Measurement of Orexin (Hypocretin) and Substance P Effects on Constitutively Active Inward Rectifier K+ Channels in Brain Neurons

Abstract Electrophysiological experiments in our laboratory have led to the discovery that the cholinergic neurons in the nucleus basalis in the rat forebrain possess constitutively active inward rectifier K + channels. Unlike cloned inward rectifier K + channels, these constitutively active inward rectifier K + channels were found to have unique properties, and thus were named “KirNB” (inward rectifier K + channels in the nucleus basalis). We found that slow excitatory transmitters, such as orexin (hypocretin) and substance P, suppress the KirNB channel, resulting in neuronal excitation. Furthermore, it was discovered that suppression of KirNB channels by these transmitters is through protein kinase C (PKC). This chapter describes detailed electrophysiological techniques for investigating the effects of orexin and substance P on constitutively active KirNB channels. For this purpose, we also present a method for culturing nucleus basalis cholinergic neurons in which KirNB channels exist. Then, we describe the procedures through which PKC has been determined to mediate inhibition of KirNB channels by orexin and substance P. There are probably many other transmitters which may produce effects on KirNB channels. This chapter will enable researchers to investigate the effects of such transmitters on KirNB channels and their roles in neuronal functions.