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

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 microM Pb2+, uptake reached a plateau level (approximately 45% of initial fluorescence) in 4 min and was partially antagonized by 25 microM 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.

Role of Calcium Channels in Heavy Metal Toxicity

“…More recently other types of VDCC, different from the classical DHP-sensitive L-type, have been implicated both in Cd [19, 33] and Pb [23] uptake. Moreover, Pb has been shown to use different pathways in several systems [34–37], and it is largely independent of VDCC even in neurons [23, 24]. …”

“…In astroglia cell models, the uptake of Pb was shown to be driven by store-depletion-activated channels [38] and by two distinct pH-sensitive transport mechanisms [39]. VDCC-independent uptake of Pb by cerebellar granule neurons was inhibited by La [24], and this observation can provide some clue as for the mechanism of permeation. The involvement of a La-sensitive store-depletion activated channel, as proposed in other systems [36, 37, 40], is unlikely because Pb influx did not require drainage of the stores.…”

“…Another possibility is that Pb 2+ enters the neuron via reverse operation of an exchanger similar (or identical) to the Na–Ca exchanger, which is also very sensitive to La block [41] and whose role in Zn permeation was demonstrated in cortical neurons [42]. Also this possibility was discarded because Na–Ca exchanger does not play a significant role when the Na concentrations are close to the physiological value, as in Esposito et al [24]. It is more probable that Pb is taken up through a La-sensitive ion carrier similar to the Zn transporter found in brain neurons [43], whose identity and features still wait full definition.…”

“…In cerebellar granule neurons, Pb uses at least three pathways of permeation and besides voltage-dependent calcium channels (VDCC) and NMDA-activated channels, is rapidly taken up through passive transport system [23, 24]. …”

Cell-Biological Requirements for the Generation of Dentate Gyrus Granule Neurons

“…Immature DG granule cells show a clear GABA ergic, rather than glutamatergic, phenotype (Schmidt-Hieber et al, 2004 ; Esposito A. et al, 2005 ; Pedroni et al, 2014 ). Transient production of GABA (expression of glutamic acid decarboxylase) and release of GABA from granule cells during maturation has been observed (Cabezas et al, 2013 ) and may persist, in attenuated form, into adulthood (Gutiérrez, 2016 ).…”