressed in SpVc and measured by fiber photometry in response to noxious heat. The heatmap shows the sensor response to 10 consecutive pinches with the average and 95% confidence intervals of all 10 responses depicted in the trace below. In vitro verification of the sensor response to exogenous application of 50 and 100 μM serotonin (thin and thick blue lines) is shown in the inset. (B) Average of pinch (t = 0) evoked serotonin transients in SpVc from naïve mice and from mice 3 to 5 days after CFA injection (n = 5 per group). There is no difference in the response, measured as area under the curve (AUC), between sham and CFA mice. p > 0.05, t-test.

Figure 3:

Tonic serotonin levels are elevated in SpVc in a mouse model of neuropathic pain. Concentrations of serotonin in isolated SpVc tissue were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Group sizes: n = 9 sham and n=11 CCI, p = 0.018, unpaired t-test.