Brainstem ADCYAP1+ neurons control multiple aspects of sickness behaviour.

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels.

Ultrasound has been used to non-invasively manipulate neuronal functions in humans and other animals. However, this approach is limited as it has been challenging to target specific cells within the brain or body. Here, we identify human Transient Receptor Potential A1 (hsTRPA1) as a candidate that confers ultrasound sensitivity to mammalian cells. Ultrasound-evoked gating of hsTRPA1 specifically requires its N-terminal tip region and cholesterol interactions; and target cells with an intact actin cytoskeleton, revealing elements of the sonogenetic mechanism. Next, we use calcium imaging and electrophysiology to show that hsTRPA1 potentiates ultrasound-evoked responses in primary neurons. Furthermore, unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to c-fos expression and contralateral limb responses in response to ultrasound delivered through an intact skull. Collectively, we demonstrate that hsTRPA1-based sonogenetics can effectively manipulate neurons within the intact mammalian brain, a method that could be used across species.

Neuropathic Pain After Spinal Cord Injury: Challenges and Research Perspectives.

Neuropathic pain is a debilitating consequence of spinal cord injury (SCI) that remains difficult to treat because underlying mechanisms are not yet fully understood. In part, this is due to limitations of evaluating neuropathic pain in animal models in general, and SCI rodents in particular. Though pain in patients is primarily spontaneous, with relatively few patients experiencing evoked pains, animal models of SCI pain have primarily relied upon evoked withdrawals. Greater use of operant tasks for evaluation of the affective dimension of pain in rodents is needed, but these tests have their own limitations such that additional studies of the relationship between evoked withdrawals and operant outcomes are recommended. In preclinical SCI models, enhanced reflex withdrawal or pain responses can arise from pathological changes that occur at any point along the sensory neuraxis. Use of quantitative sensory testing for identification of optimal treatment approach may yield improved identification of treatment options and clinical trial design. Additionally, a better understanding of the differences between mechanisms contributing to at- versus below-level neuropathic pain and neuropathic pain versus spasticity may shed insights into novel treatment options. Finally, the role of patient characteristics such as age and sex in pathogenesis of neuropathic SCI pain remains to be addressed.

Analysis of the behavioral, cellular and molecular characteristics of pain in severe rodent spinal cord injury.

Human SCI is frequently associated with chronic pain that is severe and refractory to medical therapy. Most rodent models used to assess pain outcomes in SCI apply moderate injuries to lower thoracic spinal levels, whereas the majority of human lesions are severe in degree and occur at cervical or upper thoracic levels. To better model and understand mechanisms associated with chronic pain after SCI, we subjected adult rats to T3 severe compression or complete transection lesions, and examined pain-related behaviors for three months. Within one week after injury, rats developed consistent forepaw pain-related behaviors including increased spontaneous lifts, tactile allodynia and cold sensitivity that persisted for three months. Place escape avoidance testing confirmed that withdrawal of the forepaws from a von Frey stimulus represented active pain-related aversion. Spontaneous and evoked pain-related measures were attenuated by gabapentin, further indicating that these behaviors reflect development of pain. Spinal level of injury was relevant: rats with T11 severe SCI did not exhibit forepaw pain-related behaviors. Immunoblotting and immunofluorescence of C6-C8 spinal dorsal horn, reflecting sensory innervation of the forepaw, revealed: 1) expansion of CGRP immunoreactivity in lamina I/II; 2) increased GAP-43 expression; and 3) increased IBA1, GFAP and connexin-43 expression. These findings indicate that aberrant pain fiber sprouting and gliopathy occur after severe SCI. Notably, satellite glial cells (SGCs) in C6-C8 DRGs exhibited increases in GFAP and connexin-43, suggesting ongoing peripheral sensitization. Carbenoxolone, a gap junction inhibitor, and specific peptide inhibitors of connexin-43, ameliorated established tactile allodynia after severe SCI. Collectively, severe T3 SCI successfully models persistent pain states and could constitute a useful model system for examining candidate translational pain therapies after SCI.

Local Joint inflammation and histone citrullination in a murine model of the transition from preclinical autoimmunity to inflammatory arthritis.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are characteristic of rheumatoid arthritis (RA). However, their presence years before the onset of clinical RA is perplexing. Although multiple putative citrullinated antigens have been identified, no studies have demonstrated the specific capacity of these antigens to initiate inflammatory arthritis. This study was undertaken to recapitulate the transition from preclinical to clinical RA and to demonstrate the capacity of local citrullination to facilitate this transition. METHODS: We performed proteomic analysis of activated human neutrophils to identify citrullinated proteins, including those targeted as part of the RA immune response. Using enzyme-linked immunosorbent assay, we compared RA and osteoarthritis synovial fluid for levels of citrullinated histone H2B and its immune complex. Using macrophage activation assays, we assessed the effect of histone citrullination on immunostimulatory capacity and evaluated the stimulatory capacity of native and citrullinated H2B immune complexes. Finally, we assessed the potential for anti-citrullinated H2B antibodies to mediate arthritis in vivo. RESULTS: We identified robust targeting of neutrophil-derived citrullinated histones by the ACPA immune response. More than 90% of the RA patients had anti-citrullinated H2B antibodies. Histone citrullination increased innate immunostimulatory capacity, and immune complexes containing citrullinated histones activated macrophage cytokine production and propagated neutrophil activation. Finally, we demonstrated that immunization with H2B was arthritogenic, but only in the setting of underlying articular inflammation. CONCLUSION: Our findings indicate that citrullinated histones, specifically citrullinated H2B, are an antigenic target of the ACPA immune response. Furthermore, local generation of citrullinated antigen during low-grade articular inflammation provides a mechanistic model for the conversion from preclinical autoimmunity to inflammatory arthritis.