Contribution of central sensitization to the pain-related abnormal activity in neuropathic rats

The possibility of different contributions from peripheral and central sensitization to distinct neuropathic pain syndromes has been studied in rats with chronic constriction of the sciatic nerve (CCI), showing positive behavioral signs of neuropathic pain. In anesthetized, paralyzed rats extracellular recordings were performed in the spinal sciatic afferent territory (L5-L6), ipsilateral to the injured nerve, from wide dynamic range (WDR) neurons. The spontaneous activity and the responses to noxious stimuli applied to the proper area, i.e., the skin innervated by the constricted sciatic nerve, and to "inappropriate" areas, like the tail and the area of skin supplied by the contralateral sciatic and saphenous nerves, were analyzed before and after input from the constricted nerve was reversibly blocked at the ganglionic level by local anesthetic. The neurons discharged spontaneously with high frequencies, and responded to the stimulation of proper and "inappropriate" areas with high frequency discharge and prolonged afterdischarges. During the ganglionic block, confirmed by the lack of responses to proper area stimulation, the WDR neuron background activity was significantly reduced; the responses to all "inappropriate" afferences were present, the frequency discharges being comparable to the preblock ones while the afterdischarges were significantly shorter. Since the efficacy of "inappropriate" inputs is related to neuronal sensitization, the persistence of these responses indicates that central neurons remain sensitized during peripheral block. In view of the relationship between the examined spontaneous and stimulated activities and neuropathic pain symptoms, the data suggest that central sensitization contributes with different drive strength to such symptoms, playing a crucial role in extraterritorial pain.     

Contralateral input modulates the excitability of dorsal horn neurons involved in noxious signal processes. Potential role in neuronal sensitization

Wide Dynamic Range (WDR) neurons in the spinal cord receive inputs from the contralateral side that, under normal conditions, are ineffective in generating an active response. These inputs are effective when the target WDRs change their excitability conditions. To further reveal the mechanisms supporting this effectiveness shift, we investigated the weight of the excitation of the contralateral neurons on the target WDR responses. In the circuit of presynaptic (sending) and postsynaptic (receiving) neurons in crossed spinal connections the fibres that form the presynaptic neurons impinge on postsynaptic neurons can be considered the final relay of this contralateral pathway. The enhancement of the presynaptic neuron excitability may thus modify the efficacy of the contralateral input. Pairs of neurons each on a side of the spinal cord, at the L5–L6 lumbar level were simultaneously recorded in intact, anaesthetized, paralysed rats. The excitatory aminoacid NMDA and strychnine, the antagonist of the inhibitory aminoacid glycine, were iontophoretically administrated to presynaptic neurons to increase their excitability. Before and during the drug administration, spontaneous and noxious-evoked activities of the neurons were analysed. During the iontophoresis of the two substances we found that noxious stimuli applied to the receptive field of presynaptic neurons activated up to 50% of the previously unresponsive postsynaptic neurons on the opposite side. Furthermore, the neurons on both sides of the spinal cord showed significantly increased spontaneous activity and amplified responses to ipsilateral noxious stimulation. These findings indicate that the contralateral input participates in the circuit dynamics of spinal nociceptive transmission, by modulating the excitability of the postsynaptic neurons. A possible functional role of such a nociceptive transmission circuit in neuronal sensitization following unilateral nerve injury is hypothesized.     

A physiological and molecular study of the effects of nickel deficiency and phenylphosphorodiamidate (PPD) application on urea metabolism in oilseed rape (Brassica napus L.)

Background and aims

Urea is the major nitrogen (N) form supplied as fertilizer in agriculture. However, urease, a nickel-dependent enzyme, allows plants to use external or internally generated urea as a nitrogen source. Since a urease inhibitor is frequently applied in conjunction with urea fertilizer, the N-metabolism of plants may be affected. The aim of this study was to determine physiological and molecular effects of nickel deficiency and a urease inhibitor on urea uptake and assimilation in oilseed rape.

Methods

Plants were grown on hydroponic solution with urea as the sole N source under three treatments: plants treated with nickel (+Ni) as a control, without nickel (?Ni) and with nickel and phenylphosphorodiamidate (+Ni+PPD). Urea transport and assimilation were investigated.

Results

The results show that Ni-deficiency or PPD supply led to reduced growth and reduced ¹⁵N-uptake from urea. This effect was more pronounced in PPD-treated plants, which accumulated high amounts of urea and ammonium. Thus, Ni-deficiency or addition of PPD, limit the availability of N and decreased shoot and root amino acid content. The up-regulation of BnDUR3 in roots indicated that this gene is a component of the stress response to nitrogen-deficiency. A general decline of glutamine synthetase (GS) activity and activation of glutamate dehydrogenase (GDH) and increases in its expression level were observed in control plants. At the same time, in (?N) or (+Ni+PPD) treated plants, no increases in GS or GDH activities and expression level were found.

Conclusions

Overall results showed that plants require Ni as a nutrient (while most widely used nutrient solutions are devoid of Ni), whether they are grown with or without a urea supply, and that urease inhibitors may have deleterious effects at least in hydroponic grown oilseed rape.     

Loss of Starch Granule Initiation Has a Deleterious Effect on the Growth of Arabidopsis Plants Due to an Accumulation of ADP-Glucose

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.The metabolism of starch plays an essential role in the physiology of plants. Starch breakdown provides the plant with carbon skeletons and energy when the photosynthetic machinery is inactive (transitory starch) or in the processes of germination and sprouting (storage starch). Deficiencies in the accumulation of transitory starch in Arabidopsis (Arabidopsis thaliana) have been described previously, specifically in mutants affected in the plastidial phosphoglucomutase (PGM1) or the small subunit (APS1) of the ADP-Glc pyrophosphorylase (AGPase). While they are described as “starchless,” they actually contain small amounts of starch (1%–2% of the wild-type levels; Streb et al., 2009) and share similar phenotypic alterations, such as growth retardation when cultivated under a short-day photoregime and increased levels of soluble sugars during the light phase and reduced levels during the night (Caspar et al., 1985; Lin et al., 1988b; Schulze et al., 1991). Carbon partitioning is altered in these plants. As photosynthate cannot be accumulated as starch, it is diverted via hexose phosphates in the cytosol to the synthesis of Suc, which accumulates together with the hexose sugars, Glc and Fru (Caspar et al., 1985). In Arabidopsis, there are five starch synthase isoforms: one granule-bound starch synthase and four soluble starch synthases: SS1, SS2, SS3, and SS4. We have described previously an Arabidopsis mutant plant lacking SS3 and SS4 that is also severely affected in the accumulation of starch (Szydlowski et al., 2009). SS4 is involved in the initiation of the starch granule and controls the number of granules per chloroplast (Roldán et al., 2007). The elimination of SS3 in an ss4 background leads to an absence of starch in most of the chloroplasts, despite the fact that SS1 and SS2 are still present and total starch synthase activity is only reduced by 35% (Szydlowski et al., 2009). However, a very small proportion of chloroplasts of this mutant plant contain a single huge starch granule, which is also a characteristic of chloroplasts in the ss4 single mutant (D’Hulst and Mérida, 2012). Thus, like aps1 and pgm1, ss3/ss4 plants contain only small amounts of starch. However, unlike aps1 or pgm1 plants, most of the cells of this mutant have empty chloroplasts, without starch (Szydlowski et al., 2009).In this work, we have analyzed the phenotypic effects of the impaired starch accumulation of ss3/ss4 plants. We show that this mutant displays phenotypic changes that are not found in other mutants with very low levels of starch, such as aps1 or pgm1 plants. We provide evidence that extremely high levels of ADP-Glc accumulate in the ss3/ss4 plants. Using reverse genetics to block the pathway of starch synthesis upstream of the starch synthases reduced the level of ADP-Glc in ss3/ss4 plants and reverted the other phenotypic traits. This suggests that ADP-Glc accumulation is the causal factor behind the chlorotic and stunted growth phenotypes of the ss3/ss4 mutant.     

The plant Selaginella moellendorffii possesses enzymes for synthesis and hydrolysis of the compatible solutes mannosylglycerate and glucosylglycerate

A mannosylglycerate synthase (MgS) gene detected in the genome of Selaginella moellendorffii was expressed in E. coli and the recombinant enzyme was purified and characterized. A remarkable and unprecedented feature of this enzyme was the ability to efficiently synthesize mannosylglycerate (MG) and glucosylglycerate (GG) alike, with maximal activity at 50 °C, pH 8.0 and with Mg²⁺ as reaction enhancer. We have also identified a novel glycoside hydrolase gene in this plant’s genome, which was functionally confirmed to be highly specific for the hydrolysis of MG and GG and named MG hydrolase (MgH), due to its homology with bacterial MgHs. The recombinant enzyme was maximally active at 40 °C and at pH 6.0–6.5. The activity was independent of cations, but Mn²⁺ was a strong stimulator. Regardless of these efficient enzymatic resources we could not detect MG or GG in S. moellendorffii or in the extracts of five additional Selaginella species. Herein, we describe the properties of the first eukaryotic enzymes for the synthesis and hydrolysis of the compatible solutes, MG and GG.