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INTRODUCTION |
Aluminum is the most abundant metal, and one of the most common elements, in the earth's crust. Despite being found in most body tissues, aluminum has no known physiological function. Furthermore, accumulation of aluminum within the body, via ingestion and/or inhalation, can result in neurotoxicity. Renal hemodialysis patients, who can have 15 times the brain content of aluminum of normal subjects (Berlyne 1989
), manifest multiple cognitive impairments including decreases in visual memory, attention, concentration, and frontal lobe functions (Alfrey et al. 1976
; Berlyne 1989
; Bolla et al. 1992
). Other studies have implicated aluminum in several other neuropathologic conditions including Guamanian amyotrophic lateral sclerosis-parkinsonism dementia and Alzheimer's disease (e.g., Perl et al. 1982
). Aluminum is reportedly concentrated in the neurofibrillary tangles and amyloid plaques of patients with Alzheimer's disease (Candy et al. 1986
). Furthermore, the risk of developing Alzheimer's disease is higher after consumption of drinking water with elevated aluminum concentrations (Rifat 1994
). An interesting, but controversial, hypothesis regarding the etiology of Alzheimer's disease is that a virus or exogenous toxic material (e.g., aluminum) gains access to the CNS by using the olfactory mucosa as a portal of entry. Because the olfactory system is the only portion of the CNS that has direct exposure to the external environment, it is uniquely capable of the uptake and transneuronal spread of such exogenous substances. This hypothesis is further supported by studies demonstrating that exposure to intranasal aluminum results in uptake into the brain and distribution along olfactory pathways (e.g., Perl and Good 1987
). After intranasal exposure, excess accumulation of aluminum has been documented in the olfactory bulb, cortex, hippocampus, enthorhinal area, and white matter (Zatta et al. 1993
).
Despite these results, the existence of a causal relationship between aluminum and neurodegenerative disorders, such as Alzheimer's disease, remains unclear (Doll 1993
; Rifat 1994
). Some of the reason for the continuing debate may relate to the relative paucity of available information regarding potential cellular mechanisms that may underlie aluminum's contribution to neurotoxicity and/or neuropathology. Although several preliminary observations have been made, even these data are controversial. Reportedly, aluminum can inhibit some voltage-gated ion channels (e.g., Platt and Busselberg 1994
) as well as glutamate receptor-mediated currents in rat hippocampal neurons (Platt et al. 1994
). Yet, the reported effects of aluminum on synaptic pathways that use amino acid transmitters (including glutamate) are conflicting. Although Platt et al. (1995)
reported that aluminum impairs long-term potentiation (LTP) in the hippocampus, Gilbert and Shafer (1996)
reported that aluminum has no effect on hippocampal LTP.
The present study was designed to clarify potential direct effects of aluminum on neuronal membrane properties as well as on several types of amino acid receptors on olfactory bulb neurons. The context for the experiments was the reported links among various neuropathologies, metals (aluminum, zinc), the olfactory system, and pathology of neurons that use or express the amino acid transmitters glutamate,
-aminobutyric acid (GABA), and glycine (for review see Greenamyre and Maragos 1993
). The choice of olfactory bulb culture and electrophysiology as an experimental preparation was based on several factors. First, culture allows one to investigate synaptic mechanisms of action by the coapplication of aluminum and various amino acid receptor agonists in an isolated setting. The significance of using the olfactory bulb is underscored by the above-stated hypothesis as well as Ferreyra and Barragan's (1989) proposal that Alzheimer's associated pathology begins in olfactory regions. This theory is based on the finding that olfactory dysfunction is among the first symptoms of Alzheimer's disease, often involving a loss of olfactory perception and discrimination. Furthermore, the initial neuropathology associated with Alzheimer's disease
the formation of senile plaques and neurofibrillary tangles, the degeneration of mitral cells, a decrease in the density of dopamine receptors, and a decrease in tissue volume
occurs in the olfactory bulb (Doty 1991
; Talamo et al. 1989
). A final reason to pursue these studies in the olfactory bulb is the unusually high accumulation of aluminum in this brain region. In contrast to the reported range of aluminum concentrations in cortical tissue of only 1-14 µg/g, aluminum concentrations in rat olfactory bulb range from 15 to 250 µg/g (Domingo et al. 1996
).
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METHODS |
Cell cultures
Preparation of primary cultures of olfactory bulb neurons was similar to that previously described (Trombley and Westbrook 1990
). Olfactory bulbs were dissected from embryonic day 18 to postnatal day 3 rat pups, stripped of meninges, and incubated at 37°C for 45-60 min in a Ca/ethylenediaminetetraacetic acid (EDTA)-buffered solution containing 20 U/ml papain (Worthington) and 1 mM cysteine. After enzyme inactivation with bovine serum albumin (2.5 mg/ml), the tissue was gently triturated and the cell suspension plated on a confluent layer of olfactory bulb astrocytes. The neuronal growth medium contains 90% Minimal Essential Medium (MEM, GIBCO) with 10% fetal calf serum, 6 g/l glucose, and a nutrient supplement (Serum Extender, Collaborative Research). Cytosine-
-D-arabinofuranoside (10
5 M) was added 1 day after plating neurons to prevent overgrowth of background cells. Electrophysiological recordings were performed after 1-35 days in culture.
Electrophysiology
Voltage- and current-clamp recordings were performed at room temperature with the culture dish mounted on the stage of an inverted Nikon phase-contrast microscope as a recording chamber. The dish was perfused at 0.5-2.0 ml/min with a solution containing the following (in mM): 162.5 NaCl, 2.5 KCl, 2 CaCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10 glucose, and 1 or 0 MgCl2, and also 1 µM glycine. The osmolarity was 325 mosM and the pH was adjusted to 7.3 with NaOH. Patch electrodes were pulled from borosilicate glass, fire polished, and filled with a solution containing (in mM) 145 KMeSO4 or CsCl, 1 MgCl2, 10 HEPES, 4 Mg-ATP, 0.5 Mg-guanosine 5'triphosphate (GTP), and 1.1 or 11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 7.2 and osmolarity 310. Electrode resistances were 4-6 M
. Drugs were diluted in the recording solution and delivered from drug reservoirs by a flow-pipe perfusion system, consisting of an array of eight 400-µm-ID glass barrels fed by gravity. The flow pipes were aligned with the recorded neuron by using a hydraulic manipulator, and the flow was controlled with pinch clamps. Neurons were always perfused with fast flow from one barrel containing control solution except during the application of drugs. The full concentration of the barrel solution reached the cell in 100-150 ms. This determination was made by using saturating concentrations of amino acid receptor agonists and measurements of peak response times.
Whole cell recordings were made from mitral/tufted (M/T) cells with an Axoclamp 2B amplifier (Axon Instruments). Membrane currents were recorded in the discontinuous, single-electrode voltage-clamp mode at a switch frequency of 8-12 kHz. The recorded current was filtered at 1-3 kHz and digitized at 5-10 kHz. Membrane voltage was recorded unfiltered in current-clamp mode.
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RESULTS |
Aluminum has no marked direct effects on neurons
Stable whole cell recordings were obtained from cultured M/T cells by using patch electrodes filled with a KMeSO4-based solution. In current-clamp mode, brief (5 ms) depolarizing current steps evoked overshooting action potentials with 3-4 ms half-widths (Fig. 1A). Aluminum (100 µM) had no effect on action-potential duration or amplitude (n = 6).

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| FIG. 1.
Aluminum had no apparent direct effect on olfactory bulb neurons. A: aluminum did not alter the shape or amplitude of evoked action potentials. B: under voltage clamp at 60 mV, flow-pipe application of 100 µM aluminum did not evoke a membrane current. C: aluminum did not alter the current evoked by a voltage ramp from 75 to +25 mV. D: aluminum had no marked effects on the family of inward and outward currents evoked by 10-mV, 50-ms depolarizing voltage steps. E: aluminum did not reduce spontaneous excitatory synaptic activity. A KMeSO4-based intracellular solution was used in each experiment.
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Potential direct effects of aluminum on olfactory bulb M/T cells were examined further in voltage-clamp mode. Aluminum (100 µM) did not evoke a membrane current when applied during voltage-clamp recording at holding potentials of
85 to
60 mV (Fig. 1B, n = 13). Voltage ramps were used in a second set of experiments to control for the possibility that the holding potentials in the previous experiments were near the reversal potential for a current evoked by aluminum. While using the KMeSO4-based intracellular solution, the voltage was ramped to +25 mV at 100 mV/s from an initial holding potential of
75 mV. The resulting membrane current was compared with the current evoked by the same voltage ramp in the presence of 100 µM aluminum. Aluminum did not alter the shape of evoked current; as shown in Fig. 1C, the two currents overlap completely (n = 5). Potential effects of aluminum on macroscopic voltage-gated currents also were assessed. Under voltage-clamp, a series of 10-mV, 50-ms depolarizing steps beginning at
70 mV resulted in a family of inward and outward membrane currents. The family of currents consisted of a transient inward current followed by an transient outward current and a sustained outward current. The kinetics and voltage sensitivity of these currents correspond well with previously described tetrodotoxin-sensitive sodium currents and A-type and delayed rectifier-type potassium currents (Hille 1992
; Trombley and Westbrook 1991
). Application of aluminum had no marked effects on this family of macroscopic currents (Fig. 1D, n = 5).
Aluminum has no effect on glutamate receptor-mediated currents
The results from several reports suggest that aluminum may affect amino acid receptors. Platt et al. (1994)
have suggested that aluminum can inhibit glutamate receptor-mediated currents in rat hippocampal neurons. As mentioned in the previous section, there also exists some controversy as to whether aluminum affects LTP (Glibert and Shafer 1996; Platt et al. 1995
). Therefore we tested the hypothesis that aluminum can alter amino acid receptor function by examining the effects of aluminum on whole cell currents evoked by agonists for several types of amino acid receptors.
The first agonist selected was glutamate; this was applied at 500 µM in the absence of extracellular magnesium to activate all subtypes of ionotropic glutamate receptors, including N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (non-NMDA) receptors (Patneau and Mayer 1990
). Under voltage clamp, at a holding potential of
60 mV, 500 µM glutamate evoked an inward membrane current. Aluminum (10-100 µM) applied during the middle of the current evoked by glutamate had no detectable effect (Fig. 2; n = 8). Additionally, NMDA and kainate were used to further isolate glutamate receptor subtypes. Aluminum (10-100 µM) also had no effect on NMDA receptor- or on kainate receptor-mediated currents (Fig. 2, n = 8). Consistent with these results, aluminum (100 µM) had no effect on spontaneous glutamate-mediated excitatory synaptic activity (Fig. 1E, n = 4).

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| FIG. 2.
Aluminum selectively modulates -aminobutyric acid (GABA)-evoked currents. Under voltage clamp at 60 mV, 100 µM aluminum was applied during the middle of the current evoked by glutamate, kainate, N-methyl-D-aspartate (NMDA), glycine, or GABA. Aluminum dramatically potentiated the current evoked by GABA, but had no effect on currents evoked by the other amino acid receptor agonists. A CsCl-based intracellular solution was used in each experiment.
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At low µM concentrations, aluminum selectively potentiates GABAA receptors
Because Ma and Narahashi (1993)
reported that aluminum slightly depressed the current evoked by GABA in rat dorsal root ganglion neurons, the effects of aluminum also were examined on currents evoked by the inhibitory amino acids GABA and glycine. Whole cell chloride currents evoked by GABA or glycine were inward at
60 mV and reversed near 0 mV, because with the CsCl electrode solution, the intracellular and extracellular chloride concentrations were approximately equimolar. These currents correspond to previously described, and pharmacologically distinct, GABAA receptor- and glycine receptor-mediated currents in olfactory bulb neurons (Trombley and Shepherd 1994
). Coapplication of 100 µM aluminum had no effect on the current evoked by 100-300 µM glycine (n = 12), but dramatically potentiated the current evoked by 30 µM GABA (Fig. 2). In 23 of 34 neurons, 10-100 µM aluminum potentiated the GABA receptor-mediated current by 10-400% (Fig. 3).

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| FIG. 3.
Aluminum has a biphasic effect on GABA-evoked currents. At concentrations <100 µM, aluminum potentiated the current in most neurons. As shown in the 1st panel, even 10 µM aluminum could potentiate the current in some neurons. At aluminum concentrations >300 µM, the current was blocked. Only the blocking effect was observed in some neurons. CsCl-based intracellular solution; holding potential = 60 mV.
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These effects of aluminum on GABA-evoked currents were biphasic and concentration dependent. At low concentrations (
100 µM), aluminum potentiated the current in 23 neurons, had no effect in 7 neurons, and slightly blocked the current in 4 neurons. At higher concentrations (
300 µM), aluminum either blocked the current (n = 12) or had no effect (n = 3). Yet even at the highest concentration tested (1,000 µM), aluminum failed to block all of the current (Fig. 3, 70 ± 23% block, n = 4). GABA-mediated currents in some neurons were very sensitive to aluminum. In the cell shown in Fig. 3, 10 µM aluminum potentiated the current by 60%. In some cells, the potentiating action of aluminum was highly repeatable. In others, however, the effects progressively declined with subsequent application. The blocking effects of aluminum were less subject to variation.
Aluminum did not change the reversal potential of the GABA-evoked current, suggesting that the potentiation was not due to an alteration in the ionic selectivity of the channel. Furthermore, neither the potentiating effects, nor the blocking effects, were voltage dependent. While using approximately equimolar intracellular and extracellular chloride, the effects of aluminum were examined on inward currents at hyperpolarizing potentials and on outward currents at depolarizing potentials. Aluminum at 100 µM was equally effective at potentiating the inward and outward currents (Fig. 4). At 300 µM, aluminum blocked the inward and outward current to the same extent (Fig. 4), suggesting that it was not acting as a pore or channel blocker or through surface-charge screening.

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| FIG. 4.
Effects of aluminum were not voltage dependent. Inward currents were evoked by 30 µM GABA from a holding potential of 30 mV, and outward currents from a holding potential of +30 mV. Intracellular and extracellular chloride concentrations were equimolar. Aluminum at 100 µM potentiated inward and outward currents to a similar extent. Aluminum at 300 µM also blocked inward and outward currents to a similar extent. CsCl-based intracellular solution.
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The effects of aluminum on the peak current, before desensitization, were compared with the effects of aluminum on the steady-state desensitized current. As seen in Fig. 5, when aluminum was applied simultaneously with GABA, aluminum potentiated the peak current by only 18% but potentiated the steady-state current by 241% (Fig. 5B, n = 5). The degree of potentiation of the steady-state current when GABA and aluminum were applied simultaneously was similar to the degree of potentiation observed when aluminum was applied during the middle of the desensitized current evoked by GABA (235%; Fig. 5D). In Fig. 5E the control GABA-evoked current is overlaid with the aluminum-potentiated current. The current amplitudes have been normalized. As was typical of the potentiated response, the whole cell current showed slower desensitization kinetics and the final steady-state amplitude was greater.

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| FIG. 5.
Comparison of the effects of aluminum on the peak vs. steady-state current evoked by GABA. A: current evoked by 30 µM GABA alone. B: when applied simultaneously with GABA, aluminum (100 µM) slightly increased the peak amplitude of the GABA-evoked current (before receptor desensitization), but had a much greater potentiating effect on the desensitized steady-state component. C: recovery from aluminum. D: potentiating effect of aluminum, when applied during the middle of the desensitized steady-state GABA-evoked current, was similar in amplitude to that in B. E, bottom panel: aluminum potentiates the GABA-evoked current by slowing the rate and extent of receptor desensitization. The current traces from A and B were overlaid and the amplitudes normalized. Shaded region represents the potentiation by aluminum. CsCl-based intracellular solution.
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DISCUSSION |
The present results demonstrate that aluminum selectively modulates GABAA receptor-mediated currents. At low concentrations, aluminum potentiated GABA-evoked currents; at higher concentrations, aluminum blocked GABA-evoked currents. In some neurons, aluminum only blocked the GABA-evoked current and potentiation was not observed. These results also suggest that aluminum has little effect on basic membrane properties, macroscopic voltage-gated currents, or currents evoked by the amino acid receptor agonists glutamate, kainate, NMDA, and glycine.
The mechanism of action of aluminum at GABA receptors has not been completely determined. At low concentrations, aluminum may enhance GABA-evoked currents through decreasing the rate of receptor desensitization. This hypothesis is based on the finding that aluminum had much less effect on the peak current than on the desensitized steady-state current. Under the recording conditions used, the full concentration of the agonist reached the cell in 100-150 ms. Some receptor desensitization likely occurred before reaching peak current amplitude and may have contributed to the slight potentiation observed with aluminum. A more detailed analysis using ideal drug delivery parameters (such as very fast application of saturating concentrations of agonist) is required to further elucidate these mechanisms. The fact that the effects of aluminum were not significantly voltage-dependent indicates that allosteric modulation of the receptor, rather than surface charge screening or binding within the channel, likely was responsible for the observed effects. Together these results may suggest that GABA receptors express two allosteric binding sites for aluminum: a high-affinity site that is potentiating and a low-affinity site that is inhibiting. At high concentrations the effects of the low-affinity site dominate and the current is blocked. The additional finding that aluminum at intermediate concentrations had no apparent effect on the current suggests a balance between the actions of these two sites.
It is noteworthy that the observed effects of aluminum on GABA-mediated currents are similar to the reported effects of zinc on glycine-evoked currents. At low concentrations, zinc potentiates glycine-evoked currents, whereas higher concentrations of zinc block glycine-evoked currents (Bloomenthal et al. 1994
; Trombley and Shepherd 1996
). Also paralleling the present findings, potentiation and inhibition of glycine-evoked currents by zinc appear to be due to separate binding sites, with different affinities for zinc, located on the alpha subunit of the glycine receptor (Laube et al. 1995
). That aluminum only blocked the GABA-evoked current in some neurons may suggest differences in the GABAA receptor subunit composition, with some neurons expressing receptors that only have binding sites that mediate the blocking action of aluminum.
Other reports, some conflicting, on the variety of effects of aluminum on neuronal functioning provide additional context for the interpretation of the present results. Platt et al. (1994)
concluded that the effects of aluminum on glutamate receptors were nonspecific because high concentrations of aluminum (mM) were used in their experiments, and aluminum blocked the currents mediated by all glutamate ionotropic receptors (NMDA and AMPA/kainate receptors). In contrast, the concentration range of aluminum used in the present experiments (100-300 µM) did not cause a nonspecific block of glutamate receptors. Furthermore, the present results suggest that aluminum binds to specific allosteric sites, because its effects are selective, not voltage-dependent, and even 10 µM aluminum can potentiate GABA-evoked currents.
It also has been reported by Platt et al. (1995)
that aluminum impairs LTP in vivo and in vitro. In their study, 100 µM aluminum reduced both the amplitude and duration of LTP in the CA1 region. These findings are consistent with those of Provan and Yokel (1992)
who suggest that aluminum inhibits [14C]glutamate release from hippocampal slices with an IC50 of 40 µM through effects on calcium channels, G-proteins, and protein kinase C. However, the results of another in vitro study suggest that 100 µM aluminum has no effect on the magnitude nor longevity of LTP in CA1 (Gilbert and Shafer 1996
). Furthermore, these investigators report that aluminum had no effect on calcium-dependent [14C]glutamate release from hippocampal slices.
Several additional studies have addressed the effects of aluminum on voltage-gated ion channels. For example, it has been reported that aluminum blocks voltage-gated calcium channel currents with an IC50 near 85 µM (Busselberg 1995
; Platt and Busselberg 1994
). Aluminum appears to act on the channel in the open state (Busselberg 1995
), irreversibly blocking the channel (Platt and Busselberg 1994
). These effects of aluminum on calcium channel currents also appear to be pH-dependent (Busselberg et al. 1993
; Platt et al. 1993
), i.e., low pH increases the sensitivity of the channel to aluminum. Busselberg et al. (1993)
have suggested that although aluminum blocks calcium channel currents, it has little effect on sodium or potassium channels. In contrast, Kanazirska et al. (1997)
have reported that although 100 µM aluminum has little affect on potassium channels from rat hippocampal neurons, it inhibits sodium channels.
Aluminum did not have any marked effect on macroscopic voltage-gated currents in the present study. In these experiments the extracellular solution contained a normal concentration of calcium (2 mM), and sodium and potassium currents were not blocked. Under these conditions sodium and potassium currents were large and 100 µM aluminum had no apparent effect. However, calcium channel currents would be small under these conditions; hence, a small effect of aluminum would be difficult to detect. However, aluminum (100 µM) did not reduce spontaneous glutamate-mediated excitatory synaptic transmission (Fig. 1E), suggesting that aluminum had little effect on presynaptic calcium channels and subsequent release of glutamate.
It is not clear why there is such disparity in the literature on the effects of aluminum. It is possible that the variety of results is due, in part, to the chemistry of aluminum in solution and different experimental protocols. In solution, aluminum has complex effects on pH and forms a mixture of aluminum hydroxides. These hydroxides (and alterations in pH) can exert their own independent effects (e.g., Meiri and Shimoni 1991
), thus complicating the interpretation of results. In the present experiments, adequate buffering prevented changes in pH, but aluminum hydroxides may still have contributed to the effects of aluminum because at physiological pH the dominant species of aluminum is Al(OH)3 (Zhang and Columbini 1989). A decrease in pH, as seen in some neurodegenerative diseases, would cause a shift toward free ionic aluminum (Zhang and Columbini 1989), potentially contributing to observed neuropathology.
Because most synaptic pathways in the mammalian CNS use amino acid transmitters, understanding mechanisms that alter amino acid receptor function is critical to our comprehension of both normal and pathological synaptic physiology. Recent advances in the treatment of neuropathological conditions have increasingly involved alterations in synaptic function. Thus the development of future therapeutic strategies depends on progress in our understanding of synaptic physiology. Although an etiologic role for aluminum in neuropathological conditions including Alzheimer's disease remains controversial, altered and/or elevated concentrations of aluminum in the brain have been associated with a variety of cognitive impairments (e.g., Berlyne 1989
; Perl et al. 1982
). Some researchers have hypothesized that aluminum may not be a direct cause of impairment, but rather factors associated with these conditions may allow metals (e.g., aluminum, zinc) to initiate or perpetuate deleterious effects (Cuajungco and Lees 1997
). A potential means by which metals may cause such effects is the aberrant modulation of synaptic pathways that use amino acid transmitters. The present results indicate that aluminum can alter the function of GABAA receptors. Because GABA is the dominant inhibitory transmitter in the brain, such alterations in GABAA receptor function may lead to widespread changes in inhibitory circuits that may contribute to neuropathology.