Two-Photon Analysis of Lead Accumulation in Rat Cerebellar Granule Neurons

Authors: Esposito, Alessandro¹; Robello, Mauro²; Pellistri, Francesca¹; Marchetti, Carla³

Source: Neurochemical Research, Volume 30, Number 8, August 2005 , pp. 949-954(6)

Publisher: Springer

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Abstract:

Lead (Pb²⁺) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb²⁺binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease of the fluorescence emission, which occurred even in resting condition. In the presence of 20 μM Pb²⁺, uptake reached a plateau level (≍45% of initial fluorescence) in 4 min and was partially antagonized by 25 μM lanthanum. Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous saturation was due to inactivation of the transport system. Intracellular Pb²⁺ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free Pb²⁺ sufficient to inactivate the transport system is close to 50 pM.

Keywords: Indo-1; lanthanum; metal neurotoxicity; metal transporters; multiphoton excitation fluorescence; quenching

Document Type: Research article

DOI: http://dx.doi.org/10.1007/s11064-005-5980-y

Affiliations: 1: INFM, Dipartimento di Fisica, Università di Genova, via Dodecaneso, 33, 16146, Genova, Italy, 2: INFM, Dipartimento di Fisica, Università di Genova, via Dodecaneso, 33, 16146, Genova, Italy, Email: robello@fisica.unige.it 3: Istituto di Biofisica, CNR, via De Marini 6, 16149, Genova, Italy,

Publication date: 2005-08-01