Multisite silicon probes enable simultaneous recording of spontaneous and evoked activity in multiple isolated C-fibres in rat saphenous nerve.

BACKGROUND: Recordings of electrical activity in nerves have provided valuable insights into normal function and pathological behaviours of the nervous system. Current high-resolution techniques (e.g. teased fibre recordings) typically utilise electrodes with a single recording site, capturing the activity of a single isolated neuron per recording. NEW METHOD: We conducted proof-of-principle C-fibre recordings in the saphenous nerve of urethane-anaesthetised adult Wistar rats using 32-channel multisite silicon electrodes. Data was acquired using the OpenEphys recording system and clustered offline with Kilosort 2.5. RESULTS: In single recordings in 5 rats, 32 units with conduction velocities in the C-fibre range (< 1 m/s) were identified via constant latency responses and classified using activity dependent slowing. In two animals, 6 C-fibres (5 classified as nociceptors) were well isolated after clustering. Their activity could be tracked throughout the recording - including during periods of spontaneous activity. Axonal conduction velocities were calculated from spontaneous activity and/or low frequency electrical stimulation using only the differences in action potential latency as it propagated past multiple probe sites. COMPARISON WITH EXISTING METHODS: Single electrode approaches have a low data yield and generating group data for specific fibre types is challenging as it requires multiple experimental subjects and recording sessions. This is particularly true when the experimental targets are the small, unmyelinated C-fibres carrying nociceptive information. CONCLUSIONS: We demonstrate that multisite recordings can greatly increase experimental yields and enhance fibre identification. The approach is of particular utility when coupled with clustering analysis. Multisite probes and analysis approaches constitute a valuable new toolbox for researchers studying the peripheral nervous system.

Transgenic Virus Resistance in Crop-Wild Cucurbita pepo Does Not Prevent Vertical Transmission of Zucchini yellow mosaic virus.

Zucchini yellow mosaic virus (ZYMV) is an economically important pathogen of cucurbits that is transmitted both horizontally and vertically. Although ZYMV is seed-transmitted in Cucurbita pepo, the potential for seed transmission in virus-resistant transgenic cultivars is not known. We crossed and backcrossed a transgenic squash cultivar with wild C. pepo, and determined whether seed-to-seedling transmission of ZYMV was possible in seeds harvested from transgenic backcrossed C. pepo. We then compared these transmission rates to those of non-transgenic (backcrossed and wild) C. pepo. The overall seed-to-seedling transmission rate in ZYMV was similar to those found in previous studies (1.37%), with no significant difference between transgenic backcrossed (2.48%) and non-transgenic (1.03%) backcrossed and wild squash. Fewer transgenic backcrossed plants had symptom development (7%) in comparison with all non-transgenic plants (26%) and may be instrumental in preventing yield reduction due to ZYMV. Our study shows that ZYMV is seed transmitted in transgenic backcrossed squash, which may affect the spread of ZYMV via the movement of ZYMV-infected seeds. Deep genome sequencing of the seed-transmitted viral populations revealed that 23% of the variants found in this study were present in other vertically transmitted ZYMV populations, suggesting that these variants may be necessary for seed transmission or are distributed geographically via seeds.

Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine.

Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections.

Zucchini yellow mosaic virus (ZYMV, Potyvirus): vertical transmission, seed infection and cryptic infections.

The role played by seed transmission in the evolution and epidemiology of viral crop pathogens remains unclear. We determined the seed infection and vertical transmission rates of zucchini yellow mosaic virus (ZYMV), in addition to undertaking Illumina sequencing of nine vertically transmitted ZYMV populations. We previously determined the seed-to-seedling transmission rate of ZYMV in Cucurbita pepo ssp. texana (a wild gourd) to be 1.6%, and herein observed a similar rate (1.8%) in the subsequent generation. We also observed that the seed infection rate is substantially higher (21.9%) than the seed-to-seedling transmission rate, suggesting that a major population bottleneck occurs during seed germination and seedling growth. In contrast, that two thirds of the variants present in the horizontally transmitted inoculant population were also present in the vertically transmitted populations implies that the bottleneck at vertical transmission may not be particularly severe. Strikingly, all of the vertically infected plants were symptomless in contrast to those infected horizontally, suggesting that vertical infection may be cryptic. Although no known virulence determining mutations were observed in the vertically infected samples, the 5' untranslated region was highly variable, with at least 26 different major haplotypes in this region compared to the two major haplotypes observed in the horizontally transmitted population. That the regions necessary for vector transmission are retained in the vertically infected populations, combined with the cryptic nature of vertical infection, suggests that seed transmission may be a significant contributor to the spread of ZYMV.

Deep sequencing reveals persistence of intra- and inter-host genetic diversity in natural and greenhouse populations of zucchini yellow mosaic virus.

The genetic diversity present in populations of RNA viruses is likely to be strongly modulated by aspects of their life history, including mode of transmission. However, how transmission mode shapes patterns of intra- and inter-host genetic diversity, particularly when acting in combination with de novo mutation, population bottlenecks and the selection of advantageous mutations, is poorly understood. To address these issues, this study performed ultradeep sequencing of zucchini yellow mosaic virus in a wild gourd, Cucurbita pepo ssp. texana, under two infection conditions: aphid vectored and mechanically inoculated, achieving a mean coverage of approximately 10 ,000×. It was shown that mutations persisted during inter-host transmission events in both the aphid vectored and mechanically inoculated populations, suggesting that the vector-imposed transmission bottleneck is not as extreme as previously supposed. Similarly, mutations were found to persist within individual hosts, arguing against strong systemic bottlenecks. Strikingly, mutations were seen to go to fixation in the aphid-vectored plants, suggestive of a major fitness advantage, but remained at low frequency in the mechanically inoculated plants. Overall, this study highlights the utility of ultradeep sequencing in providing high-resolution data capable of revealing the nature of virus evolution, particularly as the full spectrum of genetic diversity within a population may not be uncovered without sequence coverage of at least 2500-fold.

A de novo transcriptome assembly of Lucilia sericata (Diptera: Calliphoridae) with predicted alternative splices, single nucleotide polymorphisms and transcript expression estimates.

The blow fly Lucilia sericata (Diptera: Calliphoridae) (Meigen) is a nonmodel organism with no reference genome that is associated with numerous areas of research spanning the ecological, evolutionary, medical, veterinary and forensic sciences. To facilitate scientific discovery in this species, the transcriptome was assembled from more than six billion bases of Illumina and twenty-one million bases of 454 sequence derived from embryonic, larval, pupal, adult and larval salivary gland libraries. The assembly was carried out in a manner that enabled identification of putative single nucleotide polymorphisms (SNPs) and alternative splices, and that provided expression estimates for various life history stages and for salivary tissue. The assembled transcriptome was also used to identify transcribed transposable elements in L. sericata. The results of this study will enable blow fly biologists, dipterists and comparative genomicists to more rapidly develop and test molecular and genetic hypotheses, especially those regarding blow fly development and salivary gland biology.

Spontaneous firing in C-fibers and increased mechanical sensitivity in A-fibers of knee joint-associated mechanoreceptive primary afferent neurones during MIA-induced osteoarthritis in the rat.

OBJECTIVE: Osteoarthritis (OA) pain mechanisms are poorly understood. We used the monosodium iodoacetate (MIA) model of knee OA to characterize changes in excitability during the course of OA in different classes of mechanosensitive afferents projecting to joint-associated tissues, and examine whether these afferent responses and pain behavior are correlated. METHODS: Rats were injected intra-articularly with MIA (1mg in 50 µl). Hind-limb weight bearing was studied 3 (MIA3) and 14 (MIA14) days after MIA, followed by deep anesthesia and teased-nerve-fiber recordings. Spontaneous activity (SA) and mechanically evoked responses of A- and C-mechanosensitive fibers (AM and CM respectively, probably nociceptive) innervating tissues associated with the ipsilateral knee joint were examined. RESULTS: MIA3 and MIA14 rats exhibited reduced ipsilateral weight bearing. SA (>0.02 impulses/s) occurred in ∼50% of CMs from MIA rats vs 0% in normals. SA firing rates in CMs were significantly higher than normal; decreased weight bearing was correlated with increased CM SA rates. Neither percentages of AMs with SA (20%) nor their firing rates (0-0.01 impulses/s) significantly increased after MIA. In contrast, in MIA rats AMs, but not CMs, exhibited decreased mechanical thresholds and increased firing rates in response to suprathreshold mechanical stimulation. CONCLUSIONS: These findings of increased SA firing rate in CMs but not AMs and increased mechanical sensitivity of AMs, but not CMs, have not previously been reported. These are two distinct important physiological mechanisms that may underpin spontaneous pain (CMs) and stimulus-evoked pain (AMs) in OA. Our data contribute to a mechanism-based understanding of OA pain.

Transient receptor potential channel A1 and noxious cold responses in rat cutaneous nociceptors.

The role of transient receptor potential channel A1 (TRPA1) in noxious cold sensation remains unclear. Some data support the hypothesis that TRPA1 is a transducer of noxious cold whilst other data contest it. In this study we investigated the role of TRPA1 in cold detection in cutaneous nociceptors in vivo using complementary experimental approaches. We used noxious withdrawal reflex electromyography, and single fibre recordings in vivo, to test the hypothesis that TRPA1-expressing primary afferents mediate noxious cold responses in anaesthetised rats. TRPV1 and TRPM8 agonists sensitise their cognate receptors to heat and cold stimuli respectively. Herein we show that the TRPA1 agonist cinnamaldehyde applied to the skin in anaesthetised rats did not sensitise noxious cold evoked hind limb withdrawal. In contrast, cinnamaldehyde did sensitise the C fibre-mediated noxious heat withdrawal, indicated by a significant drop in the withdrawal temperature. TRPA1 agonist thus sensitised the noxious reflex withdrawal to heat, but not cold. Thermal stimuli also sensitise transient receptor potential (TRP) channels to agonist. Activity evoked by capsaicin in teased primary afferent fibres showed a significant positive correlation with receptive field temperature, in both normal and Freund's complete adjuvant-induced cutaneous inflammation. Altering the temperature of the receptive field did not modulate TRPA1 agonist evoked-activity in cutaneous primary afferents, in either normal or inflamed skin. In addition, block of the TRPA1 channel with Ruthenium Red did not inhibit cold evoked activity in either cinnamaldehyde sensitive or insensitive cold responsive nociceptors. In cinnamaldehyde-sensitive-cold-sensitive afferents, although TRPA1 agonist-evoked activity was totally abolished by Ruthenium Red, cold evoked activity was unaffected by channel blockade. We conclude that these results do not support the hypothesis that TRPA1-expressing cutaneous afferents play an important role in noxious cold responses.