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An immunocytochemical double-staining method was applied in order to study the co-localisation of nitric oxide synthase (NOS) with three calcium-binding proteins, calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) in the claustrum of the rat during the first 4 months of life (postnatal days: P0–P120). The co-localisation of NOS/PV and NOS/CB is reported. These neurons fall into the category of non-pyramidal cells. Double-labelled NOS/CB neurons are observed in the claustrum starting from P4, whereas double-labelled NOS/PV neurons are observed from P14 onwards. The percentages of double-labelled neurons increase in relation to the age. Double-labelled NOS/CB and NOS/PV neurons, although they do not constitute a numerous population, play an important role in the process of maturation of the claustrum. This is confirmed by the occurrence of these types of neurons at definite stages of maturation and by the increase in their number.
Immunohistochemical study of the cholinergic innervation of the parvalbuminand calbindin-containing cells in the hippocampus was conducted on 30 rat brains of various postnatal ages: P0, P4, P7, P14, P21, P30, P60 and P180. Sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and parvalbumin (PV) or calbindin (CB) were analysed using confocal laser-scanning microscope. Obtained data demonstrate that the pattern of cholinergic innervation of calbindin- and parvalbumin-immunoreactive hippocampal neurones shows some differences. During development as well as in the adult species cholinergic terminals preferentially innervate CB-containing neurones, while cholinergic terminals on PV-containing cells were observed rarely. Cholinergic endings on the CB-ir neurones are localised both on their somata and dendrites, whereas on PV-ir cells they form synaptic contact predominantly with processes. In spite of the unquestionable cholinergic influence particularly on CB-ir cells, the number of cholinergic endings suggests that this input seems not to be crucial for the activity of the studied cell populations.
Recent decades has brought significant advances in our knowledge of the chemical coding and function of enteric neurons. Calcium ions are important second messenger involved in many aspects of neuron physiology. In the present study, we analyzed immunohistochemically the presence of calcium binding proteins (calretinin and calbindin) in various subpopulations of enteric neurons from the ovine duodenum. Ten percent of submucous neurons were immunoreactive (IR) to calretinin. The presence of calretinin was not detected in myenteric neurons. Calretinin-expressing nerve fibres were found in both myenteric and submucous ganglia, between the circular and longitudinal smooth muscle layers and in the lamina muscularis mucosae. Calretinin-IR submucous neurons did not exhibit the presence of SP, NPY and VIP. Co-localization of calretinin and serotonin was found only in a small number of submucous neurons. Calbindin was expressed in 35% of myenteric neurons and in 60% of submucous neurons. Nerve fibres containing calbindin were localized in myenteric and submucous ganglia where they frequently formed basket-like formations. Calbindin-positive nerve fibres emerging from myenteric ganglia ran between the circular and longitudinal smooth muscle layers. Immunoreactivity to calbindin was also visualized in the lamina muscularis mucosae, around mucosal glands and blood vessels. None of calbindin-IR myenteric neurons revealed immunoreactivity to SP, NPY, VIP and serotonin. Virtually all calbindin-expressing submucous neurons were SP-positive. In moderate numbers of submucous perikarya, co-incidence of calbindin and NPY, calbindin and VIP or calbindin and serotonin was observed. We conclude that in the ovine duodenum, the expression of calretinin and calbindin is species specific. Co-localization studies and distribution patterns indicate that in the duodenum of the sheep, calretinin and calbindin may be present in several functional subclasses of enteric neurons.
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