A new cytochemical method for ultrastructural localization of Co2+ in rat hippocampal CA1 pyramidal neurons in vitro

• Published in: Journal of neuroscience methods 2004-05, Vol.135 (1-2), p.1-8
• Main Authors: Tanaka, E, Ishii, K, Akagi, T, Hirai, K, Motelica-Heino, I, Katayama, Y, Higashi, H, Hashikawa, T, Tsuji, S
• Format: Article
• Language: English
• Subjects: 2-Amino-5-phosphonovalerate - pharmacology; 6-Cyano-7-nitroquinoxaline-2,3-dione - pharmacology; Afferent Pathways - drug effects; Afferent Pathways - physiology; Anesthetics, Local - pharmacology; Animals; Cobalt - metabolism; Electric Stimulation - methods; Electrophysiology - methods; Excitatory Amino Acid Antagonists - pharmacology; Excitatory Postsynaptic Potentials - drug effects; Glutamic Acid - pharmacology; Hippocampus - drug effects; Hippocampus - metabolism; Hippocampus - ultrastructure; Histocytochemistry - methods; In Vitro Techniques; Life Sciences; Male; Microscopy, Electron - methods; Neurobiology; Neurons and Cognition; Pyramidal Cells - drug effects; Pyramidal Cells - metabolism; Pyramidal Cells - ultrastructure; Rats; Rats, Wistar; Synapses - drug effects; Synapses - ultrastructure; Synaptic Transmission - drug effects; Tetrodotoxin - pharmacology
• ISSN: 0165-0270
• DOI: 10.1016/j.jneumeth.2003.11.008

This paper describes new cytochemical method for the ultrastructural localization of Co(2+) following blockade of synaptic transmission. In the CA1 region of rat hippocampal slices, electrical stimulation of the Schaffer collaterals elicited field excitatory postsynaptic potentials (fEPSPs). The fEPSPs were completely blocked within 2 min after the addition of Co(2+) (2 mM). The slice was then fixed and precipitated Co(2+) was examined by means of a solution containing 2.5% glutaraldehyde and 10 mM K(3)[Fe(3+)(CN)(6)] in 90 mM NaCl. Electron spectroscopic imaging confirmed Co in the precipitate. The precipitates were found as clusters on the membranes of the fine apical dendrites and their spine heads of CA1 pyramidal neurons. No clustered precipitate was found when slices were treated: (1) without Co(2+); (2) after recovery from the Co(2+)-induced blockade of fEPSPs; (3) without electrical stimulation of the Schaffer collaterals; and (4) with dl-2-amino-5-phosphonopentanoate and 6-cyano-7-nitroquinoxaline-2,3-dione. After administrating glutamate (5 mM) in the presence of tetrodotoxin (1 microM) and Co(2+), precipitates were found on dendritic membranes and spine heads. These results indicate that the Schaffer collaterals stimulation induces the binding of Co(2+) on CA1 pyramidal neuron membrane.