Contribution of mechanoreceptors to spinal cord injury-induced mechanical allodynia.

ABSTRACT: Evidence from previous studies supports the concept that spinal cord injury (SCI)-induced neuropathic pain (NP) has its neural roots in the peripheral nervous system. There is uncertainty about how and to which degree mechanoreceptors contribute. Sensorimotor activation-based interventions (eg, treadmill training) have been shown to reduce NP after experimental SCI, suggesting transmission of pain-alleviating signals through mechanoreceptors. The aim of the present study was to understand the contribution of mechanoreceptors with respect to mechanical allodynia in a moderate mouse contusion SCI model. After genetic ablation of tropomyosin receptor kinase B expressing mechanoreceptors before SCI, mechanical allodynia was reduced. The identical genetic ablation after SCI did not yield any change in pain behavior. Peptidergic nociceptor sprouting into lamina III/IV below injury level as a consequence of SCI was not altered by either mechanoreceptor ablation. However, skin-nerve preparations of contusion SCI mice 7 days after injury yielded hyperexcitability in nociceptors, not in mechanoreceptors, which makes a substantial direct contribution of mechanoreceptors to NP maintenance unlikely. Complementing animal data, quantitative sensory testing in human SCI subjects indicated reduced mechanical pain thresholds, whereas the mechanical detection threshold was not altered. Taken together, early mechanoreceptor ablation modulates pain behavior, most likely through indirect mechanisms. Hyperexcitable nociceptors seem to be the main drivers of SCI-induced NP. Future studies need to focus on injury-derived factors triggering early-onset nociceptor hyperexcitability, which could serve as targets for more effective therapeutic interventions.

Neuron-astrocyte metabolic coupling facilitates spinal plasticity and maintenance of inflammatory pain.

Long-lasting pain stimuli can trigger maladaptive changes in the spinal cord, reminiscent of plasticity associated with memory formation. Metabolic coupling between astrocytes and neurons has been implicated in neuronal plasticity and memory formation in the central nervous system, but neither its involvement in pathological pain nor in spinal plasticity has been tested. Here we report a form of neuroglia signalling involving spinal astrocytic glycogen dynamics triggered by persistent noxious stimulation via upregulation of the Protein Targeting to Glycogen (PTG) in spinal astrocytes. PTG drove glycogen build-up in astrocytes, and blunting glycogen accumulation and turnover by Ptg gene deletion reduced pain-related behaviours and promoted faster recovery by shortening pain maintenance in mice. Furthermore, mechanistic analyses revealed that glycogen dynamics is a critically required process for maintenance of pain by facilitating neuronal plasticity in spinal lamina 1 neurons. In summary, our study describes a previously unappreciated mechanism of astrocyte-neuron metabolic communication through glycogen breakdown in the spinal cord that fuels spinal neuron hyperexcitability.

SUMOylation Modulates Reactive Oxygen Species (ROS) Levels and Acts as a Protective Mechanism in the Type 2 Model of Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is the prevalent type of peripheral neuropathy; it primarily impacts extremity nerves. Its multifaceted nature makes the molecular mechanisms of diabetic neuropathy intricate and incompletely elucidated. Several types of post-translational modifications (PTMs) have been implicated in the development and progression of DPN, including phosphorylation, glycation, acetylation and SUMOylation. SUMOylation involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, and it plays a role in various cellular processes, including protein localization, stability, and function. While the specific relationship between high blood glucose and SUMOylation is not extensively studied, recent evidence implies its involvement in the development of DPN in type 1 diabetes. In this study, we investigated the impact of SUMOylation on the onset and progression of DPN in a type 2 diabetes model using genetically modified mutant mice lacking SUMOylation, specifically in peripheral sensory neurons (SNS-Ubc9-/-). Behavioural measurement for evoked pain, morphological analyses of nerve fibre loss in the epidermis, measurement of reactive oxygen species (ROS) levels, and antioxidant molecules were analysed over several months in SUMOylation-deficient and control mice. Our longitudinal analysis at 30 weeks post-high-fat diet revealed that SNS-Ubc9-/- mice exhibited earlier and more pronounced thermal and mechanical sensation loss and accelerated intraepidermal nerve fibre loss compared to control mice. Mechanistically, these changes are associated with increased levels of ROS both in sensory neuronal soma and in peripheral axonal nerve endings in SNS-Ubc9-/- mice. In addition, we observed compromised detoxifying potential, impaired respiratory chain complexes, and reduced levels of protective lipids in sensory neurons upon deletion of SUMOylation in diabetic mice. Importantly, we also identified mitochondrial malate dehydrogenase (MDH2) as a SUMOylation target, the activity of which is negatively regulated by SUMOylation. Our results indicate that SUMOylation is an essential neuroprotective mechanism in sensory neurons in type 2 diabetes, the deletion of which causes oxidative stress and an impaired respiratory chain, resulting in energy depletion and subsequent damage to sensory neurons.

Mast cells link immune sensing to antigen-avoidance behaviour.

The physiological functions of mast cells remain largely an enigma. In the context of barrier damage, mast cells are integrated in type 2 immunity and, together with immunoglobulin E (IgE), promote allergic diseases. Allergic symptoms may, however, facilitate expulsion of allergens, toxins and parasites and trigger future antigen avoidance1-3. Here, we show that antigen-specific avoidance behaviour in inbred mice4,5 is critically dependent on mast cells; hence, we identify the immunological sensor cell linking antigen recognition to avoidance behaviour. Avoidance prevented antigen-driven adaptive, innate and mucosal immune activation and inflammation in the stomach and small intestine. Avoidance was IgE dependent, promoted by Th2 cytokines in the immunization phase and by IgE in the execution phase. Mucosal mast cells lining the stomach and small intestine rapidly sensed antigen ingestion. We interrogated potential signalling routes between mast cells and the brain using mutant mice, pharmacological inhibition, neural activity recordings and vagotomy. Inhibition of leukotriene synthesis impaired avoidance, but overall no single pathway interruption completely abrogated avoidance, indicating complex regulation. Collectively, the stage for antigen avoidance is set when adaptive immunity equips mast cells with IgE as a telltale of past immune responses. On subsequent antigen ingestion, mast cells signal termination of antigen intake. Prevention of immunopathology-causing, continuous and futile responses against per se innocuous antigens or of repeated ingestion of toxins through mast-cell-mediated antigen-avoidance behaviour may be an important arm of immunity.

Primary somatosensory cortex bidirectionally modulates sensory gain and nociceptive behavior in a layer-specific manner.

The primary somatosensory cortex (S1) is a hub for body sensation of both innocuous and noxious signals, yet its role in somatosensation versus pain is debated. Despite known contributions of S1 to sensory gain modulation, its causal involvement in subjective sensory experiences remains elusive. Here, in mouse S1, we reveal the involvement of cortical output neurons in layers 5 (L5) and 6 (L6) in the perception of innocuous and noxious somatosensory signals. We find that L6 activation can drive aversive hypersensitivity and spontaneous nocifensive behavior. Linking behavior to neuronal mechanisms, we find that L6 enhances thalamic somatosensory responses, and in parallel, strongly suppresses L5 neurons. Directly suppressing L5 reproduced the pronociceptive phenotype induced by L6 activation, suggesting an anti-nociceptive function for L5 output. Indeed, L5 activation reduced sensory sensitivity and reversed inflammatory allodynia. Together, these findings reveal a layer-specific and bidirectional role for S1 in modulating subjective sensory experiences.

Prefrontal engrams of long-term fear memory perpetuate pain perception.

A painful episode can lead to a life-long increase in an individual's experience of pain. Fearful anticipation of imminent pain could play a role in this phenomenon, but the neurobiological underpinnings are unclear because fear can both suppress and enhance pain. Here, we show in mice that long-term associative fear memory stored in neuronal engrams in the prefrontal cortex determines whether a painful episode shapes pain experience later in life. Furthermore, under conditions of inflammatory and neuropathic pain, prefrontal fear engrams expand to encompass neurons representing nociception and tactile sensation, leading to pronounced changes in prefrontal connectivity to fear-relevant brain areas. Conversely, silencing prefrontal fear engrams reverses chronically established hyperalgesia and allodynia. These results reveal that a discrete subset of prefrontal cortex neurons can account for the debilitating comorbidity of fear and chronic pain and show that attenuating the fear memory of pain can alleviate chronic pain itself.

Quanty-cFOS, a Novel ImageJ/Fiji Algorithm for Automated Counting of Immunoreactive Cells in Tissue Sections.

Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes' expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called 'Quanty-cFOS', with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells.

Layer-specific pain relief pathways originating from primary motor cortex.

The primary motor cortex (M1) is involved in the control of voluntary movements and is extensively mapped in this capacity. Although the M1 is implicated in modulation of pain, the underlying circuitry and causal underpinnings remain elusive. We unexpectedly unraveled a connection from the M1 to the nucleus accumbens reward circuitry through a M1 layer 6-mediodorsal thalamus pathway, which specifically suppresses negative emotional valence and associated coping behaviors in neuropathic pain. By contrast, layer 5 M1 neurons connect with specific cell populations in zona incerta and periaqueductal gray to suppress sensory hypersensitivity without altering pain affect. Thus, the M1 employs distinct, layer-specific pathways to attune sensory and aversive-emotional components of neuropathic pain, which can be exploited for purposes of pain relief.

Prolonged Suppression of Neuropathic Hypersensitivity upon Neurostimulation of the Posterior Insula in Mice.

Neurostimulation-based therapeutic approaches are emerging as alternatives to pharmacological drugs, but need further development to optimize efficacy and reduce variability. Despite its key relevance to pain, the insular cortex has not been explored in cortical neurostimulation approaches. Here, we developed an approach to perform repetitive transcranial direct current stimulation of the posterior insula (PI tDCS) and studied its impact on sensory and aversive components of neuropathic pain and pain-related anxiety and the underlying neural circuitry in mice using behavioral methods, pharmacological interventions and the expression of the activity-induced gene product, Fos. We observed that repetitive PI tDCS strongly attenuates the development of neuropathic mechanical allodynia and also reverses chronically established mechanical and cold allodynia for several weeks post-treatment by employing descending opioidergic antinociceptive pathways. Pain-related anxiety, but not pain-related aversion, were inhibited by PI tDCS. These effects were associated with a long-term suppression in the activity of key areas involved in pain modulation, such as the cingulate, prefrontal and motor cortices. These data uncover the significant potential of targeting the insular cortex with the objective of pain relief and open the way for more detailed mechanistic analyses that will contribute to improving cortical neurostimulation therapies for use in the clinical management of pain.

Genetic Mouse Models to Study Pancreatic Cancer-Induced Pain and Reduction in Well-Being.

In addition to the poor prognosis, excruciating abdominal pain is a major challenge in pancreatic cancer. Neurotropism appears to be the underlying mechanism leading to neuronal invasion. However, there is a lack of animal models suitable for translationally bridging in vitro findings with clinical trials. We characterized KPC (KrasG12D/+; Trp53R172H/+; P48-Cre) and KPPC (KrasG12D/+; Trp53R172H/R172H; P48-Cre) mice with genetically determined pancreatic ductal adenocarcinoma (PDAC) and compared them with an orthotopic pancreatic cancer mouse model, healthy littermates and human tissue. We analyzed behavioral correlates of cancer-associated pain and well-being, and studied neuronal remodeling and cytokine expression. Histologically, we found similarities between KPC and KPPC tissue with human samples. Compared to healthy littermates, we detect nerve fiber hypertrophy, which was not restricted to a certain fiber type. Interestingly, while KPPC mice showed significantly reduced well-being, KPC mice emerged to be better suited for studying long-lasting cancer pain that emerges over a slow course of tumor progression. To address the neuroinflammatory correlate of loss of well-being, we studied cytokine levels in KPPC mice and observed a significant upregulation of CXCL16, TNFRSF5, CCL24, CXCL1, CCL22, CLL20 and CX2CL1. In summary, we demonstrate that the KPC mouse model is best suited to studying cancer pain, whereas the KPPC model can be employed to study cancer-associated reduction in well-being.

Cholinergic basal forebrain nucleus of Meynert regulates chronic pain-like behavior via modulation of the prelimbic cortex.

The basal nucleus of Meynert (NBM) subserves critically important functions in attention, arousal and cognition via its profound modulation of neocortical activity and is emerging as a key target in Alzheimer's and Parkinson's dementias. Despite the crucial role of neocortical domains in pain perception, however, the NBM has not been studied in models of chronic pain. Here, using in vivo tetrode recordings in behaving mice, we report that beta and gamma oscillatory activity is evoked in the NBM by noxious stimuli and is facilitated at peak inflammatory pain-like behavior. Optogenetic and chemogenetic cell-specific, reversible manipulations of NBM cholinergic-GABAergic neurons reveal their role in endogenous control of nociceptive hypersensitivity, which are manifest via projections to the prelimbic cortex, resulting in layer 5-mediated antinociception. Our data unravel the importance of the NBM in top-down control of neocortical processing of pain-like behavior.

Protecting against summation of pain.

Temporal summation in the spinal cord is linked to pathological pain. In a translational genetic association study in this issue of Neuron, Trendafilova et al. (2022) identify the sodium-calcium exchanger 3 as a negative regulator of temporal summation and hypersensitivity via its modulation of calcium homeostasis.

Sounding out pain.

A circuit for sound-induced analgesia has been found in the mouse brain.

Identification and quantification of nociceptive Schwann cells in mice with and without Streptozotocin-induced diabetes.

Specialized cutaneous Schwann cells (SCs), termed nociceptive SCs, were recently discovered. Their function is not fully understood, but they are believed not only to support peripheral axons in mouse skin by forming a mesh-like neural-glio networking structure in subepidermal area, but also contributing to transduction of mechanical sensation and neuropathic pain. Diabetic neuropathy (DPN) is one of the most common complication of diabetes, however, the mechanisms behind painful and painless DPN remain unclear. Using a mouse model of DPN, we want to investigate if there are quantitative differences in nociceptive SC density between the condition of hyperglycemia-induced sensory abnormalities and control condition and at which stage in the disease the damage occurs. Here, we developed a set of counting rules for nociceptive SCs based on immunofluorescent staining, and applied the method to quantify the density of nociceptive SCs in control mice (n = 10), mice with nociceptive hypersensitivity at early diabetic stage (n = 5), and mice with sensory hyposensitivity at late diabetic stage (n = 5) in the Streptozotocin (STZ) model of type 1 diabetes. Nociceptive SCs were identified as S100+/Sox10+/DAPI+ cells abutting to peripheral nerves, with the somas located within 25 µm depth in the subepidermal area and outside glands and large fiber bundles. Hypersensitive diabetic mice had decreased nociceptive SC density, despite having normal epidermal nerve fiber density, compared with age-matched control mice (P = 0.023). In contrast, there was a reduction in intraepidermal nerve fiber density but no difference in nociceptive SC density between hyposensitive diabetic mice and the age-matched control mice. This study provides a detailed description of how to identify and quantify nociceptive SC and demonstrates that nociceptive SC density declines before nerve fiber deterioration, which supports previous observations that nociceptive SCs are critical for maintenance of cutaneous sensory nerves.

Neuropathic pain caused by miswiring and abnormal end organ targeting.

Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together1-3. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury4-7-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients.

Organic anion transporter 1 is an HDAC4-regulated mediator of nociceptive hypersensitivity in mice.

Persistent pain is sustained by maladaptive changes in gene transcription resulting in altered function of the relevant circuits; therapies are still unsatisfactory. The epigenetic mechanisms and affected genes linking nociceptive activity to transcriptional changes and pathological sensitivity are unclear. Here, we found that, among several histone deacetylases (HDACs), synaptic activity specifically affects HDAC4 in murine spinal cord dorsal horn neurons. Noxious stimuli that induce long-lasting inflammatory hypersensitivity cause nuclear export and inactivation of HDAC4. The development of inflammation-associated mechanical hypersensitivity, but neither acute nor basal sensitivity, is impaired by the expression of a constitutively nuclear localized HDAC4 mutant. Next generation RNA-sequencing revealed an HDAC4-regulated gene program comprising mediators of sensitization including the organic anion transporter OAT1, known for its renal transport function. Using pharmacological and molecular tools to modulate OAT1 activity or expression, we causally link OAT1 to persistent inflammatory hypersensitivity in mice. Thus, HDAC4 is a key epigenetic regulator that translates nociceptive activity into sensitization by regulating OAT1, which is a potential target for pain-relieving therapies.

Presynaptic NMDARs on spinal nociceptor terminals state-dependently modulate synaptic transmission and pain.

Postsynaptic NMDARs at spinal synapses are required for postsynaptic long-term potentiation and chronic pain. However, how presynaptic NMDARs (PreNMDARs) in spinal nociceptor terminals control presynaptic plasticity and pain hypersensitivity has remained unclear. Here we report that PreNMDARs in spinal nociceptor terminals modulate synaptic transmission in a nociceptive tone-dependent manner. PreNMDARs depresses presynaptic transmission in basal state, while paradoxically causing presynaptic potentiation upon injury. This state-dependent modulation is dependent on Ca2+ influx via PreNMDARs. Small conductance Ca2+-activated K+ (SK) channels are responsible for PreNMDARs-mediated synaptic depression. Rather, tissue inflammation induces PreNMDARs-PKG-I-dependent BDNF secretion from spinal nociceptor terminals, leading to SK channels downregulation, which in turn converts presynaptic depression to potentiation. Our findings shed light on the state-dependent characteristics of PreNMDARs in spinal nociceptor terminals on modulating nociceptive transmission and revealed a mechanism underlying state-dependent transition. Moreover, we identify PreNMDARs in spinal nociceptor terminals as key constituents of activity-dependent pain sensitization.

Data management strategy for a collaborative research center.

The importance of effective research data management (RDM) strategies to support the generation of Findable, Accessible, Interoperable, and Reusable (FAIR) neuroscience data grows with each advance in data acquisition techniques and research methods. To maximize the impact of diverse research strategies, multidisciplinary, large-scale neuroscience research consortia face a number of unsolved challenges in RDM. While open science principles are largely accepted, it is practically difficult for researchers to prioritize RDM over other pressing demands. The implementation of a coherent, executable RDM plan for consortia spanning animal, human, and clinical studies is becoming increasingly challenging. Here, we present an RDM strategy implemented for the Heidelberg Collaborative Research Consortium. Our consortium combines basic and clinical research in diverse populations (animals and humans) and produces highly heterogeneous and multimodal research data (e.g., neurophysiology, neuroimaging, genetics, behavior). We present a concrete strategy for initiating early-stage RDM and FAIR data generation for large-scale collaborative research consortia, with a focus on sustainable solutions that incentivize incremental RDM while respecting research-specific requirements.

Neurogenesis in the adult brain functionally contributes to the maintenance of chronic neuropathic pain.

Maladaptive adult neurogenesis in the mammalian brain has been associated with diverse behaviors including disrupted learning, negative mood disorders and psychiatric conditions. However, its functional role in the generation and maintenance of chronic pathological pain has not yet been elucidated. Using an inducible genetic deletion in vivo mouse model, different behavioural paradigms and home cage monitoring systems, we show that an absence of adult neurogenesis does not impact the development of neuropathic injury-induced peripheral nociceptive hypersensitivity, but rather promotes the recovery of pathological pain as well as improves parameters associated with the state of well-being of the injured mice. These results provide a mechanistic insight into the mechanisms of chronic pain and implicate neurogenic processes as a potential therapeutic target for reducing pain and improving the quality of life for patients.