Regulation Of Intracellular Ca²⁺ Signaling For Cultured Hypothalamic Neurons By Insulin-like Growth Factor-1

The hypothalamus serves as a major connection between the central nervous systemand the endocrine system,and it is intricately involved in integrating the multitude ofsignals controlling both growth and metabolism.Endocrine dysfunction may play apathophysiological role in age-related function disorders and aging pathology,so somespecialists think that aging may initiate from hypothalamus.The calcium homeostasisimbalance hypothesis postulated that sustained changes in the regulation of intracellularCa²⁺concentration ([Ca²⁺]_i)are the major cause of brain aging.Aging may cause theincrease of the [Ca²⁺]_i through many mechanisms.These alterations would in turn affect thesynaptic transduction,neurotransmitter release and signal transduction,thus cause aging.In adult brain,both insulin-like growth factor 1 (IGF-1)and neuropeptide Y (NPY)have coordinated effects on the regulation of metabolic,reproductive,neuroprotectiveaction and synaptic plasticity.Several findings indicate that there is a close interactionbetween IGF-1 and NPY neurons in hypothalamus arcuate nucleus (ARC).IGF-1 isthought to regulate neuroendocrine by modulating the synthesis and release of NPY.Inneurons,voltage-gated calcium channels (VGCCs)is known to involve in the control ofmembrane excitability and neurotransmitter release.However,modulation mechanismsunderlying the effects of IGF-1 on VGCCs in hypothalamic NPY neurons are poorlyunderstood.Long-term culture of neurons may be as a model for neuronal development,aging anddeath.So in this report,we examined the regulation of IGF-1 on VGCCs using whole-cellpatch clamp technique in long-term cultured rat hypothalamic NPY neurons,which we identified by morphologic features and immunocytochemical identification at the end ofrecording.Preincubation of IGF-1 for 24 h significantly increased the peak amplitude ofhigh voltage-activated calcium currents (I_HVA)in NPY neurons of ARC.The potentiationof I_HVAwas inhibited by pretreatment with the phosphatidylino 3-kinase (PI3-k)inhibitor.In addition,the effect was counteracted by the L-type calcium channel antagonistnifedipine.The modulation effects of IGF-1 on VGCCs in NPY neurons were greater atmiddle stage in culture (DIV 7d and 14d)than early (DIV 3d)and late (DIV 21d)stage inculture.Double-label immunofluorescence demonstrated that IGF-1 increased theexpression of L-type calcium channels in NPY neurons.These findings suggested thatIGF-1 enhanced I_HVAin NPY neurons via activation of PI3-k,and L-type calcium channelswere involved in the stimulation effect of IGF-1.The expression changes of calciumchannels may contribute to the effect of IGF-1.The stimulations of I_HVAmay play key rolesin regulating NPY neurons activity,and subsequently their physiological functions inducedby IGF-1.Section 1IGF-1 modulated calcium currents in hypothalarnic NPY neurons inan age-dependent wayBackground:The neuro-endocrine-immune interrelationship hypothesis postulated agingare initiated from hypothalamus.Long-term primary neuronal culture is a powerful tool forisolating cellular and molecular mechanisms of neuronal development,aging and death.IGF-1 and NPY have coordinated effects on the regulation of metabolic,reproductive andneuroprotective action.IGF-1 is thought to regulate neuroendocrine by modulating thesynthesis and release of NPY.In neurons,VGCCs is known to be involved in the control ofmembrane excitability and neurotransmitter release.Here,we investigated the effects of IGF-1 on I_HVAin hypothalamic NPY neurons and the relationship to the days in vitro.Methods:(1)The immunocytochemical method was adopted to identify NPY neurons inthe rat hypothalamus.(2)The whole-cell patch clamp technique was used to explore theeffects of IGF-1 on I_HVAin long-term cultured hypothalamic NPY neurons,which weidentified by morphologic features and immunocytochemical identification at the end ofrecording.Results:(1)The NPY neurons are typically small and medium neurons with triangular orspindle-shaped perikaryons.Most of them have 1-3 slender,poorly ramified primarydendrites.(2)IGF-1 enhanced I_HVAin NPY neurons at all stages in culture.The modulationeffects of IGF-1 on I_HVAin NPY neurons were greater at middle stage in culture (DIV 7dand 14d)than early (DIV 3d)and late (DIV 21d)stage in culture.Conclusions:IGF-1-sensitive inhibition or stimulation of VGCCs would be responsible forthe decreased or increased excitability in corresponding neurons,which,at least in part,contributed to the regulation of neuroendocrine of hypothalamus by IGF-1.Section 2Effects of IGF-1 on calcium channels of NPY neurons in thehypothalamic areuate nucleusBackground:NPY neurons in the arcuate nucleus of hypothalamus are intricately involvedin controlling metabolism,growth,reproduction and appetite.IGF-1 is thought to regulateneuroendocrine by modulating the synthesis and release of NPY.VGCCs play an importantrole in the regulation of neuron membrane excitability and neurotransmitter release.Here,we tested the modulations of VGCCs by IGF-1 in DIV 7d NPY neurons using patch-clamptechniques.Furthermore,we explored the signal pathway mediating these effects.Methods:(1)The immunocytochemical method was adopted to identify NPY neurons in the rat hypothalamus.(2)Whole-cell patch clamp technique was used to examine theeffects of IGF-1 on VGCCs in DIV 7d NPY neurons and the signal pathway mediatingthese effects.(3)Double-label immunofluorescence was used to investigate the influence ofIGF-1 on expression of the calcium channels in NPY neurons.Results:(1)The NPY neurons are typically small and medium neurons with triangular orspindle-shaped perikaryons.Most of them have 1-3 slender,poorly ramified primarydendrites.(2)IGF-1 enhanced the amplitude of I_HVAin DIV 7d NPY neurons and slightlyshifted the steady-state activation curve to negative potentials,however,the steady-stateinactivation curve was not altered.The potentiation of I_HVAwas inhibited by pretreatmentwith the phosphatidylino 3-kinase (PI3-k)inhibitor.In addition,the effect was counteractedby the L-type calcium channel antagonist nifedipine.(3)IGF-1 increased the expression ofL-type calcium channels in NPY neurons.Conclusions:IGF-1 enhanced I_HVAin NPY neurons via activation of PI3-k,and L-typecalcium channels were involved in the stimulation effect of IGF-1.The expression changesof calcium channels may contribute to the effect of IGF-1.