| The hypothalamus serves as a major connection between the central nervous system (CNS) and the endocrine systems, and it has an important role in controlling physiological activities and homeostatic mechanisms. The adipocyte-derived hormone leptin and the pancreaticβcell-derived hormone insulin function as afferent signals to the hypothalamus in an endocrine feedback loop that regulates body adiposity. They act in hypothalamic centers to modulate the function of specific neuronal subtypes, such as NPY neurons, by modifying neuronal electrical activity. BK channel is one kind of the K+ channels. In CNS, BK channels are present widely in the dendrites, axons, and synaptic terminals. In the duration of action potential, BK channels can be activated by cell membrane depolarization and increase of intracellular Ca2+ concentration and then produce fast afterherpolarization directly. So the main function of BK channels is to control the duration of action potential, reduce the discharge frequency of neurons, decrease the excitability of neurons, hence influence neuronal signaling and neurotransmitter release. The change of channel function will significantly influence the activity of neurons and the normal function of brain. However, the diffusive distribution of NPY neurons in hypothalamus made it difficult to study the electrophysiological feature of a single neuron. To investigate the intrinsic activity of these neurons and their responses to insulin and leptin, we used a combination of morphologic features and immunocytochemical technique to identify the NPY neurons and record whole-cell BK currents on them by using whole-cell patch clamp technique.Section 1: Effects of insulin on BK channels in cultured rat hypothalamic NPY neuronsAim: NPY containing neurons in hypothalamus are principal neuropeptide modulators and exert potent effects on feeding and metabolism. Many studies have shown that insulin can inhibit the activity of NPY neurons. BK channels play an important role in the regulation of membrane excitability. Here, we tested the possible modulations of BK channels by insulin in primary cultured NPY neurons using patch clamp techniques, and if so, to specify the signal transduction mechanisms in this modulated actions of insulin.Methods: (1) The immunocytochemical method was adopted to identify feeding-regulating NPY neurons in the rat hypothalamus. (2) Whole-cell patch clamp technique was used to examine the effects of insulin on BK channels in NPY neurons, which 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 slenderly, poorly ramified primary dendrites. (2) Application of insulin (0.1~30 nM) increased the BK currents in a concentration-dependent manner. 3 nM insulin shifted I-V curve of BK currents upward, especially at potential from +20 mV to +60 mV. 3 nM insulin also shifted the activation curve of BK currents to the left. (3) The increasing effects of insulin on BK currents could be antagonized by the inhibition of IR tyrosine kinase and PI3-k, but not MAPK.Conclusion: Insulin increased the BK currents in a concentration-dependent manner, and the effects of insulin depended on IR and intracellular signaling via PI3-k.Section 2: Effects of leptin on BK channels in cultured rat hypothalamic NPY neuronsAim: Leptin has similar physiological functions as insulin. In CNS, leptin can also inhibit the activity of NPY neurons. In this experiment, we tested the possible modulations of BK channels by leptin in primary cultured NPY neurons using patch-clamp techniques, and if so, to specify the signal transduction mechanisms in this modulated actions of leptin, and observe whether the effects are as similar as insulin.Methods: (1) The immunocytochemical method was adopted to identify feeding-regulating NPY neurons in the rat hypothalamus. (2) Whole-cell patch clamp technique was used to examine the effects of leptin on BK channels in NPY neurons, which identified by morphologic features and immunocytochemical identification at the end of recording.Results: (1) Leptin had identical effects as insulin on BK channels in NPY neurons. Administration of leptin increased BK currents in a concentration-dependent manner. 30 nM leptin shifted I-V curve of BK currents upward, especially at potential from +20 mV to +60 mV, but had no effect on the activation threshold potential of BK channels. 30 nM leptin also shifted the activation curve of BK currents leftward with no change in slope factor. (2) The experiment also showed leptin-mediated activation of BK channels was attenuated by an inhibitor of JAK2 and PI3-k, but not MAPK.Conclusion: Leptin increased the BK currents in a concentration-dependent manner, and the effects of leptin depended on LEPR and intracellular signaling via PI3-k.Section 3: Interaction between insulin and leptin on BK channels in cultured rat hypothalamic NPY neuronsAim: In terms of both insulin and leptin exist in the hypothalamus simultaneously and there exists"crosstalk"between the signaling pathways, we next examined the effects of insulin combined with leptin on the BK channels and the interaction between them. Methods: (1) The immunocytochemical method was adopted to identify feeding-regulating NPY neurons in the rat hypothalamus. (2) Whole-cell patch clamp technique was used to examine the effects of insulin in combination with leptin on BK channels in NPY neurons, which identified by morphologic features and immunocytochemical identification at the end of recording, and examine whether one hormone pre-treatment can influence the effects of the other.Results: (1) Bath application of 3 nM insulin in combination with 30 nM leptin significantly increased the amplitude of BK currents, and their effect can also be significantly antagonized by pre-treatment of neurons with PI3-k inhibitor. But the effect of cooperative action was similar to the effect of 3 nM insulin or 30 nM leptin alone. (2) Pretreatment with insulin (3 nM, 5 min) clearly abolished leptin-induced acute stimulation of BK channels and 30 nM leptin pre-treatment also abolished insulin-induced stimulation.Conclusion: The effect of insulin and leptin together was similar to that of insulin or leptin alone, and leptin or insulin pretreatment canceled the other hormone's increasing effects on BK currents. |
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