This study suggests that human LC neurons contain heavy metals quite commonly. These LC heavy metals are, however, more likely to be found in patients with MND than controls. In the majority of MND patients, heavy metals were detected in spinal motor neurons as well. The AMG silver amplification technique used detects the sulphides or selenides of mercury, silver, and bismuth. After silver was removed chemically from the tissues, AMG staining was still present, indicating that either mercury and bismuth was likely to be causing the staining. Human exposure to bismuth is rare, occurring mostly after consumption of bismuth-containing medications
[12]. Therefore the metal seen on AMG in this study is most likely to be mercury, a known neurotoxicant with numerous natural and anthropogenic sources
[13].
The locus ceruleus is a potential pathway for toxicants to enter the brain
Experimental animal studies show that circulating metal toxicants enter motor neurons selectively, probably via retrograde axonal transport from the neuromuscular junction
[14]. However, only recently has it been shown that a heavy metal, inorganic mercury, enters LC neurons selectively as well
[2]. This entry into the LC probably occurs because the LC innervates the great bulk of CNS microvessels
[15], and so is exposed to a large volume of circulating blood. In fact, a rough calculation indicates that, with the number of normal human LC neurons being 32,000
[16] and the total capillary length of the brain being 640 kilometres
[17], each LC neuron on average innervates a 20 meter length of capillary
[18]. The LC contains numerous neurotransmitters in addition to noradrenaline
[19], and re-uptake of these neurotransmitters at capillary terminals could be harnessed by a number of toxicants to enter the LC via recycling mimicry
[20].
Age and heavy metals in the locus ceruleus
In our previous study, we found no heavy metals in infant motor neurons, and suggested that metals were likely to accumulate in motor neurons during aging
[3]. In the present study, there was no correlation between increasing age and heavy metal content of the LC. This raises the possibility that toxicant exposure in these individuals was episodic, rather than continuous, in nature. Exposure may occur early in life, as evidenced by one 26 year-old man without neurological disease already having heavy metals within his LC. One possible reason for this early LC uptake of toxicant is that stressor-induced upregulation of the LC promotes bursts of toxicant intake into the nucleus
[2].
The amount of intracellular heavy metal varies widely between adjacent locus ceruleus neurons
A question that cannot be answered by this study was why only some LC neurons contained heavy metal staining, with densely-stained neurons usually found adjacent to neurons with no AMG staining. This phenomenon was also seen in a man who injected himself with a large dose of metallic mercury
[2]. Topographic outputs of the rodent and primate LC to various brain regions have been described, but not at the level of individual LC neurons. Possibilities are that the toxicant-containing LC neurons have a particularly large output to the capillary bed and so are exposed to greater amounts of circulating toxicant. Another possibility is that specific stressors, e.g., those requiring the LC to activate the motor system, result in the uptake of toxicants into LC neurons that are activated to deal with those specific stressors.
Could toxicants in the locus ceruleus trigger MND?
We were not able to detect heavy metals in cortical motor neurons, despite these neurons being affected in ALS, and being shown to contain mercury after exposure to metallic mercury
[2]. This could be because of a survivor effect, with the toxicant-containing cortical motor neurons dying early and leaving only toxicant-free motor neurons intact. Secondly, toxicants within the LC may reduce noradrenaline output to the cortical motor neurons and secondarily damage cortical motor neurons, without the need for a toxicant to be within cortical motor neurons. After being activated by a stressor, LC neurons supply noradrenaline directly to motor neurons, as well as to microvessels and glial cells, all of which have noradrenaline surface receptors (Figure
7).
Recent studies indicate that many CNS cells have noradrenaline receptors, so reduced noradrenaline output can cause a number of deleterious changes to neurons
[19, 21]. These include changes that are implicated in MND, such as blood–brain barrier permeability to toxicants or inflammatory agents
[22], decreased astrocytic function leading to increased synaptic glutamate
[23], reduced trophic support to the neuron
[24], microglial activation and inflammation
[25], and oligodendrocyte dysfunction causing impaired myelination
[26]. Finally, it has been suggested that the LC could transfer toxicants from the circulation to cortical motor neurons
[2], since the LC makes direct contact with both capillaries and motor neurons. A further discussion on how toxic damage to the LC could result in different neurological conditions has recently been published
[18].
No technique currently exists to reliably measure the concentration of toxicants such as heavy metals within individual neurons, which raises the question of how to assess how much heavy metal is in LC neurons. The lack of obvious neuronal loss in the metal-containing LCs of our MND patients could lead to the presumption that the amount of heavy metal was insufficient to damage the neurons. However, it has previously been shown that mouse spinal motor neurons that contain a non-toxic dose of inorganic mercury suffer axonal shrinkage, without any loss of numbers of motor neuron cell bodies
[9]. This indicates that a low dose of a heavy metal can cause neuronal damage without cell body loss, and by extrapolation suggests a loss of noradrenaline from LC terminals could occur without the loss of toxicant-containing LC cell bodies. Of interest, one of the few quantitative studies of the LC in MND has shown neuronal shrinkage of LC cell bodies without cell loss
[27], though no unbiased quantitative studies of cell numbers in the LC of MND patients have been reported. Even a moderate level of LC neuronal damage could have a deleterious effect on spinal motor neurons, since the long LC axons extending down to the spinal cord would be particularly susceptible to toxic disturbances in their parent cell bodies
[28].
AMG can detect only a few heavy metals, but environmental exposures usually involve different types of toxicants, often simultaneously. For example, cigarette smoke, a possible risk factor for MND
[29], contains 4,800 identified compounds, including metal toxicants
[30]. The heavy metals identified in this study may not therefore be a cause of neurotoxicity, but may merely be a marker that indicates how readily a number of toxicants are entering LC and motor neurons.
Although we have suggested here that heavy metals in the LC could play a part in the pathogenesis of MND, an alternative explanation for the presence of heavy metals in the LC is that metals enter the LC after the start of the disease. Stress-induced uptake of toxicants could take place after the onset of MND, because having a disease such as MND is likely to be highly stressful. The finding of heavy metals in the LC could therefore be a result of having the disease, rather than being a trigger for the disease.
Heavy metal staining in extraocular muscle neurons
Extraocular muscles are affected late or not at all in MND
[31]. Extraocular muscle neurons contained the least heavy metal staining of all the motor neurons in the present study, raising the possibility that one reason they are spared is because they take up smaller toxicant loads. This may be because their neuromuscular junctions are different to those of other muscles
[32]. Curiously, 2 out of 3 Parkinson’s disease patients had heavy metals in their extraocular muscle neurons; whether this is related to the oculomotor problems associated with Parkinson’s disease
[33] would require further study.
Phenotypic variation in familial and sporadic MND: do toxicants play a part?
Heavy metal-containing neurons were found in this study in familial, and not only sporadic, MND. One possibility for this is that FMND-associated mutations increase the neuronal uptake of toxicants, possibly by altering the permeability of the blood–brain barrier as has been suggested for SOD1
[34], TARDBP and ANG mutations
[35]. Other FMND mutations could damage mechanisms that protect neurons from toxicants, as has been suggested for a number of MND genetic variants
[36].
The presence of toxicants within LC and motor neurons could also explain the intra-familial variations in age of disease onset and phenotype that is seen in FMND, as well as the inter-patient variations commonly found in sporadic MND. For example, we found that the heavy metal content varied between hypoglossal and spinal motor neurons, a possible reason why bulbar symptoms can appear early, late or not at all in MND.
Heavy metal staining in non-MND disease controls
With the recent finding of LC damage in multiple sclerosis
[37] it is of interest that the LC of one multiple sclerosis patient contained heavy metals. The heavy metal staining within the LC of one individual with alcoholism accords with the finding by some workers that LC neurons are damaged by alcohol excess
[38].
Identification and quantification of heavy metals in neurons
Ideally, AMG should be followed up with some elemental method to confirm the identity of the heavy metal in the tissue. This has been achievable in humans exposed to large amounts mercury or bismuth, where enough of the metal was present in the tissues for quantitative methods to be of use
[12]. However, AMG, being an amplification technique, is more sensitive to the presence of toxicant metals than other currently available techniques of metal detection (such as neutron activation analysis, proton-induced X-ray emission, atomic absorption spectrophotometry, and electron emission X-ray spectrophotometry), though the lower amount of heavy metal that can be detected with AMG has not been ascertained
[10]. Unfortunately, microprobe elemental analyses performed on formalin-fixed paraffin sections are considered to be unreliable
[39]. In our study, only a small proportion of the total number of cells in the pons, brain stem and spinal cord had AMG staining, which suggests only an in situ microprobe technique, carried out on frozen sections, would have a chance of detecting toxicants in the LC. Motor neuron loss in most MND cases was too severe to be able to identify toxicants using elemental analysis.
AMG has been used extensively to study the distribution of metal toxicants in experimental animals, used occasionally in humans with known heavy metal exposure
[2, 12, 40], but used only rarely in humans with no known toxicant exposure
[11]. Humans differ from most non-primate animals in having extensive neuromelanin pigmentation in catecholaminergic neurons, as well as having lipofuscin in many aging neurons, including motor neurons
[41]. It is therefore important to ensure that the AMG staining seen in human neurons is related to heavy metal staining, and not to some unknown reaction within pigmented neurons. The fact that no heavy metal staining was seen in any neuromelanin-containing neurons of the substantia nigra, or in other neurons containing high levels of lipofuscin, indicates that these pigments themselves are unlikely to be the source of the AMG staining.
The heavy metals in this study localised preferentially to the neuromelanin of LC neurons, and to the lipofuscin of motor neurons, probably because these pigments can bind metals
[41]. Neuromelanin in the substantia nigra of neurologically-normal individuals has an affinity for a number of physiologic and exogenous metal ions, including mercury
[42]. It has been suggested that the neuromelanin of the substantia nigra could play different roles in Parkinson’s disease, firstly as a protectant by scavenging toxic substances such as exogenous metals and pesticides, and later as a destructive agent when cell death releases the neuromelanin
[43]. Neuromelanin granules are probably not membrane-bound, and toxicants in unbound neuromelanin would be free to interact with cellular constituents
[41].
Quantitation of the metal content of neuromelanin isolated from the substantia nigra of neurologically-normal individuals shows how neuromelanin can sequester toxic metals that arise from environmental exposure, with many-fold accumulations of mercury (1:96) and lead (1:1408) compared to surrounding tissue (no corresponding data are available for LC neuromelanin)
[44]. Of note, however, was the absence of stainable heavy metals in any substantia nigra neurons in our study, despite the probability of age-related heavy metal sequestration by neuromelanin in these neurons. This suggests that the AMG staining we did see in the LC, especially in densely-stained neurons, represents a concentration of heavy metal that could be physiologically active. Indirect evidence of the potential toxicity of the level of AMG staining seen is also given by the findings from a man who injected himself with a large amount of metallic mercury
[2], after which similar AMG staining of many LC neurons was seen, comparable in particular to the densely-stained neurons in the present study.
Study limitations
Our study has a number of limitations: (1) The number of controls who had both LC and spinal cord tissue available was limited, since spinal cord tissue was usually removed at post mortem from controls only when clinically indicated, and LC-containing blocks were not available from all potential controls. (2) Only one (variable) level of the LC was available for examination, so we were unable to determine if differences in heavy metal content were present in rostral, middle and caudal parts of the LC; of note, cell loss in the LC in Alzheimer's and Parkinson's diseases has been found to vary along the length of the LC
[45]. (3) We could not reliably detect the subceruleus, which in primates is thought to innervate spinal motor neurons
[46]. (4) No histories of toxicant exposure were available, so we do not know if any individuals had exposure to higher than normal levels of mercury (e.g., living near an incinerator or a coal-burning power station) or had been taking bismuth-containing medications. (5) No psychological, psychiatric, or stressful life events histories were available, so we were unable to determine levels of stress before the onset of MND. This is of potential importance, since it has been suggested that stress, by activating the LC, could increase the uptake of toxicants into LC neurons
[2]. (6) As noted above, we were unable to identify the type of heavy metal using paraffin sections, though this is likely to be inorganic mercury since human exposure to bismuth is limited.
Future studies
The finding of potential disease-causing toxicants in a CNS region that is not morphologically damaged by MND has implications for further neurotoxicological investigations into this disease. Attempts to locate toxicants in MND tissues have generally been unsuccessful, probably because toxicant-containing motor neurons have largely disappeared by the time of death, and bulk analysis of tissue is unlikely to detect toxicants affecting a small percentage of cells. An intact LC, however, is compact and easily located, and would be an ideal candidate region for newer microanalysis methods that can detect a range of toxicants
[47].
The heavy metals within LC neurons in the present study did not appear to cause structural damage to the neurons, and only minor changes in LC neuronal size have been reported in MND
[27, 48]. There are however a number of neurodegenerative and psychiatric disorders where structural damage to the LC with associated noradrenaline deficits have been described. Chief among these are Parkinson’s disease, Alzheimer’s disease, major depression and bipolar disorder
[18, 49, 50]. In Alzheimer's disease it has been suggested that the LC is the first region of the brain to be involved by the disease, and at a very early age
[51]. Environmental toxicants have been associated with all these disorders, and a search for toxicants within the LC of these patients could yield interesting results.
