Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine

Background: Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. Results: Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. Conclusions: Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway

Substance P-expressing excitatory interneurons in the mouse superficial dorsal horn provide a propriospinal input to the lateral spinal nucleus

The superficial dorsal horn (laminae I and II) of the spinal cord contains numerous excitatory and inhibitory interneurons, and recent studies have shown that each of these groups can be divided into several neurochemically distinct populations. Although it has long been known that some neurons in this region have intersegmental (propriospinal) axonal projections, there have been conflicting reports concerning the number of propriospinal cells and the extent of their axons. In addition, little is known about the neurochemical phenotype of propriospinal neurons or about the termination pattern of their axons. In the present study we show, using retrograde tracing, that around a third of lamina I–II neurons in the lumbar enlargement project at least five segments cranially. Substance P-expressing excitatory neurons are over-represented among these cells, accounting for one-third of the propriospinal neurons. In contrast, inhibitory interneurons and excitatory PKCγ neurons are both under-represented among the retrogradely labelled cells. By combining viral vector-mediated Cre-dependent anterograde tracing with immunocytochemistry, we provide evidence that the lateral spinal nucleus (LSN), rather than the superficial dorsal horn, is the main target for axons belonging to propriospinal substance P-expressing neurons. These findings help to resolve the discrepancies between earlier studies and have implications for the role of the LSN in pain mechanisms

Functional differences between neurochemically defined populations of inhibitory interneurons in the rat spinal dorsal horn

In order to understand how nociceptive information is processed in the spinal dorsal horn we need to unravel the complex synaptic circuits involving interneurons, which constitute the vast majority of the neurons in laminae I–III. The main limitation has been the difficulty in defining functional populations among these cells. We have recently identified 4 nonoverlapping classes of inhibitory interneuron, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) and parvalbumin, in the rat spinal cord. In this study we demonstrated that these form distinct functional populations that differ in terms of sst2A receptor expression and in their responses to painful stimulation. The sst2A receptor was expressed by nearly all of the nNOS- and galanin-containing inhibitory interneurons but by few of those with NPY and none of the parvalbumin cells. Many galanin- and NPY-containing cells exhibited phosphorylated extracellular signal-regulated kinases (pERK) after mechanical, thermal or chemical noxious stimuli, but very few nNOS-containing cells expressed pERK after any of these stimuli. However, many nNOS-positive inhibitory interneurons up-regulated Fos after noxious thermal stimulation or injection of formalin, but not after capsaicin injection; nor did parvalbumin cells express either activity-dependent marker after any of these stimuli. These results suggest that interneurons belonging to the NPY, nNOS and galanin populations are involved in attenuating pain, and for NPY and nNOS cells this is likely to result from direct inhibition of nociceptive projection neurons. They also suggest that the nociceptive inputs to the nNOS cells differ from those to the galanin and NPY populations

Evidence against AMPA receptor-lacking glutamatergic synapses in the superficial dorsal horn of the rat spinal cord

Pure NMDA receptor (NMDAr)-mediated EPSCs, thought to correspond to "silent" glutamatergic synapses that lack AMPA receptors (AMPArs), have been observed in superficial spinal dorsal horn of neonatal but not adult rats. Recent anatomical studies suggest that AMPArs are present at virtually all glutamatergic synapses in this region in adults. We used antigen retrieval to examine colocalization of AMPArs and PSD-95 (a marker for glutamatergic synapses) in laminae I–II of neonatal and adult rats. We found a high degree of colocalization in all cases, which suggests that AMPArs are present in the great majority of glutamatergic synapses even in neonatal animals. We therefore reexamined evidence for silent synapses by performing blind whole-cell recordings from superficial dorsal horn neurons in slices from neonatal or adult rats, with focal stimulation to activate glutamatergic synapses. On some occasions in both neonatal (10 of 109, 9%) and adult (9 of 77, 12%) slices, NMDAr-mediated EPSCs were observed when the holding potential was raised to +50 mV at a stimulus strength that had failed to evoke AMPAr-mediated EPSCs. However, in all cases tested, AMPAr-mediated EPSCs were then observed when the cell was returned to –70 mV; this and other properties of the EPSCs suggest that they do not represent genuine silent synapses. When compared with previous findings, our results indicate that the appearance of silent synapses depends on experimental protocol. This suggests that pure NMDAr-mediated EPSCs seen in previous studies do not correspond to AMPAr-lacking synapses but result from another mechanism, for example, loss of labile AMPArs from recently formed synapses

Effect of enzymatic modification on the biological activity and nutritive value of cow and buffalo casein

Buffalo and cow milk caseins were submitted to hydrolysis either with á -chymotrypsin or with pepsin. Enzymatic peptide modification (EPM) was carried out by using L-methionine ethyl ester in the reaction mixture. As catalyst, á -chymotrypsin or pepsin was used. The incorporation of methionine in to the peptide chains in the presence of á -chymotrypsin showed an optimum value at 0.14 g Met added to the reaction mixture/1 g hydrolysate in both cases. In the case of pepsin used as catalyst, the optimal Met-enrichment was at 0.14 g Met added to the reaction mixture/1 g buffalo casein hydrolysate and at 0.34 g Met/1 g cow casein hydrolysate. The covalent nature of the amino acid incorporation was confirmed by SDS - polyacryl amide gel electrophoresis in the presence of urea. Electrophoretic patterns of the products indicate that transpeptidation plays an essential role in the EPM reaction. Antigenic character of the EPM- products was investigated in vitro by competitive indirect ELISA. Enzymatic peptide modification with methionine enrichment seems to be an efficient method for the reduction of the antigenic/potential allergenic character and for the improvement of the nutritive value of buffalo and cow milk caseins

Investigation of antinutritive components in Hungarian potato cultivars depending on production technology

We have investigated the Total Glycoalcaloid (TGA), nitrite, and nitrate contents of some Hungarian and foreign potato cultivars in relation to the effect of different combination of fertilisers and green manure, late blight management strategies (none, programmed, or prediction based spraying), and irrigation regime for three years. The Hungarian cultivars have exotic potato species like S. acaule, S. demissum, S. stoloniferum, S. vernei, or S. tub. ssp. andigenum in their genetic background as sources of resistance genes. No effect of fertilisers or irrigation was found on the level of glycoalkaloids and nitrate contents, which were influenced mostly by the genotype and season. In conclusion, the absolute amount and the presence of different antinutritive components of potato tubers were influenced by the technology, genotype, and season in a complex manner. These results in general prove that ware potato production utilising intensive commercial agrotechnical practices and common cultivars is safe regarding the nitrate and TGA content of tubers

Preprotachykinin A (PPTA) is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli

The superficial dorsal horn, which is the main target for nociceptive and pruritoceptive primary afferents, contains a high density of excitatory interneurons. Our understanding of their roles in somatosensory processing has been restricted by the difficulty of distinguishing functional populations among these cells. We recently defined three non-overlapping populations among the excitatory neurons, based on the expression of neurotensin, neurokinin B (NKB) and gastrin-releasing peptide (GRP). Here we identify and characterise another population: neurons that express the tachykinin peptide substance P. We show with immunocytochemistry that its precursor protein (preprotachykinin A, PPTA) can be detected in ~14% of lamina I-II neurons, and these are concentrated in the outer part of lamina II. Over 80% of the PPTA-positive cells lack the transcription factor Pax2 (which determines an inhibitory phenotype), and these account for ~15% of the excitatory neurons in this region. They are different from the neurotensin, NKB or GRP neurons, although many of them contain somatostatin, which is widely expressed among superficial dorsal horn excitatory interneurons. We show that many of these cells respond to noxious thermal and mechanical stimuli, and to intradermal injection of pruritogens. Finally, we demonstrate that these cells can also be identified in a knock-in Cre mouse line (Tac1Cre), although our findings suggest that there is an additional population of neurons that transiently express PPTA. This population of substance P-expressing excitatory neurons is likely to play an important role in transmission of signals that are perceived as pain and itch

Neuronal circuitry for pain processing in the dorsal horn

Neurons in the spinal dorsal horn process sensory information, which is then transmitted to several brain regions, including those responsible for pain perception. The dorsal horn provides numerous potential targets for the development of novel analgesics and is thought to undergo changes that contribute to the exaggerated pain felt after nerve injury and inflammation. Despite its obvious importance, we still know little about the neuronal circuits that process sensory information, mainly because of the heterogeneity of the various neuronal components that make up these circuits. Recent studies have begun to shed light on the neuronal organization and circuitry of this complex region

Phosphorylation of ERK in neurokinin 1 receptor-expressing neurons in laminae III and IV of the rat spinal dorsal horn following noxious stimulation

BACKGROUND: There is a population of large neurons with cell bodies in laminae III and IV of the spinal dorsal horn which express the neurokinin 1 receptor (NK1r) and have dendrites that enter the superficial laminae. Although it has been shown that these are all projection neurons and that they are innervated by substance P-containing (nociceptive) primary afferents, we know little about their responses to noxious stimuli. In this study we have looked for phosphorylation of extracellular signal-regulated kinases (ERKs) in these neurons in response to different types of noxious stimulus applied to one hindlimb of anaesthetised rats. The stimuli were mechanical (repeated pinching), thermal (immersion in water at 52°C) or chemical (injection of 2% formaldehyde). RESULTS: Five minutes after each type of stimulus we observed numerous cells with phosphorylated ERK (pERK) in laminae I and IIo, together with scattered positive cells in deeper laminae. We found that virtually all of the lamina III/IV NK1r-immunoreactive neurons contained pERK after each of these stimuli and that in the great majority of cases there was internalisation of the NK1r on the dorsal dendrites of these cells. In addition, we also saw neurons in lamina III that were pERK-positive but lacked the NK1r, and these were particularly evident in animals that had had the pinch stimulus. CONCLUSION: Our results demonstrate that lamina III/IV NK1r-immunoreactive neurons show receptor internalisation and ERK phosphorylation after mechanical, thermal or chemical noxious stimuli

Multi-objective genetic algorithm applied to spectroscopic ellipsometry of organic-inorganic hybrid planar waveguides

The applicably of multi-objective optimization to ellipsometric data analysis is presented and a method to handle complex ellipsometric problems such as multi sample or multi angle analysis using multi-objective optimization is described. The performance of a multi-objective genetic algorithm (MOGA) is tested against a single objective common genetic algorithm (CGA). The procedure is applied to the characterization (refractive index and thickness) of planar waveguides intended for the production of optical components prepared sol-gel derived organic-inorganic hybrids, so-called di-ureasils, modified with zirconium tetrapropoxide, Zr(OPr(n))(4) deposited on silica on silicon substrates. The results show that for the same initial conditions, MOGA performs better than the CGA, showing a higher success rate in the task of finding the best final solution. (C) 2010 Optical Society of AmericaFCTFEDERPTDC/CTM/72093/2006SFRH/BD/41943/2007MP070