Emergence of functional sensory subtypes as defined by transient receptor potential channel expression

The existence of heterogeneous populations of dorsal root ganglion (DRG) neurons conveying different somatosensory information is the basis for the perception of touch, temperature, and pain. A differential expression of transient receptor potential (TRP) cation channels contributes to this functional heterogeneity. However, little is known about the development of functionally diverse neuronal subpopulations. Here, we use calcium imaging of acutely dissociated mouse sensory neurons and quantitative reverse transcription PCR to show that TRP cation channels emerge in waves, with the diversification of functional groups starting at embryonic day 12.5 (E12.5) and extending well into the postnatal life. Functional responses of voltage-gated calcium channels were present in DRG neurons at E11.5 and reached adult levels by E14.5. Responses to capsaicin, menthol, and cinnamaldehyde were first seen at E12.5, E16.5, and postnatal day 0 (P0), when the mRNA for TRP cation channel, subfamily V, member 1 (TRPV1), TRP cation channel, subfamily M, member 8 (TRPM8), and TRP cation channel, subfamily A, member 1 (TRPA1), respectively, was first detected. Cold-sensitive neurons were present before the expression or functional responses of TRPM8 or TRPA1. Our data support a lineage relationship in which TRPM8- and TRPA1-expressing sensory neurons derive from the population of TRPV1-expressing neurons. The TRPA1 subpopulation of neurons emerges independently in two distinct classes of nociceptors: around birth in the peptidergic population and after P14 in the nonpeptidergic class. This indicates that neurons with similar receptive properties can be generated in different sublineages at different developmental stages. This study describes for the first time the emergence of functional subtypes of sensory neurons, providing new insight into the development of nociception and thermoreception

A Local Action of Neurotrophin-3 Prevents the Death of Proliferating Sensory Neuron Precursor Cells

AbstractThe role of neurotrophin-3 (NT-3) in early development of the dorsal root ganglion was investigated. Excessive cell death in the dorsal root ganglion of mice that carry a deleted NT-3 gene (NT-3−/− mice) preceded the period of programmed cell death, detected by the TUNEL method, and caused a reduction in the number of proliferating precursors without altering the proportion of proliferating cells to total number of neurons. Furthermore, the majority of proliferating cells detected by bromodeoxyuridine incorporation also stained with the TUNEL method. NT-3 mRNA was expressed locally in the embryonic, but not the postnatal dorsal root ganglion. Most cultured early embryonic NT-3−/− neurons died in the absence of exogenous NT-3 as did the wild-type neurons when cultured with NT-3 neutralizing antibodies, suggesting that NT-3 acts locally to prevent the death of proliferating sensory precursor cells during neurogenesis. Thus, NT-3 may inflict constraints on the number of proliferating precursor cells and thereby affect the number of neurons generated during development of the peripheral nervous system

The boundary cap: a source of neural crest stem cells that generate multiple sensory neuron subtypes

The boundary cap (BC) is a transient neural crest-derived group of cells located at the dorsal root entry zone (DREZ) that have been shown to differentiate into sensory neurons and glia in vivo. We find that when placed in culture, BC cells self-renew, show multipotency in clonal cultures and express neural crest stem cell (NCSCs) markers. Unlike sciatic nerve NCSCs, the BC-NCSC (bNCSCs) generates sensory neurons upon differentiation. The bNCSCs constitute a common source of cells for functionally diverse types of neurons, as a single bNCSC can give rise to several types of nociceptive and thermoreceptive sensory neurons. Our data suggests that BC cells comprise a source of multipotent sensory specified stem cells that persist throughout embryogenesis

The effect of cattle slurry in combination with nitrate and the nitrification inhibitor dicyandiamide on in situ nitrous oxide and dinitrogen emissions

peer-reviewedA field study was conducted to determine the effect of the nitrification inhibitor dicyandiamide (DCD) on N2O and N2 emissions after cattle slurry (CS) application in the presence of nitrate (NO3) fertiliser on seven different occasions (between March 2009 and March 2011). N2O emissions from CS in the presence of NO3 fertiliser were very high (0.4–8.7% of applied N) over a 20-day period, under mild moist conditions. Emissions were significantly larger from the CS treatment compared to an NH4+-N source, supplying the same rate of N as in the slurry. This study supports the view that organic fertilisers should not be applied at the same time as nitrate-based fertilisers, as significant increases in N2O emissions occur. The average N2O mole fraction (N2O/(N2O + N2)) over all seven application dates was 0.34 for CSNO3 compared to 0.24 for the NH4ClNO3 treatment, indicating the dominance of N2 emissions. The rate of nitrification in CSNO3 was slower than in NH4ClNO3, and DCD was found to be an effective nitrification inhibitor in both treatments. However, as N2O emissions were found to be predominantly associated with the NO3 pool, the effect of DCD in lowering N2O emissions is limited in the presence of a NO3 fertiliser. To obtain the maximum cost-benefit of DCD in lowering N2O emissions, under mild moist conditions, it should not be applied to a nitrate containing fertiliser (e.g. ammonium nitrate or calcium ammonium nitrate), and therefore the application of DCD should be restricted to ammonium-based organic or synthetic fertilisers.This research was funded by the Irish National Development Plan, through the Research Stimulus Fund (RSF 07 519), administered by the Irish Department of Agriculture, Food and the Marine

Nitrate leaching losses and the fate of 15N fertilizer in perennial intermediate wheatgrass and annual wheat — A field study

Perennial grains, such as the intermediate wheatgrass (Thinopyrum intermedium) (IWG), may reduce negative environmental effects compared to annual grain crops. Their permanent, and generally larger, root systems are likely to retain nitrogen (N) better, decreasing harmful losses of N and improving fertilizer N use efficiency, but there have been no comprehensive N fertilizer recovery studies in IWG to date. We measured fertilizer N recovery with stable isotope tracers in crop biomass and soil, soil N mineralization and nitrification, and nitrate leaching in IWG and annual wheat in a replicated block field experiment. Nitrate leaching was drastically reduced in IWG (0.1 and 3.1 kg N ha−1 yr−1) in its third and fourth year since establishment, compared with 5.6 kg N ha−1 yr−1 in annual wheat and 41.0 kg N ha−1 yr−1 in fallow respectively. There were no differences in net N mineralization or nitrification between IWG and annual wheat, though there was generally more inorganic N in the soil profile of annual wheat. More 15N fertilizer was recovered in the straw and all depths of the roots and soils in IWG than annual wheat. However, annual wheat recovered much more 15N fertilizer in the seeds compared to IWG, which had lower grain yields. 15N-labeled fertilizer contributed little

Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart

<p>Abstract</p> <p>Background</p> <p>Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance <it>ex vivo</it>, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.</p> <p>Results</p> <p>Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for <it>ex vivo </it>culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.</p> <p>Conclusion</p> <p>The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.</p

Neurotrophin-3-enhanced nerve regeneration selectively improves recovery of muscle fibers expressing myosin heavy chains 2b

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation. In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 μm, controls 70.8 μm; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types. This study indicates that NT-3–enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro–muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury

Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects.By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spa¨ tzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases

Striking parallels between carotid body glomus cell and adrenal chromaffin cell development

Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson’s disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are ‘émigrés’ from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the ‘émigré’ hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest