소뇌 신경 세포의 연접부 가소성과 NA+-Ca2+ 교환이동체의 역할

초록

Introduction : Cerebellar long-term depression (LTD) is a cellular model system of information storage that has been proposed to underlie several forms of motor learning. Cerebellar LTD is produced when climbing fiber and parallel fiber inputs to a Purkinje neuron are stimulated conjunctively at low frequencies and is expressed as an attenuation of parallel fiber Purkinje neuron synaptic strength. Variations in intracellular calcium activity ([Ca2+i]) play crucial roles in information processing in Purkinje neurons. Purkinje neurons have powerful systems to modulate [Ca2+i], such as Ca2+-binding proteins, intracellular stores, Ca2+-ATPase and Na+-Ca2+ exchanger. Na+-Ca2+ exchanger has been shown to participate in the regulation of Ca2+ homeostasis and secretion in neuronal cells (Reuter & Porzig, 1995). Although the presence of Na+-Ca2+ exchanger in Purkinje neurons has been identified with antibodies against mRNA encoding the cardiac type of exchanger, the physiological role of Na+-Ca2+ exchanger in Purkinje neurons are not well understood, especially in cerebellar synaptic plasticity. Thus, in this study, we investigated the intracellular Ca2+ metabolism with cellular Ca2+ imaging technique with isolated rat Purkinje neurons. Methods : Cerebellar neuronal cultures were prepared from Sprague Dawley rats of postnatal day 1 (p1) and maintained in serum-free DMEM/F-12 that contain supplements, according to a published for Purkinje cell culture (Furuya et al., 1998) with slight modification. Acutely dissociated cells were prepared from S. D. rats of postnatal day 10 (Womack et al., 2000). Cultured cells were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100. Cells were blocked with 1% BSA solution, and then incubated with a monoclonal antibody calbindin D-28k. After cells were washed, cells were incubated with rhodamine-conjugated anti-mouse secondary antibody or ABC-kits and DAB was used for the staining. For intracellular Ca2+ measureme