A growing number of reports suggest that elevated levels of extracellular Alzheimer's ␤-amyloid protein alter the homeostasis of free [Ca 2؉ ] i in different cell types of the mammalian brain. In line with these results, we have previously shown that A␤P[1-40] forms cation-selective channels (Ca 2؉ included) across artificial planar bilayers formed from acidic phospholipids and across excised membrane patches from immortalized hypothalamic GnRH neurons (GT1-7 cells), suggesting that the nonregulated Ca 2؉ -influx through these spontaneously formed "amyloid channels" may provide a mechanism to explain its toxicity (1). We have now found and report here that the application of A␤P[1-40] to GT1-7 neurons consistently elevates [Ca 2؉ ] i levels. We also found that human islet amylin and the prion protein fragment (PrP106 -126), peptides that acquire ␤-pleated sheet conformation in water solutions and have been reported to form ion channels across planar bilayer membranes, also increase cytosolic free calcium in GT1-7 neurons. Searching for protective agents, we found that soluble cholesterol, known to decrease the fluidity of the cell membrane, inhibits A␤P[1-40]-evoked [Ca 2؉ ] i rise. These results suggest that unregulated Ca 2؉ entry across amyloid channels may be a common mechanism causing cell death, not only in diseases of the third age, including Alzheimer's disease and type 2 diabetes mellitus, but also in prion-induced diseases. Alzheimer's disease (AD)1 is a dementia characterized by the loss of mental and physical functions and the presence in the brain of protein deposits called extracellular plaques and intracellular aggregates called fibril tangles (2, 3). The principal constituents of these deposits are peptides, 39 -43 residues long, termed amyloid ␤-proteins (A␤P), that exhibit distinct domains that acquire ␤-pleated sheet structure and ␣-helical architecture. These peptides originate from the proteolytic degradation by secretases of the amyloid precursor protein defined by a locus on chromosome 21 (4, 5). It is well established that ␤-amyloid peptides in water solutions acquire ␤-pleated sheet architecture and form insoluble aggregates. Earlier studies showed that application of A␤P[1-40] to either primary cultures of rat hippocampal neurons (6) or hippocampal slice cultures (7) had toxic effects. These results provide support for the idea that A␤P[1-40] might be the causal agent of neuronal death in AD (8). Interestingly, islets of Langerhans of patients with non-insulin-dependent diabetes mellitus, which like AD is an age-dependent disease, also exhibit protein deposits. The major constituent of the pancreatic islet plaques is an amyloid peptide termed amylin (9). It has also been reported that human islet amylin (37-amino acid residues), found in protein deposits within islets of Langerhans of patients with non-insulindependent diabetes mellitus, is toxic to both cultured islet cells (10) and rat hippocampal neurons (11) and also induces apoptosis in rat cortical neurons (12). Although there i...

We have previously shown that the 40-residue peptide termed amyloid beta-protein (A beta P[1-40]) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether A beta P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A beta P[1-40] leads to the spontaneous formation of cation-selective channels. With Cs+ as the main cation in both the external as well as the internal saline, the amplitude of the A beta P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid beta-protein in solution, can block the flow of Cs+ through the A beta P[1-40] channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A beta P[1-40] channel pore. These properties of the A beta P[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid beta-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid beta-protein and neuronal membranes also occur in vivo, lending further support to the idea that A beta P[1-40] channel formation might be a mechanism of amyloid beta-protein neurotoxicity.

BackgroundAcetamiprid (ACE) and imidacloprid (IMI) belong to a new, widely used class of pesticide, the neonicotinoids. With similar chemical structures to nicotine, neonicotinoids also share agonist activity at nicotinic acetylcholine receptors (nAChRs). Although their toxicities against insects are well established, their precise effects on mammalian nAChRs remain to be elucidated. Because of the importance of nAChRs for mammalian brain function, especially brain development, detailed investigation of the neonicotinoids is needed to protect the health of human children. We aimed to determine the effects of neonicotinoids on the nAChRs of developing mammalian neurons and compare their effects with nicotine, a neurotoxin of brain development.Methodology/Principal FindingsPrimary cultures of cerebellar neurons from neonatal rats allow for examinations of the developmental neurotoxicity of chemicals because the various stages of neurodevelopment—including proliferation, migration, differentiation, and morphological and functional maturation—can be observed in vitro. Using these cultures, an excitatory Ca2+-influx assay was employed as an indicator of neural physiological activity. Significant excitatory Ca2+ influxes were evoked by ACE, IMI, and nicotine at concentrations greater than 1 µM in small neurons in cerebellar cultures that expressed the mRNA of the α3, α4, and α7 nAChR subunits. The firing patterns, proportion of excited neurons, and peak excitatory Ca2+ influxes induced by ACE and IMI showed differences from those induced by nicotine. However, ACE and IMI had greater effects on mammalian neurons than those previously reported in binding assay studies. Furthermore, the effects of the neonicotinoids were significantly inhibited by the nAChR antagonists mecamylamine, α-bungarotoxin, and dihydro-β-erythroidine.Conclusions/SignificanceThis study is the first to show that ACE, IMI, and nicotine exert similar excitatory effects on mammalian nAChRs at concentrations greater than 1 µM. Therefore, the neonicotinoids may adversely affect human health, especially the developing brain.

1. In Mg(2+)-free external solution, rat cortical neurons in cultured networks entered a stable firing mode, consisting of regular bursts of action potentials superimposed on long-lasting depolarizations. The average separation between bursts varied from culture to culture, but was usually between 5 and 20 s. The distribution of burst intervals followed a Gaussian or normal distribution, with a standard deviation of typically 10% of the average burst period. 2. A gradually depolarizing pacemaker potential was never observed between bursts, but the threshold for action potentials during the quiescent phase was > or = 10 mV above the resting potential. No progressive change in conductance or excitability was observed during the quiescent period. Intracellular stimulation of action potentials did not reproduce the long-lasting depolarization. 3. Switching from current clamp to voltage clamp at the resting potential revealed large postsynaptic currents, mainly excitatory but with a small inhibitory component, at the same phase and frequency as the spike bursts, showing that periodic synaptic input is responsible for the burst-depolarizations. The current could be eliminated by local application of 2-amino-5-phosphonovaleric acid (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the postsynaptic cell. In the presence of tetrodotoxin, irregular miniature excitatory postsynaptic currents were observed. 4. A fluorescent calcium indicator (fluo-3, 100 microM) was included in the whole-cell pipette solution, to allow simultaneous electrical and calcium measurements in the same cell. In current clamp, transient intracellular calcium increases were found, which were synchronized to the spike bursts. The Ca2+ rise lasted as long as the action potential burst, and was followed by an exponential decay considerably slower than that of the membrane potential. Calcium transients disappeared during voltage clamp at the resting potential, suggesting that calcium influx through voltage-dependent calcium channels greatly exceeds that through synaptic channels. 5. Multisite Ca2+ recording, after loading with fluo-3 acetoxymethyl (AM) ester, revealed that the onsets of burst-related calcium transients were synchronized in all active cells of each view-field, to within approximately 20 ms. Occasionally, secondary rhythms were observed in which only a subset of cells participated. The times to peak and the decay times of calcium transients varied among synchronized cells. 6. The pharmacology of the burst-related calcium transients was investigated by bath application of a variety of compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

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