William D. Atchison

The role of environmental mercury, lead and pesticide exposure in development of amyotrophic lateral sclerosis

Exposure to an environmental toxicant as a risk factor in the development of amyotrophic lateral sclerosis (ALS) was first hinted at (demonstrated) in the Chamorro indigenous people of Guam. During the 1950s and 1960s these indigenous people presented an extremely high incidence of ALS which was presumed to be associated with the consumption of flying fox and cycad seeds. No other strong association between ALS and environmental toxicants has since been reported, although circumstantial epidemiological evidence has implicated exposure to heavy metals such as lead and mercury, industrial solvents and pesticides especially organophosphates and certain occupations such as playing professional soccer. Given that only approximately 10% of all ALS diagnosis have a genetic basis, a gene-environmental interaction provides a plausible explanation for the other 90% of cases. This mini-review provides an overview of our current knowledge of environmental etiologies of ALS with emphasis on the effects of mercury, lead and pesticides as potential risk factors in developing ALS. Epidemiologic and experimental evidence from animal models investigating the possible association between exposure to environmental toxicant and ALS disease has proven inconclusive. Nonetheless, there are indications that there may be causal links, and a need for more research.

Effects of Toxic Environmental Contaminants on Voltage-Gated Calcium Channel Function: From Past to Present

Voltage-gated Ca2+ channels are targets of the number of naturally occurring toxins, therapeutic agents as well as environmental toxicants. Because of similarities of their chemical structure to Ca2+ in terms of hydrated ionic radius, electron orbital configuration, or other chemical properties, polyvalent cations from aluminum to zinc variously interact with multiple types of voltage-gated Ca2+ channels. These nonphysiological metals have been used to study the structure and function of the Ca2+ channel, especially its permeability characteristics. Two nonphysiological cations, Pb2+ and Hg2+, as well as their organic derivatives, are environmental neurotoxicants which are highly potent Ca2+ channel blockers. These metals also apparently gain intracellular access in part by permeating through Ca2+ channels. In this review the history of Ca2+ channel block produced by Pb2+ and Hg2+ as well as other nonphysiological cations is traced. In particular the characteristics of Ca2+ channel block induced by these environmental neurotoxic metals and the consequences of this action for neuronal function are discussed.

Effects of neurotoxicants on synaptic transmission: lessons learned from electrophysiological studies.

A number of environmentally-important neurotoxicants affect chemical synaptic transmission in the peripheral and central nervous system. These include heavy metals such as lead, mercury, cadmium and tin; organophosphates; pyrethroid insecticides, and 2,5-hexanedione. Electrophysiological techniques including intracellular microelectrode recording of nerve-evoked and spontaneously occurring synaptic potentials, iontophoresis of neurotransmitter, and voltage clamp of presynaptic and postsynaptic membrane ionic current have proven to be especially useful in analyzing the cellular mechanisms by which these toxicants affect neurotransmission. The process of synaptic transmission can be broadly subdivided into those processes associated with transmitter synthesis, storage and release and sometimes termination of transmitter action (presynaptic processes), and those processes associated with binding of transmitter to its receptors on the receiving cell, activation of the receptor-associated ionic channel and degradation of chemical transmitter (postsynaptic processes). The processes associated with release of neurotransmitter are the target of a number of naturally-occurring toxins and environmentally important toxicants. General mechanisms by which these agents disrupt presynaptic processes associated with transmission include: prevention or disruption of axonal excitability (pyrethroid insecticides); disruption of calcium-dependent neurotransmitter release (heavy metals, antibiotics, certain snake and spider venom toxins, botulinum toxin); and disruption of intracellular buffering of calcium (heavy metals), Mechanisms by which these agents may disrupt postsynaptic processes include effects on transmitter degradation (organophosphates) or effects on the postsynaptic membrane receptors or associated ionic channels (organophosphates, antibiotics, and perhaps pyrethroids). Microelectrode studies have shown that cadmium, lead and mercury (organic and inorganic forms) suppress release of neurotransmitter by presynaptic mechanisms and increase spontaneous discharge of transmitter quanta from the presynaptic nerve terminal. This has led to the suggestion that a component of synaptic toxicity of these agents entails block of Ca entry into and buffering by the presynaptic nerve terminals. Conventional and patch voltage clamp studies have been used to measure effects of neurotoxicants on ionic currents carried through voltage-sensitive and receptor-operated ionic channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Methylmercury-Induced Increase of Intracellular Ca2+ Increases Spontaneous Synaptic Current Frequency in Rat Cerebellar Slices

The relationship between increased intracellular calcium concentration ([Ca2+]i) and changes in spontaneous synaptic current frequency caused by the neurotoxicant methylmercury (MeHg) was examined in Purkinje cells of cerebellar slices using confocal microscopy and whole-cell recording. MeHg (10–100 μM) stimulated and then suppressed completely the frequency of spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). Current amplitude was also initially increased. The same MeHg concentrations markedly increased fluorescence of the Ca2+ indicator Fluo-4 throughout the molecular layer as well as the granule cells. No changes in fluorescence occurred in Purkinje cell soma, although fluorescence increased in their subplasmalemmal shell. Simultaneous confocal imaging and whole-cell recording revealed that time to onset of MeHg-induced increase in fluorescence in the molecular layer correlated with that of increased sEPSC and sIPSC frequency in Purkinje cells. Pretreatment with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) significantly suppressed the MeHg-induced increase in sIPSC frequency, further suggesting that MeHg-induced elevation of [Ca2+]i is partially responsible for its early stimulatory effects on spontaneous synaptic responses. However when spontaneous synaptic currents ceased with MeHg, Fluo-4 fluorescence remained elevated. Thus synaptic transmission cessation is apparently not related to changes in [Ca2+]i. It may result from effects of MeHg on transmitter release or sensitivity of postsynaptic receptors. The lack of effect of MeHg on Purkinje cell somal fluorescence reinforces that they are more resistant to MeHg-induced elevations of [Ca2+]i than other cells, including cerebellar granule cells.

Ca2+ channels as targets of neurological disease: Lambert-Eaton Syndrome and other Ca2+ channelopathies.

In the nervous system, voltage-gated Ca2+ channels regulate numerous processes critical to neuronal function including secretion of neurotransmitters, initiation of action potentials in dendritic regions of some neurons, growth cone elongation, and gene expression. Because of the critical role which Ca2+ channels play in signaling processes within the nervous system, disruption of their function will lead to profound disturbances in neuronal function. Voltage-gated Ca2+ channels are the targets of several relatively rare neurological or neuromuscular diseases resulting from spontaneously-occurring mutations in genes encoding for parts of the channel proteins, or from autoimmune attack on the channel protein responses. Mutations in CACNA1A, which encodes for the α1A subunit of P/Q-type Ca2+ channels, lead to symptoms seen in familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6. Conversely, autoimmune attack on Ca2+ channels at motor axon terminals causes peripheral cholinergic nerve dysfunction observed in Lambert–Eaton Myasthenic Syndrome (LEMS), the best studied of the disorders targeting voltage-gated Ca2+ channels. LEMS is characterized by decreased evoked quantal release of acetylcholine (ACh) and disruption of the presynaptic active zones, the sites at which ACh is thought to be released. LEMS is generally believed to be due to circulating antibodies directed specifically at the Ca2+ channels located at or near the active zone of motor nerve terminals (P/Q-type) and hence involved in the release of ACh. However, other presynaptic proteins have also been postulated to be targets of the autoantibodies. LEMS has a high degree of coincidence (∼60%) with small cell lung cancer; the remaining 40% of patients with LEMS have no detectable tumor. Diagnosis of LEMS relies on characteristic patterns of electromyographic changes; these changes are observable at neuromuscular junctions of muscle biopsies from patients with LEMS. In the majority of LEMS patients, those having detectable tumor, the disease is thought to occur as a result of immune response directed initially against voltage-gated Ca2+ channels found on the lung tumor cells. In these patients, effective treatment of the underlying tumor generally causes marked improvement of the symptoms of LEMS as well. Animal models of LEMS can be generated by chronic administration of plasma, serum or immunoglobulin G to mice. These models have helped dramatically in our understanding of the pathogenesis of LEMS. This “passive transfer” model mimics the electrophysiological and ultrastructural findings seen in muscle biopsies of patients with LEMS. In this model, we have shown that the reduction in amplitude of Ca2+ currents through P/Q-type channels is followed by “unmasking” of an L-type Ca2+ current not normally found at the motor nerve terminal which participates in release of ACh from terminals of mice treated with plasma from patients with LEMS. It is unclear what mechanisms underlie the development of this novel L-type Ca2+ current involved in release of ACh at motor nerve terminals during passive transfer of LEMS.

Bay K 8644 increases release of acetylcholine at the murine neuromuscular junction

In this study we describe the stimulatory effects of submicromolar concentrations of BAY K 8644 on neurally evoked and spontaneous release of transmitter at the vertebrate neuromuscular junction, a model cholinergic synapse. BAY K 8644 increases mean quantal content. This effect is blocked by pretreatment with the dihydropyridine (DHP) antagonist, nimodipine, but nimodipine itself had no effect on quantal content. Furthermore, BAY K 8644 induces a marked increase in spontaneous quantal release of transmitter as measured by miniature endplate potential frequency. These results are important in that they indicate that DHP-sensitive Ca2+ channels exist at motor nerve terminals and when activated can participate in transmitter release; second, they imply that these elements do not normally participate in transmitter release; third, they indicate a functional effect of DHPs on neurons under somewhat more relevant physiological conditions than those imposed by prolonged K+-induced depolarization, a technique used to evoke transmitter release in subcellular systems and fourth, they indicate an effect of BAY K 8644 on spontaneous release of transmitter, an effect not reported in previous neurochemical experiments.

Methylmercury‐Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F‐NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca2+ ([Ca2+]i) and another endogenous polyvalent cation in both synaptosomes and NG108‐15 cells. In synaptosomes, the elevation in [Ca2+]i was strictly dependent on extracellular Ca2+ (Ca2+e); similarly, in NG108‐15 cells, a component of the elevations in [Ca2+]i was Ca2+e dependent. The MeHg‐induced elevations in endogenous polyvalent cation concentration were independent of Ca2+e in synaptosomes and NG108‐15 cells. The pattern of alterations in fura‐2 fluorescence suggested the endogenous polyvalent cation may be Zn2+. Using 19F‐NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (5F‐BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn2+ concentration ([Zn2+]i). In buffer containing 200 µM EGTA to prevent the Ca2+e‐dependent elevations in [Ca2+]i, the [Zn2+]i was 1.37 ± 0.20 nM; following a 40‐min exposure to MeHg‐free buffer [Zn2+]i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn2+]i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn2+]i to 3.99 ± 0.68 nM. No Zn2+ peak was observed in synaptosomes treated with the cell‐permeant heavy metal chelator N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca2+]i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40‐min exposure to MeHg‐free buffer. [Ca2+]i was 498 ± 28 or 492 ± 53 nM during a 40‐min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca2+]i differed significantly from either pretreatment levels or buffer‐treated controls.

Methylmercury Differentially Affects GABAA Receptor‐Mediated Spontaneous IPSCs in Purkinje and Granule Cells of Rat Cerebellar Slices

Using whole‐cell recording techniques we compared effects of the environmental cerebellar neurotoxicant methylmercury (MeHg) on spontaneous IPSCs (sIPSCs) of both Purkinje and granule cells in cerebellar slices of the rat. In Purkinje cells, bath application of 10, 20 or 100 μM MeHg initially increased then suppressed the frequency of sIPSCs to zero. In granule cells, the initial increase in frequency was not observed in ≈50 % of cells examined, but suppression of sIPSCs by MeHg occurred in every cell tested. For both cells, time to onset of effects of MeHg was inversely related to the concentration; moreover, the pattern of changes in mIPSCs induced by MeHg in the presence of tetrodotoxin was similar to that in sIPSCs. For each concentration of MeHg, it took 2–3 times longer to block sIPSCs in Purkinje cells than it did in granule cells. MeHg also initially increased then decreased amplitudes of sIPSCs to block in both cells; again the response was more variable in granule cells. In most Purkinje and some granule cells, MeHg induced a giant, slow inward current during the late stages of exposure. Appearance of this current appeared to be MeHg concentration dependent, and the direction of current flow was reversed by changing the holding potentials. Reduction of the [Cl−] in the internal solution caused inwardly directed, but not outwardly directed giant currents to disappear, suggesting that this current is a Cl−‐mediated response. However, bicuculline and picrotoxin failed to block it. MeHg apparently acts at both presynaptic and postsynaptic sites to alter GABAA receptor‐mediated inhibitory synaptic transmission. GABAA receptors in granule cells appear to be more sensitive to block by MeHg than are those in Purkinje cells, although the general patterns of effects on the two cells are similar.

Passive transfer of Lambert-Eaton syndrome to mice induces dihydropyridine sensitivity of neuromuscular transmission

Lambert‐Eaton myasthenic syndrome (LEMS) is a paraneoplastic disorder in which autoantibodies apparently target the voltage‐gated Ca2+ channels that regulate acetylcholine (ACh) release at motor nerve terminals. P/Q‐type Ca2+ channels are primarily involved in ACh release at mammalian neuromuscular junctions. Passive transfer of LEMS to mice by repeated administration of plasma from LEMS patients reduces the amplitude of the perineurial P/Q‐type current, and unmasks a dihydropyridine (DHP)‐sensitive L‐type Ca2+ current at the motor nerve terminal. The present study sought to determine if this DHP‐sensitive component contributes to ACh release. Mice were treated for 30 days with plasma from healthy human controls or patients with LEMS. For some studies, diaphragms from naive mice were incubated with LEMS or control human plasma for 2 or 24 h. End‐plate potentials (EPPs) and miniature end‐plate potentials (MEPPs) were recorded from neuromuscular junctions in the hemidiaphragm. Treatment of mice with LEMS plasma evoked the characteristic electrophysiological signs of LEMS: reduced quantal content and facilitation of EPP amplitudes at high‐frequency stimulation. Quantal content was also reduced in muscles incubated acutely with LEMS plasma. Nimodipine, a DHP‐type blocker of L‐type Ca2+ channels, did not significantly affect the quantal content of muscles treated for 2 or 24 h with either control or LEMS plasma, or following chronic treatment with control plasma. However, following 30 days treatment with LEMS plasma, nimodipine significantly reduced the remaining quantal content to 57.7 ± 3.3% of pre‐nimodipine control levels. Thus, DHP‐sensitive Ca2+ channels become involved in synaptic transmission at the mouse neuromuscular junction after chronic, but not acute treatment with LEMS plasma. However, reductions in quantal release of ACh occur even after very short periods of exposure to LEMS plasma. As such, development of the L‐type Ca2+ channel contribution to ACh release during passive transfer of LEMS appears to occur only after quantal release is significantly impaired for an extended duration, suggesting that an adaptive response of the ACh release apparatus occurs in LEMS.

Effect of alteration of nerve terminal Ca2+ regulation on increased spontaneous quantal release of acetylcholine by methyl mercury

Agents known to disrupt intraterminal Ca2+ buffering, N,N-dimethylamino-8-octyl-3,4,5-trimethoxybenzoate (TMB-8), 25 microM; caffeine, 7.5 mM; N,N-bis(3,4-dimethoxyphenethyl)-N-methylamine (YS035), 180 microM; ouabain, 200 microM; and dantrolene, 50 microM, were tested for the ability to alter effects of methyl mercury (MeHg) on spontaneous quantal release of acetylcholine (ACh) at the rat neuromuscular junction. In particular, we sought to determine whether any of the above agents could prevent the MeHg-induced increase of spontaneous release of ACh, an effect measured electrophysiologically as increased frequency of miniature end-plate potentials (MEPPs). MEPPs were recorded continuously from myofibers of the rat hemidiaphragm using conventional, intracellular recording techniques during pretreatment with an inhibitor of Ca2+ regulation and subsequently with the inhibitor plus MeHg (100 microM). When given alone, caffeine and ouabain, which release Ca2+ from the smooth endoplasmic reticulum and mitochondria, respectively, increased MEPP frequency in a biphasic manner. Following pretreatment, concomitant application of MeHg with caffeine or ouabain increased MEPP frequency after a brief latent period to peak values of 53 and 92 Hz, respectively. TMB-8 and dantrolene, putative inhibitors of Ca2+ release from smooth endoplasmic reticulum, differed in their effects on MEPP frequency; TMB-8 alone decreased MEPP frequency to approximately 10% of drug-free control, whereas dantrolene did not significantly alter control MEPP frequency. Subsequent concomitant application of MeHg with TMB-8 or dantrolene increased MEPP frequency to peak values of 40 and 100 Hz after 17 and 30 min, respectively. YS035, a putative inhibitor of mitochondrial uptake and release of Ca2+, decreased MEPP frequency to less than 10% of control after 15 min when given alone. Application of MeHg following YS035 pretreatment failed to increase MEPP frequency for up to 90 min. YS035 did not mask a MeHg effect by blocking postsynaptic sensitivity to ACh or preventing its release since subsequent treatment with La3+ (2 mM) after YS035 had abolished spontaneous release, increased MEPP frequency within 5 min. Thus, of the five inhibitors of nerve terminal Ca2+ regulation tested, only YS035 prevented the stimulatory action of MeHg on MEPP frequency. Results of the present study suggest that release of Ca2+ from nerve terminal mitochondria contributes to the increased MEPP frequency caused by MeHg while release of Ca2+ from smooth endoplasmic reticulum may not.