Differentiated pattern of sodium channel expression in dissociated Purkinje neurons maintained in long-term culture.
Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.
Fry
Mark
M
Department of Physiology, Queen's University, Kingston, Ontario, Canada. fryw@post.queensu.ca
Boegle
Aimee K
AK
Maue
Robert A
RA
D000818:Animals; D001369:Axons / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation / Q000379:methods; D004622:Embryo, Mammalian; D005260:Female; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D011247:Pregnancy; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011689:Purkinje Cells / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015222:Sodium Channels / Q000145:classification / Q000235:genetics / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; D021122:Protein Subunits; D012333:RNA, Messenger; D015222:Sodium Channels; D049452:Green Fluorescent Proteins