Parabrachial Calca neurons drive nociplasticity.

Pain that persists beyond the time required for tissue healing and pain that arises in the absence of tissue injury, collectively referred to as nociplastic pain, are poorly understood phenomena mediated by plasticity within the central nervous system. The parabrachial nucleus (PBN) is a hub that relays aversive sensory information and appears to play a role in nociplasticity. Here, by preventing PBN Calca neurons from releasing neurotransmitters, we demonstrate that activation of Calca neurons is necessary for the manifestation and maintenance of chronic pain. Additionally, by directly stimulating Calca neurons, we demonstrate that Calca neuron activity is sufficient to drive nociplasticity. Aversive stimuli of multiple sensory modalities, such as exposure to nitroglycerin, cisplatin, or lithium chloride, can drive nociplasticity in a Calca-neuron-dependent manner. Aversive events drive nociplasticity in Calca neurons in the form of increased activity and excitability; however, neuroplasticity also appears to occur in downstream circuitry.

Parabrachial Calca neurons drive nociplasticity.

Pain that persists beyond the time required for tissue healing and pain that arises in the absence of tissue injury are poorly understood phenomena mediated by plasticity within the central nervous system. The parabrachial nucleus (PBN) is a hub that relays aversive sensory information and appears to play a role in nociplasticity. Here, by preventing PBN Calca neurons from releasing neurotransmitter or directly stimulating them we demonstrate that activation of Calca neurons is both necessary for the manifestation of chronic pain after nerve ligation and is sufficient to drive nociplasticity in wild-type mice. Aversive stimuli such as exposure to nitroglycerin, cisplatin, or LiCl can drive nociplasticity in a Calca-neuron-dependent manner. Calcium fluorescence imaging reveals that nitroglycerin activates PBN Calca neurons and potentiates their responses to mechanical stimulation. The activity and excitability of Calca neurons increased for several days after aversive events, but prolonged nociplasticity likely occurs in downstream circuitry.

A novel small molecule, AS1, reverses the negative hedonic valence of noxious stimuli.

BACKGROUND: Pain is the primary reason people seek medical care, with chronic pain affecting ~ 20% of people in the USA. However, many existing analgesics are ineffective in treating chronic pain, while others (e.g., opioids) have undesirable side effects. Here, we describe the screening of a small molecule library using a thermal place aversion assay in larval zebrafish to identify compounds that alter aversion to noxious thermal stimuli and could thus serve as potential analgesics. RESULTS: From our behavioral screen, we discovered a small molecule, Analgesic Screen 1 (AS1), which surprisingly elicited attraction to noxious painful heat. When we further explored the effects of this compound using other behavioral place preference assays, we found that AS1 was similarly able to reverse the negative hedonic valence of other painful (chemical) and non-painful (dark) aversive stimuli without being inherently rewarding. Interestingly, targeting molecular pathways canonically associated with analgesia did not replicate the effects of AS1. A neuronal imaging assay revealed that clusters of dopaminergic neurons, as well as forebrain regions located in the teleost equivalent of the basal ganglia, were highly upregulated in the specific context of AS1 and aversive heat. Through a combination of behavioral assays and pharmacological manipulation of dopamine circuitry, we determined that AS1 acts via D1 dopamine receptor pathways to elicit this attraction to noxious stimuli. CONCLUSIONS: Together, our results suggest that AS1 relieves an aversion-imposed "brake" on dopamine release, and that this unique mechanism may provide valuable insight into the development of new valence-targeting analgesic drugs, as well as medications for other valence-related neurological conditions, such as anxiety and post-traumatic stress disorder (PTSD).

Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.

Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.

A zebrafish and mouse model for selective pruritus via direct activation of TRPA1.

Little is known about the capacity of lower vertebrates to experience itch. A screen of itch-inducing compounds (pruritogens) in zebrafish larvae yielded a single pruritogen, the TLR7 agonist imiquimod, that elicited a somatosensory neuron response. Imiquimod induced itch-like behaviors in zebrafish distinct from those induced by the noxious TRPA1 agonist, allyl isothiocyanate. In the zebrafish, imiquimod-evoked somatosensory neuronal responses and behaviors were entirely dependent upon TRPA1, while in the mouse TRPA1 was required for the direct activation of somatosensory neurons and partially responsible for behaviors elicited by this pruritogen. Imiquimod was found to be a direct but weak TRPA1 agonist that activated a subset of TRPA1 expressing neurons. Imiquimod-responsive TRPA1 expressing neurons were significantly more sensitive to noxious stimuli than other TRPA1 expressing neurons. Together, these results suggest a model for selective itch via activation of a specialized subpopulation of somatosensory neurons with a heightened sensitivity to noxious stimuli.

An ancient neurotrophin receptor code; a single Runx/Cbfß complex determines somatosensory neuron fate specification in zebrafish.

In terrestrial vertebrates such as birds and mammals, neurotrophin receptor expression is considered fundamental for the specification of distinct somatosensory neuron types where TrkA, TrkB and TrkC specify nociceptors, mechanoceptors and proprioceptors/mechanoceptors, respectively. In turn, Runx transcription factors promote neuronal fate specification by regulating neurotrophin receptor and sensory receptor expression where Runx1 mediates TrkA+ nociceptor diversification while Runx3 promotes a TrkC+ proprioceptive/mechanoceptive fate. Here, we report in zebrafish larvae that orthologs of the neurotrophin receptors in contrast to terrestrial vertebrates mark overlapping and distinct subsets of nociceptors suggesting that TrkA, TrkB and TrkC do not intrinsically promote nociceptor, mechanoceptor and proprioceptor/mechanoceptor neuronal fates, respectively. While we find that zebrafish Runx3 regulates nociceptors in contrast to terrestrial vertebrates, it shares a conserved regulatory mechanism found in terrestrial vertebrate proprioceptors/mechanoceptors in which it promotes TrkC expression and suppresses TrkB expression. We find that Cbfß, which enhances Runx protein stability and affinity for DNA, serves as an obligate cofactor for Runx in neuronal fate determination. High levels of Runx can compensate for the loss of Cbfß, indicating that in this context Cbfß serves solely as a signal amplifier of Runx activity. Our data suggests an alteration/expansion of the neurotrophin receptor code of sensory neurons between larval teleost fish and terrestrial vertebrates, while the essential roles of Runx/Cbfß in sensory neuron cell fate determination while also expanded are conserved.