Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons.

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.

Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons.

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify 3 clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 & ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.

A critical brainstem relay for mediation of diffuse noxious inhibitory controls.

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.

Distinct brainstem to spinal cord noradrenergic pathways inversely regulate spinal neuronal activity.

Brainstem to spinal cord noradrenergic pathways include a locus coeruleus origin projection and diffuse noxious inhibitory controls. While both pathways are traditionally viewed as exerting an inhibitory effect on spinal neuronal activity, the locus coeruleus was previously shown to have a facilitatory influence on thermal nocioception according to the subpopulation of coerulean neurons activated. Coupled with knowledge of its functional modular organisation and the fact that diffuse noxious inhibitory controls are not expressed in varied animal models of chronicity, we hypothesized a regulatory role for the locus coeruleus on non-coerulean, discrete noradrenergic cell group(s). We implemented locus coeruleus targeting strategies by microinjecting canine adenovirus encoding for channelrhodopsin-2 under a noradrenaline-specific promoter in the spinal cord (retrogradely labelling a coeruleospinal module) or the locus coeruleus itself (labelling the entire coerulean module). Coeruleospinal module optoactivation abolished diffuse noxious inhibitory controls (two-way ANOVA, P < 0.0001), which were still expressed following locus coeruleus neuronal ablation. We propose that the cerulean system interacts with, but does not directly govern, diffuse noxious inhibitory controls. This mechanism may underlie the role of the locus coeruleus as a 'chronic pain generator'. Pinpointing the functionality of discrete top-down pathways is crucial for understanding sensorimotor modulation in health and disease.

The impact of bone cancer on the peripheral encoding of mechanical pressure stimuli.

Skeletal metastases are frequently accompanied by chronic pain that is mechanoceptive in nature. Mechanistically, cancer-induced bone pain (CIBP) is mediated by peripheral sensory neurons innervating the cancerous site, the cell bodies of which are housed in the dorsal root ganglia (DRG). How these somatosensory neurons encode sensory information in CIBP remains only partly explained. Using a validated rat model, we first confirmed cortical bone destruction in CIBP but not sham-operated rats (day 14 after surgery, designated "late"-stage bone cancer). This occurred with behavioural mechanical hypersensitivity (Kruskal-Wallis H for independent samples; CIBP vs sham-operated, day 14; P < 0.0001). Next, hypothesising that the proportion and phenotype of primary afferents would be altered in the disease state, dorsal root ganglia in vivo imaging of genetically encoded calcium indicators and Markov Cluster Analysis were used to analyse 1748 late-stage CIBP (n = 10) and 757 sham-operated (n = 9), neurons. Distinct clusters of responses to peripheral stimuli were revealed. In CIBP rats, upon knee compression of the leg ipsilateral to the tumour, (1) 3 times as many sensory afferents responded (repeated-measures analysis of variance: P < 0.0001 [vs sham]); (2) there were significantly more small neurons responding (Kruskal-Wallis for independent samples (vs sham): P < 0.0001); and (3) approximately 13% of traced tibial cavity afferents responded (no difference observed between CIBP and sham-operated animals). We conclude that an increased sensory afferent response is present in CIBP rats, and this is likely to reflect afferent recruitment from outside of the bone rather than increased intraosseous afferent activity.