A critical brainstem relay for mediation of diffuse noxious inhibitory controls

Abstract The CNS houses naturally occurring pathways that project from the brain to modulate spinal neuronal activity. The noradrenergic locus coeruleus (the A6 nucleus) originates such a descending control whose influence on pain modulation encompasses an interaction with a spinally projecting non-cerulean noradrenergic cell group. Hypothesizing the origin of an endogenous pain inhibitory pathway, our aim was to identify this cell group. A5 and A7 noradrenergic nuclei also spinally project. We probed their activity using an array of optogenetic manipulation techniques during in vivo electrophysiological experimentation. Interestingly, noxious stimulus evoked spinal neuronal firing was decreased upon opto-activation of A5 neurons (two-way ANOVA with Tukey post hoc, P < 0.0001). Hypothesizing that this may reflect activity in the noradrenergic diffuse noxious inhibitory control circuit, itself activated upon application of a conditioning stimulus, we opto-inhibited A5 neurons with concurrent conditioning stimulus application. Surprisingly, no spinal neuronal inhibition was observed; activity in the diffuse noxious inhibitory control circuit was abolished (two-way ANOVA, P < 0.0001). We propose that the A5 nucleus is a critical relay nucleus for mediation of diffuse noxious inhibitory controls. Given the plasticity of diffuse noxious inhibitory controls in disease, and its back and forward clinical translation, our data reveal a potential therapeutic target.


Introduction
The descending pain modulatory system (DPMS) comprises pathways that (i) emerge from distinct origin nuclei; and thus (ii) are subserved by discrete neuroanatomical frameworks. Housed in the brainstem, the A5, A6 and A7 nuclei all contain spinally-projecting noradrenergic neurons. 1,2 The A6 nucleus, better known as the locus coeruleus (LC), originates an endogenous analgesic circuit historically linked to activation of spinal α 2 -adrenoceptors (ARs). The role of brainstem noradrenergic nuclei in pain modulatory processing is complex as evidence by the potentiated inhibitory effect of spinal α 2 -ARs antagonism on pain-related behaviours, 3 opposing α 2 -AR-mediated facilitatory signalling in the brainstem, 4 and the modular functional organization of the LC coupled with its role as a chronic pain generator. 5,6 We recently demonstrated that LC-modulation of spinal wide dynamic range (WDR) neuronal nociceptive processing is linked to an interaction with a non-cerulean noradrenergic cell group. 7 Proposing that this interaction may underlie the role of the LC as a chronic pain generator, our present study sought to identify this non-cerulean noradrenergic cell group. Since WDR neurons govern plasticity in the transmission centre and are thus placed front and centre stage of mechanisms that can initiate the development of persistent pain, we examined their electrophysiological properties upon spatial and genetic manipulation of A5, A6 and A7 nuclei. Our aim was to evidence a critical brainstem relay for a hitherto undefined descending inhibitory pathway.

Spinal cord in vivo electrophysiology
In vivo electrophysiology was performed on animals weighing 240-300 g as previously described. 10 Physiological homeostasis was monitored throughout. Extracellular single-unit activity of spinal WDR neurons in deep laminae IV/V was measured. Mechanical stimuli (8,26 and 60 g von Frey filaments) and von Frey filaments with concurrent ipsilateral calibrated noxious ear pinch [to trigger diffuse noxious inhibitory controls (DNIC) 10 ], were applied to the receptive field for 10 s per stimulus. DNIC are quantified as the inhibitory effect of noxious ear pinch on WDR neuronal firing (% of inhibition after ear pinch). Following baseline control data collection, 100 μg atipamezole (an α 2 -AR antagonist), or 20 μg prazosin hydrochloride (α 1 -AR antagonist) was administered topically on the lumbar spinal cord surface.

Optogenetics
ChR2 was activated using a 450 nm laser (20 ms pulse at 5 Hz, 238 mW/mm 2 ). Continuous irradiation (400 mW/mm 2 ) was used to activate GtACR2. Continuous 637 nm laser (160 mW/mm 2 ) was used to activate Jaws. Light power density was measured at the tip of the implantable 200 μm fibre cannula. 3 Spinal WDR neurons were characterized by three stable baseline responses followed by three optically modulated responses. For combined optogenetics and spinal pharmacology, after collecting three stable baseline and three stable optoactivation responses, a drug was applied on the cord surface. On completion, animals were sacrificed by isoflurane overdose and transcardially perfused with saline followed by 4% paraformaldehyde.

Quantification and statistical analysis
Typically, three WDR neurons were characterized per preparation (n), and data were collected from at least six rats per group (N ). Pharmacological investigation was performed on one neuron per animal. Statistical analysis was performed either on n for populational studies, or N for pharmacological studies. Uncorrected twoway repeated-measures (RM) ANOVA with the Tukey post hoc was used to assess von Frey and DNIC responses in the baseline conditions. For pharmacological experiments, Geisser-Greenhouse correction was used for RM-ANOVA. GraphPad Prism was used to analyse the data. P < 0.05 was considered significant.

Data availability
Means of stimulus-evoked responses for each studied neuron are provided in the Supplementary material. Full length Spike2-format recordings as well as other data are available upon request.

Inhibition of spinally projecting A5 neurons abolishes DNIC
Following robust labelling of the A5-A7 brainstem nuclei with the CAV vectors each nucleus was selectively illuminated in separate animals via a sterotaxically implanted optic fibre positioned 200 µm above the target nucleus (Supplementary Fig. 2A-C). Optic fibres used to target each nucleus were paint-coated except for the tip, to ensure minimum light off target effects around the fibre. Using this spatially and genetically restricted approach, we found that inhibition of no nuclei affected basal mechanically-evoked ac-
Subsequently, using spatially and genetically restricted expression of red light (637 nm)-driven inward inhibitory chloride ion pump (Jaws), we verified that, as before, nucleus inhibition did not affect basal mechanically-evoked activity of spinal DDH-WDR  Fig. 4A, C, D and F). Next, we adopted a previously optimized approach for CAV-mediated delivery of ChR2 to spinally projecting neurons from all three nuclei. 3,7,13 After confirming a similar labelling pattern as for the GtACR2 constructs ( Fig. 4G and Supplementary Fig. 5A and B), we optoactivated spinally projecting ChR2-expressing A5 neurons, with pulsed 450 nm laser light (5 Hz, 20 ms square-wave pulses at 238 mW/mm 2 ). A5 optoactivation

Discussion
Herein we investigated the impact of spatially and genetically restricted optical manipulation of descending projections from noradrenergic A5, A6 and A7 brainstem nuclei on spinal WDR neuronal activity. While inhibition of any singular nucleus did not inhibit basal WDR neuronal activity, activation of an excitatory opsin in the pontine A5 nucleus reduced the firing rate of spinal WDR neurons in a manner that was reversed by antagonism of the spinal α 2 -ARs.
This led us to consider the mechanisms by which the body inhibits pain in an endogenous manner. DNIC, a naturally occurring pain-inhibiting pathway, is subserved by noradrenergic transmission via spinal α 2 -ARs. 7,10,23 This pathway is not tonically active but rather is evoked by application of a CS concurrent to stimulation of WDR neuronal peripheral receptive fields. The origin of DNIC was initially postulated, following a series of lesioning experiments, 24,25 to be the medullary reticular dorsal nucleus (MdD). 26 However, a recent genetic, anatomically and functionally precise investigation revealed that activation of the MdD Tac1 + neurons facilitates thermal pain reflexes. 27 Further, the MdD is non-catecholaminergic. Interestingly, we found that upon activation of an inhibitory opsin on A5 neurons, WDR firing rates were no longer inhibited in response  to application of a CS. Cumulatively, our data lead us to propose that the spinal cord projection site of a pathway that governs naturally occurring analgesia is the pontine A5 noradrenergic cell group, the origin of DNIC (Supplementary Fig. 5F).
Interestingly, optoinhibition of the DLF suggested the presence of a tonic noradrenergic inhibitory control restricted to innocuous mechanical stimuli. This result requires further investigation; given that inhibition of no individual nucleus replicated this effect, one interpretation is that there is an underlying interplay between the A-nucleus to spinal cord pathways. This could represent a homeostatic mechanism. For example, activation of the ipsilateral A6 is proalgesic via an interaction with superficial dorsal horn astrocytes expressing α 1 -ARs. 28 Reciprocity between DPMS circuits that govern DNIC's expression (and other modulatory controls) is highly likely and, in some cases, already evidenced. 7 The fact that the DNIC pathway specifically inhibits the activity of WDR neurons, a cell group which (i) are so named because of their ability to respond differentially over a range of stimulus intensities; and (ii) famously underpin the gate control theory of pain, highlights that defining the functionality of pathways that directly modulate WDR activity is key for better understanding of the pain circuitry; delineation in health is necessary before dysfunction in disease may be pinpointed. Insight regarding brainstem and spinal α 2 -AR-mediated mechanisms, specifically linking DNIC attenuation to impairment of descending noradrenergic modulation from the LC in a rodent model of joint inflammatory pain, 29 highlights the need to investigate governance of effects subsequent to A-nucleus activation in health and disease. Since screening for dysfunction in controls such as DNIC is clinically possible, tailored patient approaches could be on the horizon. Further preclinical investigation of the A5 nucleus is warranted.