title: Imaging calcium dynamics in the nervous system by means of ballistic delivery of indicators.
abstract: The use of fluorescence-based calcium indicators has, over the years, unraveled important calcium-dependent mechanisms underlying neuronal function and development. However, difficulties associated with the loading of calcium indicators have limited their widespread use, particularly for the study of neuronal processing in the adult nervous system. Here, we show that in the central and peripheral nervous systems, populations of neurons and their processes, including dendritic spines and filopodia, can be labeled rapidly and efficiently by delivering calcium indicator-coated particles using a 'gene gun'. Importantly, neuronal labeling occurred both in vitro and in vivo, and across a wide range of ages and preparations. The labeled cells demonstrate spontaneous and evoked calcium transients, indicating that particle-mediated delivery is not deleterious to neuronal function. Furthermore, unlike loading with patch pipettes, cytoplasmic content is preserved following ballistic loading. This enables the study of calcium-dependent second messenger pathways without loss of signaling components. The ballistic delivery of calcium indicators thus opens up many new avenues for further exploration of the structure and function of the nervous system from single spines to neuronal networks.
authors.affiliation: Department of Neuroscience, Nobel Institute for Neurophysiology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Petronella
authors.initials: P
authors.lastname: Kettunen
authors.forename: Jay
authors.initials: J
authors.lastname: Demas
authors.forename: Christian
authors.initials: C
authors.lastname: Lohmann
authors.forename: Narayanan
authors.initials: N
authors.lastname: Kasthuri
authors.forename: Yandao
authors.initials: Y
authors.lastname: Gong
authors.forename: Rachel O L
authors.initials: RO
authors.lastname: Wong
authors.forename: Wen-Biao
authors.initials: WB
authors.lastname: Gan
mesh_terms: D000818:Animals; D019470:Biolistics; D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D002642:Chick Embryo; D046508:Culture Techniques; D005456:Fluorescent Dyes; D016257:Fura-2; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D009930:Organic Chemicals; D017933:Peripheral Nervous System / Q000166:cytology / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology / Q000502:physiology; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000502:physiology; D014414:Tungsten
chemical_list: D005456:Fluorescent Dyes; C403124:Oregon Green BAPTA-dextran; D009930:Organic Chemicals; C071336:calcium green; D002118:Calcium; D016257:Fura-2; D014414:Tungsten