Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons

BACKGROUND: Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain. RESULTS: Bath-applied glutamate robustly increased [Ca2+]i in 14.4 ± 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3) 75% of cells that responded to locally-applied glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals. CONCLUSIONS: Bulk-loading fura-2 AM into spinal cord slices is a successful means for determining glutamate-evoked Ca2+ mobilization in naïve adult dorsal horn neurons. AMPA receptors mediate the majority of these responses. Peripheral neuropathic injury potentiates Ca2+ signaling in dorsal horn

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP+ neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP+ cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP+ DRG neurons expressed TRPV1, ~25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP+ neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα+ DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8+/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons

Forecasts for Dark Energy Measurements with Future HI Surveys

We use two independent methods to forecast the dark energy measurements achievable by combining future galaxy redshift surveys based on the radio HI emission line with Cosmic Microwave Background (CMB) data from the {\sl Planck} satellite. In the first method we focus on the `standard ruler' provided by the baryon acoustic oscillation (BAO) length scale. In the second method we utilize additional information encoded in the galaxy power spectrum including galaxy bias from velocity-space distortions and the growth of cosmic structure. We find that a radio synthesis array with about 10 per cent of the collecting area of the Square Kilometre Array (SKA), equipped with a wide ($10-100 ~ {\rm deg}^2$) field-of-view, would have the capacity to perform a $20{,}000 ~ {\rm deg}^2$ redshift survey to a maximum redshift $z_{\rm max} \sim 0.8$ and thereby produce dark energy measurements that are competitive with surveys likely to be undertaken by optical telescopes around 2015. There would then be powerful arguments for adding collecting area to such a `Phase-1' SKA because of the square-law scaling of survey speed with telescope sensitivity for HI surveys, compared to the linear scaling for optical redshift surveys. The full SKA telescope should, by performing a $20{,}000 ~ {\rm deg}^2$ HI redshift survey to $z_{\rm max} \sim 2$ around 2020, yield an accurate measurement of cosmological parameters independent of CMB datasets. Combining CMB ({\sl Planck}) and galaxy power spectrum (SKA) measurements will drive errors in the dark energy equation-of-state parameter $w$ well below the 1 per cent level. The major systematic uncertainty in these forecasts is the lack of direct information about the mass function of high-redshift HI-emitting galaxies.Comment: 19 pages; 2 tables; 18 figures. accepted by MNRA

Enhanced histamine-induced itch in diacylglycerol kinase iota knockout mice

Subsets of small-diameter dorsal root ganglia (DRG) neurons detect pruritogenic (itch-causing) and algogenic (pain-causing) stimuli and can be activated or sensitized by chemical mediators. Many of these chemical mediators activate receptors that are coupled to lipid hydrolysis and diacylglycerol (DAG) production. Diacylglycerol kinase iota (DGKI) can phos-phorylate DAG and is expressed at high levels in small-diameter mouse DRG neurons. Given the importance of these neurons in sensing pruritogenic and algogenic chemicals, we sought to determine if loss of DGKI impaired responses to itch- or pain-producing stimuli. Using male and female Dgki-knockout mice, we found that in vivo sensitivity to histamine—but not other pruritogens—was enhanced. In contrast, baseline pain sensitivity and pain sensitization following inflammatory or neuropathic injury were equivalent between wild type and Dgki-/- mice. In vitro calcium responses in DRG neurons to histamine was enhanced, while responses to algogenic ligands were unaffected by Dgki deletion. These data suggest Dgki regulates sensory neuron and behavioral responses to histamine, without affecting responses to other pruritogenic or algogenic agents

Ecto-5'-Nucleotidase (CD73) Inhibits Nociception by Hydrolyzing AMP to Adenosine in Nociceptive Circuits

Ecto-5’-nucleotidase (NT5E, CD73) is a membrane-anchored protein that hydrolyzes extracellular adenosine 5’-monophosphate (AMP) to adenosine in diverse tissues but has not been directly studied in nociceptive neurons. We found that NT5E was located on peptidergic and nonpeptidergic nociceptive neurons in dorsal root ganglia (DRG) and on axon terminals in lamina II (the substantia gelatinosa) of spinal cord. NT5E was also located on epidermal keratinocytes, cells of the dermis and on nociceptive axon terminals in the epidermis. Following nerve injury, NT5E protein and AMP histochemical staining were coordinately reduced in lamina II. In addition, AMP hydrolytic activity was reduced in DRG neurons and spinal cord of Nt5e−/− mice. The antinociceptive effects of AMP, when combined with the adenosine kinase inhibitor 5-iodotubericidin, were reduced by ~50% in Nt5e−/− mice and were eliminated in Adenosine A1 receptor (A1R, Adora1) knockout mice. Additionally, Nt5e−/− mice displayed enhanced sensitivity in the tail immersion assay, in the complete Freund's adjuvant (CFA) model of inflammatory pain and in the spared nerve injury (SNI) model of neuropathic pain. Collectively, our data indicate that the ectonucleotidase NT5E regulates nociception by hydrolyzing AMP to adenosine in nociceptive circuits and represents a new molecular target for the treatment of chronic pain. Moreover, our data suggest NT5E is well localized to regulate nucleotide signaling between skin cells and sensory axons

Pushmepullyou: An efficient micro-swimmer

The swimming of a pair of spherical bladders that change their volumes and mutual distance is efficient at low Reynolds numbers and is superior to other models of artificial swimmers. The change of shape resembles the wriggling motion known as {\it metaboly} of certain protozoa.Comment: Minor rephrasing and changes in style; short explanations adde

Spinal macrophages resolve nociceptive hypersensitivity after peripheral injury

Peripheral nerve injury induces long-term pro-inflammatory responses in spinal cord glial cells that facilitate neuropathic pain, but the identity of endogenous cells that resolve spinal inflammation has not been determined. Guided by single-cell RNA sequencing (scRNA-seq), we found that MRC1+ spinal cord macrophages proliferated and upregulated the anti-inflammatory mediator Cd163 in mice following superficial injury (SI; nerve intact), but this response was blunted in nerve-injured animals. Depleting spinal macrophages in SI animals promoted microgliosis and caused mechanical hypersensitivity to persist. Conversely, expressing Cd163 in spinal macrophages increased Interleukin 10 expression, attenuated micro- and astrogliosis, and enduringly alleviated mechanical and thermal hypersensitivity in nerve-injured animals. Our data indicate that MRC1+ spinal macrophages actively restrain glia to limit neuroinflammation and resolve mechanical pain following a superficial injury. Moreover, we show that spinal macrophages from nerve-injured animals mount a dampened anti-inflammatory response but can be therapeutically coaxed to promote long-lasting recovery of neuropathic pain

Testing KiDS cross-correlation redshifts with simulations

Measuring cosmic shear in wide-field imaging surveys requires accurate knowledge of the redshift distribution of all sources. The clustering-redshift technique exploits the angular cross-correlation of a target galaxy sample with unknown redshifts and a reference sample with known redshifts. It represents an attractive alternative to colour-based methods of redshift calibration. Here we test the performance of such clustering redshift measurements using mock catalogues that resemble the Kilo-Degree Survey (KiDS). These mocks are created from the MICE simulation and closely mimic the properties of the KiDS source sample and the overlapping spectroscopic reference samples. We quantify the performance of the clustering redshifts by comparing the cross-correlation results with the true redshift distributions in each of the five KiDS photometric redshift bins. Such a comparison to an informative model is necessary due to the incompleteness of the reference samples at high redshifts. Clustering mean redshifts are unbiased at |Δz|< 0.006 under these conditions. The redshift evolution of the galaxy bias of the reference and target samples represents one of the most important systematic errors when estimating clustering redshifts. It can be reliably mitigated at this level of precision using auto-correlation measurements and self-consistency relations, and will not become a dominant source of systematic error until the arrival of Stage-IV cosmic shear surveys. Using redshift distributions from a direct colour-based estimate instead of the true redshift distributions as a model for comparison with the clustering redshifts increases the biases in the mean to up to |Δz|∼0.04. This indicates that the interpretation of clustering redshifts in real-world applications will require more sophisticated (parameterised) models of the redshift distribution in the future. If such better models are available, the clustering-redshift technique promises to be a highly complementary alternative to other methods of redshift calibration

Testing KiDS cross-correlation redshifts with simulations

Measuring cosmic shear in wide-field imaging surveys requires accurate knowledge of the redshift distribution of all sources. The clustering-redshift technique exploits the angular cross-correlation of a target galaxy sample with unknown redshifts and a reference sample with known redshifts, and is an attractive alternative to colour-based methods of redshift calibration. We test the performance of such clustering redshift measurements using mock catalogues that resemble the Kilo-Degree Survey (KiDS). These mocks are created from the MICE simulation and closely mimic the properties of the KiDS source sample and the overlapping spectroscopic reference samples. We quantify the performance of the clustering redshifts by comparing the cross-correlation results with the true redshift distributions in each of the five KiDS photometric redshift bins. Such a comparison to an informative model is necessary due to the incompleteness of the reference samples at high redshifts. Clustering mean redshifts are unbiased at $|\Delta z|<0.006$ under these conditions. The redshift evolution of the galaxy bias can be reliably mitigated at this level of precision using auto-correlation measurements and self-consistency relations, and will not become a dominant source of systematic error until the arrival of Stage-IV cosmic shear surveys. Using redshift distributions from a direct colour-based estimate instead of the true redshift distributions as a model for comparison with the clustering redshifts increases the biases in the mean to up to $|\Delta z|\sim0.04$. This indicates that the interpretation of clustering redshifts in real-world applications will require more sophisticated (parameterised) models of the redshift distribution in the future. If such better models are available, the clustering-redshift technique promises to be a highly complementary alternative to other methods of redshift calibration.Comment: 21 pages, 18 figures, 10 tables, submitted to A&

Enhanced nociception in angelman syndrome model mice

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the maternal UBE3A allele. The maternal UBE3A allele is expressed in nearly all neurons of the brain and spinal cord, whereas the paternal UBE3A allele is repressed by an extremely long antisense transcript (UBE3A-ATS). Little is known about expression of UBE3A in the peripheral nervous system, where loss of maternal UBE3A might contribute to AS phenotypes. Here we sought to examine maternal and paternal Ube3a expression in DRGs neurons and to evaluate whether nociceptive responses were affected in AS model mice (global deletion of maternal Ube3a allele; Ube3am+/p+). We found that most large-diameter proprioceptive and mechanosensitive DRG neurons expressed maternal Ube3a and paternal Ube3a-ATS. In contrast, most small-diameter neurons expressed Ube3a biallelically and had low to undetectable levels of Ube3a-ATS. Analysis of single-cell DRG transcriptomes further suggested that Ube3a is expressed monoallelically in myelinated large-diameter neurons and biallelically in unmyelinated small-diameter neurons. Behavioral responses to some noxious thermal and mechanical stimuli were enhanced in male and female AS model mice; however, nociceptive responses were not altered by the conditional deletion of maternal Ube3a in the DRG. These data suggest that the enhanced nociceptive responses in AS model mice are due to loss of maternal Ube3a in the central, but not peripheral, nervous system. Our study provides new insights into sensory processing deficits associated with AS