In vivo spectrometric calcium flux recordings of intrinsic Caudate-Putamen cells and transplanted IMR-32 neuroblastoma cells using miniature fiber optrodes in anesthetized and awake rats and monkeys.
A method is described to enable the recording of transient intracellular calcium changes in deep brain structures in anesthetized and awake animals using a fluorescent indicator combined with in vivo optical detection methods. Optrodes were fabricated using a bifurcated fiber-optic cable with an attached infusion guide cannula. After intracranial implantation of an optrode, animals were prepared in the following manner, (1) rats (intra-striatal) and monkeys (intra-putamen) were infused with the fluorescent calcium indicator, Oregon Green, to load intrinsic cells; or (2) rats were intra-striatally transplanted with a slurry of dye-loaded IMR-32 neuroblastoma cells via pipette ejection. Excitation light from an argon-ion laser was launched through the optrode and passed into the tissue. The resulting calcium-induced fluorescence signals were captured by the optrode, then detected and processed by externalized photomultiplier- and CCD-based spectrometer electronics. In approximately 25% of all intrinsic cell recordings, the baseline fluorescence intensity was relatively stable over time whereas in the remainder, large amplitude oscillations were observed with a frequency in the range of 0.5-2 Hz. These Ca(2+) transients were inhibited by local infusion of 10 microM omega-conotoxin MVIIC and 1 microM TTX. Extracellular electrophysiological recordings that were made adjacent to the optrode tip revealed that the Ca(2+) oscillations were in phase with the burst firing of striatal neurons. This suggested that the optical signals had a neuronal origin, most likely from medium spiny neurons. Baseline fluorescence intensity increased during infusion of high [K(+)](o), the calcium ionophore, A-23187, or during temporary bilateral carotid artery occlusion. Monkey (Saimiri sciureus) putamen recordings also affirmed the presence of similar calcium-related transients in a non-human primate. In the transplant preparations, the IMR-32 cells displayed a stable, non-oscillating baseline fluorescence. They were similarly responsive to high [K(+)](o) challenge and appeared viable for at least several hours. Similar optical recording approaches might be applied to monitor other fluorescent, chemiluminescent or bioluminescent events from almost any brain structure. Moreover, transplanted transfected cells expressing a single specific receptor or ion-channel protein may effectively serve as biosensing elements for the measurement of extracellular neurochemical signaling.
Duff Davis
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Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., 2800 Plymouth Road, 48105, Ann Arbor, MI, USA. davism@aa.wl.com
Schmidt
J J
JJ
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D002421:Caudate Nucleus / Q000166:cytology / Q000378:metabolism*; D001330:Electronic Data Processing / Q000379:methods*; D004594:Electrophysiology / Q000379:methods*; D005336:Fiber Optic Technology / Q000379:methods*; D005456:Fluorescent Dyes; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D055100:Optical Fibers; D011189:Potassium Chloride / Q000494:pharmacology; D011699:Putamen / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D012453:Saimiri; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D014851:Wakefulness / Q000502:physiology
D005456:Fluorescent Dyes; D007473:Ion Channels; D007476:Ionophores; D011189:Potassium Chloride