| Partâ… Effects of culture time on intracellular Ca2+ signals in primary cultured hypothalamic neuronsAim: It is well established that aging leads to a progressive increase of basal [Ca2+]i , especially the decline of the ability of neurons to handle a load of [Ca2+]i induced by various insults. The neuro-endocrine-immune interrelationship hypothesis postulated aging initiates from hypothalamus. Long-term primary neuronal culture is a powerful tool for isolating cellular and molecular mechanisms of neuronal development, aging and death. Our researches have focused on the hypothalamus, and a given load of [Ca2+]i was assessed by challenging the neurons with KCl-depalarization. Here we explored the age-related alterations in response to excessive [Ca2+]i.Methods: We evaluated the Ca2+ homeostatic mechanisms of young, adult, mid-aged and old-aged hypothalamic neurons using Fura-2 based calcium imaging techniques and whole-cell patch clamp techniques. (1)In calcium imaging investigations, in order to explore the effects of culture time on alterations in Ca2+ homeostatic mechanisms, the ability of long-term cultured hypothalamic neurons to handle a given load of Ca2+ was assessed by chanllenging the neurons with 25 mM KCl. (2)In patch clamp tests, the current amplitudes and memmbrane capacities of VGCCs were compared among the four groups.Results: (1) Stimulation with 25mM KCl produced an equal peak of Ca2+ transient in rat hypothalamic neurons over long term in culture, but the recovery curves varied in the neurons on different stages in culture. As the days in vitro were increased, the speed and the degree of recovery for [Ca2+]i exhibited reduction. (2) In patch clamp tests, the current amplitudes and the membrane capacities of VGCCs were increased with the increasing days in vitro, and the L-type calcium currents performed more relatively significant.Conclusion: In hypothalamic neurons, the deficits of Ca2+ homeostatic mechanisms are age-related. The adult, mid-aged, and old-aged neurons had a significant delayed recovery of [Ca2+]i in comparision young neurons (P<0.05). The increase of L-type Ca2+ currents may be involved in the alteration of Ca2+ homeostatic mechanisms.Partâ…¡Leptin modulates Ca2+ signals in hypothalamic neurons in an age-dependent mannerAim: Leptin, the protein encoded by the obese (ob) gene, is secreted from adipose tissue and is thought to act in the central nervous system to regulate food intake and energy expenditure. Intracellular calcium is a ubquitous sencond messenger. In neurons, it is known to be involved in the control of neuron membrane excitability, neurotransmitter release and synaptic activity. Here, we investigated the effects of leptin on the intracellular Ca2+ signals in hypothalamic neurons and the relationship to the days in vitro.Methods: (1) Fura-2 based calcium imaging technique was used to examinate the effects of leptin on the basal [Ca2+]i, and the modulation effects of leptin on KCl-evoked [Ca2+]i increase in long-term cultured hypothalamic neurons. (2) The whole-cell patch clamp technique was used to explore the effects of leptin on the VGCC in long-term cultured hypothalamic neurons. (3) Immunofluorescence and Western blot analysis were used to investigate the influence of culture time on expression of the leptin receptors in hypothalamic neurons. Results: (1) Leptin failed to affect the basal [Ca2+]i in recorded hypothalamic neurons, but could decrease the KCl-evoked [Ca2+]i transient. The maximal inhibition effects of leptin were produced at days in vitro culture (DIV) 16d, and in young and old-aged neurons, the influences of leptin were reduced. (2)The modulation effects of leptin on VGCC in hypothalamic neurons also correlated to the culture time. Leptin performed greater effects on middle stage in culture (DIV 9d and 16d) than early (DIV 3d) and late (DIV 23d) stage in culture. (3) The expressions of leptin receptor were augmented gradually with the culture time in adult and mid-aged neurons, however, in old-aged neurons, the expressions of leptin receptors were decreased.Conclusion: The expression alteration of leptin receptors in long-term cultured hypothalmic neurons may contribute to the relationship between the effects of leptin and the culture time.Partâ…¢Leptin regulates calcium channels of NPY and POMC neurons by activation of different signal pathwaysAim: Proopiomelanocortin (POMC) and Neuropeptide Y (NPY) containing neurons in hypothalamus are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, and they exert opposing effects on feeding and metabolism. VGCCs play an important role in the regulation of membrane excitability, however, the direct effects of leptin on VGCCs in NPY and POMC neurons have not yet been elucidated. Here, we tested the possible modulations of VGCCs by leptin in primary cultured NPY and POMC neurons using patch-clamp techniques, and if so, to specify the signal transduction mechanisms in this modulated actions of leptin.Methods: (1) The immunocytochemical method was adopted to identify feeding-regulating NPY and POMC neurons in the rat hypothalamus. (2) Whole-cell patch clamp technique was used to examine the effects of leptin on VGCC in NPY and POMC neurons, which we identified by morphologic features and immunocytochemical identification at the end of recording.Results:(1)The NPY neurons are typically small and medium neurons with triangular or spindle-shaped perikaryons. Most of them have 1-3 slender, poorly ramified primary dendrites. POMC neurons are medium and large sizes, displaying a quadrangular or multipolar perikaryon with a variable number of primary dendrites, well-ramified dendrites. (2) Leptin directly inhibited IHVA in NPY neurons via leptin receptor (LEPR)-JAK2-MAPK pathways, whereas evoked IHVA in POMC neurons by LEPR-JAK2-PI3-k pathways.Conclusion: The different downstream kinase pathways provide a unifying hypothesis to explain how this hormone exerts opposing effects on the activity of these 2 key subsets of neurons. |
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