Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein.
Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5' regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl-D-aspartate receptors.
Díez-García
Javier
J
Laboratory for Neuronal Circuit Dynamics, RIKEN Brain Science Institute, 2-1 Hirosawa, Saitama 351-0198, Japan.
Matsushita
Shinichi
S
Mutoh
Hiroki
H
Nakai
Junichi
J
Ohkura
Masamichi
M
Yokoyama
Jennifer
J
Dimitrov
Dimitar
D
Knöpfel
Thomas
T
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002531:Cerebellum / Q000166:cytology*; D002867:Chromones / Q000494:pharmacology; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009412:Nerve Fibers / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011810:Quinoxalines / Q000494:pharmacology; D051670:Shaw Potassium Channels
C117789:7-(hydroxyimino)cyclopropan(b)chromen-1a-carbxoylic acid ethyl ester; D002867:Chromones; D018691:Excitatory Amino Acid Antagonists; D008164:Luminescent Proteins; D009479:Neuropeptides; D024642:Potassium Channels, Voltage-Gated; D011810:Quinoxalines; D051670:Shaw Potassium Channels; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; D015763:2-Amino-5-phosphonovalerate; D002118:Calcium