In vivo calcium imaging shows that satellite glial cells have increased activity in painful states.

Satellite glial cells are important for proper neuronal function of primary sensory neurons for which they provide homeostatic support. Most research on satellite glial cell function has been performed with in vitro studies, but recent advances in calcium imaging and transgenic mouse models have enabled this first in vivo study of single-cell satellite glial cell function in mouse models of inflammation and neuropathic pain. We found that in naïve conditions, satellite glial cells do not respond in a time-locked fashion to neuronal firing. In painful inflammatory and neuropathic states, we detected time-locked signals in a subset of satellite glial cells, but only with suprathreshold stimulation of the sciatic nerve. Surprisingly, therefore, we conclude that most calcium signals in satellite glial cells seem to develop at arbitrary intervals not directly linked to neuronal activity patterns. More in line with expectations, our experiments also revealed that the number of active satellite glial cells was increased under conditions of inflammation or nerve injury. This could reflect the increased requirement for homeostatic support across dorsal root ganglion neuron populations, which are more active during such painful states.

Assessing spontaneous sensory neuron activity using in vivo calcium imaging.

ABSTRACT: Heightened spontaneous activity in sensory neurons is often reported in individuals living with chronic pain. It is possible to study this activity in rodents using electrophysiology, but these experiments require great skill and can be prone to bias. Here, we have examined whether in vivo calcium imaging with GCaMP6s can be used as an alternative approach. We show that spontaneously active calcium transients can be visualised in the fourth lumbar dorsal root ganglion (L4 DRG) through in vivo imaging in a mouse model of inflammatory pain. Application of lidocaine to the nerve, between the inflamed site and the DRG, silenced spontaneous firing and revealed the true baseline level of calcium for spontaneously active neurons. We used these data to train a machine learning algorithm to predict when a neuron is spontaneously active. We show that our algorithm is accurate in 2 different models of pain: intraplantar complete Freund adjuvant and antigen-induced arthritis, with accuracies of 90.0% ±1.2 and 85.9% ±2.1, respectively, assessed against visual inspection by an experienced observer. The algorithm can also detect neuronal activity in imaging experiments generated in a different laboratory using a different microscope configuration (accuracy = 94.0% ±2.2). We conclude that in vivo calcium imaging can be used to assess spontaneous activity in sensory neurons and provide a Google Colaboratory Notebook to allow anyone easy access to our novel analysis tool, for the assessment of spontaneous neuronal activity in their own imaging setups.

Enhancing research culture in academia: a spotlight on early career researchers.

This editorial highlights common challenges faced by early career researchers (ECRs) who play a crucial role in our research community. We propose that enhancing the experiences of ECRs will yield benefits to the entire scientific community and we give practical suggestions on how such improvements may be achieved.

Sodium-calcium exchanger-3 regulates pain "wind-up": From human psychophysics to spinal mechanisms.

Repeated application of noxious stimuli leads to a progressively increased pain perception; this temporal summation is enhanced in and predictive of clinical pain disorders. Its electrophysiological correlate is "wind-up," in which dorsal horn spinal neurons increase their response to repeated nociceptor stimulation. To understand the genetic basis of temporal summation, we undertook a GWAS of wind-up in healthy human volunteers and found significant association with SLC8A3 encoding sodium-calcium exchanger type 3 (NCX3). NCX3 was expressed in mouse dorsal horn neurons, and mice lacking NCX3 showed normal, acute pain but hypersensitivity to the second phase of the formalin test and chronic constriction injury. Dorsal horn neurons lacking NCX3 showed increased intracellular calcium following repetitive stimulation, slowed calcium clearance, and increased wind-up. Moreover, virally mediated enhanced spinal expression of NCX3 reduced central sensitization. Our study highlights Ca2+ efflux as a pathway underlying temporal summation and persistent pain, which may be amenable to therapeutic targeting.

Encoding of cutaneous stimuli by lamina I projection neurons.

ABSTRACT: Lamina I of the dorsal horn, together with its main output pathway, lamina I projection neurons, has long been implicated in the processing of nociceptive stimuli, as well as the development of chronic pain conditions. However, the study of lamina I projection neurons is hampered by technical challenges, including the low throughput and selection biases of traditional electrophysiological techniques. Here we report on a technique that uses anatomical labelling strategies and in vivo imaging to simultaneously study a network of lamina I projection neurons in response to electrical and natural stimuli. Although we were able to confirm the nociceptive involvement of this group of cells, we also describe an unexpected preference for innocuous cooling stimuli. We were able to characterize the thermal responsiveness of these cells in detail and found cooling responses decline when exposed to stable cold temperatures maintained for more than a few seconds, as well as to encode the intensity of the end temperature, while heating responses showed an unexpected reliance on adaptation temperatures.

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.

Cortical Mechanisms of Single-Pulse Transcranial Magnetic Stimulation in Migraine.

Single-pulse transcranial magnetic stimulation (sTMS) of the occipital cortex is an effective migraine treatment. However, its mechanism of action and cortical effects of sTMS in migraine are yet to be elucidated. Using calcium imaging and GCaMP-expressing mice, sTMS did not depolarise neurons and had no effect on vascular tone. Pre-treatment with sTMS, however, significantly affected some characteristics of the cortical spreading depression (CSD) wave, the correlate of migraine aura. sTMS inhibited spontaneous neuronal firing in the visual cortex in a dose-dependent manner and attenuated L-glutamate-evoked firing, but not in the presence of GABAA/B antagonists. In the CSD model, sTMS increased the CSD electrical threshold, but not in the presence of GABAA/B antagonists. We first report here that sTMS at intensities similar to those used in the treatment of migraine, unlike traditional sTMS applied in other neurological fields, does not excite cortical neurons but it reduces spontaneous cortical neuronal activity and suppresses the migraine aura biological substrate, potentially by interacting with GABAergic circuits.

Large Scale In Vivo Recording of Sensory Neuron Activity with GCaMP6.

Greater emphasis on the study of intact cellular networks in their physiological environment has led to rapid advances in intravital imaging of the central nervous system (CNS), while the peripheral system remains largely unexplored. To assess large networks of sensory neurons, we selectively label primary afferents with GCaMP6s in male and female C57bl/6 mice and visualize their functional responses to peripheral stimulation in vivo. We show that we are able to monitor the activity of hundreds of sensory neurons simultaneously, with sufficient sensitivity to detect, in most cases, single action potentials with a typical rise time of around 200 ms, and an exponential decay with a time constant of approximately 700 ms. With this technique we are able to characterize the responses of large populations of sensory neurons to innocuous and noxious mechanical and thermal stimuli under normal and inflammatory conditions. We demonstrate that the majority of primary afferents are polymodal with between 50-80% of thermally sensitive DRG neurons responding also to noxious mechanical stimulation. We also specifically assess the small population of peripheral cold neurons and demonstrate significant sensitization to cooling after a model of sterile and persistent inflammation, with significantly increased sensitivity already at decreases of 5°C when compared to uninflamed responses. This not only reveals interesting new insights into the (patho)physiology of the peripheral nervous system but also demonstrates the sensitivity of this imaging technique to physiological changes in primary afferents.

Immune or Genetic-Mediated Disruption of CASPR2 Causes Pain Hypersensitivity Due to Enhanced Primary Afferent Excitability.

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/- mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability.

Peripheral inflammatory pain sensitisation is independent of mast cell activation in male mice.

The immune and sensory systems are known for their close proximity and interaction. Indeed, in a variety of pain states, a myriad of different immune cells are activated and recruited, playing a key role in neuronal sensitisation. During inflammatory pain it is thought that mast cells (MC) are one of the immune cell types involved in this process, but so far the evidence outlining their direct effect on neuronal cells remains unclear. To clarify whether MC are involved in inflammatory pain states, we used a transgenic mouse line (Mctp5Cre-iDTR) in which MC could be depleted in an inducible manner by administration of diphtheria toxin. Our results show that ablation of MC in male mice did not result in any change in mechanical and thermal hypersensitivity in the CFA model of inflammatory pain. Similarly, edema and temperature triggered by CFA inflammation at the injection site remained identical in MC depleted mice compared with their littermate controls. In addition, we show that Mctp5Cre-iDTR mice display normal levels of mechanical hypersensitivity after local injection of nerve growth factor (NGF), a factor well characterised to produce peripheral sensitisation and for being upregulated upon injury and inflammation. We also demonstrate that NGF treatment in vitro does not lead to an increased level of tumor necrosis factor-α in bone marrow-derived MC. Furthermore, our qRT-PCR data reveal that MC express negligible levels of NGF receptors, thereby explaining the lack of response to NGF. Together, our data suggest that MC do not play a direct role in peripheral sensitisation during inflammatory conditions.

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis.

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective.